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Sample records for cell protein tyrosine

  1. Structure determination of T-cell protein-tyrosine phosphatase

    DEFF Research Database (Denmark)

    Iversen, L.F.; Møller, K. B.; Pedersen, A.K.;

    2002-01-01

    Protein-tyrosine phosphatase 1B (PTP1B) has recently received much attention as a potential drug target in type 2 diabetes. This has in particular been spurred by the finding that PTP1B knockout mice show increased insulin sensitivity and resistance to diet-induced obesity. Surprisingly, the highly...... homologous T cell protein-tyrosine phosphatase (TC-PTP) has received much less attention, and no x-ray structure has been provided. We have previously co-crystallized PTP1B with a number of low molecular weight inhibitors that inhibit TC-PTP with similar efficiency. Unexpectedly, we were not able to co...... the high degree of functional and structural similarity between TC-PTP and PTP1B, we have been able to identify areas close to the active site that might be addressed to develop selective inhibitors of each enzyme....

  2. Protein tyrosine nitration

    Science.gov (United States)

    Chaki, Mounira; Leterrier, Marina; Barroso, Juan B

    2009-01-01

    Nitric oxide metabolism in plant cells has a relative short history. Nitration is a chemical process which consists of introducing a nitro group (-NO2) into a chemical compound. in biological systems, this process has been found in different molecules such as proteins, lipids and nucleic acids that can affect its function. This mini-review offers an overview of this process with special emphasis on protein tyrosine nitration in plants and its involvement in the process of nitrosative stress. PMID:19826215

  3. Protein-tyrosine phosphatases in zebrafish gastrulation

    NARCIS (Netherlands)

    van Eekelen, M.J.L.

    2011-01-01

    Protein tyrosine phosphorylation plays a key role in relaying external stimuli and signals into the cell towards the appropriate responses. This process is mediated by protein-tyrosine kinases adding a phosphor group to a tyrosine residue and protein-tyrosine phosphatases removing a phosphor group f

  4. Quantitative profiling of spreading-coupled protein tyrosine phosphorylation in migratory cells.

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    Xie, Yajun; Wang, Jinlong; Zhang, Yuanya; Liu, Xiaofei; Wang, Xiaorong; Liu, Kehui; Huang, Xiahe; Wang, Yingchun

    2016-01-01

    Protein tyrosine phosphorylation is an important mechanism that regulates cytoskeleton reorganization and cell spreading of migratory cells. A number of cytoskeletal proteins are known to be tyrosine phosphorylated (pY) in different cellular processes. However, the profile of pY proteins during different stages of cell spreading has not been available. Using immunoafffinity enrichment of pY proteins coupled with label free quantitative proteomics, we quantitatively identified 447 pY proteins in the migratory ECV-304 cells at the early spreading (adhesion) and the active spreading stages. We found that pY levels of the majority of the quantified proteins were significantly increased in the active spreading stage compared with the early spreading stage, suggesting that active cell spreading is concomitant with extra tyrosine phosphorylation. The major categories of proteins impacted by tyrosine phosphorylation are involved in cytoskeleton and focal adhesion regulation, protein translation and degradation. Our findings, for the first time, dissect the cell spreading-specific pY signals from the adhesion induced pY signals, and provide a valuable resource for the future mechanistic research regarding the regulation of cell spreading. PMID:27554326

  5. Bacterial Protein-Tyrosine Kinases

    DEFF Research Database (Denmark)

    Shi, Lei; Kobir, Ahasanul; Jers, Carsten;

    2010-01-01

    in exopolysaccharide production, virulence, DNA metabolism, stress response and other key functions of the bacterial cell. BY-kinases act through autophosphorylation (mainly in exopolysaccharide production) and phosphorylation of other proteins, which have in most cases been shown to be activated by tyrosine......Bacteria and Eukarya share essentially the same family of protein-serine/threonine kinases, also known as the Hanks-type kinases. However, when it comes to protein-tyrosine phosphorylation, bacteria seem to have gone their own way. Bacterial protein-tyrosine kinases (BY-kinases) are bacterial...... and highlighted their importance in bacterial physiology. Having no orthologues in Eukarya, BY-kinases are receiving a growing attention from the biomedical field, since they represent a particularly promising target for anti-bacterial drug design....

  6. Immunohistochemical Analysis of IA-2 Family of Protein Tyrosine Phosphatases in Rat Gastrointestinal Endocrine Cells

    OpenAIRE

    Gomi, Hiroshi; Kubota-Murata, Chisato; Yasui, Tadashi; Tsukise, Azuma; Torii, Seiji

    2013-01-01

    Islet-associated protein–2 (IA-2) and IA-2β (also known as phogrin) are unique neuroendocrine-specific protein tyrosine phosphatases (PTPs). The IA-2 family of PTPs was originally identified from insulinoma cells and discovered to be major autoantigens in type 1 diabetes. Despite its expression in the neural and canonical endocrine tissues, data on expression of the IA-2 family of PTPs in gastrointestinal endocrine cells (GECs) are limited. Therefore, we immunohistochemically investigated the...

  7. Incorporation of Ortho- and Meta-Tyrosine Into Cellular Proteins Leads to Erythropoietin-Resistance in an Erythroid Cell Line

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    Esztella Mikolás

    2014-04-01

    Full Text Available Background/Aims: Erythropoietin-resistance is an unsolved concern in the treatment of renal anaemia. We aimed to investigate the possible role of ortho- and meta-tyrosine - the hydroxyl free radical products of L-phenylalanine - in the development of erythropoietin-resistance. Methods: TF-1 erythroblast cell line was used. Cell concentration was determined on day 1; 2 and 3 by two independent observers simultaneously in Bürker cell counting chambers. Protein concentration was determined with colorimetric method. Para-, ortho- and meta-tyrosine levels were measured using reverse phase-HPLC with fluorescence detection. Using Western blot method activating phosphorylation of STAT5 and ERK1/2 were investigated. Results: We found a time- and concentration-dependent decrease of erythropoietin-induced proliferative activity in case of ortho- and meta-tyrosine treated TF-1 erythroblasts, compared to the para-tyrosine cultured cells. Decreased erythropoietin-response could be regained with a competitive dose of para-tyrosine. Proteins of erythroblasts treated by ortho- or meta-tyrosine had lower para-tyrosine and higher ortho- or meta-tyrosine content. Activating phosphorylation of ERK and STAT5 due to erythropoietin was practically prevented by ortho- or meta-tyrosine treatment. Conclusion: According to this study elevated ortho- and meta-tyrosine content of erythroblasts may lead to the dysfunction of intracellular signaling, resulting in erythropoietin-hyporesponsiveness.

  8. Inhibition of receptor tyrosine kinase signalling by small molecule agonist of T-cell protein tyrosine phosphatase

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    Tähtinen Siri

    2010-01-01

    Full Text Available Abstract Background T-cell protein tyrosine phosphatase (TCPTP/TC45 is a ubiquitously expressed intra-cellular non-receptor protein tyrosine phosphatase involved in the negative regulation of several cancer relevant cellular signalling pathways. We have previously shown that interaction between the α-cytoplasmic tail of α1β1 integrin and TCPTP activates TCPTP by disrupting an inhibitory intra-molecular bond in TCPTP. Thus, inhibition of the regulatory interaction in TCPTP is a desirable strategy for TCPTP activation and attenuation of oncogenic RTK signalling. However, this is challenging with low molecular weight compounds. Methods We developed a high-throughput compatible assay to analyse activity of recombinant TCPTP in vitro. Using this assay we have screened 64280 small molecules to identify novel agonists for TCPTP. Dose-dependent response to TCPTP agonist was performed using the in vitro assay. Inhibition effects and specificity of TCPTP agonists were evaluated using TCPTP expressing and null mouse embryonic fibroblasts. Western blot analysis was used to evaluate attenuation of PDGFRβ and EGFR phosphorylation. Inhibition of VEGF signalling was analysed with VEGF-induced endothelial cell sprouting assays. Results From the screen we identified six TCPTP agonists. Two compounds competed with α1-cytoplasmic domain for binding to TCPTP, suggesting that they activate TCPTP similar to α1-cyt by disrupting the intra-molecular bond in TCPTP. Importantly, one of the compounds (spermidine displayed specificity towards TCPTP in cells, since TCPTP -/- cells were 43-fold more resistant to the compound than TCPTP expressing cells. This compound attenuates PDGFRβ and VEGFR2 signalling in cells in a TCPTP-dependent manner and functions as a negative regulator of EGFR phosphorylation in cancer cells. Conclusions In this study we showed that small molecules mimicking TCPTP-α1 interaction can be used as TCPTP agonists. These data provide the first

  9. Role of Cbl-associated protein/ponsin in receptor tyrosine kinase signaling and cell adhesion

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    Ritva Tikkanen

    2012-10-01

    Full Text Available The Cbl-associated protein/ponsin (CAP is an adaptor protein that contains a so-called Sorbin homology (SoHo domain and three Src homology 3 (SH3 domains which are engaged in diverse protein-protein interactions. CAP has been shown to function in the regulation of the actin cytoskeleton and cell adhesion and to be involved in the differentiation of muscle cells and adipocytes. In addition, it participates in signaling pathways through several receptor tyrosine kinases such as insulin and neurotrophin receptors. In the last couple of years, several studies have shed light on the details of these processes and identified novel interaction partners of CAP. In this review, we summarize these recent findings and provide an overview on the function of CAP especially in cell adhesion and membrane receptor signaling.

  10. Activation of protein tyrosine kinase p72syk by Fc epsilon RI aggregation in rat basophilic leukemia cells. p72syk is a minor component but the major protein tyrosine kinase of pp72.

    Science.gov (United States)

    Minoguchi, K; Benhamou, M; Swaim, W D; Kawakami, Y; Kawakami, T; Siraganian, R P

    1994-06-17

    Aggregation of the high affinity IgE receptors (Fc epsilon RI) on rat basophilic leukemia RBL-2H3 cells results in protein tyrosine phosphorylations. Previously we reported that there is prominent tyrosine phosphorylation of approximately 72-kDa proteins (pp72) and that the tyrosine kinase p72syk is one component of pp72. Here we studied further the relationship of p72syk to pp72. The aggregation of Fc epsilon RI induced the activation of p72syk which was parallel to its tyrosine phosphorylation. By in vitro kinase assay of immune complexes purified with anti-phosphotyrosine antibodies, p72syk was the major pp72 tyrosine kinase. However, by immunoblotting with anti-phosphotyrosine antibodies, p72syk was a minor component of pp72. The heterogeneous nature of pp72 was indicated by different studies. Under optimum conditions of one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis, pp72 consisted of a heterogeneous group of 69-, 71-, and 72-kDa tyrosine-phosphorylated proteins. There were differences in the tyrosine phosphorylation of these proteins in cells activated in the absence of extracellular calcium or when stimulation was with the calcium ionophore A23187 or with phorbol myristate acetate. One of the proteins migrating at 69 kDa was p72syk. By two-dimensional gel electrophoresis pp72 was found to consist of multiple tyrosine-phosphorylated protens including 71-80-kDa proteins that associate with p53/56lyn. A 75-kDa tyrosine-phosphorylated protein, different from pp72, was identified as p75HS1 (SPY75). These results demonstrate the heterogeneous nature of the pp72 and that p72syk is activated after Fc epsilon RI aggregation. PMID:7515887

  11. Recruitment of SHP-1 protein tyrosine phosphatase and signalling by a chimeric T-cell receptor-killer inhibitory receptor

    DEFF Research Database (Denmark)

    Christensen, M D; Geisler, C

    2000-01-01

    Receptors expressing the immunoreceptor tyrosine-based inhibitory motif (ITIM) in their cytoplasmic tail play an important role in the negative regulation of natural killer and B-cell activation. A subpopulation of T cells expresses the ITIM containing killer cell inhibitory receptor (KIR), which...... recognize MHC class I molecules. Following coligation of KIR with an activating receptor, the tyrosine in the ITIM is phosphorylated and the cytoplasmic protein tyrosine phosphatase SHP-1 is recruited to the ITIM via its SH2 domains. It is still not clear how SHP-1 affects T-cell receptor (TCR) signalling....... In this study, we constructed a chimeric TCR-KIR receptor. We demonstrated that SHP-1 is recruited to the chimeric TCR-KIR receptor following T-cell stimulation with either anti-TCR monoclonal antibody (MoAb) or superantigen. However, in spite of this we could not detect any effect of SHP-1 on TCR signalling...

  12. Receptor-type Protein Tyrosine Phosphatase β Regulates Met Phosphorylation and Function in Head and Neck Squamous Cell Carcinoma

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    Yiru Xu

    2012-11-01

    Full Text Available Head and neck squamous cell carcinoma (HNSCC is the sixth most common cancer and has a high rate of mortality. Emerging evidence indicates that hepatocyte growth factor receptor (or Met pathway plays a pivotal role in HNSCC metastasis and resistance to chemotherapy. Met function is dependent on tyrosine phosphorylation that is under direct control by receptor-type protein tyrosine phosphatase β (RPTP-β. We report here that RPTP-β expression is significantly downregulated in HNSCC cells derived from metastatic tumors compared to subject-matched cells from primary tumors. Knockdown of endogenous RPTP-β in HNSCC cells from primary tumor potentiated Met tyrosine phosphorylation, downstream mitogen-activated protein (MAP kinase pathway activation, cell migration, and invasion. Conversely, restoration of RPTP-β expression in cells from matched metastatic tumor decreased Met tyrosine phosphorylation and downstream functions. Furthermore, we observed that six of eight HNSCC tumors had reduced levels of RPTP-β protein in comparison with normal oral tissues. Collectively, the results demonstrate the importance of RPTP-β in tumor biology of HNSCC through direct dephosphorylation of Met and regulation of downstream signal transduction pathways. Reduced RPTP-β levels, with or without Met overexpression, could promote Met activation in HNSCC tumors.

  13. Protein tyrosine phosphatase PTP1 negatively regulates Dictyostelium STATa and is required for proper cell-type proportioning.

    Science.gov (United States)

    Early, A; Gamper, M; Moniakis, J; Kim, E; Hunter, T; Williams, J G; Firtel, R A

    2001-04-01

    The protein tyrosine phosphatase PTP1, which mediates reversible phosphorylation on tyrosine, has been shown to play an important regulatory role during Dictyostelium development. Mutants lacking PTP1 develop more rapidly than normal, while strains that overexpress PTP1 display aberrant morphology. However, the signalling pathways involved have not been characterised. In reexamining these strains, we have found that there is an inverse correlation between levels of PTP1 activity, the extent of tyrosine phosphorylation on Dictyostelium STATa after treatment with cAMP, and the proportion of the slug population exhibiting STATa nuclear enrichment in vivo. This suggests that PTP1 acts to attenuate the tyrosine phosphorylation of STATa and downstream STATa-mediated pathways. Consistent with this, we show that when PTP1 is overexpressed, there is increased expression of a prestalk cell marker at the slug posterior, a phenocopy of STATa null slugs. In ptp1 null strains, STATa tyrosine phosphorylation and nuclear enrichment in the slug anterior is increased. There is also a change in the prestalk to prespore cell ratio. Synergy experiments suggest that this is due to a cell-autonomous defect in forming the subset of prespore cells that are located in the anterior prespore region.

  14. Role of lymphocyte-specific protein tyrosine kinase (LCK) in the expansion of glioma-initiating cells by fractionated radiation

    International Nuclear Information System (INIS)

    Research highlights: → Activation of Lymphocyte-specific protein tyrosine kinase (LCK) is involved in the fractionated radiation-induced expansion of glioma stem-like cells. → Inhibition of LCK prevents acquisition of fractionated radiation-induced resistance to chemotherapeutic treatment. → LCK activity is critical for the maintenance of self-renewal in glioma stem-like cells. -- Abstract: Brain cancers frequently recur or progress as focal masses after treatment with ionizing radiation. Radiation used to target gliomas may expand the cancer stem cell population and enhance the aggressiveness of tumors; however, the mechanisms underlying the expansion of cancer stem cell population after radiation have remained unclear. In this study, we show that LCK (lymphocyte-specific protein tyrosine kinase) is involved in the fractionated radiation-induced expansion of the glioma-initiating cell population and acquisition of resistance to anticancer treatments. Fractionated radiation caused a selective increase in the activity of LCK, a Src family non-receptor tyrosine kinase. The activities of other Src family kinases Src, Fyn, and Lyn were not significantly increased. Moreover, knockdown of LCK expression with a specific small interfering RNA (siRNA) effectively blocked fractionated radiation-induced expansion of the CD133+ cell population. siRNA targeting of LCK also suppressed fractionated radiation-induced expression of the glioma stem cell marker proteins CD133, Nestin, and Musashi. Expression of the known self-renewal-related proteins Notch2 and Sox2 in glioma cells treated with fractionated radiation was also downregulated by LCK inhibition. Moreover, siRNA-mediated knockdown of LCK effectively restored the sensitivity of glioma cells to cisplatin and etoposide. These results indicate that the non-receptor tyrosine kinase LCK is critically involved in fractionated radiation-induced expansion of the glioma-initiating cell population and decreased cellular

  15. α-Ketoacids as precursors for phenylalanine and tyrosine labelling in cell-based protein overexpression.

    Science.gov (United States)

    Lichtenecker, Roman J; Weinhäupl, Katharina; Schmid, Walther; Konrat, Robert

    2013-12-01

    (13)C-α-ketoacid metabolic precursors of phenylalanine and tyrosine effectively enter the metabolism of a protein overexpressing E. coli strain to label Phe- and Tyr-residues devoid of any cross-labelling. The methodology gives access to highly selective labelling patterns as valuable tools in protein NMR spectroscopy without the need of (15)N-chiral amino acid synthesis using organic chemistry.

  16. Astragaloside Ⅱ triggers T cell activation through regulation of CD45 protein tyrosine phosphatase activity

    Institute of Scientific and Technical Information of China (English)

    Chun-ping WAN; Li-xin GAO; Li-fei HOU; Xiao-qian YANG; Pei-lan HE; Yi-fu YANG; Wei TANG

    2013-01-01

    Aim:To investigate the immunomodulating activity of astragalosides,the active compounds from a traditional tonic herb Astragalus membranaceus Bge,and to explore the molecular mechanisms underlying the actions,focusing on CD45 protein tyrosine phosphatase (CD45 PTPase),which plays a critical role in T lymphocyte activation.Methods:Primary splenocytes and T cells were prepared from mice.CD45 PTPase activity was assessed using a colorimetric assay.Cell proliferation was measured using a [3H]-thymidine incorporation assay.Cytokine proteins and mRNAs were examined with ELISA and RT-PCR,respectively.Activation markers,including CD25 and CD69,were analyzed using flow cytometry.Activation of LCK (Tyr505) was detected using Western blot analysis.Mice were injected with the immunosuppressant cyclophosphamide (CTX,80 mg/kg),and administered astragaloside Ⅱ (50 mg/kg).Results:Astragaloside Ⅰ,Ⅱ,Ⅲ,and Ⅳ concentration-dependently increased the CD45-mediated of pNPP/OMFP hydrolysis with the EC50 values ranged from 3.33 to 10.42 μg/mL.Astragaloside Ⅱ (10 and 30 μg/mL) significantly enhanced the proliferation of primary splenocytes induced by ConA,alloantigen or anti-CD3.Astragaloside Ⅱ (30 μg/mL) significantly increased IL-2 and IFN-y secretion,upregulated the mRNA levels of IFN-y and T-bet in primary splenocytes,and promoted CD25 and CD69 expression on primary CD4+T cells upon TCR stimulation.Furthermore,astragaloside Ⅱ (100 ng/mL) promoted CD45-mediated dephosphorylation of LCK (Tyr505) in primary T cells,which could be blocked by a specific CD45 PTPase inhibitor.In CTX-induced immunosuppressed mice,oral administration of astragaloside Ⅱ restored the proliferation of splenic T cells and the production of IFN-Y and IL-2.However,astragaloside Ⅱ had no apparent effects on B cell proliferation.Conclusion:Astragaloside Ⅱ enhances T cell activation by regulating the activity of CD45 PTPase,which may explain why Astragalus membranaceus Bge is used as a tonic

  17. Identification of tyrosine-phosphorylated proteins associated with metastasis and functional analysis of FER in human hepatocellular carcinoma cells

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    Wang Yan

    2009-10-01

    Full Text Available Abstract Background- Aberrant activity of tyrosine-phosphorylated proteins is commonly associated with HCC metastasis. Cell signaling events driven by these proteins are implicated in numerous processes that alter cancer cell behavior. Exploring the activities and signaling pathways of these proteins in HCC metastasis may help in identifying new candidate molecules for HCC-targeted therapy. Methods- Hep3B (a nonmetastatic HCC cell line and MHCC97H (a highly metastatic HCC cell line were used in this study, and the tyrosine-phosphorylated proteins expressed in these cell lines were profiled by a phosphoproteomics technique based on LC-MS/MS. Protein-protein interaction and functional clustering analyses were performed to determine the activities of the identified proteins and the signaling pathways closely related to HCC metastasis. Results- In both cell lines, a total of 247 phosphotyrosine (pTyr proteins containing 281 pTyr sites were identified without any stimulation. The involvement of almost 30% of these in liver or liver cancer has not been reported previously. Biological process clustering analysis indicated that pTyr proteins involved in cell motility, migration, protein autophosphorylation, cell-cell communication, and antiapoptosis functions were overexpressed during metastasis. Pathway clustering analysis revealed that signaling pathways such as those involved in EGFR signaling, cytokine- and chemokine-mediated signal transduction, and the PI3K and JAK-STAT cascades were significantly activated during HCC metastasis. Moreover, noncanonical regulation of the JNK cascade might also provide new targets for HCC metastasis. After comparing the pTyr proteins that were differentially expressed during HCC cell metastasis, we selected FER, a nonreceptor tyrosine kinase, and validated its role in terms of both expression and function. The data confirmed that FER might play a critical role in the invasion and metastasis of HCC. Conclusion- The

  18. Protein tyrosine kinases p53/56lyn and p72syk in MHC class I-mediated signal transduction in B lymphoma cells

    DEFF Research Database (Denmark)

    Pedersen, Anders Elm; Bregenholt, S; Skov, S;

    1998-01-01

    Crosslinking of major histocompatibility complex class I (MHC-I) molecules on the surface of human B lymphoma cells was shown to induce protein tyrosine phosphorylation and mobilization of intracellular free calcium. Immunoprecipitations indicated that the protein tyrosine kinases p53/56lyn and p72......syk are among the tyrosine-phosphorylated proteins. The kinetics of phosphorylation of these kinases after MHC-I crosslinking differ from the kinetics observed after crosslinking of the B cell antigen receptor (BCR). Additional experiments were performed with chicken lyn- and syk-negative DT40 B cells...... and the results indicate that these two kinases have different substrate specificity and regulate intracellular free calcium differently in response to MHC-I crosslinking. In addition MHC-I crosslinking of a sIgM-negative DT40 chicken B cell variant results in less activity of tyrosine kinases and less...

  19. Src family protein tyrosine kinases induce autoactivation of Bruton's tyrosine kinase.

    OpenAIRE

    Mahajan, S.; Fargnoli, J.; Burkhardt, A L; Kut, S A; Saouaf, S J; Bolen, J B

    1995-01-01

    Bruton's tyrosine kinase (Btk) is tyrosine phosphorylated and enzymatically activated following ligation of the B-cell antigen receptor. These events are temporally regulated, and Btk activation follows that of various members of the Src family of protein tyrosine kinases, thus raising the possibility that Src kinases participate in the Btk activation process. We have evaluated the mechanism underlying Btk enzyme activation and have explored the potential regulatory relationship between Btk a...

  20. Implication of protein tyrosine phosphatase 1B in MCF-7 cell proliferation and resistance to 4-OH tamoxifen

    Energy Technology Data Exchange (ETDEWEB)

    Blanquart, Christophe; Karouri, Salah-Eddine [Institut Cochin, Universite Paris Descartes, CNRS (UMR 8104), Paris (France); Inserm, U567, Paris (France); Issad, Tarik, E-mail: tarik.issad@inserm.fr [Institut Cochin, Universite Paris Descartes, CNRS (UMR 8104), Paris (France); Inserm, U567, Paris (France)

    2009-10-02

    The protein tyrosine phosphatase 1B (PTP1B) and the T-cell protein tyrosine phosphatase (TC-PTP) were initially thought to be mainly anti-oncogenic. However, overexpression of PTP1B and TC-PTP has been observed in human tumors, and recent studies have demonstrated that PTP1B contributes to the appearance of breast tumors by modulating ERK pathway. In the present work, we observed that decreasing the expression of TC-PTP or PTP1B in MCF-7 cells using siRNA reduced cell proliferation without affecting cell death. This reduction in proliferation was associated with decreased ERK phosphorylation. Moreover, selection of tamoxifen-resistant MCF-7 cells, by long-term culture in presence of 4-OH tamoxifen, resulted in cells that display overexpression of PTP1B and TC-PTP, and concomitant increase in ERK and STAT3 phosphorylation. siRNA experiments showed that PTP1B, but not TC-PTP, is necessary for resistance to 4-OH tamoxifen. Therefore, our work indicates that PTP1B could be a relevant therapeutic target for treatment of tamoxifen-resistant breast cancers.

  1. A novel role of protein tyrosine kinase2 in mediating chloride secretion in human airway epithelial cells.

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    Lihua Liang

    Full Text Available Ca(2+ activated Cl(- channels (CaCC are up-regulated in cystic fibrosis (CF airway surface epithelia. The presence and functional properties of CaCC make it a possible therapeutic target to compensate for the deficiency of Cl(- secretion in CF epithelia. CaCC is activated by an increase in cytosolic Ca(2+, which not only activates epithelial CaCCs, but also inhibits epithelial Na(+ hyperabsorption, which may also be beneficial in CF. Our previous study has shown that spiperone, a known antipsychotic drug, activates CaCCs and stimulates Cl(- secretion in polarized human non-CF and CF airway epithelial cell monolayers in vitro, and in Cystic Fibrosis Transmembrane Conductance Regulator (CFTR knockout mice in vivo. Spiperone activates CaCC not by acting in its well-known role as an antagonist of either 5-HT2 or D2 receptors, but through a protein tyrosine kinase-coupled phospholipase C-dependent pathway. Moreover, spiperone independently activates CFTR through a novel mechanism. Herein, we performed a mass spectrometry analysis and identified the signaling molecule that mediates the spiperone effect in activating chloride secretion through CaCC and CFTR. Proline-rich tyrosine kinase 2 (PYK2 is a non-receptor protein tyrosine kinase, which belongs to the focal adhesion kinase family. The inhibition of PYK2 notably reduced the ability of spiperone to increase intracellular Ca(2+ and Cl(- secretion. In conclusion, we have identified the tyrosine kinase, PYK2, as the modulator, which plays a crucial role in the activation of CaCC and CFTR by spiperone. The identification of this novel role of PYK2 reveals a new signaling pathway in human airway epithelial cells.

  2. ADHESION-INDUCE PROTEIN TYROSINE PHOSPHORY-LATION IS ASSOCIATED WITH INVASIVE AND METASTATIC POTENTIALS IN B16-BL6 MELANOMA CELLS

    Institute of Scientific and Technical Information of China (English)

    Yan Chunhong; Han Rui

    1998-01-01

    Objective: The interaction of cancer cell with extracellular matrix (ECM) happens as an earlier and specific event in the invasive and metastatic cascade. To explore the key element(s) in cancer metastasis and observe the cell-ECM interaction and its role. Methods:To interrupt the cell-ECM interaction by suppression of adhesion-induced protein tyrosine phosphorylation with protein tyrosine kinase inhibitor genistein in B16-B16mouse melanoma cells. Results: When B16-BL6 cells attached to Matrigel, a solubilized basement membrane preparation from EHS sarcoma, a 125 kDa protein increased its phosphotyrosine content dramatically. In contrast, when the cells were pretreated with 20μM or 30μM genistein for 3 days, it was revealed a less increase in the phosphotyrosine content of this 125 kDa protein inresponse to cell attachment to ECM was revealed with immunoblot analysis. Accompanied by the lower level of adhesion-induced protein tyrosine phosphorylation the genistein-treated cells exhibited a decrease in their capabilities of adhesion to Matrigel and invasion through reconstituted basement membrane. The potentials of and forming lung metastatic nodules were also shown to be decreased dramatically in these genistein-treated cells.Conclusion: It was suggested that protein tyrosine phosphorylation in cell-ECM interaction might be associated with invasive and metastatic potentials in cancer cells.

  3. MHC-I-induced apoptosis in human B-lymphoma cells is dependent on protein tyrosine and serine/threonine kinases

    DEFF Research Database (Denmark)

    Pedersen, Anders Elm; Bregenholt, S; Johansen, B;

    1999-01-01

    B lymphoma cells, is dependent on protein tyrosine kinases and the phosphatidylinositol 3 (PI-3) kinase. Functional studies showed that MHC-I crosslinking induced almost complete inhibition of the spontaneous proliferation of the B lymphoma cells as early as 6 h post-crosslinking and apoptosis 24 h...... post-crosslinking. Preincubation with either protein tyrosine kinase or protein serine/threonine kinase inhibitors reduced the MHC-I-induced apoptosis to background levels, whereas inhibition of PI-3 kinase had no effect. These data demonstrate a pivotal role for protein tyrosine and serine....../threonine kinases in MHC-I-mediated apoptosis in human B-cells and suggest the presence of several MHC-I signaling pathways leading to diverse effects in these cells....

  4. Loss of Protein Tyrosine Phosphatase Receptor J Expression Predicts an Aggressive Clinical Course in Patients with Esophageal Squamous Cell Carcinoma.

    Science.gov (United States)

    Qiao, Dongfeng; Li, Ming; Pu, Juan; Wang, Wanwei; Zhu, Weiguo; Liu, Haiyan

    2016-07-01

    Protein Tyrosine Phosphatase Receptor J (PTPRJ) has been reported to be a tumor suppressor in various human cancers. The aim of this study was to investigate the clinical significance of PTPRJ in ESCC patients and its effects on biological behaviors of ESCC cells. PTPRJ expression, at mRNA and protein levels, were respectively detected by quantitative real-time PCR, western blot and immunohistochemistry, based on 106 newly diagnosed ESCC patients. The associations between PTPRJ expression and clinicopathological characteristics of ESCC patients were statistically analyzed. Then, the effects of PTPRJ in migration and invasion were determined by wound healing and transwell assays based on ESCC cell line transfected with siRNA or expression vector of PTPRJ. Expression of PTPRJ at mRNA and protein levels were both significantly lower in ESCC tissues than those in normal esophageal mucosa. Immunohistochemistry showed that PTPRJ protein was localized in the cytoplasm of cancer cells in ESCC tissues. In addition, PTPRJ downregulation was found to be closely correlated with advanced tumor stage (P = 0.01) and poor differentiation (P = 0.03). Moreover, knockdown of PTPRJ in KYSE510 cells could significantly promote cell migration and invasion (both P ESCC patients. PTPRJ may function as a tumor suppressor and play an important role in the regulation of ESCC cell motility, suggesting its potentials as a therapeutic agent for human ESCC.

  5. A tyrosine-rich cell surface protein in the diatom Amphora coffeaeformis identified through transcriptome analysis and genetic transformation.

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    Matthias T Buhmann

    Full Text Available Diatoms are single-celled eukaryotic microalgae that are ubiquitously found in almost all aquatic ecosystems, and are characterized by their intricately structured SiO2 (silica-based cell walls. Diatoms with a benthic life style are capable of attaching to any natural or man-made submerged surface, thus contributing substantially to both microbial biofilm communities and economic losses through biofouling. Surface attachment of diatoms is mediated by a carbohydrate- and protein- based glue, yet no protein involved in diatom underwater adhesion has been identified so far. In the present work, we have generated a normalized transcriptome database from the model adhesion diatom Amphora coffeaeformis. Using an unconventional bioinformatics analysis we have identified five proteins that exhibit unique amino acid sequences resembling the amino acid composition of the tyrosine-rich adhesion proteins from mussel footpads. Establishing the first method for the molecular genetic transformation of A. coffeaeformis has enabled investigations into the function of one of these proteins, AC3362, through expression as YFP fusion protein. Biochemical analysis and imaging by fluorescence microscopy revealed that AC3362 is not involved in adhesion, but rather plays a role in biosynthesis and/or structural stability of the cell wall. The methods established in the present study have paved the way for further molecular studies on the mechanisms of underwater adhesion and biological silica formation in the diatom A. coffeaeformis.

  6. Natural compounds as a source of protein tyrosine phosphatase inhibitors : Application to the rational design of small-molecule derivatives

    NARCIS (Netherlands)

    Ferreira, Carmen V.; Justo, Giselle Z.; Souza, Ana C. S.; Queiroz, Karla C. S.; Zambuzzi, William F.; Aoyama, Hiroshi; Peppelenbosch, Maikel P.

    2006-01-01

    Reversible phosphorylation of tyrosine residues is a key regulatory mechanism for numerous cellular events. Protein tyrosine kinases and protein tyrosine phosphatases (PTPs) have a pivotal role in regulating both normal cell physiology and pathophysiology. Accordingly, deregulated activity of both p

  7. Temporal differences in the activation of three classes of non-transmembrane protein tyrosine kinases following B-cell antigen receptor surface engagement.

    Science.gov (United States)

    Saouaf, S J; Mahajan, S; Rowley, R B; Kut, S A; Fargnoli, J; Burkhardt, A L; Tsukada, S; Witte, O N; Bolen, J B

    1994-09-27

    We evaluated in WEHI 231 B cells the time-dependent responses of Lyn, Blk, Btk, Syk, and three members of the Jak family of protein tyrosine kinases following antibody-mediated surface engagement of the B-cell antigen receptor. Our results show that the enzyme activities of Lyn and Blk were stimulated within seconds of antigen receptor engagement and correlated with the initial tyrosine phosphorylation of the Ig alpha and Ig beta subunits of the B-cell antigen receptor. Btk enzyme activity was also transiently stimulated and was maximal at approximately 5 min after B-cell receptor surface binding. Syk activity gradually increased to a maximum at 10-30 min following receptor ligation and was found to parallel the association of Syk with the tyrosine phosphorylated Ig alpha and Ig beta subunits of the receptor. While the specific activities of the Jak1, Jak2, and Tyk2 protein tyrosine kinases were unaltered following B-cell receptor ligation, the abundance of Jak1 and Jak2 were increased 3- to 4-fold within 10 min of receptor engagement. These results demonstrate that multiple families of non-transmembrane protein tyrosine kinases are temporally regulated during the process of B-cell antigen receptor-initiated intracellular signal transduction. PMID:7524079

  8. SH-2-containing protein tyrosine phosphatase 1 is required for IL-4-induced IL-4R expression in spleen cells

    Institute of Scientific and Technical Information of China (English)

    CAO Xin; HUANG Zan; FAN Jingyi

    2005-01-01

    To investigate the role of SH-2-containing protein tyrosine phosphatase 1, SHP-1, in IL-4-induced IL-4 receptor (IL-4R) expression, we examined IL-4 receptor α-chain (IL-4Rα) mRNA expression in Na3VO4-treated wild type (WT) spleen cells and measured IL-4R mRNA in IL-4-stimulated spleen cells of viable motheaten mice (mev/mev). It is found that IL-4-induced IL-4R mRNA expression was impaired in Na3VO4-treated WT spleen cells and IL-4-stimulated mev/mev spleen cells. Here we show that the impaired IL-4-induced IL-4RαmRNA expression was due to reduced expression of IL-4R that led to impaired STAT6 signaling. We further demonstrate that reduction of IL-4Rαprotein expression in mev/mev spleen cells was due to alteration in cell compositions. In mev/mev spleen, the percentages of CD4+, CD8+, and CD19+ cells expressing relatively high levels of IL-4R were reduced dramatically while the percentages of Mac-1+ and Gr-1+ cells with relative low levels of IL-4R increased greatly. Despite the profound effect of reduced expression of IL-4R protein, the IL-4RαmRNA expression was comparable in spleen cells of littermate control mice (+/() and mev/mev mice and no differences were found in B cells, T cells, and macrophages, suggesting cell type-specific downregulation of IL-4R expression in macrophages through a posttranscriptional mechanism. Our study suggests that SHP-1 is required for IL-4-meidated function and indirectly regulates IL-4-meidated function in spleen cells by affecting hematopoiesis.

  9. Src family protein tyrosine kinases induce autoactivation of Bruton's tyrosine kinase.

    Science.gov (United States)

    Mahajan, S; Fargnoli, J; Burkhardt, A L; Kut, S A; Saouaf, S J; Bolen, J B

    1995-10-01

    Bruton's tyrosine kinase (Btk) is tyrosine phosphorylated and enzymatically activated following ligation of the B-cell antigen receptor. These events are temporally regulated, and Btk activation follows that of various members of the Src family of protein tyrosine kinases, thus raising the possibility that Src kinases participate in the Btk activation process. We have evaluated the mechanism underlying Btk enzyme activation and have explored the potential regulatory relationship between Btk and Src protein kinases. We demonstrate in COS transient-expression assays that Btk can be activated through intramolecular autophosphorylation at tyrosine 551 and that Btk autophosphorylation is required for Btk catalytic functions. Coexpression of Btk with members of the Src family of protein tyrosine kinases, but not Syk, led to Btk tyrosine phosphorylation and activation. Using a series of point mutations in Blk (a representative Src protein kinase) and Btk, we show that Src kinases activate Btk through an indirect mechanism that requires membrane association of the Src enzymes as well as functional Btk SH3 and SH2 domains. Our results are compatible with the idea that Src protein tyrosine kinases contribute to Btk activation by indirectly stimulating Btk intramolecular autophosphorylation. PMID:7565679

  10. Expression of protein tyrosine phosphatase alpha (RPTPalpha) in human breast cancer correlates with low tumor grade, and inhibits tumor cell growth in vitro and in vivo

    DEFF Research Database (Denmark)

    Ardini, E; Agresti, R; Tagliabue, E;

    2000-01-01

    of Src family kinases, and regulation of integrin signaling, cell adhesion, and growth factor responsiveness. To explore its potential contribution to human neoplasia, we surveyed RPTPalpha protein levels in primary human breast cancer. We found RPTPalpha levels to vary widely among tumors, with 29......% of cases manifesting significant overexpression. High RPTPalpha protein levels correlated significantly with low tumor grade and positive estrogen receptor status. Expression of RPTPalpha in breast carcinoma cells led to growth inhibition, associated with increased accumulation in G0 and G1, and delayed......Tyrosine phosphorylation is controlled by a balance of tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs). Whereas the contribution of PTKs to breast tumorigenesis is the subject of intense scrutiny, the potential role of PTPs is poorly known. RPTPalpha is implicated in the activation...

  11. Fulvestrant-induced cell death and proteasomal degradation of estrogen receptor α protein in MCF-7 cells require the CSK c-Src tyrosine kinase.

    Directory of Open Access Journals (Sweden)

    Wei-Lan Yeh

    Full Text Available Fulvestrant is a representative pure antiestrogen and a Selective Estrogen Receptor Down-regulator (SERD. In contrast to the Selective Estrogen Receptor Modulators (SERMs such as 4-hydroxytamoxifen that bind to estrogen receptor α (ERα as antagonists or partial agonists, fulvestrant causes proteasomal degradation of ERα protein, shutting down the estrogen signaling to induce proliferation arrest and apoptosis of estrogen-dependent breast cancer cells. We performed genome-wide RNAi knockdown screenings for protein kinases required for fulvestrant-induced apoptosis of the MCF-7 estrogen-dependent human breast caner cells and identified the c-Src tyrosine kinase (CSK, a negative regulator of the oncoprotein c-Src and related protein tyrosine kinases, as one of the necessary molecules. Whereas RNAi knockdown of CSK in MCF-7 cells by shRNA-expressing lentiviruses strongly suppressed fulvestrant-induced cell death, CSK knockdown did not affect cytocidal actions of 4-hydroxytamoxifen or paclitaxel, a chemotherapeutic agent. In the absence of CSK, fulvestrant-induced proteasomal degradation of ERα protein was suppressed in both MCF-7 and T47D estrogen-dependent breast cancer cells whereas the TP53-mutated T47D cells were resistant to the cytocidal action of fulvestrant in the presence or absence of CSK. MCF-7 cell sensitivities to fulvestrant-induced cell death or ERα protein degradation was not affected by small-molecular-weight inhibitors of the tyrosine kinase activity of c-Src, suggesting possible involvement of other signaling molecules in CSK-dependent MCF-7 cell death induced by fulvestrant. Our observations suggest the importance of CSK in the determination of cellular sensitivity to the cytocidal action of fulvestrant.

  12. Temporal differences in the activation of three classes of non-transmembrane protein tyrosine kinases following B-cell antigen receptor surface engagement.

    OpenAIRE

    Saouaf, S J; Mahajan, S.; Rowley, R B; Kut, S A; Fargnoli, J.; Burkhardt, A L; Tsukada, S; Witte, O. N.; Bolen, J B

    1994-01-01

    We evaluated in WEHI 231 B cells the time-dependent responses of Lyn, Blk, Btk, Syk, and three members of the Jak family of protein tyrosine kinases following antibody-mediated surface engagement of the B-cell antigen receptor. Our results show that the enzyme activities of Lyn and Blk were stimulated within seconds of antigen receptor engagement and correlated with the initial tyrosine phosphorylation of the Ig alpha and Ig beta subunits of the B-cell antigen receptor. Btk enzyme activity wa...

  13. Selective expression of a protein-tyrosine kinase, p56lyn, in hematopoietic cells and association with production of human T-cell lymphotropic virus type I

    International Nuclear Information System (INIS)

    This paper reports the identification of the lyn gene product, a member of the src-related family of protein-tyrosine kinases, and its expression in hematopoietic cells. A lyn-specific sequence (Arg-25 to Ala-119 of the protein) was expressed in Escherichia coli as a fusion protein with β-galactosidase. Antiserum raised against the fusion protein immunoprecipitated a 56-kDa protein from human B lymphocytes. Incubation of the immunoprecipitate with [γ-32P]ATP resulted in the phosphorylation of this protein at tyrosine residues. Immunohistological and immunoblotting analyses showed that the lyn gene product was expressed in lymphatic tissues (spleen and tonsil) and in adult lung, which contains many macrophages. Furthermore, both the transcripts and the protein products of the lyn gene accumulated in macrophages/monocytes, platelets, and B lymphocytes but were not expressed appreciably in granulocytes, erythrocytes, or T lymphocytes, suggesting that lyn gene products function primarily in certain differentiated cells of lymphoid and myeloid lineages

  14. A complex between contactin-1 and the protein tyrosine phosphatase PTPRZ controls the development of oligodendrocyte precursor cells

    Energy Technology Data Exchange (ETDEWEB)

    Lamprianou, Smaragda; Chatzopoulou, Elli; Thomas, Jean-Léon; Bouyain, Samuel; Harroch, Sheila (IP-Korea); (UPMC); (UMKC)

    2013-09-23

    The six members of the contactin (CNTN) family of neural cell adhesion molecules are involved in the formation and maintenance of the central nervous system (CNS) and have been linked to mental retardation and neuropsychiatric disorders such as autism. Five of the six CNTNs bind to the homologous receptor protein tyrosine phosphatases gamma (PTPRG) and zeta (PTPRZ), but the biological roles of these interactions remain unclear. We report here the cocrystal structure of the carbonic anhydrase-like domain of PTPRZ bound to tandem Ig repeats of CNTN1 and combine these structural data with binding assays to show that PTPRZ binds specifically to CNTN1 expressed at the surface of oligodendrocyte precursor cells. Furthermore, analyses of glial cell populations in wild-type and PTPRZ-deficient mice show that the binding of PTPRZ to CNTN1 expressed at the surface of oligodendrocyte precursor cells inhibits their proliferation and promotes their development into mature oligodendrocytes. Overall, these results implicate the PTPRZ/CNTN1 complex as a previously unknown modulator of oligodendrogenesis.

  15. Crk adaptor protein-induced phosphorylation of Gab1 on tyrosine 307 via Src is important for organization of focal adhesions and enhanced cell migration

    Institute of Scientific and Technical Information of China (English)

    Takuya Watanabe; Masumi Tsuda; Yoshinori Makino; Tassos Konstantinou; Hiroshi Nishihara; Tokifumi Majima; Akio Minami; Stephan M Feller; Shinya Tanaka

    2009-01-01

    Upon growth factor stimulation, the scaffold protein, Gabl, is tyrosine phosphorylated and subsequently the adaptor protein, Crk, transmits signals from Gabl. We have previously shown that Crk overexpression, which is detectable in various human cancers, induces tyrosine phosphorylation of Gab1 without extraceilular stimuli. In the present study, the underlying mechanisms were further investigated. Mutational analyses of Crkll demonstrated that the SH2 domain, but not the SH3(N) or the regulatory Y221 residue of Crkll, is critical for the induction of Gabl-Y307 phosphorylation. SH2 mutation of Crkll also decreased the interaction with Gab1. In GST pull-down assay, Crk-SH2 bound to wild-type Gabl, whereas Crk-SH3(N) interacted with the Gabl mutant, which lacks the clus-tered tyrosine region (residues 242-410). Tyrosine phosphorylation of Gabl was induced by all Crk family proteins, but not other SH2-containing signalling adaptors. Src-family kinase inhibitor, PP2, abrogates Crk-induced tyrosine phosphorylations of Gabl. Y307 phosphorylation was undetectable in fibroblasts lacking Src, Yes, and Fyn, even upon overexpression of Crk, whereas cells lacking only Yes and Fyn still contained Gabl with phosphorylated Y307. Furthermore, Crk induced the phosphorylation of Src-Y416; accordingly the interaction between Crk and Csk was increased. The GabI-Y307F mutant failed to localize near the plasma membrane even upon HGF stimulation and decreased cell migration. Moreover, Gabl-Y307F disturbed the localization of Crk, FAK, and paxiilin, which are the typical components of focal adhesions. Taken together, these results indicate that Crk facilitates tyrosine phosphory-lation of Gabl-Y307 through Src, contributing to the organization of focal adhesions and enhanced cell migration, thereby possibly promoting human cancer development.

  16. Redox Regulation of Protein Tyrosine Phosphatase Activity by Hydroxyl Radical

    OpenAIRE

    Meng, Fan-Guo; Zhang, Zhong-Yin

    2012-01-01

    Substantial evidence suggests that transient production of reactive oxygen species (ROS) such as hydrogen peroxide (H2O2) is an important signaling event triggered by the activation of various cell surface receptors. Major targets of H2O2 include protein tyrosine phosphatases (PTPs). Oxidation of the active site Cys by H2O2 abrogates PTP catalytic activity, thereby potentially furnishing a mechanism to ensure optimal tyrosine phosphorylation in response to a variety of physiological stimuli. ...

  17. Suppression of PKG by PDGF or nitric oxide in differentiated aortic smooth muscle cells: obligatory role of protein tyrosine phosphatase 1B

    OpenAIRE

    Zhuang, Daming; Balani, Poonam; Pu, Qinghua; Thakran, Shalini; Hassid, Aviv

    2010-01-01

    Treatment of aortic smooth muscle cells with PDGF induces the upregulation of protein tyrosine phosphatase 1B (PTP1B). PTP1B, in turn, decreases the function of several growth factor receptors, thus completing a negative feedback loop. Studies have reported that PDGF induces the downregulation of PKG as part of a repertoire of dedifferentiation of vascular smooth muscle cells. Other studies have reported that chronic nitric oxide (NO) treatment also induces the downregulation of PKG. In the p...

  18. Regulation of tumor cell migration by protein tyrosine phosphatase (PTP)-proline-, glutamate-, serine-,and threonine-rich sequence (PEST)

    Institute of Scientific and Technical Information of China (English)

    Yanhua Zheng; Zhimin Lu

    2013-01-01

    Protein tyrosine phosphatase (PTP)-proline-,glutamate-,serine-,and threonine-rich sequence (PEST) is ubiquitously expressed and is a critical regulator of cell adhesion and migration.PTP-PEST activity can be regulated transcriptionally via gene deletion or mutation in several types of human cancers or via post-translational modifications,including phosphorylation,oxidation,and caspase-dependent cleavage.PTP-PEST interacts with and dephosphorylates cytoskeletal and focal adhesion-associated proteins.Dephosphorylation of PTP-PEST substrates regulates their enzymatic activities and/or their interaction with other proteins and plays an essential role in the tumor cell migration process.

  19. THE EFFECTS OF HUMAN CHORIONIC GONADOTROPIN AND TYROSINE PROTEIN KINASE ON THE GROWTHOF HYBRIDOMA CELLS

    Institute of Scientific and Technical Information of China (English)

    HANShou-Wei; LIUShah-Ling; CAOZe-Yi; CHENMan-Ling

    1989-01-01

    In recent years, the production and development of receptor monoclonal antibodies (McAB) have been attentively studied. Wc observed the effects of human ehorionicgonadotropin (HCG) and tyrosinc protein kinase (TPK) on the growth of two hybridoma

  20. Protein tyrosine phosphatase 1B deficiency ameliorates murine experimental colitis via the expansion of myeloid-derived suppressor cells.

    Directory of Open Access Journals (Sweden)

    Jing Zhang

    Full Text Available Protein tyrosine phosphatase 1B (PTP1B is a key molecule in modulating low-degree inflammatory conditions such as diabetes. The role of PTP1B in other chronic inflammations, however, remains unknown. Here, we report that PTP1B deficiency ameliorates Dextran Sulfate Sodium (DSS-induced murine experimental colitis via expanding CD11b(+Gr-1(+ myeloid-derived suppressor cells (MDSCs. Employing DSS-induced murine experimental colitis as inflammatory animal model, we found that, compared with wild-type littermates, PTP1B-null mice demonstrated greater resistance to DSS-induced colitis, as reflected by slower weight-loss, greater survival rates and decreased PMN and macrophage infiltration into the colon. The evidence collectively also demonstrated that the resistance of PTP1B-null mice to DSS-induced colitis is based on the expansion of MDSCs. First, PTP1B-null mice exhibited a greater frequency of MDSCs in the bone marrow (BM, peripheral blood and spleen when compared with wild-type littermates. Second, PTP1B levels in BM leukocytes were significantly decreased after cells were induced into MDSCs by IL-6 and GM-CSF, and the MDSC induction occurred more rapidly in PTP1B-null mice than in wild-type littermates, suggesting PTP1B as a negative regulator of MDSCs. Third, the adoptive transfer of MDSCs into mice with DSS-colitis significantly attenuated colitis, which accompanies with a decreased serum IL-17 level. Finally, PTP1B deficiency increased the frequency of MDSCs from BM cells likely through enhancing the activities of signal transducer and activator of transcription 3 (STAT3 and Janus kinase 2 (JAK2. In conclusion, our study provides the first evidences that PTP1B deficiency ameliorates murine experimental colitis via expanding MDSCs.

  1. Chlamydia trachomatis serovar L2 induces protein tyrosine phosphorylation during uptake by HeLa cells

    DEFF Research Database (Denmark)

    Birkelund, Svend; Johnsen, H; Christiansen, Gunna

    1994-01-01

    inactive. Attachment of EBs to host cells is medicated by a heparan sulfate-like glycosaminoglycan. Following attachment, the EB is internalized within a membrane-bound vesicle, and during the first 8 h of infection the vesicles are transported to a perinuclear location where they aggregate and fuse...

  2. Receptor tyrosine phosphatase R-PTP-alpha is tyrosine-phosphorylated and associated with the adaptor protein Grb2

    DEFF Research Database (Denmark)

    Su, J; Batzer, A; Sap, J

    1994-01-01

    -phosphorylated and associated in vivo with the Grb2 protein. This association can be reproduced in stably and transiently transfected cells, as well as in vitro using recombinant Grb2 protein. Association requires the presence of an intact SH2 domain in Grb2, as well as tyrosine phosphorylation of R-PTP-alpha. This observation......Receptor tyrosine phosphatases (R-PTPases) have generated interest because of their suspected involvement in cellular signal transduction. The adaptor protein Grb2 has been implicated in coupling receptor tyrosine kinases to Ras. We report that a ubiquitous R-PTPase, R-PTP-alpha, is tyrosine...... links a receptor tyrosine phosphatase with a key component of a central cellular signalling pathway and provides a basis for addressing R-PTP-alpha function....

  3. Enzyme kinetic characterization of protein tyrosine phosphatases

    DEFF Research Database (Denmark)

    Peters, Günther H.J.; Branner, S.; Møller, K. B.;

    2003-01-01

    Protein tyrosine phosphatases (PTPs) play a central role in cellular signaling processes, resulting in an increased interest in modulating the activities of PTPs. We therefore decided to undertake a detailed enzyme kinetic evaluation of various transmembrane and cytosolic PTPs (PTPalpha, PTPbeta...

  4. Conditional Knockout of Src Homology 2 Domain-containing Protein Tyrosine Phosphatase-2 in Myeloid Cells Attenuates Renal Fibrosis after Unilateral Ureter Obstruction

    Institute of Scientific and Technical Information of China (English)

    Jing-Fei Teng; Kai Wang; Yao Li; Fa-Jun Qu; Qing Yuan; Xin-Gang Cui; Quan-Xing Wang

    2015-01-01

    Background:Src homology 2 domain-containing protein tyrosine phosphatase-2 (SHP-2) is a kind of intracellular protein tyrosine phosphatase.Studies have revealed its roles in various disease,however,whether SHP-2 involves in renal fibrosis remains unclear.The aim of this study was to explore the roles of myeloid cells SHP-2 in renal interstitial fibrosis.Methods:Myeloid cells SHP-2 gene was conditionally knocked-out (CKO) in mice using loxP-Cre system,and renal interstitial fibrosis was induced by unilateral ureter obstruction (UUO).The total collagen deposition in the renal interstitium was assessed using picrosirius red stain.F4/80 immunostaing was used to evaluate macrophage infiltration in renal tubular interstitium.Quantitative real-time polymerase chain reaction and enzyme linked immunosorbent assay were used to analyze the production of cytokines in the kidney.Transferase-mediated dUTP nick-end labeling stain was used to assess the apoptotic renal tubular epithelial cells.Results:Src homology 2 domain-containing protein tyrosine phosphatase-2 gene CKO in myeloid cells significantly reduced collagen deposition in the renal interstitium after UUO.Macrophage infiltration was evidently decreased in renal tubular interstitium of SHP-2 CKO mice.Meanwhile,the production of pro-inflammatory cytokines was significantly suppressed in SHP-2 CKO mice.However,no significant difference was observed in the number of apoptotic renal tubular epithelial cells between wild-type and SHP-2 CKO mice.Conclusions:Our observations suggested that SHP-2 in myeloid cells plays a pivotal role in the pathogenesis of renal fibrosis,and that silencing of SHP-2 gene in myeloid cells may protect renal from inflammatory damage and prevent renal fibrosis after renal injury.

  5. Protein tyrosine phosphatase µ (PTP µ or PTPRM, a negative regulator of proliferation and invasion of breast cancer cells, is associated with disease prognosis.

    Directory of Open Access Journals (Sweden)

    Ping-Hui Sun

    Full Text Available BACKGROUND: PTPRM has been shown to exhibit homophilic binding and confer cell-cell adhesion in cells including epithelial and cancer cells. The present study investigated the expression of PTPRM in breast cancer and the biological impact of PTPRM on breast cancer cells. DESIGN: Expression of PTPRM protein and gene transcript was examined in a cohort of breast cancer patients. Knockdown of PTPRM in breast cancer cells was performed using a specific anti-PTPRM transgene. The impact of PTPRM knockdown on breast cancer was evaluated using in vitro cell models. RESULTS: A significant decrease of PTPRM transcripts was seen in poorly differentiated and moderately differentiated tumours compared with well differentiated tumours. Patients with lower expression of PTPRM had shorter survival compared with those which had a higher level of PTPRM expression. Knockdown of PTPRM increased proliferation, adhesion, invasion and migration of breast cancer cells. Furthermore, knockdown of PTPRM in MDA-MB-231 cells resulted in increased cell migration and invasion via regulation of the tyrosine phosphorylation of ERK and JNK. CONCLUSIONS: Decreased expression of PTPRM in breast cancer is correlated with poor prognosis and inversely correlated with disease free survival. PTPRM coordinated cell migration and invasion through the regulation of tyrosine phosphorylation of ERK and JNK.

  6. The human cytomegalovirus UL11 protein interacts with the receptor tyrosine phosphatase CD45, resulting in functional paralysis of T cells.

    Directory of Open Access Journals (Sweden)

    Ildar Gabaev

    2011-12-01

    Full Text Available Human cytomegalovirus (CMV exerts diverse and complex effects on the immune system, not all of which have been attributed to viral genes. Acute CMV infection results in transient restrictions in T cell proliferative ability, which can impair the control of the virus and increase the risk of secondary infections in patients with weakened or immature immune systems. In a search for new immunomodulatory proteins, we investigated the UL11 protein, a member of the CMV RL11 family. This protein family is defined by the RL11 domain, which has homology to immunoglobulin domains and adenoviral immunomodulatory proteins. We show that pUL11 is expressed on the cell surface and induces intercellular interactions with leukocytes. This was demonstrated to be due to the interaction of pUL11 with the receptor tyrosine phosphatase CD45, identified by mass spectrometry analysis of pUL11-associated proteins. CD45 expression is sufficient to mediate the interaction with pUL11 and is required for pUL11 binding to T cells, indicating that pUL11 is a specific CD45 ligand. CD45 has a pivotal function regulating T cell signaling thresholds; in its absence, the Src family kinase Lck is inactive and signaling through the T cell receptor (TCR is therefore shut off. In the presence of pUL11, several CD45-mediated functions were inhibited. The induction of tyrosine phosphorylation of multiple signaling proteins upon TCR stimulation was reduced and T cell proliferation was impaired. We therefore conclude that pUL11 has immunosuppressive properties, and that disruption of T cell function via inhibition of CD45 is a previously unknown immunomodulatory strategy of CMV.

  7. Stimulation of receptor protein-tyrosine phosphatase alpha activity and phosphorylation by phorbol ester

    DEFF Research Database (Denmark)

    den Hertog, J; Sap, J; Pals, C E;

    1995-01-01

    Receptor Protein-Tyrosine Phosphatase alpha (RPTP alpha) is a transmembrane protein with two cytoplasmic catalytic protein-tyrosine phosphatase (PTP) domains and a relatively short (123 amino acids) extracellular domain. Here we report that treatment of transfected cells that express RPTP alpha...

  8. cDNA isolated from a human T-cell library encodes a member of the protein-tyrosine-phosphatase family

    International Nuclear Information System (INIS)

    A human peripheral T-cell cDNA library was screened with two labeled synthetic oligonucleotides encoding regions of a human placenta protein-tyrosine-phosphatase. One positive clone was isolated and the nucleotide sequence was determined. It contained 1,305 base pairs of open reading frame followed by a TAA stop codon and 978 base pairs of 3' untranslated end, although a poly(A)+ tail was not found. An initiator methionine residue was predicted at position 61, which would result in a protein of 415 amino acid residues. This was supported by the synthesis of a Mr 48,000 protein in an in vitro reticulocyte lysate translation system using RNA transcribed from the cloned cDNA and T7 RNA polymerase. The deduced amino acid sequence was compared to other known proteins revealing 65% identity to the low Mr PTPase 1B isolated from placenta. In view of the high degree of similarity, the T-cell cDNA likely encodes a newly discovered protein-tyrosine-phosphatase, thus expanding this family of genes

  9. Receptor-like protein-tyrosine phosphatase alpha specifically inhibits insulin-increased prolactin gene expression

    DEFF Research Database (Denmark)

    Jacob, K K; Sap, J; Stanley, F M

    1998-01-01

    A physiologically relevant response to insulin, stimulation of prolactin promoter activity in GH4 pituitary cells, was used as an assay to study the specificity of protein-tyrosine phosphatase function. Receptor-like protein-tyrosine phosphatase alpha (RPTPalpha) blocks the effect of insulin to i...

  10. From immune response to cancer : a spot on the low molecular weight protein tyrosine phosphatase

    NARCIS (Netherlands)

    Souza, A. C. S.; Azoubel, S.; Queiroz, K. C. S.; Peppelenbosch, M. P.; Ferreira, C. V.

    2009-01-01

    Reversible tyrosine phosphorylation is a key posttranslational regulatory modification of proteins in all eukaryotic cells in normal and pathological processes. Recently a pivotal janus-faced biological role of the low molecular weight protein tyrosine phosphatase (LMWPTP) has become clear. On the o

  11. Protein tyrosine kinase inhibitors modify kainic acid-induced epileptiform activity and mossy fiber sprouting but do not protect against limbic cell death

    Directory of Open Access Journals (Sweden)

    C.M. Queiroz

    2008-05-01

    Full Text Available Intrahippocampal administration of kainic acid (KA induces synaptic release of neurotrophins, mainly brain-derived neurotrophic factor, which contributes to the acute neuronal excitation produced by the toxin. Two protein tyrosine kinase inhibitors, herbimycin A and K252a, were administered intracerebroventricularly, in a single dose, to attenuate neurotrophin signaling during the acute effects of KA, and their role in epileptogenesis was evaluated in adult, male Wistar rats weighing 250-300 g. The latency for the first Racine stage V seizure was 90 ± 8 min in saline controls (N = 4 which increased to 369 ± 71 and 322 ± 63 min in animals receiving herbimycin A (1.74 nmol, N = 4 and K252a (10 pmol, N = 4, respectively. Behavioral alterations were accompanied by diminished duration of EEG paroxysms in herbimycin A- and K252a-treated animals. Notwithstanding the reduction in seizure severity, cell death (60-90% of cell loss in KA-treated animals in limbic regions was unchanged by herbimycin A and K252a. However, aberrant mossy fiber sprouting was significantly reduced in the ipsilateral dorsal hippocampus of K252a-treated animals. In this model of temporal lobe epilepsy, both protein kinase inhibitors diminished the acute epileptic activity triggered by KA and the ensuing morphological alterations in the dentate gyrus without diminishing cell loss. Our current data indicating that K252a, but not herbimycin, has an influence over KA-induced mossy fiber sprouting further suggest that protein tyrosine kinase receptors are not the only factors which control this plasticity. Further experiments are necessary to elucidate the exact signaling systems associated with this K252a effect.

  12. PROTEN TYROSINE PHOSPHATASE ACTIVITY IN RAT ASCITES HEPATOMA CELLS

    Directory of Open Access Journals (Sweden)

    M.Saadat

    1998-10-01

    Full Text Available Protein tyrosine phosphatases (PTPases regulate tyrosine phosphorylation of target proteins involved in several aspects of cellular functions. Enzyme activities of the PTPases in cytosolic and particulate fractions of rat ascites hepatoma cell lines were determined and compared with those of normal rat liver. Our present data revealed that although there was no neoplatic-specific alteration of the PTPase activity in examined hepatomas, the activity in particulate fractions of island type of hepatomas was remarkably decreased compared with either rat liver or free type hepatomas.

  13. Phospho-tyrosine dependent protein–protein interaction network

    Science.gov (United States)

    Grossmann, Arndt; Benlasfer, Nouhad; Birth, Petra; Hegele, Anna; Wachsmuth, Franziska; Apelt, Luise; Stelzl, Ulrich

    2015-01-01

    Post-translational protein modifications, such as tyrosine phosphorylation, regulate protein–protein interactions (PPIs) critical for signal processing and cellular phenotypes. We extended an established yeast two-hybrid system employing human protein kinases for the analyses of phospho-tyrosine (pY)-dependent PPIs in a direct experimental, large-scale approach. We identified 292 mostly novel pY-dependent PPIs which showed high specificity with respect to kinases and interacting proteins and validated a large fraction in co-immunoprecipitation experiments from mammalian cells. About one-sixth of the interactions are mediated by known linear sequence binding motifs while the majority of pY-PPIs are mediated by other linear epitopes or governed by alternative recognition modes. Network analysis revealed that pY-mediated recognition events are tied to a highly connected protein module dedicated to signaling and cell growth pathways related to cancer. Using binding assays, protein complementation and phenotypic readouts to characterize the pY-dependent interactions of TSPAN2 (tetraspanin 2) and GRB2 or PIK3R3 (p55γ), we exemplarily provide evidence that the two pY-dependent PPIs dictate cellular cancer phenotypes. PMID:25814554

  14. Distinct Mechanisms of Receptor and Nonreceptor Tyrosine Kinase Activation by Reactive Oxygen Species in Vascular Smooth Muscle Cells: Role of Metalloprotease and Protein Kinase C-δ

    OpenAIRE

    Frank, Gerald D.; Mifune, Mizuo; Inagami, Tadashi; Ohba, Motoi; Sasaki, Terukatsu; Higashiyama, Shigeki; Dempsey, Peter J; Eguchi, Satoru

    2003-01-01

    Reactive oxygen species (ROS) are implicated in cardiovascular diseases. ROS, such as H2O2, act as second messengers to activate diverse signaling pathways. Although H2O2 activates several tyrosine kinases, including the epidermal growth factor (EGF) receptor, JAK2, and PYK2, in vascular smooth muscle cells (VSMCs), the intracellular mechanism by which ROS activate these tyrosine kinases remains unclear. Here, we identified two distinct signaling pathways required for receptor and nonreceptor...

  15. Protein tyrosine phosphatases involved in signaling of the ABA-induced H2O2generation in guard cells of Vicia faba L.

    Institute of Scientific and Technical Information of China (English)

    SHI Wuliang; JlA Wensuo; LIU Xin; ZHANG Shuqiu

    2004-01-01

    Although protein tyrosine phosphatases (PTPases) play an important role in signal transduction in animal cells, little is known about the function of PTPases in higher plants. Hydrogen peroxide (H2O2) and mitogen-activated protein kinases (MAPKs) are the critical components of ABA signaling pathway in guard cells. PTPase is an important regulator of MAPK, which is believed to mediate ABA-induced H2O2 generation in guard cells of Viciafaba L. Here, we investigate the possible role of PTPases in stomatal movement process. Phenylarsine oxide (PAO), a specific inhibitor of PTPases, could prevent ABA or H2O2-induced stomatal closure of Vicia faba L; furthermore, it could promote opening of the stomata closed by ABA or H2O2. The activity of PTPases can be effectively inhibited by PAO and H2O2. DTT had no effect on the PAO-induced inhibition of PTPases activity, but it could relieve the inhibition of H2O2 on PTPases activity. PAO could also inhibit the ABA-induced H2O2 generation in guard cells of Vicia faba L. These results suggested that PTPases is a critical signaling component in ABA-induced stomatal closure, and serve as targets for H2O2 lying on the signaling pathways downstream of ABA induced H2O2 generation.

  16. Protein tyrosine kinase inhibition and cell proliferation: is the [3H]-thymidine uptake assay representative of the T-lymphocyte proliferation rate?

    Science.gov (United States)

    Spinozzi, F; Pagliacci, M C; Agea, E; Migliorati, G; Riccardi, C; Bertotto, A; Nicoletti, I

    1995-01-01

    T-cell growth is controlled to a large degree by extracellular signals that bind to specific receptors on the surface of cells. A number of these receptors have intrinsic protein tyrosine kinase (PTK) activity. Their action on second messenger generation, and thus on cell proliferation, has been indirectly demonstrated by the decrease in [3H]-thymidine (TdR) uptake that follows co-stimulation of T-cells with mitogens and PTK inhibitors such as genistein (GEN). In this paper we report that the [3H]-TdR uptake assay is not a valid and reliable tool for investigating the proliferative activity of certain T-cell lines. In fact, a concomitant assessment of both [3H]-TdR uptake and cell cycle progression demonstrated that GEN is able to block G2/M progression of Jurkat T-lymphocytes even at doses (5 micrograms/ml) that do not influence [3H]-TdR uptake. Pretreatment with sodium o-vanadate (100 nM) could not reverse the GEN-related cell cycle perturbation, but was able to restore optimal [3H]-TdR uptake. Finally, GEN treatment was able to induce concentration-dependent apoptotic cell death of Jurkat T-cells. The control of cell activation, proliferation and programmed cell death is undoubtedly influenced by receptor-associated PTKs. The final effect on cell survival is almost entirely dependent on the activation state of the cell. The [3H]-TdR uptake assay seems to be inadequate for a correct interpretation of the expected results. PMID:7655707

  17. Protein tyrosine kinase, JNK, and ERK involvement in p seudolaric acid B-induced apoptosis of human breast cancer MCF-7 cells

    Institute of Scientific and Technical Information of China (English)

    Jing-hua YU; Hong-jun WANG; Xiang-ru LI; Shin-ichi TASHIRO; Satoshi ONODERA; Takashi IKEJIMA

    2008-01-01

    Aim:To investigate the apoptotic mechanism ofpseudolaric acid B (PAB) in hu-man breast cancer MCF-7 cells. Methods: 3-(4,5-Dimethylthiazol-2-yl)-2, 5-di-phenyltetrazolium bromide analysis and morphological changes were applied to detect apoptosis. The percentage of apoptotic and necrotic cells were calculated by the lactate dehydrogenase activity-based cytotoxicity assay, and the protein expression was examined by Western blot analysis. Results: PAB and/or the mitogen-activated protein kinases, including p38, c-Jun-N-terrninal kinase (JNK) and extracellular signal-regulated kinase (ERK), did not participate in necrosis. P38 had no obvious function on apoptosis after 4 μmol/L PAB treatment for 36 h, but PAB induced JNK phosphorylation and inhibited ERK phosphorylation in the apoptotic process. In this study the inhibitor of protein tyrosine kinase (PTK) genistein inverted the inhibitory effect of PAB, instead promoting the survival of MCF-7 cells. Like genistein, another PTK inhibitor AG1024 had a similar ef-fect on PAB-treated MCF-7 cells, indicating that PAB activated PTK to induce apoptosis. Together with PAB, genistein increased the expression of p-ERK, and decreased the expressions of JNK and p-JNK in PAB-treated MCF-7 cells at 36 h. And it is considered that the p-ERK and p-JNK were active patterns of ERK and JNK, respectively. Conclusion: PTK were upstream of ERK and JNK, and PTK induced apoptosis through activating JNK and inactivating ERK in PAB-treated MCF-7 cells.

  18. Protein tyrosine adduct in humans self-poisoned by chlorpyrifos

    International Nuclear Information System (INIS)

    Studies of human cases of self-inflicted poisoning suggest that chlorpyrifos oxon reacts not only with acetylcholinesterase and butyrylcholinesterase but also with other blood proteins. A favored candidate is albumin because in vitro and animal studies have identified tyrosine 411 of albumin as a site covalently modified by organophosphorus poisons. Our goal was to test this proposal in humans by determining whether plasma from humans poisoned by chlorpyrifos has adducts on tyrosine. Plasma samples from 5 self-poisoned humans were drawn at various time intervals after ingestion of chlorpyrifos for a total of 34 samples. All 34 samples were analyzed for plasma levels of chlorpyrifos and chlorpyrifos oxon (CPO) as a function of time post-ingestion. Eleven samples were analyzed for the presence of diethoxyphosphorylated tyrosine by mass spectrometry. Six samples yielded diethoxyphosphorylated tyrosine in pronase digests. Blood collected as late as 5 days after chlorpyrifos ingestion was positive for CPO-tyrosine, consistent with the 20-day half-life of albumin. High plasma CPO levels did not predict detectable levels of CPO-tyrosine. CPO-tyrosine was identified in pralidoxime treated patients as well as in patients not treated with pralidoxime, indicating that pralidoxime does not reverse CPO binding to tyrosine in humans. Plasma butyrylcholinesterase was a more sensitive biomarker of exposure than adducts on tyrosine. In conclusion, chlorpyrifos oxon makes a stable covalent adduct on the tyrosine residue of blood proteins in humans who ingested chlorpyrifos. - Highlights: • Chlorpyrifos-poisoned patients have adducts on protein tyrosine. • Diethoxyphosphate-tyrosine does not lose an alkyl group. • Proteins in addition to AChE and BChE are modified by organophosphates

  19. Protein tyrosine adduct in humans self-poisoned by chlorpyrifos

    Energy Technology Data Exchange (ETDEWEB)

    Li, Bin, E-mail: binli@unmc.edu [Eppley Institute, University of Nebraska Medical Center, Omaha, NE 68198-5950 (United States); Eyer, Peter, E-mail: peter.eyer@lrz.uni-muenchen.de [Walther-Straub-Institut Für Pharmakologie und Toxikologie, Ludwig-Maximilians-Universität München, 80336 München (Germany); Eddleston, Michael, E-mail: M.Eddleston@ed.ac.uk [Clinical Pharmacology Unit, University of Edinburgh, Edinburgh (United Kingdom); Jiang, Wei, E-mail: wjiang@unmc.edu [Eppley Institute, University of Nebraska Medical Center, Omaha, NE 68198-5950 (United States); Schopfer, Lawrence M., E-mail: lmschopf@unmc.edu [Eppley Institute, University of Nebraska Medical Center, Omaha, NE 68198-5950 (United States); Lockridge, Oksana, E-mail: olockrid@unmc.edu [Eppley Institute, University of Nebraska Medical Center, Omaha, NE 68198-5950 (United States)

    2013-06-15

    Studies of human cases of self-inflicted poisoning suggest that chlorpyrifos oxon reacts not only with acetylcholinesterase and butyrylcholinesterase but also with other blood proteins. A favored candidate is albumin because in vitro and animal studies have identified tyrosine 411 of albumin as a site covalently modified by organophosphorus poisons. Our goal was to test this proposal in humans by determining whether plasma from humans poisoned by chlorpyrifos has adducts on tyrosine. Plasma samples from 5 self-poisoned humans were drawn at various time intervals after ingestion of chlorpyrifos for a total of 34 samples. All 34 samples were analyzed for plasma levels of chlorpyrifos and chlorpyrifos oxon (CPO) as a function of time post-ingestion. Eleven samples were analyzed for the presence of diethoxyphosphorylated tyrosine by mass spectrometry. Six samples yielded diethoxyphosphorylated tyrosine in pronase digests. Blood collected as late as 5 days after chlorpyrifos ingestion was positive for CPO-tyrosine, consistent with the 20-day half-life of albumin. High plasma CPO levels did not predict detectable levels of CPO-tyrosine. CPO-tyrosine was identified in pralidoxime treated patients as well as in patients not treated with pralidoxime, indicating that pralidoxime does not reverse CPO binding to tyrosine in humans. Plasma butyrylcholinesterase was a more sensitive biomarker of exposure than adducts on tyrosine. In conclusion, chlorpyrifos oxon makes a stable covalent adduct on the tyrosine residue of blood proteins in humans who ingested chlorpyrifos. - Highlights: • Chlorpyrifos-poisoned patients have adducts on protein tyrosine. • Diethoxyphosphate-tyrosine does not lose an alkyl group. • Proteins in addition to AChE and BChE are modified by organophosphates.

  20. Pervanadate-induced adhesion of CD4+ T cell to fibronectin is associated with tyrosine phosphorylation of paxillin.

    Science.gov (United States)

    Miron, S; Kachalsky, S G; Hershkoviz, R; Lider, O

    1997-09-01

    The initial stages of T cell activation involve tyrosine protein kinase-mediated intracellular signaling events. Integrin-mediated adhesion of CD4+ T lymphocytes to extracellular matrix glycoproteins, such as fibronectin, is an activation-dependent process. The involvement of tyrosine protein kinases in the adhesion of CD4+ T cells to fibronectin was examined using pervanadate, a protein-tyrosine phosphatase inhibitor. Pervanadate induced the adhesion of human CD4+ T cells to immobilized fibronectin in a beta1 integrin-mediated fashion, and adhesion was associated with an increase of protein tyrosine phosphorylation. Tyrosine protein kinase inhibitors abrogated both T cell adhesion and intracellular protein tyrosine phosphorylation. Participation of cytoskeletal proteins in the pervanadate-induced T cell adhesion was indicated because cytoskeleton disruption by cytochalasin B inhibited cell adhesion to fibronectin. We demonstrate that the cytoskeletal protein paxillin underwent time-dependent tyrosine phosphorylation simultaneously with pervanadate-induced T cell adhesion to fibronectin. Tyrosine phosphorylation of paxillin was related to cell adhesion, since pretreatment of T cells with cytochalasin B abrogated both adhesion and phosphorylation. This study demonstrates a correlation between activation of protein tyrosine kinases, tyrosine phosphorylation of paxillin, and integrin-mediated T cell adhesion to extracellular matrix glycoproteins. PMID:9307082

  1. MicroRNA-194 promotes the growth, migration, and invasion of ovarian carcinoma cells by targeting protein tyrosine phosphatase nonreceptor type 12

    Science.gov (United States)

    Liang, Tian; Li, Liru; Cheng, Yan; Ren, Chengcheng; Zhang, Guangmei

    2016-01-01

    Ovarian carcinoma is the most lethal gynecologic malignancy among women. Ovarian cancer metastasis is the main reason for poor prognosis. MicroRNAs (miRNAs) have been shown to play an important role in tumorigenesis and metastasis in various cancers by affecting the expression of their targets. In this study, we explored the role of miR-194 in ovarian cancer. Real-time polymerase chain reaction assays showed that miR-194 was significantly upregulated in ovarian cancer tissues. Overexpression of miR-194 in ovarian cancer cells promotes cell proliferation, migration, and invasion; in contrast, inhibition of the expression of miR-194 has the opposite effects. Meanwhile, bioinformatics tools were used to identify protein tyrosine phosphatase nonreceptor type 12 (PTPN12) as a potential target of miR-194. The luciferase assay showed that miR-194 directly binds to the 3′-untranslated region of PTPN12. Western blot analysis and quantitative real-time polymerase chain reaction assay revealed that PTPN12 expression was negatively associated with miR-194 expression in both ovarian cancer tissues and cells. Thus, we conclude that miR-194 targets PTPN12 and functions as an oncogene in ovarian cancer cells. This novel pathway may provide a new insight to explain ovarian cancer development and metastasis. PMID:27486333

  2. Overexpression of protein tyrosine phosphatase-alpha (PTP-alpha) but not PTP-kappa inhibits translocation of GLUT4 in rat adipose cells

    DEFF Research Database (Denmark)

    Cong, L N; Chen, H; Li, Y;

    1999-01-01

    Protein tyrosine phosphatases (PTPases) are likely to play important roles in insulin action. We recently demonstrated that the nontransmembrane PTPase PTP1B can act as a negative modulator of insulin-stimulated translocation of GLUT4. We now examine the role of PTP-alpha and PTP-kappa (two trans......-stimulated glucose transport....

  3. ABNORMAL PROTEIN TYROSINE KINASES ASSOCIATED WITH HUMAN HAEMATOLOGICAL MALIGNANCIES

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Objective: To survey the role of protein tyrosine kinases (PTKs) in the pathogenesis of several hematopoietic malignancies. Methods: By reviewing the published laboratory and clinical studies on PTK-related oncoproteins and their causative role in some leukemias and lymphomas. Results: Protein tyrosine kinases are key participants in signal transduction pathways that regulate cellular growth, activation and differentiations. Aberrant PTK activity resulting from gene mutation (often accompanying chromosome translocation) plays an etiologic role in several clonal hematopoietic malignancies. For example, the PTK product of the BCR-ABL fusion gene resulting from the t (9; 22) translocation exhibits several fold higher tyrosine kinase activity than the product of the ABL gene. Evidence suggests that the BCR-ABL oncoprotein alone is sufficient to case chronic myelogenous leukemia (CML) and other Ph positive acute leukemia. PTK over-activity resulting from chromosomal translocations creating TEL-ABL, TEL-JAK2 and TEL-PDGFR( fusion proteins plays an important role in the pathogenesis of other types of leukemia. Another example occurs in anaplastic large cell lymphoma (ALCL). Experimental and clinical evidences indicate that translocations involving ALK gene on chromosome 2p23, most commonly resulting in an NPM-ALK fusion oncogene, result in constitutive activation of ALK and cause ALCL. This group of lymphomas is now named ALK positive lymphoma or ALKoma. Conclusion: Genetic lesions creating aberrant fusion proteins that result in excessive PTK activity are increasingly being recognized as central to the pathogenesis of hemotopoietic malignancies. These chimeric PTK molecules represent attractive disease-specific targets against which new classes therapeutic agents are being developed.

  4. Cloning of two members of the SIRP alpha family of protein tyrosine phosphatase binding proteins in cattle that are expressed on monocytes and a subpopulation of dendritic cells and which mediate binding to CD4 T cells.

    Science.gov (United States)

    Brooke, G P; Parsons, K R; Howard, C J

    1998-01-01

    Recent experimental studies have greatly clarified the function of cell surface molecules in the induction and modulation of T cell responses by antigen-presenting cells (APC). However, the differences in ability to stimulate T cells evident for different types and subpopulations of the same APC, such as dendritic cell subsets, is less well understood. This report details an investigation of an antigen expressed on monocytes that is also expressed on a subset of cattle afferent lymph veiled cells (ALVC). A cDNA library derived from cattle monocytes was screened with monoclonal antibodies (mAb) for expression in COS-7 cells. Using separate mAb for screening, two cDNA were cloned, the sequences of which showed a single long open reading frame encoding a predicted type I glycoprotein of 506 amino acids that contained three immunoglobulin superfamily domains and a long 112-amino acid cytoplasmic tail. We have termed this antigen MyD-1, reflecting its myeloid and dendritic cell distribution. Analysis of the EMBL database revealed that the molecule is a member of the recently described family of signal regulatory proteins (SIRP). The outeremost Ig domain was of the adhesion/receptor I-type, suggesting that MyD-1 might bind to a ligand on another cell. Evidence for this was subsequently obtained by demonstrating that COS-7 cells transfected with MyD-1 cDNA bound CD4 T cells and this binding was blocked by specific mAb. The potential importance of this interaction was supported by the finding that the proliferation of resting memory CD4 T cells to ovalbumin-pulsed monocytes was significantly reduced in the presence of mAb to MyD-1. A role for the molecule in the modulation of the monocyte/dendritic APC response is also predicted from the existence of multiple potential tyrosine phosphorylation sites in the cytoplasmic domain, including the presence of an immunoreceptor tyrosine-based inhibitory motif (ITIM) and the observation that the SIRP alpha family members have been

  5. The tyrosine phosphatase SHP-1 regulates hypoxia inducible factor-1α (HIF-1α protein levels in endothelial cells under hypoxia.

    Directory of Open Access Journals (Sweden)

    Stefan K Alig

    Full Text Available The tyrosine phosphatase SHP-1 negatively influences endothelial function, such as VEGF signaling and reactive oxygen species (ROS formation, and has been shown to influence angiogenesis during tissue ischemia. In ischemic tissues, hypoxia induced angiogenesis is crucial for restoring oxygen supply. However, the exact mechanism how SHP-1 affects endothelial function during ischemia or hypoxia remains unclear. We performed in vitro endothelial cell culture experiments to characterize the role of SHP-1 during hypoxia.SHP-1 knock-down by specific antisense oligodesoxynucleotides (AS-Odn increased cell growth as well as VEGF synthesis and secretion during 24 hours of hypoxia compared to control AS-Odn. This was prevented by HIF-1α inhibition (echinomycin and apigenin. SHP-1 knock-down as well as overexpression of a catalytically inactive SHP-1 (SHP-1 CS further enhanced HIF-1α protein levels, whereas overexpression of a constitutively active SHP-1 (SHP-1 E74A resulted in decreased HIF-1α levels during hypoxia, compared to wildtype SHP-1. Proteasome inhibition (MG132 returned HIF-1α levels to control or wildtype levels respectively in these cells. SHP-1 silencing did not alter HIF-1α mRNA levels. Finally, under hypoxic conditions SHP-1 knock-down enhanced intracellular endothelial reactive oxygen species (ROS formation, as measured by oxidation of H2-DCF and DHE fluorescence.SHP-1 decreases half-life of HIF-1α under hypoxic conditions resulting in decreased cell growth due to diminished VEGF synthesis and secretion. The regulatory effect of SHP-1 on HIF-1α stability may be mediated by inhibition of endothelial ROS formation stabilizing HIF-1α protein. These findings highlight the importance of SHP-1 in hypoxic signaling and its potential as therapeutic target in ischemic diseases.

  6. Chemical inhibition of bacterial protein tyrosine phosphatase suppresses capsule production.

    Science.gov (United States)

    Standish, Alistair J; Salim, Angela A; Zhang, Hua; Capon, Robert J; Morona, Renato

    2012-01-01

    Capsule polysaccharide is a major virulence factor for a wide range of bacterial pathogens, including Streptococcus pneumoniae. The biosynthesis of Wzy-dependent capsules in both gram-negative and -positive bacteria is regulated by a system involving a protein tyrosine phosphatase (PTP) and a protein tyrosine kinase. However, how the system functions is still controversial. In Streptococcus pneumoniae, a major human pathogen, the system is present in all but 2 of the 93 serotypes found to date. In order to study this regulation further, we performed a screen to find inhibitors of the phosphatase, CpsB. This led to the observation that a recently discovered marine sponge metabolite, fascioquinol E, inhibited CpsB phosphatase activity both in vitro and in vivo at concentrations that did not affect the growth of the bacteria. This inhibition resulted in decreased capsule synthesis in D39 and Type 1 S. pneumoniae. Furthermore, concentrations of Fascioquinol E that inhibited capsule also lead to increased attachment of pneumococci to a macrophage cell line, suggesting that this compound would inhibit the virulence of the pathogen. Interestingly, this compound also inhibited the phosphatase activity of the structurally unrelated gram-negative PTP, Wzb, which belongs to separate family of protein tyrosine phosphatases. Furthermore, incubation with Klebsiella pneumoniae, which contains a homologous phosphatase, resulted in decreased capsule synthesis. Taken together, these data provide evidence that PTPs are critical for Wzy-dependent capsule production across a spectrum of bacteria, and as such represents a valuable new molecular target for the development of anti-virulence antibacterials.

  7. Chemical inhibition of bacterial protein tyrosine phosphatase suppresses capsule production.

    Directory of Open Access Journals (Sweden)

    Alistair J Standish

    Full Text Available Capsule polysaccharide is a major virulence factor for a wide range of bacterial pathogens, including Streptococcus pneumoniae. The biosynthesis of Wzy-dependent capsules in both gram-negative and -positive bacteria is regulated by a system involving a protein tyrosine phosphatase (PTP and a protein tyrosine kinase. However, how the system functions is still controversial. In Streptococcus pneumoniae, a major human pathogen, the system is present in all but 2 of the 93 serotypes found to date. In order to study this regulation further, we performed a screen to find inhibitors of the phosphatase, CpsB. This led to the observation that a recently discovered marine sponge metabolite, fascioquinol E, inhibited CpsB phosphatase activity both in vitro and in vivo at concentrations that did not affect the growth of the bacteria. This inhibition resulted in decreased capsule synthesis in D39 and Type 1 S. pneumoniae. Furthermore, concentrations of Fascioquinol E that inhibited capsule also lead to increased attachment of pneumococci to a macrophage cell line, suggesting that this compound would inhibit the virulence of the pathogen. Interestingly, this compound also inhibited the phosphatase activity of the structurally unrelated gram-negative PTP, Wzb, which belongs to separate family of protein tyrosine phosphatases. Furthermore, incubation with Klebsiella pneumoniae, which contains a homologous phosphatase, resulted in decreased capsule synthesis. Taken together, these data provide evidence that PTPs are critical for Wzy-dependent capsule production across a spectrum of bacteria, and as such represents a valuable new molecular target for the development of anti-virulence antibacterials.

  8. Cancer Cell-derived Exosomes Induce Mitogen-activated Protein Kinase-dependent Monocyte Survival by Transport of Functional Receptor Tyrosine Kinases.

    Science.gov (United States)

    Song, Xiao; Ding, Yanping; Liu, Gang; Yang, Xiao; Zhao, Ruifang; Zhang, Yinlong; Zhao, Xiao; Anderson, Gregory J; Nie, Guangjun

    2016-04-15

    Tumor-associated macrophages (TAM) play pivotal roles in cancer initiation and progression. Monocytes, the precursors of TAMs, normally undergo spontaneous apoptosis within 2 days, but can subsist in the inflammatory tumor microenvironment for continuous survival and generation of sufficient TAMs. The mechanisms underlying tumor-driving monocyte survival remain obscure. Here we report that cancer cell-derived exosomes were crucial mediators for monocyte survival in the inflammatory niche. Analysis of the survival-promoting molecules in monocytes revealed that cancer cell-derived exosomes activated Ras and extracellular signal-regulated kinases in the mitogen-activated protein kinase (MAPK) pathway, resulting in the prevention of caspase cleavage. Phosphorylated receptor tyrosine kinases (RTKs), such as phosphorylated epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER-2), were abundantly expressed in cancer cell-derived exosomes. Knock-out of EGFR or/and HER-2, or alternatively, inhibitors against their phosphorylation significantly disturbed the exosome-mediated activation of the MAPK pathway, inhibition of caspase cleavage, and increase in survival rate in monocytes. Moreover, the deprived survival-stimulating activity of exosomes due to null expression of EGFR and HER-2 could be restored by activation of another RTK, insulin receptor. Overall, our study uncovered a mechanism of tumor-associated monocyte survival and demonstrated that cancer cell-derived exosomes can stimulate the MAPK pathway in monocytes through transport of functional RTKs, leading to inactivation of apoptosis-related caspases. This work provides insights into the long sought question on monocyte survival prior to formation of plentiful TAMs in the tumor microenvironment.

  9. Emerging issues in receptor protein tyrosine phosphatase function: lifting fog or simply shifting?

    DEFF Research Database (Denmark)

    Petrone, A; Sap, J

    2000-01-01

    Transmembrane (receptor) tyrosine phosphatases are intimately involved in responses to cell-cell and cell-matrix contact. Several important issues regarding the targets and regulation of this protein family are now emerging. For example, these phosphatases exhibit complex interactions with signal...

  10. Antigen receptor signaling: integration of protein tyrosine kinase functions.

    Science.gov (United States)

    Tamir, I; Cambier, J C

    1998-09-17

    Antigen receptors on T and B cells function to transduce signals leading to a variety of biologic responses minimally including antigen receptor editing, apoptotic death, developmental progression, cell activation, proliferation and survival. The response to antigen depends upon antigen affinity and valence, involvement of coreceptors in signaling and differentiative stage of the responding cell. The requirement that these receptors integrate signals that drive an array of responses may explain their evolved structural complexity. Antigen receptors are composed of multiple subunits compartmentalized to provide antigen recognition and signal transduction function. In lieu of on-board enzymatic activity these receptors rely on associated Protein Tyrosine Kinases (PTKs) for their signaling function. By aggregating the receptors, and hence their appended PTKs, antigens induce PTK transphosphorylation, activating them to phosphorylate the receptor within conserved motifs termed Immunoreceptor Tyrosine-based Activation Motifs (ITAMs) found in transducer subunits. The tyrosyl phosphorylated ITAMs then interact with Src Homology 2 (SH2) domains within the PTKs leading to their further activation. As receptor phosphorylation is amplified, other effectors, such as Shc, dock by virtue of SH2 binding, and serve, in-turn, as substrates for these PTKs. This sequence of events not only provides a signal amplification mechanism by combining multiple consecutive steps with positive feedback, but also allows for signal diversification by differential recruitment of effectors that provide access to distinct parallel downstream signaling pathways. The subject of antigen receptor signaling has been recently reviewed in depth (DeFranco, 1997; Kurosaki, 1997). Here we discuss the biochemical basis of antigen receptor signal transduction, using the B cell receptor (BCR) as a paradigm, with specific emphasis on the involved PTKs. We review several specific mechanisms by which responses

  11. Cloning and expression of catalytic domain of Abl protein tyrosine kinase gene in E. coli

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Protein tyrosine kinases (PTKs) regulate cell proliferation, differentiation and are involved in signal transduction. Uncontrolled signaling from receptor tyrosine kinases to intracellular tyrosine kinases can lead to inflamma tory responses and diseases such as cancer and atherosclerosis. Thus, inhibitors that block the activity of tyrosine kinases or the signaling pathways of PTKs activation could be assumed as the potential candidate for drug development. On this assumption, we cloned and expressed the Abl PTK gene in E. coli, and purified the PTK, which was used to screen the PTK inhibitors from the extracts of Chinese herbs. The catalytic domain sequence of PTK gene was amplified by PCR us ing the cDNA of abl from Abelson murine leukemia virus as template. The amplified fragment was then cloned into the GST-tagged expression vector pGEX2T. The recombinant plasmid was transformed into host cell E. coli DH5α and was induced to express PTK protein. The expression of the protein was detected using SDS-PAGE. The result showed that a specific protein was induced to express after 12 min induction, and reached peak level about 40% of the host total pro tein after 4 h induction. The molecular weight of the fusion protein was about 58 kD. The purified GST-PTK fusion pro tein presented higher activity for tyrosine phosphorylation.

  12. Expression of Tyrosine Hydroxylase is Negatively Regulated Via Prion Protein.

    Science.gov (United States)

    da Luz, Marcio Henrique Mello; Glezer, Isaias; Xavier, Andre Machado; da Silva, Marcelo Alberti Paiva; Pino, Jessica Monteiro Volejnik; Zamith, Thiago Panaro; Vieira, Taynara Fernanda; Antonio, Bruno Brito; Antunes, Hanna Karen Moreira; Martins, Vilma Regina; Lee, Kil Sun

    2016-07-01

    Cellular prion protein (PrP(C)) is a glycoprotein of the plasma membrane that plays pleiotropic functions by interacting with multiple signaling complexes at the cell surface. Recently, a number of studies have reported the involvement of PrP(C) in dopamine metabolism and signaling, including its interactions with tyrosine hydroxylase (TH) and dopamine receptors. However, the outcomes reported by independent studies are still debatable. Therefore in this study, we investigated the effects of PrP(C) on the TH expression during the differentiation of N2a cells with dibutyryl-cAMP, a well-known cAMP analog that activates TH transcription. Upon differentiation, TH was induced with concomitant reduction of PrP(C) at protein level, but not at mRNA level. shRNA-mediated PrP(C) reduction increased the basal level of TH at both mRNA and protein levels without dibutyryl-cAMP treatment. This phenotype was reversed by re-expression of PrP(C). PrP(C) knockdown also potentiated the effect of dibutyryl-cAMP on TH expression. Our findings suggest that PrP(C) has suppressive effects on TH expression. As a consequence, altered PrP(C) functions may affect the regulation of dopamine metabolism and related neurological disorders.

  13. Adipocyte-Specific Protein Tyrosine Phosphatase 1B Deletion Increases Lipogenesis, Adipocyte Cell Size and is a Minor Regulator of Glucose Homeostasis

    OpenAIRE

    Carl Owen; Alicja Czopek; Abdelali Agouni; Louise Grant; Robert Judson; Lees, Emma K; George D Mcilroy; Olga Göransson; Andy Welch; Bence, Kendra K.; Kahn, Barbara B.; Neel, Benjamin G.; Nimesh Mody; Mirela Delibegović

    2012-01-01

    Protein tyrosine phosphatase 1B (PTP1B), a key negative regulator of leptin and insulin signaling, is positively correlated with adiposity and contributes to insulin resistance. Global PTP1B deletion improves diet-induced obesity and glucose homeostasis via enhanced leptin signaling in the brain and increased insulin signaling in liver and muscle. However, the role of PTP1B in adipocytes is unclear, with studies demonstrating beneficial, detrimental or no effect(s) of adipose-PTP1B-deficiency...

  14. Purification and Characterization of PRL Protein Tyrosine Phosphatases

    Institute of Scientific and Technical Information of China (English)

    LI Zhao-fa; WANG Yan; LI Qing-shan; ZHAO Zhi-zhuang Joe; FU Xue-qi; LI Yu-lin; LI Yi-lei

    2005-01-01

    PRLs constitute a subfamily of protein tyrosine phosphatases(PTPs). In the present paper are reported the molecular cloning, expression, purification, and characterization of all the three members of the PRL enzymes in human and the only PRL in C.elegans. These enzymes were expressed as glutathione S-transferase(GST) fusion proteins in DE3pLysS E.coli cells, and the recombinant fusion proteins were purified on glutathione-Sepharose affinity columns. Having been cleaved with thrombin, GST-free enzymes were further purified on an S-100 Sepharose gel filtration column. The purified proteins show single polypeptide bands on SDS-polyacrylamide gel electrophoresis. With para-nitrophenyl phosphate(p-NPP) as a substrate, PRLs exhibit classical Michaelis-Menten kinetics with Vmax values two orders of magnitude smaller than those of classic PTPs. The responses of PRLs to ionic strength, metal ions and phosphatase inhibitors are similar to those of other characterized PTPs, but their optimal pH values are different. These data thus reveal distinct common biochemical properties of PRL subfamily PTPs as well.

  15. Protein tyrosine phosphatases expression during development of mouse superior colliculus.

    Science.gov (United States)

    Reinhard, Jacqueline; Horvat-Bröcker, Andrea; Illes, Sebastian; Zaremba, Angelika; Knyazev, Piotr; Ullrich, Axel; Faissner, Andreas

    2009-12-01

    Protein tyrosine phosphatases (PTPs) are key regulators of different processes during development of the central nervous system. However, expression patterns and potential roles of PTPs in the developing superior colliculus remain poorly investigated. In this study, a degenerate primer-based reverse transcription-polymerase chain reaction (RT-PCR) approach was used to isolate seven different intracellular PTPs and nine different receptor-type PTPs (RPTPs) from embryonic E15 mouse superior colliculus. Subsequently, the expression patterns of 11 PTPs (TC-PTP, PTP1C, PTP1D, PTP-MEG2, PTP-PEST, RPTPJ, RPTPε, RPTPRR, RPTPσ, RPTPκ and RPTPγ) were further analyzed in detail in superior colliculus from embryonic E13 to postnatal P20 stages by quantitative real-time RT-PCR, Western blotting and immunohistochemistry. Each of the 11 PTPs exhibits distinct spatiotemporal regulation of mRNAs and proteins in the developing superior colliculus suggesting their versatile roles in genesis of neuronal and glial cells and retinocollicular topographic mapping. At E13, additional double-immunohistochemical analysis revealed the expression of PTPs in collicular nestin-positive neural progenitor cells and RC-2-immunoreactive radial glia cells, indicating the potential functional importance of PTPs in neurogenesis and gliogenesis. PMID:19727691

  16. Eliminating tyrosine sequence variants in CHO cell lines producing recombinant monoclonal antibodies.

    Science.gov (United States)

    Feeney, Lauren; Carvalhal, Veronica; Yu, X Christopher; Chan, Betty; Michels, David A; Wang, Yajun Jennifer; Shen, Amy; Ressl, Jan; Dusel, Brendon; Laird, Michael W

    2013-04-01

    Amino acid sequence variants are defined as unintended amino acid sequence changes that contribute to product variation with potential impact to product safety, immunogenicity, and efficacy. Therefore, it is important to understand the propensity for sequence variant (SV) formation during the production of recombinant proteins for therapeutic use. During the development of clinical therapeutic products, several monoclonal antibodies (mAbs) produced from Chinese Hamster Ovary (CHO) cells exhibited SVs at low levels (≤3%) in multiple locations throughout the mAbs. In these examples, the cell culture process depleted tyrosine, and the tyrosine residues in the recombinant mAbs were replaced with phenylalanine or histidine. In this work, it is demonstrated that tyrosine supplementation eliminated the tyrosine SVs, while early tyrosine starvation significantly increased the SV level in all mAbs tested. Additionally, it was determined that phenylalanine is the amino acid preferentially misincorporated in the absence of tyrosine over histidine, with no other amino acid misincorporated in the absence of tyrosine, phenylalanine, and histidine. The data support that the tyrosine SVs are due to mistranslation and not DNA mutation, most likely due to tRNA(Tyr) mischarging due to the structural similarities between tyrosine and phenylalanine.

  17. Involvement of Src tyrosine kinase and protein kinase C in the expression of macrophage migration inhibitory factor induced by H2O2 in HL-1 mouse cardiac muscle cells

    International Nuclear Information System (INIS)

    Macrophage migration inhibitory factor (MIF), a pleiotropic cytokine, plays an important role in the pathogenesis of atrial fibrillation; however, the upstream regulation of MIF in atrial myocytes remains unclear. In the present study, we investigated whether and how MIF is regulated in response to the renin-angiotensin system and oxidative stress in atrium myocytes (HL-1 cells). MIF protein and mRNA levels in HL-1 cells were assayed using immunofluorescence, real-time PCR, and Western blot. The result indicated that MIF was expressed in the cytoplasm of HL-1 cells. Hydrogen peroxide (H2O2), but not angiotensin II, stimulated MIF expression in HL-1 cells. H2O2-induced MIF protein and gene levels increased in a dose-dependent manner and were completely abolished in the presence of catalase. H2O2-induced MIF production was completely inhibited by tyrosine kinase inhibitors genistein and PP1, as well as by protein kinase C (PKC) inhibitor GF109203X, suggesting that redox-sensitive MIF production is mediated through tyrosine kinase and PKC-dependent mechanisms in HL-1 cells. These results suggest that MIF is upregulated by HL-1 cells in response to redox stress, probably by the activation of Src and PKC

  18. Involvement of Src tyrosine kinase and protein kinase C in the expression of macrophage migration inhibitory factor induced by H{sub 2}O{sub 2} in HL-1 mouse cardiac muscle cells

    Energy Technology Data Exchange (ETDEWEB)

    Rao, F. [Department of Cardiology, Guangdong General Hospital, Guangdong Cardiovascular Institute, Guangdong Academy of Medical Sciences, Guangzhou (China); Research Center of Medical Sciences, Guangdong General Hospital, Guangzhou (China); Guangdong Academy of Medical Sciences, Guangzhou (China); Deng, C.Y. [Research Center of Medical Sciences, Guangdong General Hospital, Guangzhou (China); Guangdong Academy of Medical Sciences, Guangzhou (China); Zhang, Q.H.; Xue, Y.M. [Department of Cardiology, Guangdong General Hospital, Guangdong Cardiovascular Institute, Guangdong Academy of Medical Sciences, Guangzhou (China); Guangdong Academy of Medical Sciences, Guangzhou (China); Xiao, D.Z.; Kuang, S.J.; Lin, Q.X.; Shan, Z.X.; Liu, X.Y.; Zhu, J.N. [Research Center of Medical Sciences, Guangdong General Hospital, Guangzhou (China); Guangdong Academy of Medical Sciences, Guangzhou (China); Yu, X.Y. [Department of Cardiology, Guangdong General Hospital, Guangdong Cardiovascular Institute, Guangdong Academy of Medical Sciences, Guangzhou (China); Research Center of Medical Sciences, Guangdong General Hospital, Guangzhou (China); Guangdong Academy of Medical Sciences, Guangzhou (China); Wu, S.L. [Department of Cardiology, Guangdong General Hospital, Guangdong Cardiovascular Institute, Guangdong Academy of Medical Sciences, Guangzhou (China); Guangdong Academy of Medical Sciences, Guangzhou (China)

    2013-09-06

    Macrophage migration inhibitory factor (MIF), a pleiotropic cytokine, plays an important role in the pathogenesis of atrial fibrillation; however, the upstream regulation of MIF in atrial myocytes remains unclear. In the present study, we investigated whether and how MIF is regulated in response to the renin-angiotensin system and oxidative stress in atrium myocytes (HL-1 cells). MIF protein and mRNA levels in HL-1 cells were assayed using immunofluorescence, real-time PCR, and Western blot. The result indicated that MIF was expressed in the cytoplasm of HL-1 cells. Hydrogen peroxide (H{sub 2}O{sub 2}), but not angiotensin II, stimulated MIF expression in HL-1 cells. H{sub 2}O{sub 2}-induced MIF protein and gene levels increased in a dose-dependent manner and were completely abolished in the presence of catalase. H{sub 2}O{sub 2}-induced MIF production was completely inhibited by tyrosine kinase inhibitors genistein and PP1, as well as by protein kinase C (PKC) inhibitor GF109203X, suggesting that redox-sensitive MIF production is mediated through tyrosine kinase and PKC-dependent mechanisms in HL-1 cells. These results suggest that MIF is upregulated by HL-1 cells in response to redox stress, probably by the activation of Src and PKC.

  19. Redox and zinc signalling pathways converging on protein tyrosine phosphatases.

    Science.gov (United States)

    Bellomo, Elisa; Hogstrand, Christer; Maret, Wolfgang

    2014-10-01

    Zinc ions, though redox-inert, have either pro-antioxidant or pro-oxidant functions at critical junctures in redox metabolism and redox signalling. They are released from cells and in cells, e.g. from metallothionein, a protein that transduces redox signals into zinc signals (1). The released zinc ions inhibit enzymes such as protein tyrosine phosphatases (PTPs), key regulatory enzymes of cellular phosphorylation signalling. The Ki(Zn) value for inhibition of receptor PTPB is 21pM (2). The binding is about as tight as the binding of zinc to zinc metalloenzymes and suggests tonic zinc inhibition. PTP1-B (PTPN1), an enzyme regulating the insulin and leptin receptors and involved in cancer and diabetes pathobiochemistry, has a Ki(Zn) value of about 5nM (3). Zinc ions bind to the enzyme in the closed conformation when additional metal-binding ligands are brought into the vicinity of the active site. In contrast, redox reactions target cysteines in the active sites of PTPs in the open conformation. This work provides a molecular basis how hydrogen peroxide and free zinc ions generated by growth factor signalling stimulate phosphorylation signalling differentially. (Supported by the Biotechnology and Biological Sciences Research Council UK, grant BB/K001442/1.). PMID:26461422

  20. Bacillus subtilis strain deficient for the protein-tyrosine kinase PtkA exhibits impaired DNA replication

    DEFF Research Database (Denmark)

    Petranovic, Dina; Michelsen, Ole; Zahradka, K;

    2007-01-01

    Bacillus subtilis has recently come into the focus of research on bacterial protein-tyrosine phosphorylation, with several proteins kinases, phosphatases and their substrates identified in this Gram-positive model organism. B. subtilis protein-tyrosine phosphorylation system PtkA/PtpZ was previou...... microscopy. B. subtilis cells lacking the kinase PtkA accumulated extra chromosome equivalents, exhibited aberrant initiation mass for DNA replication and an unusually long D period....

  1. Palmitylation of Src family tyrosine kinases regulates functional interaction with a B cell substrate.

    Science.gov (United States)

    Saouaf, S J; Wolven, A; Resh, M D; Bolen, J B

    1997-05-19

    Palmitylation of Src family tyrosine kinases has been shown to play a role in directing their membrane localization. Here we demonstrate that palmitylation can also regulate recognition and tyrosine phosphorylation of the B cell Src kinase substrate Ig alpha. Blk and Src, which are not palmitylated, phosphorylate co-expressed Ig alpha in Cos cells, whereas palmitylated Src kinases do not. Addition of a palmitylation site to Blk abrogates its phosphorylation of the substrate, while mutation of Fyn's palmitylation sites results in recognition and phosphorylation of Ig alpha. These results indicate that palmitylation, a reversible protein modification, aids in regulating recognition of physiologic substrates by Src family tyrosine kinases. PMID:9177269

  2. Identification of nitrated tyrosine residues of protein kinase G-Iα by mass spectrometry.

    Science.gov (United States)

    Lu, Jingshan; Yao, Ikuko; Shimojo, Masahito; Katano, Tayo; Uchida, Hitoshi; Setou, Mitsutoshi; Ito, Seiji

    2014-02-01

    The nitration of tyrosine to 3-nitrotyrosine is an oxidative modification of tyrosine by nitric oxide and is associated with many diseases, and targeting of protein kinase G (PKG)-I represents a potential therapeutic strategy for pulmonary hypertension and chronic pain. The direct assignment of tyrosine residues of PKG-I has remained to be made due to the low sensitivity of the current proteomic approach. In order to assign modified tyrosine residues of PKG-I, we nitrated purified PKG-Iα expressed in insect Sf9 cells by use of peroxynitrite in vitro and analyzed the trypsin-digested fragments by matrix-assisted laser desorption/ionization-time of flight mass spectrometry and liquid chromatography-tandem mass spectrometry. Among the 21 tyrosine residues of PKG-Iα, 16 tyrosine residues were assigned in 13 fragments; and six tyrosine residues were nitrated, those at Y71, Y141, Y212, Y336, Y345, and Y567, in the peroxynitrite-treated sample. Single mutation of tyrosine residues at Y71, Y212, and Y336 to phenylalanine significantly reduced the nitration of PKG-Iα; and four mutations at Y71, Y141, Y212, and Y336 (Y4F mutant) reduced it additively. PKG-Iα activity was inhibited by peroxynitrite in a concentration-dependent manner from 30 μM to 1 mM, and this inhibition was attenuated in the Y4F mutant. These results demonstrated that PKG-Iα was nitrated at multiple tyrosine residues and that its activity was reduced by nitration of these residues.

  3. Sch proteins are localized on endoplasmic reticulum membranes and are redistributed after tyrosine kinase receptor activation

    DEFF Research Database (Denmark)

    Lotti, L V; Lanfrancone, L; Migliaccio, E;

    1996-01-01

    area of the cell and mostly associated with the cytosolic side of rough endoplasmic reticulum membranes. Upon epidermal growth factor treatment and receptor tyrosine kinase activation, the immunolabeling became peripheral and was found to be associated with the cytosolic surface of the plasma membrane......The intracellular localization of Shc proteins was analyzed by immunofluorescence and immunoelectron microscopy in normal cells and cells expressing the epidermal growth factor receptor or the EGFR/erbB2 chimera. In unstimulated cells, the immunolabeling was localized in the central perinuclear....... The rough endoplasmic reticulum localization of Shc proteins in unstimulated cells and their massive recruitment to the plasma membrane, endocytic structures, and peripheral cytosol following receptor tyrosine kinase activation could account for multiple putative functions of the adaptor protein....

  4. Outer membrane protein A (OmpA of Shigella flexneri 2a induces TLR2-mediated activation of B cells: involvement of protein tyrosine kinase, ERK and NF-κB.

    Directory of Open Access Journals (Sweden)

    Rajsekhar Bhowmick

    Full Text Available B cells are critically important in combating bacterial infections and their differentiation into plasma cells and memory cells aids bacterial clearance and long-lasting immunity conferred by essentially all vaccines. Outer membrane protein A (OmpA of Shigella flexneri 2a has been demonstrated to induce the production of IgG and IgA in vivo following immunization of mice through intranasal route, but the direct involvement of B cells in OmpA-mediated immune regulation was not determined. Consequently, we investigated whether OmpA can modulate B cell functions and identified the molecular events involved in OmpA-induced B cell immune response in vitro. We show that OmpA of S. flexneri 2a activates B cells to produce protective cytokines, IL-6 and IL-10 as well as facilitates their differentiation into antibody secreting cells (ASCs. The immunostimulatory properties of OmpA are attributed to the increased surface expression of MHCII and CD86 on B cells. We also report here that B cell activation by OmpA is mediated strictly through recognition by TLR2, resulting in initiation of cascades of signal transduction events, involving increased phosphorylation of protein tyrosine kinases (PTKs, ERK and IκBα, leading to nuclear translocation of NF-κB. Importantly, a TLR2 antibody diminishes OmpA-induced upregulation of MHCII and CD86 on B cell surface as well as significantly inhibits B cell differentiation and cytokine secretion. Furthermore, we illustrate that B cell differentiation into ASCs and induction of cytokine secretion by OmpA are dependent on PTKs activity. Moreover, we identify that OmpA-induced B cell differentiation is entirely dependent on ERK pathway, whereas both NF-κB and ERK are essential for cytokine secretion by B cells. Overall, our data demonstrate that OmpA of S. flexneri 2a amplifies TLR signaling in B cells and triggers B cell immune response, which is critical for the development of an effective adaptive immunity to an

  5. Protein-tyrosine phosphorylation in Bacillus subtilis: a 10-year retrospective

    Directory of Open Access Journals (Sweden)

    Josef eDeutscher

    2015-01-01

    Full Text Available The discovery of tyrosine-phosphorylated proteins in Bacillus subtilis in the year 2003 was followed by a decade of intensive research activity. Here we provide an overview of the lessons learned in that period. While the number of characterized kinases and phosphatases involved in reversible protein-tyrosine phosphorylation in B. subtilis has remained essentially unchanged, the number of proteins known to be targeted by this post-translational modification has increased dramatically. This is mainly due to phosphoproteomics and interactomics studies, which were instrumental in identifying new tyrosine-phosphorylated proteins. Despite their structural similarity, the two B. subtilis protein-tyrosine kinases (BY-kinases, PtkA and PtkB (EpsB, seem to accomplish different functions in the cell. The PtkB is encoded by a large operon involved in exopolysaccharide production, and its main role appears to be the control of this process. The PtkA seems to have a more complex role; it phosphorylates and regulates a large number of proteins involved in the DNA, fatty acid and carbon metabolism and engages in physical interaction with other types of kinases (Ser/Thr kinases, leading to mutual phosphorylation. PtkA also seems to respond to several activator proteins, which direct its activity towards different substrates. In that respect PtkA seems to function as a highly connected signal integration device.

  6. Protein-Tyrosine Kinase Signaling in the Biological Functions Associated with Sperm

    Directory of Open Access Journals (Sweden)

    Takashi W. Ijiri

    2012-01-01

    Full Text Available In sexual reproduction, two gamete cells (i.e., egg and sperm fuse (fertilization to create a newborn with a genetic identity distinct from those of the parents. In the course of these developmental processes, a variety of signal transduction events occur simultaneously in each of the two gametes, as well as in the fertilized egg/zygote/early embryo. In particular, a growing body of knowledge suggests that the tyrosine kinase Src and/or other protein-tyrosine kinases are important elements that facilitate successful implementation of the aforementioned processes in many animal species. In this paper, we summarize recent findings on the roles of protein-tyrosine phosphorylation in many sperm-related processes (from spermatogenesis to epididymal maturation, capacitation, acrosomal exocytosis, and fertilization.

  7. SOCS proteins in regulation of receptor tyrosine kinase signaling

    DEFF Research Database (Denmark)

    Kazi, Julhash U.; Kabir, Nuzhat N.; Flores Morales, Amilcar;

    2014-01-01

    signaling mediated by RTKs must be tightly regulated by interacting proteins including protein-tyrosine phosphatases and ubiquitin ligases. The suppressors of cytokine signaling (SOCS) family proteins are well-known negative regulators of cytokine receptors signaling consisting of eight structurally similar...... proteins, SOCS1-7, and cytokine-inducible SH2-containing protein (CIS). A key feature of this family of proteins is the presence of an SH2 domain and a SOCS box. Recent studies suggest that SOCS proteins also play a role in RTK signaling. Activation of RTK results in transcriptional activation of SOCS......-encoding genes. These proteins associate with RTKs through their SH2 domains and subsequently recruit the E3 ubiquitin machinery through the SOCS box, and thereby limit receptor stability by inducing ubiquitination. In a similar fashion, SOCS proteins negatively regulate mitogenic signaling by RTKs. It is also...

  8. Protein tyrosine nitration in higher plants grown under natural and stress conditions

    Science.gov (United States)

    Corpas, Francisco J.; Palma, José M.; del Río, Luis A.; Barroso, Juan B.

    2013-01-01

    Protein tyrosine nitration is a post-translational modification (PTM) mediated by reactive nitrogen species (RNS) that is linked to nitro-oxidative damages in plant cells. During the last decade, the identification of proteins undergoing this PTM under adverse environmental conditions has increased. However, there is also a basal endogenous nitration which seems to have a regulatory function. The technological advances in proteome analysis have allowed identifying these modified proteins and have shown that the number and identity of the nitrated proteins change among plant species, analysed organs and growing/culture conditions. In this work, the current knowledge of protein tyrosine nitration in higher plants under different situations is reviewed. PMID:23444154

  9. Protein tyrosine phosphatase SAP-1 protects against colitis through regulation of CEACAM20 in the intestinal epithelium.

    Science.gov (United States)

    Murata, Yoji; Kotani, Takenori; Supriatna, Yana; Kitamura, Yasuaki; Imada, Shinya; Kawahara, Kohichi; Nishio, Miki; Daniwijaya, Edwin Widyanto; Sadakata, Hisanobu; Kusakari, Shinya; Mori, Munemasa; Kanazawa, Yoshitake; Saito, Yasuyuki; Okawa, Katsuya; Takeda-Morishita, Mariko; Okazawa, Hideki; Ohnishi, Hiroshi; Azuma, Takeshi; Suzuki, Akira; Matozaki, Takashi

    2015-08-01

    Intestinal epithelial cells contribute to regulation of intestinal immunity in mammals, but the detailed molecular mechanisms of such regulation have remained largely unknown. Stomach-cancer-associated protein tyrosine phosphatase 1 (SAP-1, also known as PTPRH) is a receptor-type protein tyrosine phosphatase that is localized specifically at microvilli of the brush border in gastrointestinal epithelial cells. Here we show that SAP-1 ablation in interleukin (IL)-10-deficient mice, a model of inflammatory bowel disease, resulted in a marked increase in the severity of colitis in association with up-regulation of mRNAs for various cytokines and chemokines in the colon. Tyrosine phosphorylation of carcinoembryonic antigen-related cell adhesion molecule (CEACAM) 20, an intestinal microvillus-specific transmembrane protein of the Ig superfamily, was greatly increased in the intestinal epithelium of the SAP-1-deficient animals, suggesting that this protein is a substrate for SAP-1. Tyrosine phosphorylation of CEACAM20 by the protein tyrosine kinase c-Src and the consequent association of CEACAM20 with spleen tyrosine kinase (Syk) promoted the production of IL-8 in cultured cells through the activation of nuclear factor-κB (NF-κB). In addition, SAP-1 and CEACAM20 were found to form a complex through interaction of their ectodomains. SAP-1 and CEACAM20 thus constitute a regulatory system through which the intestinal epithelium contributes to intestinal immunity.

  10. Protein tyrosine phosphatases expression during development of mouse superior colliculus

    OpenAIRE

    Reinhard, Jacqueline; Horvat-Bröcker, Andrea; Illes, Sebastian; Zaremba, Angelika; Knyazev, Piotr; Ullrich, Axel; Faissner, Andreas

    2009-01-01

    Protein tyrosine phosphatases (PTPs) are key regulators of different processes during development of the central nervous system. However, expression patterns and potential roles of PTPs in the developing superior colliculus remain poorly investigated. In this study, a degenerate primer-based reverse transcription-polymerase chain reaction (RT-PCR) approach was used to isolate seven different intracellular PTPs and nine different receptor-type PTPs (RPTPs) from embryonic E15 mouse superior col...

  11. Bombesin, vasopressin, and endothelin rapidly stimulate tyrosine phosphorylation in intact Swiss 3T3 cells

    Energy Technology Data Exchange (ETDEWEB)

    Zachary, I.; Gil, J.; Lehmann, W.; Sinnett-Smith, J.; Rozengurt, E. (Imperial Cancer Research Fund, London (England))

    1991-06-01

    The mitogenic neuropeptides bombesin and vasopressin markedly increased tyrosine and serine phosphorylation of multiple substrates in quiescent Swiss 3T3 fibroblasts, including two major bands of M{sub r} 90,000 and 115,000. Tyrosine phosphorylation of these proteins was increased as judged by immunoprecipitation of {sup 32}P{sub i}-labeled cells and immunoblotting of unlabeled cells with monoclonal antiphosphotyrosine antibodies, elution with phenyl phosphate, and phospho amino acid analysis. Phosphotyrosyl proteins generated by bombesin and vasopressin did not correspond either by apparent molecular weight or by immunological and biochemical criteria to several known tyrosine kinase substrates, including phospholipase C{sub {gamma}}, the microtubule-associated protein 2 kinase, GTPase-activating protein, or phosphatidylinositol kinase. The effect was rapid (within seconds), concentration dependent, and inhibited by specific receptor antagonists for both bombesin and vasopressin. The endothelin-related peptide, vasoactive intestinal contractor, also elicited a rapid and concentration-dependent tyrosine/serine phosphorylation of a similar set of substrates. These results demonstrate that neuropeptides, acting through receptors linked to GTP-binding proteins, stimulate tyrosine phosphorylation of a common set of substrates in quiescent Swiss 3T3 cells and suggest the existence of an additional signal transduction pathway in neuropeptide-induced mitogenesis.

  12. Modulation of protein tyrosine phosphorylation in gastric mucosa during re-epithelization processes

    Institute of Scientific and Technical Information of China (English)

    Olena; V; Bogdanova; Larysa; I; Kot; Kateryna; V; Lavrova; Volodymyr; B; Bogdanov; Erica; K; Sloan; Tetyana; V; Beregova; Ludmyla; I; Ostapchenko

    2010-01-01

    AIM:To investigate the role of protein tyrosine phosphorylation in gastric wound formation and repair following ulceration. METHODS:Gastric lesions were induced in rats using restraint cold stress.To investigate the effect of oxidative and nitrosative cell stress on tyrosine phosphorylation during wound repair,total activity of protein tyrosine kinase(PTK),protein tyrosine phosphatase (PTP),antioxidant enzymes,nitric oxide synthase (NOS), 2’,5’-oligoadenylate synthetase,hydroxyl radical and zinc levels were assayed in parallel. RESULTS:Ulcer provocation induced an immediate decrease in tyrosine kinase(40% in plasma membranes and 56% in cytosol,(P<0.05) and phosphatase activity (threefold in plasma membranes and 3.3-fold in cytosol),followed by 2.3-2.4-fold decrease (P<0.05) in protein phosphotyrosine content in the gastric mucosa. Ulceration induced no immediate change in superoxide dismutase (SOD) activity,30% increase (P<0.05) in catalase activity,2.3-fold inhibition (P<0.05) of glutathione peroxidase,3.3-fold increase (P<0.05) in hydroxyl radical content,and 2.3-fold decrease (P<0.05) in zinc level in gastric mucosa.NOS activity was three times higher in gastric mucosa cells after cold stress. Following ulceration,PTK activity increased in plasma membranes and reached a maximum on day 4 after stress (twofold increase,P<0.05),but remained inhibited(1.6-3-fold decrease on days 3,4 and 5,P<0.05) in the cytosol.Tyrosine phosphatases remained inhibited both in membranes and cytosol(1.5-2.4-fold,P< 0.05).NOS activity remained increased on days 1,2 and 3(3.8-,2.6-,2.2-fold,respectively,P<0.05).Activity of SOD increased 1.6 times(P<0.05)days 4 and 5 after stress.Catalase activity normalized after day 2. Glutathione peroxidase activity and zinc level decreased (3.3-and 2-fold,respectively,P<0.05)on the last day. Activity of 2’,5’-oligoadenylate synthethase increased 2.8-fold (P<0.05) at the beginning,and 1.6-2.3-fold (P<0.05) during ulcer recuperation

  13. Tyrosine-selective protein alkylation using pi-allylpalladium complexes.

    Science.gov (United States)

    Tilley, S David; Francis, Matthew B

    2006-02-01

    A new protein modification reaction has been developed based on a palladium-catalyzed allylic alkylation of tyrosine residues. This technique employs electrophilic pi-allyl intermediates derived from allylic acetate and carbamate precursors and can be used to modify proteins in aqueous solution at room temperature. To facilitate the detection of modified proteins using SDS-PAGE analysis, a fluorescent allyl acetate was synthesized and coupled to chymotrypsinogen A and bacteriophage MS2. The tyrosine selectivity of the reaction was confirmed through trypsin digest analysis. The utility of the reaction was demonstrated by using taurine-derived carbamates as water solubilizing groups that are cleaved upon protein functionalization. This solubility switching technique was used to install hydrophobic farnesyl and C(17) chains on chymotrypsinogen A in water using little or no cosolvent. Following this, the C(17) alkylated proteins were found to associate with lipid vesicles. In addition to providing a new protein modification strategy targeting an under-utilized amino acid side chain, this method provides convenient access to synthetic lipoproteins. PMID:16433516

  14. Expression and Characterization of Catalytic Domain of T Cell Protein Tyrosine Phosphatase(ΔTC-PTP)——Immunohistochemical Study of ΔTC-PTP Expression in Non-small Cell Lung Carcinomas

    Institute of Scientific and Technical Information of China (English)

    ZHU Zhi-cheng; SUN Mei; ZHANG Xing-yi; LIU Ke-xiang; SHI Dong-lei; LI Jin-dong; SU Ji-quan; XU Yue-chi; FU Xue-qi

    2007-01-01

    This study objective was to express and characterize the catalytic domain of the human T cell protein tyrosine phosphatase(ΔTC-PTP) and to study immunohistochemically the expression of ΔTC-PTP in human non-small cell lung cancers. ΔTC-PTP gene was PCR amplified with the cDNA of human TC-PTP as template, and cloned into the pT7 expression vector. The recombinant pT7-ΔTC-PTP was expressed in E.coli Rosetta(DE3) host cells and purified. The enzymatic characteristics of ΔTC-PTP including enzyme activity and kinetics assay were measured. The antiserum was prepared by immunizing rabbit with the purified recombinant ΔTC-PTP. Rabbit polyclonal antibody against ΔTC-PTP was purified by PVDF immobilized antigen affinity chromatography. Immunohistochemical staining of lung cancer tissues was performed with antibody against ΔTC-PTP protein. ΔTC-PTP gene was correctly cloned, expressed, and purified. The recombinant ΔTC-PTP had a highly catalytic activity of PTPase. Squamous cell lung carcinoma showed a significantly higher expression rate of ΔTC-PTP(76.92%, 10/13) than adenocarcinoma(57.14%, 4/7) and normal lung tissue(20%, 1/5). This study represents the first demonstration that ΔTC-PTP is highly expressed in human squamous cell lung carcinomas. In addition, this study provides an important basis for further studying the biological function of TC-PTP and its relationship with lung carcinomas and other diseases.

  15. Protein tyrosine phosphatase SHP-1 sensitizes EGFR/HER-2 positive breast cancer cells to trastuzumab through modulating phosphorylation of EGFR and HER-2

    Directory of Open Access Journals (Sweden)

    Wu YF

    2015-09-01

    Full Text Available Yifen Wu,1,2,* Rong Li,3,* Junyi Zhang,4 Gang Wang,5 Bin Liu,6 Xiaofang Huang,7 Tao Zhang,7 Rongcheng Luo8 1Traditional Chinese Medicine-Integrated Hospital, Southern Medical University, Guangzhou, 2Department of Oncology, Dongguan People’s Hospital, Dongguan, 3Department of Oncology, Nanfang Hospital, 4Department of Oncology, Traditional Chinese Medicine-Integrated Hospital, Southern Medical University, Guangzhou, 5Department of Radiology, Dongguan People’s Hospital, Dongguan, 6Second Affiliated Hospital of Guangzhou Medical College, 7College of Traditional Chinese medicine, Southern Medical University, 8Cancer Center, Traditional Chinese Medicine-Integrated Hospital, Southern Medical University, Guangzhou, Guangdong, People’s Republic of China *These authors contributed equally to this work Background: Trastuzumab resistance in HER-2 positive breast cancer cells is closely related to overexpression of both epidermal growth factor receptor (EGFR and human epidermal receptor (HER-2. SHP-1 has been demonstrated to downregulate tyrosine kinase activity including EGFR via its phosphatase function, but its effect on HER-2 activity is still unknown. Here, we examined the hypothesis that SHP-1 enhances the anticancer efficacy of trastuzumab in EGFR/HER-2 positive breast cancer cells through combining dual inhibition of EGFR and HER-2.Methods: Trastuzumab-resistant breast cancer SKBr-3 cells were generated by long-term in vitro culture of SKBr-3cells in the presence of trastuzumab. The SHP-1 was ectopically expressed by stable transfection. The activity and expression of EGFR, HER-2, and downstream signaling pathways were tested by Western blot. Cell viability was examined by the 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT assay, and apoptosis was examined by flow cytometry. The binding between SHP-1 and EGFR/HER-2 was evaluated by immunoprecipitation assay and bimolecular fluorescence complementation. The effects of SHP-1

  16. The tyrosine phosphatase SHP2 associates with CUB domain-containing protein-1 (CDCP1, regulating its expression at the cell surface in a phosphorylation-dependent manner.

    Directory of Open Access Journals (Sweden)

    Leslie Yewakon Gandji

    Full Text Available CUB domain-containing protein-1 (CDCP1 is a transmembrane glycoprotein that is phosphorylated by SRC family kinases (SFK before recruiting and activating PKCδ. CDCP1 is overproduced in many cancers. It promotes metastasis and resistance to anoïkis. The robust production of CDCP1 would be associated with stemness and has been proposed as a novel prognosis marker. The natural transmembrane location of CDCP1 makes it an ideal therapeutic target and treatments based on the use of appropriate antibodies are currently being evaluated. However, we still know very little about the molecular fate of CDCP1 and its downstream signaling events. Improvements in our understanding of the molecular events occurring downstream of CDCP1 are required to make use of changes of CDCP1 production or functions for therapeutic purposes. By the mean of co-immunoprecipitation and affinity precipitation we show here, for the first time, that CDCP1 interacts directly, with the cytosolic tyrosine phosphatase SHP2. Point mutants of CDCP1 show that residues Y734 and Y743 are responsible for its interaction with SHP2. It may therefore compete with SFK. We also demonstrate that a shRNA-mediated down regulation of SHP2 is associated with a stronger CDCP1 phosphorylation and an impairment of antibody-mediated CDCP1 internalization.

  17. Down-regulation of BDNF in cell and animal models increases striatal-enriched protein tyrosine phosphatase 61 (STEP61 ) levels.

    Science.gov (United States)

    Xu, Jian; Kurup, Pradeep; Azkona, Garikoitz; Baguley, Tyler D; Saavedra, Ana; Nairn, Angus C; Ellman, Jonathan A; Pérez-Navarro, Esther; Lombroso, Paul J

    2016-01-01

    Brain-derived neurotrophic factor (BDNF) regulates synaptic strengthening and memory consolidation, and altered BDNF expression is implicated in a number of neuropsychiatric and neurodegenerative disorders. BDNF potentiates N-methyl-D-aspartate receptor function through activation of Fyn and ERK1/2. STriatal-Enriched protein tyrosine Phosphatase (STEP) is also implicated in many of the same disorders as BDNF but, in contrast to BDNF, STEP opposes the development of synaptic strengthening. STEP-mediated dephosphorylation of the NMDA receptor subunit GluN2B promotes internalization of GluN2B-containing NMDA receptors, while dephosphorylation of the kinases Fyn, Pyk2, and ERK1/2 leads to their inactivation. Thus, STEP and BDNF have opposing functions. In this study, we demonstrate that manipulation of BDNF expression has a reciprocal effect on STEP61 levels. Reduced BDNF signaling leads to elevation of STEP61 both in BDNF(+/-) mice and after acute BDNF knockdown in cortical cultures. Moreover, a newly identified STEP inhibitor reverses the biochemical and motor abnormalities in BDNF(+/-) mice. In contrast, increased BDNF signaling upon treatment with a tropomyosin receptor kinase B agonist results in degradation of STEP61 and a subsequent increase in the tyrosine phosphorylation of STEP substrates in cultured neurons and in mouse frontal cortex. These findings indicate that BDNF-tropomyosin receptor kinase B signaling leads to degradation of STEP61 , while decreased BDNF expression results in increased STEP61 activity. A better understanding of the opposing interaction between STEP and BDNF in normal cognitive functions and in neuropsychiatric disorders will hopefully lead to better therapeutic strategies. Altered expression of BDNF and STEP61 has been implicated in several neurological disorders. BDNF and STEP61 are known to regulate synaptic strengthening, but in opposite directions. Here, we report that reduced BDNF signaling leads to elevation of STEP61 both in

  18. Tyrosine Sulfation as a Protein Post-Translational Modification

    Directory of Open Access Journals (Sweden)

    Yuh-Shyong Yang

    2015-01-01

    Full Text Available Integration of inorganic sulfate into biological molecules plays an important role in biological systems and is directly involved in the instigation of diseases. Protein tyrosine sulfation (PTS is a common post-translational modification that was first reported in the literature fifty years ago. However, the significance of PTS under physiological conditions and its link to diseases have just begun to be appreciated in recent years. PTS is catalyzed by tyrosylprotein sulfotransferase (TPST through transfer of an activated sulfate from 3'-phosphoadenosine-5'-phosphosulfate to tyrosine in a variety of proteins and peptides. Currently, only a small fraction of sulfated proteins is known and the understanding of the biological sulfation mechanisms is still in progress. In this review, we give an introductory and selective brief review of PTS and then summarize the basic biochemical information including the activity and the preparation of TPST, methods for the determination of PTS, and kinetics and reaction mechanism of TPST. This information is fundamental for the further exploration of the function of PTS that induces protein-protein interactions and the subsequent biochemical and physiological reactions.

  19. Protein tyrosine phosphatase non-receptor type 2 and inflammatory bowel disease

    OpenAIRE

    Spalinger, Marianne R.; MCCOLE, DECLAN F.; Rogler, Gerhard; Scharl, Michael

    2016-01-01

    Genome wide association studies have associated single nucleotide polymorphisms within the gene locus encoding protein tyrosine phosphatase non-receptor type 2 (PTPN2) with the onset of inflammatory bowel disease (IBD) and other inflammatory disorders. Expression of PTPN2 is enhanced in actively inflamed intestinal tissue featuring a marked up-regulation in intestinal epithelial cells. PTPN2 deficient mice suffer from severe intestinal and systemic inflammation and display aberrant innate and...

  20. Protein Tyrosine Phosphatase 1B Inhibitors from Plantago asiatica

    Institute of Scientific and Technical Information of China (English)

    CUI Long; LEE Hyun-sun; AHN Jong-seog; YUAN Guang-xin; SUN Ya-nan

    2011-01-01

    Objective To identify the active compounds for protein tyrosine phosphatase 1B (PTP1B) from the seeds of Plantago asiatica. Methods Bioassay-guided fractionation resulted in the isolation of iridoid glucosides (1-5) with PTP1B inhibitory activity. Results Five compounds were identified as desacetylhookerioside (1), melittoside (2), geniposidic acid (3), 10-O-acetyl-geniposidic acid (4), and alpinoside (5). Conclusion Isolated compounds 3-5 inhibit PTP1B with IC50 values ranged from (16.3 ± 1.1) to (19.8 ± 1.2) μmol/L.

  1. Adipocyte-specific protein tyrosine phosphatase 1B deletion increases lipogenesis, adipocyte cell size and is a minor regulator of glucose homeostasis.

    Directory of Open Access Journals (Sweden)

    Carl Owen

    Full Text Available Protein tyrosine phosphatase 1B (PTP1B, a key negative regulator of leptin and insulin signaling, is positively correlated with adiposity and contributes to insulin resistance. Global PTP1B deletion improves diet-induced obesity and glucose homeostasis via enhanced leptin signaling in the brain and increased insulin signaling in liver and muscle. However, the role of PTP1B in adipocytes is unclear, with studies demonstrating beneficial, detrimental or no effect(s of adipose-PTP1B-deficiency on body mass and insulin resistance. To definitively establish the role of adipocyte-PTP1B in body mass regulation and glucose homeostasis, adipocyte-specific-PTP1B knockout mice (adip-crePTP1B(-/- were generated using the adiponectin-promoter to drive Cre-recombinase expression. Chow-fed adip-crePTP1B(-/- mice display enlarged adipocytes, despite having similar body weight/adiposity and glucose homeostasis compared to controls. High-fat diet (HFD-fed adip-crePTP1B(-/- mice display no differences in body weight/adiposity but exhibit larger adipocytes, increased circulating glucose and leptin levels, reduced leptin sensitivity and increased basal lipogenesis compared to controls. This is associated with decreased insulin receptor (IR and Akt/PKB phosphorylation, increased lipogenic gene expression and increased hypoxia-induced factor-1-alpha (Hif-1α expression. Adipocyte-specific PTP1B deletion does not beneficially manipulate signaling pathways regulating glucose homeostasis, lipid metabolism or adipokine secretion in adipocytes. Moreover, PTP1B does not appear to be the major negative regulator of the IR in adipocytes.

  2. Redox regulation of protein tyrosine phosphatase 1B involves a sulphenyl-amide intermediate.

    Science.gov (United States)

    Salmeen, Annette; Andersen, Jannik N; Myers, Michael P; Meng, Tzu-Ching; Hinks, John A; Tonks, Nicholas K; Barford, David

    2003-06-12

    The second messenger hydrogen peroxide is required for optimal activation of numerous signal transduction pathways, particularly those mediated by protein tyrosine kinases. One mechanism by which hydrogen peroxide regulates cellular processes is the transient inhibition of protein tyrosine phosphatases through the reversible oxidization of their catalytic cysteine, which suppresses protein dephosphorylation. Here we describe a structural analysis of the redox-dependent regulation of protein tyrosine phosphatase 1B (PTP1B), which is reversibly inhibited by oxidation after cells are stimulated with insulin and epidermal growth factor. The sulphenic acid intermediate produced in response to PTP1B oxidation is rapidly converted into a previously unknown sulphenyl-amide species, in which the sulphur atom of the catalytic cysteine is covalently linked to the main chain nitrogen of an adjacent residue. Oxidation of PTP1B to the sulphenyl-amide form is accompanied by large conformational changes in the catalytic site that inhibit substrate binding. We propose that this unusual protein modification both protects the active-site cysteine residue of PTP1B from irreversible oxidation to sulphonic acid and permits redox regulation of the enzyme by promoting its reversible reduction by thiols.

  3. Clustering of Helicobacter pylori VacA in lipid rafts, mediated by its receptor, receptor-like protein tyrosine phosphatase beta, is required for intoxication in AZ-521 Cells

    DEFF Research Database (Denmark)

    Nakayama, Masaaki; Hisatsune, Jyunzo; Yamasaki, Eiki;

    2006-01-01

    Helicobacter pylori vacuolating cytotoxin, VacA, induces multiple effects on epithelial cells through different cellular events: one involves pore formation, leading to vacuolation, mitochondrial damage, and apoptosis, and the second involves cell signaling, resulting in stimulation of proinflamm......Helicobacter pylori vacuolating cytotoxin, VacA, induces multiple effects on epithelial cells through different cellular events: one involves pore formation, leading to vacuolation, mitochondrial damage, and apoptosis, and the second involves cell signaling, resulting in stimulation...... of proinflammatory responses and cell detachment. Our recent data demonstrated that VacA uses receptor-like protein tyrosine phosphatase beta (RPTPbeta) as a receptor, of which five residues (QTTQP) at positions 747 to 751 are involved in binding. In AZ-521 cells, which mainly express RPTPbeta, VacA, after binding...... to RPTPbeta in non-lipid raft microdomains on the cell surface, is localized with RPTPbeta in lipid rafts in a temperature- and VacA concentration-dependent process. Methyl-beta-cyclodextrin (MCD) did not block binding to RPTPbeta but inhibited translocation of VacA with RPTPbeta to lipid rafts and all...

  4. Role of Protein Phosphorylation and Tyrosine Phosphatases in the Adrenal Regulation of Steroid Synthesis and Mitochondrial Function.

    Science.gov (United States)

    Paz, Cristina; Cornejo Maciel, Fabiana; Gorostizaga, Alejandra; Castillo, Ana F; Mori Sequeiros García, M Mercedes; Maloberti, Paula M; Orlando, Ulises D; Mele, Pablo G; Poderoso, Cecilia; Podesta, Ernesto J

    2016-01-01

    In adrenocortical cells, adrenocorticotropin (ACTH) promotes the activation of several protein kinases. The action of these kinases is linked to steroid production, mainly through steroidogenic acute regulatory protein (StAR), whose expression and activity are dependent on protein phosphorylation events at genomic and non-genomic levels. Hormone-dependent mitochondrial dynamics and cell proliferation are functions also associated with protein kinases. On the other hand, protein tyrosine dephosphorylation is an additional component of the ACTH signaling pathway, which involves the "classical" protein tyrosine phosphatases (PTPs), such as Src homology domain (SH) 2-containing PTP (SHP2c), and members of the MAP kinase phosphatase (MKP) family, such as MKP-1. PTPs are rapidly activated by posttranslational mechanisms and participate in hormone-stimulated steroid production. In this process, the SHP2 tyrosine phosphatase plays a crucial role in a mechanism that includes an acyl-CoA synthetase-4 (Acsl4), arachidonic acid (AA) release and StAR induction. In contrast, MKPs in steroidogenic cells have a role in the turn-off of the hormonal signal in ERK-dependent processes such as steroid synthesis and, perhaps, cell proliferation. This review analyzes the participation of these tyrosine phosphates in the ACTH signaling pathway and the action of kinases and phosphatases in the regulation of mitochondrial dynamics and steroid production. In addition, the participation of kinases and phosphatases in the signal cascade triggered by different stimuli in other steroidogenic tissues is also compared to adrenocortical cell/ACTH and discussed.

  5. Tyrosine phosphorylation profiling in FGF-2 stimulated human embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    Vanessa M Y Ding

    Full Text Available The role of fibroblast growth factor-2 (FGF-2 in maintaining undifferentiated human embryonic stem cells (hESC was investigated using a targeted phosphoproteomics approach to specifically profile tyrosine phosphorylation events following FGF-2 stimulation. A cumulative total number of 735 unique tyrosine phosphorylation sites on 430 proteins were identified, by far the largest inventory to date for hESC. Early signaling events in FGF-2 stimulated hESC were quantitatively monitored using stable isotope dimethyl labeling, resulting in temporal tyrosine phosphorylation profiles of 316 unique phosphotyrosine peptides originating from 188 proteins. Apart from the rapid activation of all four FGF receptors, trans-activation of several other receptor tyrosine kinases (RTKs was observed as well as induced tyrosine phosphorylation of downstream proteins such as PI3-K, MAPK and several Src family members. Both PI3-K and MAPK have been linked to hESC maintenance through FGF-2 mediated signaling. The observed activation of the Src kinase family members by FGF-2 and loss of pluripotent marker expression post Src kinase inhibition may point to the regulation of cytoskeletal and actin depending processes to maintain undifferentiated hESC.

  6. Expression of protein-tyrosine phosphatases in the major insulin target tissues

    DEFF Research Database (Denmark)

    Norris, K; Norris, F; Kono, D H;

    1997-01-01

    Protein-tyrosine phosphatases (PTPs) are key regulators of the insulin receptor signal transduction pathway. We have performed a detailed analysis of PTP expression in the major human insulin target tissues or cells (liver, adipose tissue, skeletal muscle and endothelial cells). To obtain...... PTPs. Surprisingly, PTP-LAR, previously suggested to be a major regulator of the insulin receptor tyrosine kinase, was expressed in extremely low levels in skeletal muscle, whereas the related receptor-type PTP-sigma and PTP-alpha were expressed in relatively high levels in all four tissues. The low...... a representative picture, all tissues were analyzed by PCR using three different primer sets corresponding to conserved regions of known PTPs. A total of 24 different PTPs were identified. A multiprobe RNase protection assay was developed to obtain a semiquantitative measure of the expression levels of selected...

  7. Bruton tyrosine kinase inhibitors: a promising novel targeted treatment for B cell lymphomas

    OpenAIRE

    Aalipour, Amin; Advani, Ranjana H.

    2013-01-01

    Constitutive or aberrant signalling of the B cell receptor signalling cascade has been implicated in the propagation and maintenance of a variety of B cell malignancies. Small molecule inhibitors of Bruton tyrosine kinase (BTK), a protein early in this cascade and specifically expressed in B cells, have emerged as a new class of targeted agents. There are several BTK inhibitors, including ONO-WG-307, LFM-A13, dasatinib, CC-292, and PCI-32765 (ibrutinib), in preclinical and/or clinical develop...

  8. Dynamic recruitment of protein tyrosine phosphatase PTPD1 to EGF stimulation sites potentiates EGFR activation.

    Directory of Open Access Journals (Sweden)

    Pedro Roda-Navarro

    Full Text Available Balanced activity of protein tyrosine kinases and phosphatases (PTPs controls tyrosine phosphorylation levels and, consequently, is needed to prevent pathologies like cancer. Phosphatase activity is tightly regulated in space and time. Thus, in order to understand how phospho-tyrosine signalling is regulated, the intracellular dynamics of PTPs should be investigated. Here, we have studied the intracellular dynamics of PTPD1, a FERM (four-point-one, ezrin, radixin, moesin domain-containing PTP that is over expressed in cancer cells and potentiates EGFR signalling. Whereas PTPD1 was excluded from E-cadherin rich cell-cell adhesions in epithelial cell monolayers, it diffused from the cytoplasm to those membranes in contact with the extracellular medium. Localisation of PTPD1 at the plasma membrane was mediated by its FERM domain and enabled the formation of EGFR/PTPD1-containing signalling complexes that pre-existed at the plasma membrane before EGF stimulation. PTPD1 and EGFR transiently co-localised at EGF stimulation sites until the formation of macropinosomes containing active species of EGFR. Interference of PTPD1 expression caused a decrease in EGFR phosphorylated species at the periphery of the cell. Presented data suggest that the transient formation of dynamic PTPD1/EGFR signalling complexes strengthens EGF signalling by promoting the spatial propagation of EGFR phosphorylated species.

  9. Protein-tyrosine-phosphatase SHPTP2 couples platelet-derived growth factor receptor beta to Ras.

    OpenAIRE

    Bennett, A.M.; Tang, T. L.; SUGIMOTO, S; Walsh, C T; Neel, B G

    1994-01-01

    Protein-tyrosine-phosphatase SHPTP2 (Syp/PTP-1D/PTP2C) is the homologue of the Drosophila corkscrew (csw) gene product, which transmits positive signals from receptor tyrosine kinases. Likewise, SHPTP2 has been implicated in positive signaling from platelet-derived growth factor receptor beta (PDGFR). Upon PDGF stimulation, SHPTP2 binds to the PDGFR and becomes tyrosine-phosphorylated. We have identified tyrosine-542 (pY542TNI) as the major in vivo site of SHPTP2 tyrosine phosphorylation. The...

  10. The tyrosine phosphatase Shp2 interacts with NPM-ALK and regulates anaplastic lymphoma cell growth and migration

    DEFF Research Database (Denmark)

    Voena, Claudia; Conte, Chiara; Ambrogio, Chiara;

    2007-01-01

    Anaplastic large cell lymphomas (ALCL) are mainly characterized by the reciprocal translocation t(2;5)(p23;q35) that involves the anaplastic lymphoma kinase (ALK) gene and generates the fusion protein NPM-ALK with intrinsic tyrosine kinase activity. NPM-ALK triggers several signaling cascades......, leading to increased cell growth, resistance to apoptosis, and changes in morphology and migration of transformed cells. To search for new NPM-ALK interacting molecules, we developed a mass spectrometry-based proteomic approach in HEK293 cells expressing an inducible NPM-ALK and identified the tyrosine...... phosphatase Shp2 as a candidate substrate. We found that NPM-ALK was able to bind Shp2 in coprecipitation experiments and to induce its phosphorylation in the tyrosine residues Y542 and Y580 both in HEK293 cells and ALCL cell lines. In primary lymphomas, antibodies against the phosphorylated tyrosine Y542...

  11. Structural basis for inhibition of the protein tyrosine phosphatase 1B by phosphotyrosine peptide mimetics

    NARCIS (Netherlands)

    Groves, M R; Yao, Z J; Roller, P P; Burke, T R; Barford, D

    1998-01-01

    Protein tyrosine phosphatases regulate diverse cellular processes and represent important targets for therapeutic intervention in a number of diseases. The crystal structures of protein tyrosine phosphatase 1B (PTP1B) in complex with small molecule inhibitors based upon two classes of phosphotyrosin

  12. G1 cell cycle arrest due to the inhibition of erbB family receptor tyrosine kinases does not require the retinoblastoma protein

    International Nuclear Information System (INIS)

    The erbB receptor family (EGFr, erbB-2, erbB-3, and erbB-4) consists of transmembrane glycoproteins that transduce extracellular signals to the nucleus when activated. erbB family members are widely expressed in epithelial, mesenchymal, and neuronal cells and contribute to the proliferation, differentiation, migration, and survival of these cell types. The present study evaluates the effects of erbB family signaling on cell cycle progression and the role that pRB plays in regulating this process. ErbB family RTK activity was inhibited by PD 158780 in the breast epithelial cell line MCF10A. PD 158780 (0.5 μM) inhibited EGF-stimulated and heregulin-stimulated autophosphorylation and caused a G1 cell cycle arrest within 24 h, which correlated with hypophosporylation of pRB. MCF10A cells lacking functional pRB retained the ability to arrest in G1 when treated with PD 158780. Both cell lines showed induction of p27KIP1 protein when treated with PD 158780 and increased association of p27KIP1 with cyclin E-CDK2. Furthermore, CDK2 kinase activity was dramatically inhibited with drug treatment. Changes in other pRB family members were noted with drug treatment, namely a decrease in p107 and an increase in p130. These findings show that the G1 arrest induced through inhibition of erbB family RTK activity does not require functional pRB

  13. Examination of tyrosine/adenine stacking interactions in protein complexes.

    Science.gov (United States)

    Copeland, Kari L; Pellock, Samuel J; Cox, James R; Cafiero, Mauricio L; Tschumper, Gregory S

    2013-11-14

    The π-stacking interactions between tyrosine amino acid side chains and adenine-bearing ligands are examined. Crystalline protein structures from the protein data bank (PDB) exhibiting face-to-face tyrosine/adenine arrangements were used to construct 20 unique 4-methylphenol/N9-methyladenine (p-cresol/9MeA) model systems. Full geometry optimization of the 20 crystal structures with the M06-2X density functional theory method identified 11 unique low-energy conformations. CCSD(T) complete basis set (CBS) limit interaction energies were estimated for all of the structures to determine the magnitude of the interaction between the two ring systems. CCSD(T) computations with double-ζ basis sets (e.g., 6-31G*(0.25) and aug-cc-pVDZ) indicate that the MP2 method overbinds by as much as 3.07 kcal mol(-1) for the crystal structures and 3.90 kcal mol(-1) for the optimized structures. In the 20 crystal structures, the estimated CCSD(T) CBS limit interaction energy ranges from -4.00 to -6.83 kcal mol(-1), with an average interaction energy of -5.47 kcal mol(-1), values remarkably similar to the corresponding data for phenylalanine/adenine stacking interactions. Geometry optimization significantly increases the interaction energies of the p-cresol/9MeA model systems. The average estimated CCSD(T) CBS limit interaction energy of the 11 optimized structures is 3.23 kcal mol(-1) larger than that for the 20 crystal structures.

  14. Family of receptor-linked protein tyrosine phosphatases in humans and Drosophila

    International Nuclear Information System (INIS)

    To understand the regulation of cell proliferation by tyrosine phosphorylation, characterization of protein tyrosine phosphatases is essential. The human genes LCA (leukocyte common antigen) and LAR encode putative receptor-linked PTPases. By using consensus sequence probes, two additional receptor-linked PTPase genes, DLAR and DPTP, were isolated from Drosophila melanogaster. The extra-cellular segments of both DLAR and DPTP are composed of multiple immunoglobulin-like domains and fibronectin type III-like domains. The cytoplasmic region of DLAR and DPTP, as well as human LCA and LAR, are composed of two tandemly repeated PTPase domains. PTPase activities of immunoprecipitated LCA and LAR were demonstrated by measuring the release of phosphate from a 32P-labeled [Tyr(P)]peptide. Furthermore, the cytoplasmic domains of LCA, LAR, DLAR, and DPTP, expressed in Escherichia coli, have PTPase activity. Site-directed mutagenesis showed that a conserved cysteine residue is essential for PTPase activity

  15. Crystallization and preliminary X-ray diffraction analysis of rat protein tyrosine phosphatase η

    Energy Technology Data Exchange (ETDEWEB)

    Matozo, Huita C.; Nascimento, Alessandro S.; Santos, Maria A. M. [Instituto de Física de São Carlos, Departamento de Física e Informática, Universidade de São Paulo, Avenida Trabalhador São Carlense 400, CEP 13566-590 São Carlos, SP (Brazil); Iuliano, Rodolfo [Dipartimento di Medicina Sperimentale e Clinica, Facoltà di Medicina e Chirurgia, Università di Catanzaro, 88100 Catanzaro (Italy); Fusco, Alfredo [Dipartimento di Biologia e Patologia Cellulare e Molecolare, c/o Instituto di Endocrinologia ed Oncologia Sperimentale del CNR, Facolta di Medicina e Chirurgia, Università degli Studi di Napoli ‘Federico II’, Via Pansini 5, 80131 Naples (Italy); NOGEC (Naples Oncogenomocs Center)-CEINGE, Biotecnologie Avanzate, Via Comunale Margherita 482, 80145 Naples (Italy); Polikarpov, Igor, E-mail: ipolikarpov@if.sc.usp.br [Instituto de Física de São Carlos, Departamento de Física e Informática, Universidade de São Paulo, Avenida Trabalhador São Carlense 400, CEP 13566-590 São Carlos, SP (Brazil); Laboratório Nacional de Luz Síncrotron, Campinas, SP (Brazil)

    2006-09-01

    In this study, the catalytic domain of rat protein tyrosine phosphatase η was produced in Escherichia coli in soluble form and purified to homogeneity. Crystals were obtained by the hanging-drop vapour-diffusion method. The rat protein tyrosine phosphatase η (rPTPη) is a cysteine-dependent phosphatase which hydrolyzes phosphoester bonds in proteins and other molecules. rPTPη and its human homologue DEP-1 are involved in neoplastic transformations. Thus, expression of the protein is reduced in all oncogene-transformed thyroid cell lines and is absent in highly malignant thyroid cells. Moreover, consistent with the suggested tumour suppression role of PTPη, inhibition of the tumorigenic process occurs after its exogenous reconstitution, suggesting that PTPη might be important for gene therapy of cancers. In this study, the catalytic domain of rPTPη was produced in Escherichia coli in soluble form and purified to homogeneity. Crystals were obtained by the hanging-drop vapour-diffusion method. Diffraction data were collected to 1.87 Å resolution. The crystal belongs to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 46.46, b = 63.07, c = 111.64 Å, and contains one molecule per asymmetric unit.

  16. 1H-2,3-Dihydroperimidine Derivatives: A New Class of Potent Protein Tyrosine Phosphatase 1B Inhibitors

    Directory of Open Access Journals (Sweden)

    Wen-Long Wang

    2013-12-01

    Full Text Available A series of 1H-2,3-dihydroperimidine derivatives was designed, synthesized, and evaluated as a new class of inhibitors of protein tyrosine phosphatase 1B (PTP1B with IC50 values in the micromolar range. Compounds 46 and 49 showed submicromolar inhibitory activity against PTP1B, and good selectivity (3.48-fold and 2.10-fold respectively over T-cell protein tyrosine phosphatases (TCPTP. These results have provided novel lead compounds for the design of inhibitors of PTP1B as well as other PTPs.

  17. RhoA/phosphatidylinositol 3-kinase/protein kinase B/mitogen-activated protein kinase signaling after growth arrest-specific protein 6/mer receptor tyrosine kinase engagement promotes epithelial cell growth and wound repair via upregulation of hepatocyte growth factor in macrophages.

    Science.gov (United States)

    Lee, Ye-Ji; Park, Hyun-Jung; Woo, So-Youn; Park, Eun-Mi; Kang, Jihee Lee

    2014-09-01

    Growth arrest-specific protein 6 (Gas6)/Mer receptor tyrosine kinase (Mer) signaling modulates cytokine secretion and helps to regulate the immune response and apoptotic cell clearance. Signaling pathways that activate an epithelial growth program in macrophages are still poorly defined. We report that Gas6/Mer/RhoA signaling can induce the production of epithelial growth factor hepatic growth factor (HGF) in macrophages, which ultimately promotes epithelial cell proliferation and wound repair. The RhoA/protein kinase B (Akt)/mitogen-activated protein (MAP) kinases, including p38 MAP kinase, extracellular signal-regulated protein kinase, and Jun NH2-terminal kinase axis in RAW 264.7 cells, was identified as Gas6/Mer downstream signaling pathway for the upregulation of HGF mRNA and protein. Conditioned medium from RAW 264.7 cells that had been exposed to Gas6 or apoptotic cells enhanced epithelial cell proliferation of the epithelial cell line LA-4 and wound closure. Cotreatment with an HGF receptor-blocking antibody or c-Met antagonist downregulated this enhancement. Inhibition of Mer with small interfering RNA (siRNA) or the RhoA/Rho kinase pathway by RhoA siRNA or Rho kinase pharmacologic inhibitor suppressed Gas6-induced HGF mRNA and protein expression in macrophages and blocked epithelial cell proliferation and wound closure induced by the conditioned medium. Our data provide evidence that macrophages can be reprogrammed by Gas6 to promote epithelial proliferation and wound repair via HGF, which is induced by the Mer/RhoA/Akt/MAP kinase pathway. Thus, defects in Gas6/Mer/RhoA signaling in macrophages may delay tissue repair after injury to the alveolar epithelium.

  18. Protein tyrosine phosphatase receptor delta acts as a neuroblastoma tumor suppressor by destabilizing the aurora kinase a oncogene

    Directory of Open Access Journals (Sweden)

    Meehan Maria

    2012-02-01

    Full Text Available Abstract Background Protein tyrosine phosphatase receptor delta (PTPRD is a member of a large family of protein tyrosine phosphatases which negatively regulate tyrosine phosphorylation. Neuroblastoma is a major childhood cancer arising from precursor cells of the sympathetic nervous system which is known to acquire deletions and alterations in the expression patterns of PTPRD, indicating a potential tumor suppressor function for this gene. The molecular mechanism, however, by which PTPRD renders a tumor suppressor effect in neuroblastoma is unknown. Results As a molecular mechanism, we demonstrate that PTPRD interacts with aurora kinase A (AURKA, an oncogenic protein that is over-expressed in multiple forms of cancer, including neuroblastoma. Ectopic up-regulation of PTPRD in neuroblastoma dephosphorylates tyrosine residues in AURKA resulting in a destabilization of this protein culminating in interfering with one of AURKA's primary functions in neuroblastoma, the stabilization of MYCN protein, the gene of which is amplified in approximately 25 to 30% of high risk neuroblastoma. Conclusions PTPRD has a tumor suppressor function in neuroblastoma through AURKA dephosphorylation and destabilization and a downstream destabilization of MYCN protein, representing a novel mechanism for the function of PTPRD in neuroblastoma.

  19. Protein tyrosine phosphatase receptor delta acts as a neuroblastoma tumor suppressor by destabilizing the aurora kinase a oncogene

    LENUS (Irish Health Repository)

    Meehan, Maria

    2012-02-05

    Abstract Background Protein tyrosine phosphatase receptor delta (PTPRD) is a member of a large family of protein tyrosine phosphatases which negatively regulate tyrosine phosphorylation. Neuroblastoma is a major childhood cancer arising from precursor cells of the sympathetic nervous system which is known to acquire deletions and alterations in the expression patterns of PTPRD, indicating a potential tumor suppressor function for this gene. The molecular mechanism, however, by which PTPRD renders a tumor suppressor effect in neuroblastoma is unknown. Results As a molecular mechanism, we demonstrate that PTPRD interacts with aurora kinase A (AURKA), an oncogenic protein that is over-expressed in multiple forms of cancer, including neuroblastoma. Ectopic up-regulation of PTPRD in neuroblastoma dephosphorylates tyrosine residues in AURKA resulting in a destabilization of this protein culminating in interfering with one of AURKA\\'s primary functions in neuroblastoma, the stabilization of MYCN protein, the gene of which is amplified in approximately 25 to 30% of high risk neuroblastoma. Conclusions PTPRD has a tumor suppressor function in neuroblastoma through AURKA dephosphorylation and destabilization and a downstream destabilization of MYCN protein, representing a novel mechanism for the function of PTPRD in neuroblastoma.

  20. Cross-phosphorylation of bacterial serine/threonine and tyrosine protein kinases on key regulatory residues

    Directory of Open Access Journals (Sweden)

    Lei eShi

    2014-09-01

    Full Text Available Bacteria possess protein serine/threonine and tyrosine kinases which resemble eukaryal kinases in their capacity to phosphorylate multiple substrates. We hypothesized that the analogy might extend further, and bacterial kinases may also undergo mutual phosphorylation and activation, which is currently considered as a hallmark of eukaryal kinase networks. In order to test this hypothesis, we explored the capacity of all members of four different classes of serine/threonine and tyrosine kinases present in the firmicute model organism Bacillus subtilis to phosphorylate each other in vitro and interact with each other in vivo. The interactomics data suggested a high degree of connectivity among all types of kinases, while phosphorylation assays revealed equally wide-spread cross-phosphorylation events. Our findings suggest that the Hanks-type kinases PrkC, PrkD and YabT exhibit the highest capacity to phosphorylate other B. subtilis kinases, while the BY-kinase PtkA and the two-component-like kinases RsbW and SpoIIAB show the highest propensity to be phosphorylated by other kinases. Analysis of phosphorylated residues on several selected recipient kinases suggests that most cross-phosphorylation events concern key regulatory residues. Therefore, cross-phosphorylation events are very likely to influence the capacity of recipient kinases to phosphorylate substrates downstream in the signal transduction cascade. We therefore conclude that bacterial serine/threonine and tyrosine kinases probably engage in a network-type behavior previously described only in eukaryal cells.

  1. Protein tyrosine phosphatase receptor type z negatively regulates oligodendrocyte differentiation and myelination.

    Directory of Open Access Journals (Sweden)

    Kazuya Kuboyama

    Full Text Available BACKGROUND: Fyn tyrosine kinase-mediated down-regulation of Rho activity through activation of p190RhoGAP is crucial for oligodendrocyte differentiation and myelination. Therefore, the loss of function of its counterpart protein tyrosine phosphatase (PTP may enhance myelination during development and remyelination in demyelinating diseases. To test this hypothesis, we investigated whether Ptprz, a receptor-like PTP (RPTP expressed abuntantly in oligodendrocyte lineage cells, is involved in this process, because we recently revealed that p190RhoGAP is a physiological substrate for Ptprz. METHODOLOGY/PRINCIPAL FINDINGS: We found an early onset of the expression of myelin basic protein (MBP, a major protein of the myelin sheath, and early initiation of myelination in vivo during development of the Ptprz-deficient mouse, as compared with the wild-type. In addition, oligodendrocytes appeared earlier in primary cultures from Ptprz-deficient mice than wild-type mice. Furthermore, adult Ptprz-deficient mice were less susceptible to experimental autoimmune encephalomyelitis (EAE induced by active immunization with myelin/oligodendrocyte glycoprotein (MOG peptide than were wild-type mice. After EAE was induced, the tyrosine phosphorylation of p190RhoGAP increased significantly, and the EAE-induced loss of MBP was markedly suppressed in the white matter of the spinal cord in Ptprz-deficient mice. Here, the number of T-cells and macrophages/microglia infiltrating into the spinal cord did not differ between the two genotypes after MOG immunization. All these findings strongly support the validity of our hypothesis. CONCLUSIONS/SIGNIFICANCE: Ptprz plays a negative role in oligodendrocyte differentiation in early central nervous system (CNS development and remyelination in demyelinating CNS diseases, through the dephosphorylation of substrates such as p190RhoGAP.

  2. Protein tyrosine phosphatase 1B inhibitors isolated from Artemisia roxburghiana.

    Science.gov (United States)

    Shah, Muhammad Raza; Ishtiaq; Hizbullah, Syed Muhammad; Habtemariam, Solomon; Zarrelli, Armando; Muhammad, Akhtar; Collina, Simona; Khan, Inamulllah

    2016-08-01

    Artemisia roxburghiana is used in traditional medicine for treating various diseases including diabetes. The present study was designed to evaluate the antidiabetic potential of active constituents by using protein tyrosine phosphatase 1B (PTP1B) as a validated target for management of diabetes. Various compounds were isolated as active principles from the crude methanolic extract of aerial parts of A. roxburghiana. All compounds were screened for PTP1B inhibitory activity. Molecular docking simulations were performed to investigate the mechanism behind PTP1B inhibition of the isolated compound and positive control, ursolic acid. Betulinic acid, betulin and taraxeryl acetate were the active PTP1B principles with IC50 values 3.49 ± 0.02, 4.17 ± 0.03 and 87.52 ± 0.03 µM, respectively. Molecular docking studies showed significant molecular interactions of the triterpene inhibitors with Gly220, Cys215, Gly218 and Asp48 inside the active site of PTP1B. The antidiabetic activity of A. roxburghiana could be attributed due to PTP1B inhibition by its triterpene constituents, betulin, betulinic acid and taraxeryl acetate. Computational insights of this study revealed that the C-3 and C-17 positions of the compounds needs extensive optimization for the development of new lead compounds. PMID:26118418

  3. Screening of anti-hypoxia/reoxygenation agents by an in vitro model. Part 1: Natural inhibitors for protein tyrosine kinase activated by hypoxia/reoxygenation in cultured human umbilical vein endothelial cells.

    Science.gov (United States)

    Zhang, Y W; Morita, I; Shao, G; Yao, X S; Murota, S

    2000-03-01

    Protein tyrosine kinase (PTK) signaling pathways play important roles in ischemia/reperfusion (I/R) or hypoxia/reoxygenation (H/R) injuries. Inhibition of PTK activation can protect against I/R- or H/R-induced damages. As one part of our work for seeking bioactive compounds from natural sources against I/R or H/R, in the present study we examined the effects of 54 compounds purified from various traditional Chinese herbs on H/R-induced PTK activation by means of an in vitro H/R model in cultured human umbilical vein endothelial cells (HUVEC). The results demonstrated that an increase in PTK activation was induced after 2 h of reoxygenation. Compounds 2 (macrostemososide A), 3 (laxogenin-3-O-beta-D-xylopyranosyl-(1-->4)-alpha-L-arabinopyra nosyl- (1-->6)-beta-D-glucopyranoside), 4 (chinenoside II), 7 (ginsenoside-Rd), 52 (icariin), 53 (icariside), and 54 (icaritin) showed relatively obvious inhibition on this H/R-induced PTK activation. Compounds 5 (beta-sitosterol) and 6 (daucosterine), especially 5, completely blocked such an increased activation of PTK induced by H/R. On the contrary, compound 29 (isocumarine) significantly promoted PTK activation further. Moreover, the effects of these compounds on PTK activation were dose-dependent. PMID:10763582

  4. Unbiased identification of substrates of protein tyrosine phosphatase ptp-3 in C. elegans.

    Science.gov (United States)

    Mitchell, Christopher J; Kim, Min-Sik; Zhong, Jun; Nirujogi, Raja Sekhar; Bose, Anjun K; Pandey, Akhilesh

    2016-06-01

    The leukocyte antigen related (LAR) family of receptor-like protein tyrosine phosphatases has three members in humans - PTPRF, PTPRD and PTPRS - that have been implicated in diverse processes including embryonic development, inhibition of cell growth and axonal guidance. Mutations in the LAR family are associated with developmental defects such as cleft palate as well as various cancers including breast, neck, lung, colon and brain. Although this family of tyrosine phosphatases is important for many developmental processes, little is known of their substrates. This is partially due to functional redundancy within the LAR family, as deletion of a single gene in the LAR family does not have an appreciable phenotype, but a dual knockout is embryonically lethal in mouse models. To circumvent the inability to knockout multiple members of the LAR family in mouse models, we used a knockout of ptp-3, which is the only known ortholog of the LAR family in Caenorhabditis elegans and allows for the study of the LAR family at the organismal level. Using SILAC-based quantitative phosphoproteomics, we identified 255 putative substrates of ptp-3, which included four of the nine known annotated substrates of the LAR family. A motif analysis of the identified phosphopeptides allowed for the determination of sequences that appear to be preferentially dephosphorylated. Finally, we discovered that kinases were overrepresented in the list of identified putative substrates and tyrosine residues whose phosphorylation is known to increase kinase activity were dephosphorylated by ptp-3. These data are suggestive of ptp-3 as a potential negative regulator of several kinase families, such as the mitogen activated kinases (MAPKs), and multiple tyrosine kinases including FER, MET, and NTRK2. PMID:27067626

  5. Pharmacophore modeling for protein tyrosine phosphatase 1B inhibitors.

    Science.gov (United States)

    Bharatham, Kavitha; Bharatham, Nagakumar; Lee, Keun Woo

    2007-05-01

    A three dimensional chemical feature based pharmacophore model was developed for the inhibitors of protein tyrosine phosphatase 1B (PTP1B) using the CATALYST software, which would provide useful knowledge for performing virtual screening to identify new inhibitors targeted toward type II diabetes and obesity. A dataset of 27 inhibitors, with diverse structural properties, and activities ranging from 0.026 to 600 microM, was selected as a training set. Hypol, the most reliable quantitative four featured pharmacophore hypothesis, was generated from a training set composed of compounds with two H-bond acceptors, one hydrophobic aromatic and one ring aromatic features. It has a correlation coefficient, RMSD and cost difference (null cost-total cost) of 0.946, 0.840 and 65.731, respectively. The best hypothesis (Hypol) was validated using four different methods. Firstly, a cross validation was performed by randomizing the data using the Cat-Scramble technique. The results confirmed that the pharmacophore models generated from the training set were valid. Secondly, a test set of 281 molecules was scored, with a correlation of 0.882 obtained between the experimental and predicted activities. Hypol performed well in correctly discriminating the active and inactive molecules. Thirdly, the model was investigated by mapping on two PTP1B inhibitors identified by different pharmaceutical companies. The Hypol model correctly predicted these compounds as being highly active. Finally, docking simulations were performed on few compounds to substantiate the role of the pharmacophore features at the binding site of the protein by analyzing their binding conformations. These multiple validation approaches provided confidence in the utility of this pharmacophore model as a 3D query for virtual screening to retrieve new chemical entities showing potential as potent PTP1B inhibitors.

  6. Characterization of the interactions between the active site of a protein tyrosine kinase and a divalent metal activator

    Directory of Open Access Journals (Sweden)

    Ayrapetov Marina K

    2005-11-01

    Full Text Available Abstract Background Protein tyrosine kinases are important enzymes for cell signalling and key targets for anticancer drug discovery. The catalytic mechanisms of protein tyrosine kinase-catalysed phosphorylation are not fully understood. Protein tyrosine kinase Csk requires two Mg2+ cations for activity: one (M1 binds to ATP, and the other (M2 acts as an essential activator. Results Experiments in this communication characterize the interaction between M2 and Csk. Csk activity is sensitive to pH in the range of 6 to 7. Kinetic characterization indicates that the sensitivity is not due to altered substrate binding, but caused by the sensitivity of M2 binding to pH. Several residues in the active site with potential of binding M2 are mutated and the effect on metal activation studied. An active mutant of Asn319 is generated, and this mutation does not alter the metal binding characteristics. Mutations of Glu236 or Asp332 abolish the kinase activity, precluding a positive or negative conclusion on their role in M2 coordination. Finally, the ability of divalent metal cations to activate Csk correlates to a combination of ionic radius and the coordination number. Conclusion These studies demonstrate that M2 binding to Csk is sensitive to pH, which is mainly responsible for Csk activity change in the acidic arm of the pH response curve. They also demonstrate critical differences in the metal activator coordination sphere in protein tyrosine kinase Csk and a protein Ser/Thr kinase, the cAMP-dependent protein kinase. They shed light on the physical interactions between a protein tyrosine kinase and a divalent metal activator.

  7. Implication of a protein-tyrosine-phosphatase in human lung cancer.

    Science.gov (United States)

    Gaits, F; Li, R Y; Ragab, A; Selves, J; Ragab-Thomas, J M; Chap, H

    1994-07-01

    Protein tyrosyl phosphorylation plays an essential role in regulating cellular events such as proliferation, differentiation and oncogenesis. The recent characterization of the family of protein tyrosine phosphatases (PTPases) suggests that dephosphorylation might be a crucial event in these phenomena. One of the functions of PTPases is to reverse the effect of protein tyrosine kinases (PTKases), many of which are oncogenes, suggesting that they may act as tumor suppressors as described for HPTP gamma. In order to investigate the implication in lung cancer of HPTP beta, a receptor PTPase, we have developed a semi-quantitative method derived from primer-directed reverse transcription (RT) and subsequent polymerase chain reaction (PCR) with 32P-labelled nucleotide. We have demonstrated that the expression of HPTP beta mRNA was dramatically decreased in lung adenocarcinomas and lung malpighian carcinomas as compared to normal lung tissue. In addition, HPTP beta was not expressed in the pulmonar adenocarcinoma cell line A427, which proliferates in a deregulated way. These results suggest that the loss of expression of HPTP beta might play a role in neoplasic transformation and thus this molecule could act as a tumor suppressor factor. PMID:7981622

  8. Implication of a protein-tyrosine-phosphatase in human lung cancer.

    Science.gov (United States)

    Gaits, F; Li, R Y; Ragab, A; Selves, J; Ragab-Thomas, J M; Chap, H

    1994-07-01

    Protein tyrosyl phosphorylation plays an essential role in regulating cellular events such as proliferation, differentiation and oncogenesis. The recent characterization of the family of protein tyrosine phosphatases (PTPases) suggests that dephosphorylation might be a crucial event in these phenomena. One of the functions of PTPases is to reverse the effect of protein tyrosine kinases (PTKases), many of which are oncogenes, suggesting that they may act as tumor suppressors as described for HPTP gamma. In order to investigate the implication in lung cancer of HPTP beta, a receptor PTPase, we have developed a semi-quantitative method derived from primer-directed reverse transcription (RT) and subsequent polymerase chain reaction (PCR) with 32P-labelled nucleotide. We have demonstrated that the expression of HPTP beta mRNA was dramatically decreased in lung adenocarcinomas and lung malpighian carcinomas as compared to normal lung tissue. In addition, HPTP beta was not expressed in the pulmonar adenocarcinoma cell line A427, which proliferates in a deregulated way. These results suggest that the loss of expression of HPTP beta might play a role in neoplasic transformation and thus this molecule could act as a tumor suppressor factor.

  9. A versatile spectrophotometric protein tyrosine phosphatase assay based on 3-nitrophosphotyrosine containing substrates

    NARCIS (Netherlands)

    van Ameijde, Jeroen; Overvoorde, John; Knapp, Stefan; den Hertog, Jeroen; Ruijtenbeek, Rob; Liskamp, Rob M J

    2014-01-01

    A versatile assay for protein tyrosine phosphatases (PTP) employing 3-nitrophosphotyrosine containing peptidic substrates is described. These therapeutically important phosphatases feature in signal transduction pathways. The assay involves spectrophotometric detection of 3-nitrotyrosine production

  10. Growth inhibition by tyrosine kinase inhibitors in mesothelioma cell lines.

    Science.gov (United States)

    Nutt, Joyce E; O'Toole, Kieran; Gonzalez, David; Lunec, John

    2009-06-01

    Clinical outcome following chemotherapy for malignant pleural mesothelioma is poor and improvements are needed. This preclinical study investigates the effect of five tyrosine kinase inhibitors (PTK787, ZD6474, ZD1839, SU6668 and SU11248) on the growth of three mesothelioma cell lines (NCI H226, NCI H28 and MSTO 211H), the presence of growth factor receptors and inhibition of their downstream signalling pathways. GI50 values were determined: ZD6474 and SU11248, mainly VEGFR2 inhibitors, gave the lowest GI50 across all cell lines (3.5-6.9 microM) whereas ZD1839 gave a GI50 in this range only in H28 cells. All cell lines were positive for EGFR, but only H226 cells were positive for VEGFR2 by Western blotting. ZD6474 and ZD1839 inhibited EGF-induced phosphorylation of EGFR, AKT and ERK, whereas VEGF-induced phosphorylation of VEGFR2 was completely inhibited with 0.1 microM SU11248. VEGFR2 was detected in tumour samples by immunohistochemistry. VEGFR2 tyrosine kinase inhibitors warrant further investigation in mesothelioma. PMID:19318229

  11. Tyrosine phosphorylation of platelet endothelial cell adhesion molecule-1 (PECAM-1, CD31) in mechanically stimulated vascular endothelial cells.

    Science.gov (United States)

    Osawa, M; Masuda, M; Harada, N; Lopes, R B; Fujiwara, K

    1997-03-01

    Fluid flow triggers signal transducing events, modulates gene expression, and remodels cytoskeletal structures in vascular endothelial cells (ECs). However, the primary steps of mechanoreception are still unknown. We have recently reported that a glycoprotein is rapidly tyrosine-phosphorylated in bovine ECs exposed to fluid flow or osmotic shock. Here were cloned a 3.4 kb cDNA encoding this protein and found that this was bovine PECAM-1. The tyrosine-phosphorylation level of PECAM-1 immunoprecipitated from mechanically stimulated bovine or human ECs increased. The PECAM-1 phosphorylation was not induced by reagents that triggered Ca2+ mobilization in ECs. An autophosphorylatable band comigrating with c-Src was co-immunoprecipitated with anti-PECAM-1, and c-Src phosphorylated and bound to a GST fusion protein containing the PECAM-1 cytoplasmic domain. A spliced mRNA form lacking amino acid residues 703-721 in the cytoplasmic domain was also expressed in bovine ECs, c-Src neither phosphorylated nor bound to the fusion protein containing the spliced PECAM-1 cytoplasmic domain which lacked one (Tyr 713) of the six tyrosine residues in the PECAM-1 cytoplasmic domain. These results suggest that the YSEI motif containing Tyr 713 is the Src phosphorylation/binding site. Our study is the first demonstration of inducible tyrosine phosphorylation of PECAM-1 and suggests involvement of PECAM-1 and Src family kinases in the sensing/signal transduction of mechanical stimuli in ECs. PMID:9084985

  12. Mechanisms underlying the inhibitory effects of arsenic compounds on protein tyrosine phosphatase (PTP)

    Energy Technology Data Exchange (ETDEWEB)

    Rehman, Kanwal [Department of Pharmacology, Toxicology, and Biochemical Pharmaceutics, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058 (China); Chen, Zhe [Zhejiang Hospital of Traditional Chinese Medicine, Zhejiang Chinese Medical University, Hangzhou (China); Wang, Wen Wen; Wang, Yan Wei [Department of Pharmacology, Toxicology, and Biochemical Pharmaceutics, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058 (China); Sakamoto, Akira [Graduate School of Pharmaceutical Sciences, Chiba University, Chiba 260‐8675 (Japan); Zhang, Yan Fang [Department of Pharmacology, Toxicology, and Biochemical Pharmaceutics, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058 (China); Naranmandura, Hua, E-mail: narenman@zju.edu.cn [Department of Pharmacology, Toxicology, and Biochemical Pharmaceutics, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058 (China); Suzuki, Noriyuki [Graduate School of Pharmaceutical Sciences, Chiba University, Chiba 260‐8675 (Japan)

    2012-09-15

    Arsenic binding to biomolecules is considered one of the major toxic mechanisms, which may also be related to the carcinogenic risks of arsenic in humans. At the same time, arsenic is also known to activate the phosphorylation-dependent signaling pathways including the epidermal growth factor receptor, the mitogen-activated protein kinase and insulin/insulin-like growth factor-1 pathways. These signaling pathways originate at the level of receptor tyrosine kinases whose phosphorylation status is regulated by opposing protein tyrosine phosphatase (PTP) activity. Reversible tyrosine phosphorylation, which is governed by the balanced action of protein tyrosine kinases and phosphatases, regulates important signaling pathways that are involved in the control of cell proliferation, adhesion and migration. In the present study, we have focused on the interaction of cellular PTPs with toxic trivalent arsenite (iAs{sup III}) and its intermediate metabolites such as monomethylarsonous acid (MMA{sup III}) and dimethylarsinous acid (DMA{sup III}) in vitro, and then determined the arsenic binding site in PTP by the use of recombinant PTPs (e.g., PTP1B and CD45). Interestingly, the activities of PTP1B (cytoplasm-form) or CD45 (receptor-linked form) were observed to be strongly inhibited by both methylated metabolites (i.e., MMA{sup III} and DMA{sup III}) but not by iAs{sup III}. Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) has clearly confirmed that the organic intermediate, DMA{sup III} directly bound to the active site cysteine residue of PTP1B (e.g., Cys215), resulting in inhibition of enzyme activity. These results suggest that arsenic exposure may disturb the cellular signaling pathways through PTP inactivation. Highlights: ► This study focused on the interaction of PTPs with trivalent arsenicals in vitro. ► We for the first time confirmed that DMA{sup III} strongly inhibited activity of PTP1B. ► DMA{sup III} directly

  13. Tyrosine administration decreases glutathione and stimulates lipid and protein oxidation in rat cerebral cortex.

    Science.gov (United States)

    Sgaravatti, Angela M; Magnusson, Alessandra S; de Oliveira, Amanda S; Rosa, Andréa P; Mescka, Caroline Paula; Zanin, Fernanda R; Pederzolli, Carolina D; Wyse, Angela T S; Wannmacher, Clóvis M D; Wajner, Moacir; Dutra-Filho, Carlos Severo

    2009-09-01

    Tyrosine levels are abnormally elevated in tissues and physiological fluids of patients with inborn errors of tyrosine catabolism especially in tyrosinemia type II which is caused by deficiency of tyrosine aminotransferase (TAT) and provokes eyes, skin and central nervous system disturbances. We have recently reported that tyrosine promoted oxidative stress in vitro but the exact mechanisms of brain damage in these disorder are poorly known. In the present study, we investigated the in vivo effect of L-tyrosine (500 mg/Kg) on oxidative stress indices in cerebral cortex homogenates of 14-day-old Wistar rats. A single injection of L-tyrosine decreased glutathione (GSH) and thiol-disulfide redox state (SH/SS ratio) while thiobarbituric acid-reactive substances, protein carbonyl content and glucose-6-phosphate dehydrogenase activity were enhanced. In contrast, the treatment did not affect ascorbic acid content, and the activities of superoxide dismutase, catalase and glutathione peroxidase. These results indicate that acute administration of L-tyrosine may impair antioxidant defenses and stimulate oxidative damage to lipids and proteins in cerebral cortex of young rats in vivo. This suggests that oxidative stress may represent a pathophysiological mechanism in hypetyrosinemic patients.

  14. The role for protein tyrosine phosphatases in regulating the immune system: implications for chronic intestinal inflammation

    OpenAIRE

    Spalinger, Marianne R.; MCCOLE, DECLAN F.; Rogler, Gerhard; Scharl, Michael

    2015-01-01

    Current hypothesis suggests that genetic, immunological and bacterial factors contribute essentially to the pathogenesis of inflammatory bowel disease (IBD). Variations within the gene loci encoding protein tyrosine phosphatases (PTPs) have been associated with the onset of IBD. PTPs modulate the activity of their substrates by dephosphorylation of tyrosine residues and are critical for the regulation of fundamental cellular signalling processes.Evidence emerges that expression levels of PTPN...

  15. Role of protein tyrosine phosphatases in regulating the immune system: implications for chronic intestinal inflammation

    OpenAIRE

    Spalinger, Marianne R.; MCCOLE, DECLAN F.; Rogler, Gerhard; Scharl, Michael

    2015-01-01

    Current hypothesis suggests that genetic, immunological, and bacterial factors contribute essentially to the pathogenesis of inflammatory bowel disease. Variations within the gene loci encoding protein tyrosine phosphatases (PTPs) have been associated with the onset of inflammatory bowel disease. PTPs modulate the activity of their substrates by dephosphorylation of tyrosine residues and are critical for the regulation of fundamental cellular signaling processes. Evidence emerges that express...

  16. EFFECT OF ACTIVE COMPOUNDS ISOLATED FROM PTERIS SEMIPINNATA L ON DNA TOPOISOMERASES AND TYROSINE PROTEIN KINASE AND EXPRESSION OF C-MYC IN LUNG ADENOCARCINOMA CELLS

    Institute of Scientific and Technical Information of China (English)

    李金华; 梁念慈; 莫丽儿; 张晓; 何承伟

    2001-01-01

    Objective: To study the effect of active compound 6F and A from Pteris semipinnata L.(PsL) on the activities of DNA topoisomerase (TOPO) I and II, activities of cytosolic and membrane TPK, and expression of oncogene c-myc in lung adenocarcinoma cells. Methods: The effect of compound 6F and A on activities of cytosolic and membrane TPK was measured by scintillation counting; the effect of compound A on expression of oncogene c-myc was determined by flow cytometry indirect fluorimetry. Results: compound 6F and A could inhibit the activities of TOPO I, and they strongly inhibited the TOPO II in 0.01 mg/L and 10.0 mg/L respectively. Compound A slightly inhibited the activities of membrane TPK, but not the cytosolic one. Compound A could inhibit the expression of oncogene c-myc. Conclusion: Topoisomerases are target of compound 6F and A. Compound A could slightly inhibit the activities of TPK, and showed an inhibitory effect on the expression of oncogene c-myc.

  17. Protein tyrosine phosphatase non-receptor type 22 modulates NOD2-induced cytokine release and autophagy.

    Directory of Open Access Journals (Sweden)

    Marianne R Spalinger

    Full Text Available BACKGROUND: Variations within the gene locus encoding protein tyrosine phosphatase non-receptor type 22 (PTPN22 are associated with the risk to develop inflammatory bowel disease (IBD. PTPN22 is involved in the regulation of T- and B-cell receptor signaling, but although it is highly expressed in innate immune cells, its function in other signaling pathways is less clear. Here, we study whether loss of PTPN22 controls muramyl-dipeptide (MDP-induced signaling and effects in immune cells. MATERIAL & METHODS: Stable knockdown of PTPN22 was induced in THP-1 cells by shRNA transduction prior to stimulation with the NOD2 ligand MDP. Cells were analyzed for signaling protein activation and mRNA expression by Western blot and quantitative PCR; cytokine secretion was assessed by ELISA, autophagosome induction by Western blot and immunofluorescence staining. Bone marrow derived dendritic cells (BMDC were obtained from PTPN22 knockout mice or wild-type animals. RESULTS: MDP-treatment induced PTPN22 expression and activity in human and mouse cells. Knockdown of PTPN22 enhanced MDP-induced activation of mitogen-activated protein kinase (MAPK-isoforms p38 and c-Jun N-terminal kinase as well as canonical NF-κB signaling molecules in THP-1 cells and BMDC derived from PTPN22 knockout mice. Loss of PTPN22 enhanced mRNA levels and secretion of interleukin (IL-6, IL-8 and TNF in THP-1 cells and PTPN22 knockout BMDC. Additionally, loss of PTPN22 resulted in increased, MDP-mediated autophagy in human and mouse cells. CONCLUSIONS: Our data demonstrate that PTPN22 controls NOD2 signaling, and loss of PTPN22 renders monocytes more reactive towards bacterial products, what might explain the association of PTPN22 variants with IBD pathogenesis.

  18. Influence of berberine on protein tyrosine kinase of erythrocyte insulin receptors from type 2 diabetes mellitus

    Institute of Scientific and Technical Information of China (English)

    Xianglei Deng; Xinrong Li; Chenggong Tian

    2005-01-01

    Objective: Bererine has been used to treat type 2 diabetes mellitus in Chinese traditional medicine because of its hypoglycemic effect. In this report, we compared the intrinsic tyrosine kinase activities of erythrocyte insulin receptors from type 2 diabetes mellitus with or without stimulation by berberine in vitro. Methods: Preparations containing insulin receptors were obtained from soluble human erythrocytes, and the insulin receptors were partially purified by affinity chromatography. The tyrosine kinase activity was measured by the exogenous substrate phosphorylation. Results: Both the membrane tyrosine kinase activity and the purified receptor tyrosine kinase activity from diabetics decreased significantly compared with those of normal individuals (reduced by 67.4 % and 47.2 %, respectively).After incubation with berberine, there is a statistical difference in the activity of membrane tyrosine kinase for diabetic patients (a 150% increase). Bererine had no effect on the tyrosine kinase activity of purified insulin receptors. Conclusion: We concluded from these results that berberine was able to improve the insulin sensitivity by increasing the protein tyrosine kinase activity of membrane-bound insulin receptors from type 2 diabetes mellitus.

  19. 磷酸化蛋白质组学鉴定 PTPLAD1调控的结肠癌细胞酪氨酸磷酸化蛋白质%Phosphoproteomics to analyze PTPLAD1-regulated tyrosine-phosphoryla-ted proteins in colon cancer cells

    Institute of Scientific and Technical Information of China (English)

    胡阳; 杨杰; 余汝媛; 汪洋

    2015-01-01

    目的:用磷酸化蛋白质组学的方法鉴定并分析结肠癌细胞中含蛋白酪氨酸磷酸酶样A结构域蛋白1( PTPLAD1)调控的酪氨酸磷酸化蛋白。方法:运用siRNA技术沉默PTPLAD1的表达,于细胞培养条件下运用氨基酸稳定同位素标记技术( stable lsotope labeling with amino acids in cell culture, SILAC)标记细胞,用酪氨酸磷酸化抗体免疫沉淀富集酪氨酸磷酸化蛋白,用LTQ-OrbitrapXL质谱鉴定因敲低PTPLAD1所出现的差异表达的酪氨酸磷酸化蛋白,进一步利用Ingenuity Pathway Analysis ( IPA)软件对这些差异蛋白进行生物信息学分析。结果:用real-time PCR筛选得到有效的siRNA干扰片段,Western blot验证其干扰效果及有效干扰时间。质谱鉴定PTPLAD1调控的差异酪氨酸磷酸化蛋白共20个,其中显著上调的8个,显著下调的10个,主要为转录因子及肿瘤标志物相关蛋白。 IPA软件的结果表明PTPLAD1调控的差异酪氨酸磷酸化蛋白的功能主要与器官发育分化、维持组织分化类型及细胞凋亡、增殖相关。结论:成功鉴定出PTPLAD1调控的差异酪氨酸磷酸化蛋白,可以为后续研究PTP-LAD1在结肠癌的发生发展中的作用及机理提供基础。%AIM:To identify and analyze tyrosine-phosphorylated proteins regulated by protein tyrosine phos-phatase-like A domain containing protein 1 ( PTPLAD1) in colon cancer cells by phosphoproteomics.METHODS: The expression of PTPLAD1 in colon cancer cell line HCT-116 was knocked down by small interfering RNAs, and the differenti-al expression of tyrosine-phosphorylated proteins in response to the konckdown of PTPLAD1 in HCT-116 cells was identified by stable isotope labeling with amino acid in cell culture ( SILAC) , coupled with the tyrosine phosphorylation antibody im-munoprecipitation and LC-MS/MS analysis.The Ingenuity Pathway Analysis ( IPA) software was employed for bioinformat-ics analysis on the

  20. Functional Analysis of Protein Tyrosine Phosphatases in Thrombosis and Hemostasis.

    Science.gov (United States)

    Rahmouni, Souad; Hego, Alexandre; Delierneux, Céline; Wéra, Odile; Musumeci, Lucia; Tautz, Lutz; Oury, Cécile

    2016-01-01

    Platelets are small blood cells derived from cytoplasmic fragments of megakaryocytes and play an essential role in thrombosis and hemostasis. Platelet activation depends on the rapid phosphorylation and dephosphorylation of key signaling molecules, and a number of kinases and phosphatases have been identified as major regulators of platelet function. However, the investigation of novel signaling proteins has suffered from technical limitations due to the anucleate nature of platelets and their very limited levels of mRNA and de novo protein synthesis. In the past, experimental methods were restricted to the generation of genetically modified mice and the development of specific antibodies. More recently, novel (phospho)proteomic technologies and pharmacological approaches using specific small-molecule inhibitors have added additional capabilities to investigate specific platelet proteins.In this chapter, we report methods for using genetic and pharmacological approaches to investigate the function of platelet signaling proteins. While the described experiments focus on the role of the dual-specificity phosphatase 3 (DUSP3) in platelet signaling, the presented methods are applicable to any signaling enzyme. Specifically, we describe a testing strategy that includes (1) aggregation and secretion experiments with mouse and human platelets, (2) immunoprecipitation and immunoblot assays to study platelet signaling events, (3) detailed protocols to use selected animal models in order to investigate thrombosis and hemostasis in vivo, and (4) strategies for utilizing pharmacological inhibitors on human platelets. PMID:27514813

  1. The tyrosine phosphatase Shp2 interacts with NPM-ALK and regulates anaplastic lymphoma cell growth and migration.

    Science.gov (United States)

    Voena, Claudia; Conte, Chiara; Ambrogio, Chiara; Boeri Erba, Elisabetta; Boccalatte, Francesco; Mohammed, Shabaz; Jensen, Ole N; Palestro, Giorgio; Inghirami, Giorgio; Chiarle, Roberto

    2007-05-01

    Anaplastic large cell lymphomas (ALCL) are mainly characterized by the reciprocal translocation t(2;5)(p23;q35) that involves the anaplastic lymphoma kinase (ALK) gene and generates the fusion protein NPM-ALK with intrinsic tyrosine kinase activity. NPM-ALK triggers several signaling cascades, leading to increased cell growth, resistance to apoptosis, and changes in morphology and migration of transformed cells. To search for new NPM-ALK interacting molecules, we developed a mass spectrometry-based proteomic approach in HEK293 cells expressing an inducible NPM-ALK and identified the tyrosine phosphatase Shp2 as a candidate substrate. We found that NPM-ALK was able to bind Shp2 in coprecipitation experiments and to induce its phosphorylation in the tyrosine residues Y542 and Y580 both in HEK293 cells and ALCL cell lines. In primary lymphomas, antibodies against the phosphorylated tyrosine Y542 of Shp2 mainly stained ALK-positive cells. In ALCL cell lines, Shp2-constitutive phosphorylation was dependent on NPM-ALK, as it significantly decreased after short hairpin RNA (shRNA)-mediated NPM-ALK knock down. In addition, only the constitutively active NPM-ALK, but not the kinase dead NPM-ALK(K210R), formed a complex with Shp2, Gab2, and growth factor receptor binding protein 2 (Grb2), where Grb2 bound to the phosphorylated Shp2 through its SH2 domain. Shp2 knock down by specific shRNA decreased the phosphorylation of extracellular signal-regulated kinase 1/2 and of the tyrosine residue Y416 in the activation loop of Src, resulting in impaired ALCL cell proliferation and growth disadvantage. Finally, migration of ALCL cells was reduced by Shp2 shRNA. These findings show a direct involvement of Shp2 in NPM-ALK lymphomagenesis, highlighting its critical role in lymphoma cell proliferation and migration.

  2. Free Fatty Acids Inhibit Protein Tyrosine Phosphatase 1B and Activate Akt

    Directory of Open Access Journals (Sweden)

    Eisuke Shibata

    2013-09-01

    Full Text Available Background/Aims: Accumulating evidence has suggested that free fatty acids (FFAs interact with protein kinases and protein phosphatases. The present study examined the effect of FFAs on protein phosphatases and Akt. Methods: Activities of protein phosphatase 1 (PP1, protein phosphatase 2A (PP2A, and protein tyrosine phosphatase 1B (PTP1B were assayed under the cell-free conditions. Phosphorylation of Akt was monitored in MSTO-211H human malignant pleural mesothelioma cells without and with knocking-down phosphatidylinositol 3 kinase (PI3K or 3-phosphoinositide-dependent protein kinase-1 (PDK1. Results: In the cell-free assay, unsaturated FFAs (uFFAs such as oleic, linoleic and linolenic acid and saturated FFAs (sFFAs such as stearic, palmitic, myristic, and behenic acid markedly reduced PTP1B activity, with the potential for uFFAs greater than that for sFFAs. All the investigated sFFAs inhibited PP2A activity, but otherwise no inhibition was obtained with uFFAs. Both uFFAs and sFFAs had no effect on PP1 activity. Oleic acid phosphorylated Akt both on Thr308 and Ser473, while stearic acid phosphorylated Akt on Thr308 alone. The effects of oleic and stearic acid on Akt phosphorylation were abrogated by the PI3K inhibitor wortmannin or the PDK1 inhibitor BX912 and also by knocking-down PI3K or PDK1. Conclusion: The results of the present study indicate that uFFAs and sFFAs could activate Akt through a pathway along a PI3K/PDK1/Akt axis in association with PTP1B inhibition.

  3. Nonreceptor protein tyrosine kinase involvement in signal transduction and immunodeficiency disease.

    Science.gov (United States)

    Saouaf, S J; Burkhardt, A L; Bolen, J B

    1995-09-01

    The nonreceptor protein tyrosine kinases (PTKs) have been grouped into 10 different enzyme families based on predicted amino acid sequences. As the number of enzymes belonging to the nonreceptor class of PTK is increasing, one challenge is to determine how these various classes of PTKs interact within the cell to promote signal transduction. Herein, the activation of four classes of nonreceptor PTKs is discussed in relation to their interactions with each other as well as with other signaling molecules during the process of lymphocyte surface antigen receptor-mediated activation. Recent findings of nonreceptor PTK loss-of-function mutations in different immunodeficiency diseases has revealed the important contribution of this group of enzymes to lymphocyte development. PMID:7554458

  4. Structural basis of interaction between protein tyrosine phosphatase PCP-2 and β-catenin

    Institute of Scientific and Technical Information of China (English)

    HE; Yaqin; YAN; Hexin; DONG; Hui; ZHANG; Peng; TANG; Liang

    2005-01-01

    PCP-2 is a member of receptor-like protein tyrosine phosphatase of the MAM domain family. To investigate which part of PCP-2 was involved in its interaction with β-catenin, we constructed various deletion mutants of PCP-2. These PCP-2 mutants and wild-type PCP-2 were co-transfected into BHK-21 cells with β-catenin individually. An in vivo binding assay revealed that the expression of wild-type PCP-2, PCP-2△C1C2 (deleted PCP-2 without both PTP domains) and PCP-2△C2 (deleted PCP-2 without the second PTP domain) could be immunoprecipitated by anti-catenin antibody in every co-transfection, but PCP-2 EXT (deleted PCP-2 without the juxtamembrane region and both PTP domains) was missing, which implied that PCP-2 and β-catenin could associate directly and the juxtamembrane region in PCP-2 was sufficient for the process.

  5. CDPKs are dual-specificity protein kinases and tyrosine autophosphorylation attenuates kinase activity

    Science.gov (United States)

    Calcium-dependent protein kinases (CDPKs or CPKs) are classified as serine/threonine protein kinases but we made the surprising observation that soybean CDPK' and several Arabidopsis isoforms (AtCPK4 and AtCPK34) could also autophosphorylate on tyrosine residues. In studies with His6-GmCDPK', we ide...

  6. Role of protein tyrosine phosphatases in regulating the immune system: implications for chronic intestinal inflammation.

    Science.gov (United States)

    Spalinger, Marianne R; McCole, Declan F; Rogler, Gerhard; Scharl, Michael

    2015-03-01

    Current hypothesis suggests that genetic, immunological, and bacterial factors contribute essentially to the pathogenesis of inflammatory bowel disease. Variations within the gene loci encoding protein tyrosine phosphatases (PTPs) have been associated with the onset of inflammatory bowel disease. PTPs modulate the activity of their substrates by dephosphorylation of tyrosine residues and are critical for the regulation of fundamental cellular signaling processes. Evidence emerges that expression levels of PTPN2, PTPN11, and PTPN22 are altered in actively inflamed intestinal tissue. PTPN2 seems to be critical for protecting intestinal epithelial barrier function, regulating innate and adaptive immune responses and finally for maintaining intestinal homeostasis. These observations have been confirmed in PTPN2 knockout mice in vivo. Those animals are clearly more susceptible to intestinal and systemic inflammation and feature alterations in innate and adaptive immune responses. PTPN22 controls inflammatory signaling in lymphocytes and mononuclear cells resulting in aberrant cytokine secretion pattern and autophagosome formation. PTPN22 deficiency in vivo results in more severe colitis demonstrating the relevance of PTPN22 for intestinal homeostasis in vivo. Of note, loss of PTPN22 promotes mitogen-activated protein kinase-induced cytokine secretion but limits secretion of nuclear factor κB-associated cytokines and autophagy in mononuclear cells. Loss of PTPN11 is also associated with increased colitis severity in vivo. In summary, dysfunction of those PTPs results in aberrant and uncontrolled immune responses that result in chronic inflammatory conditions. This way, it becomes more and more evident that dysfunction of PTPs displays an important factor in the pathogenesis of chronic intestinal inflammation, in particular inflammatory bowel disease.

  7. Role of protein tyrosine phosphatases in regulating the immune system: implications for chronic intestinal inflammation.

    Science.gov (United States)

    Spalinger, Marianne R; McCole, Declan F; Rogler, Gerhard; Scharl, Michael

    2015-03-01

    Current hypothesis suggests that genetic, immunological, and bacterial factors contribute essentially to the pathogenesis of inflammatory bowel disease. Variations within the gene loci encoding protein tyrosine phosphatases (PTPs) have been associated with the onset of inflammatory bowel disease. PTPs modulate the activity of their substrates by dephosphorylation of tyrosine residues and are critical for the regulation of fundamental cellular signaling processes. Evidence emerges that expression levels of PTPN2, PTPN11, and PTPN22 are altered in actively inflamed intestinal tissue. PTPN2 seems to be critical for protecting intestinal epithelial barrier function, regulating innate and adaptive immune responses and finally for maintaining intestinal homeostasis. These observations have been confirmed in PTPN2 knockout mice in vivo. Those animals are clearly more susceptible to intestinal and systemic inflammation and feature alterations in innate and adaptive immune responses. PTPN22 controls inflammatory signaling in lymphocytes and mononuclear cells resulting in aberrant cytokine secretion pattern and autophagosome formation. PTPN22 deficiency in vivo results in more severe colitis demonstrating the relevance of PTPN22 for intestinal homeostasis in vivo. Of note, loss of PTPN22 promotes mitogen-activated protein kinase-induced cytokine secretion but limits secretion of nuclear factor κB-associated cytokines and autophagy in mononuclear cells. Loss of PTPN11 is also associated with increased colitis severity in vivo. In summary, dysfunction of those PTPs results in aberrant and uncontrolled immune responses that result in chronic inflammatory conditions. This way, it becomes more and more evident that dysfunction of PTPs displays an important factor in the pathogenesis of chronic intestinal inflammation, in particular inflammatory bowel disease. PMID:25581833

  8. The use of the tyrosine phosphatase antagonist orthovanadate in the study of a cell proliferation inhibitor

    Science.gov (United States)

    Enebo, D. J.; Hanek, G.; Fattaey, H. K.; Johnson, T. C.; Spooner, B. S. (Principal Investigator)

    1993-01-01

    Incubation of murine fibroblasts with orthovanadate, a global tyrosine phosphatase inhibitor, was shown to confer a "pseudo-transformed" phenotype with regard to cell morphology and growth characteristics. This alteration was manifested by both an increasing refractile appearance of the cells, consistent with many transformed cell lines, as well as an increase in maximum cell density was attained. Despite the abrogation of cellular tyrosine phosphatase activity, orthovanadate-treated cells remained sensitive to the biological activity of a naturally occurring sialoglycopeptide (SGP) cell surface proliferation inhibitor. The results indicated that tyrosine phosphatase activity, inhibited by orthovanadate, was not involved in the signal transduction pathway of the SGP.

  9. A protein tyrosine phosphatase-like protein from baculovirus has RNA 5'-triphosphatase and diphosphatase activities.

    Science.gov (United States)

    Takagi, T; Taylor, G S; Kusakabe, T; Charbonneau, H; Buratowski, S

    1998-08-18

    The superfamily of protein tyrosine phosphatases (PTPs) includes at least one enzyme with an RNA substrate. We recently showed that the RNA triphosphatase domain of the Caenorhabditis elegans mRNA capping enzyme is related to the PTP enzyme family by sequence similarity and mechanism. The PTP most similar in sequence to the capping enzyme triphosphatase is BVP, a dual-specificity PTP encoded by the Autographa californica nuclear polyhedrosis virus. Although BVP previously has been shown to have modest tyrosine and serine/threonine phosphatase activity, we find that it is much more potent as an RNA 5'-phosphatase. BVP sequentially removes gamma and beta phosphates from the 5' end of triphosphate-terminated RNA, leaving a 5'-monophosphate end. The activity was specific for polynucleotides; nucleotide triphosphates were not hydrolyzed. A mutant protein in which the active site cysteine was replaced with serine was inactive. Three other dual-specificity PTPs (VH1, VHR, and Cdc14) did not exhibit detectable RNA phosphatase activity. Therefore, capping enzyme and BVP are members of a distinct PTP-like subfamily that can remove phosphates from RNA. PMID:9707557

  10. Regulation of brown fat adipogenesis by protein tyrosine phosphatase 1B.

    Directory of Open Access Journals (Sweden)

    Kosuke Matsuo

    Full Text Available Protein-tyrosine phosphatase 1B (PTP1B is a physiological regulator of insulin signaling and energy balance, but its role in brown fat adipogenesis requires additional investigation.To precisely determine the role of PTP1B in adipogenesis, we established preadipocyte cell lines from wild type and PTP1B knockout (KO mice. In addition, we reconstituted KO cells with wild type, substrate-trapping (D/A and sumoylation-resistant (K/R PTP1B mutants, then characterized differentiation and signaling in these cells. KO, D/A- and WT-reconstituted cells fully differentiated into mature adipocytes with KO and D/A cells exhibiting a trend for enhanced differentiation. In contrast, K/R cells exhibited marked attenuation in differentiation and lipid accumulation compared with WT cells. Expression of adipogenic markers PPARγ, C/EBPα, C/EBPδ, and PGC1α mirrored the differentiation pattern. In addition, the differentiation deficit in K/R cells could be reversed completely by the PPARγ activator troglitazone. PTP1B deficiency enhanced insulin receptor (IR and insulin receptor substrate 1 (IRS1 tyrosyl phosphorylation, while K/R cells exhibited attenuated insulin-induced IR and IRS1 phosphorylation and glucose uptake compared with WT cells. In addition, substrate-trapping studies revealed that IRS1 is a substrate for PTP1B in brown adipocytes. Moreover, KO, D/A and K/R cells exhibited elevated AMPK and ACC phosphorylation compared with WT cells.These data indicate that PTP1B is a modulator of brown fat adipogenesis and suggest that adipocyte differentiation requires regulated expression of PTP1B.

  11. Receptor protein tyrosine phosphatase alpha is essential for hippocampal neuronal migration and long-term potentiation

    DEFF Research Database (Denmark)

    Petrone, Angiola; Battaglia, Fortunato; Wang, Cheng;

    2003-01-01

    (RPTPalpha) regulates SRC family kinases, potassium channels and NMDA receptors. Here, we report that absence of RPTPalpha compromises correct positioning of pyramidal neurons during development of mouse hippocampus. Thus, RPTPalpha is a novel member of the functional class of genes that control radial......Despite clear indications of their importance in lower organisms, the contributions of protein tyrosine phosphatases (PTPs) to development or function of the mammalian nervous system have been poorly explored. In vitro studies have indicated that receptor protein tyrosine phosphatase alpha...

  12. Identification and analysis of a novel protein-tyrosine kinase from bovine thymus

    International Nuclear Information System (INIS)

    A cytosolic protein-tyrosine kinase has been identified and purified to near homogeneity from calf thymus by using the phosphorylation of the tyrosine-containing peptide angiotensin I as an assay. Specific peptide phosphorylating activity was enhanced by carrying out the assay at high ionic strength (2M NaCl). The inclusion of NaCl at this concentration acts to stimulate endogenous protein-tyrosine kinase activity while simultaneously inhibiting other endogenous kinases. The purification procedure involved extraction of the enzyme from calf-thymus and sequential chromatography on columns of DEAE-cellulose, heparin-agarose, casein-sepharose, butylagarose, and Sephadex G-75. Analysis of the most highly purified preparations by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single Coomassie blue-stained band of 41 KDa. This molecular weight was consistent with results obtained from gel filtration, indicating that the enzyme exists as a monomer. The enzyme has also been found to catalyze an autophosphorylation reaction. Incubation of the enzyme with Mn2+ and [γ-32P]ATP led to its modification on a tyrosine residue. Phosphopeptide mapping experiments indicated that the 41 KDa kinase was distinct from p56, the major membrane-associated protein-tyrosine kinase in T lymphocytes

  13. Activation of the low molecular weight protein tyrosine phosphatase in keratinocytes exposed to hyperosmotic stress.

    Directory of Open Access Journals (Sweden)

    Rodrigo A Silva

    Full Text Available Herein, we provide new contribution to the mechanisms involved in keratinocytes response to hyperosmotic shock showing, for the first time, the participation of Low Molecular Weight Protein Tyrosine Phosphatase (LMWPTP activity in this event. We reported that sorbitol-induced osmotic stress mediates alterations in the phosphorylation of pivotal cytoskeletal proteins, particularly Src and cofilin. Furthermore, an increase in the expression of the phosphorylated form of LMWPTP, which was followed by an augment in its catalytic activity, was observed. Of particular importance, these responses occurred in an intracellular milieu characterized by elevated levels of reduced glutathione (GSH and increased expression of the antioxidant enzymes glutathione peroxidase and glutathione reductase. Altogether, our results suggest that hyperosmostic stress provides a favorable cellular environment to the activation of LMWPTP, which is associated with increased expression of antioxidant enzymes, high levels of GSH and inhibition of Src kinase. Finally, the real contribution of LMWPTP in the hyperosmotic stress response of keratinocytes was demonstrated through analysis of the effects of ACP1 gene knockdown in stressed and non-stressed cells. LMWPTP knockdown attenuates the effects of sorbitol induced-stress in HaCaT cells, mainly in the status of Src kinase, Rac and STAT5 phosphorylation and activity. These results describe for the first time the participation of LMWPTP in the dynamics of cytoskeleton rearrangement during exposure of human keratinocytes to hyperosmotic shock, which may contribute to cell death.

  14. Protein-protein interactions in crystals of the human receptor-type protein tyrosine phosphatase ICA512 ectodomain.

    Directory of Open Access Journals (Sweden)

    María E Primo

    Full Text Available ICA512 (or IA-2 is a transmembrane protein-tyrosine phosphatase located in secretory granules of neuroendocrine cells. Initially, it was identified as one of the main antigens of autoimmune diabetes. Later, it was found that during insulin secretion, the cytoplasmic domain of ICA512 is cleaved and relocated to the nucleus, where it stimulates the transcription of the insulin gene. The role of the other parts of the receptor in insulin secretion is yet to be unveiled. The structures of the intracellular pseudocatalytic and mature extracellular domains are known, but the transmembrane domain and several intracellular and extracellular parts of the receptor are poorly characterized. Moreover the overall structure of the receptor remains to be established. We started to address this issue studying by X-ray crystallography the structure of the mature ectodomain of ICA512 (ME ICA512 and variants thereof. The variants and crystallization conditions were chosen with the purpose of exploring putative association interfaces, metal binding sites and all other structural details that might help, in subsequent works, to build a model of the entire receptor. Several structural features were clarified and three main different association modes of ME ICA512 were identified. The results provide essential pieces of information for the design of new experiments aimed to assess the structure in vivo.

  15. Protein-Protein Interactions in Crystals of the Human Receptor-Type Protein Tyrosine Phosphatase ICA512 Ectodomain

    Energy Technology Data Exchange (ETDEWEB)

    Primo M. E.; Jakoncic J.; Noguera M.E.; Risso V.A.; Sosa L.; Sica M.P.; Solimena M.; Poskus E. and Ermacora M.

    2011-09-15

    ICA512 (or IA-2) is a transmembrane protein-tyrosine phosphatase located in secretory granules of neuroendocrine cells. Initially, it was identified as one of the main antigens of autoimmune diabetes. Later, it was found that during insulin secretion, the cytoplasmic domain of ICA512 is cleaved and relocated to the nucleus, where it stimulates the transcription of the insulin gene. The role of the other parts of the receptor in insulin secretion is yet to be unveiled. The structures of the intracellular pseudocatalytic and mature extracellular domains are known, but the transmembrane domain and several intracellular and extracellular parts of the receptor are poorly characterized. Moreover the overall structure of the receptor remains to be established. We started to address this issue studying by X-ray crystallography the structure of the mature ectodomain of ICA512 (ME ICA512) and variants thereof. The variants and crystallization conditions were chosen with the purpose of exploring putative association interfaces, metal binding sites and all other structural details that might help, in subsequent works, to build a model of the entire receptor. Several structural features were clarified and three main different association modes of ME ICA512 were identified. The results provide essential pieces of information for the design of new experiments aimed to assess the structure in vivo.

  16. Stearic Acid Serves as a Potent Inhibitor of Protein Tyrosine Phosphatase 1B

    Directory of Open Access Journals (Sweden)

    Ayako Tsuchiya

    2013-11-01

    Full Text Available Background/Aims: Free fatty acids (FFAs are implicated in diverse signal transduction pathways. The present study investigated the effects of the saturated FFA stearic acid on protein tyrosine phosphatase 1B (PTP1B activity, Akt activity, and glucose uptake into cells relevant to insulin signal. Methods: PTP1B activity was assayed under the cell-free conditions. Phosphorylation of insulin receptor and Akt and glucose uptake into cells were monitored in differentiated 3T3-L1-GLUT4myc adipocytes. Results: In the cell-free PTP1B assay, stearic acid suppressed PTP1B activity in a concentration (1-30 µM-dependent manner. For 3T3-L1-GLUT4myc adipocytes insulin phosphorylated insulin receptor at Tyr1185 and Akt at Thr308 and Ser473 in a concentration (100 fM-100 nM-dependent manner and stimulated glucose uptake into cells in a concentration (0.1-100 nM-dependent manner. Stearic acid (30 µM significantly increased insulin-induced phosphorylation of insulin receptor at Tyr1185, but insulin-induced phosphorylation of Akt was not significantly enhanced. Stearic acid (30 µM by itself promoted glucose uptake into adipocytes. Conclusion: The results of the present study indicate that stearic acid serves as a potent PTP1B inhibitor, possibly causing an enhancement in the insulin receptor signaling to stimulate glucose uptake into adipocytes.

  17. Identification of protein tyrosine phosphatase 1B and casein as substrates for 124-v-Mos

    Directory of Open Access Journals (Sweden)

    Stabel Silvia

    2002-04-01

    Full Text Available Abstract Background The mos proto-oncogene encodes a cytoplasmic serine/threonine-specific protein kinase with crucial function during meiotic cell division in vertebrates. Based on oncogenic amino acid substitutions the viral derivative, 124-v-Mos, displays constitutive protein kinase activity and functions independent of unknown upstream effectors of mos protein kinase. We have utilized this property of 124-v-Mos and screened for novel mos substrates in immunocomplex kinase assays in vitro. Results We generated recombinant 124-v-Mos using the baculovirus expression system in Spodoptera frugiperda cells and demonstrated constitutive kinase activity by the ability of 124-v-Mos to auto-phosphorylate and to phosphorylate vimentin, a known substrate of c-Mos. Using this approach we analyzed a panel of acidic and basic substrates in immunocomplex protein kinase assays and identified novel in vitro substrates for 124-v-Mos, the protein tyrosine phosphatase 1B (PTP1B, alpha-casein and beta-casein. We controlled mos-specific phosphorylation of PTP1B and casein in comparative assays using a synthetic kinase-inactive 124-v-Mos mutant and further, tryptic digests of mos-phosphorylated beta-casein identified a phosphopeptide specifically targeted by wild-type 124-v-Mos. Two-dimensional phosphoamino acid analyses showed that 124-v-mos targets serine and threonine residues for phosphorylation in casein at a 1:1 ratio but auto-phosphorylation occurs predominantly on serine residues. Conclusion The mos substrates identified in this study represent a basis to approach the identification of the mos-consensus phosphorylation motif, important for the development of specific inhibitors of the Mos protein kinase.

  18. Functional activation of the T-cell antigen receptor induces tyrosine phosphorylation of phospholipase C-gamma 1.

    OpenAIRE

    Weiss, A; Koretzky, G; Schatzman, R C; Kadlecek, T

    1991-01-01

    Stimulation of the T-cell antigen receptor (TCR), which itself is not a protein-tyrosine kinase (PTK), activates a PTK and phospholipase C (PLC). Using the human T-cell leukemic line Jurkat and normal peripheral blood lymphocytes, we demonstrate that stimulation of the TCR specifically induces the recovery of PLC activity in eluates from anti-phosphotyrosine immunoprecipitates. Stimulation of the human muscarinic receptor, subtype 1, when expressed in Jurkat activates PLC through a guanine nu...

  19. DMPD: Macrophage-stimulating protein and RON receptor tyrosine kinase: potentialregulators of macrophage inflammatory activities. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 12472665 Macrophage-stimulating protein and RON receptor tyrosine kinase: potential...:545-53. (.png) (.svg) (.html) (.csml) Show Macrophage-stimulating protein and RON receptor tyrosine kinase:... potentialregulators of macrophage inflammatory activities. PubmedID 12472665 Title Macro

  20. The Dtk receptor tyrosine kinase, which binds protein S, is expressed during hematopoiesis.

    Science.gov (United States)

    Crosier, P S; Freeman, S A; Orlic, D; Bodine, D M; Crosier, K E

    1996-02-01

    Dtk (Tyro 3/Sky/Rse/Brt/Tif) belongs to a recently recognized subfamily of receptor tyrosine kinases that also includes Ufo (Axl/Ark) and Mer (Eyk). Ligands for Dtk and Ufo have been identified as protein S and the related molecule Gas6, respectively. This study examined expression of Dtk during ontogeny of the hematopoietic system and compared the pattern of expression with that of Ufo. Both receptors were abundantly expressed in differentiating embryonic stem cells, yolk sac blood islands, para-aortic splanchnopleural mesoderm, fractionated AA4+ fetal liver cells, and fetal thymus from day 14 until birth. Although Ufo was expressed at moderate levels in adult bone marrow, expression of Dtk in this tissue was barely detectable. In adult bone marrow subpopulations fractionated using counterflow centrifugal elutriation, immunomagnetic bead selection for lineage-depletion and FACS sorting for c-kit expression, very low levels of Dtk and/or Ufo were detected in some cell fractions. These results suggest that Dtk and Ufo are likely to be involved in the regulation of hematopoiesis, particularly during the embryonic stages of blood cell development.

  1. The c-Met receptor tyrosine kinase inhibitor MP470 radiosensitizes glioblastoma cells

    International Nuclear Information System (INIS)

    Glioblastoma multiforme (GBM) is resistant to current cytotoxic therapies, in part because of enhanced DNA repair. Activation of the receptor tyrosine kinase c-Met has been shown to protect cancer cells from DNA damage. We hypothesized that inhibiting c-Met would decrease this protection and thus sensitize resistant tumor cells to the effects of radiation therapy. Eight human GBM cell lines were screened for radiosensitivity to the small-molecule c-Met inhibitor MP470 with colony-count assays. Double-strand (ds) DNA breaks was quantified by using antibodies to gamma H2AX. Western blotting demonstrate expression of RAD51, glycogen synthase kinase (GSK)-3β, and other proteins. A murine xenograft tumor flank model was used for in vivo radiosensitization studies. MP470 reduced c-Met phosphorylation and enhanced radiation-induced cell kill by 0.4 logs in SF767 cells. Cells pretreated with MP470 had more ds DNA damage than cells treated with radiation alone. Mechanistically, MP470 was shown to inhibit dsDNA break repair and increase apoptosis. MP470 influences various survival and DNA repair related proteins such as pAKT, RAD51 and GSK3β. In vivo, the addition of MP470 to radiation resulted in a tumor-growth-delay enhancement ratio of 2.9 over radiation alone and extended survival time. GBM is a disease site where radiation is often used to address both macroscopic and microscopic disease. Despite attempts at dose escalation outcomes remain poor. MP470, a potent small-molecule tyrosine kinase inhibitor of c-Met, radiosensitized several GBM cell lines both in vitro and in vivo, and may help to improve outcomes for patients with GBM

  2. Residue 182 influences the second step of protein-tyrosine phosphatase-mediated catalysis

    DEFF Research Database (Denmark)

    Pedersen, A.K.; Guo, X.; Møller, K.B.;

    2004-01-01

    Previous enzyme kinetic and structural studies have revealed a critical role for Asp(181) (PTP1B numbering) in PTP (protein-tyrosine phosphatase)-mediated catalysis. In the E-P (phosphoenzyme) formation step, Asp(181) functions as a general acid, while in the E-P hydrolysis step it acts as a gene...

  3. In vitro characterization of the Bacillus subtilis protein tyrosine phosphatase YwqE

    DEFF Research Database (Denmark)

    Mijakovic, Ivan; Musumeci, Lucia; Tautz, Lutz;

    2005-01-01

    Both gram-negative and gram-positive bacteria possess protein tyrosine phosphatases (PTPs) with a catalytic Cys residue. In addition, many gram-positive bacteria have acquired a new family of PTPs, whose first characterized member was CpsB from Streptococcus pneumoniae. Bacillus subtilis contains...

  4. Low molecular weight protein tyrosine phosphatase (LMWPTP) upregulation mediates malignant potential in colorectal cancer

    NARCIS (Netherlands)

    E. Hoekstra (Elmer); L.L. Kodach (Liudmila L.); A. Mooppilmadham Das (Asha); R.R. Ruela-de-Sousa (Roberta); C.V. Ferreira (Carmen); J.C. Hardwick (James); C.J. van der Woude (Janneke); M.P. Peppelenbosch (Maikel); T.L.M. ten Hagen (Timo); G.M. Fuhler (Gwenny)

    2015-01-01

    textabstractPhosphatases have long been regarded as tumor suppressors, however there is emerging evidence for a tumor initiating role for some phosphatases in several forms of cancer. Low Molecular Weight Protein Tyrosine Phosphatase (LMWPTP; acid phosphatase 1 [ACP1]) is an 18 kDa enzyme that influ

  5. Protein tyrosine phosphatase non-receptor type 2 and inflammatory bowel disease.

    Science.gov (United States)

    Spalinger, Marianne R; McCole, Declan F; Rogler, Gerhard; Scharl, Michael

    2016-01-21

    Genome wide association studies have associated single nucleotide polymorphisms within the gene locus encoding protein tyrosine phosphatase non-receptor type 2 (PTPN2) with the onset of inflammatory bowel disease (IBD) and other inflammatory disorders. Expression of PTPN2 is enhanced in actively inflamed intestinal tissue featuring a marked up-regulation in intestinal epithelial cells. PTPN2 deficient mice suffer from severe intestinal and systemic inflammation and display aberrant innate and adaptive immune responses. In particular, PTPN2 is involved in the regulation of inflammatory signalling cascades, and critical for protecting intestinal epithelial barrier function, regulating innate and adaptive immune responses, and finally for maintaining intestinal homeostasis. On one hand, dysfunction of PTPN2 has drastic effects on innate host defence mechanisms, including increased secretion of pro-inflammatory cytokines, limited autophagosome formation in response to invading pathogens, and disruption of the intestinal epithelial barrier. On the other hand, PTPN2 function is crucial for controlling adaptive immune functions, by regulating T cell proliferation and differentiation as well as maintaining T cell tolerance. In this way, dysfunction of PTPN2 contributes to the manifestation of IBD. The aim of this review is to present an overview of recent findings on the role of PTPN2 in intestinal homeostasis and the impact of dysfunctional PTPN2 on intestinal inflammation. PMID:26811645

  6. Diagnosis and prognosis of male infertility in mammal: the focusing of tyrosine phosphorylation and phosphotyrosine proteins.

    Science.gov (United States)

    Kwon, Woo-Sung; Rahman, Md Saidur; Pang, Myung-Geol

    2014-11-01

    Male infertility refers to the inability of a man to achieve a pregnancy in a fertile female. In more than one-third of cases, infertility arises due to the male factor. Therefore, developing strategies for the diagnosis and prognosis of male infertility is critical. Simultaneously, a satisfactory model for the cellular mechanisms that regulate normal sperm function must be established. In this regard, tyrosine phosphorylation is one of the most common mechanisms through which several signal transduction pathways are adjusted in spermatozoa. It regulates the various aspects of sperm function, for example, motility, hyperactivation, capacitation, the acrosome reaction, fertilization, and beyond. Several recent large-scale studies have identified the proteins that are phosphorylated in spermatozoa to acquire fertilization competence. However, most of these studies are basal and have not presented an overall mechanism through which tyrosine phosphorylation regulates male infertility. In this review, we focus of this mechanism, discussing most of the tyrosine-phosphorylated proteins in spermatozoa that have been identified to date. We categorized tyrosine-phosphorylated proteins in spermatozoa that regulate male infertility using MedScan Reader (v5.0) and Pathway Studio (v9.0).

  7. Tyrosine 129 of the murine gammaherpesvirus M2 protein is critical for M2 function in vivo.

    Directory of Open Access Journals (Sweden)

    Udaya S Rangaswamy

    Full Text Available A common strategy shared by all known gammaherpesviruses is their ability to establish a latent infection in lymphocytes--predominantly in B cells. In immunocompromised patients, such as transplant recipients or AIDS patients, gammaherpesvirus infections can lead to the development of lymphoproliferative disease and lymphoid malignancies. The human gamma-herpesviruses, EBV and KSHV, encode proteins that are capable of modulating the host immune signaling machinery, thereby subverting host immune responses. Murine gamma-herpesvirus 68 (MHV68 infection of laboratory strains of mice has proven to be useful small-animal model that shares important pathogenic strategies with the human gamma-herpesviruses. The MHV68 M2 protein is known to manipulate B cell signaling and, dependent on route and dose of virus inoculation, plays a role in both the establishment of latency and virus reactivation. M2 contains two tyrosines that are targets for phosphorylation, and have been shown to interact with the B cell signaling machinery. Here we describe in vitro and in vivo studies of M2 mutants which reveals that while both tyrosines Y120 and Y129 are required for M2 induction of IL-10 expression from primary murine B cells in vitro, only Y129 is critical for reactivation from latency and plasma cell differentiation in vivo.

  8. Tyrosine 129 of the murine gammaherpesvirus M2 protein is critical for M2 function in vivo.

    Science.gov (United States)

    Rangaswamy, Udaya S; O'Flaherty, Brigid M; Speck, Samuel H

    2014-01-01

    A common strategy shared by all known gammaherpesviruses is their ability to establish a latent infection in lymphocytes--predominantly in B cells. In immunocompromised patients, such as transplant recipients or AIDS patients, gammaherpesvirus infections can lead to the development of lymphoproliferative disease and lymphoid malignancies. The human gamma-herpesviruses, EBV and KSHV, encode proteins that are capable of modulating the host immune signaling machinery, thereby subverting host immune responses. Murine gamma-herpesvirus 68 (MHV68) infection of laboratory strains of mice has proven to be useful small-animal model that shares important pathogenic strategies with the human gamma-herpesviruses. The MHV68 M2 protein is known to manipulate B cell signaling and, dependent on route and dose of virus inoculation, plays a role in both the establishment of latency and virus reactivation. M2 contains two tyrosines that are targets for phosphorylation, and have been shown to interact with the B cell signaling machinery. Here we describe in vitro and in vivo studies of M2 mutants which reveals that while both tyrosines Y120 and Y129 are required for M2 induction of IL-10 expression from primary murine B cells in vitro, only Y129 is critical for reactivation from latency and plasma cell differentiation in vivo.

  9. Palmitoylation of protease-activated receptor-1 regulates adaptor protein complex-2 and -3 interaction with tyrosine-based motifs and endocytic sorting.

    Science.gov (United States)

    Canto, Isabel; Trejo, JoAnn

    2013-05-31

    Protease-activated receptor-1 (PAR1) is a G protein-coupled receptor for the coagulant protease thrombin. Thrombin binds to and cleaves the N terminus of PAR1, generating a new N terminus that functions as a tethered ligand that cannot diffuse away. In addition to rapid desensitization, PAR1 trafficking is critical for the regulation of cellular responses. PAR1 displays constitutive and agonist-induced internalization. Constitutive internalization of unactivated PAR1 is mediated by the clathrin adaptor protein complex-2 (AP-2), which binds to a distal tyrosine-based motif localized within the C-terminal tail (C-tail) domain. Once internalized, PAR1 is sorted from endosomes to lysosomes via AP-3 interaction with a second C-tail tyrosine motif proximal to the transmembrane domain. However, the regulatory processes that control adaptor protein recognition of PAR1 C-tail tyrosine-based motifs are not known. Here, we report that palmitoylation of PAR1 is critical for regulating proper utilization of tyrosine-based motifs and endocytic sorting. We show that PAR1 is basally palmitoylated at highly conserved C-tail cysteines. A palmitoylation-deficient PAR1 mutant is competent to signal and exhibits a marked increase in constitutive internalization and lysosomal degradation compared with wild type receptor. Intriguingly, enhanced constitutive internalization of PAR1 is mediated by AP-2 and requires the proximal tyrosine-based motif rather than the distal tyrosine motif used by wild type receptor. Moreover, palmitoylation-deficient PAR1 displays increased degradation that is mediated by AP-3. These findings suggest that palmitoylation of PAR1 regulates appropriate utilization of tyrosine-based motifs by adaptor proteins and endocytic trafficking, processes that are critical for maintaining appropriate expression of PAR1 at the cell surface. PMID:23580642

  10. STRIATAL-ENRICHED PROTEIN TYROSINE PHOSPHATASE (STEP) KNOCKOUT MICE HAVE ENHANCED HIPPOCAMPAL MEMORY

    OpenAIRE

    Venkitaramani, Deepa V.; Moura, Paula J.; Picciotto, Marina R.; Lombroso, Paul J.

    2011-01-01

    STEP is a brain-specific phosphatase that opposes synaptic strengthening by the regulation of key synaptic signaling proteins. Previous studies suggest a possible role for STriatal-Enriched protein tyrosine Phosphatase (STEP) in learning and memory. To demonstrate the functional importance of STEP in learning and memory, we generated STEP knockout (KO) mice and examined the effect of deletion of STEP on behavioral performance, as well as the phosphorylation and expression of its substrates. H...

  11. Protein Tyrosine Phosphatase-1B Negatively Impacts Host Defense against Pseudomonas aeruginosa Infection.

    Science.gov (United States)

    Yue, Lei; Xie, Zhongping; Li, Hua; Pang, Zheng; Junkins, Robert D; Tremblay, Michel L; Chen, Xiaochun; Lin, Tong-Jun

    2016-05-01

    Pseudomonas aeruginosa is a major opportunistic pathogen in immune-compromised individuals. Mechanisms governing immune responses to P. aeruginosa infection remain incompletely defined. Herein, we demonstrate that protein tyrosine phosphatase-1B (PTP1B) is a critical negative regulator in P. aeruginosa infection. PTP1B-deficient mice display greatly enhanced bacterial clearance and reduced disease scores, which are accompanied by increased neutrophil infiltration and cytokine production. Interestingly, PTP1B deficiency mainly up-regulates the production of interferon-stimulated response elements-regulated cytokines and chemokines, including chemokine ligand 5 (regulated on activation normal T cell expressed and secreted), CXCL10 (interferon γ-inducible protein 10), and interferon-β production. Further studies reveal that PTP1B deficiency leads to increased interferon regulatory factor 7 (IRF7) expression and activation. These findings demonstrate a novel regulatory mechanism of the immune response to P. aeruginosa infection through PTP1B-IRF7 interaction. This novel PTP1B-IRF7-interferon-stimulated response elements pathway may have broader implications in Toll-like receptor-mediated innate immunity. PMID:27105736

  12. Protein Tyrosine Phosphatases Mediate the Signaling Pathway of Stomatal Closure of Vicia faba L.

    Institute of Scientific and Technical Information of China (English)

    Wu-Liang SHI; Xin LIU; Wen-Suo JIA; Shu-Qiu ZHANG

    2005-01-01

    The regulation of stomatal movement is one of the most important signaling networks in plants.The H+-ATPase at the plasma membrane of guard cells plays a critical role in the stomata opening, while there are some conflicting results regarding the effectiveness of the plasma membrane H+-ATPase inhibitor,vanadate, in inhibiting stomata opening. We observed that 2 mmol/L vanadate hardly inhibited light-stimulated stomata opening in epidermal peels of Viciafaba L., but significantly inhibited dark- and ABA-induced stomatal closure. These results cannot be explained with the previous findings that H+-ATPase was inhibited by vanadate. In view of the fact that vanadate is an inhibitor of protein tyrosine phosphatases (PTPases),we investigated whether the stomatal movement regulated by vanadate is through the regulation of PTPase.As expected, phenylarsine oxide (PAO), a specific inhibitor of PTPase, has very similar effects and even more effective than vanadate. Typical PTPase activity was found in guard cells of V. faba; moreover, the phosphatase activity could be inhibited by both vanadate and PAO. These results not only provide a novel explanation for conflicting results about vanadate modulating stomatal movement, but also provide further evidence for the involvement of PTPases in modulating signal transduction of stomatal movement.

  13. Fluorescence lifetimes of tyrosine residues in cytochrome c'' as local probes to study protein unfolding.

    Science.gov (United States)

    Noronha, Melinda; Santos, Raquel; Paci, Emanuele; Santos, Helena; Maçanita, António L

    2009-04-01

    Time-resolved fluorescence spectroscopy was used to show that multiple tyrosine residues of a protein can serve as localized probes of structural changes during thermal unfolding. Cytochrome c'' from Methylophilus methylotrophus, which has four tyrosine residues, was chosen as a model protein. The procedure involved, first, the assignment of the experimental decay times to the tyrosine residues, followed by the interpretation of the changes in the decay times and pre-exponential coefficients with temperature. We found that the fluorescence decays of cytochrome c'' are double-exponential from 23 to 80 degrees C, with decay times much shorter than those of the parent compound N-acetyl-tyrosinamide; this quenching was ascribed to dipole-dipole energy transfer from the tyrosine residues to the heme. The tyrosine-heme distances (R) and theoretical decay times, tau(comp), were estimated for each tyrosine residue. The analysis of the simulated decay generated with tau(comp), showed that a double-exponential fit is sufficient to describe the four decay times with two pre-exponential coefficients close to values observed from the experimental decay. Therefore, the decay times at 23 degrees C could be assigned to the individual tyrosine residues as tau(1) to Tyr-10 and Tyr-23 (at 20.3 A) and tau(2) to Tyr-12 and Tyr-115 (at 12-14 A). On the basis of this assignment and MD simulations, the temperature dependence of the decay times and pre-exponential coefficients suggest that upon unfolding, Tyr-12 is displaced from the heme, with loss of the structure of alpha-helix I. Moreover, Tyr-115 remains close to the heme and the structure in this region of the protein is not altered significantly. Altogether the data support the view that the protein core, comprising the heme and the four alpha-helices II to V, is clearly more stable than the remaining region that includes alpha-helix I and the loop between residues 19-27.

  14. Hypoxia-induced protein binding to O2-responsive sequences on the tyrosine hydroxylase gene.

    Science.gov (United States)

    Norris, M L; Millhorn, D E

    1995-10-01

    We reported recently that the gene that encodes tyrosine hydroxylase (TH), the rate-limiting enzyme in the biosynthesis of catecholamines, is regulated by hypoxia in the dopaminergic cells of the mammalian carotid body (Czyzyk-Krzeska, M. F., Bayliss, D. A., Lawson, E. E. & Millhorn, D. E. (1992) J. Neurochem. 58, 1538-1546) and in pheochromocytoma (PC12) cells (Czyzyk-Krzeska, M. F., Furnari, B. A., Lawson, E. E. & Millhorn, D. E. (1994) J. Biol. Chem. 269, 760-764). Regulation of this gene during low O2 conditions occurs at both the level of transcription and RNA stability. Increased transcription during hypoxia is regulated by a region of the proximal promoter that extends from -284 to + 27 bases, relative to transcription start site. The present study was undertaken to further characterize the sequences that confer O2 responsiveness of the TH gene and to identify hypoxia-induced protein interactions with these sequences. Results from chloramphenicol acetyltransferase assays identified a region between bases -284 and -150 that contains the essential sequences for O2 regulation. This region contains a number of regulatory elements including AP1, AP2, and HIF-1. Gel shift assays revealed enhanced protein interactions at the AP1 and HIF-1 elements of the native gene. Further investigations using supershift and shift-Western analysis showed that c-Fos and JunB bind to the AP1 element during hypoxia and that these protein levels are stimulated by hypoxia. Mutation of the AP1 sequence prevented stimulation of transcription of the TH-chloramphenicol acetyltransferase reporter gene by hypoxia. PMID:7559551

  15. MALDI mass sequencing and biochemical characterization of Setaria cervi protein tyrosine phosphatase.

    Science.gov (United States)

    Rai, Reeta; Singh, Neetu; Elesela, Srikanth; Tiwari, Savitri; Rathaur, Sushma

    2013-01-01

    A 30-kDa acid phosphatase with protein tyrosine phosphatase activity was identified in Setaria cervi (ScPTP). The enzyme was purified to homogeneity using three-step column chromatography. Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) analysis of purified ScPTP yielded a total of eight peptides matching most closely to phosphoprotein phosphatase of Ricinus communis (RcPP). A hydrophilicity plot of RcPP revealed the presence of these peptides in the hydrophilic region, suggesting their antigenic nature. The substrate specificity of ScPTP with ortho-phospho-L-tyrosine and inhibition with sodium orthovanadate and ammonium molybdate affirmed it as a protein tyrosine phosphatase. ScPTP was also found to be tartrate resistant. The Km and Vmax were 6.60 mM and 83.3 μM/ml/min, respectively, with pNPP and 8.0 mM and 111 μM/ml/min, respectively, with ortho-phospho-L-tyrosine as the substrate. The Ki value with sodium orthovanadate was calculated to be 16.10 mM. Active site modification with DEPC, EDAC and pHMB suggested the presence of histidine, cysteine and aspartate at its active site. Thus, on the basis of MALDI-TOF and biochemical studies, it was confirmed that purified acid phosphatase is a PTP. PMID:23052758

  16. Multiple angiopoietin recombinant proteins activate the Tie1 receptor tyrosine kinase and promote its interaction with Tie2

    OpenAIRE

    Saharinen, Pipsa; Kerkelä, Katja; Ekman, Niklas; Marron, Marie; Brindle, Nicholas; Lee, Gyun Min; Augustin, Hellmut; Koh, Gou Young; Alitalo, Kari

    2005-01-01

    The Tie1 receptor tyrosine kinase was isolated over a decade ago, but so far no ligand has been found to activate this receptor. Here, we have examined the potential of angiopoietins, ligands for the related Tie2 receptor, to mediate Tie1 activation. We show that a soluble Ang1 chimeric protein, COMP-Ang1, stimulates Tie1 phosphorylation in endothelial cells with similar kinetics and angiopoietin dose dependence when compared with Tie2. The phosphorylation of overexpressed Tie1 was weakly ind...

  17. A Rapid Lateral Flow Immunoassay for the Detection of Tyrosine Phosphatase-Like Protein IA-2 Autoantibodies in Human Serum

    OpenAIRE

    Ingrid Kikkas; Roberto Mallone; Etienne Larger; Hervé Volland; Nathalie Morel

    2014-01-01

    Type 1 diabetes (T1D) results from the destruction of pancreatic insulin-producing beta cells and is strongly associated with the presence of islet autoantibodies. Autoantibodies to tyrosine phosphatase-like protein IA-2 (IA-2As) are considered to be highly predictive markers of T1D. We developed a novel lateral flow immunoassay (LFIA) based on a bridging format for the rapid detection of IA-2As in human serum samples. In this assay, one site of the IA-2As is bound to HA-tagged-IA-2, which is...

  18. Identification of the insulin receptor tyrosine residues undergoing insulin-stimulated phosphorylation in intact rat hepatoma cells

    International Nuclear Information System (INIS)

    Tyr(P)-containing proteins were purified from extracts of insulin-treated rat hepatoma cells (H4-II-E-C3) by antiphosphotyrosine immunoaffinity chromatography. Two major insulin-stimulated, Tyr(P) proteins were recovered: an M/sub r/ 95,000 protein (identified as the insulin receptor β subunit by its immunoprecipitation by a patient-derived anti-insulin receptor serum and several anti-insulin receptor (peptide) anti-sera) and an M/sub r/ 180,000 protein. After purification and tryptic digestion of the M/sub r/ 95,000 protein, tryptic peptides containing Tyr (P) were purified by sequential antiphosphotyrosine immunoaffinity, reversed-phase, anion-exchange chromatography. Approximately 80% of all β subunit [32P]Tyr(P) resides on two tryptic peptides: 50-60% of [32P]Tyr(P) is found on the tryptic peptide Asp-Ile-Try-Glu-Thr-Asp-Try-Try-Arg from the tyrosine kinase domain. A second tryptic peptide is located near the carboxyl terminus; this contains 20-30% of β subunit [32P]Tyr(P) and is identified primarily in a double phosphorylated form. In a summary, the insulin-stimulated tyrosine phosphorylation of the insulin receptor in intact rat hepatoma cells involves at least 6 of the 13 tyrosine residues located on the β subunit intracellular extension. These tyrosines are clustered in several domains in a distribution virtually identical to that previously found for partially purified human insulin receptor autophosphorylated in vitro in the presence of insulin

  19. Interferon signaling is dependent on specific tyrosines located within the intracellular domain of IFNAR2c. Expression of IFNAR2c tyrosine mutants in U5A cells.

    Science.gov (United States)

    Wagner, T Charis; Velichko, Sharlene; Vogel, David; Rani, M R Sandhya; Leung, Stewart; Ransohoff, Richard M; Stark, George R; Perez, H Daniel; Croze, Ed

    2002-01-11

    Type I interferons (IFNs) are cytokines that play a central role in mediating antiviral, antiproliferative, and immunomodulatory activities in virtually all cells. These activities are entirely dependent on the interaction of IFNs with their particular cell surface receptor. In this report, we identify two specific tyrosine residues located within the cytoplasmic domain of IFNAR2c that are obligatory for IFN-dependent signaling. Various IFNAR2c tyrosine mutants were expressed in a human lung fibroscarcoma cell line lacking IFNAR2c (U5A). Stable clones expressing these mutants were analyzed for their ability to induce STAT1 and STAT2 activation, ISGF3 transcriptional complex formation, gene expression, and cell growth regulation in response to stimulation with type I IFNs. The replacement of all seven cytoplasmic tyrosine residues of IFNAR2c with phenylalanine resulted in a receptor unable to respond to IFN stimulation. Substitution of single tyrosines at amino acid residue 269, 316, 318, 337, or 512 with phenylalanine had no effect on IFN-dependent signaling, suggesting that no single tyrosine is essential for IFN receptor-mediated signaling. In addition, IFNAR2c retaining five proximal tyrosines residues (269, 306, 316, 318, and 337) or either two distal tyrosine residues (411 or 512) continued to be responsive to IFN stimulation. Surprisingly, the presence of only a single tyrosine at either position 337 or 512 was sufficient to restore a complete IFN response. These results indicate that IFN-dependent signaling proceeds through the redundant usage of two tyrosine residues in the cytoplasmic domain of IFNAR2c.

  20. Pancreatic Protein Tyrosine Phosphatase 1B Deficiency Exacerbates Acute Pancreatitis in Mice.

    Science.gov (United States)

    Bettaieb, Ahmed; Koike, Shinichiro; Chahed, Samah; Bachaalany, Santana; Griffey, Stephen; Sastre, Juan; Haj, Fawaz G

    2016-08-01

    Acute pancreatitis (AP) is a common and devastating gastrointestinal disorder that causes significant morbidity. The disease starts as local inflammation in the pancreas that may progress to systemic inflammation and complications. Protein tyrosine phosphatase 1B (PTP1B) is implicated in inflammatory signaling, but its significance in AP remains unclear. To investigate whether PTP1B may have a role in AP, we used pancreas PTP1B knockout (panc-PTP1B KO) mice and determined the effects of pancreatic PTP1B deficiency on cerulein- and arginine-induced acute pancreatitis. We report that PTP1B protein expression was increased in the early phase of AP in mice and rats. In addition, histological analyses of pancreas samples revealed enhanced features of AP in cerulein-treated panc-PTP1B KO mice compared with controls. Moreover, cerulein- and arginine-induced serum amylase and lipase were significantly higher in panc-PTP1B KO mice compared with controls. Similarly, pancreatic mRNA and serum concentrations of the inflammatory cytokines IL-1B, IL-6, and tumor necrosis factor-α were increased in panc-PTP1B KO mice compared with controls. Furthermore, panc-PTP1B KO mice exhibited enhanced cerulein- and arginine-induced NF-κB inflammatory response accompanied with increased mitogen-activated protein kinases activation and elevated endoplasmic reticulum stress. Notably, these effects were recapitulated in acinar cells treated with a pharmacological inhibitor of PTP1B. These findings reveal a novel role for pancreatic PTP1B in cerulein- and arginine-induced acute pancreatitis. PMID:27461362

  1. Rational design of allosteric-inhibition sites in classical protein tyrosine phosphatases

    Science.gov (United States)

    Chio, Cynthia M.; Yu, Xiaoling; Bishop, Anthony C.

    2015-01-01

    Protein tyrosine phosphatases (PTPs), which catalyze the dephosphorylation of phosphotyrosine in protein substrates, are critical regulators of metazoan cell signaling and have emerged as potential drug targets for a range of human diseases. Strategies for chemically targeting the function of individual PTPs selectively could serve to elucidate the signaling roles of these enzymes and would potentially expedite validation of the therapeutic promise of PTP inhibitors. Here we report a novel strategy for the design of non-natural allosteric-inhibition sites in PTPs; these sites, which can be introduced into target PTPs through protein engineering, serve to sensitize target PTPs to potent and selective inhibition by a biarsenical small molecule. Building on the recent discovery of a naturally occurring cryptic allosteric site in wild-type Src-homology-2 domain containing PTP (Shp2) that can be targeted by biarsenical compounds, we hypothesized that Shp2’s unusual sensitivity to biarsenicals could be strengthened through rational design and that the Shp2-specific site could serve as a blueprint for the introduction of non-natural inhibitor sensitivity in other PTPs. Indeed, we show here that the strategic introduction of a cysteine residue at a position removed from the Shp2 active site can serve to increase the potency and selectivity of the interaction between Shp2’s allosteric site and the biarsenical inhibitor. Moreover, we find that “Shp2-like” allosteric sites can be installed de novo in PTP enzymes that do not possess naturally occurring sensitivity to biarsenical compounds. Using primary-sequence alignments to guide our enzyme engineering, we have successfully introduced allosteric-inhibition sites in four classical PTPs—PTP1B, PTPH-1, FAP-1, and HePTP—from four different PTP subfamilies, suggesting that our sensitization approach can likely be applied widely across the classical PTP family to generate biarsenical-responsive PTPs. PMID:25828055

  2. Transient expression of protein tyrosine phosphatases encoded in Cotesia plutellae bracovirus inhibits insect cellular immune responses

    Science.gov (United States)

    Ibrahim, Ahmed M. A.; Kim, Yonggyun

    2008-01-01

    Several immunosuppressive factors are associated with parasitism of an endoparasitoid wasp, Cotesia plutellae, on the diamondback moth, Plutella xylostella. C. plutellae bracovirus (CpBV) encodes a large number of putative protein tyrosine phosphatases (PTPs), which may play a role in inhibiting host cellular immunity. To address this inhibitory hypothesis of CpBV-PTPs, we performed transient expression of individual CpBV-PTPs in hemocytes of the beet armyworm, Spodoptera exigua, and analyzed their cellular immune responses. Two different forms of CpBV-PTPs were chosen and cloned into a eukaryotic expression vector under the control of the p10 promoter of baculovirus: one with the normal cysteine active site (CpBV-PTP1) and the other with a mutated active site (CpBV-PTP5). The hemocytes transfected with CpBV-PTP1 significantly increased in PTP activity compared to control hemocytes, but those with CpBV-PTP5 exhibited a significant decrease in the PTP activity. All transfected hemocytes exhibited a significant reduction in both cell spreading and encapsulation activities compared to control hemocytes. Co-transfection of CpBV-PTP1 together with its double-stranded RNA reduced the messenger RNA (mRNA) level of CpBV-PTP1 and resulted in recovery of both hemocyte behaviors. This is the first report demonstrating that the polydnaviral PTPs can manipulate PTP activity of the hemocytes to interrupt cellular immune responses.

  3. A Novel Molecular Diagnostic of Glioblastomas: Detection of an Extracellular Fragment of Protein Tyrosine Phosphatase μ

    Directory of Open Access Journals (Sweden)

    Susan M. Burden-Gulley

    2010-04-01

    Full Text Available We recently found that normal human brain and low-grade astrocytomas express the receptor protein tyrosine phosphatase mu (PTPμ and that the more invasive astrocytomas, glioblastoma multiforme (GBM, downregulate full-length PTPμ expression. Loss of PTPμ expression in GBMs is due to proteolytic cleavage that generates an intracellular and potentially a cleaved and released extracellular fragment of PTPμ. Here, we identify that a cleaved extracellular fragment containing the domains required for PTPμ-mediated adhesion remains associated with GBM tumor tissue. We hypothesized that detection of this fragment would make an excellent diagnostic tool for the localization of tumor tissue within the brain. To this end, we generated a series of fluorescently tagged peptide probes that bind the PTPμ fragment. The peptide probes specifically recognize GBM cells in tissue sections of surgically resected human tumors. To test whether the peptide probes are able to detect GBM tumors in vivo, the PTPμ peptide probes were tested in both mouse flank and intracranial xenograft human glioblastoma tumor model systems. The glial tumors were molecularly labeled with the PTPμ peptide probes within minutes of tail vein injection using the Maestro FLEX In Vivo Imaging System. The label was stable for at least 3 hours. Together, these results indicate that peptide recognition of the PTPμ extracellular fragment provides a novel molecular diagnostic tool for detection of human glioblastomas. Such a tool has clear translational applications and may lead to improved surgical resections and prognosis for patients with this devastating disease.

  4. The expression andprognostic value ofprotein tyrosine kinase 6 inearly-stage cervical squamous cell cancer

    Institute of Scientific and Technical Information of China (English)

    XiaoJingWang; YingXiong; ZeBiaoMa; JianChuanXia; YanFangLi

    2016-01-01

    Background:Protein tyrosine kinase 6 (PTK6) is overexpressed in many epithelial tumors and predicts poor progno‑sis. However, PTK6 expression status and its role in cervical squamous cell cancer are unknown. This study aimed to investigate the expression level and clinical signiifcance of PTK6 in early‑stage cervical squamous cell cancer. Methods:Quantitative reverse transcription‑polymerase chain reaction (qRT‑PCR) and western blotting analysis were performed to detect PTK6 mRNA and protein expression levels in 10 freshly frozen, early‑stage cervical squamous cell cancer specimens and adjacent non‑tumorous cervical tissues. The expression of PTK6 was detected using immuno‑histochemical staining in 150 formalin‑ifxed, paraffn‑embedded, early‑stage cervical squamous cell cancer sections and 10 normal cervical tissue sections. Results:The mRNA and protein levels of PTK6 in cancer tissues were higher than those in adjacent non‑tumorous cervical tissues. Immunohistochemical analysis showed that PTK6 was not expressed in normal cervical tissues but was overexpressed in the cytoplasm of cervical squamous cell cancer cells. The level of PTK6 expression was signiif‑cantly associated with tumor grade (P=0.020). The 5‑year overall survival rate of patients with high PTK6 expression was lower than that of patients with low PTK6 expression (81.3% vs. 96.2%,P=0.008). Multivariate Cox regression analysis showed that the expression level of PTK6 in cervical squamous cell cancer was an independent prognostic factor for patient survival (hazard ratio=5.999, 95% conifdence interval 1.622–22.191,P Conclusions:PTK6 is overexpressed in cervical squamous cell cancer. Increased PTK6 expression is associated with reduced 5‑year overall survival. PTK6 expression is an independent prognostic predictor for cervical cancer.

  5. Identification of a human src homology 2-containing protein-tyrosine-phosphatase: a putative homolog of Drosophila corkscrew.

    OpenAIRE

    Freeman, R. M.; Plutzky, J; Neel, B G

    1992-01-01

    src homology 2 (SH2) domains direct binding to specific phosphotyrosyl proteins. Recently, SH2-containing protein-tyrosine-phosphatases (PTPs) were identified. Using degenerate oligonucleotides and the PCR, we have cloned a cDNA for an additional PTP, SH-PTP2, which contains two SH2 domains and is expressed ubiquitously. When expressed in Escherichia coli, SH-PTP2 displays tyrosine-specific phosphatase activity. Strong sequence similarity between SH-PTP2 and the Drosophila gene corkscrew (csw...

  6. Src family protein tyrosine kinase regulates the basolateral K channel in the distal convoluted tubule (DCT) by phosphorylation of KCNJ10 protein.

    Science.gov (United States)

    Zhang, Chengbiao; Wang, Lijun; Thomas, Sherin; Wang, Kemeng; Lin, Dao-Hong; Rinehart, Jesse; Wang, Wen-Hui

    2013-09-01

    The loss of function of the basolateral K channels in the distal nephron causes electrolyte imbalance. The aim of this study is to examine the role of Src family protein tyrosine kinase (SFK) in regulating K channels in the basolateral membrane of the mouse initial distal convoluted tubule (DCT1). Single-channel recordings confirmed that the 40-picosiemen (pS) K channel was the only type of K channel in the basolateral membrane of DCT1. The suppression of SFK reversibly inhibited the basolateral 40-pS K channel activity in cell-attached patches and decreased the Ba(2+)-sensitive whole-cell K currents in DCT1. Inhibition of SFK also shifted the K reversal potential from -65 to -43 mV, suggesting a role of SFK in determining the membrane potential in DCT1. Western blot analysis showed that KCNJ10 (Kir4.1), a key component of the basolateral 40-pS K channel in DCT1, was a tyrosine-phosphorylated protein. LC/MS analysis further confirmed that SFK phosphorylated KCNJ10 at Tyr(8) and Tyr(9). The single-channel recording detected the activity of a 19-pS K channel in KCNJ10-transfected HEK293T cells and a 40-pS K channel in the cells transfected with KCNJ10+KCNJ16 (Kir.5.1) that form a heterotetramer in the basolateral membrane of the DCT. Mutation of Tyr(9) did not alter the channel conductance of the homotetramer and heterotetramer. However, it decreased the whole-cell K currents, the probability of finding K channels, and surface expression of KCNJ10 in comparison to WT KCNJ10. We conclude that SFK stimulates the basolateral K channel activity in DCT1, at least partially, by phosphorylating Tyr(9) on KCNJ10. We speculate that the modulation of tyrosine phosphorylation of KCNJ10 should play a role in regulating membrane transport function in DCT1.

  7. A receptor tyrosine kinase, UFO/Axl, and other genes isolated by a modified differential display PCR are overexpressed in metastatic prostatic carcinoma cell line DU145.

    Science.gov (United States)

    Jacob, A N; Kalapurakal, J; Davidson, W R; Kandpal, G; Dunson, N; Prashar, Y; Kandpal, R P

    1999-01-01

    We have used a modified differential display PCR protocol for isolating 3' restriction fragments of cDNAs specifically expressed or overexpressed in metastatic prostate carcinoma cell line DU145. Several cDNA fragments were identified that matched to milk fat globule protein, UFO/Axl, a receptor tyrosine kinase, human homologue of a Xenopus maternal transcript, laminin and laminin receptor, human carcinoma-associated antigen, and some expressed sequence tags. The transcript for milk fat globule protein, a marker protein shown to be overexpressed in breast tumors, was elevated in DU145 cells. The expression of UFO/Axl, a receptor tyrosine kinase, was considerably higher in DU145 cells as compared to normal prostate cells and prostatic carcinoma cell line PC-3. The overexpression of UFO oncogene in DU145 cells is discussed in the context of prostate cancer metastasis.

  8. A novel role for protein tyrosine phosphatase 1B as a positive regulator of neuroinflammation

    OpenAIRE

    Song, Gyun Jee; Jung, Myungsu; Kim, Jong-Heon; Park, Hana; Rahman, Md. Habibur; Zhang, Sheng; Zhang, Zhong-Yin; Park, Dong Ho; Kook, Hyun; Lee, In-Kyu; Suk, Kyoungho

    2016-01-01

    Background Protein tyrosine phosphatase 1B (PTP1B) is a member of the non-transmembrane phosphotyrosine phosphatase family. Recently, PTP1B has been proposed to be a novel target of anti-cancer and anti-diabetic drugs. However, the role of PTP1B in the central nervous system is not clearly understood. Therefore, in this study, we sought to define PTP1B’s role in brain inflammation. Methods PTP1B messenger RNA (mRNA) and protein expression levels were examined in mouse brain and microglial cel...

  9. Transgenic mice expressing yellow fluorescent protein under control of the human tyrosine hydroxylase promoter.

    Science.gov (United States)

    Choi, Eun Yang; Yang, Jae Won; Park, Myung Sun; Sun, Woong; Kim, Hyun; Kim, Seung U; Lee, Myung Ae

    2012-10-01

    Pathogenesis of Parkinson's disease and related catecholaminergic neurological disorders is closely associated with changes in the levels of tyrosine hydroxylase (TH). Therefore, investigation of the regulation of the TH gene system should assist in understanding the pathomechanisms involved in these neurological disorders. To identify regulatory domains that direct human TH expression in the central nervous system (CNS), we generated two transgenic mouse lines in which enhanced yellow fluorescent protein (EYFP) is expressed under the control of either 3.2-kb (hTHP-EYFP construct) human TH promoter or 3.2-kb promoter with 2-kb 3'-flanking regions (hTHP-ex3-EYFP construct) of the TH gene. In the adult transgenic mouse brain, the hTHP-EYFP construct directs neuron-specific EYFP expression in various CNS areas, such as olfactory bulb, striatum, interpeduncular nucleus, cerebral cortex, hippocampus, and particularly dentate gyrus. Although these EYFP-positive cells were identified as mature neurons, few EYFP-positive cells were TH-positive neurons. On the other hand, we could detect the EYFP mRNA expression in a subset of neurons in the olfactory bulb, midbrain, and cerebellum, in which expression of endogenous TH is enriched, with hTHP-ex3-EYFP transgenic mice. These results indicate that the 3.2-kb sequence upstream of the TH gene is not sufficient for proper expression and that the 2-kb sequence from the translation start site to exon 3 is necessary for expression of EYFP in a subset of catecholaminergic neurons. PMID:22714400

  10. Tyrosine kinases in rheumatoid arthritis

    Directory of Open Access Journals (Sweden)

    Kobayashi Akiko

    2011-08-01

    Full Text Available Abstract Rheumatoid arthritis (RA is an inflammatory, polyarticular joint disease. A number of cellular responses are involved in the pathogenesis of rheumatoid arthritis, including activation of inflammatory cells and cytokine expression. The cellular responses involved in each of these processes depends on the specific signaling pathways that are activated; many of which include protein tyrosine kinases. These pathways include the mitogen-activated protein kinase pathway, Janus kinases/signal transducers and activators transcription pathway, spleen tyrosine kinase signaling, and the nuclear factor κ-light-chain-enhancer of activated B cells pathway. Many drugs are in development to target tyrosine kinases for the treatment of RA. Based on the number of recently published studies, this manuscript reviews the role of tyrosine kinases in the pathogenesis of RA and the potential role of kinase inhibitors as new therapeutic strategies of RA.

  11. Phosphoproteomics identified Endofin, DCBLD2, and KIAA0582 as novel tyrosine phosphorylation targets of EGF signaling and Iressa in human cancer cells

    DEFF Research Database (Denmark)

    Chen, Yunhao; Low, Teck-Yew; Choong, Lee-Yee;

    2007-01-01

    With the completion of the human genome project, analysis of enriched phosphotyrosyl proteins from epidermal growth factor (EGF)-induced phosphotyrosine proteome permits the identification of novel downstream substrates of the EGF receptor (EGFR). Using cICAT-based LC-MS/MS method, we identified...... and relatively quantified the tyrosine phosphorylation levels of 21 proteins between control and EGF-treated A431 human cervical cancer cells. Of these, Endofin, DCBLD2, and KIAA0582 were validated to be novel tyrosine-phosphorylation targets of EGF signaling and Iressa, a highly selective inhibitor of EGFR...

  12. Imipramine protects retinal ganglion cells from oxidative stress through the tyrosine kinase receptor B signaling pathway

    Institute of Scientific and Technical Information of China (English)

    Ming-lei Han; Guo-hua Liu; Jin Guo; Shu-juan Yu; Jing Huang

    2016-01-01

    Retinal ganglion cell (RGC) degeneration is irreversible in glaucoma and tyrosine kinase receptor B (TrkB)-associated signaling pathways have been implicated in the process. In this study, we attempted to examine whether imipramine, a tricyclic antidepressant, may protect hydrogen peroxide (H2O2)-induced RGC degeneration through the activation of the TrkB pathway in RGC-5 cell lines. RGC-5 cell lines were pre-treated with imipramine 30 minutes before exposure to H2O2. Western blot assay showed that in H2O2-damaged RGC-5 cells, imipramine activated TrkB pathways through extracellular signal-regulated protein kinase/TrkB phosphorylation. TUNEL staining assay also demonstrated that imipramine ameliorated H2O2-induced apoptosis in RGC-5 cells. Finally, TrkB-IgG intervention was able to reverse the protective effect of imipramine on H2O2-induced RGC-5 apoptosis. Imipramine therefore protects RGCs from oxidative stress-induced apoptosis through the TrkB signaling pathway.

  13. Expression of tetraspan protein CD63 activates protein-tyrosine kinase (PTK) and enhances the PTK-induced inhibition of ROMK channels.

    NARCIS (Netherlands)

    Lin, D.; Kamsteeg, E.J.; Zhang, Y.; Jin, Y.; Sterling, H.; Yue, P.; Roos, M.; Duffield, A.; Spencer, J.; Caplan, M.; Wang, W.H.

    2008-01-01

    In the present study, we tested the role of CD63 in regulating ROMK1 channels by protein-tyrosine kinase (PTK). Immunocytochemical staining shows that CD63 and receptor-linked tyrosine phosphatase alpha (RPTPalpha) are expressed in the cortical collecting duct and outer medulla collecting duct. Immu

  14. Treatment of Breast Cancer Cells by IGF1R Tyrosine Kinase Inhibitor Combined with Conventional Systemic Drugs

    NARCIS (Netherlands)

    Hartog, H.; Van der Graaf, W. T. A.; Boezen, H. M.; Wesseling, J.

    2012-01-01

    Aim: Insulin-like growth factor-1 receptor (IGF1R) is a tyrosine kinase receptor mediating cell growth and survival of cancer cells. We studied responses to IGF1R tyrosine kinase inhibitor NVP-AEW541 combined with conventional systemic drugs in breast cancer cell lines of different clinical subtype.

  15. Treatment of breast cancer cells by IGF1R tyrosine kinase inhibitor combined with conventional systemic drugs.

    NARCIS (Netherlands)

    Hartog, H.; Graaf, W.T.A. van der; Boezen, H.M.; Wesseling, J.

    2012-01-01

    AIM: Insulin-like growth factor-1 receptor (IGF1R) is a tyrosine kinase receptor mediating cell growth and survival of cancer cells. We studied responses to IGF1R tyrosine kinase inhibitor NVP-AEW541 combined with conventional systemic drugs in breast cancer cell lines of different clinical subtype.

  16. Integrin engagement mediates tyrosine dephosphorylation on platelet-endothelial cell adhesion molecule 1.

    OpenAIRE

    Lu, T T; Yan, L G; Madri, J. A.

    1996-01-01

    Platelet-endothelial cell adhesion molecule 1 (PECAM-1, CD31) is a 130-kDa member of the immunoglobulin gene superfamily expressed on endothelial cells, platelets, neutrophils, and monocytes and plays a role during endothelial cell migration. Phosphoamino acid analysis and Western blot analysis with anti-phosphotyrosine antibody show that endothelial PECAM-1 is tyrosine-phosphorylated. Phosphorylation is decreased with endothelial cell migration on fibronectin and collagen and with cell sprea...

  17. PTPRT regulates the interaction of Syntaxin-binding protein 1 with Syntaxin 1 through dephosphorylation of specific tyrosine residue

    Energy Technology Data Exchange (ETDEWEB)

    Lim, So-Hee; Moon, Jeonghee [Biomedical Proteomics Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 305-806 (Korea, Republic of); Lee, Myungkyu [Bionanotechnology Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 305-806 (Korea, Republic of); Lee, Jae-Ran, E-mail: leejr@kribb.re.kr [Biomedical Proteomics Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 305-806 (Korea, Republic of)

    2013-09-13

    Highlights: •PTPRT is a brain-specific, expressed, protein tyrosine phosphatase. •PTPRT regulated the interaction of Syntaxin-binding protein 1 with Syntaxin 1. •PTPRT dephosphorylated the specific tyrosine residue of Syntaxin-binding protein 1. •Dephosphorylation of Syntaxin-binding protein 1 enhanced the interaction with Syntaxin 1. •PTPRT appears to regulate the fusion of synaptic vesicle through dephosphorylation. -- Abstract: PTPRT (protein tyrosine phosphatase receptor T), a brain-specific tyrosine phosphatase, has been found to regulate synaptic formation and development of hippocampal neurons, but its regulation mechanism is not yet fully understood. Here, Syntaxin-binding protein 1, a key component of synaptic vesicle fusion machinery, was identified as a possible interaction partner and an endogenous substrate of PTPRT. PTPRT interacted with Syntaxin-binding protein 1 in rat synaptosome, and co-localized with Syntaxin-binding protein 1 in cultured hippocampal neurons. PTPRT dephosphorylated tyrosine 145 located around the linker between domain 1 and 2 of Syntaxin-binding protein 1. Syntaxin-binding protein 1 directly binds to Syntaxin 1, a t-SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) protein, and plays a role as catalysts of SNARE complex formation. Syntaxin-binding protein 1 mutant mimicking non-phosphorylation (Y145F) enhanced the interaction with Syntaxin 1 compared to wild type, and therefore, dephosphorylation of Syntaxin-binding protein 1 appeared to be important for SNARE-complex formation. In conclusion, PTPRT could regulate the interaction of Syntaxin-binding protein 1 with Syntaxin 1, and as a result, the synaptic vesicle fusion appeared to be controlled through dephosphorylation of Syntaxin-binding protein 1.

  18. Identification of genomic regions that interact with a viable allele of the Drosophila protein tyrosine phosphatase corkscrew.

    OpenAIRE

    Firth, L; Manchester, J; Lorenzen, J A; Baron, M.; Perkins, L A

    2000-01-01

    Signaling by receptor tyrosine kinases (RTKs) is critical for a multitude of developmental decisions and processes. Among the molecules known to transduce the RTK-generated signal is the nonreceptor protein tyrosine phosphatase Corkscrew (Csw). Previously, Csw has been demonstrated to function throughout the Drosophila life cycle and, among the RTKs tested, Csw is essential in the Torso, Sevenless, EGF, and Breathless/FGF RTK pathways. While the biochemical function of Csw remains to be unamb...

  19. A Cell-Based Protein-Protein Interaction Method Using a Permuted Luciferase Reporter

    OpenAIRE

    Eishingdrelo, Haifeng; Cai, Jidong; Weissensee, Paul; Sharma, Praveen; Tocci, Michael J; Wright, Paul S

    2011-01-01

    We have developed a novel cell-based protein-protein interaction assay method. The method relies on conversion of an inactive permuted luciferase containing a Tobacco Etch Virus protease (TEV) cleavage sequence fused onto protein (A) to an active luciferase upon interaction and cleavage by another protein (B) fused with the TEV protease. We demonstrate assay applicability for ligand-induced protein-protein interactions including G-protein coupled receptors, receptor tyrosine kinases and nucle...

  20. Tyrosine 402 Phosphorylation of Pyk2 Is Involved in Ionomycin-Induced Neurotransmitter Release

    OpenAIRE

    Zhao Zhang; Yun Zhang; Zheng Mou; Shifeng Chu; Xiaoyu Chen; Wenbin He; Xiaofeng Guo; Yuhe Yuan; Masami Takahashi; Naihong Chen

    2014-01-01

    Protein tyrosine kinases, which are highly expressed in the central nervous system, are implicated in many neural processes. However, the relationship between protein tyrosine kinases and neurotransmitter release remains unknown. In this study, we found that ionomycin, a Ca²⁺ ionophore, concurrently induced asynchronous neurotransmitter release and phosphorylation of a non-receptor protein tyrosine kinase, proline-rich tyrosine kinase 2 (Pyk2), in clonal rat pheochromocytoma PC12 cells and ce...

  1. A novel protein tyrosine phosphatase 1B inhibitor from Tinospora sinensis

    OpenAIRE

    Prasoon Gupta; Upasana Sharma; Gupta, Praveen K.; Rakesh Maurya

    2012-01-01

    Bioassay-directed fractionation led to the identification of a new compound, 4-hydroxy-heptadec-6-enoic acid ethyl ester (1) together with three known compounds (2-4) from Tinospora sinensis. The structure of 1 was determined by analysis of spectroscopic data. The isolated compounds were evaluated for their protein tyrosine phosphatase 1B (PTP1B) inhibitory activity. Compounds 1 and 2 displayed significant inhibitory activity with IC 50 values 61.1 and 74.2 μM, respectively

  2. Water molecule network and active site flexibility of apo protein tyrosine phosphatase 1B

    DEFF Research Database (Denmark)

    Pedersen, A.K.; Peters, Günther H.J.; Møller, K.B.;

    2004-01-01

    Protein tyrosine phosphatase 1B (PTP1B) plays a key role as a negative regulator of insulin and leptin signalling and is therefore considered to be an important molecular target for the treatment of type 2 diabetes and obesity. Detailed structural information about the structure of PTP1B, including...... the conformation and flexibility of active-site residues as well as the water-molecule network, is a key issue in understanding ligand binding and enzyme kinetics and in structure-based drug design. A 1.95 Angstrom apo PTP1B structure has been obtained, showing four highly coordinated water molecules...

  3. A genomic perspective on protein tyrosine phosphatases: gene structure, pseudogenes, and genetic disease linkage

    DEFF Research Database (Denmark)

    Andersen, Jannik N; Jansen, Peter G; Echwald, Søren M;

    2004-01-01

    The protein tyrosine phosphatases (PTPs) are now recognized as critical regulators of signal transduction under normal and pathophysiological conditions. In this analysis we have explored the sequence of the human genome to define the composition of the PTP family. Using public and proprietary...... and provide predicted amino acid sequences for four human PTPs that are currently defined by fragments only. Finally, we correlated each PTP locus with genetic disease markers and identified 4 PTPs that map to known susceptibility loci for type 2 diabetes and 19 PTPs that map to regions frequently deleted...

  4. Molecular dynamics simulations of protein-tyrosine phosphatase 1B. I. Ligand-induced changes in the protein motions

    DEFF Research Database (Denmark)

    Peters, Günther H. J.; Frimurer, T.M.; Andersen, J.N.;

    1999-01-01

    Activity of enzymes, such as protein tyrosine phosphatases (PTPs), is often associated with structural changes in the enzyme, resulting in selective and stereospecific reactions with the substrate. To investigate the effect of a substrate on the motions occurring in PTPs, we have performed...... in the protein region, where the N-terminal of the substrate is located, and in the loop region Val(198-)Gly(209). Displacements in the latter loop are associated with the motions in the WPD loop, which contains a catalytically important aspartic acid. Estimation of the pK(a) of the active-site cysteine along...

  5. IDENTIFICATION OF SPECIFIC PEPTIDE LIGANDS FOR B-LYMPHOMA CELL AND ITS EFFECT ON TYROSINE PHOSPHORYLATION AND CELL APOPTOSIS

    Institute of Scientific and Technical Information of China (English)

    宋良文; 马宪梅; 崔雪梅; 李扬; 王晓民

    2004-01-01

    Objective To search novel method for diagnosis and therapy of B-lymphoma, specific small molecular peptide ligands against binding site of tumor cells were screened and its effects on signal transduction and cell apoptosis were tested. Methods Specific peptide ligands were screened by binding with site of human B lymphoma cell (OC1LY8) using peptide-bead libraries. The identified peptides were characterized with responsible cells by rebinding test. The role of tyrosine phosphorylation of peptide ligand was tested by Western blot;and its apoptosispromoting role was observed by confocal fluorescent microscope. Results Specific peptide ligand was able to bind specifically to site on cell surface and enter into cytoplasm. Tetrameric peptide ligand was able to strongly trigger signal transduction resulting in tyrosine phosphorylation and cellular apoptosis in OC1LY8 cell line.Conclusion Screened peptide ligand can effectively bind with OC1LY8 cell, stimulate cellular tyrosine phosphorylation and induce cellular apoptosis.

  6. ER-bound protein tyrosine phosphatase PTP1B interacts with Src at the plasma membrane/substrate interface.

    Directory of Open Access Journals (Sweden)

    Melisa C Monteleone

    Full Text Available PTP1B is an endoplasmic reticulum (ER anchored enzyme whose access to substrates is partly dependent on the ER distribution and dynamics. One of these substrates, the protein tyrosine kinase Src, has been found in the cytosol, endosomes, and plasma membrane. Here we analyzed where PTP1B and Src physically interact in intact cells, by bimolecular fluorescence complementation (BiFC in combination with temporal and high resolution microscopy. We also determined the structural basis of this interaction. We found that BiFC signal is displayed as puncta scattered throughout the ER network, a feature that was enhanced when the substrate trapping mutant PTP1B-D181A was used. Time-lapse and co-localization analyses revealed that BiFC puncta did not correspond to vesicular carriers; instead they localized at the tip of dynamic ER tubules. BiFC puncta were retained in ventral membrane preparations after cell unroofing and were also detected within the evanescent field of total internal reflection fluorescent microscopy (TIRFM associated to the ventral membranes of whole cells. Furthermore, BiFC puncta often colocalized with dark spots seen by surface reflection interference contrast (SRIC. Removal of Src myristoylation and polybasic motifs abolished BiFC. In addition, PTP1B active site and negative regulatory tyrosine 529 on Src were primary determinants of BiFC occurrence, although the SH3 binding motif on PTP1B also played a role. Our results suggest that ER-bound PTP1B dynamically interacts with the negative regulatory site at the C-terminus of Src at random puncta in the plasma membrane/substrate interface, likely leading to Src activation and recruitment to adhesion complexes. We postulate that this functional ER/plasma membrane crosstalk could apply to a wide array of protein partners, opening an exciting field of research.

  7. Targeting Bruton's tyrosine kinase signaling as an emerging therapeutic agent of B-cell malignancies

    OpenAIRE

    Xia, Bing; QU, FULIAN; Yuan, Tian; Zhang, Yizhuo

    2015-01-01

    It is becoming increasingly evident that B-cell receptor (BCR) signaling is central to the development and function of B cells. BCR signaling has emerged as a pivotal pathway and a key driver of numerous B-cell lymphomas. Disruption of BCR signaling can be lethal to malignant B cells. Recently, kinase inhibitors that target BCR signaling have induced notable clinical responses. These inhibitors include spleen tyrosine kinase, mammalian target of rapamycin, phosphoinositide 3′-kinase and Bruto...

  8. The SHP-2 tyrosine phosphatase: Signaling mechanisms and biological functions

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Cellular biological activities are tightly controlled by intracellular signaling processes initiated by extracellular signals.Protein tyrosine phosphatases, which remove phosphate groups from phosphorylated signaling molecules, play equally important tyrosine roles as protein tyrosine kinases in signal transduction.SHP-2, a cytoplasmic SH2 domain containing protein tyrosine phosphatase, is involved in the signaling pathways of a variety of growth factors and cytokines. Recent studies have clearly demonstrated that this phosphatase plays an important role in transducing signal relay from the cell surface to the nucleus, and is a critical intracellular regulator in mediating cell proliferation and differentiation.

  9. Src-Like adaptor protein (SLAP) binds to the receptor tyrosine kinase Flt3 and modulates receptor stability and downstream signaling.

    Science.gov (United States)

    Kazi, Julhash U; Rönnstrand, Lars

    2012-01-01

    Fms-like tyrosine kinase 3 (Flt3) is an important growth factor receptor in hematopoiesis. Gain-of-function mutations of the receptor contribute to the transformation of acute myeloid leukemia (AML). Src-like adaptor protein (SLAP) is an interaction partner of the E3 ubiquitin ligase Cbl that can regulate receptor tyrosine kinases-mediated signal transduction. In this study, we analyzed the role of SLAP in signal transduction downstream of the type III receptor tyrosine kinase Flt3. The results show that upon ligand stimulation SLAP stably associates with Flt3 through multiple phosphotyrosine residues in Flt3. SLAP constitutively interacts with oncogenic Flt3-ITD and co-localizes with Flt3 near the cell membrane. This association initiates Cbl-dependent receptor ubiquitination and degradation. Depletion of SLAP expression by shRNA in Flt3-transfected Ba/F3 cells resulted in a weaker activation of FL-induced PI3K-Akt and MAPK signaling. Meta-analysis of microarray data from patient samples suggests that SLAP mRNA is differentially expressed in different cancers and its expression was significantly increased in patients carrying the Flt3-ITD mutation. Thus, our data suggest a novel role of SLAP in different cancers and in modulation of receptor tyrosine kinase signaling apart from its conventional role in regulation of receptor stability.

  10. Src-Like adaptor protein (SLAP binds to the receptor tyrosine kinase Flt3 and modulates receptor stability and downstream signaling.

    Directory of Open Access Journals (Sweden)

    Julhash U Kazi

    Full Text Available Fms-like tyrosine kinase 3 (Flt3 is an important growth factor receptor in hematopoiesis. Gain-of-function mutations of the receptor contribute to the transformation of acute myeloid leukemia (AML. Src-like adaptor protein (SLAP is an interaction partner of the E3 ubiquitin ligase Cbl that can regulate receptor tyrosine kinases-mediated signal transduction. In this study, we analyzed the role of SLAP in signal transduction downstream of the type III receptor tyrosine kinase Flt3. The results show that upon ligand stimulation SLAP stably associates with Flt3 through multiple phosphotyrosine residues in Flt3. SLAP constitutively interacts with oncogenic Flt3-ITD and co-localizes with Flt3 near the cell membrane. This association initiates Cbl-dependent receptor ubiquitination and degradation. Depletion of SLAP expression by shRNA in Flt3-transfected Ba/F3 cells resulted in a weaker activation of FL-induced PI3K-Akt and MAPK signaling. Meta-analysis of microarray data from patient samples suggests that SLAP mRNA is differentially expressed in different cancers and its expression was significantly increased in patients carrying the Flt3-ITD mutation. Thus, our data suggest a novel role of SLAP in different cancers and in modulation of receptor tyrosine kinase signaling apart from its conventional role in regulation of receptor stability.

  11. Tyrosine phosphorylation of the Helicobacter pylori CagA antigen after cag-driven host cell translocation.

    Science.gov (United States)

    Stein, M; Rappuoli, R; Covacci, A

    2000-02-01

    Helicobacter pylori strains associated with severe tissue damage and inflammation possess a unique genetic locus, cag, containing 31 genes originating from a distant event of horizontal transfer and retained as a pathogenicity island. The cag system is an Helicobacter-specific type IV secretion engine involved in cellular responses like induction of pedestals, secretion of IL-8, and phosphorylation of proteic targets. It has previously been reported that cocultivation of epithelial cells with Helicobacter pylori triggers signal transduction and tyrosine phosphorylation of a 145-kDa putative host cell protein. Herein, we demonstrate that this protein is not derived from the host but rather is the bacterial immunodominant antigen CagA, a virulence factor commonly expressed in peptic ulcer disease and thought to be an orphan of a specific biological function. Thus, CagA is delivered into the epithelial cells by the cag type IV secretion system where it is phosphorylated on tyrosine residues by an as yet unidentified host cell kinase and wired to eukaryotic signal transduction pathways and cytoskeletal plasticity. PMID:10655519

  12. Multisite tyrosine phosphorylation of the N-terminus of Mint1/X11α by Src kinase regulates the trafficking of amyloid precursor protein.

    Science.gov (United States)

    Dunning, Christopher J R; Black, Hannah L; Andrews, Katie L; Davenport, Elizabeth C; Conboy, Michael; Chawla, Sangeeta; Dowle, Adam A; Ashford, David; Thomas, Jerry R; Evans, Gareth J O

    2016-05-01

    Mint/X11 is one of the four neuronal trafficking adaptors that interact with amyloid precursor protein (APP) and are linked with its cleavage to generate β-amyloid peptide, a key player in the pathology of Alzheimer's disease. How APP switches between adaptors at different stages of the secretory pathway is poorly understood. Here, we show that tyrosine phosphorylation of Mint1 regulates the destination of APP. A canonical SH2-binding motif ((202) YEEI) was identified in the N-terminus of Mint1 that is phosphorylated on tyrosine by C-Src and recruits the active kinase for sequential phosphorylation of further tyrosines (Y191 and Y187). A single Y202F mutation in the Mint1 N-terminus inhibits C-Src binding and tyrosine phosphorylation. Previous studies observed that co-expression of wild-type Mint1 and APP causes accumulation of APP in the trans-Golgi. Unphosphorylatable Mint1 (Y202F) or pharmacological inhibition of Src reduced the accumulation of APP in the trans-Golgi of heterologous cells. A similar result was observed in cultured rat hippocampal neurons where Mint1(Y202F) permitted the trafficking of APP to more distal neurites than the wild-type protein. These data underline the importance of the tyrosine phosphorylation of Mint1 as a critical switch for determining the destination of APP. The regulation of amyloid precursor protein (APP) trafficking is poorly understood. We have discovered that the APP adapter, Mint1, is phosphorylated by C-Src kinase. Mint1 causes APP accumulation in the trans-Golgi network, whereas inhibition of Src or mutation of Mint1-Y202 permits APP recycling. The phosphorylation status of Mint1 could impact on the pathological trafficking of APP in Alzheimer's disease. PMID:26865271

  13. Proteomic identification of plasma protein tyrosine phosphatase alpha and fibronectin associated with liver fluke, Opisthorchis viverrini, infection.

    Directory of Open Access Journals (Sweden)

    Jarinya Khoontawad

    Full Text Available Opisthorchiasis caused by Opisthorchis viverrini induces periductal fibrosis via host immune/inflammatory responses. Plasma protein alteration during host-parasite interaction-mediated inflammation may provide potential diagnostic and/or prognostic biomarkers. To search for target protein changes in O. viverrini-infected hamsters, a 1-D PAGE gel band was trypsin-digested and analyzed by a LC-MS/MS-based proteomics approach in the plasma profile of infected hamsters, and applied to humans. Sixty seven proteins were selected for further analysis based on at least two unique tryptic peptides with protein ID score >10 and increased expression at least two times across time points. These proteins have not been previously identified in O. viverrini-associated infection. Among those, proteins involved in structural (19%, immune response (13%, cell cycle (10% and transcription (10% were highly expressed. Western blots revealed an expression level of protein tyrosine phosphatase alpha (PTPα which reached a peak at 1 month and subsequently tended to decrease. Fibronectin significantly increased at 1 month and tended to increase with time, supporting proteomic analysis. PTPα was expressed in the cytoplasm of inflammatory cells, while fibronectin was observed mainly in the cytoplasm of fibroblasts and the extracellular matrix at periductal fibrosis areas. In addition, these protein levels significantly increased in the plasma of O. viverrini-infected patients compared to healthy individuals, and significantly decreased at 2-months post-treatment, indicating their potential as disease markers. In conclusion, our results suggest that plasma PTPα and fibronectin may be associated with opisthorchiasis and the hamster model provides the basis for development of novel diagnostic markers in the future.

  14. L-1-C-11-tyrosine PET in patients with laryngeal carcinomas : Comparison of standardized uptake value and protein synthesis rate

    NARCIS (Netherlands)

    de Boer, [No Value; Pruim, J; van der Laan, BFAM; Que, TH; Willemsen, ATM; Albers, FWJ; Vaalburg, W

    2003-01-01

    PET with L-1-C-11-tyrosine (TYR) can measure and quantify increased protein synthesis in tumor tissue in vivo. For quantification of the protein synthesis rate (PSR), arterial cannulation with repeated blood sampling to obtain the plasma input function and a dynamic TYR PET study to calculate a time

  15. Asymmetric Receptor Contact is Required for Tyrosine Autophosphorylation of Fibroblast Growth Factor Receptor in Living Cells

    Energy Technology Data Exchange (ETDEWEB)

    Bae, J.; Boggon, T; Tomé, F; Mandiyan, V; Lax, I; Schlessinge, J

    2010-01-01

    Tyrosine autophosphorylation of receptor tyrosine kinases plays a critical role in regulation of kinase activity and in recruitment and activation of intracellular signaling pathways. Autophosphorylation is mediated by a sequential and precisely ordered intermolecular (trans) reaction. In this report we present structural and biochemical experiments demonstrating that formation of an asymmetric dimer between activated FGFR1 kinase domains is required for transphosphorylation of FGFR1 in FGF-stimulated cells. Transphosphorylation is mediated by specific asymmetric contacts between the N-lobe of one kinase molecule, which serves as an active enzyme, and specific docking sites on the C-lobe of a second kinase molecule, which serves a substrate. Pathological loss-of-function mutations or oncogenic activating mutations in this interface may hinder or facilitate asymmetric dimer formation and transphosphorylation, respectively. The experiments presented in this report provide the molecular basis underlying the control of transphosphorylation of FGF receptors and other receptor tyrosine kinases.

  16. Tyrosine phosphorylation of 3BP2 is indispensable for the interaction with VAV3 in chicken DT40 cells

    International Nuclear Information System (INIS)

    Adaptor protein c-Abl SH3 domain-binding protein-2 (3BP2) is known to play regulatory roles in immunoreceptor-mediated signal transduction. We have previously demonstrated that Tyr174, Tyr183 and Tyr446 in mouse 3BP2 are predominantly phosphorylated by Syk, and the phosphorylation of Tyr183 and the Src homology 2 (SH2) domain of mouse 3BP2 are critical for B cell receptor (BCR)-induced activation of nuclear factor of activated T cells (NFAT) in human B cells. In this report, we have shown that Syk, but not Abl family protein-tyrosine kinases, is critical for BCR-mediated tyrosine phosphorylation of 3BP2 in chicken DT40 cells. Mutational analysis showed that Tyr174, Tyr183 and Tyr426 of chicken 3BP2 are the major phosphorylation sites by Syk and the SH2 domain of 3BP2 is critical for tyrosine phosphorylation. In addition, phosphorylation of Tyr426 is required for the inducible interaction with the SH2 domain of Vav3. Moreover, the expression of the mutant form of 3BP2 in which Tyr426 was substituted to Phe resulted in the reduction in BCR-mediated Rac1 activation, when compared with the case of wild-type. Altogether, these data suggest that 3BP2 is involved in the activation of Rac1 through the regulation of Vav3 by Syk-dependent phosphorylation of Tyr426 following BCR stimulation. - Highlights: • 3BP2 is phosphorylated by Syk, but not Abl family kinases in BCR signaling. • Tyr183 and Tyr426 in chicken 3BP2 are the major phosphorylation sites by Syk. • The SH2 domain of 3BP2 is critical for tyrosine phosphorylation of 3BP2. • Phosphorylation of Tyr426 in 3BP2 is required for the inducible binding with Vav3. • 3BP2 is involved in the regulation of BCR-mediated Rac1 activation

  17. Tyrosine phosphorylation of 3BP2 is indispensable for the interaction with VAV3 in chicken DT40 cells

    Energy Technology Data Exchange (ETDEWEB)

    Chihara, Kazuyasu [Division of Genome Science and Microbiology, Department of Pathological Sciences, Faculty of Medical Sciences, Fukui 910-1193 (Japan); Organization for Life Science Advancement Programs, University of Fukui, Fukui 910-1193 (Japan); Kimura, Yukihiro [Division of Genome Science and Microbiology, Department of Pathological Sciences, Faculty of Medical Sciences, Fukui 910-1193 (Japan); Division of Otorhinolaryngology Head and Neck Surgery, Department of Sensory and Locomotor Medicine, Faculty of Medical Sciences, Fukui 910-1193 (Japan); Honjoh, Chisato [Division of Genome Science and Microbiology, Department of Pathological Sciences, Faculty of Medical Sciences, Fukui 910-1193 (Japan); Third Department of Internal Medicine, Faculty of Medical Sciences, Fukui 910-1193 (Japan); Yamauchi, Shota; Takeuchi, Kenji [Division of Genome Science and Microbiology, Department of Pathological Sciences, Faculty of Medical Sciences, Fukui 910-1193 (Japan); Organization for Life Science Advancement Programs, University of Fukui, Fukui 910-1193 (Japan); Sada, Kiyonao, E-mail: ksada@u-fukui.ac.jp [Division of Genome Science and Microbiology, Department of Pathological Sciences, Faculty of Medical Sciences, Fukui 910-1193 (Japan); Organization for Life Science Advancement Programs, University of Fukui, Fukui 910-1193 (Japan)

    2014-03-10

    Adaptor protein c-Abl SH3 domain-binding protein-2 (3BP2) is known to play regulatory roles in immunoreceptor-mediated signal transduction. We have previously demonstrated that Tyr{sup 174}, Tyr{sup 183} and Tyr{sup 446} in mouse 3BP2 are predominantly phosphorylated by Syk, and the phosphorylation of Tyr{sup 183} and the Src homology 2 (SH2) domain of mouse 3BP2 are critical for B cell receptor (BCR)-induced activation of nuclear factor of activated T cells (NFAT) in human B cells. In this report, we have shown that Syk, but not Abl family protein-tyrosine kinases, is critical for BCR-mediated tyrosine phosphorylation of 3BP2 in chicken DT40 cells. Mutational analysis showed that Tyr{sup 174}, Tyr{sup 183} and Tyr{sup 426} of chicken 3BP2 are the major phosphorylation sites by Syk and the SH2 domain of 3BP2 is critical for tyrosine phosphorylation. In addition, phosphorylation of Tyr{sup 426} is required for the inducible interaction with the SH2 domain of Vav3. Moreover, the expression of the mutant form of 3BP2 in which Tyr{sup 426} was substituted to Phe resulted in the reduction in BCR-mediated Rac1 activation, when compared with the case of wild-type. Altogether, these data suggest that 3BP2 is involved in the activation of Rac1 through the regulation of Vav3 by Syk-dependent phosphorylation of Tyr{sup 426} following BCR stimulation. - Highlights: • 3BP2 is phosphorylated by Syk, but not Abl family kinases in BCR signaling. • Tyr183 and Tyr426 in chicken 3BP2 are the major phosphorylation sites by Syk. • The SH2 domain of 3BP2 is critical for tyrosine phosphorylation of 3BP2. • Phosphorylation of Tyr426 in 3BP2 is required for the inducible binding with Vav3. • 3BP2 is involved in the regulation of BCR-mediated Rac1 activation.

  18. Non–lineage/stage-restricted effects of a gain-of-function mutation in tyrosine phosphatase Ptpn11 (Shp2) on malignant transformation of hematopoietic cells

    OpenAIRE

    Xu, Dan; Liu, Xia; Yu, Wen-Mei; Meyerson, Howard J.; Guo, Caiying; Gerson, Stanton L.; Qu, Cheng-Kui

    2011-01-01

    Activating mutations in protein tyrosine phosphatase 11 (Ptpn11) have been identified in childhood acute leukemias, in addition to juvenile myelomonocytic leukemia (JMML), which is a myeloproliferative disorder (MPD). It is not clear whether activating mutations of this phosphatase play a causal role in the pathogenesis of acute leukemias. If so, the cell origin of leukemia-initiating stem cells (LSCs) remains to be determined. Ptpn11E76K mutation is the most common and most active Ptpn11 mut...

  19. Stress-evoked tyrosine phosphorylation of signal regulatory protein α regulates behavioral immobility in the forced swim test.

    Science.gov (United States)

    Ohnishi, Hiroshi; Murata, Takaaki; Kusakari, Shinya; Hayashi, Yuriko; Takao, Keizo; Maruyama, Toshi; Ago, Yukio; Koda, Ken; Jin, Feng-Jie; Okawa, Katsuya; Oldenborg, Per-Arne; Okazawa, Hideki; Murata, Yoji; Furuya, Nobuhiko; Matsuda, Toshio; Miyakawa, Tsuyoshi; Matozaki, Takashi

    2010-08-01

    Severe stress induces changes in neuronal function that are implicated in stress-related disorders such as depression. The molecular mechanisms underlying the response of the brain to stress remain primarily unknown, however. Signal regulatory protein alpha (SIRPalpha) is an Ig-superfamily protein that undergoes tyrosine phosphorylation and binds the protein tyrosine phosphatase Shp2. Here we show that mice expressing a form of SIRPalpha that lacks most of the cytoplasmic region manifest prolonged immobility (depression-like behavior) in the forced swim (FS) test. FS stress induced marked tyrosine phosphorylation of SIRPalpha in the brain of wild-type mice through activation of Src family kinases. The SIRPalpha ligand CD47 was important for such SIRPalpha phosphorylation, and CD47-deficient mice also manifested prolonged immobility in the FS test. Moreover, FS stress-induced tyrosine phosphorylation of both the NR2B subunit of the NMDA subtype of glutamate receptor and the K+-channel subunit Kvbeta2 was regulated by SIRPalpha. Thus, tyrosine phosphorylation of SIRPalpha is important for regulation of depression-like behavior in the response of the brain to stress.

  20. A tyrosine-phosphorylated 55-kilodalton motility-associated bovine sperm protein is regulated by cyclic adenosine 3',5'-monophosphates and calcium.

    Science.gov (United States)

    Vijayaraghavan, S; Trautman, K D; Goueli, S A; Carr, D W

    1997-06-01

    Sperm motility is regulated by protein phosphorylation. We have recently shown that a serine/threonine phosphatase system is involved in motility regulation. Two of the components of the phosphatase system, GSK-3 and PP1gamma2, are regulated by tyrosine phosphorylation. During our investigation of sperm tyrosine-phosphorylated proteins we discovered a 55-kDa protein whose tyrosine phosphorylation correlates closely to the motility state of sperm. This protein is tyrosine phosphorylated to a much higher degree in motile caudal than in immotile caput epididymal sperm. Motility inhibition of caudal epididymal sperm by protein kinase A (PKA) anchoring inhibition or by ionomycin-induced calcium overload led to the virtual disappearance of tyrosine phosphorylation of the 55-kDa protein. Conversely, treatment of sperm with motility activators, isobutylmethylxanthine or 8-bromo-cAMP, resulted in increased tyrosine phosphorylation of the protein. The protein was present in the soluble 100 000 x g supernatants of sperm extracts and was heat labile. Chromatography through diethylaminoethyl-cellulose and Western blot analysis showed that this 55-kDa protein is not a regulatory subunit of PKA or alpha-tubulin. Our results represent the identification of a soluble protein whose tyrosine phosphorylation varies directly with motility and suggest that motility regulation may involve cross talk between PKA, calcium, and tyrosine kinase pathways. PMID:9166697

  1. Role of Receptor Protein Tyrosine Phosphatase γ in Sensing Extracellular CO2 and HCO3.

    Science.gov (United States)

    Zhou, Yuehan; Skelton, Lara A; Xu, Lumei; Chandler, Margaret P; Berthiaume, Jessica M; Boron, Walter F

    2016-09-01

    Regulation of blood pH-critical for virtually every facet of life-requires that the renal proximal tubule (PT) adjust its rate of H(+) secretion (nearly the same as the rate of HCO3 (-) reabsorption, JHCO3 ) in response to changes in blood [CO2] and [HCO3 (-)]. Yet CO2/HCO3 (-) sensing mechanisms remain poorly characterized. Because receptor tyrosine kinase inhibitors render JHCO3 in the PT insensitive to changes in CO2 concentration, we hypothesized that the structural features of receptor protein tyrosine phosphatase-γ (RPTPγ) that are consistent with binding of extracellular CO2 or HCO3 (-) facilitate monitoring of blood CO2/HCO3 (-) concentrations. We now report that PTs express RPTPγ on blood-facing membranes. Moreover, RPTPγ deletion in mice eliminated the CO2 and HCO3 (-) sensitivities of JHCO3 as well as the normal defense of blood pH during whole-body acidosis. Thus, RPTPγ appears to be a novel extracellular CO2/HCO3 (-) sensor critical for pH homeostasis. PMID:26839367

  2. Hepatocyte growth factor regulated tyrosine kinase substrate in the peripheral development and function of B-cells

    Energy Technology Data Exchange (ETDEWEB)

    Nagata, Takayuki [Department of Pharmacy, Iwaki Meisei University, 5-5-1 Chuodai Iino, Iwaki, Fukushima 970-8551 (Japan); Department of Microbiology and Immunology, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai 980-8575 (Japan); Murata, Kazuko, E-mail: murata-k@iwakimu.ac.jp [Department of Pharmacy, Iwaki Meisei University, 5-5-1 Chuodai Iino, Iwaki, Fukushima 970-8551 (Japan); Murata, Ryo [Department of Pharmacy, Iwaki Meisei University, 5-5-1 Chuodai Iino, Iwaki, Fukushima 970-8551 (Japan); Sun, Shu-lan [Department of Microbiology and Immunology, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai 980-8575 (Japan); Saito, Yutaro; Yamaga, Shuhei [Department of Pharmacy, Iwaki Meisei University, 5-5-1 Chuodai Iino, Iwaki, Fukushima 970-8551 (Japan); Tanaka, Nobuyuki; Tamai, Keiichi [Division of Immunology, Miyagi Cancer Research Institute, 47-1 Nodayama, Medeshima-Shiode, Natori 981-1293 (Japan); Moriya, Kunihiko [Department of Pediatrics, Tohoku University School of Medicine, 1-1 Seiryo-machi, Aoba-ku, Sendai 980-8575 (Japan); Kasai, Noriyuki [Institute for Animal Experimentation, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai 980-8575 (Japan); Sugamura, Kazuo [Division of Immunology, Miyagi Cancer Research Institute, 47-1 Nodayama, Medeshima-Shiode, Natori 981-1293 (Japan); Ishii, Naoto [Department of Microbiology and Immunology, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai 980-8575 (Japan)

    2014-01-10

    Highlights: •ESCRT-0 protein regulates the development of peripheral B-cells. •BCR expression on cell surface should be controlled by the endosomal-sorting system. •Hrs plays important roles in responsiveness to Ag stimulation in B lymphocytes. -- Abstract: Hepatocyte growth factor (HGF)-regulated tyrosine kinase substrate (Hrs) is a vesicular sorting protein that functions as one of the endosomal-sorting proteins required for transport (ESCRT). Hrs, which binds to ubiquitinated proteins through its ubiquitin-interacting motif (UIM), contributes to the lysosomal transport and degradation of ubiquitinated membrane proteins. However, little is known about the relationship between B-cell functions and ESCRT proteins in vivo. Here we examined the immunological roles of Hrs in B-cell development and functions using B-cell-specific Hrs-deficient (Hrs{sup flox/flox};mb1{sup cre/+}:Hrs-cKO) mice, which were generated using a cre-LoxP recombination system. Hrs deficiency in B-cells significantly reduced T-cell-dependent antibody production in vivo and impaired the proliferation of B-cells treated in vitro with an anti-IgM monoclonal antibody but not with LPS. Although early development of B-cells in the bone marrow was normal in Hrs-cKO mice, there was a significant decrease in the number of the peripheral transitional B-cells and marginal zone B-cells in the spleen of Hrs-cKO mice. These results indicate that Hrs plays important roles during peripheral development and physiological functions of B lymphocytes.

  3. MHC class I signaling in T cells leads to tyrosine kinase activity and PLC-gamma 1 phosphorylation

    DEFF Research Database (Denmark)

    Skov, S; Odum, Niels; Claesson, M H

    1995-01-01

    phosphorylation and the subsequent calcium response. The early tyrosine kinase activity was found to be dependent on expression of the TCR/CD3 complex and the CD45 molecule on the surface of the T cells. Furthermore, MHC-I cross-linking was shown to tyrosine phosphorylate PLC-gamma 1 (phospholipase C-gamma 1...

  4. In vivo modification of tyrosine residues in recombinant mussel adhesive protein by tyrosinase co-expression in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Choi Yoo

    2012-10-01

    Full Text Available Abstract Background In nature, mussel adhesive proteins (MAPs show remarkable adhesive properties, biocompatibility, and biodegradability. Thus, they have been considered promising adhesive biomaterials for various biomedical and industrial applications. However, limited production of natural MAPs has hampered their practical applications. Recombinant production in bacterial cells could be one alternative to obtain useable amounts of MAPs, although additional post-translational modification of tyrosine residues into 3,4-dihydroxyphenyl-alanine (Dopa and Dopaquinone is required. The superior properties of MAPs are mainly attributed to the introduction of quinone-derived intermolecular cross-links. To solve this problem, we utilized a co-expression strategy of recombinant MAP and tyrosinase in Escherichia coli to successfully modify tyrosine residues in vivo. Results A recombinant hybrid MAP, fp-151, was used as a target for in vivo modification, and a dual vector system of pET and pACYC-Duet provided co-expression of fp-151 and tyrosinase. As a result, fp-151 was over-expressed and mainly obtained from the soluble fraction in the co-expression system. Without tyrosinase co-expression, fp-151 was over-expressed in an insoluble form in inclusion bodies. The modification of tyrosine residues in the soluble-expressed fp-151 was clearly observed from nitroblue tetrazolium staining and liquid-chromatography-mass/mass spectrometry analyses. The purified, in vivo modified, fp-151 from the co-expression system showed approximately 4-fold higher bulk-scale adhesive strength compared to in vitro tyrosinase-treated fp-151. Conclusion Here, we reported a co-expression system to obtain in vivo modified MAP; additional in vitro tyrosinase modification was not needed to obtain adhesive properties and the in vivo modified MAP showed superior adhesive strength compared to in vitro modified protein. It is expected that this co-expression strategy will accelerate

  5. Receptor tyrosine kinase (c-Kit) inhibitors: a potential therapeutic target in cancer cells

    Science.gov (United States)

    Abbaspour Babaei, Maryam; Kamalidehghan, Behnam; Saleem, Mohammad; Huri, Hasniza Zaman; Ahmadipour, Fatemeh

    2016-01-01

    c-Kit, a receptor tyrosine kinase, is involved in intracellular signaling, and the mutated form of c-Kit plays a crucial role in occurrence of some cancers. The function of c-Kit has led to the concept that inhibiting c-Kit kinase activity can be a target for cancer therapy. The promising results of inhibition of c-Kit for treatment of cancers have been observed in some cancers such as gastrointestinal stromal tumor, acute myeloid leukemia, melanoma, and other tumors, and these results have encouraged attempts toward improvement of using c-Kit as a capable target for cancer therapy. This paper presents the findings of previous studies regarding c-Kit as a receptor tyrosine kinase and an oncogene, as well as its gene targets and signaling pathways in normal and cancer cells. The c-Kit gene location, protein structure, and the role of c-Kit in normal cell have been discussed. Comprehending the molecular mechanism underlying c-Kit-mediated tumorogenesis is consequently essential and may lead to the identification of future novel drug targets. The potential mechanisms by which c-Kit induces cellular transformation have been described. This study aims to elucidate the function of c-Kit for future cancer therapy. In addition, it has c-Kit inhibitor drug properties and their functions have been listed in tables and demonstrated in schematic pictures. This review also has collected previous studies that targeted c-Kit as a novel strategy for cancer therapy. This paper further emphasizes the advantages of this approach, as well as the limitations that must be addressed in the future. Finally, although c-Kit is an attractive target for cancer therapy, based on the outcomes of treatment of patients with c-Kit inhibitors, it is unlikely that Kit inhibitors alone can lead to cure. It seems that c-Kit mutations alone are not sufficient for tumorogenesis, but do play a crucial role in cancer occurrence. PMID:27536065

  6. Expression, purification and characterization of recombinant protein tyrosine phosphatase from Thermus thermophilus HB27

    Institute of Scientific and Technical Information of China (English)

    Yejing Wang; Fanguo Meng; Yingmei Zhang

    2009-01-01

    The low-molecular-weight protein tyrosine phospha-tases (PTPase) exist ubiquitously in prokaryotes and eukaryotes and play important roles in the regulation of physiological activities. We report here the expression, purification and characterization of an active and soluble PTPase from Thermus thermophilus HB27 in Escherichia coli. This PTPase has an optimum pH range of 2.8-4.8 when using p-nitrophenyl phos-phate as the substrate. The thermal inactivation results indicate a high thermal stability of this enzyme, with the optimum temperature of 75℃ for activity. It can be activated by Mn2+, Mg2+, Ca2+, Ba2+, and Ni2+, but inhibited by Zn2+, Cu2+, Cl-, and SO2-4. These results suggest that this heat-resistant PTPase may play impor-tant roles in vivo in the adaptation of the microorgan-ism to extreme temperatures and specific nutritional conditions.

  7. Knockout mice reveal a role for protein tyrosine phosphatase H1 in cognition

    Directory of Open Access Journals (Sweden)

    Ardizzone Michele

    2008-08-01

    Full Text Available Abstract Background The present study has investigated the protein tyrosine phosphatase H1 (PTPH1 expression pattern in mouse brain and its impact on CNS functions. Methods We have previously described a PTPH1-KO mouse, generated by replacing the PTP catalytic and the PDZ domain with a LacZ neomycin cassette. PTPH1 expression pattern was evaluated by LacZ staining in the brain and PTPH1-KO and WT mice (n = 10 per gender per genotype were also behaviorally tested for CNS functions. Results In CNS, PTPH1 is expressed during development and in adulthood and mainly localized in hippocampus, thalamus, cortex and cerebellum neurons. The behavioral tests performed on the PTPH1-KO mice showed an impact on working memory in male mice and an impaired learning performance at rotarod in females. Conclusion These results demonstrate for the first time a neuronal expression of PTPH1 and its functionality at the level of cognition.

  8. Association between receptor protein-tyrosine phosphatase RPTPalpha and the Grb2 adaptor. Dual Src homology (SH) 2/SH3 domain requirement and functional consequences

    DEFF Research Database (Denmark)

    Su, J; Yang, L T; Sap, J

    1996-01-01

    Receptor protein-tyrosine phosphatase RPTPalpha is found associated in vivo with the adaptor protein Grb2. Formation of this complex, which contains no detectable levels of Sos, is known to depend on a C-terminal phosphorylated tyrosine residue (Tyr798) in RPTPalpha and on the Src homology (SH) 2...

  9. Bypass of hexavalent chromium-induced growth arrest by a protein tyrosine phosphatase inhibitor: Enhanced survival and mutagenesis

    International Nuclear Information System (INIS)

    Although the consequences of genotoxic injury include cell cycle arrest and apoptosis, cell survival responses after genotoxic injury can produce intrinsic death-resistance and contribute to the development of a transformed phenotype. Protein tyrosine phosphatases (PTPs) are integral components of key survival pathways, and are responsible for their inactivation, while PTP inhibition is often associated with enhanced cell proliferation. Our aim was to elucidate signaling events that modulate cell survival after genotoxin exposure. Diploid human lung fibroblasts (HLF) were treated with Cr(VI) (as Na2CrO4), the soluble oxyanionic dissolution product of certain particulate chromates, which are well-documented human respiratory carcinogens. In vitro soluble Cr(VI) induces a wide spectrum of DNA damage, in both the presence and absence of a broad-range PTP inhibitor, sodium orthovanadate (SOV). Notably, SOV abrogated Cr(VI)-induced clonogenic lethality. The enhanced survival of Cr(VI)-exposed cells after SOV treatment was predominantly due to a bypass of cell cycle arrest, as there was no effect of the PTP inhibitor on Cr-induced apoptosis. Moreover, the SOV effect was not due to decreased Cr uptake as evidenced by unchanged Cr-DNA adduct burden. Additionally, the bypass of Cr-induced growth arrest by SOV was accompanied by a decrease in Cr(VI)-induced expression of cell cycle inhibiting genes, and an increase in Cr(VI)-induced expression of cell cycle promoting genes. Importantly, SOV resulted in an increase in forward mutations at the HPRT locus, supporting the hypothesis that PTP inhibition in the presence of certain types of DNA damage may lead to increased genomic instability, via bypass of cell cycle checkpoints

  10. A protein-binding domain, EH, identified in the receptor tyrosine kinase substrate Eps15 and conserved in evolution

    DEFF Research Database (Denmark)

    Wong, W T; Schumacher, C; Salcini, A E;

    1995-01-01

    In this report we structurally and functionally define a binding domain that is involved in protein association and that we have designated EH (for Eps15 homology domain). This domain was identified in the tyrosine kinase substrate Eps15 on the basis of regional conservation with several heteroge...

  11. Endogenous 3, 4- Dihydroxyphenylalanine and Dopaquinone Modifications on Protein Tyrosine: links to mitochondrially derived oxidative stress via hydroxyl radical

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Xu; Monroe, Matthew E.; Chen, Baowei; Chin, Mark H.; Heibeck, Tyler H.; Schepmoes, Athena A.; Yang, Feng; Petritis, Brianne O.; Camp, David G.; Pounds, Joel G.; Jacobs, Jon M.; Smith, Desmond J.; Bigelow, Diana J.; Smith, Richard D.; Qian, Weijun

    2010-06-02

    Oxidative modifications of protein tyrosines have been implicated in multiple human diseases. Among these modifications, elevations in levels of 3, 4-dihydroxyphenylalanine (DOPA), a major product of hydroxyl radical addition to tyrosine, has been observed in a number of pathologies. Here we report the first global proteome survey of endogenous site-specific modifications, i.e, DOPA and its further oxidation product dopaquinone (DQ) in mouse brain and heart tissues. Results from LC-MS/MS analyses included 203 and 71 DOPA-modified tyrosine sites identified from brain and heart, respectively, with a false discovery rate of ~1%; while only a few nitrotyrosine containing peptides, a more commonly studied marker of oxidative stress, were detectable, suggesting the much higher abundance for DOPA modification as compared with tyrosine nitration. Moreover, 57 and 29 DQ modified peptides were observed from brain and heart, respectively; nearly half of these peptides were also observed with DOPA modification on the same sites. For both tissues, these modifications are preferentially found in mitochondrial proteins with metal-binding properties, consistent with metal catalyzed hydroxyl radical formation from mitochondrial superoxide and hydrogen peroxide. These modifications also link to a number of mitochondria-associated and other signaling pathways. Furthermore, many of the modification sites were common sites of previously reported tyrosine phosphorylation suggesting potential disruption of signaling pathways. Structural aspects of DOPA-modified tyrosine sequences are distinct from those of nitrotyrosines suggesting that each type of modifications provides a marker for different in vivo reactive chemistries and can be used to predict sensitive protein targets. Collectively, the results suggest that these modifications are linked with mitochondrially-derived oxidative stress, and may serve as sensitive markers for disease pathologies.

  12. Involvement of β3A Subunit of Adaptor Protein-3 in Intracellular Trafficking of Receptor-like Protein Tyrosine Phosphatase PCP-2

    Institute of Scientific and Technical Information of China (English)

    Hui DONG; Hong YUAN; Weirong JIN; Yan SHEN; Xiaojing XU; Hongyang WANG

    2007-01-01

    PCP-2 is a human receptor-like protein tyrosine phosphatase and a member of the MAM domain family cloned in human pancreatic adenocarcinoma cells. Previous studies showed that PCP-2 directly interacted with β-catenin through the juxtamembrane domain, dephosphorylated β-catenin and played an important role in the regulation of cell adhesion. Recent study showed that PCP-2 was also involved in the repression of β-catenin-induced transcriptional activity. Here we describe the interactions of PCP-2 with the β3A subunit of adaptor protein (AP)-3 and sorting nexin (SNX) 3. These protein complexes were detected using the yeast two-hybrid assay with the juxtamembrane and membrane-proximal catalytic domain of PCP-2 as "bait". Both AP-3 and SNX3 are molecules involved in intracellular trafficking of membrane receptors. The association between the β3A subunit of AP-3 and PCP-2 was further confirmed in mammalian cells. Our results suggested a possible mechanism of intracellular trafficking of PCP-2 mediated by AP-3 and SNX3 which might participate in the regulation of PCP-2 functions.

  13. Low molecular weight protein tyrosine phosphatase: Multifaceted functions of an evolutionarily conserved enzyme.

    Science.gov (United States)

    Caselli, Anna; Paoli, Paolo; Santi, Alice; Mugnaioni, Camilla; Toti, Alessandra; Camici, Guido; Cirri, Paolo

    2016-10-01

    Originally identified as a low molecular weight acid phosphatase, LMW-PTP is actually a protein tyrosine phosphatase that acts on many phosphotyrosine-containing cellular proteins that are primarily involved in signal transduction. Differences in sequence, structure, and substrate recognition as well as in subcellular localization in different organisms enable LMW-PTP to exert many different functions. In fact, during evolution, the LMW-PTP structure adapted to perform different catalytic actions depending on the organism type. In bacteria, this enzyme is involved in the biosynthesis of group 1 and 4 capsules, but it is also a virulence factor in pathogenic strains. In yeast, LMW-PTPs dephosphorylate immunophilin Fpr3, a peptidyl-prolyl-cis-trans isomerase member of the protein chaperone family. In humans, LMW-PTP is encoded by the ACP1 gene, which is composed of three different alleles, each encoding two active enzymes produced by alternative RNA splicing. In animals, LMW-PTP dephosphorylates a number of growth factor receptors and modulates their signalling processes. The involvement of LMW-PTP in cancer progression and in insulin receptor regulation as well as its actions as a virulence factor in a number of pathogenic bacterial strains may promote the search for potent, selective and bioavailable LMW-PTP inhibitors. PMID:27421795

  14. Inhibition of Setaria cervi protein tyrosine phosphatases by Phenylarsine oxide: A proteomic and biochemical study.

    Science.gov (United States)

    Singh, Neetu; Wadhawan, Mohit; Tiwari, Savitri; Kumar, Ranjeet; Rathaur, Sushma

    2016-07-01

    Phenylarsine oxide (PAO), a specific protein tyrosine phosphatase (PTP) inhibitor significantly decreased the motility and viability of Setaria cervi ultimately leading to its death. The PTP activity present in the cytosolic and detergent soluble fractions as well as on surface of these parasites was significantly inhibited by PAO. A marked alteration in protein spots abundance after proteomic analysis showed 14 down-regulated and 9 upregulated spots in the treated parasites as compared to the control. The PTP inhibition led to increase in the cytosolic and mitochondrial calpain activity in these parasites. PAO also blocked the ATP generation in the parasite depicted by reduced activity of phosphoglycerate kinase and expression of enolase. An increased ROS level, induced lipid peroxidation/protein carbonyl formation and decreased activity of different antioxidant enzymes like thioredoxin reductase, glutathione reductase and glutathione transferases was also observed in the PAO treated parasites. PAO, thus disturbs the overall homeostasis of the filarial parasite by inhibiting PTPs. Thereby suggesting that these molecules could be used as a good chemotherapeutic target for lymphatic filariasis. PMID:26965172

  15. Regulation of gene expression for tyrosine hydroxylase in oxygen sensitive cells by hypoxia.

    Science.gov (United States)

    Millhorn, D E; Raymond, R; Conforti, L; Zhu, W; Beitner-Johnson, D; Filisko, T; Genter, M B; Kobayashi, S; Peng, M

    1997-02-01

    Carotid body type I cells and the O2 sensitive pheochromocytoma (PC12) cells release dopamine during hypoxia. Reduced O2 tension causes inhibition of an outward rectifying the O2-sensitive potassium (K) channel in the O2-sensitive pheochromocytoma (PC12) cell line, which leads to membrane depolarization and increased intracellular free Ca2+. We found that removal of Ca2+ from the extracellular milieu, inhibition of voltage-dependent Ca2+ channels, and chelation of intracellular Ca2+ prevents full activation of the TH gene expression during hypoxia. These findings suggest that membrane depolarization and regulation of intracellular free Ca2+ are critical signal transduction events that regulate expression of the TH gene in PC12 cells during hypoxia. Gene expression of tyrosine hydroxylase (TH), the rate-limiting enzyme in the biosynthesis of dopamine, is stimulated by reduced O2 tension in both type I cells and PC12 cells. The increase in TH gene expression in PC12 cells during hypoxia is due to increases in both the rate of transcription and mRNA stability. Analysis of reporter-gene constructs revealed that increased transcription of the TH gene during hypoxia is regulated by a region of the proximal promoter that extends from -284 to -150 bases, relative to the transcription start site. This region of the gene contains a number of cis-acting regulatory elements including AP1, AP2 and hypoxia-inducible factor (HIF-1). Competition assays revealed that hypoxia-induced binding occurs at both the AP1 and HIF-1 sites. Results from super-shift and shift Western assays showed that a heterodimer consisting of c-Fos and JunB binds to the AP1 site during hypoxia. Mutagenesis experiments revealed that the AP1 site is required for increased transcription of the TH gene during hypoxia. We also found that the genes that encode the c-Fos and JunB transcription factor proteins are regulated by reduced O2 tension. PMID:9027733

  16. Ribosomal Protein S6 Kinase (RSK-2 as a central effector molecule in RON receptor tyrosine kinase mediated epithelial to mesenchymal transition induced by macrophage-stimulating protein

    Directory of Open Access Journals (Sweden)

    Zhang Rui-Wen

    2011-05-01

    Full Text Available Abstract Background Epithelial to mesenchymal transition (EMT occurs during cancer cell invasion and malignant metastasis. Features of EMT include spindle-like cell morphology, loss of epithelial cellular markers and gain of mesenchymal phenotype. Activation of the RON receptor tyrosine kinase by macrophage-stimulating protein (MSP has been implicated in cellular EMT program; however, the major signaling determinant(s responsible for MSP-induced EMT is unknown. Results The study presented here demonstrates that RSK2, a downstream signaling protein of the Ras-Erk1/2 pathway, is the principal molecule that links MSP-activated RON signaling to complete EMT. Using MDCK cells expressing RON as a model, a spindle-shape based screen was conducted, which identifies RSK2 among various intracellular proteins as a potential signaling molecule responsible for MSP-induced EMT. MSP stimulation dissociated RSK2 with Erk1/2 and promoted RSK2 nuclear translocation. MSP strongly induced RSK2 phosphorylation in a dose-dependent manner. These effects relied on RON and Erk1/2 phosphorylation, which is significantly potentiated by transforming growth factor (TGF-β1, an EMT-inducing cytokine. Specific RSK inhibitor SL0101 completely prevented MSP-induced RSK phosphorylation, which results in inhibition of MSP-induced spindle-like morphology and suppression of cell migration associated with EMT. In HT-29 cancer cells that barely express RSK2, forced RSK2 expression results in EMT-like phenotype upon MSP stimulation. Moreover, specific siRNA-mediated silencing of RSK2 but not RSK1 in L3.6pl pancreatic cancer cells significantly inhibited MSP-induced EMT-like phenotype and cell migration. Conclusions MSP-induced RSK2 activation is a critical determinant linking RON signaling to cellular EMT program. Inhibition of RSK2 activity may provide a therapeutic opportunity for blocking RON-mediated cancer cell migration and subsequent invasion.

  17. Protein synthesis rate measured with L-[1-C-11]tyrosine positron emission tomography correlates with mitotic activity and MIB-1 antibody-detected proliferation in human soft tissue sarcomas

    NARCIS (Netherlands)

    Plaat, B; Kole, A; Mastik, M; Hoekstra, H; Molenaar, W; Vaalburg, W

    1999-01-01

    Protein synthesis rate (PSR can be assessed in vivo using positron emission tomography with L-[1-C-11]tyrosine (TYR-PET). Biological activity of soft tissue sarcomas (STS) can be measured in vitro by the mitotic rate and number of proliferating cells. In STS the grade of malignancy, in which the mit

  18. Effects of platelet inhibitors on propyl gallate-induced platelet aggregation, protein tyrosine phosphorylation, and platelet factor 3 activation.

    Science.gov (United States)

    Xiao, Hongyan; Kovics, Richard; Jackson, Van; Remick, Daniel G

    2004-04-01

    Propyl gallate (PG) is a platelet agonist characterized by inducing platelet aggregation, protein tyrosine phosphorylation, and platelet factor 3 activity. The mechanisms of platelet activation following PG stimulation were examined by pre-incubating platelets with well-defined platelet inhibitors using platelet aggregation, protein tyrosine phosphorylation, activated plasma clotting time, and annexin V binding by flow cytometry. PG-induced platelet aggregation and tyrosine phosphorylation of multiple proteins were substantially abolished by aspirin, apyrase, and abciximab (c7E3), suggesting that PG is associated with activation of platelet cyclooxygenase 1, adenosine phosphate receptors, and glycoprotein IIb/IIIa, respectively. The phosphorylation of the cytoskeletal enzyme pp60(c-src) increased following PG stimulation, but was blunted by pre-incubation of platelets with aspirin, apyrase, and c7E3, suggesting that tyrosine kinase is important for the signal transduction of platelet aggregation. Propyl gallate also activates platelet factor 3 by decreasing the platelet coagulation time and increasing platelet annexin V binding. Platelet incubation with aspirin, apyrase, and c7E3 did not alter PG-induced platelet coagulation and annexin V binding. The results suggest that platelet factor 3 activation and membrane phosphotidylserine expression were not involved with activation of platelet cyclooxygenase, adenosine phosphate receptors, and glycoprotein IIb/IIIa. PG is unique in its ability to stimulate platelet aggregation and coagulation simultaneously, and platelet inhibitors in this study affect only platelet aggregation but not platelet coagulation. PMID:15060414

  19. Prognostic value of protein tyrosine kinase 6 (PTK6) for long-term survival of breast cancer patients.

    Science.gov (United States)

    Aubele, M; Walch, A K; Ludyga, N; Braselmann, H; Atkinson, M J; Luber, B; Auer, G; Tapio, S; Cooke, T; Bartlett, J M S

    2008-10-01

    The cytoplasmic tyrosine kinase PTK6 (BRK) shows elevated expression in approximately two-thirds of primary breast tumours, and is implicated in EGF receptor-dependent signalling and epithelial tumorigenesis. Using immunohistochemistry, we performed a retrospective study on 426 archival breast cancer samples from patients with long-term follow-up and compared the protein expression levels of PTK6, the HER receptors, Sam68 (a substrate of PTK6), and signalling proteins including MAP kinase (MAPK), phosphorylated MAPK (P-MAPK), and PTEN. We show that PTK6 expression is of significant prognostic value in the outcome of breast carcinomas. In multivariate analysis, the disease-free survival of patients of >or=240 months was directly associated with the protein expression level of PTK6 (Pproteins, we used protein extracts from the T47D cell line for immunoprecipitation and western blot analysis. By this, interactions could be demonstrated between PTK6 and MAPK, P-MAPK, HER2/neu, HER3, HER4, PTEN, and Sam68. On the basis of these results, we suggest that PTK6 may serve as a future target for the development of novel treatments in breast cancer.

  20. Purification and Characterization of the Catalytic Domain of Protein Tyrosine Phosphatase SHP-1 and the Preparation of Anti-△SHP-1 Antibodies

    Institute of Scientific and Technical Information of China (English)

    LI Wan-nan; ZHUANG Yan; LI He; SUN Ying; FU Yao; WU Xiao-xia; ZHAO Zhi-zhuang; FU Xue-qi

    2008-01-01

    This study is focused on the expression of an SH2 domain-truncated form of protein tyrosine phosphatase SHP-1(designated △SHP-1) and the preparation of its polyelonal antibodies.A cDNA fragment encoding △SHP-1 was amplified by PCR and then cloned into the pT7 expression vector.The recombinant pT7-△SHP-1 plasmid was used to transform Rosetta(DE3) E.coll cells.△SHP-1 was distributed in the exclusion body of E.coll cell extracts and was purified through a two-column chromatographic procedure.The purified enzyme exhibited an expected molecular weight on SDS-gels and HPLC gel filtration columns.It possesses robust tyrosine phosphatase activity and shows typical enzymatic characteristics of classic tyrosine phosphatases.To generate polyclonal anti-△SHP-1 antibodies,purified recombinant △SHP-1 was used to immunize a rabbit.The resultant anti-serum was subjected to purification on △SHP-1 antigen affinity chromatography.The purified polyclonal antibody displayed a high sensitivity and specificity toward △SHP-1.This study thus provides the essential materials for further investigating the biological function and pathological implication of SHP-1 and screening the inhibitors and activators of the enzyme for therapeutic drug development.

  1. Application of SAIL phenylalanine and tyrosine with alternative isotope-labeling patterns for protein structure determination

    Energy Technology Data Exchange (ETDEWEB)

    Takeda, Mitsuhiro [Nagoya University, Structural Biology Research Center, Graduate School of Science (Japan); Ono, Akira M.; Terauchi, Tsutomu [SAIL Technologies Co., Inc. (Japan); Kainosho, Masatsune, E-mail: kainosho@nmr.chem.metro-u.ac.j [Nagoya University, Structural Biology Research Center, Graduate School of Science (Japan)

    2010-01-15

    The extensive collection of NOE constraint data involving the aromatic ring signals is essential for accurate protein structure determination, although it is often hampered in practice by the pervasive signal overlapping and tight spin couplings for aromatic rings. We have prepared various types of stereo-array isotope labeled phenylalanines ({epsilon}- and {zeta}-SAIL Phe) and tyrosine ({epsilon}-SAIL Tyr) to overcome these problems (Torizawa et al. 2005), and proven that these SAIL amino acids provide dramatic spectral simplification and sensitivity enhancement for the aromatic ring NMR signals. In addition to these SAIL aromatic amino acids, we recently synthesized {delta}-SAIL Phe and {delta}-SAIL Tyr, which allow us to observe and assign {delta}-{sup 13}C/{sup 1}H signals very efficiently. Each of the various types of SAIL Phe and SAIL Tyr yields well-resolved resonances for the {delta}-, {epsilon}- or {zeta}-{sup 13}C/{sup 1}H signals, respectively, which can readily be assigned by simple and robust pulse sequences. Since the {delta}-, {epsilon}-, and {zeta}-proton signals of Phe/Tyr residues give rise to complementary NOE constraints, the concomitant use of various types of SAIL-Phe and SAIL-Tyr would generate more accurate protein structures, as compared to those obtained by using conventional uniformly {sup 13}C, {sup 15}N-double labeled proteins. We illustrated this with the case of an 18.2 kDa protein, Escherichia coli peptidyl-prolyl cis-trans isomerase b (EPPIb), and concluded that the combined use of {zeta}-SAIL Phe and {epsilon}-SAIL Tyr would be practically the best choice for protein structural determinations.

  2. Voltage sensitive phosphatases: emerging kinship to protein tyrosine phosphatases from structure-function research

    Directory of Open Access Journals (Sweden)

    Kirstin eHobiger

    2015-02-01

    Full Text Available The transmembrane protein Ci-VSP from the ascidian Ciona intestinalis was described as first member of a fascinating family of enzymes, the voltage sensitive phosphatases (VSPs. Ci-VSP and its voltage-activated homologs from other species are stimulated by positive membrane potentials and dephosphorylate the head groups of negatively charged phosphoinositide phosphates (PIPs. In doing so, VSPs act as control centers at the cytosolic membrane surface, because they intervene in signaling cascades that are mediated by PIP lipids. The characteristic motif CX5RT/S in the active site classifies VSPs as members of the huge family of cysteine-based protein tyrosine phosphatases (PTPs. Although PTPs have already been well characterized regarding both, structure and function, their relationship to VSPs has drawn only limited attention so far. Therefore, the intention of this review is to give a short overview about the extensive knowledge about PTPs in relation to the facts known about VSPs. Here, we concentrate on the structural features of the catalytic domain which are similar between both classes of phosphatases and their consequences for the enzymatic function. By discussing results obtained from crystal structures, molecular dynamics simulations, and mutagenesis studies, a possible mechanism for the catalytic cycle of VSPs is presented based on that one proposed for PTPs. In this way, we want to link the knowledge about the catalytic activity of VSPs and PTPs.

  3. Bruton’s tyrosine kinase inhibitors and their clinical potential in the treatment of B-cell malignancies: focus on ibrutinib

    OpenAIRE

    Aalipour, Amin; Advani, Ranjana H.

    2014-01-01

    Aberrant signaling of the B-cell receptor pathway has been linked to the development and maintenance of B-cell malignancies. Bruton’s tyrosine kinase (BTK), a protein early in this pathway, has emerged as a new therapeutic target in a variety of such malignancies. Ibrutinib, the most clinically advanced small molecule inhibitor of BTK, has demonstrated impressive tolerability and activity in a range of B-cell lymphomas which led to its recent approval for relapsed mantle cell lymphoma and chr...

  4. PAH- and PCB-induced Alterations of Protein Tyrosine Kinase and Cytokine Gene Transcription in Harbor Seal (Phoca Vitulina PBMC

    Directory of Open Access Journals (Sweden)

    Jennifer C. C. Neale

    2005-01-01

    Full Text Available Mechanisms underlying in vitro immunomodulatory effects of polycyclic aromatic hydrocarbons (PAHs and polychlorinated biphenyls (PCBs were investigated in harbor seal peripheral leukocytes, via real-time PCR. We examined the relative genetic expression of the protein tyrosine kinases (PTKs Fyn and Itk, which play a critical role in T cell activation, and IL-2, a cytokine of central importance in initiating adaptive immune responses. IL-1, the macrophage-derived pro-inflammatory cytokine of innate immunity, was also included as a measure of macrophage function. Harbor seal PBMC were exposed to the prototypic immunotoxic PAH benzo[a]pyrene (BaP, 3,3',4,4',5,5'-hexachlorobiphenyl (CB-169, a model immunotoxic PCB, or DMSO (vehicle control. Exposure of Con A-stimulated harbor seal PBMC to both BaP and CB-169 produced significantly altered expression in all four targets relative to vehicle controls. The PTKs Fyn and Itk were both up-regulated following exposure to BaP and CB-169. In contrast, transcripts for IL-2 and IL-1 were decreased relative to controls by both treatments. Our findings are consistent with those of previous researchers working with human and rodent systems and support a hypothesis of contaminant-altered lymphocyte function mediated (at least in part by disruption of T cell receptor (TCR signaling and cytokine production.

  5. Bacterial single-stranded DNA-binding proteins are phosphorylated on tyrosine

    DEFF Research Database (Denmark)

    Mijakovic, Ivan; Petranovic, Dina; Macek, B;

    2006-01-01

    by kinase YwqD and phosphatase YwqE. Phosphorylation of B.subtilis SSB increased binding almost 200-fold to single-stranded DNA in vitro. Tyrosine phosphorylation of B.subtilis, S.coelicolor and Escherichia coli SSBs occured while they were expressed in E.coli, indicating that tyrosine phosphorylation...

  6. Synthesis of aromatic (13)C/(2)H-α-ketoacid precursors to be used in selective phenylalanine and tyrosine protein labelling.

    Science.gov (United States)

    Lichtenecker, R J

    2014-10-14

    Recent progress in protein NMR spectroscopy revealed aromatic residues to be valuable information sources for performing structure and motion analysis of high molecular weight proteins. However, the applied NMR experiments require tailored isotope labelling patterns in order to regulate spin-relaxation pathways and optimize magnetization transfer. We introduced a methodology to use α-ketoacids as metabolic amino acid precursors in cell-based overexpression of phenylalanine and/or tyrosine labelled proteins in a recent publication, which we have now developed further by providing synthetic routes to access the corresponding side-chain labelled precursors. The target compounds allow for selective introduction of (13)C-(1)H spin systems in a highly deuterated chemical environment and feature alternating (12)C-(13)C-(12)C ring-patterns. The resulting isotope distribution is especially suited to render straightforward (13)C spin relaxation experiments possible, which provide insight into the dynamic properties of the corresponding labelled proteins.

  7. Glycoxidised LDL induced the upregulation of Axl receptor tyrosine kinase and its ligand in mouse mesangial cells.

    Directory of Open Access Journals (Sweden)

    Young Sook Kim

    Full Text Available AIM/HYPOTHESIS: Low-density lipoprotein (LDL is subjected to glycoxidation in diabetes, and a novel signalling mechanism by which glycoxidised LDL functions in glomerular mesangial cells remains to be ascertained. METHODS: We performed gene expression analysis in mouse glomerular mesangial cells treated with LDL modified by glycation and oxidation (GO-LDL, 100 µg/ml for 48 h by using DNA microarray analysis and quantitative real-time PCR. We examined the GO-LDL-specific changes in gene and protein expression in mesangial cells and glomeruli of type 2 diabetic Zucker diabetic fatty (ZDF rats. RESULTS: By microarray profiling, we noted that GO-LDL treatment increased Axl receptor tyrosine kinase (Axl mRNA expression (∼2.5-fold, p<0.05 compared with normal LDL (N-LDL treatment in mesangial cells. Treatment with GO-LDL also increased the protein levels of Axl and its ligand Gas6 as measured by Western blotting. These increases were inhibited by neutralising Axl receptor-specific antibody. Silencing Gas6 by siRNA inhibited GO-LDL-induced Axl expression in mesangial cells. Axl and Gas6 protein were also increased in cells cultured in high glucose (30 mM or methylglyoxal (200 µM. Gas6 treatment increased the expression and secretion of TGF-β1 protein, a key regulator of extracellular matrix expression in the glomeruli of diabetic kidneys. Immunohistochemical analyses of glomeruli from 20-week-old ZDF rats exhibited increased Axl protein expression. Rottlerin, a selective PKC-δ inhibitor, completely blocked Gas6-induced TGF-β1 expression. CONCLUSIONS/INTERPRETATION: These data suggest that LDL modified by glycoxidation may mediate Axl/Gas6 pathway activation, and this mechanism may play a significant role in the pathogenesis of diabetic nephropathy.

  8. Tyrosine phosphorylation of transcriptional coactivator WW-domain binding protein 2 regulates estrogen receptor α function in breast cancer via the Wnt pathway.

    Science.gov (United States)

    Lim, Shen Kiat; Orhant-Prioux, Magali; Toy, Weiyi; Tan, Kah Yap; Lim, Yoon Pin

    2011-09-01

    WW-binding protein 2 (WBP2) has been demonstrated in different studies to be a tyrosine kinase substrate, to activate estrogen receptor α (ERα)/progesterone receptor (PR) transcription, and to play a role in breast cancer. However, the role of WBP2 tyrosine phosphorylation in regulating ERα function and breast cancer biology is unknown. Here, we established WBP2 as a tyrosine phosphorylation target of estrogen signaling via EGFR crosstalk. Using dominant-negative, constitutively active mutants, RNAi, and pharmacological studies, we demonstrated that phosphorylation of WBP2 at Tyr192 and Tyr231 could be regulated by c-Src and c-Yes kinases. We further showed that abrogating WBP2 phosphorylation impaired >60% of ERα reporter activity, putatively by blocking nuclear entry of WBP2 and its interaction with ERα. Compared to vector control, overexpression of WBP2 and its phospho-mimic mutant in MCF7 cells resulted in larger tumors in mice, induced loss of cell-cell adhesion, and enhanced cell proliferation, anchorage-independent growth, migration, and invasion in both estrogen-dependent and -independent manners, events of which could be substantially abolished by overexpression of the phosphorylation-defective mutant. Hormone independence of cells expressing WBP2 phospho-mimic mutant was associated with heightened ERα and Wnt reporter activities. Wnt/β-catenin inhibitor FH535 blocked phospho-WBP2-mediated cancer cell growth more pronouncedly than tamoxifen and fulvestrant, in part by reducing the expression of ERα. Wnt pathway is likely to be a critical component in WBP2-mediated breast cancer biology.

  9. Overall survival after immunotherapy, tyrosine kinase inhibitors and surgery in treatment of metastatic renal cell cancer

    DEFF Research Database (Denmark)

    de Lichtenberg, Trine Honnens; Hermann, Gregers G.; Rorth, Mikael;

    2014-01-01

    , stage, performance status and white cell blood count were related to poor OS. Using multivariate analyses to adjust for risk predictors the difference in OS disappeared. Median OS before and after introduction of TKIs was 16 months and 14 months, respectively (p = 0.189). Memorial Sloan Kettering Cancer......Abstract Objective. The aim of this study was to evaluate overall survival (OS) after treatment of metastatic renal cell carcinoma (mRCC) following the introduction of tyrosine kinase inhibitors (TKIs) and mammalian target of rapamycin (mTOR) inhibitors. Material and methods. One-hundred and forty...

  10. EGFR tyrosine kinase inhibitory peptide attenuates Helicobacter pylori-mediated hyper-proliferation in AGS enteric epithelial cells

    International Nuclear Information System (INIS)

    Helicobacter pylori infection is one of the most critical causes of stomach cancer. The current study was conducted to explore the protective effects of an isolated active peptide H-P-6 (Pro-Gln-Pro-Lys-Val-Leu-Asp-Ser) from microbial hydrolysates of Chlamydomonas sp. against H. pylori-induced carcinogenesis. The peptide H-P-6 has effectively suppressed H. pylori-induced hyper-proliferation and migration of gastric epithelial cells (AGS). However, the peptide did not inhibit the viability of the bacteria or invasion into AGS cells. Therefore, the effect of the peptide on regulating H. pylori-induced molecular signaling was investigated. The results indicated that H. pylori activates the EGFR tyrosine kinase signaling and nuclear translocation of the β-catenin. The EGFR activation has led to the up-regulation of PI3K/Akt signaling pathway. Moreover, the nuclear translocation levels of β-catenin were significantly increased as a result of Akt mediated down-regulation of GSK3/β protein levels in the cytoplasm. Both of these consequences have resulted in increased expression of cell survival and migration related genes such as c-Myc, cyclin-D, MMP-2 and matrilysin. Interestingly, the isolated peptide potently inhibited H. pylori-mediated EGFR activation and thereby down-regulated the subsequent P13K/Akt signaling leading to β-catenin nuclear translocation. The effect of the peptide was confirmed with the use of EGFR tyrosine kinase inhibitor AG1487 and molecular docking studies. Collectively this study identifies a potent peptide which regulates the H. pylori-induced hyper-proliferation and migration of AGS cells at molecular level. - Highlights: • Chlamydomonas sp. derived peptide H-P-6 inhibits H. pylori-induced pathogenesis. • H-P-6 suppresses H. pylori-induced hyper-proliferation and migration of AGS cells. • The peptide inhibits H. pylori-induced EGFR activation

  11. EGFR tyrosine kinase inhibitory peptide attenuates Helicobacter pylori-mediated hyper-proliferation in AGS enteric epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Himaya, S.W.A. [Marine Bio-Process Research Center, Pukyong National University, Nam-Gu, Busan, 608-737 (Korea, Republic of); Dewapriya, Pradeep [Department of Chemistry, Pukyong National University, Nam-Gu, Busan, 608-737 (Korea, Republic of); Kim, Se-Kwon, E-mail: sknkim@pknu.ac.kr [Marine Bio-Process Research Center, Pukyong National University, Nam-Gu, Busan, 608-737 (Korea, Republic of); Department of Chemistry, Pukyong National University, Nam-Gu, Busan, 608-737 (Korea, Republic of)

    2013-06-15

    Helicobacter pylori infection is one of the most critical causes of stomach cancer. The current study was conducted to explore the protective effects of an isolated active peptide H-P-6 (Pro-Gln-Pro-Lys-Val-Leu-Asp-Ser) from microbial hydrolysates of Chlamydomonas sp. against H. pylori-induced carcinogenesis. The peptide H-P-6 has effectively suppressed H. pylori-induced hyper-proliferation and migration of gastric epithelial cells (AGS). However, the peptide did not inhibit the viability of the bacteria or invasion into AGS cells. Therefore, the effect of the peptide on regulating H. pylori-induced molecular signaling was investigated. The results indicated that H. pylori activates the EGFR tyrosine kinase signaling and nuclear translocation of the β-catenin. The EGFR activation has led to the up-regulation of PI3K/Akt signaling pathway. Moreover, the nuclear translocation levels of β-catenin were significantly increased as a result of Akt mediated down-regulation of GSK3/β protein levels in the cytoplasm. Both of these consequences have resulted in increased expression of cell survival and migration related genes such as c-Myc, cyclin-D, MMP-2 and matrilysin. Interestingly, the isolated peptide potently inhibited H. pylori-mediated EGFR activation and thereby down-regulated the subsequent P13K/Akt signaling leading to β-catenin nuclear translocation. The effect of the peptide was confirmed with the use of EGFR tyrosine kinase inhibitor AG1487 and molecular docking studies. Collectively this study identifies a potent peptide which regulates the H. pylori-induced hyper-proliferation and migration of AGS cells at molecular level. - Highlights: • Chlamydomonas sp. derived peptide H-P-6 inhibits H. pylori-induced pathogenesis. • H-P-6 suppresses H. pylori-induced hyper-proliferation and migration of AGS cells. • The peptide inhibits H. pylori-induced EGFR activation.

  12. The role of oestrogen receptor {alpha} in human thyroid cancer: contributions from coregulatory proteins and the tyrosine kinase receptor HER2.

    LENUS (Irish Health Repository)

    Kavanagh, Dara O

    2012-02-01

    Epidemiological, clinical, and molecular studies suggest a role for oestrogen in thyroid cancer. How oestrogen mediates its effects and the consequence of it on clinical outcome has not been fully elucidated. The participation of coregulatory proteins in modulating oestrogen receptor (ER) function and input of crosstalk with the tyrosine kinase receptor HER2 was investigated. Oestrogen induced cell proliferation in the follicular thyroid cancer (FTC)-133 cells, but not in the anaplastic 8305C cell line. Knockdown of the coactivator steroid receptor coactivator (SRC)-1 inhibited FTC-133 basal, but not oestrogen induced, cell proliferation. Oestrogen also increased protein expression of SRC-1 and the ER target gene cyclin D1 in the FTC-133 cell line. ERalpha, ERbeta, the coregulatory proteins SRC-1 and nuclear corepressor (NCoR), and the tyrosine kinase receptor HER2 were localised by immunohistochemistry and immnofluorescence in paraffin-embedded tissue from thyroid tumour patients (n=111). ERalpha was colocalised with both SRC-1 and NCoR to the nuclei of the tumour epithelial cells. Expression of ERalpha and NCoR was found predominantly in non-anaplastic tumours and was significantly associated with well-differentiated tumours and reduced incidence of disease recurrence. In non-anaplastic tumours, HER2 was significantly associated with SRC-1, and these proteins were associated with poorly differentiated tumours, capsular invasion and disease recurrence. Totally, 87% of anaplastic tumours were positive for SRC-1. Kaplan-Meier estimates of disease-free survival indicated that in thyroid cancer, SRC-1 strongly correlates with reduced disease-free survival (P<0.001), whereas NCoR predicted increased survival (P<0.001). These data suggest opposing roles for the coregulators SRC-1 and NCoR in thyroid tumour progression.

  13. Efficacy of ponatinib against ABL tyrosine kinase inhibitor-resistant leukemia cells

    Energy Technology Data Exchange (ETDEWEB)

    Okabe, Seiichi, E-mail: okabe@tokyo-med.ac.jp; Tauchi, Tetsuzo; Tanaka, Yuko; Ohyashiki, Kazuma

    2013-06-07

    Highlights: •Efficacy of ponatinib against ABL tyrosine kinase inhibitor-resistant leukemia cells okabe et al. •Imatinib or nilotinib resistance was involved Src family kinase. •The BCR-ABL point mutation (E334V) was highly resistant to imatinib or nilotinib. •Ponatinib was a powerful strategy against imatinib or nilotinib resistant Ph-positive cells. -- Abstract: Because a substantial number of patients with chronic myeloid leukemia acquire resistance to ABL tyrosine kinase inhibitors (TKIs), their management remains a challenge. Ponatinib, also known as AP24534, is an oral multi-targeted TKI. Ponatinib is currently being investigated in a pivotal phase 2 clinical trial. In the present study, we analyzed the molecular and functional consequences of ponatinib against imatinib- or nilotinib-resistant (R) K562 and Ba/F3 cells. The proliferation of imatinib- or nilotinib-resistant K562 cells did not decrease after treatment with imatinib or nilotinib. Src family kinase Lyn was activated. Point mutation Ba/F3 cells (E334 V) were also highly resistant to imatinib and nilotinib. Treatment with ponatinib for 72 h inhibited the growth of imatinib- and nilotinib-resistant cells. The phosphorylation of BCR-ABL, Lyn, and Crk-L was reduced. This study demonstrates that ponatinib has an anti-leukemia effect by reducing ABL and Lyn kinase activity and this information may be of therapeutic relevance.

  14. Regulation of Platelet Derived Growth Factor Signaling by Leukocyte Common Antigen-related (LAR) Protein Tyrosine Phosphatase: A Quantitative Phosphoproteomics Study.

    Science.gov (United States)

    Sarhan, Adil R; Patel, Trushar R; Creese, Andrew J; Tomlinson, Michael G; Hellberg, Carina; Heath, John K; Hotchin, Neil A; Cunningham, Debbie L

    2016-06-01

    Intracellular signaling pathways are reliant on protein phosphorylation events that are controlled by a balance of kinase and phosphatase activity. Although kinases have been extensively studied, the role of phosphatases in controlling specific cell signaling pathways has been less so. Leukocyte common antigen-related protein (LAR) is a member of the LAR subfamily of receptor-like protein tyrosine phosphatases (RPTPs). LAR is known to regulate the activity of a number of receptor tyrosine kinases, including platelet-derived growth factor receptor (PDGFR). To gain insight into the signaling pathways regulated by LAR, including those that are PDGF-dependent, we have carried out the first systematic analysis of LAR-regulated signal transduction using SILAC-based quantitative proteomic and phosphoproteomic techniques. We haveanalyzed differential phosphorylation between wild-type mouse embryo fibroblasts (MEFs) and MEFs in which the LAR cytoplasmic phosphatase domains had been deleted (LARΔP), and found a significant change in abundance of phosphorylation on 270 phosphosites from 205 proteins because of the absence of the phosphatase domains of LAR. Further investigation of specific LAR-dependent phosphorylation sites and enriched biological processes reveal that LAR phosphatase activity impacts on a variety of cellular processes, most notably regulation of the actin cytoskeleton. Analysis of putative upstream kinases that may play an intermediary role between LAR and the identified LAR-dependent phosphorylation events has revealed a role for LAR in regulating mTOR and JNK signaling. PMID:27074791

  15. Surface-tuned electron transfer and electrocatalysis of hexameric tyrosine-coordinated heme protein.

    Science.gov (United States)

    Peng, Lei; Utesch, Tillmann; Yarman, Aysu; Jeoung, Jae-Hun; Steinborn, Silke; Dobbek, Holger; Mroginski, Maria Andrea; Tanne, Johannes; Wollenberger, Ulla; Scheller, Frieder W

    2015-05-11

    Molecular modeling, electrochemical methods, and quartz crystal microbalance were used to characterize immobilized hexameric tyrosine-coordinated heme protein (HTHP) on bare carbon or on gold electrodes modified with positively and negatively charged self-assembled monolayers (SAMs), respectively. HTHP binds to the positively charged surface but no direct electron transfer (DET) is found due to the long distance of the active sites from the electrode surfaces. At carboxyl-terminated surfaces, the neutrally charged bottom of HTHP can bind to the SAM. For this "disc" orientation all six hemes are close to the electrode and their direct electron transfer should be efficient. HTHP on all negatively charged SAMs showed a quasi-reversible redox behavior with rate constant ks values between 0.93 and 2.86 s(-1) and apparent formal potentials ${E{{0{^{\\prime }}\\hfill \\atop {\\rm app}\\hfill}}}$ between -131.1 and -249.1 mV. On the MUA/MU-modified electrode, the maximum surface concentration corresponds to a complete monolayer of the hexameric HTHP in the disc orientation. HTHP electrostatically immobilized on negatively charged SAMs shows electrocatalysis of peroxide reduction and enzymatic oxidation of NADH. PMID:25825040

  16. Molecular dynamics simulations of interaction between protein-tyrosine phosphatase 1B and a bidentate inhibitor

    Institute of Scientific and Technical Information of China (English)

    Gui-xia LIU; Jin-zhi TAN; Chun-ying NIU; Jian-hua SHEN; Xiao-min LUO; Xu SHEN; Kai-xian CHEN; Hua-liang JIANG

    2006-01-01

    Aim: To investigate the dynamic properties of protein-tyrosine phosphatase (PTP)1B and reveal the structural factors responsible for the high inhibitory potency and selectivity of the inhibitor SNA for PTP1B. Methods: We performed molecular dynamics (MD) simulations using a long time-scale for both PTP1B and PTP1B complexed with the inhibitor SNA, the most potent and selective PTP1B inhibitor reported to date. The trajectories were analyzed by using principal component analysis. Results: Trajectory analyses showed that upon binding the ligand, the flexibility of the entire PTP1B molecule decreases. The most notable change is the movement of the WPD-loop. Our simulation results also indicated that electrostatic interactions contribute more to PTP1B-SNA complex conformation than the van der Waals interactions, and that Lys41, Arg47, and Asp48 play important roles in determining the conformation of the inhibitor SNA and in the potency and selectivity of the inhibitor. Of these, Arg47 contributed most. These results were in agreement with previous experimental results. Conclusion: The information presented here suggests that potent and selective PTP1B inhibitors can be designed by targeting the surface residues, for example the region containing Lys41,Arg47, and Asp48, instead of the second phosphate binding site (besides the active phosphate binding site).

  17. Contribution of casein kinase 2 and spleen tyrosine kinase to CFTR trafficking and protein kinase A-induced activity.

    Science.gov (United States)

    Luz, Simão; Kongsuphol, Patthara; Mendes, Ana Isabel; Romeiras, Francisco; Sousa, Marisa; Schreiber, Rainer; Matos, Paulo; Jordan, Peter; Mehta, Anil; Amaral, Margarida D; Kunzelmann, Karl; Farinha, Carlos M

    2011-11-01

    Previously, the pleiotropic "master kinase" casein kinase 2 (CK2) was shown to interact with CFTR, the protein responsible for cystic fibrosis (CF). Moreover, CK2 inhibition abolished CFTR conductance in cell-attached membrane patches, native epithelial ducts, and Xenopus oocytes. CFTR possesses two CK2 phosphorylation sites (S422 and T1471), with unclear impact on its processing and trafficking. Here, we investigated the effects of mutating these CK2 sites on CFTR abundance, maturation, and degradation coupled to effects on ion channel activity and surface expression. We report that CK2 inhibition significantly decreased processing of wild-type (wt) CFTR, with no effect on F508del CFTR. Eliminating phosphorylation at S422 and T1471 revealed antagonistic roles in CFTR trafficking: S422 activation versus T1471 inhibition, as evidenced by a severe trafficking defect for the T1471D mutant. Notably, mutation of Y512, a consensus sequence for the spleen tyrosine kinase (SYK) possibly acting in a CK2 context adjacent to the common CF-causing defect F508del, had a strong effect on both maturation and CFTR currents, allowing the identification of this kinase as a novel regulator of CFTR. These results reinforce the importance of CK2 and the S422 and T1471 residues for regulation of CFTR and uncover a novel regulation of CFTR by SYK, a recognized controller of inflammation.

  18. Identification of the tyrosine-protein phosphatase non-receptor type 2 as a rheumatoid arthritis susceptibility locus in europeans.

    Directory of Open Access Journals (Sweden)

    Joanna E Cobb

    Full Text Available OBJECTIVES: Genome-wide association studies have facilitated the identification of over 30 susceptibility loci for rheumatoid arthritis (RA. However, evidence for a number of potential susceptibility genes have not so far reached genome-wide significance in studies of Caucasian RA. METHODS: A cohort of 4286 RA patients from across Europe and 5642 population matched controls were genotyped for 25 SNPs, then combined in a meta-analysis with previously published data. RESULTS: Significant evidence of association was detected for nine SNPs within the European samples. When meta-analysed with previously published data, 21 SNPs were associated with RA susceptibility. Although SNPs in the PTPN2 gene were previously reported to be associated with RA in both Japanese and European populations, we show genome-wide evidence for a different SNP within this gene associated with RA susceptibility in an independent European population (rs7234029, P = 4.4×10(-9. CONCLUSIONS: This study provides further genome-wide evidence for the association of the PTPN2 locus (encoding the T cell protein tyrosine phosphastase with Caucasian RA susceptibility. This finding adds to the growing evidence for PTPN2 being a pan-autoimmune susceptibility gene.

  19. Identification of the Tyrosine-Protein Phosphatase Non-Receptor Type 2 as a Rheumatoid Arthritis Susceptibility Locus in Europeans

    Science.gov (United States)

    Cobb, Joanna E.; Plant, Darren; Flynn, Edward; Tadjeddine, Meriem; Dieudé, Philippe; Cornélis, François; Ärlestig, Lisbeth; Dahlqvist, Solbritt Rantapää; Goulielmos, George; Boumpas, Dimitrios T.; Sidiropoulos, Prodromos; Krintel, Sophine B.; Ørnbjerg, Lykke M.; Hetland, Merete L.; Klareskog, Lars; Haeupl, Thomas; Filer, Andrew; Buckley, Christopher D.; Raza, Karim; Witte, Torsten; Schmidt, Reinhold E.; FitzGerald, Oliver; Veale, Douglas; Eyre, Stephen; Worthington, Jane

    2013-01-01

    Objectives Genome-wide association studies have facilitated the identification of over 30 susceptibility loci for rheumatoid arthritis (RA). However, evidence for a number of potential susceptibility genes have not so far reached genome-wide significance in studies of Caucasian RA. Methods A cohort of 4286 RA patients from across Europe and 5642 population matched controls were genotyped for 25 SNPs, then combined in a meta-analysis with previously published data. Results Significant evidence of association was detected for nine SNPs within the European samples. When meta-analysed with previously published data, 21 SNPs were associated with RA susceptibility. Although SNPs in the PTPN2 gene were previously reported to be associated with RA in both Japanese and European populations, we show genome-wide evidence for a different SNP within this gene associated with RA susceptibility in an independent European population (rs7234029, P = 4.4×10−9). Conclusions This study provides further genome-wide evidence for the association of the PTPN2 locus (encoding the T cell protein tyrosine phosphastase) with Caucasian RA susceptibility. This finding adds to the growing evidence for PTPN2 being a pan-autoimmune susceptibility gene. PMID:23840476

  20. A rapid lateral flow immunoassay for the detection of tyrosine phosphatase-like protein IA-2 autoantibodies in human serum.

    Directory of Open Access Journals (Sweden)

    Ingrid Kikkas

    Full Text Available Type 1 diabetes (T1D results from the destruction of pancreatic insulin-producing beta cells and is strongly associated with the presence of islet autoantibodies. Autoantibodies to tyrosine phosphatase-like protein IA-2 (IA-2As are considered to be highly predictive markers of T1D. We developed a novel lateral flow immunoassay (LFIA based on a bridging format for the rapid detection of IA-2As in human serum samples. In this assay, one site of the IA-2As is bound to HA-tagged-IA-2, which is subsequently captured on the anti-HA-Tag antibody-coated test line on the strip. The other site of the IA-2As is bound to biotinylated IA-2, allowing the complex to be visualized using colloidal gold nanoparticle-conjugated streptavidin. For this study, 35 serum samples from T1D patients and 44 control sera from non-diabetic individuals were analyzed with our novel assay and the results were correlated with two IA-2A ELISAs. Among the 35 serum samples from T1D patients, the IA-2A LFIA, the in-house IA-2A ELISA and the commercial IA-2A ELISA identified as positive 21, 29 and 30 IA-2A-positive sera, respectively. The major advantages of the IA-2A LFIA are its rapidity and simplicity.

  1. Functional dissection of T. brucei Protein Tyrosine Phosphatase 1 and investigation of its development as a therapeutic target

    OpenAIRE

    Ruberto, Irene

    2011-01-01

    Trypanosoma brucei undergoes developmentally regulated morphological and biochemical changes during its life cycle, being transmitted between the mammalian host and the tsetse fly. It is generally recognized that cellular responses to environmental changes are mediated through signalling pathways, but our understanding of trypanosome signal transduction during differentiation is limited. Protein Tyrosine Phosphatase 1 (TbPTP1) is the one of the few factors identified to b...

  2. Identification of New Substrates of the Protein-tyrosine Phosphatase PTP1B by Bayesian Integration of Proteome Evidence*

    Science.gov (United States)

    Ferrari, Emanuela; Tinti, Michele; Costa, Stefano; Corallino, Salvatore; Nardozza, Aurelio Pio; Chatraryamontri, Andrew; Ceol, Arnaud; Cesareni, Gianni; Castagnoli, Luisa

    2011-01-01

    There is growing evidence that tyrosine phosphatases display an intrinsic enzymatic preference for the sequence context flanking the target phosphotyrosines. On the other hand, substrate selection in vivo is decisively guided by the enzyme-substrate connectivity in the protein interaction network. We describe here a system wide strategy to infer physiological substrates of protein-tyrosine phosphatases. Here we integrate, by a Bayesian model, proteome wide evidence about in vitro substrate preference, as determined by a novel high-density peptide chip technology, and “closeness” in the protein interaction network. This allows to rank candidate substrates of the human PTP1B phosphatase. Ultimately a variety of in vitro and in vivo approaches were used to verify the prediction that the tyrosine phosphorylation levels of five high-ranking substrates, PLC-γ1, Gab1, SHP2, EGFR, and SHP1, are indeed specifically modulated by PTP1B. In addition, we demonstrate that the PTP1B-mediated dephosphorylation of Gab1 negatively affects its EGF-induced association with the phosphatase SHP2. The dissociation of this signaling complex is accompanied by a decrease of ERK MAP kinase phosphorylation and activation. PMID:21123182

  3. IGF-IR tyrosine kinase interacts with NPM-ALK oncogene to induce survival of T-cell ALK+ anaplastic large-cell lymphoma cells.

    Science.gov (United States)

    Shi, Ping; Lai, Raymond; Lin, Quan; Iqbal, Abid S; Young, Leah C; Kwak, Larry W; Ford, Richard J; Amin, Hesham M

    2009-07-01

    Type I insulin-like growth factor receptor (IGF-IR) tyrosine kinase plays important roles in the pathogenesis of several malignancies. Although it promotes the growth of stimulated hematopoietic cells, a direct role of IGF-IR in malignant lymphoma has not been identified. Anaplastic lymphoma kinase-positive anaplastic large-cell lymphoma (ALK(+) ALCL) is a unique type of T-cell lymphoma. Approximately 85% of ALK(+) ALCL cases harbor the translocation t(2;5)(p23;q35), which generates the chimeric oncogene NPM-ALK. In the present study, we explored a possible role of IGF-IR in ALK(+) ALCL. Our results demonstrate that IGF-IR and IGF-I are widely expressed in ALK(+) ALCL cell lines and primary tumors. Importantly, we identified novel reciprocal functional interactions between IGF-IR and NPM-ALK. Antagonism of IGF-IR decreased the viability, induced apoptosis and cell-cycle arrest, and decreased proliferation and colony formation of ALK(+) ALCL cell lines. These effects could be explained by alterations of cell survival regulatory proteins downstream of IGF-IR signaling. Our findings improve current understanding of the biology of IGF-IR and NPM-ALK and have significant therapeutic implications as they identify IGF-IR signaling as a potential therapeutic target in ALK(+) ALCL and possibly other types of malignant lymphoma.

  4. The phosphorylated C-terminus of cAR1 plays a role in cell-type-specific gene expression and STATa tyrosine phosphorylation.

    Science.gov (United States)

    Briscoe, C; Moniakis, J; Kim, J Y; Brown, J M; Hereld, D; Devreotes, P N; Firtel, R A

    2001-05-01

    cAMP receptors mediate some signaling pathways via coupled heterotrimeric G proteins, while others are G-protein-independent. This latter class includes the activation of the transcription factors GBF and STATa. Within the cellular mounds formed by aggregation of Dictyostelium, micromolar levels of cAMP activate GBF function, thereby inducing the transcription of postaggregative genes and initiating multicellular differentiation. Activation of STATa, a regulator of culmination and ecmB expression, results from cAMP receptor-dependent tyrosine phosphorylation and nuclear localization, also in mound-stage cells. During mound development, the cAMP receptor cAR1 is in a low-affinity state and is phosphorylated on multiple serine residues in its C-terminus. This paper addresses possible roles of cAMP receptor phosphorylation in the cAMP-mediated stimulation of GBF activity, STATa tyrosine phosphorylation, and cell-type-specific gene expression. To accomplish this, we have expressed cAR1 mutants in a strain in which the endogenous cAMP receptors that mediate postaggregative gene expression in vivo are deleted. We then examined the ability of these cells to undergo morphogenesis and induce postaggregative and cell-type-specific gene expression and STATa tyrosine phosphorylation. Analysis of cAR1 mutants in which the C-terminal tail is deleted or the ligand-mediated phosphorylation sites are mutated suggests that the cAR1 C-terminus is not essential for GBF-mediated postaggregative gene expression or STATa tyrosine phosphorylation, but may play a role in regulating cell-type-specific gene expression and morphogenesis. A mutant receptor, in which the C-terminal tail is constitutively phosphorylated, exhibits constitutive activation of STATa tyrosine phosphorylation in pulsed cells in suspension and a significantly impaired ability to induce cell-type-specific gene expression. The constitutively phosphorylated receptor also exerts a partial dominant negative effect on

  5. NPM-ALK oncogenic tyrosine kinase controls T-cell identity by transcriptional regulation and epigenetic silencing in lymphoma cells.

    Science.gov (United States)

    Ambrogio, Chiara; Martinengo, Cinzia; Voena, Claudia; Tondat, Fabrizio; Riera, Ludovica; di Celle, Paola Francia; Inghirami, Giorgio; Chiarle, Roberto

    2009-11-15

    Transformed cells in lymphomas usually maintain the phenotype of the postulated normal lymphocyte from which they arise. By contrast, anaplastic large cell lymphoma (ALCL) is a T-cell lymphoma with aberrant phenotype because of the defective expression of the T-cell receptor and other T-cell-specific molecules for still undetermined mechanisms. The majority of ALCL carries the translocation t(2;5) that encodes for the oncogenic tyrosine kinase NPM-ALK, fundamental for survival, proliferation, and migration of transformed T cells. Here, we show that loss of T-cell-specific molecules in ALCL cases is broader than reported previously and involves most T-cell receptor-related signaling molecules, including CD3epsilon, ZAP70, LAT, and SLP76. We further show that NPM-ALK, but not the kinase-dead NPM-ALK(K210R), downregulated the expression of these molecules by a STAT3-mediated gene transcription regulation and/or epigenetic silencing because this downregulation was reverted by treating ALCL cells with 5-aza-2-deoxycytidine or by knocking down STAT3 through short hairpin RNA. Finally, NPM-ALK increased the methylation of ZAP70 intron 1-exon 2 boundary region, and both NPM-ALK and STAT3 regulated the expression levels of DNA methyltransferase 1 in transformed T cells. Thus, our data reveal that oncogene-deregulated tyrosine kinase activity controls the expression of molecules that determine T-cell identity and signaling.

  6. Nuclear localization of lymphocyte-specific protein tyrosine kinase (Lck) and its role in regulating LIM domain only 2 (Lmo2) gene

    Energy Technology Data Exchange (ETDEWEB)

    Venkitachalam, Srividya; Chueh, Fu-Yu [Department of Microbiology and Immunology, H. M. Bligh Cancer Research Laboratories, Chicago Medical School, Rosalind Franklin University of Medicine and Science, North Chicago, IL 60064 (United States); Yu, Chao-Lan, E-mail: chaolan.yu@rosalindfranklin.edu [Department of Microbiology and Immunology, H. M. Bligh Cancer Research Laboratories, Chicago Medical School, Rosalind Franklin University of Medicine and Science, North Chicago, IL 60064 (United States)

    2012-01-20

    Highlights: Black-Right-Pointing-Pointer Lmo2 expression is elevated in Lck-transformed cells. Black-Right-Pointing-Pointer Both endogenous and exogenous Lck localize in the nucleus. Black-Right-Pointing-Pointer Nuclear Lck is active in Lck-transformed cells. Black-Right-Pointing-Pointer Lck binds to the promoter region of Lmo2 gene in vivo. Black-Right-Pointing-Pointer In contrast to JAK2, Lck does not increase histone H3 phosphorylation on Tyr 41. -- Abstract: LIM domain only protein 2 (Lmo2) is a transcription factor that plays a critical role in the development of T-acute lymphoblastic leukemia (T-ALL). A previous report established a link between Lmo2 expression and the nuclear presence of oncogenic Janus kinase 2 (JAK2), a non-receptor protein tyrosine kinase. The oncogenic JAK2 kinase phosphorylates histone H3 on Tyr 41 that leads to the relief of Lmo2 promoter repression and subsequent gene expression. Similar to JAK2, constitutive activation of lymphocyte-specific protein tyrosine kinase (Lck) has been implicated in lymphoid malignancies. However, it is not known whether oncogenic Lck regulates Lmo2 expression through a similar mechanism. We show here that Lmo2 expression is significantly elevated in T cell leukemia LSTRA overexpressing active Lck kinase and in HEK 293 cells expressing oncogenic Y505FLck kinase. Nuclear localization of active Lck kinase was confirmed in both Lck-transformed cells by subcellular fractionation and immunofluorescence microscopy. More importantly, in contrast to oncogenic JAK2, oncogenic Lck kinase does not result in significant increase in histone H3 phosphorylation on Tyr 41. Instead, chromatin immunoprecipitation experiment shows that oncogenic Y505FLck kinase binds to the Lmo2 promoter in vivo. This result raises the possibility that oncogenic Lck may activate Lmo2 promoter through direct interaction.

  7. Protein modification in the post-mating spermatophore of the signal crayfish Pacifastacus leniusculus: insight into the tyrosine phosphorylation in a non-motile spermatozoon.

    Science.gov (United States)

    Niksirat, Hamid; Vancová, Marie; Andersson, Liselotte; James, Peter; Kouba, Antonín; Kozák, Pavel

    2016-09-01

    After mating, spermatophores of signal crayfish are stored on the body of the female for a period before fertilization. This study compared the post-mating protein profile and pattern of protein tyrosine phosphorylation of the signal crayfish spermatophore to that of the freshly ejaculated spermatophore and found substantial differences. Two major bands of tyrosine-phosphorylated proteins of molecular weights 10 and 50kDa were observed in the freshly ejaculated spermatophore of the signal crayfish. While the tyrosine-phosphorylated protein band with molecular weight 10kDa was formed by protein(s) of similar pH, the band with molecular weight of 50kDa consisted of proteins of varying pH. In the post-mating spermatophore, the band with molecular weight of 50kDa was not detected, and an increase in the level of protein tyrosine phosphorylation was observed in the 10kDa band. The microtubular radial arms of the spermatozoon showed a positive reaction to an anti-tyrosine antibody conjugated with gold particles in both the freshly ejaculated and post-mating spermatophores. In conclusion, the male gamete of the signal crayfish undergoes molecular modification during post-mating storage on the body of the female including changes in the level of protein expression and protein tyrosine phosphorylation. Structural similarity of the radial arms in the crayfish immotile spermatozoon with flagellum, which is the main site of protein tyrosine phosphorylation in the mammalian motile spermatozoa, raises questions regarding evolution and function of such organelles across the animal kingdom that must be addressed in the future studies. PMID:27481552

  8. Receptor tyrosine phosphatase beta is expressed in the form of proteoglycan and binds to the extracellular matrix protein tenascin

    DEFF Research Database (Denmark)

    Barnea, G; Grumet, M; Milev, P;

    1994-01-01

    immunohistochemical studies indicated that both RPTP beta and the extracellular matrix protein tenascin are localized in similar regions of the central nervous system. We have performed co-aggregation assays with red and green Co-vaspheres coated with tenascin and 3F8 PG, respectively, showing that the extracellular...... domain of RPTP beta (3F8 PG) binds specifically to tenascin. The interaction between a receptor tyrosine phosphatase and an extracellular matrix protein may have a role in development of the mammalian central nervous system....

  9. Dark rearing maintains tyrosine hydroxylase expression in retinal amacrine cells following optic nerve transection

    Institute of Scientific and Technical Information of China (English)

    Wei Wan; Zhenghai Liu; Xiaosheng Wang; Xuegang Luo

    2012-01-01

    The present study examined changes in retinal tyrosine hydroxylase (TH) expression in rats having undergone optic nerve transection and housed under a normal day/night cycle or in the dark. The aim was to investigate the effects of amacrine cells on axonal regeneration in retinal ganglion cells and on the synapses that transmit visual signals. The results revealed that retinal TH expression gradually decreased following optic nerve transection in rats housed under a normal day/night cycle, reaching a minimum at 5 days. In contrast, retinal TH expression decreased to a minimum at 1 day following optic nerve transection in dark reared rats, gradually increasing afterward and reaching a normal level at 5-7 days. The number of TH-positive synaptic particles correlated with the TH levels, indicating that dark rearing can help maintain TH expression during the synaptic degeneration stage (5-7 days after optic nerve injury) in retinal amacrine cells.

  10. Effect of Src Tyrosine Kinase Inhibition on Secretion of MMP-2 and MMP-9 by Non-small Cell Lung Cancer Cells

    Directory of Open Access Journals (Sweden)

    Rui ZHENG

    2011-01-01

    Full Text Available Background and objective Src tyrosine kinase and matrix metalloproteinase play the pivotal roles in lung cancer invasion and metastasis. The aim of this study is to evaluate the effect of Src tyrosine kinase inhibition on secretion of matrix metalloproteinase 2 (MMP-2 and matrix metalloproteinase 9 (MMP-9 by non-small cell lung cancer (NSCLC cells. Methods ELISA was used to examine the activity of MMP-2 and MMP-9 produced by NSCLC cells (PC14PE6, H226, PC-9, A549 as well as the effect of Src tyrosine kinase inhibition on secretion of MMP-2 and MMP-9 by NSCLC cells. Boyden chamber assay was used to assess the effect of Src tyrosine kinase inhibition on invasion of NSCLC cells in vitro. Results The levels of MMP-2 and MMP-9 in PC14PE6 and H226 cells were high, whereas the level of MMP-9 in A549 cell was low. MMP-2 and MMP-9 levels in PC-9 cell could not be detected. Src tyrosine kinase inhibitor obviously decreased the secretion of MMP-9 by PC14PE6, H226 and A549 cells, as well as MMP-2 by PC14PE6 cells in a dose-dependent manner. 10 μM Src tyrosine kinase inhibitor suppressed the secretion of MMP-9 by H226 and A549 cells, as wells as MMP-2 by PC14PE6 cells by more than 50%, while the same concentration of Src tyrosine kinase inhibitor almost had no effect on the level of MMP-2 in H226 cell. Invasiveness of NSCLC cells was suppressed by Src tyrosine kinase inhibitor in a dose-dependent manner, though there was minor difference in degree of the inhibition among four cell lines. 3 μM Src tyrosine kinase inhibitor suppressed the cell invasiveness of PC14PE6, H226, A549 and PC-9 cells by 79.1%, 68.09%, 90.96% and 96.98%, respectively (P < 0.001. Conclusion Inhibition of Src tyrosine kinase could suppress the invasion of NSCLC cells as well as the secretion of MMP-2 and MMP-9 by NSCLC cells in vitro. MMP-2 and MMP-9 were involved in regulating cell migration and invasion.

  11. Mandibular Jaw Bone Regeneration Using Human Dental Cell-Seeded Tyrosine-Derived Polycarbonate Scaffolds.

    Science.gov (United States)

    Zhang, Weibo; Zhang, Zheng; Chen, Shuang; Macri, Lauren; Kohn, Joachim; Yelick, Pamela C

    2016-07-01

    Here we present a new model for alveolar jaw bone regeneration, which uses human dental pulp cells (hDPCs) combined with tyrosine-derived polycarbonate polymer scaffolds [E1001(1k)] containing beta-tricalcium phosphate (β-TCP) [E1001(1k)/β-TCP]. E1001(1k)/β-TCP scaffolds (5 mm diameter × 1 mm thickness) were fabricated to fit a 5 mm rat mandibular ramus critical bone defect. Five experimental groups were examined in this study: (1) E1001(1k)/β-TCP scaffolds seeded with a high density of hDPCs, 5.0 × 10(5) hDPCs/scaffold (CH); (2) E1001(1k)/β-TCP scaffolds seeded with a lower density of hDPCs, 2.5 × 10(5) hDPCs/scaffold (CL); (3) acellular E1001(1k)/β-TCP scaffolds (SA); (4) acellular E1001(1k)/β-TCP scaffolds supplemented with 4 μg recombinant human bone morphogenetic protein-2 (BMP); and (5) empty defects (EDs). Replicate hDPC-seeded and acellular E1001(1k)/β-TCP scaffolds were cultured in vitro in osteogenic media for 1 week before implantation for 3 and 6 weeks. Live microcomputed tomography (μCT) imaging at 3 and 6 weeks postimplantation revealed robust bone regeneration in the BMP implant group. CH and CL groups exhibited similar uniformly distributed mineralized tissue coverage throughout the defects, but less than the BMP implants. In contrast, SA-treated defects exhibited sparse areas of mineralized tissue regeneration. The ED group exhibited slightly reduced defect size. Histological analyses revealed no indication of an immune response. In addition, robust expression of dentin and bone differentiation marker expression was observed in hDPC-seeded scaffolds, whereas, in contrast, BMP and SA implants exhibited only bone and not dentin differentiation marker expression. hDPCs were detected in 3-week but not in 6-week hDPC-seeded scaffold groups, indicating their survival for at least 3 weeks. Together, these results show that hDPC-seeded E1001(1k)/β-TCP scaffolds support the rapid regeneration of osteo

  12. Small tyrosine kinase inhibitors interrupt EGFR signaling by interacting with erbB3 and erbB4 in glioblastoma cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Carrasco-Garcia, Estefania; Saceda, Miguel [Instituto de Biologia Molecular y Celular, Universidad Miguel Hernandez, 03202 Elche (Alicante) (Spain); Unidad de Investigacion, Hospital General Universitario de Elche, 03203 Elche (Alicante) (Spain); Grasso, Silvina; Rocamora-Reverte, Lourdes; Conde, Mariano; Gomez-Martinez, Angeles [Instituto de Biologia Molecular y Celular, Universidad Miguel Hernandez, 03202 Elche (Alicante) (Spain); Garcia-Morales, Pilar [Instituto de Biologia Molecular y Celular, Universidad Miguel Hernandez, 03202 Elche (Alicante) (Spain); Unidad de Investigacion, Hospital General Universitario de Elche, 03203 Elche (Alicante) (Spain); Ferragut, Jose A. [Instituto de Biologia Molecular y Celular, Universidad Miguel Hernandez, 03202 Elche (Alicante) (Spain); Martinez-Lacaci, Isabel, E-mail: imlacaci@umh.es [Instituto de Biologia Molecular y Celular, Universidad Miguel Hernandez, 03202 Elche (Alicante) (Spain); Unidad AECC de Investigacion Traslacional en Cancer, Hospital Universitario Virgen de la Arrixaca, 30120 Murcia (Spain)

    2011-06-10

    Signaling through the epidermal growth factor receptor (EGFR) is relevant in glioblastoma. We have determined the effects of the EGFR inhibitor AG1478 in glioblastoma cell lines and found that U87 and LN-229 cells were very sensitive to this drug, since their proliferation diminished and underwent a marked G{sub 1} arrest. T98 cells were a little more refractory to growth inhibition and A172 cells did not undergo a G{sub 1} arrest. This G{sub 1} arrest was associated with up-regulation of p27{sup kip1}, whose protein turnover was stabilized. EGFR autophosphorylation was blocked with AG1478 to the same extent in all the cell lines. Other small-molecule EGFR tyrosine kinase inhibitors employed in the clinic, such as gefitinib, erlotinib and lapatinib, were able to abrogate proliferation of glioblastoma cell lines, which underwent a G{sub 1} arrest. However, the EGFR monoclonal antibody, cetuximab had no effect on cell proliferation and consistently, had no effect on cell cycle either. Similarly, cetuximab did not inhibit proliferation of U87 {Delta}EGFR cells or primary glioblastoma cell cultures, whereas small-molecule EGFR inhibitors did. Activity of downstream signaling molecules of EGFR such as Akt and especially ERK1/2 was interrupted with EGFR tyrosine kinase inhibitors, whereas cetuximab treatment could not sustain this blockade over time. Small-molecule EGFR inhibitors were able to prevent phosphorylation of erbB3 and erbB4, whereas cetuximab only hindered EGFR phosphorylation, suggesting that EGFR tyrosine kinase inhibitors may mediate their anti-proliferative effects through other erbB family members. We can conclude that small-molecule EGFR inhibitors may be a therapeutic approach for the treatment of glioblastoma patients.

  13. Protein tyrosine phosphatase is possibly involved in cellular signal transduction and the regulation of ABA accumulation in response to water deficit in Maize L. coleoptile

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    Water deficit-induced ABA accumulation is an ideal model or "stimulus-response" system to investigate cellular stress signaling in plant cells, using such a model the cellular stress signaling triggered by water deficit was investigated in Maize L. coleoptile. Water deficit-induced ABA accumulation was sensitively blocked by NaVO3, a potent inhibitor both to plasma membrane H+-ATPase (PM-H+- ATPase) and protein tyrosine phosphatase (PTPase). However, while PM- H+-ATPase activity was unaffected under water deficit and PM- H+-ATPase activator did not induce an ABA accumulation instead of water deficit, water deficit induced an increase in the protein phosphatase activity, and furthermore, ABA accumulation was inhibited by PAO, a specific inhibitor of PTPase. These results indicate that protein phosphtases may be involved in the cellular signaling in response to water deficit. Further studies identified at least four species of protein phosphtase as assayed by using pNPP as substrate, among which one component was especially sensitive to NaVO3. The NaVO3-sensitive enzyme was purified and finally showed a protein band about 66 kD on SDS/PAGE. The purified enzyme showed a great activity to some specific PTPase substrates at pH 6.0. In addition to NaVO3, the enzyme was also sensitive to some other PTPase inhibitors such as Zn2+ and MO33+, but not to Ca2+ and Mg2+, indicating that it might be a protein tyrosine phosphatase. Interestingly, the purified enzyme could be deactivated by some reducing agent DTT, which was previously proved to be an inhibitor of water deficit-induced ABA accumulation. This result further proved that PTPase might be involved in the cellular signaling of ABA accumulation in response to water deficit.

  14. Apoptosis-related molecular differences for response to tyrosin kinase inhibitors in drug-sensitive and drug-resistant human bladder cancer cells

    Directory of Open Access Journals (Sweden)

    Jixia Li

    2013-01-01

    Full Text Available Context: The epidermal growth factor receptor (EGFR family is reportedly overexpressed in bladder cancer, and tyrosine kinaseinhibitors (TKIs have been suggested as treatment. Gefitinib is a selective inhibitor of the EGFR and lapatinib is a dual inhibitor of both the EGFR and HER2 (human EGFR type 2 receptor. Both compounds compete with the binding of adenosine triphosphate (ATP to the tyrosine kinase domain of the respective receptors to inhibit receptor autophosphorylation causing suppression of signal transduction. Unfortunately, resistance to these inhibitors is a major clinical problem. Aims: To compare the apoptosis signaling pathway(s induced by gefitinib and lapatinib, in UM-UC-5 (drug-sensitive and UM-UC-14 (drug-resistant bladder cancer cells and to identify molecular differences that might be useful predictors of their efficacy. Materials and Methods: Cell proliferation, cell cycle and apoptosis assay were used to detect the effect of TKIs on UM-UC-5 and UM-UC-14 cells. Molecular differences for response to TKIs were examined by protein array. Results: TKIs strongly inhibited cell proliferation and induced cell cycle G1 arrest and apoptosis in UM-UC-5 cells. Most notable apoptosis molecular differences included decreased claspin, trail, and survivin by TKIs in the sensitive cells. In contrast, TKIs had no effect on resistant cells. Conclusions: Claspin, trail, and survivin might be used to determine the sensitivity of bladder cancers to TKIs.

  15. Alr5068, a Low-Molecular-Weight protein tyrosine phosphatase, is involved in formation of the heterocysts polysaccharide layer in the cyanobacterium Anabaena sp. PCC 7120.

    Science.gov (United States)

    Tan, Hui; Wan, Shuang; Liu, Pi-Qiong; Wang, Li; Zhang, Cheng-Cai; Chen, Wen-Li

    2013-10-01

    The filamentous cyanobacterium Anabaena sp. PCC 7120 forms nitrogen-fixing heterocysts after deprivation of combined nitrogen. Under such conditions, vegetative cells provide heterocysts with photosynthate and receive fixed nitrogen from the latter. Heterocyst envelope contains a glycolipid layer and a polysaccharide layer to restrict the diffusion of oxygen into heterocysts. Low-Molecular-Weight protein tyrosine phosphatases (LMW-PTPs) are involved in the biosynthesis of exopolysaccharides in bacteria. Alr5068, a protein from Anabaena sp. PCC 7120, shows significant sequence similarity with LMW-PTPs. In this study we characterized the enzymatic properties of Alr5068 and showed that it can dephosphorylate several autophosphorylated tyrosine kinases (Alr2856, Alr3059 and All4432) of Anabaena sp. PCC 7120 in vitro. Several conserved residues among LMW-PTPs are shown to be essential for the phosphatase activity of Alr5068. Overexpression of alr5068 results in a strain unable to survive under diazotrophic conditions, with the formation of morphologically mature heterocysts detached from the filaments. Overexpression of an alr5068 allele that lost phosphatase activity led to the formation of heterocyst with an impaired polysaccharide layer. The alr5068 gene was upregulated after nitrogen step-down and its mutation affected the expression of hepA and hepC, two genes necessary for the formation of the heterocyst envelope polysaccharide (HEP) layer. Our results suggest that Alr5068 is associated with the production of HEP in Anabaena sp. PCC 7120.

  16. The alternative splice variant of protein tyrosine kinase 6 negatively regulates growth and enhances PTK6-mediated inhibition of β-catenin.

    Directory of Open Access Journals (Sweden)

    Patrick M Brauer

    Full Text Available Protein tyrosine kinase 6 (PTK6, also called breast tumor kinase (BRK, is expressed in epithelial cells of various tissues including the prostate. Previously it was shown that PTK6 is localized to epithelial cell nuclei in normal prostate, but becomes cytoplasmic in human prostate tumors. PTK6 is also primarily cytoplasmic in the PC3 prostate adenocarcinoma cell line. Sequencing revealed expression of wild type full-length PTK6 transcripts in addition to an alternative transcript lacking exon 2 in PC3 cells. The alternative transcript encodes a 134 amino acid protein, referred to here as ALT-PTK6, which shares the first 77 amino acid residues including the SH3 domain with full length PTK6. RT-PCR was used to show that ALT-PTK6 is coexpressed with full length PTK6 in established human prostate and colon cell lines, as well as in primary cell lines derived from human prostate tissue and tumors. Although interaction between full-length PTK6 and ALT-PTK6 was not detected, ALT-PTK6 associates with the known PTK6 substrates Sam68 and β-catenin in GST pull-down assays. Coexpression of PTK6 and ALT-PTK6 led to suppression of PTK6 activity and reduced association of PTK6 with tyrosine phosphorylated proteins. While ALT-PTK6 alone did not influence β-catenin/TCF transcriptional activity in a luciferase reporter assay, it enhanced PTK6-mediated inhibition of β-catenin/TCF transcription by promoting PTK6 nuclear functions. Ectopic expression of ALT-PTK6 led to reduced expression of the β-catenin/TCF targets Cyclin D1 and c-Myc in PC3 cells. Expression of tetracycline-inducible ALT-PTK6 blocked the proliferation and colony formation of PC3 cells. Our findings suggest that ALT-PTK6 is able to negatively regulate growth and modulate PTK6 activity, protein-protein associations and/or subcellular localization. Fully understanding functions of ALT-PTK6 and its impact on PTK6 signaling will be critical for development of therapeutic strategies that target PTK6

  17. Cardiac sodium channel Na(v)1.5 interacts with and is regulated by the protein tyrosine phosphatase PTPH1

    DEFF Research Database (Denmark)

    Jespersen, Thomas; Gavillet, Bruno; van Bemmelen, Miguel X;

    2006-01-01

    In order to identify proteins interacting with the cardiac voltage-gated sodium channel Na(v)1.5, we used the last 66 amino acids of the C-terminus of the channel as bait to screen a human cardiac cDNA library. We identified the protein tyrosine phosphatase PTPH1 as an interacting protein. Pull...

  18. Regulation of the EphA2 kinase by the low molecular weight tyrosine phosphatase induces transformation.

    Science.gov (United States)

    Kikawa, Keith D; Vidale, Derika R; Van Etten, Robert L; Kinch, Michael S

    2002-10-18

    Intracellular signaling by protein tyrosine phosphorylation is generally understood to govern many aspects of cellular behavior. The biological consequences of this signaling pathway are important because the levels of protein tyrosine phosphorylation are frequently elevated in cancer cells. In the classic paradigm, tyrosine kinases promote tumor cell growth, survival, and invasiveness, whereas tyrosine phosphatases negatively regulate these same behaviors. Here, we identify one particular tyrosine phosphatase, low molecular weight tyrosine phosphatase (LMW-PTP), which is frequently overexpressed in transformed cells. We also show that overexpression of LMW-PTP is sufficient to confer transformation upon non-transformed epithelial cells. Notably, we show that the EphA2 receptor tyrosine kinase is a prominent substrate for LMW-PTP and that the oncogenic activities of LMW-PTP result from altered EphA2 expression and function. These results suggest a role for LMW-PTP in transformation progression and link its oncogenic potential to EphA2.

  19. Targeting the MET receptor tyrosine kinase in non-small cell lung cancer: emerging role of tivantinib

    International Nuclear Information System (INIS)

    MET receptor tyrosine kinase and its natural ligand, hepatocyte growth factor, have been implicated in a variety of cancers, including non-small cell lung cancer (NSCLC). Mechanisms by which cellular deregulation of MET occurs include overexpression, genomic amplification, mutation, or alternative splicing. MET overexpression or activation is a known cause of acquired resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors in NSCLC. Inhibition of MET signaling in these EGFR tyrosine kinase inhibitor-resistant cells may potentially restore sensitivity to EGFR inhibitors. Tivantinib (ARQ 197), reported as a small-molecule MET inhibitor, has demonstrated antitumor activity in early clinical studies. This review focuses on MET and lung cancer, the clinical development of tivantinib, the clinical trials of tivantinib in NSCLC to date, its current/emerging role in the management of NSCLC, and future directions

  20. Hypoxia stimulates binding of a cytoplasmic protein to a pyrimidine-rich sequence in the 3'-untranslated region of rat tyrosine hydroxylase mRNA.

    Science.gov (United States)

    Czyzyk-Krzeska, M F; Dominski, Z; Kole, R; Millhorn, D E

    1994-04-01

    Reduced oxygen tension (hypoxia) induces a 3-fold increase in stability of mRNA for tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine synthesis, in the pheochromocytoma (PC12) clonal cell line. To investigate the possibility that RNA-protein interactions are involved in mediating this increase in stability, RNA gel shift assays were performed using different fragments of labeled TH mRNA and the S-100 fraction of PC12 cytoplasmic protein extracts. We identified a sequence within the 3'-untranslated region of TH mRNA that binds cytoplasmic protein. RNase T1 mapping revealed that the protein was bound to a 28 nucleotide long sequence that is located between bases 1551-1579 of TH mRNA. Moreover, protein binding to this fragment was prevented with an antisense oligonucleotide directed against bases 1551-1579 and subsequent RNase H digestion. This fragment of the 3'-untranslated region of TH mRNA is rich in pyrimidine nucleotides, and the binding of cytoplasmic protein to this fragment was reduced by competition with other polypyrimidine sequences including poly(C) but not poly(U) polymers. The binding of the protein to TH mRNA was increased when cytoplasmic proteins were extracted from PC12 cells exposed to hypoxia (5% O2) for 24 h. Electrophoresis of the UV cross-linked RNA-protein complex on SDS-polyacrylamide gel electrophoresis revealed a complex of 74 kDa. The potential role of this protein-TH mRNA interaction in regulation of TH mRNA stability during hypoxia is discussed. PMID:7908289

  1. Uptake mechanisms of L-3-[I-125]iodo-alpha-methyl-tyrosine in a human small-cell lung cancer cell line : comparison with L-1-[C-14]tyrosine

    NARCIS (Netherlands)

    Jager, PL; De Vries, EGE; Piers, DA; Timmer-Bosscha, H

    2001-01-01

    The radiolabelled amino acid analogue L-3-[I-125]iodo-alpha-methyl-tyrosine (IMT) is under evaluation in brain tumours, where it reflects amino acid transport activity, but is also taken up in many other tumour types. This study investigated the uptake mechanism of IMT in tumour cells not derived fr

  2. Improved Glucose Homeostasis in Mice with Muscle-Specific Deletion of Protein-Tyrosine Phosphatase 1B▿

    OpenAIRE

    Delibegovic, Mirela; Bence, Kendra K.; Mody, Nimesh; Hong, Eun-Gyoung; Ko, Hwi Jin; Jason K Kim; Kahn, Barbara B.; Neel, Benjamin G

    2007-01-01

    Obesity and type 2 diabetes are characterized by insulin resistance. Mice lacking the protein-tyrosine phosphatase PTP1B in all tissues are hypersensitive to insulin but also have diminished fat stores. Because adiposity affects insulin sensitivity, the extent to which PTP1B directly regulates glucose homeostasis has been unclear. We report that mice lacking PTP1B only in muscle have body weight and adiposity comparable to those of controls on either chow or a high-fat diet (HFD). Muscle trig...

  3. Protein tyrosine phosphatase controls breast cancer invasion through the expression of matrix metalloproteinase-9

    Directory of Open Access Journals (Sweden)

    Bo-Mi Hwang

    2013-11-01

    Full Text Available The expression of matrix metalloproteinases (MMPs produced bycancer cells has been associated with the high potential ofmetastasis in several human carcinomas, including breast cancer.Several pieces of evidence demonstrate that protein tyrosinephosphatases (PTP have functions that promote cell migrationand metastasis in breast cancer. We analyzed whether PTPinhibitor might control breast cancer invasion through MMPexpression. Herein, we investigate the effect of 4-hydroxy-3,3-dimethyl-2H benzo[g]indole-2,5(3H-dione (BVT948, anovel PTP inhibitor, on 12-O-tetradecanoyl phorbol-13-acetate(TPA-induced MMP-9 expression and cell invasion in MCF-7cells. The expression of MMP-9 and cell invasion increased afterTPA treatment, whereas TPA-induced MMP-9 expression and cellinvasion were decreased by BVT948 pretreatment. Also, BVT948suppressed NF-κB activation in TPA-treated MCF-7 cells.However, BVT948 didn’t block TPA-induced AP-1 activation inMCF-7 cells. Our results suggest that the PTP inhibitor blocksbreast cancer invasion via suppression of the expression ofMMP-9. [BMB Reports 2013; 46(11: 533-538

  4. Down-regulated expression of the protein-tyrosine phosphatase 1B (PTP1B) is associated with aggressive clinicopathologic features and poor prognosis in hepatocellular carcinoma

    International Nuclear Information System (INIS)

    Highlights: ► PTP1B protein showed decreased expression in 67.79% of the HCC patients. ► Low PTP1B expression predicts poor prognosis of HCC. ► Low PTP1B expression is correlated with expansion of OV6+ tumor-initiating cells. ► Down-regulation of PTP1B is associated with activation of Wnt/β-Catenin signaling. -- Abstract: The protein-tyrosine phosphatase 1B (PTP1B) is a classical non-transmembrane protein tyrosine phosphatase that plays a key role in metabolic signaling and can exert both tumor suppressing and tumor promoting effects in different cancers depending on the substrate involved and the cellular context. However, the expression level and function of PTP1B in hepatocellular carcinoma (HCC) remain unclear. In this study, PTP1B expression was detected by immunohistochemistry in normal liver tissue (n = 16) and hepatocellular carcinoma (n = 169). The correlations between PTP1B expression level and clinicopathologic features and patient survival were also analyzed. One hundred and eleven of 169 HCC patients (65.7%) had negative or low PTP1B expression in tumorous tissues, whereas normal tissues always expressed strong PTP1B. Decreased PTP1B expression was significantly associated with aggressive clinicopathologic features and poor prognosis. Immunohistochemistry also showed that low PTP1B expression level was correlated with high percentage of OV6+ tumor-initiating cells (T-ICs) and high frequency of nuclear β-Catenin expression in HCC specimens. Our findings demonstrate for the first time that the loss of inhibitory effect of PTP1B may contribute to progression and invasion of HCC through activation of Wnt/β-Catenin signaling and expansion of liver T-ICs. PTP1B may serve as a valuable prognostic biomarker and potential therapeutic target in HCC.

  5. The Tyrosine Kinome Dictates Breast Cancer Heterogeneity and Therapeutic Responsiveness.

    Science.gov (United States)

    Ha, Jacqueline R; Siegel, Peter M; Ursini-Siegel, Josie

    2016-09-01

    Phospho-tyrosine signaling networks control numerous biological processes including cellular differentiation, cell growth and survival, motility, and invasion. Aberrant regulation of the tyrosine kinome is a hallmark of malignancy and influences all stages of breast cancer progression, from initiation to the development of metastatic disease. The success of specific tyrosine kinase inhibitors strongly validates the clinical relevance of tyrosine phosphorylation networks in breast cancer pathology. However, a significant degree of redundancy exists within the tyrosine kinome. Numerous receptor and cytoplasmic tyrosine kinases converge on a core set of signaling regulators, including adaptor proteins and tyrosine phosphatases, to amplify pro-tumorigenic signal transduction pathways. Mutational activation, amplification, or overexpression of one or more components of the tyrosine kinome represents key contributing events responsible for the tumor heterogeneity that is observed in breast cancers. It is this molecular heterogeneity that has become the most significant barrier to durable clinical responses due to the development of therapeutic resistance. This review focuses on recent literature that supports a prominent role for specific components of the tyrosine kinome in the emergence of unique breast cancer subtypes and in shaping breast cancer plasticity, sensitivity to targeted therapies, and the eventual emergence of acquired resistance. J. Cell. Biochem. 117: 1971-1990, 2016. © 2016 Wiley Periodicals, Inc.

  6. The Tyrosine Kinome Dictates Breast Cancer Heterogeneity and Therapeutic Responsiveness.

    Science.gov (United States)

    Ha, Jacqueline R; Siegel, Peter M; Ursini-Siegel, Josie

    2016-09-01

    Phospho-tyrosine signaling networks control numerous biological processes including cellular differentiation, cell growth and survival, motility, and invasion. Aberrant regulation of the tyrosine kinome is a hallmark of malignancy and influences all stages of breast cancer progression, from initiation to the development of metastatic disease. The success of specific tyrosine kinase inhibitors strongly validates the clinical relevance of tyrosine phosphorylation networks in breast cancer pathology. However, a significant degree of redundancy exists within the tyrosine kinome. Numerous receptor and cytoplasmic tyrosine kinases converge on a core set of signaling regulators, including adaptor proteins and tyrosine phosphatases, to amplify pro-tumorigenic signal transduction pathways. Mutational activation, amplification, or overexpression of one or more components of the tyrosine kinome represents key contributing events responsible for the tumor heterogeneity that is observed in breast cancers. It is this molecular heterogeneity that has become the most significant barrier to durable clinical responses due to the development of therapeutic resistance. This review focuses on recent literature that supports a prominent role for specific components of the tyrosine kinome in the emergence of unique breast cancer subtypes and in shaping breast cancer plasticity, sensitivity to targeted therapies, and the eventual emergence of acquired resistance. J. Cell. Biochem. 117: 1971-1990, 2016. © 2016 Wiley Periodicals, Inc. PMID:27392311

  7. Molecular dynamics simulations of protein-tyrosine phosphatase 1B: II. Substrate-enzyme interactions and dynamics

    DEFF Research Database (Denmark)

    Peters, Günther H.j.; Frimurer, T. M.; Andersen, J. N.;

    2000-01-01

    for catalysis. Analysis of the individual enzyme-substrate interaction energies revealed that mainly electrostatic forces contribute to binding. Indeed, calculation of the electrostatic field of the enzyme reveals that only the field surrounding the binding pocket is positive, while the remaining protein......Molecular dynamics simulations of protein tyrosine phosphatase 1B (PTP1B) complexed with the phosphorylated peptide substrate DADEpYL and the free substrate have been conducted to investigate 1) the physical forces involved in substrate-protein interactions, 2) the importance of enzyme...... and substrate flexibility for binding, 3) the electrostatic properties of the enzyme, and 4) the contribution from solvation. The simulations were performed for 1 ns, using explicit water molecules. The last 700 ps of the trajectories was used for analysis determining enthalpic and entropic contributions...

  8. Regulation of embryonic neurotransmitter and tyrosine hydroxylase protein levels by ascorbic acid

    OpenAIRE

    Meredith, M. Elizabeth; May, James M.

    2013-01-01

    Scope: Ascorbic acid (ascorbate) is required to recycle tetrahydrobiopterin, which is necessary for neurotransmitter synthesis by the rate-limiting enzymes tyrosine and tryptophan hydroxylases. We sought to determine whether ascorbate might regulate embryonic brain cortex monoamine synthesis utilizing transgenic mouse models with varying intracellular ascorbate levels. Methods and Results: In embryos lacking the sodium-dependent vitamin C transporter 2 (SVCT2), very low levels of brain ascorb...

  9. Functional interaction between nonreceptor tyrosine kinase c-Abl and SR-Rich protein RBM39.

    Science.gov (United States)

    Mai, Sanyue; Qu, Xiuhua; Li, Ping; Ma, Qingjun; Liu, Xuan; Cao, Cheng

    2016-04-22

    RBM39, also known as splicing factor HCC1.4, acts as a transcriptional coactivator for the steroid nuclear receptors JUN/AP-1, ESR1/ER-α and ESR2/ER-β. RBM39 is involved in the regulation of the transcriptional responses of these steroid nuclear receptors and promotes transcriptional initiation. In this paper, we report that RBM39 interacts with the nonreceptor tyrosine kinase c-Abl. Both the Src homology (SH) 2 and SH3 domains of c-Abl interact with RBM39. The major tyrosine phosphorylation sites on RBM39 that are phosphorylated by c-Abl are Y95 and Y99, as demonstrated by liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) and mutational analysis. c-Abl was shown boost the transcriptional coactivation activity of RBM39 for ERα and PRβ in a tyrosine kinase-dependent manner. The results suggest that mammalian c-Abl plays an important role in steroid hormone receptor-mediated transcription by regulating RBM39.

  10. PROLACTIN-INDUCED TYROSINE PHOSPHORYLATION, ACTIVATION AND RECEPTOR ASSOCIATION OF FOCAL ADHESION KINASE (FAK) IN MAMMARY EPITHELIAL CELLS

    Science.gov (United States)

    Prolactin-Induced Tyrosine Phosphorylation, Activation and ReceptorAssociation of Focal Adhesion Kinase (FAK) in Mammary Epithelial Cells. Suzanne E. Fenton1 and Lewis G. Sheffield2. 1U.S. Environmental ProtectionAgency, MD-72, Research Triangle Park, NC 27711, and

  11. Tyrosine phosphorylation and protein degradation control the transcriptional activity of WRKY involved in benzylisoquinoline alkaloid biosynthesis

    Science.gov (United States)

    Yamada, Yasuyuki; Sato, Fumihiko

    2016-01-01

    Benzylisoquinoline alkaloids (BIQ) are among the most structurally diverse and pharmaceutically valuable secondary metabolites. A plant-specific WRKY-type transcription factor, CjWRKY1, was isolated from Coptis japonica and identified as a transcriptional activator of BIQ biosynthesis. However, the expression of CjWRKY1 gene alone was not sufficient for the activation of genes encoding biosynthetic enzymes. Here, we report the importance of post-translational regulation of CjWRKY1 in BIQ biosynthesis. First, we detected the differential accumulation of CjWRKY1 protein in two cell lines with similar CjWRKY1 gene expression but different levels of accumulated alkaloids. Further investigation of the WRKY protein identified the phosphorylation of the WRKYGQK core domain at Y115. The CjWRKYY115E phosphorylation-mimic mutant showed loss of nuclear localization, DNA-binding activity, and transactivation activity compared to wild-type CjWRKY1. Rapid degradation of the CjWRKY1 protein was also confirmed following treatment with inhibitors of the 26S proteasome and protease inhibitors. The existence of two independent degradation pathways as well as protein phosphorylation suggests the fine-tuning of CjWRKY1 activities is involved in the regulation of biosynthesis of BIQs. PMID:27552928

  12. The roles of the conserved tyrosine in the β2-α2 loop of the prion protein.

    Science.gov (United States)

    Huang, Danzhi; Caflisch, Amedeo

    2015-01-01

    Prions cause neurodegenerative diseases for which no cure exists. Despite decades of research activities the function of the prion protein (PrP) in mammalians is not known. Moreover, little is known on the molecular mechanisms of the self-assembly of the PrP from its monomeric state (cellular PrP, PrP(C)) to the multimeric state. The latter state includes the toxic species (scrapie PrP, PrP(Sc)) knowledge of which would facilitate the development of drugs against prion diseases. Here we analyze the role of a tyrosine residue (Y169) which is strictly conserved in mammalian PrPs. Nuclear magnetic resonance (NMR) spectroscopy studies of many mammalian PrP(C) proteins have provided evidence of a conformational equilibrium between a 3(10)-helical turn and a type I β turn conformation in the β2-α2 loop (residues 165-175). In vitro cell-free experiments of the seeded conversion of PrP(C) indicate that non-aromatic residues at position 169 reduce the formation of proteinase K-resistant PrP. Recent molecular dynamics (MD) simulations of monomeric PrP and several single-point mutants show that Y169 stabilizes the 3(10)-helical turn conformation more than single-point mutants at position 169 or residues in contact with it. In the 3(10)-helical turn conformation the hydrophobic and aggregation-prone segment 169-YSNQNNF-175 is buried and thus not-available for self-assembly. From the combined analysis of simulation and experimental results it emerges that Y169 is an aggregation gatekeeper with a twofold role. Mutations related to 3 human prion diseases are interpreted on the basis of the gatekeeper role in the monomeric state. Another potential role of the Y169 side chain is the stabilization of the ordered aggregates, i.e., reduction of frangibility of filamentous protofibrils and fibrils, which is likely to reduce the generation of toxic species.

  13. Combined effects of EGFR tyrosine kinase inhibitors and vATPase inhibitors in NSCLC cells

    Energy Technology Data Exchange (ETDEWEB)

    Jin, Hyeon-Ok [KIRAMS Radiation Biobank, Korea Institute of Radiological and Medical Sciences, 75 Nowon-ro, Nowon-gu, Seoul, 139–706 (Korea, Republic of); Hong, Sung-Eun [Division of Radiation Cancer Research, Korea Institute of Radiological and Medical Sciences, 75 Nowon-ro, Nowon-gu, Seoul, 139–706 (Korea, Republic of); Kim, Chang Soon [Department of Microbiological Engineering, Kon-Kuk University, 120 Neungdong-ro, Gwangjin-gu, Seoul, 143–701 (Korea, Republic of); Park, Jin-Ah; Kim, Jin-Hee; Kim, Ji-Young; Kim, Bora [KIRAMS Radiation Biobank, Korea Institute of Radiological and Medical Sciences, 75 Nowon-ro, Nowon-gu, Seoul, 139–706 (Korea, Republic of); Chang, Yoon Hwan; Hong, Seok-Il; Hong, Young Jun [Department of Laboratory Medicine, Korea Cancer Center Hospital, Korea Institute of Radiological and Medical Sciences, 75 Nowon-ro, Nowon-gu, Seoul, 139–706 (Korea, Republic of); Park, In-Chul, E-mail: parkic@kirams.re.kr [Division of Radiation Cancer Research, Korea Institute of Radiological and Medical Sciences, 75 Nowon-ro, Nowon-gu, Seoul, 139–706 (Korea, Republic of); Lee, Jin Kyung, E-mail: jklee@kirams.re.kr [KIRAMS Radiation Biobank, Korea Institute of Radiological and Medical Sciences, 75 Nowon-ro, Nowon-gu, Seoul, 139–706 (Korea, Republic of); Department of Laboratory Medicine, Korea Cancer Center Hospital, Korea Institute of Radiological and Medical Sciences, 75 Nowon-ro, Nowon-gu, Seoul, 139–706 (Korea, Republic of)

    2015-08-15

    Despite excellent initial clinical responses of non-small cell lung cancer (NSCLC) patients to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs), many patients eventually develop resistance. According to a recent report, vacuolar H + ATPase (vATPase) is overexpressed and is associated with chemotherapy drug resistance in NSCLC. We investigated the combined effects of EGFR TKIs and vATPase inhibitors and their underlying mechanisms in the regulation of NSCLC cell death. We found that combined treatment with EGFR TKIs (erlotinib, gefitinib, or lapatinib) and vATPase inhibitors (bafilomycin A1 or concanamycin A) enhanced synergistic cell death compared to treatments with each drug alone. Treatment with bafilomycin A1 or concanamycin A led to the induction of Bnip3 expression in an Hif-1α dependent manner. Knock-down of Hif-1α or Bnip3 by siRNA further enhanced cell death induced by bafilomycin A1, suggesting that Hif-1α/Bnip3 induction promoted resistance to cell death induced by the vATPase inhibitors. EGFR TKIs suppressed Hif-1α and Bnip3 expression induced by the vATPase inhibitors, suggesting that they enhanced the sensitivity of the cells to these inhibitors by decreasing Hif-1α/Bnip3 expression. Taken together, we conclude that EGFR TKIs enhance the sensitivity of NSCLC cells to vATPase inhibitors by decreasing Hif-1α/Bnip3 expression. We suggest that combined treatment with EGFR TKIs and vATPase inhibitors is potentially effective for the treatment of NSCLC. - Highlights: • Co-treatment with EGFR TKIs and vATPase inhibitors induces synergistic cell death • EGFR TKIs enhance cell sensitivity to vATPase inhibitors via Hif-1α downregulation • Co-treatment of these inhibitors is potentially effective for the treatment of NSCLC.

  14. Retroviral transfer of a human tyrosine hydroxylase cDNA in various cell lines: regulated release of dopamine in mouse anterior pituitary AtT-20 cells.

    OpenAIRE

    Horellou, P; Guibert, B; Leviel, V; Mallet, J

    1989-01-01

    Little is known about the molecular events mediating neurotransmitter release, a crucial step in synaptic transmission. In this paper, the biosynthesis and release of L-beta-3,4-dihydroxyphenylalanine (L-DOPA) and dopamine were analyzed in three heterologous cell lines after retroviral-mediated gene transfer of tyrosine hydroxylase (EC 1.14.16.2), the rate-limiting enzyme in catecholamine synthesis. A recombinant retrovirus encoding human tyrosine hydroxylase type I as well as neomycin-resist...

  15. Looking for a needle in a haystack: Cellular proteins that may interact with the tyrosine-based sorting signal of the TGEV S protein.

    Science.gov (United States)

    Trincone, Anna; Schwegmann-Weßels, Christel

    2015-04-16

    The spike protein S of transmissible gastroenteritis virus, an Alphacoronavirus, contains a tyrosine-based sorting signal that is responsible for ERGIC retention and may be important for a correct viral assembly process. To find out whether the S protein interacts with cellular proteins via this sorting signal, a pulldown assay with GST fusion proteins was performed. Filamin A has been identified as a putative interaction candidate. Immunofluorescence assays confirmed a co-localization between the TGEV S protein and filamin A. Further experiments have to be performed to prove a significant impact of filamin A on TGEV infection. Different approaches of several researchers for the identification of cellular interaction candidates relevant for coronavirus replication are summarized. These results may help in the future to identify the role of cellular proteins during coronavirus assembly at the ER-Golgi intermediate compartment. PMID:25481285

  16. Tyrosylprotein sulfotransferase-1 and tyrosine sulfation of chemokine receptor 4 are induced by Epstein-Barr virus encoded latent membrane protein 1 and associated with the metastatic potential of human nasopharyngeal carcinoma.

    Directory of Open Access Journals (Sweden)

    Juan Xu

    Full Text Available The latent membrane protein 1 (LMP1, which is encoded by the Epstein-Barr virus (EBV, is an important oncogenic protein that is closely related to carcinogenesis and metastasis of nasopharyngeal carcinoma (NPC, a prevalent cancer in China. We previously reported that the expression of the functional chemokine receptor CXCR4 is associated with human NPC metastasis. In this study, we show that LMP1 induces tyrosine sulfation of CXCR4 through tyrosylprotein sulfotransferase-1 (TPST-1, an enzyme that is responsible for catalysis of tyrosine sulfation in vivo, which is likely to contribute to the highly metastatic character of NPC. LMP1 could induce tyrosine sulfation of CXCR4 and its associated cell motility and invasiveness in a NPC cell culture model. In contrast, the expression of TPST-1 small interfering RNA reversed LMP1-induced tyrosine sulfation of CXCR4. LMP1 conveys signals through the epidermal growth factor receptor (EGFR pathway, and EGFR-targeted siRNA inhibited the induction of TPST-1 by LMP1. We used a ChIP assay to show that EGFR could bind to the TPST-1 promoter in vivo under the control of LMP1. A reporter gene assay indicated that the activity of the TPST-1 promoter could be suppressed by deleting the binding site between EGFR and TPST-1. Finally, in human NPC tissues, the expression of TPST-1 and LMP1 was directly correlated and clinically, the expression of TPST-1 was associated with metastasis. These results suggest the up-regulation of TPST-1 and tyrosine sulfation of CXCR4 by LMP1 might be a potential mechanism contributing to NPC metastasis.

  17. Coincident signals from GPCRs and receptor tyrosine kinases are uniquely transduced by PI3Kβ in myeloid cells.

    Science.gov (United States)

    Houslay, Daniel M; Anderson, Karen E; Chessa, Tamara; Kulkarni, Suhasini; Fritsch, Ralph; Downward, Julian; Backer, Jonathan M; Stephens, Len R; Hawkins, Phillip T

    2016-01-01

    Class I phosphoinositide 3-kinases (PI3Ks) catalyze production of the lipid messenger phosphatidylinositol 3,4,5-trisphosphate (PIP3), which plays a central role in a complex signaling network regulating cell growth, survival, and movement. This network is overactivated in cancer and inflammation, and there is interest in determining the PI3K catalytic subunit (p110α, p110β, p110γ, or p110δ) that should be targeted in different therapeutic contexts. Previous studies have defined unique regulatory inputs for p110β, including direct interaction with Gβγ subunits, Rac, and Rab5. We generated mice with knock-in mutations of p110β that selectively blocked the interaction with Gβγ and investigated its contribution to the PI3K isoform dependency of receptor tyrosine kinase (RTK) and G protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptor (GPCR) responses in primary macrophages and neutrophils. We discovered a unique role for p110β in supporting synergistic PIP3 formation in response to the coactivation of macrophages by macrophage colony-stimulating factor (M-CSF) and the complement protein C5a. In contrast, we found partially redundant roles for p110α, p110β, and p110δ downstream of M-CSF alone and a nonredundant role for p110γ downstream of C5a alone. This role for p110β completely depended on direct interaction with Gβγ, suggesting that p110β transduces GPCR signals in the context of coincident activation by an RTK. The p110β-Gβγ interaction was also required for neutrophils to generate reactive oxygen species in response to the Fcγ receptor-dependent recognition of immune complexes and for their β2 integrin-mediated adhesion to fibrinogen or poly-RGD+, directly implicating heterotrimeric G proteins in these two responses. PMID:27531651

  18. Plasiatine, an Unprecedented Indole–Phenylpropanoid Hybrid from Plantago asiatica as a Potent Activator of the Nonreceptor Protein Tyrosine Phosphatase Shp2

    Science.gov (United States)

    Gao, Zhong-Hua; Shi, Yi-Ming; Qiang, Zhe; Wang, Xia; Shang, Shan-Zhai; Yang, Yan; Du, Bao-Wen; Peng, Hui-Pan; Ji, Xu; Li, Honglin; Wang, Fei; Xiao, Wei-Lie

    2016-04-01

    Plasiatine (1), isolated from the seeds of Plantago asiatica, is an unprecedented indole analogue linked to a phenylpropanoid moiety via a carbon bond that builds up a novel heteromeric construction with a C19N2 scaffold. Its structure was determined by spectroscopic data and computational evidence. Notably, experimental assay demonstrated that 1 significantly enhanced the activity of the nonreceptor protein tyrosine phosphatase Shp2 in vitro in a concentration-dependent manner with an EC50 value of 0.97 μM, and activated phosphorylation of ERK, a known target of Shp2. Moreover, plasiatine (1) promoted hepatocellular HepG2 cells migration. Molecular docking suggested that plasiatine (1) binds to the catalytic cleft of Shp2. These results identified plasiatine (1) as the first small molecule Shp2 activator, and it warrants further investigation as a novel pharmaceutical tool to study the function of Shp2 in tumorigenesis.

  19. Plasiatine, an Unprecedented Indole-Phenylpropanoid Hybrid from Plantago asiatica as a Potent Activator of the Nonreceptor Protein Tyrosine Phosphatase Shp2.

    Science.gov (United States)

    Gao, Zhong-Hua; Shi, Yi-Ming; Qiang, Zhe; Wang, Xia; Shang, Shan-Zhai; Yang, Yan; Du, Bao-Wen; Peng, Hui-Pan; Ji, Xu; Li, Honglin; Wang, Fei; Xiao, Wei-Lie

    2016-01-01

    Plasiatine (1), isolated from the seeds of Plantago asiatica, is an unprecedented indole analogue linked to a phenylpropanoid moiety via a carbon bond that builds up a novel heteromeric construction with a C19N2 scaffold. Its structure was determined by spectroscopic data and computational evidence. Notably, experimental assay demonstrated that 1 significantly enhanced the activity of the nonreceptor protein tyrosine phosphatase Shp2 in vitro in a concentration-dependent manner with an EC50 value of 0.97 μM, and activated phosphorylation of ERK, a known target of Shp2. Moreover, plasiatine (1) promoted hepatocellular HepG2 cells migration. Molecular docking suggested that plasiatine (1) binds to the catalytic cleft of Shp2. These results identified plasiatine (1) as the first small molecule Shp2 activator, and it warrants further investigation as a novel pharmaceutical tool to study the function of Shp2 in tumorigenesis. PMID:27101899

  20. Cryopreservation-induced alterations in protein tyrosine phosphorylation of spermatozoa from different portions of the boar ejaculate.

    Science.gov (United States)

    Kumaresan, A; Siqueira, A P; Hossain, M S; Bergqvist, A S

    2011-12-01

    Previous studies have shown that boar sperm quality after cryopreservation differs depending on the ejaculate fraction used and that spermatozoa contained in the first 10mL (P1) of the sperm-rich fraction (SRF) show better cryosurvival than those in the SRF-P1. Since protein tyrosine phosphorylation (PTP) in spermatozoa is related with the tolerance of spermatozoa to frozen storage and cryocapacitation, we assessed the dynamics of cryopreservation-induced PTP and intracellular calcium ([Ca(2+)]i) in spermatozoa, using flow cytometry, from P1 and SRF-P1 of the boar ejaculate at different stages of cryopreservation. Sperm kinetics, assessed using a computer-assisted semen analyzer, did not differ between P1 and SRF-P1 during cryopreservation but the decrease in sperm velocity during cryopreservation was significant (Psemen. A higher (Pboar ejaculate. However at any given step during cryopreservation the percentage of spermatozoa with PTP was comparatively higher in SRF-P1 than P1. A 32kDa tyrosine phosphorylated protein, associated with capacitation, appeared after cooling suggesting that cooling induces capacitation-like changes in boar spermatozoa. In conclusion, the study has shown that the cryopreservation process induced PTP in spermatozoa and their proportions were similar between portions of SRF. PMID:21893053

  1. Effective Penetration of Cell-permeable Peptide Mimic of Tyrosine Residue 654 Domain of β-catenin into Human Renal Tubular Epithelial Cells

    Institute of Scientific and Technical Information of China (English)

    ZENG Rui; XU Gang; HAN Min; LIU Wei; LIU Xiaocheng

    2007-01-01

    Phosphorylation of β-catenin tyrosine residue 654 plays an important role in the epithelial to myofibroblast transition (EMT). Introducing mimic peptide of tyrosine residue 654 domain of β-catenin into cells may influence phosphorylation of β-catenin tyrosine residue 654. To deliver this mimic peptide into renal epithelial cells, we used penetratin as a vector, which is a novel cell perme-able peptide, to deliver hydrophilic molecules into cells. A tyrosine 654 residue domain mimic pep-tide of β-catenin (PM) with fused penetratin was constructed, purified and then detected for the pene-tration of the mimic peptide into human renal tubular epithelial cells (HK-2). The results showed that purified fusion mimic peptide could efficiently and rapidly translocate into human renal tubular epithelial cells. It is concluded that a cell-permeable peptides mimic of tyrosine residue 654 domain of β-catenin was successfully obtained, which may provide a useful reagent for interfering the human renal tubular epithelial-mesenchymal transition.

  2. Endocytosis of Receptor Tyrosine Kinases

    Science.gov (United States)

    Goh, Lai Kuan

    2013-01-01

    Endocytosis is the major regulator of signaling from receptor tyrosine kinases (RTKs). The canonical model of RTK endocytosis involves rapid internalization of an RTK activated by ligand binding at the cell surface and subsequent sorting of internalized ligand-RTK complexes to lysosomes for degradation. Activation of the intrinsic tyrosine kinase activity of RTKs results in autophosphorylation, which is mechanistically coupled to the recruitment of adaptor proteins and conjugation of ubiquitin to RTKs. Ubiquitination serves to mediate interactions of RTKs with sorting machineries both at the cell surface and on endosomes. The pathways and kinetics of RTK endocytic trafficking, molecular mechanisms underlying sorting processes, and examples of deviations from the standard trafficking itinerary in the RTK family are discussed in this work. PMID:23637288

  3. Design of a selective insulin receptor tyrosine kinase inhibitor and its effect on glucose uptake and metabolism in intact cells

    International Nuclear Information System (INIS)

    An inhibitor of the insulin receptor tyrosine kinase (IRTK), (hydroxy-2-napthalenylmethyl)phosphonic acid, was designed and synthesized and was shown to be an inhibitor of the biological effects of insulin in vitro. With a wheat germ purified human placental insulin receptor preparation, this compound inhibited the insulin-stimulated autophosphorylation of the 95-kDa β-subunit of the insulin receptor. The ability of the kinase to phosphorylate an exogenous peptide substrate, angiotensin II, was also inhibited. Half-maximal inhibition of basal and insulin-stimulated human placental IRTK activity was found at concentrations of 150 and 100 μM, respectively, with 2 mM angiotensin II as the peptide substrate. The inhibitor was found to be specific for tyrosine kinases over serine kinases and noncompetitive with ATP. The inhibitor was converted into various (acyloxy)methyl prodrugs in order to achieve permeability through cell membranes. These prodrugs inhibited insulin-stimulated autophosphorylation of the insulin receptor 95-kDa β-subunit in intact CHO cells transfected with human insulin receptor. Inhibition of insulin-stimulated glucose oxidation in isolated rat adipocytes and 2-deoxyglucose uptake into CHO cells was observed with these prodrugs. The data provide additional evidence for the involvement of the insulin receptor tyrosine kinase in the regulation of glucose uptake and metabolism. These results and additional data reported herein suggest that this class of prodrugs and inhibitors will be useful for modulating the activity of a variety of tyrosine kinases

  4. Eps15R is a tyrosine kinase substrate with characteristics of a docking protein possibly involved in coated pits-mediated internalization

    DEFF Research Database (Denmark)

    Coda, L; Salcini, A E; Confalonieri, S;

    1998-01-01

    , and 76 kDa were specifically recognized by anti-eps15R sera. The 125-kDa species is a bona fide product of the eps15R gene, whereas p108 and p76 are most likely products of alternative splicing events. Eps15R protein(s) are tyrosine-phosphorylated following epidermal growth factor receptor activation...

  5. Cross talk of tyrosine kinases with the DNA damage signaling pathways.

    Science.gov (United States)

    Mahajan, Kiran; Mahajan, Nupam P

    2015-12-15

    Tyrosine kinases respond to extracellular and intracellular cues by activating specific cellular signaling cascades to regulate cell cycle, growth, proliferation, differentiation and survival. Likewise, DNA damage response proteins (DDR) activated by DNA lesions or chromatin alterations recruit the DNA repair and cell cycle checkpoint machinery to restore genome integrity and cellular homeostasis. Several new examples have been uncovered in recent studies which reveal novel epigenetic and non-epigenetic mechanisms by which tyrosine kinases interact with DDR proteins to dictate cell fate, i.e. survival or apoptosis, following DNA damage. These studies reveal the ability of tyrosine kinases to directly regulate the activity of DNA repair and cell cycle check point proteins by tyrosine phosphorylation. In addition, tyrosine kinases epigenetically regulate DNA damage signaling pathways by modifying the core histones as well as chromatin modifiers at critical tyrosine residues. Thus, deregulated tyrosine kinase driven epigenomic alterations have profound implications in cancer, aging and genetic disorders. Consequently, targeting oncogenic tyrosine kinase induced epigenetic alterations has gained significant traction in overcoming cancer cell resistance to various therapies. This review discusses mechanisms by which tyrosine kinases interact with DDR pathways to regulate processes critical for maintaining genome integrity as well as clinical strategies for targeted cancer therapies. PMID:26546517

  6. Mechanism of c-Met and EGFR tyrosine kinase inhibitor resistance through epithelial mesenchymal transition in non-small cell lung cancer.

    Science.gov (United States)

    Rastogi, Ichwaku; Rajanna, Supriya; Webb, Andrew; Chhabra, Gagan; Foster, Brad; Webb, Brian; Puri, Neelu

    2016-09-01

    According to currently available estimates from Cancer Research UK, 14.1 million new lung cancer cases were diagnosed and a staggering 8.2 million people worldwide died from lung cancer in 2012. EGFR and c-Met are two tyrosine kinase receptors most commonly overexpressed or mutated in Non-small Cell Lung Cancer (NSCLC) resulting in increased proliferation and survival of lung cancer cells. Tyrosine kinase inhibitors (TKIs), such as erlotinib, approved by the FDA as first/second line therapy for NSCLC patients have limited clinical efficacy due to acquired resistance. In this manuscript, we investigate and discuss the role of epithelial mesenchymal transition (EMT) in the development of resistance against EGFR and c-Met TKIs in NSCLC. Our findings show that Zeb-1, a transcriptional repressor of E-Cadherin, is upregulated in TKI-resistant cells causing EMT. We observed that TKI-resistant cells have increased gene and protein expression of EMT related proteins such as Vimentin, N-Cadherin, β-Catenin and Zeb-1, while expression of E-Cadherin, an important cell adhesion molecule, was suppressed. We also confirmed that TKI-resistant cells display mesenchymal cell type morphology, and have upregulation of β-Catenin which may regulate expression of Zeb-1, a transcriptional repressor of E-Cadherin in TKI-resistant NSCLC cells. Finally, we show that down-regulating Zeb-1 by inducing miR-200a or β-Catenin siRNA can increase drug sensitivity of TKI-resistant cells. PMID:27396618

  7. Overexpression of Tyro3 receptor tyrosine kinase leads to the acquisition of taxol resistance in ovarian cancer cells.

    Science.gov (United States)

    Lee, Chuhee

    2015-07-01

    The majority of patients with ovarian cancer are diagnosed at the advanced stages (III/IV) and their 5-year-survival rate is relatively low. One of the major causes of the poor prognosis of ovarian cancer is the development of resistance to first-line chemotherapy, including platinum and taxol. Therefore, improvements in current understanding of chemoresistance is required for the successful treatment of ovarian cancer. In the present study, taxol-resistant ovarian cancer cells, SKOV3/TR, were established by exposing parental SKOV3 cells to increasing concentrations of taxol. . Briefly, cells were treated with 1.5 nM (for 4 weeks), 3 nM (for 4 weeks), 6 nM (for 5 weeks), 12 nM (for 5 weeks) and 24 nM taxol (for 8 weeks) over 6 months. The SKOV3/TR cells were found to be smaller in size and rounder in shape compared with their parental cells. Cell viability and colony formation assays demonstrated an increase in the population doubling time of the SKOV3/TR cells, indicating a reduction in the proliferative capacity of these cells. Reverse transcription-polymerase chain reaction and western blot analysis revealed that, among the TAM receptor tyrosine kinases (RTKs), the mRNA and protein expression levels of Tyro3 RTK were increased, while those of Axl and Mer RTK were decreased in the SKOV3/TR cells. In addition, restoration of the level of Tyro3 by transfecting Tyro3-specific small interfering RNA into the SKOV3/TR cells reduced the proliferative capacity of the cells, indicating that upregulation of the expression of Tyro3 in SKOV3/TR cells may promote survival in the presence of taxol, which eventually resulted in the acquisition of resistance upon taxol treatment. The present study subsequently found that, in the SKOV3/TR cells, the level of intracellular reactive oxygen species (ROS) was elevated, and antioxidant treatment with N-acetyl cysteine (NAC) exerted more profound antiproliferative effects compared with the parental cells. The western blot analysis

  8. Third generation tyrosine kinase inhibitors and their development in advanced renal cell carcinoma.

    Science.gov (United States)

    Bukowski, Ronald M

    2012-01-01

    Angiogenesis in general and the vascular endothelial growth factor (VEGF) signaling axis in particular is a validated target in renal cell carcinoma (RCC). Clear-cell carcinoma of the kidney is now recognized as a malignancy that is sensitive to inhibitors of the VEGF pathway. Treatment options for patients with metastatic renal cell carcinoma have evolved in dramatic fashion over the past 6 years, and a new paradigm has developed. The cytokines interferon-α and interleukin-2 were previously utilized for therapy, but since December 2005, six new agents have been approved in the United States for the treatment of advanced RCC. Two are tyrosine kinase inhibitors (TKI's) including sunitinib and recently pazopanib, and the multikinase inhibitor sorafenib. The current review examines the evolving data with the next generation of TKI's, axitinib and tivozanib being developed for the treatment of advanced RCC. These agents were synthesized to provide increased target specificity and enhanced target inhibition. The preclinical and clinical data are examined, an overview of the development of these TKI's is provided, and discussion plus speculation concerning their potential roles as RCC therapy is provided.

  9. Review : Third Generation Tyrosine Kinase Inhibitors and Their Development in Advanced Renal Cell Carcinoma

    Directory of Open Access Journals (Sweden)

    Ronald M Bukowski

    2012-02-01

    Full Text Available Angiogenesis in general and the VEGF signaling axis in particular is a validated target in renal cell carcinoma. Clear cell carcinoma of the kidney is now recognized as a malignancy that is sensitive to inhibitors of the vascular endothelial growth factor pathway. Treatment options for patients with metastatic renal cell carcinoma have evolved in dramatic fashion over the past six years, and a new paradigm has developed. The cytokines interferon-α and interleukin-2 were previously utilized for therapy, but since December 2005, six new agents have been approved in the United States for the treatment of advanced RCC. Three are tyrosine kinase inhibitors (TKI’s including sunitinib, sorafenib, and recently pazopanib. The current review examines the evolving data with the next generation of TKI’s, axitinib and tivozanib being developed for the treatment of advanced RCC. These agents were synthesized to provide increased target specificity and enhanced target inhibition. The preclinical and clinical data are examined, an overview of the development of these TKI’s is provided, and discussion plus speculation concerning their potential roles as RCC therapy is provided.

  10. The receptor tyrosine kinase inhibitor amuvatinib (MP470) sensitizes tumor cells to radio- and chemo-therapies in part by inhibiting homologous recombination

    International Nuclear Information System (INIS)

    Background and purpose: RAD51 is a key protein involved in homologous recombination (HR) and a potential target for radiation- and chemotherapies. Amuvatinib (formerly known as MP470) is a novel receptor tyrosine kinase inhibitor that targets c-KIT and PDGFRα and can sensitize tumor cells to ionizing radiation (IR). Here, we studied amuvatinib mechanism on RAD51 and functional HR. Materials and methods: Protein and RNA analyses, direct repeat green fluorescent protein (DR-GFP) assay and polysomal fractioning were used to measure HR efficiency and global translation in amuvatinib-treated H1299 lung carcinoma cells. Synergy of amuvatinib with IR or mitomycin c (MMC) was assessed by clonogenic survival assay. Results: Amuvaninib inhibited RAD51 protein expression and HR. This was associated with reduced ribosomal protein S6 phosphorylation and inhibition of global translation. Amuvatinib sensitized cells to IR and MMC, agents that are selectively toxic to HR-deficient cells. Conclusions: Amuvatinib is a promising agent that may be used to decrease tumor cell resistance. Our work suggests that this is associated with decreased RAD51 expression and function and supports the further study of amuvatinib in combination with chemotherapy and radiotherapy.

  11. In situ quantification of HER2–protein tyrosine kinase 6 (PTK6) protein–protein complexes in paraffin sections from breast cancer tissues

    Science.gov (United States)

    Aubele, M; Spears, M; Ludyga, N; Braselmann, H; Feuchtinger, A; Taylor, K J; Lindner, K; Auer, G; Stering, K; Höfler, H; Schmitt, M; Bartlett, J M S

    2010-01-01

    Background: Protein tyrosine kinase 6 (PTK6; breast tumour kinase) is overexpressed in up to 86% of the invasive breast cancers, and its association with the oncoprotein human epidermal growth factor receptor 2 (HER2) was shown in vitro by co-precipitation. Furthermore, expression of PTK6 in tumours is linked with the expression of HER2. Method and results: In this study, we used the proximity ligation assay (PLA) technique on formalin-fixed paraffin sections from eighty invasive breast carcinoma tissue specimens to locate PTK6–HER2 protein–protein complexes. Proximity ligation assay signals from protein complexes were assessed quantitatively, and expression levels showed a statistically significant association with tumour size (P=0.015) and course of the cancer disease (P=0.012). Conclusion: Protein tyrosine kinase 6 forms protein complexes with HER2 in primary breast cancer tissues, which can be visualised by use of the PLA technique. Human epidermal growth factor receptor 2–PTK6 complexes are of prognostic relevance. PMID:20700126

  12. Inhibition of the receptor tyrosine kinase ROR1 by anti-ROR1 monoclonal antibodies and siRNA induced apoptosis of melanoma cells.

    Directory of Open Access Journals (Sweden)

    Mohammad Hojjat-Farsangi

    Full Text Available The receptor tyrosine kinase (RTK ROR1 is overexpressed and of importance for the survival of various malignancies, including lung adenocarcinoma, breast cancer and chronic lymphocytic leukemia (CLL. There is limited information however on ROR1 in melanoma. In the present study we analysed in seven melanoma cell lines ROR1 expression and phosphorylation as well as the effects of anti-ROR1 monoclonal antibodies (mAbs and ROR1 suppressing siRNA on cell survival. ROR1 was overexpressed at the protein level to a varying degree and phosphorylated at tyrosine and serine residues. Three of our four self-produced anti-ROR1 mAbs (clones 3H9, 5F1 and 1A8 induced a significant direct apoptosis of the ESTDAB049, ESTDAB112, DFW and A375 cell lines as well as cell death in complement dependent cytotoxicity (CDC and antibody dependent cellular cytotoxicity (ADCC. The ESTDAB081 and 094 cell lines respectively were resistant to direct apoptosis of the four anti-ROR1 mAbs alone but not in CDC or ADCC. ROR1 siRNA transfection induced downregulation of ROR1 expression both at mRNA and protein levels proceeded by apoptosis of the melanoma cells (ESTDAB049, ESTDAB112, DFW and A375 including ESTDAB081, which was resistant to the direct apoptotic effect of the mAbs. The results indicate that ROR1 may play a role in the survival of melanoma cells. The surface expression of ROR1 on melanoma cells may support the notion that ROR1 might be a suitable target for mAb therapy.

  13. Cis and trans regulatory mechanisms control AP2-mediated B cell receptor endocytosis via select tyrosine-based motifs.

    Directory of Open Access Journals (Sweden)

    Kathleen Busman-Sahay

    Full Text Available Following antigen recognition, B cell receptor (BCR-mediated endocytosis is the first step of antigen processing and presentation to CD4+ T cells, a crucial component of the initiation and control of the humoral immune response. Despite this, the molecular mechanism of BCR internalization is poorly understood. Recently, studies of activated B cell-like diffuse large B cell lymphoma (ABC DLBCL have shown that mutations within the BCR subunit CD79b leads to increased BCR surface expression, suggesting that CD79b may control BCR internalization. Adaptor protein 2 (AP2 is the major mediator of receptor endocytosis via clathrin-coated pits. The BCR contains five putative AP2-binding YxxØ motifs, including four that are present within two immunoreceptor tyrosine-based activation motifs (ITAMs. Using a combination of in vitro and in situ approaches, we establish that the sole mediator of AP2-dependent BCR internalization is the membrane proximal ITAM YxxØ motif in CD79b, which is a major target of mutation in ABC DLBCL. In addition, we establish that BCR internalization can be regulated at a minimum of two different levels: regulation of YxxØ AP2 binding in cis by downstream ITAM-embedded DCSM and QTAT regulatory elements and regulation in trans by the partner cytoplasmic domain of the CD79 heterodimer. Beyond establishing the basic rules governing BCR internalization, these results illustrate an underappreciated role for ITAM residues in controlling clathrin-dependent endocytosis and highlight the complex mechanisms that control the activity of AP2 binding motifs in this receptor system.

  14. The viral G protein-coupled receptor ORF74 unmasks phospholipase C signaling of the receptor tyrosine kinase IGF-1R.

    Science.gov (United States)

    de Munnik, Sabrina M; van der Lee, Rosan; Velders, Daniëlle M; van Offenbeek, Jody; Smits-de Vries, Laura; Leurs, Rob; Smit, Martine J; Vischer, Henry F

    2016-06-01

    Kaposi's sarcoma-associated herpesvirus (KSHV) encodes the constitutively active G protein-coupled receptor ORF74, which is expressed on the surface of infected host cells and has been linked to the development of the angioproliferative tumor Kaposi's sarcoma. Furthermore, the insulin-like growth factor (IGF)-1 receptor, a receptor tyrosine kinase, also plays an essential role in Kaposi's sarcoma growth and survival. In this study we examined the effect of the constitutively active viral receptor ORF74 on human IGF-1R signaling. Constitutive and CXCL1-induced ORF74 signaling did not transactivate IGF-1R. In contrast, IGF-1 stimulated phospholipase C (PLC) activation in an ORF74-dependent manner without affecting chemokine binding to ORF74. Inhibition of constitutive ORF74 activity by mutagenesis or the inverse agonist CXCL10, or neutralizing IGF-1R with an antibody or silencing IGF-1R expression using siRNA inhibited PLC activation by IGF-1. Transactivation of ORF74 in response to IGF-1 occurred independently of Src, PI3K, and secreted ORF74 ligands. Furthermore, tyrosine residues in the carboxyl-terminus and intracellular loop 2 of ORF74 are not essential for IGF-1-induced PLC activation. Interestingly, PLC activation in response to IGF-1 is specific for ORF74 as IGF-1 was unable to activate PLC in cells expressing the constitutively active human cytomegalovirus (HCMV)-encoded GPCR US28. Interestingly, IGF-1 does not induce β-arrestin recruitment to ORF74. The proximity ligation assay revealed close proximity between ORF74 and IGF-1R on the cell surface, but a physical interaction was not confirmed by co-immunoprecipitation. Unmasking IGF-1R signaling to PLC in response to IGF-1 is a previously unrecognized action of ORF74. PMID:26931381

  15. Insulin Phosphorylates Tyrosine Residue 464 of Tub and Translocates Tubby into the Nucleus in HIRcB Cells

    OpenAIRE

    Kim, Jin Wook; Kim, Hyeon Soo; Kim, Sang Dae; Park, Jung Yul

    2014-01-01

    Background The tubby protein has a motif that might be relevant for its action in the insulin signaling pathway. Previous studies have indicated that tubby undergoes phosphorylation on tyrosine residues in response to several stimuli and is known to localize in the nucleus as well as in the plasma membrane. However, the relationship between phosphorylation and nuclear translocation is not well understood. Here, we report that insulin directly phosphorylates tubby, which translocates into the ...

  16. Effect of Src Tyrosine Kinase Inhibition on Secretion of MMP-2 and MMP-9 by Non-small Cell Lung Cancer Cells

    OpenAIRE

    ZHENG, Rui; Qin, Xiaosong; Li, Wenjie; Kang, Jian

    2011-01-01

    Background and objective Src tyrosine kinase and matrix metalloproteinase play the pivotal roles in lung cancer invasion and metastasis. The aim of this study is to evaluate the effect of Src tyrosine kinase inhibition on secretion of matrix metalloproteinase 2 (MMP-2) and matrix metalloproteinase 9 (MMP-9) by non-small cell lung cancer (NSCLC) cells. Methods ELISA was used to examine the activity of MMP-2 and MMP-9 produced by NSCLC cells (PC14PE6, H226, PC-9, A549) as well as the effect of ...

  17. Electroacupuncture Improves Insulin Resistance by Reducing Neuroprotein Y/Agouti-Related Protein Levels and Inhibiting Expression of Protein Tyrosine Phosphatase 1B in Diet-induced Obese Rats.

    Science.gov (United States)

    Liu, Xia; He, Jun-Feng; Qu, Ya-Ting; Liu, Zhi-Jun; Pu, Qing-Yang; Guo, Sheng-Tong; Du, Jia; Jiang, Peng-Fei

    2016-04-01

    Electroacupuncture (EA) has been shown to exert beneficial effects on obesity, but the mechanism is unclear. This study investigated the effects of EA on diet-induced obese (DIO) rats. Fifty male Sprague-Dawley rats were randomly divided into low-fat diet (LFD, 10 rats) and high-fat diet (HFD, 40 rats) groups. After the DIO models had been established, successful model rats were randomly divided into HFD, EA, and orlistat (OLST) groups. The EA group received EA at Zusanli (ST36) and Quchi (LI11) for 20 minutes once per day for 28 days. The OLST group was treated with orlistat by gavage. The body weight, homeostasis model assessment-insulin resistance index, adipocyte diameters, and neuroprotein Y/agouti-related protein and protein tyrosine phosphatase 1B levels were significantly lower in the EA group than in the HFD group. The rats of the OLST group showed watery stools and yellow hairs whereas those of the EA group had regular stools and sleek coats. The effect of EA on weight loss may be related to improved insulin resistance caused by changes in the adipocyte size and by reductions in the expressions of neuroprotein Y/agouti-related protein and protein tyrosine phosphatase 1B. This study indicates that EA may be a better method of alternative therapy for treating obesity and other metabolic diseases. PMID:27079226

  18. The prognostic significance of protein tyrosine phosphatase 4A2 in breast cancer

    Directory of Open Access Journals (Sweden)

    Zhao D

    2015-07-01

    Full Text Available Duanzheng Zhao,1 Libin Guo,2,* Henrique Neves,3,* Hiu-Fung Yuen,4 Shu-Dong Zhang,5 Cian M McCrudden,6 Qing Wen,5 Jin Zhang,2 Qi Zeng,4 Hang Fai Kwok,3,5,6 Yao Lin2 1College of Continuing Education, Nanjing University of Aeronautics and Astronautics, Nanjing, Jiangsu, People’s Republic of China; 2College of Life Sciences, Fujian Normal University, Fuzhou, Fujian, People’s Republic of China; 3Faculty of Health Sciences, University of Macau, Avenida de Universidade, Taipa, Macau Special Administrative Region, People’s Republic of China; 4Institute of Molecular and Cell Biology, Biopolis Drive, Proteos, Singapore; 5Center for Cancer Research and Cell Biology, 6School of Pharmacy, Queen’s University of Belfast, Belfast, UK *These authors have contributed equally to this work Abstract: Although PTP4A3 has been shown to be a very important factor in promoting cancer progression, the role of its close family member PTP4A2 is still largely unknown. Recent reports have shown contradicting results on the role of PTP4A2 in breast cancer progression. Considering this, we aimed to investigate the prognostic value of PTP4A2 in five independent breast cancer data sets (minimum 198 patients per cohort, totaling 1,124 patients in the Gene Expression Omnibus Database. We found that high expression of PTP4A2 was a favorable prognostic marker in all five independent breast cancer data sets, as well as in the combined cohort, with a hazard ratio of 0.68 (95% confidence interval =0.56–0.83; P<0.001. Low PTP4A2 expression was associated with estrogen receptor-negative tumors and tumors with higher histological grading; furthermore, low expression was inversely correlated with the expression of genes involved in proliferation, including MKI67 and the MCM gene family encoding the minichromosome maintenance proteins. These findings suggest that PTP4A2 may play a role in breast cancer progression by dysregulating cell proliferation. PTP4A2 expression was

  19. Impaired degradation followed by enhanced recycling of epidermal growth factor receptor caused by hypo-phosphorylation of tyrosine 1045 in RBE cells

    Directory of Open Access Journals (Sweden)

    Gui Anping

    2012-05-01

    Full Text Available Abstract Background Since cholangiocarcinoma has a poor prognosis, several epidermal growth factor receptor (EGFR-targeted therapies with antibody or small molecule inhibitor treatment have been proposed. However, their effect remains limited. The present study sought to understand the molecular genetic characteristics of cholangiocarcinoma related to EGFR, with emphasis on its degradation and recycling. Methods We evaluated EGFR expression and colocalization by immunoblotting and immunofluorescence, cell surface EGFR expression by fluorescence-activated cell sorting (FACS, and EGFR ubiquitination and protein binding by immunoprecipitation in the human cholangiocarcinoma RBE and immortalized cholangiocyte MMNK-1 cell lines. Monensin treatment and Rab11a depletion by siRNA were adopted for inhibition of EGFR recycling. Results Upon stimulation with EGF, ligand-induced EGFR degradation was impaired and the expression of phospho-tyrosine 1068 and phospho-p44/42 MAPK was sustained in RBE cells as compared with MMNK-1 cells. In RBE cells, the process of EGFR sorting for lysosomal degradation was blocked at the early endosome stage, and non-degradated EGFR was recycled to the cell surface. A disrupted association between EGFR and the E3 ubiquitin ligase c-Cbl, as well as hypo-phosphorylation of EGFR at tyrosine 1045 (Tyr1045, were also observed in RBE cells. Conclusion In RBE cells, up-regulation of EGFR Tyr1045 phosphorylation is a potentially useful molecular alteration in EGFR-targeted therapy. The combination of molecular-targeted therapy determined by the characteristics of individual EGFR phosphorylation events and EGFR recycling inhibition show promise in future treatments of cholangiocarcinoma.

  20. A conditional form of Bruton's tyrosine kinase is sufficient to activate multiple downstream signaling pathways via PLC Gamma 2 in B cells

    Directory of Open Access Journals (Sweden)

    Witte Owen N

    2001-06-01

    Full Text Available Abstract Background Bruton's tyrosine kinase (Btk is essential for B cell development and function. Mutations of Btk elicit X-linked agammaglobulinemia in humans and X-linked immunodeficiency in the mouse. Btk has been proposed to participate in B cell antigen receptor-induced signaling events leading to activation of phospholipase C-γ2 (PLCγ2 and calcium mobilization. However it is unclear whether Btk activation is alone sufficient for these signaling events, and whether Btk can activate additional pathways that do not involve PLCγ2. To address such issues we have generated Btk:ER, a conditionally active form of the kinase, and expressed it in the PLCγ2-deficient DT40 B cell line. Results Activation of Btk:ER was sufficient to induce multiple B cell signaling pathways in PLCγ2-sufficient DT40 cells. These included tyrosine phosphorylation of PLCγ2, mobilization of intracellular calcium, activation of extracellular signal-regulated kinase (ERK and c-Jun NH2-terminal kinase (JNK mitogen-activated protein kinase (MAPK pathways, and apoptosis. In DT40 B cells deficient for PLCγ2, Btk:ER activation failed to induce the signaling events described above with the consequence that the cells failed to undergo apoptosis. Conclusions These data suggest that Btk:ER regulates downstream signaling pathways primarily via PLCγ2 in B cells. While it is not known whether activated Btk:ER precisely mimics activated Btk, this conditional system will likely facilitate the dissection of the role of Btk and its family members in a variety of biological processes in many different cell types.

  1. Impaired degradation followed by enhanced recycling of epidermal growth factor receptor caused by hypo-phosphorylation of tyrosine 1045 in RBE cells

    International Nuclear Information System (INIS)

    Since cholangiocarcinoma has a poor prognosis, several epidermal growth factor receptor (EGFR)-targeted therapies with antibody or small molecule inhibitor treatment have been proposed. However, their effect remains limited. The present study sought to understand the molecular genetic characteristics of cholangiocarcinoma related to EGFR, with emphasis on its degradation and recycling. We evaluated EGFR expression and colocalization by immunoblotting and immunofluorescence, cell surface EGFR expression by fluorescence-activated cell sorting (FACS), and EGFR ubiquitination and protein binding by immunoprecipitation in the human cholangiocarcinoma RBE and immortalized cholangiocyte MMNK-1 cell lines. Monensin treatment and Rab11a depletion by siRNA were adopted for inhibition of EGFR recycling. Upon stimulation with EGF, ligand-induced EGFR degradation was impaired and the expression of phospho-tyrosine 1068 and phospho-p44/42 MAPK was sustained in RBE cells as compared with MMNK-1 cells. In RBE cells, the process of EGFR sorting for lysosomal degradation was blocked at the early endosome stage, and non-degradated EGFR was recycled to the cell surface. A disrupted association between EGFR and the E3 ubiquitin ligase c-Cbl, as well as hypo-phosphorylation of EGFR at tyrosine 1045 (Tyr1045), were also observed in RBE cells. In RBE cells, up-regulation of EGFR Tyr1045 phosphorylation is a potentially useful molecular alteration in EGFR-targeted therapy. The combination of molecular-targeted therapy determined by the characteristics of individual EGFR phosphorylation events and EGFR recycling inhibition show promise in future treatments of cholangiocarcinoma

  2. Verruculides A and B, two new protein tyrosine phosphatase 1B inhibitors from an Indonesian ascidian-derived Penicillium verruculosum.

    Science.gov (United States)

    Yamazaki, Hiroyuki; Nakayama, Wataru; Takahashi, Ohgi; Kirikoshi, Ryota; Izumikawa, Yuta; Iwasaki, Kohei; Toraiwa, Kengo; Ukai, Kazuyo; Rotinsulu, Henki; Wewengkang, Defny S; Sumilat, Deiske A; Mangindaan, Remy E P; Namikoshi, Michio

    2015-08-15

    Two new merosesquiterpenes, verruculides A (1) and B (2), were isolated from a culture broth of the Indonesian ascidian-derived Penicillium verruculosum TPU1311, together with three known congeners, chrodrimanins A (3), B (4), and H (5). The structures of 1 and 2 were assigned on the basis of their spectroscopic data (1D and 2D NMR, HRMS, UV, CD, and IR). Compound 2 had a linear sesquiterpene moiety and was considered to be the derivative of the biosynthetic precursor for 1 and 3-5. Compounds 1, 3, and 5 inhibited the activity of protein tyrosine phosphatase 1B (PTP1B) with IC50 values of 8.4, 8.5, and 14.9 μM, respectively. Compound 2 showed 40% inhibition at 23.1 μM, while 4 was not active at 20.7 μM.

  3. Protein tyrosine phosphatase 1B (PTP1B)-inhibiting constituents from the leaves of Syzygium polyanthum.

    Science.gov (United States)

    Saifudin, Azis; Tanaka, Ken; Kadota, Shigetoshi; Tezuka, Yasuhiro

    2012-08-01

    A methanol extract of the leaves of Syzygium polyanthum (Wight) Walp. afforded four new acylbenzene derivatives (1-4) together with seven known compounds (5-11). The structures of 1-11 were elucidated by extensive spectroscopic methods and comparison with the literature data. The new compounds 1-3 and a known compound, campest-4-en-3-one (10), exhibited a significant protein tyrosine phosphatase 1B inhibitory activity with IC₅₀ values of 13.1 ± 0.1, 5.77 ± 0.15, 4.01 ± 0.26, and 10.4 ± 0.5 µM, respectively. The inhibitory potency of the new compounds 2 and 3 was comparable to that of a positive control RK-682 (IC₅₀, 5.51 ± 0.04 µM). PMID:22763740

  4. Use of an Anaerobic Chamber Environment for the Assay of Endogenous Cellular Protein-Tyrosine Phosphatase Activities

    Directory of Open Access Journals (Sweden)

    Zhu Li

    2002-01-01

    Full Text Available Protein-tyrosine phosphatases (PTPases have a catalytic cysteine residue whose reduced state is integral to the reaction mechanism. Since exposure to air can artifactually oxidize this highly reactive thiol, PTPase assays have typically used potent reducing agents to reactivate the enzymes present; however, this approach does not allow for the measurement of the endogenous PTPase activity directly isolated from the in vivo cellular environment. Here we provide a method for using an anaerobic chamber to preserve the activity of the total PTPase complement in a tissue lysate or of an immunoprecipitated PTPase homolog to characterize their endogenous activation state. Comparison with a sample treated with biochemical reducing agents allows the determination of the activatable (reducible fraction of the endogenous PTPase pool.

  5. Sesquiterpene dimmer (DSF-27) inhibits the release of neuroinflammatory mediators from microglia by targeting spleen tyrosine kinase (Syk) and Janus kinase 2 (Jak2): Two major non-receptor tyrosine signaling proteins involved in inflammatory events

    Energy Technology Data Exchange (ETDEWEB)

    Zeng, Ke-Wu [State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University Health Science Center, Beijing 100191 (China); Wang, Shu [State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University Health Science Center, Beijing 100191 (China); Department of Medicinal Chemistry and Pharmaceutical Analysis, Logistics College of Chinese People' s Armed Police Forces, Tianjin 300162 (China); Dong, Xin; Jiang, Yong; Jin, Hong-Wei [State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University Health Science Center, Beijing 100191 (China); Tu, Peng-Fei, E-mail: pengfeitu@vip.163.com [State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University Health Science Center, Beijing 100191 (China)

    2014-03-15

    Non-receptor protein tyrosine kinases (NRPTKs)-dependent inflammatory signal transduction cascades play key roles in immunoregulation. However, drug intervention through NRPTKs-involved immunoregulation mechanism in microglia (the major immune cells of the central nervous system) has not been widely investigated. A main aim of the present study is to elucidate the contribution of two major NRPTKs (Syk and Jak2) in neuroinflammation suppression by a bioactive sesquiterpene dimmer (DSF-27). We found that LPS-stimulated BV-2 cells activated Syk and further initiated Akt/NF-κB inflammatory pathway. This Syk-dependent Akt/NF-κB inflammatory pathway can be effectively ameliorated by DSF-27. Moreover, Jak2 was activated by LPS, which was followed by transcriptional factor Stat3 activation. The Jak2/Stat3 signal was suppressed by DSF-27 through inhibition of Jak2 and Stat3 phosphorylation, promotion of Jak/Stat3 inhibitory factors PIAS3 expression, and down-regulation of ERK and p38 MAPK phosphorylation. Furthermore, DSF-27 protected cortical and mesencephalic dopaminergic neurons against neuroinflammatory injury. Taken together, our findings indicate NRPTK signaling pathways including Syk/NF-κB and Jak2/Stat3 cascades are potential anti-neuroinflammatory targets in microglia, and may also set the basis for the use of sesquiterpene dimmer as a therapeutic approach for neuroinflammation via interruption of these pathways. - Highlights: • Sesquiterpene dimmer DSF-27 inhibits inflammatory mediators' production in microglia. • Syk-dependent Akt/NF-κB pathway is important for DSF-27's anti-inflammation activity. • Jak2/Stat3 pathway is important for DSF-27's anti-inflammation activity. • Jak2/Stat3 signaling pathway is partly regulated by ERK and p38 MAPKs and PIAS3. • DSF-27 protects neurons against microglia-mediated neuroinflammatory injury.

  6. Intestinal Epithelial Cell Tyrosine Kinase 2 Transduces IL-22 Signals To Protect from Acute Colitis.

    Science.gov (United States)

    Hainzl, Eva; Stockinger, Silvia; Rauch, Isabella; Heider, Susanne; Berry, David; Lassnig, Caroline; Schwab, Clarissa; Rosebrock, Felix; Milinovich, Gabriel; Schlederer, Michaela; Wagner, Michael; Schleper, Christa; Loy, Alexander; Urich, Tim; Kenner, Lukas; Han, Xiaonan; Decker, Thomas; Strobl, Birgit; Müller, Mathias

    2015-11-15

    In the intestinal tract, IL-22 activates STAT3 to promote intestinal epithelial cell (IEC) homeostasis and tissue healing. The mechanism has remained obscure, but we demonstrate that IL-22 acts via tyrosine kinase 2 (Tyk2), a member of the Jak family. Using a mouse model for colitis, we show that Tyk2 deficiency is associated with an altered composition of the gut microbiota and exacerbates inflammatory bowel disease. Colitic Tyk2(-/-) mice have less p-STAT3 in colon tissue and their IECs proliferate less efficiently. Tyk2-deficient primary IECs show reduced p-STAT3 in response to IL-22 stimulation, and expression of IL-22-STAT3 target genes is reduced in IECs from healthy and colitic Tyk2(-/-) mice. Experiments with conditional Tyk2(-/-) mice reveal that IEC-specific depletion of Tyk2 aggravates colitis. Disease symptoms can be alleviated by administering high doses of rIL-22-Fc, indicating that Tyk2 deficiency can be rescued via the IL-22 receptor complex. The pivotal function of Tyk2 in IL-22-dependent colitis was confirmed in Citrobacter rodentium-induced disease. Thus, Tyk2 protects against acute colitis in part by amplifying inflammation-induced epithelial IL-22 signaling to STAT3. PMID:26432894

  7. Fusicoccin A, a Phytotoxic Carbotricyclic Diterpene Glucoside of Fungal Origin, Reduces Proliferation and Invasion of Glioblastoma Cells by Targeting Multiple Tyrosine Kinases1

    Science.gov (United States)

    Bury, Marina; Andolfi, Anna; Rogister, Bernard; Cimmino, Alessio; Mégalizzi, Véronique; Mathieu, Véronique; Feron, Olivier; Evidente, Antonio; Kiss, Robert

    2013-01-01

    Glioblastoma multiforme (GBM) is a deadly cancer that possesses an intrinsic resistance to pro-apoptotic insults, such as conventional chemotherapy and radiotherapy, and diffusely invades the brain parenchyma, which renders it elusive to total surgical resection. We found that fusicoccin A, a fungal metabolite from Fusicoccum amygdali, decreased the proliferation and migration of human GBM cell lines in vitro, including several cell lines that exhibit varying degrees of resistance to pro-apoptotic stimuli. The data demonstrate that fusicoccin A inhibits GBM cell proliferation by decreasing growth rates and increasing the duration of cell division and also decreases two-dimensional (measured by quantitative video microscopy) and three-dimensional (measured by Boyden chamber assays) migration. These effects of fusicoccin A treatment translated into structural changes in actin cytoskeletal organization and a loss of GBM cell adhesion. Therefore, fusicoccin A exerts cytostatic effects but low cytotoxic effects (as demonstrated by flow cytometry). These cytostatic effects can partly be explained by the fact that fusicoccin inhibits the activities of a dozen kinases, including focal adhesion kinase (FAK), that have been implicated in cell proliferation and migration. Overexpression of FAK, a nonreceptor protein tyrosine kinase, directly correlates with the invasive phenotype of aggressive human gliomas because FAK promotes cell proliferation and migration. Fusicoccin A led to the down-regulation of FAK tyrosine phosphorylation, which occurred in both normoxic and hypoxic GBM cell culture conditions. In conclusion, the current study identifies a novel compound that could be used as a chemical template for generating cytostatic compounds designed to combat GBM. PMID:23544164

  8. Site-specific Incorporation of 3-Iodo-L-tyrosine into Proteins and Single-wavelength Anomalous Dispersion Phasing with Soft X-ray in Protein Crystallography

    Science.gov (United States)

    Murayama, Kazutaka; Sakamoto, Kensaku

    Iodine is a good anomalous scatter for radiations from in-house X-ray generators (Cu/CrKα). Non-natural amino acid, 3-iodo-L-tyrosine, is able to be site-specifically incorporated into proteins with amber suppresser tRNA and mutated TyrRS from M. jannaschii in the E. coli expression system. To determine the crystal structure of acetyl transferase from T. thermophilus, iodotyrosine-containing proteins were prepared and crystallized. Structure determination was successfully conducted with the protein variant with iodotyrosine at position 111. Anomalous signals from iodotyrosine with Cu/CrKα radiations were both sufficient to calculate clear electron density map. In the crystal structure, iodotyrosine did not significantly disturb the native structure.

  9. A feasibility study on L-[1-carbon-11]tyrosine and L-[methyl-carbon-11]methionine to assess liver protein synthesis by PET

    NARCIS (Netherlands)

    Ishiwata, K; Enomoto, K; Elsinga, PH; Senda, M; Okazumi, SI; Isono, K; Paans, AMJ; Vaalburg, W

    1996-01-01

    We studied the potential of L-[1-C-11]tyrosine ([1-C-11]Tyr) and L-[methyl-C-11]methionine ([Me-C-11]Met) as tracers for measuring protein synthesis rate (PSR) in the liver by PET and proposed their metabolic models. Methods: In the liver and plasma of control and cycloheximide-treated mice injected

  10. SRC protein tyrosine kinase, c-Jun N-terminal kinase (JNK), and NF-kappaBp65 signaling in commercial and wild-type turkey leukocytes

    Science.gov (United States)

    Studies comparing signaling in wild-type turkey (WT) leukocytes and commercial turkey (CT) leukocytes found that the activity of protein tyrosine kinases (PTK) and MAP kinases, ERK 1/2 and p38, were significantly higher in WT leukocytes compared to CT lines upon exposure to both SE and OPSE on days...

  11. Stimulated regeneration of the crushed adult rat optic nerve correlates with attenuated expression of the protein tyrosine phosphatases RPTPalpha, STEP, and LAR.

    NARCIS (Netherlands)

    Lorber, B.; Berry, M.; Hendriks, W.J.A.J.; Hertog, J.F. den; Pulido, R.; Logan, A.

    2004-01-01

    We have evaluated the spatial and temporal expression patterns of three protein tyrosine phosphatases (PTPs), receptor PTPalpha (RPTPalpha), striatal enriched phosphatase (STEP), and leucocyte common antigen-related phosphatase (LAR), in the retina and optic nerve (ON) of adult rats in which the cru

  12. Upregulation of ICAM-1 Expression on J774.2 Macrophages by Endotoxin Involves Activation of NF-κB but not Protein Tyrosine Kinase: Comparison to Induction of iNOS

    Directory of Open Access Journals (Sweden)

    Hartmut Ruetten

    1999-01-01

    Full Text Available This study compares the signal transduction pathway which leads to the upregulation of intercellular adhesion molecule-1 (ICAM-1 expression with that of the increase in the expression of inducible nitric oxide synthase (iNOS protein and activity caused by endotoxin in cultured J774.2 macrophages. Treatment of J774.2 cells with lipopolysaccharide E. coli (LPS induced a concentration-dependent increase in the expression of ICAM-1 on the cell surface within 4 h and an increase in iNOS protein and activity at 24 h. The upregulation of ICAM-1 expression on J774.2 macrophages caused by LPS was significantly inhibited by pretreatment of the cells with inhibitors of the activation of the nuclear transcription factor NF-κB, such as L-1-tosylamido-2-phenylethylchloromethyl ketone (TPCK, pyrrolidine dithiocarbamate (PDTC, rotenone or calpain inhibitor I, but not by the tyrosine kinase inhibitors, tyrphostin AG126 or genistein. In contrast, genistein or tyrphostin AG126 also prevented the induction of iNOS protein and activity in J774.2 macrophages elicited by LPS. Thus, the increase in the expression of ICAM-1 on J774.2 macrophages by endotoxin involves the activation of NFκB, but not of protein tyrosine kinase.

  13. Reduced intensity conditioning is superior to nonmyeloablative conditioning for older chronic myelogenous leukemia patients undergoing hematopoietic cell transplant during the tyrosine kinase inhibitor era

    DEFF Research Database (Denmark)

    Warlick, Erica; Ahn, Kwang Woo; Pedersen, Tanya L;

    2012-01-01

    Tyrosine kinase inhibitors (TKIs) and reduced intensity conditioning (RIC)/nonmyeloablative (NMA) conditioning hematopoietic cell transplants (HCTs) have changed the therapeutic strategy for chronic myelogenous leukemia (CML) patients. We analyzed post-HCT outcomes of 306 CML patients reported to...

  14. Small molecule receptor tyrosine kinase inhibitor of platelet-derived growth factor signaling (SU9518) modifies radiation response in fibroblasts and endothelial cells

    International Nuclear Information System (INIS)

    Several small receptor tyrosine kinase inhibitors (RTKI) have entered clinical cancer trials alone and in combination with radiotherapy or chemotherapy. The inhibitory spectrum of these compounds is often not restricted to a single target. For example Imatinib/Gleevec (primarily a bcr/abl kinase inhibitor) or SU11248 (mainly a VEGFR inhibitor) are also potent inhibitors of PDGFR and other kinases. We showed previously that PDGF signaling inhibition attenuates radiation-induced lung fibrosis in a mouse model. Here we investigate effects of SU9518, a PDGFR inhibitor combined with ionizing radiation in human primary fibroblasts and endothelial cells in vitro, with a view on utilizing RTKI for antifibrotic therapy. Protein levels of PDGFR-α/-β and phosphorylated PDGFR in fibroblasts were analyzed using western and immunocytochemistry assays. Functional proliferation and clonogenic assays were performed (i) to assess PDGFR-mediated survival and proliferation in fibroblasts and endothelial cells after SU9518 (small molecule inhibitor of PDGF receptor tyrosine kinase); (ii) to test the potency und selectivity of the PDGF RTK inhibitor after stimulation with PDGF isoforms (-AB, -AA, -BB) and VEGF+bFGF. In order to simulate in vivo conditions and to understand the role of radiation-induced paracrine PDGF secretion, co-culture models consisting of fibroblasts and endothelial cells were employed. In fibroblasts, radiation markedly activated PDGF signaling as detected by enhanced PDGFR phosphorylation which was potently inhibited by SU9518. In fibroblast clonogenic assay, SU9518 reduced PDGF stimulated fibroblast survival by 57%. Likewise, SU9518 potently inhibited fibroblast and endothelial cell proliferation. In the co-culture model, radiation of endothelial cells and fibroblast cells substantially stimulated proliferation of non irradiated fibroblasts and vice versa. Importantly, the RTK inhibitor significantly inhibited this paracrine radiation-induced fibroblast and

  15. Small molecule receptor tyrosine kinase inhibitor of platelet-derived growth factor signaling (SU9518 modifies radiation response in fibroblasts and endothelial cells

    Directory of Open Access Journals (Sweden)

    Lipson Kenneth E

    2006-03-01

    Full Text Available Abstract Background Several small receptor tyrosine kinase inhibitors (RTKI have entered clinical cancer trials alone and in combination with radiotherapy or chemotherapy. The inhibitory spectrum of these compounds is often not restricted to a single target. For example Imatinib/Gleevec (primarily a bcr/abl kinase inhibitor or SU11248 (mainly a VEGFR inhibitor are also potent inhibitors of PDGFR and other kinases. We showed previously that PDGF signaling inhibition attenuates radiation-induced lung fibrosis in a mouse model. Here we investigate effects of SU9518, a PDGFR inhibitor combined with ionizing radiation in human primary fibroblasts and endothelial cells in vitro, with a view on utilizing RTKI for antifibrotic therapy. Methods Protein levels of PDGFR-α/-β and phosphorylated PDGFR in fibroblasts were analyzed using western and immunocytochemistry assays. Functional proliferation and clonogenic assays were performed (i to assess PDGFR-mediated survival and proliferation in fibroblasts and endothelial cells after SU9518 (small molecule inhibitor of PDGF receptor tyrosine kinase; (ii to test the potency und selectivity of the PDGF RTK inhibitor after stimulation with PDGF isoforms (-AB, -AA, -BB and VEGF+bFGF. In order to simulate in vivo conditions and to understand the role of radiation-induced paracrine PDGF secretion, co-culture models consisting of fibroblasts and endothelial cells were employed. Results In fibroblasts, radiation markedly activated PDGF signaling as detected by enhanced PDGFR phosphorylation which was potently inhibited by SU9518. In fibroblast clonogenic assay, SU9518 reduced PDGF stimulated fibroblast survival by 57%. Likewise, SU9518 potently inhibited fibroblast and endothelial cell proliferation. In the co-culture model, radiation of endothelial cells and fibroblast cells substantially stimulated proliferation of non irradiated fibroblasts and vice versa. Importantly, the RTK inhibitor significantly inhibited

  16. Sesamin Modulates Tyrosine Hydroxylase, Superoxide Dismutase, Catalase, Inducible No Synthase and Interleukin-6 Expression in Dopaminergic Cells Under Mpp+-Induced Oxidative Stress

    Directory of Open Access Journals (Sweden)

    Vicky Lahaie-Collins

    2008-01-01

    Full Text Available Oxidative stress is regarded as a mediator of nerve cell death in several neurodegenerative disorders, such as Parkinson's disease. Sesamin, a lignan mainly found in sesame oil, is currently under study for its anti-oxidative and possible neuroprotective properties. We used 1-methyl-4-phenyl-pyridine (MPP+ ion, the active metabolite of the potent parkinsonism-causing toxin 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine, to produce oxidative stress and neurodegeneration in neuronal PC12 cells, which express dopamine, as well as neurofilaments. Our results show that picomolar doses of sesamin protected neuronal PC12 cells from MPP+-induced cellular death, as revealed by colorimetric measurements and production of reactive oxygen species. We also demonstrated that sesamin acted by rescuing tyrosine hydroxylase levels from MPP+-induced depletion. Sesamin, however, did not modulate dopamine transporter levels, and estrogen receptor-alpha and -beta protein expression. By examining several parameters of cell distress, we found that sesamin also elicited a strong increase in superoxide dismutase activity as well as protein expression and decreased catalase activity and the MPP+ stimulated inducible nitric oxide synthase protein expression, in neuronal PC12 cells. Finally, sesamin possessed significant anti-inflammatory properties, as disclosed by its potential to reduce MPP+-induced interleukin-6 mRNA levels in microglia. From these studies, we determined the importance of the lignan sesamin as a neuroprotective molecule and its possible role in complementary and/or preventive therapies of neurodegenerative diseases.

  17. Cloning and Expression of Intracellular Part of Receptor Protein Tyrosine Phosphatase RPTPα and Preparation of Its Polyclonal Antibodies

    Institute of Scientific and Technical Information of China (English)

    CHEN Yang; YANG Su-juan; FU Yao; WANG Jia-peng; ZHAO Zhi-zhuang; FU Xue-qi

    2008-01-01

    A DNA fragment encoding the intracellular part of tyrosine phosphatase RPTPα designated as RPTPα-2D gene was amplified by PCR from a human prostate cDNA library and cloned into the pT7 E. coli expression vector. The resulting plasmid pT7-RPTPα-2D was used to transform Rosetta DE3 E. coli cells. RPTPα-2D was predominately expressed in the insoluble inclusion body and was effectively purified using preparative electrophoresis gels. Polyclonal antibodies were obtained after immunization of a rabbit with purified RPTPα-2D. The antibodies displayed a high titer and sensitivity. This study thus provided a valuable tool for further researches on RPTPa.

  18. Expression of the recepteur d'originenantais receptor tyrosine kinase in non-small cell lung cancer and its clinical significance

    Institute of Scientific and Technical Information of China (English)

    HAN Wei-li; LI Wei-dong; HU Jian; RUSIDANMU Aizemaiti; CHEN Ling-fang; SHEN Ling; ZHENG Shu-sen

    2012-01-01

    Background Recepteur d'originenantais (RON) is a receptor tyrosine kinase (RTK) that belongs to the MET proto-oncogene family.The aim of this study was to investigate the expression of RON receptor tyrosine kinase in human non-small cell lung cancer (NSCLC) and its relationship with clinical pathology of NSCLC and prognosis.Methods RON protein expression by immunohistochemistry (IHC) in 96 NSCLC specimens was evaluated and compared with the clinical pathology and prognosis,and 20 para-neoplastic tissues were included as controls.RON mRNA and protein expression in 25 fresh tissue samples of lung cancer and 10 normal lung tissues were also analyzed by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting.Results The rate of positive RON expression differed significantly between NSCLC tissues (55.2%,53/96) and para-neoplastic tissues (5%,1/20) (P <0.001).RON protein expression was not found to be associated with gender or age.However,RON expression positively correlated with clinical TNM stage (P=0.004),histological types (P=0.001),lymph node metastasis (P=0.012) and differentiation (P=0.035).RT-PCR and Western blotting analysis also confirmed that the expression of RON mRNA and protein was significantly increased in the NSCLC tissues versus normal tissues.In addition,RON expression was associated with a poor prognosis for patients with NSCLC (P=0.045).Conclus1ons The expression of RON protein and mRNA is significant in human NSCLC and low in para-neoplastic and normal tissues.Elevated RON expression may contribute to the occurrence,progression and metastasis of NSCLC,inferring that it could be useful as a new prognostic indicator for patients with NSCLC.

  19. Inactivation and unfolding of protein tyrosine phosphatase from Thermus thermophilus HB27 during urea and guanidine hydrochloride denaturation.

    Directory of Open Access Journals (Sweden)

    Yejing Wang

    Full Text Available The effects of urea and guanidine hydrochloride (GdnHCl on the activity, conformation and unfolding process of protein tyrosine phosphatase (PTPase, a thermostable low molecular weight protein from Thermus thermophilus HB27, have been studied. Enzymatic activity assays showed both urea and GdnHCl resulted in the inactivation of PTPase in a concentration and time-dependent manner. Inactivation kinetics analysis suggested that the inactivation of PTPase induced by urea and GdnHCl were both monophasic and reversible processes, and the effects of urea and GdnHCl on PTPase were similar to that of mixed-type reversible inhibitors. Far-ultraviolet (UV circular dichroism (CD, Tryptophan and 1-anilinonaphthalene -8-sulfonic acid (ANS fluorescence spectral analyses indicated the existence of a partially active and an inactive molten globule-like intermediate during the unfolding processes induced by urea and GdnHCl, respectively. Based on the sequence alignment and the homolog Tt1001 protein structure, we discussed the possible conformational transitions of PTPase induced by urea and GdnHCl and compared the conformations of these unfolding intermediates with the transient states in bovine PTPase and its complex structures in detail. Our results may be able to provide some valuable clues to reveal the relationship between the structure and enzymatic activity, and the unfolding pathway and mechanism of PTPase.

  20. Light-switched inhibitors of protein tyrosine phosphatase PTP1B based on phosphonocarbonyl phenylalanine as photoactive phosphotyrosine mimetic.

    Science.gov (United States)

    Wagner, Stefan; Schütz, Anja; Rademann, Jörg

    2015-06-15

    Phosphopeptide mimetics containing the 4-phosphonocarbonyl phenylalanine (pcF) as a photo-active phosphotyrosine isoster are developed as potent, light-switchable inhibitors of the protein tyrosine phosphatase PTP1B. The photo-active inhibitors 6-10 are derived from phosphopeptide substrates and are prepared from the suitably protected pcF building block 12 by Fmoc-based solid phase peptide synthesis. All pcF-containing peptides are moderate inhibitors of PTP1B with KI values between 10 and 50μM. Irradiation of the inhibitors at 365nm in the presence of the protein PTP1B amplify the inhibitory activity of pcF-peptides up to 120-fold, switching the KI values of the best inhibitors to the sub-micromolar range. Photo-activation of the inhibitors results in the formation of triplet intermediates of the benzoylphosphonate moiety, which deactivate PTP1B following an oxidative radical mechanism. Deactivation of PTP1B proceeds without covalent crosslinking of the protein target with the photo-switched inhibitors and can be reverted by subsequent addition of reducing agent dithiothreitol (DTT).

  1. Progress in protein tyrosine phosphates (PTPs) related to insulin signaling pathway%蛋白酪氨酸磷酸酶在胰岛素信号转导通路中的作用

    Institute of Scientific and Technical Information of China (English)

    董敏; 刘昭前

    2011-01-01

    蛋白酪氨酸磷酸酶(protein tyrosine phosphates,PTPs)是调节胰岛素信号转导的关键酶.PTPs通过对胰岛素受体和胰岛素受体底物蛋白磷酸化和去磷酸化调控胰岛素信号转导.PTPs抑制剂是潜在的治疗糖尿病和肥胖症的靶点药物,可以延长胰岛素信号的转导,加速葡萄糖的吸收,使血糖降低.本文主要概述PTPs在胰岛素信号转导通路中的作用及其作为新的治疗糖尿病药物靶点的研究进展.%Protein tyrosine phosphates(PTPs) are key regulators of the insulin receptor signal transduction pathway. Insulin signaling is tightly regulated by the balance of IR tyrosine phosphorylation and dephosphorylat-ing. Several PTPs expressed in the major human insulin target tissues or cells and could attenuate insulin action by dephosphorylating the IR. Inhibiting several PTPs would prolong insulin sig-naling and facilitate glucose uptake and decrease blood glucose. Inhibitors of several PTPs are predicted to be the novel drug targets for type 2 diabetes and obesity treatment.

  2. RTK SLAP down: the emerging role of Src-like adaptor protein as a key player in receptor tyrosine kinase signaling.

    Science.gov (United States)

    Wybenga-Groot, Leanne E; McGlade, C Jane

    2015-02-01

    SLAP (Src like adaptor protein) contains adjacent Src homology 3 (SH3) and Src homology 2 (SH2) domains closely related in sequence to that of cytoplasmic Src family tyrosine kinases. Expressed most abundantly in the immune system, SLAP function has been predominantly studied in the context of lymphocyte signaling, where it functions in the Cbl dependent downregulation of antigen receptor signaling. However, accumulating evidence suggests that SLAP plays a role in the regulation of a broad range of membrane receptors including members of the receptor tyrosine kinase (RTK) family. In this review we highlight the role of SLAP in the ubiquitin dependent regulation of type III RTKs PDGFR, CSF-1R, KIT and Flt3, as well as Eph family RTKs. SLAP appears to bind activated type III and Eph RTKs via a conserved autophosphorylated juxtamembrane tyrosine motif in an SH2-dependent manner, suggesting that SLAP is important in regulating RTK signaling.

  3. A receptor tyrosine kinase inhibitor, Tyrphostin A9 induces cancer cell death through Drp1 dependent mitochondria fragmentation

    International Nuclear Information System (INIS)

    Highlights: → We screened and identified Tyrphostin A9, a receptor tyrosine kinase inhibitor as a strong mitochondria fission inducer. → Tyrphostin A9 treatment promotes mitochondria dysfunction and contributes to cytotoxicity in cancer cells. → Tyrphostin A9 induces apoptotic cell death through a Drp1-mediated pathway. → Our studies suggest that Tyrphostin A9 induces mitochondria fragmentation and apoptotic cell death via Drp1 dependently. -- Abstract: Mitochondria dynamics controls not only their morphology but also functions of mitochondria. Therefore, an imbalance of the dynamics eventually leads to mitochondria disruption and cell death. To identify specific regulators of mitochondria dynamics, we screened a bioactive chemical compound library and selected Tyrphostin A9, a tyrosine kinase inhibitor, as a potent inducer of mitochondrial fission. Tyrphostin A9 treatment resulted in the formation of fragmented mitochondria filament. In addition, cellular ATP level was decreased and the mitochondrial membrane potential was collapsed in Tyr A9-treated cells. Suppression of Drp1 activity by siRNA or over-expression of a dominant negative mutant of Drp1 inhibited both mitochondrial fragmentation and cell death induced by Tyrpohotin A9. Moreover, treatment of Tyrphostin A9 also evoked mitochondrial fragmentation in other cells including the neuroblastomas. Taken together, these results suggest that Tyrphostin A9 induces Drp1-mediated mitochondrial fission and apoptotic cell death.

  4. Tyrosine phosphatase SHP-2 is a regulator of p27(Kip1) tyrosine phosphorylation.

    Science.gov (United States)

    Tossidou, Irini; Dangers, Marc; Koch, Alexandra; Brandt, Dominique T; Schiffer, Mario; Kardinal, Christian

    2008-12-15

    Tyrosine phosphorylation of the cell cycle regulator p27(Kip1) plays a crucial role in its binding to cyclin dependent kinases and its subcellular localization. While Src and Bcr-Abl were shown to be responsible for tyrosine phosphorylation, no data are available on the dephosphorylation of p27(Kip1) and the phosphatase involved. Considering the associated dephosphorylation as a pivotal event in the regulation of cell cycle proteins, we focused on the tyrosine phosphatase SHP-2, which is regulated in promyelocytic leukemia cells on G-CSF stimulation. SHP-2 was thus found in association with p27(Kip1) and the G-CSF receptor, and we observed a nuclear translocation of SHP-2 on G-CSF stimulation. Using a catalytically inactive form of SHP-2 and siRNA directed against SHP-2, we could demonstrate the involvement of SHP-2 in tyrosine dephosphorylation of p27(Kip1). Moreover, SHP-2 was strongly activated on G-CSF stimulation and specifically dephosphorylated p27(Kip1) in vitro. Most importantly, we could illustrate that SHP-2 modulates p27(Kip1) stability and contributes to p27(Kip1)-mediated cell cycle progression. Taken together, our results demonstrate that SHP-2 is a key regulator of p27(Kip1) tyrosine phosphorylation.

  5. Distinct cellular properties of oncogenic KIT receptor tyrosine kinase mutants enable alternative courses of cancer cell inhibition.

    Science.gov (United States)

    Shi, Xiarong; Sousa, Leiliane P; Mandel-Bausch, Elizabeth M; Tome, Francisco; Reshetnyak, Andrey V; Hadari, Yaron; Schlessinger, Joseph; Lax, Irit

    2016-08-16

    Large genomic sequencing analysis as part of precision medicine efforts revealed numerous activating mutations in receptor tyrosine kinases, including KIT. Unfortunately, a single approach is not effective for inhibiting cancer cells or treating cancers driven by all known oncogenic KIT mutants. Here, we show that each of the six major KIT oncogenic mutants exhibits different enzymatic, cellular, and dynamic properties and responds distinctly to different KIT inhibitors. One class of KIT mutants responded well to anti-KIT antibody treatment alone or in combination with a low dose of tyrosine kinase inhibitors (TKIs). A second class of KIT mutants, including a mutant resistant to imatinib treatment, responded well to a combination of TKI with anti-KIT antibodies or to anti-KIT toxin conjugates, respectively. We conclude that the preferred choice of precision medicine treatments for cancers driven by activated KIT and other RTKs may rely on clear understanding of the dynamic properties of oncogenic mutants. PMID:27482095

  6. {delta}-Opioid receptor-stimulated Akt signaling in neuroblastoma x glioma (NG108-15) hybrid cells involves receptor tyrosine kinase-mediated PI3K activation

    Energy Technology Data Exchange (ETDEWEB)

    Heiss, Anika; Ammer, Hermann [Institute of Pharmacology, Toxicology and Pharmacy Ludwig-Maximilians-University of Munich Koeniginstrasse 16 80539 Muenchen Federal Republic of Germany (Germany); Eisinger, Daniela A., E-mail: eisinger@pharmtox.vetmed.uni-muenchen.de [Institute of Pharmacology, Toxicology and Pharmacy Ludwig-Maximilians-University of Munich Koeniginstrasse 16 80539 Muenchen Federal Republic of Germany (Germany)

    2009-07-15

    {delta}-Opioid receptor (DOR) agonists possess cytoprotective properties, an effect associated with activation of the 'pro-survival' kinase Akt. Here we delineate the signal transduction pathway by which opioids induce Akt activation in neuroblastoma x glioma (NG108-15) hybrid cells. Exposure of the cells to both [D-Pen{sup 2,5}]enkephalin and etorphine resulted in a time- and dose-dependent increase in Akt activity, as measured by means of an activation-specific antibody recognizing phosphoserine-473. DOR-mediated Akt signaling is blocked by the opioid antagonist naloxone and involves inhibitory G{sub i/o} proteins, because pre-treatment with pertussis toxin, but not over-expression of the G{sub q/11} scavengers EBP50 and GRK2-K220R, prevented this effect. Further studies with Wortmannin and LY294002 revealed that phophoinositol-3-kinase (PI3K) plays a central role in opioid-induced Akt activation. Opioids stimulate Akt activity through transactivation of receptor tyrosine kinases (RTK), because pre-treatment of the cells with inhibitors for neurotrophin receptor tyrosine kinases (AG879) and the insulin-like growth factor receptor IGF-1 (AG1024), but not over-expression of the G{beta}{gamma} scavenger phosducin, abolished this effect. Activated Akt translocates to the nuclear membrane, where it promotes GSK3 phosphorylation and prevents caspase-3 cleavage, two key events mediating inhibition of cell apoptosis and enhancement of cell survival. Taken together, these results demonstrate that in NG108-15 hybrid cells DOR agonists possess cytoprotective properties mediated by activation of the RTK/PI3K/Akt signaling pathway.

  7. A mass spectrometric-derived cell surface protein atlas.

    Directory of Open Access Journals (Sweden)

    Damaris Bausch-Fluck

    Full Text Available Cell surface proteins are major targets of biomedical research due to their utility as cellular markers and their extracellular accessibility for pharmacological intervention. However, information about the cell surface protein repertoire (the surfaceome of individual cells is only sparsely available. Here, we applied the Cell Surface Capture (CSC technology to 41 human and 31 mouse cell types to generate a mass-spectrometry derived Cell Surface Protein Atlas (CSPA providing cellular surfaceome snapshots at high resolution. The CSPA is presented in form of an easy-to-navigate interactive database, a downloadable data matrix and with tools for targeted surfaceome rediscovery (http://wlab.ethz.ch/cspa. The cellular surfaceome snapshots of different cell types, including cancer cells, resulted in a combined dataset of 1492 human and 1296 mouse cell surface glycoproteins, providing experimental evidence for their cell surface expression on different cell types, including 136 G-protein coupled receptors and 75 membrane receptor tyrosine-protein kinases. Integrated analysis of the CSPA reveals that the concerted biological function of individual cell types is mainly guided by quantitative rather than qualitative surfaceome differences. The CSPA will be useful for the evaluation of drug targets, for the improved classification of cell types and for a better understanding of the surfaceome and its concerted biological functions in complex signaling microenvironments.

  8. A mass spectrometric-derived cell surface protein atlas.

    Science.gov (United States)

    Bausch-Fluck, Damaris; Hofmann, Andreas; Bock, Thomas; Frei, Andreas P; Cerciello, Ferdinando; Jacobs, Andrea; Moest, Hansjoerg; Omasits, Ulrich; Gundry, Rebekah L; Yoon, Charles; Schiess, Ralph; Schmidt, Alexander; Mirkowska, Paulina; Härtlová, Anetta; Van Eyk, Jennifer E; Bourquin, Jean-Pierre; Aebersold, Ruedi; Boheler, Kenneth R; Zandstra, Peter; Wollscheid, Bernd

    2015-01-01

    Cell surface proteins are major targets of biomedical research due to their utility as cellular markers and their extracellular accessibility for pharmacological intervention. However, information about the cell surface protein repertoire (the surfaceome) of individual cells is only sparsely available. Here, we applied the Cell Surface Capture (CSC) technology to 41 human and 31 mouse cell types to generate a mass-spectrometry derived Cell Surface Protein Atlas (CSPA) providing cellular surfaceome snapshots at high resolution. The CSPA is presented in form of an easy-to-navigate interactive database, a downloadable data matrix and with tools for targeted surfaceome rediscovery (http://wlab.ethz.ch/cspa). The cellular surfaceome snapshots of different cell types, including cancer cells, resulted in a combined dataset of 1492 human and 1296 mouse cell surface glycoproteins, providing experimental evidence for their cell surface expression on different cell types, including 136 G-protein coupled receptors and 75 membrane receptor tyrosine-protein kinases. Integrated analysis of the CSPA reveals that the concerted biological function of individual cell types is mainly guided by quantitative rather than qualitative surfaceome differences. The CSPA will be useful for the evaluation of drug targets, for the improved classification of cell types and for a better understanding of the surfaceome and its concerted biological functions in complex signaling microenvironments. PMID:25894527

  9. TetraMabs: simultaneous targeting of four oncogenic receptor tyrosine kinases for tumor growth inhibition in heterogeneous tumor cell populations

    Science.gov (United States)

    Castoldi, Raffaella; Schanzer, Jürgen; Panke, Christian; Jucknischke, Ute; Neubert, Natalie J.; Croasdale, Rebecca; Scheuer, Werner; Auer, Johannes; Klein, Christian; Niederfellner, Gerhard; Kobold, Sebastian; Sustmann, Claudio

    2016-01-01

    Monoclonal antibody-based targeted tumor therapy has greatly improved treatment options for patients. Antibodies against oncogenic receptor tyrosine kinases (RTKs), especially the ErbB receptor family, are prominent examples. However, long-term efficacy of such antibodies is limited by resistance mechanisms. Tumor evasion by a priori or acquired activation of other kinases is often causative for this phenomenon. These findings led to an increasing number of combination approaches either within a protein family, e.g. the ErbB family or by targeting RTKs of different phylogenetic origin like HER1 and cMet or HER1 and IGF1R. Progress in antibody engineering technology enabled generation of clinical grade bispecific antibodies (BsAbs) to design drugs inherently addressing such resistance mechanisms. Limited data are available on multi-specific antibodies targeting three or more RTKs. In the present study, we have evaluated the cloning, eukaryotic expression and purification of tetraspecific, tetravalent Fc-containing antibodies targeting HER3, cMet, HER1 and IGF1R. The antibodies are based on the combination of single-chain Fab and Fv fragments in an IgG1 antibody format enhanced by the knob-into-hole technology. They are non-agonistic and inhibit tumor cell growth comparable to the combination of four parental antibodies. Importantly, TetraMabs show improved apoptosis induction and tumor growth inhibition over individual monospecific or BsAbs in cellular assays. In addition, a mimicry assay to reflect heterogeneous expression of antigens in a tumor mass was established. With this novel in vitro assay, we can demonstrate the superiority of a tetraspecific antibody to bispecific tumor antigen-binding antibodies in early pre-clinical development. PMID:27578890

  10. Oncogenic activation of the Met receptor tyrosine kinase fusion protein, Tpr-Met, involves exclusion from the endocytic degradative pathway.

    Science.gov (United States)

    Mak, H H L; Peschard, P; Lin, T; Naujokas, M A; Zuo, D; Park, M

    2007-11-01

    Multiple mechanisms of dysregulation of receptor tyrosine kinases (RTKs) are observed in human cancers. In addition to gain-of-function, loss of negative regulation also contributes to oncogenic activation of RTKs. Negative regulation of many RTKs involves their internalization and degradation in the lysosome, a process regulated through ubiquitination. RTK oncoproteins activated following chromosomal translocation, are no longer transmembrane proteins, and are predicted to escape lysosomal degradation. To test this, we used the Tpr-Met oncogene, generated following chromosomal translocation of the hepatocyte growth factor receptor (Met). Unlike Met, Tpr-Met is localized in the cytoplasm and also lacks the binding site for Cbl ubiquitin ligases. We determined whether subcellular localization of Tpr-Met, and/or loss of its Cbl-binding site, is important for oncogenic activity. Presence of a Cbl-binding site and ubiquitination of cytosolic Tpr-Met oncoproteins does not alter their transforming activity. In contrast, plasma membrane targeting allows Tpr-Met to enter the endocytic pathway, and Tpr-Met transforming activity as well as protein stability are decreased in a Cbl-dependent manner. We show that transformation by Tpr-Met is in part dependent on its ability to escape normal downregulatory mechanisms. This provides a paradigm for many RTK oncoproteins activated following chromosomal translocation.

  11. Mapping of the receptor protein-tyrosine kinase 10 to human chromosome 1q21-q23 and mouse chromosome 1H1-5 by fluorescence in situ hybridization

    Energy Technology Data Exchange (ETDEWEB)

    Edelhoff, S.; Disteche, C.M. [Univ. of Washington School of Medicine, Seattle, WA (United States); Lai, C. [Scripps Research Inst., LaJolla, CA (United States)

    1995-01-01

    Receptor protein-tyrosine kinases (PTKs) play a critical role in the transduction of signals important to cell growth, differentiation, and survival. Mutations affecting the expression of receptor PTK genes have been associated with a number of vertebrate and invertebrate developmental abnormalities, and the aberrant regulation of tyrosine phosphorylation is implicated in a variety of neoplasias. One estimate suggests that approximately 100 receptor PTK genes exist in the mammalian genome, about half of which have been identified. The tyro-10 receptor protein-tyrosine kinase, first identified in a PCR-based survey for novel tyrosine kinases in the rat nervous system, defines a new subfamily of PTKs. It exhibits a catalytic domain most closely related to those found in the trk PTK receptor subfamily, which transduces signals for nerve growth factor and the related molecules brain-derived neurotrophic factor (BDNF), neurotrophin-3, and neurotrophin-4 (NT-3 and NT-4). Trk and the related PTK receptors trkB and trkC play a critical role in the neurotrophin-dependent survival of subsets of sensory and motor neurons. The predicted tyro-10 extracellular region is, however, distinct from that of the trk subfamily and is unique except for a domain shared with the blood coagulation factors V and VIII, thought to be involved in phospholipid binding. Although tyro-10 RNA is most abundant in heart and skeletal muscle in the adult rat, it is expressed in a wide variety of tissues, including the developing and mature brain. Tyro-10 appears identical to the murine TKT sequence reported by Karn et al. and exhibits a high degree of similarity with the CaK, DDR, and Nep PTKs. A ligand for tyro-10 has not yet been identified. 10 refs., 1 fig.

  12. Murine germinal center B cells require functional Fms-like tyrosine kinase 3 signaling for IgG1 class-switch recombination.

    Science.gov (United States)

    Svensson, Mattias N D; Andersson, Karin M E; Wasén, Caroline; Erlandsson, Malin C; Nurkkala-Karlsson, Merja; Jonsson, Ing-Marie; Brisslert, Mikael; Bemark, Mats; Bokarewa, Maria I

    2015-12-01

    Switched antibody classes are important for efficient immune responses. Aberrant antibody production to otherwise harmless antigens may result in autoimmunity. The protein kinase fms-like tyrosine kinase 3 receptor (Flt3) has an important role during early B-cell development, but the role of Flt3 in peripheral B cells has not been assessed before. Herein we describe a previously unappreciated role for Flt3 in IgG1 class-switch recombination (CSR) and production. We show that Flt3 is reexpressed on B-cell lymphoma 6(+) germinal center B cells in vivo and following LPS activation of peripheral B cells in vitro. Absence of Flt3 signaling in Flt3 ligand-deficient mice results in impaired IgG1 CSR and accumulation of IgM-secreting plasma cells. On activated B cells, Flt3 is coexpressed and functions in synergy with the common-gamma chain receptor family. B cells from Flt3 ligand-deficient mice have impaired IL-4R signaling, with reduced phosphorylation of signal transducer and activator of transcription (Stat) 6, and demonstrate a failure to initiate CSR to IgG1 with low expression of γ1 germ-line transcripts, resulting in impaired IgG1 production. Thus, functional synergy between Flt3 and IL-4R signaling is critical for Stat-mediated regulation of sterile γ1 germ-line transcripts and CSR to IgG1. PMID:26627255

  13. Imatinib-dependent tyrosine phosphorylation profiling of Bcr-Abl-positive chronic myeloid leukemia cells

    OpenAIRE

    Preisinger, C.; Schwarz, J. P.; Bleijerveld, O.B.; et al

    2012-01-01

    Bcr-Abl is the major cause and pathogenetic principle of chronic myeloid leukemia (CML). Bcr-Abl results from a chromosomal translocation that fuses the bcr and abl genes, thereby generating a constitutively active tyrosine kinase, which stimulates several signaling networks required for proliferation and survival. peer-reviewed

  14. 6-Phosphogluconate dehydrogenase regulates tumor cell migration in vitro by regulating receptor tyrosine kinase c-Met

    Energy Technology Data Exchange (ETDEWEB)

    Chan, Barden, E-mail: cchan@bidmc.harvard.edu [Division of Interdisciplinary Medicine and Biotechnology, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215 (United States); VanderLaan, Paul A. [Department of Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215 (United States); Sukhatme, Vikas P. [Division of Interdisciplinary Medicine and Biotechnology, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215 (United States)

    2013-09-20

    Highlights: •Expression of 6PGD positively correlates with advancing stage of lung carcinoma. •Knockdown of 6PGD by shRNA potently inhibits c-Met tyrosine phosphorylation. •Exogenous HGF fails to restore c-Met phosphorylation in cells with 6PGD knocked down. •6PGD knockdown results in inhibition of cell migration in vitro. •Constitutively active TPR-cMet significantly restores migration of cells without 6PGD. -- Abstract: 6-Phosphogluconate dehydrogenase (6PGD) is the third enzyme in the oxidative pentose phosphate pathway (PPP). Recently, we reported that knockdown of 6PGD inhibited lung tumor growth in vitro and in a xenograft model in mice. In this study, we continued to examine the functional role of 6PGD in cancer. We show that 6PGD expression positively correlates with advancing stage of lung carcinoma. In search of functional signals related to 6PGD, we discovered that knockdown of 6PGD significantly inhibited phosphorylation of c-Met at tyrosine residues known to be critical for activity. This downregulation of c-Met phosphorylation correlated with inhibition of cell migration in vitro. Overexpression of a constitutively active c-Met specifically rescued the migration but not proliferation phenotype of 6PGD knockdown. Therefore, 6PGD appears to be required for efficient c-Met signaling and migration of tumor cells in vitro.

  15. Functional characterization of protein-tyrosine phosphatases in zebrafish development using image analysis

    NARCIS (Netherlands)

    Runtuwene, Vincent Jimmy

    2012-01-01

    During gastrulation, the cells, and consequently the organ anlagen, are repositioned according to their future arrangement along the anterio- posterior axis. The movements responsible, the convergence and extension (CE) cell movements, are crucial for normal development and defects in their mechanis

  16. Screening and analysis of Chinese herbs with liver channel tropism acting on human liver cancer cell differential expressed protein tyrosine kinase%归肝经中药作用于人肝癌细胞差异表达 蛋白酪氨酸激酶的筛选与分析

    Institute of Scientific and Technical Information of China (English)

    田雪飞; 黄晓蒂; 周青; 王志琪; 田莎; 陈园

    2015-01-01

    PTKs差异表达在化瘀消癥药对组有17种,其中9种RTKs〔受体酪氨酸激酶样孤儿受体(ROR2)、干细胞因子受体(SCFR)、间变性淋巴瘤激酶(ALK)、血小板源性生长因子受体-β(PDGFR-β)、胰岛素样生长因子-IR(IGF-IR)、ErbB2、ErbB3、EphB1、EphA2〕,1种nrPTKs〔fps/fes相关酪氨酸激酶(FER)〕,7种PTKs,包括3种RTKs(M-CSFR、FGFR2-α、EphA3)和4种nrPTKs〔乙酸激酶1(ACK1)、布鲁顿酪氨酸蛋白激酶(Btk)、非受体酪氨酸激酶ABL1(ABL1)、BMX〕;在攻毒散结药对组有7种,包括5种RTKs(M-CSFR、FGFR1、ROR2、EphB1、ErbB2)和2种nrPTKs〔缺乏C端调节与N端烷基化位点的Src相关激酶(SRMS)、FER〕.结论 具有攻毒散结作用的蜈蚣和全蝎药对比具有化瘀消癥作用的三棱和莪术药有更好的抗肝癌作用,其机制可能是通过调节PTKs信号通路.归肝经活血化瘀药对三棱和莪术可能存在独立于PTKs信号系统外的抗肝癌效应途径.%Objective To screen the Chinese drugs with liver channel tropism acting on the characteristic differential expressed protein of human liver cancer cell protein tyrosine kinase (PTKs) system by protein chip technology, and to analyze the difference in different Chinese drugs with liver channel tropism in relation to PTKs regulation. Methods Forty BALB/C nude mice were chosen; a piece of subcutaneous tumor mass was implanted into the left lobe of liver parenchyma to reduplicate the orthotopically implanted tumor models. After modeling for 10 days, the tumorigenicity was confirmed, and all the nude mice models were divided into four groups; different Chinese medicine extracts were injected intra-peritoneally into corresponding treatment groups respectively, and the methods of treatment in the 4 groups were as follows: In liver channel tropism drug pair of Huayu Xiaozhen group, rhizoma sparganii and curcuma zedoary with dosage containing crude drug 4.5 g·kg-1·d-1 was given, in liver channel tropism drug pair of Gongdu

  17. Global Conservation of Protein Status between Cell Lines and Xenografts

    Directory of Open Access Journals (Sweden)

    Julian Biau

    2016-08-01

    Full Text Available Common preclinical models for testing anticancer treatment include cultured human tumor cell lines in monolayer, and xenografts derived from these cell lines in immunodeficient mice. Our goal was to determine how similar the xenografts are compared with their original cell line and to determine whether it is possible to predict the stability of a xenograft model beforehand. We studied a selection of 89 protein markers of interest in 14 human cell cultures and respective subcutaneous xenografts using the reverse-phase protein array technology. We specifically focused on proteins and posttranslational modifications involved in DNA repair, PI3K pathway, apoptosis, tyrosine kinase signaling, stress, cell cycle, MAPK/ERK signaling, SAPK/JNK signaling, NFκB signaling, and adhesion/cytoskeleton. Using hierarchical clustering, most cell culture-xenograft pairs cluster together, suggesting a global conservation of protein signature. Particularly, Akt, NFkB, EGFR, and Vimentin showed very stable protein expression and phosphorylation levels highlighting that 4 of 10 pathways were highly correlated whatever the model. Other proteins were heterogeneously conserved depending on the cell line. Finally, cell line models with low Akt pathway activation and low levels of Vimentin gave rise to more reliable xenograft models. These results may be useful for the extrapolation of cell culture experiments to in vivo models in novel targeted drug discovery.

  18. Dominant-negative inhibition of the Axl receptor tyrosine kinase suppresses brain tumor cell growth and invasion and prolongs survival

    Science.gov (United States)

    Vajkoczy, Peter; Knyazev, Pjotr; Kunkel, Andrea; Capelle, Hans-Holger; Behrndt, Sandra; von Tengg-Kobligk, Hendrik; Kiessling, Fabian; Eichelsbacher, Uta; Essig, Marco; Read, Tracy-Ann; Erber, Ralf; Ullrich, Axel

    2006-01-01

    Malignant gliomas remain incurable brain tumors because of their diffuse-invasive growth. So far, the genetic and molecular events underlying gliomagenesis are poorly understood. In this study, we have identified the receptor tyrosine kinase Axl as a mediator of glioma growth and invasion. We demonstrate that Axl and its ligand Gas6 are overexpressed in human glioma cell lines and that Axl is activated under baseline conditions. Furthermore, Axl is expressed at high levels in human malignant glioma. Inhibition of Axl signaling by overexpression of a dominant-negative receptor mutant (AXL-DN) suppressed experimental gliomagenesis (growth inhibition >85%, P 72 days). A detailed analysis of the distinct hallmarks of glioma pathology, such as cell proliferation, migration, and invasion and tumor angiogenesis, revealed that inhibition of Axl signaling interfered with cell proliferation (inhibition 30% versus AXL-WT), glioma cell migration (inhibition 90% versus mock and AXL-WT, P < 0.05), and invasion (inhibition 62% and 79% versus mock and AXL-WT, respectively; P < 0.05). This study describes the identification, functional manipulation, in vitro and in vivo validation, and preclinical therapeutic inhibition of a target receptor tyrosine kinase mediating glioma growth and invasion. Our findings implicate Axl in gliomagenesis and validate it as a promising target for the development of approaches toward a therapy of these highly aggressive but, as yet, therapy-refractory, tumors. PMID:16585512

  19. Synthesis and protein tyrosine phosphatase 1B inhibition activities of two new synthetic bromophenols and their methoxy derivatives

    Science.gov (United States)

    Cui, Yongchao; Shi, Dayong; Hu, Zhiqiang

    2011-11-01

    3-bromo-4,5-bis(2,3-dibromo-4,5-dihydroxybenzyl)-1,2-benzenediol ( 1) is a natural bromophenol isolated from the red algae Rhodomela confervoides that exhibits significant inhibition against protein tyrosine phosphatase 1B (PTP1B). Based on its activity, we synthesized two new synthetic bromophenols and their methoxy derivatives from vanillin using the structure of natural bromophenol 1 as a scaffold. The structures of these bromophenols were elucidated from 1H NMR, 13C NMR, and high resolution electron ionization mass spectrometry as 2,3-dibromo-1-(2'-bromo-6'-(3″,4″-dimethoxybenzyl)-3',4'-dimethoxybenzyl)-4,5-dimethoxybenzene ( 2), 2,3-dibromo-1-(2'-bromo-6'-(2″-bromo-4″,5″-dimethoxybenzyl)-3',4'-dimethoxybenzyl)-4,5-dimethoxybenzene ( 3), 3,4-dibromo-5-(2'-bromo-6'-(2″-bromo-4″,5″-dihydroxybenzyl)-3',4'-dihydroxybenzyl)pyrocatechol ( 4) and 3,4-dibromo-5-(2'-bromo-6'-(3″,4″-dihydroxybenzyl)-3',4'-dihydroxybenzyl)pyrocatechol ( 5). PTP1B inhibition activities of these compounds were evaluated using a colorimetric assay, and compounds 3 and 4 demonstrated interesting activity against PTP1B.

  20. Study of Mutation in Tyrosine Protein Kinase of Insulin Receptor Gene in Patients with Polycystic Ovarian Syndrome

    Institute of Scientific and Technical Information of China (English)

    Min LI; Hong-yu QIU; Yong-yu SUN; Hong-fa LI; Yong-li CHU

    2003-01-01

    Objective To explore the molecular mechanism of insulin resistance in the patients with polycystic ovarian syndrome (PCOS)Methods Polymerase chain reaction, silver staining-single strand conformation polymorphism(PCR-SSCP) and DNA direct sequencing were used to detect the mutation of insulin receptor(INSR) gene in exon 17~21 with the abdominal wall adipose tissue from 31 patients with PCOS (PCOS Group) and 30 patients with pure hysteromyoma in reproductive lift (Control Group).Results Twenty-two variant SSCP patterns in exon 17 of INSR gene were detected. Direct sequence analysis of exon 17 showed that homozygous nonsense mutation was two alleles single nucleotide polymorphism(SNP) at the codon 1058 (CAC→CAT). Exons 18~21 were not detected with any significantly mutation. The INSR gene His1058C→T substitution collecting rate and insulin resistance were significantly higher in the PCOS group than in the control group (P=0.0293, P<0.05, P<0.01).Conclusion It is suggested that the SNP in codon 1058 of the INSR gene might be related with the insulin resistance in PCOS patients, which has hereditary tendency. And the missense mutation,nonsense mutation and frameshift mutation at exons 18~21 in tyrosine protein kinase region of INSR gene for PCOS patients were not frequently observed.

  1. Effect of feeding garlic leaf on microbial nitrogen supply, kinetics of plasma phenylalanine, tyrosine and protein synthesis in sheep.

    Science.gov (United States)

    Kamruzzaman, Md; Liang, Xi; Sekiguchi, Natsumi; Sano, Hiroaki

    2014-05-01

    The objective of the present study was to assess the feeding effects of garlic leaf on microbial N supply (MNS), turnover rates of plasma phenylalanine (PheTR) and tyrosine (TyrTR) and whole body protein synthesis (WBPS) in sheep. The sheep were fed either mixed hay (Hay-diet, as control) or hay plus garlic leaf diet (GL-diet, at a ratio of 9:1) in a crossover design each for a 21 day period. The isotope dilution method using [(2) H5 ]Phe and [(2) H2 ]Tyr was performed on the 21st day of each dietary treatment. Nitrogen intake remained similar between the diets and N absorption and N digestibility were higher (P<0.05) in the GL-diet than Hay-diet. Total purine derivatives excretion and MNS were greater (P<0.05) in the GL-diet than the Hay-diet. Plasma PheTR tended to be higher (P=0.06) during GL feeding and TyrTR did not differ between the diets. Further, WBPS tended to be greater (P=0.05) for the GL-diet compared with the Hay-diet. Hence, the present results suggest that garlic leaf may have positive effects on N metabolism by influencing MNS in sheep and could be used as a potential ruminant feed in the future.

  2. Slitrks control excitatory and inhibitory synapse formation with LAR receptor protein tyrosine phosphatases

    OpenAIRE

    Yim, Yeong Shin; Kwon, Younghee; Nam, Jungyong; Yoon, Hong In; Lee, Kangduk; Kim, Dong Goo; Kim, Eunjoon; Kim, Chul Hoon; Ko, Jaewon

    2013-01-01

    The balance between excitatory and inhibitory synaptic inputs, which is governed by multiple synapse organizers, controls neural circuit functions and behaviors. Slit- and Trk-like proteins (Slitrks) are a family of synapse organizers, whose emerging synaptic roles are incompletely understood. Here, we report that Slitrks are enriched in postsynaptic densities in rat brains. Overexpression of Slitrks promoted synapse formation, whereas RNAi-mediated knockdown of Slitrks decreased synapse dens...

  3. Ternary oxovanadium(IV) complexes of ONO-donor Schiff base and polypyridyl derivatives as protein tyrosine phosphatase inhibitors: synthesis, characterization, and biological activities.

    Science.gov (United States)

    Yuan, Caixia; Lu, Liping; Gao, Xiaoli; Wu, Yanbo; Guo, Maolin; Li, Ying; Fu, Xueqi; Zhu, Miaoli

    2009-08-01

    A series of oxovanadium complexes with mixed ligands, a tridentate ONO-donor Schiff base ligand [viz., salicylidene anthranilic acid (SAA)], and a bidentate NN ligand [viz., 2,2'-bipyridine (bpy), 1,10-phenanthroline (phen), dipyrido[3,2-d:2',3'-f]quinoxaline (dpq), dipyrido[3,2-a:2',3'-c]phenazine (dppz), or 7-methyldipyrido[3,2-a:2',3'-c]phenazine (dppm)], have been synthesized and characterized by elemental analysis, electrospray ionization mass spectrometry, UV-vis spectroscopy, Fourier transform IR spectroscopy, EPR spectroscopy, and X-ray crystallography. Crystal structures of both complexes, [V(IV)O(SAA)(bpy)].0.25bpy and [V(IV)O(SAA)(phen)].0.33H(2)O, reveal that oxovanadium(IV) is coordinated with one nitrogen and two oxygen atoms from the Schiff base and two nitrogen atoms from the bidentate planar ligands, in a distorted octahedral geometry (VO(3)N(3)). The oxidation state of V(IV) with d(1) configuration was confirmed by EPR spectroscopy. The speciation of VO-SAA-bpy in aqueous solution was investigated by potentiomtreic pH titrations, and the results revealed that the main species are two ternary complexes at a pH range of 7.0-7.4, and one is the isolated crystalline complex. The complexes have been found to be potent inhibitors against human protein tyrosine phosphatase 1B (PTP1B) (IC(50) approximately 30-61 nM), T-cell protein tyrosine phosphatase (TCPTP), and Src homology phosphatase 1 (SHP-1) in vitro. Interestingly, the [V(IV)O(SAA)(bpy)] complex selectively inhibits PTP1B over the other two phosphatases (approximate ninefold selectivity against SHP-1 and about twofold selectivity against TCPTP). Kinetics assays suggest that the complexes inhibit PTP1B in a competitive and reversible manner. These suggest that the complexes may be promising candidates as novel antidiabetic agents. PMID:19290551

  4. Regulation of alternative macrophage activation in the liver following acetaminophen intoxication by stem cell-derived tyrosine kinase

    Energy Technology Data Exchange (ETDEWEB)

    Gardner, Carol R., E-mail: cgardner@pharmacy.rutgers.edu [Department of Pharmacology and Toxicology, Rutgers University, Ernest Mario School of Pharmacy, Piscataway, NJ 08854 (United States); Hankey, Pamela [Department of Veterinary and Biomedical Science, Pennsylvania State University, University Park, PA 16802 (United States); Mishin, Vladimir; Francis, Mary [Department of Pharmacology and Toxicology, Rutgers University, Ernest Mario School of Pharmacy, Piscataway, NJ 08854 (United States); Yu, Shan [Department of Veterinary and Biomedical Science, Pennsylvania State University, University Park, PA 16802 (United States); Laskin, Jeffrey D. [Department of Environmental and Occupational Medicine, UMDNJ-Robert Wood Johnson Medical School, Piscataway, NJ 08854 (United States); Laskin, Debra L. [Department of Pharmacology and Toxicology, Rutgers University, Ernest Mario School of Pharmacy, Piscataway, NJ 08854 (United States)

    2012-07-15

    Stem cell-derived tyrosine kinase (STK) is a transmembrane receptor reported to play a role in macrophage switching from a classically activated/proinflammatory phenotype to an alternatively activated/wound repair phenotype. In the present studies, STK{sup −/−} mice were used to assess the role of STK in acetaminophen-induced hepatotoxicity as evidence suggests that the pathogenic process involves both of these macrophage subpopulations. In wild type mice, centrilobular hepatic necrosis and increases in serum transaminase levels were observed within 6 h of acetaminophen administration (300 mg/kg, i.p.). Loss of STK resulted in a significant increase in sensitivity of mice to the hepatotoxic effects of acetaminophen and increased mortality, effects independent of its metabolism. This was associated with reduced levels of hepatic glutathione, rapid upregulation of inducible nitric oxide synthase, and prolonged induction of heme oxygenase-1, suggesting excessive oxidative stress in STK{sup −/−} mice. F4/80, a marker of mature macrophages, was highly expressed on subpopulations of Kupffer cells in livers of wild type, but not STK{sup −/−} mice. Whereas F4/80{sup +} macrophages rapidly declined in the livers of wild type mice following acetaminophen intoxication, they increased in STK{sup −/−} mice. In wild type mice hepatic expression of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-12, products of classically activated macrophages, increased after acetaminophen administration. Monocyte chemotactic protein-1 (MCP-1) and its receptor, CCR2, as well as IL-10, mediators involved in recruiting and activating anti-inflammatory/wound repair macrophages, also increased in wild type mice after acetaminophen. Loss of STK blunted the effects of acetaminophen on expression of TNFα, IL-1β, IL-12, MCP-1 and CCR2, while expression of IL-10 increased. Hepatic expression of CX3CL1, and its receptor, CX3CR1 also increased in STK{sup −/−} mice

  5. Sulfone-stabilized carbanions for the reversible covalent capture of a posttranslationally-generated cysteine oxoform found in protein tyrosine phosphatase 1B (PTP1B).

    Science.gov (United States)

    Parsons, Zachary D; Ruddraraju, Kasi Viswanatharaju; Santo, Nicholas; Gates, Kent S

    2016-06-15

    Redox regulation of protein tyrosine phosphatase 1B (PTP1B) involves oxidative conversion of the active site cysteine thiolate into an electrophilic sulfenyl amide residue. Reduction of the sulfenyl amide by biological thiols regenerates the native cysteine residue. Here we explored fundamental chemical reactions that may enable covalent capture of the sulfenyl amide residue in oxidized PTP1B. Various sulfone-containing carbon acids were found to react readily with a model peptide sulfenyl amide via attack of the sulfonyl carbanion on the electrophilic sulfur center in the sulfenyl amide. Both the products and the rates of these reactions were characterized. The results suggest that capture of a peptide sulfenyl amide residue by sulfone-stabilized carbanions can slow, but not completely prevent, thiol-mediated generation of the corresponding cysteine-containing peptide. Sulfone-containing carbon acids may be useful components in the construction of agents that knock down PTP1B activity in cells via transient covalent capture of the sulfenyl amide oxoform generated during insulin signaling processes. PMID:27132865

  6. Deletion of Protein Tyrosine Phosphatase 1B (PTP1B Enhances Endothelial Cyclooxygenase 2 Expression and Protects Mice from Type 1 Diabetes-Induced Endothelial Dysfunction.

    Directory of Open Access Journals (Sweden)

    David J Herren

    Full Text Available Protein tyrosine phosphatase 1B (PTP1B dephosphorylates receptors tyrosine kinase and acts as a molecular brake on insulin signaling pathway. Conditions of metabolic dysfunction increase PTP1B, when deletion of PTP1B protects against metabolic disorders by increasing insulin signaling. Although vascular insulin signaling contributes to the control of glucose disposal, little is known regarding the direct role of PTP1B in the control of endothelial function. We hypothesized that metabolic dysfunctions increase PTP1B expression in endothelial cells and that PTP1B deletion prevents endothelial dysfunction in situation of diminished insulin secretion. Type I diabetes (T1DM was induced in wild-type (WT and PTP1B-deficient mice (KO with streptozotocin (STZ injection. After 28 days of T1DM, KO mice exhibited a similar reduction in body weight and plasma insulin levels and a comparable increase in glycemia (WT: 384 ± 20 vs. Ko: 432 ± 29 mg/dL, cholesterol and triglycerides, as WT mice. T1DM increased PTP1B expression and impaired endothelial NO-dependent relaxation, in mouse aorta. PTP1B deletion did not affect baseline endothelial function, but preserved endothelium-dependent relaxation, in T1DM mice. NO synthase inhibition with L-NAME abolished endothelial relaxation in control and T1DM WT mice, whereas L-NAME and the cyclooxygenases inhibitor indomethacin were required to abolish endothelium relaxation in T1DM KO mice. PTP1B deletion increased COX-2 expression and PGI2 levels, in mouse aorta and plasma respectively, in T1DM mice. In parallel, simulation of diabetic conditions increased PTP1B expression and knockdown of PTP1B increased COX-2 but not COX-1 expression, in primary human aortic endothelial cells. Taken together these data indicate that deletion of PTP1B protected endothelial function by compensating the reduction in NO bioavailability by increasing COX-2-mediated release of the vasodilator prostanoid PGI2, in T1DM mice.

  7. CEP-701 and CEP-751 inhibit constitutively activated RET tyrosine kinase activity and block medullary thyroid carcinoma cell growth.

    Science.gov (United States)

    Strock, Christopher J; Park, Jong-In; Rosen, Mark; Dionne, Craig; Ruggeri, Bruce; Jones-Bolin, Susan; Denmeade, Samuel R; Ball, Douglas W; Nelkin, Barry D

    2003-09-01

    All of the cases of medullary thyroid carcinoma (MTC) express the RET receptor tyrosine kinase. In essentially all of the hereditary cases and approximately 40% of the sporadic cases of MTC, the RET kinase is constitutively activated by mutation. This suggests that RET may be an effective therapeutic target for treatment of MTC. We show that the indolocarbazole derivatives, CEP-701 and CEP-751, inhibit RET in MTC cells. These compounds effectively inhibit RET phosphorylation in a dose-dependent manner at concentrations <100 nM in 0.5% serum and at somewhat higher concentrations in the presence of 16% serum. They also blocked the growth of these MTC cells in culture. CEP-751 and its prodrug, CEP-2563, also inhibited tumor growth in MTC cell xenografts. These results show that inhibiting RET can block the growth of MTC cells and may have a therapeutic benefit in MTC.

  8. Cloning, purification, crystallization and preliminary X-ray analysis of two low-molecular-weight protein tyrosine phosphatases from Vibrio cholerae

    International Nuclear Information System (INIS)

    Two protein tyrosine phosphatases, namely VcLMWPTP-1 and VcLMWPTP-2, from V. cholerae have been cloned, expressed, purified and crystallized. Low-molecular-weight protein tyrosine phosphatases (LMWPTPs) are small cytoplasmic enzymes of molecular weight ∼18 kDa that belong to the large family of protein tyrosine phosphatases (PTPs). Despite their wide distribution in both prokaryotes and eukaryotes, their exact biological role in bacterial systems is not yet clear. Two low-molecular-weight protein tyrosine phosphatases (VcLMWPTP-1 and VcLMWPTP-2) from the Gram-negative bacterium Vibrio cholerae have been cloned, overexpressed, purified by Ni2+–NTA affinity chromatography followed by gel filtration and used for crystallization. Crystals of VcLMWPTP-1 were grown in the presence of ammonium sulfate and glycerol and diffracted to a resolution of 1.6 Å. VcLMWPTP-2 crystals were grown in PEG 4000 and diffracted to a resolution of 2.7 Å. Analysis of the diffraction data showed that the VcLMWPTP-1 crystals had symmetry consistent with space group P31 and that the VcLMWPTP-2 crystals had the symmetry of space group C2. Assuming the presence of four molecules in the asymmetric unit, the Matthews coefficient for the VcLMWPTP-1 crystals was estimated to be 1.97 Å3 Da−1, corresponding to a solvent content of 37.4%. The corresponding values for the VcLMWPTP-2 crystals, assuming the presence of two molecules in the asymmetric unit, were 2.77 Å3 Da−1 and 55.62%, respectively

  9. Potential Utility of Sodium Selenate as an Adjunct to Metformin in Treating Type II Diabetes Mellitus in Rats: A Perspective on Protein Tyrosine Phosphatase

    OpenAIRE

    Rania M. Salama; Schaalan, Mona F; Elkoussi, Alaaeldin A.; Khalifa, Amani E.

    2013-01-01

    Metformin is widely regarded as the standard first-line antidiabetic agent, in terms of efficacy and safety profiles. However, in most patients with type II diabetes mellitus (T2DM), it was found that metformin alone is not enough to adequately control hyperglycemia. Thus, we designed this study with the aim to investigate the effect of sodium selenate, a protein tyrosine phosphatase (PTP) inhibitor, individually and as an adjunct to metformin, on a rat model that simulates the metabolic char...

  10. Tyrosine phosphorylation of the human guanylyl cyclase C receptor

    Indian Academy of Sciences (India)

    Rashna Bhandari; Roy Mathew; K Vijayachandra; Sandhya S Visweswariah

    2000-12-01

    Tyrosine phosphorylation events are key components of several cellular signal transduction pathways. This study describes a novel method for identification of substrates for tyrosine kinases. Co-expression of the tyrosine kinase EphB1 with the intracellular domain of guanylyl cyclase C (GCC) in Escherichia coli cells resulted in tyrosine phosphorylation of GCC, indicating that GCC is a potential substrate for tyrosine kinases. Indeed, GCC expressed in mammalian cells is tyrosine phosphorylated, suggesting that tyrosine phosphorylation may play a role in regulation of GCC signalling. This is the first demonstration of tyrosine phosphorylation of any member of the family of membrane-associated guanylyl cyclases.

  11. Comparative evaluation of bone marrow cells morpho-functional activity in chronic myeloid leukemia patients treated with tyrosine kinase inhibitors of the first and second generation

    Directory of Open Access Journals (Sweden)

    I. O. Zhaleyko

    2014-07-01

    Full Text Available The efficiency of using the culture techniques of research for monitoring the patient’s response to the treatment by tyrosine kinase inhibitors of the first and second generation is shown. Thus, the functional activity of bone marrow cells in patients having the optimal treatment response to inhibitors of tyrosine kinases was significantly lower compared with patients with the acquired resistance to the drug, and patients who had CML diagnosed for first time. Furthermore, for patients with the optimal response to the nilotinib therapy, numbers of colonies in semi-solid agar in vitro was lower, than in patients with the optimal response to imatinib. When the leukaemic cell clone becomes resistant to tyrosine kinase inhibitors, the prevalence of early cells of granulocyte-macrophage hematopoietic stem cells is observed in CFU culture which can be an important prognostic factor for choosing the appropriate treatment strategy.

  12. Mutations of the EPHB6 receptor tyrosine kinase induce a pro-metastatic phenotype in non-small cell lung cancer.

    Directory of Open Access Journals (Sweden)

    Etmar Bulk

    Full Text Available Alterations of Eph receptor tyrosine kinases are frequent events in human cancers. Genetic variations of EPHB6 have been described but the functional outcome of these alterations is unknown. The current study was conducted to screen for the occurrence and to identify functional consequences of EPHB6 mutations in non-small cell lung cancer. Here, we sequenced the entire coding region of EPHB6 in 80 non-small cell lung cancer patients and 3 tumor cell lines. Three potentially relevant mutations were identified in primary patient samples of NSCLC patients (3.8%. Two point mutations led to instable proteins. An in frame deletion mutation (del915-917 showed enhanced migration and accelerated wound healing in vitro. Furthermore, the del915-917 mutation increased the metastatic capability of NSCLC cells in an in vivo mouse model. Our results suggest that EPHB6 mutations promote metastasis in a subset of patients with non-small cell lung cancer.

  13. STAT1 is phosphorylated and downregulated by the oncogenic tyrosine kinase NPM-ALK in ALK-positive anaplastic large-cell lymphoma.

    Science.gov (United States)

    Wu, Chengsheng; Molavi, Ommoleila; Zhang, Haifeng; Gupta, Nidhi; Alshareef, Abdulraheem; Bone, Kathleen M; Gopal, Keshav; Wu, Fang; Lewis, Jamie T; Douglas, Donna N; Kneteman, Norman M; Lai, Raymond

    2015-07-16

    The tumorigenicity of most cases of ALK-positive anaplastic large-cell lymphoma (ALK+ ALCL) is driven by the oncogenic fusion protein NPM-ALK in a STAT3-dependent manner. Because it has been shown that STAT3 can be inhibited by STAT1 in some experimental models, we hypothesized that the STAT1 signaling pathway is defective in ALK+ ALCL, thereby leaving the STAT3 signaling unchecked. Compared with normal T cells, ALK+ ALCL tumors consistently expressed a low level of STAT1. Inhibition of the ubiquitin-proteasome pathway appreciably increased STAT1 expression in ALK+ ALCL cells. Furthermore, we found evidence that NPM-ALK binds to and phosphorylates STAT1, thereby promoting its proteasomal degradation in a STAT3-dependent manner. If restored, STAT1 is functionally intact in ALK+ ALCL cells, because it effectively upregulated interferon-γ, induced apoptosis/cell-cycle arrest, potentiated the inhibitory effects of doxorubicin, and suppressed tumor growth in vivo. STAT1 interfered with the STAT3 signaling by decreasing STAT3 transcriptional activity/DNA binding and its homodimerization. The importance of the STAT1/STAT3 functional interaction was further highlighted by the observation that short interfering RNA knockdown of STAT1 significantly decreased apoptosis induced by STAT3 inhibition. Thus, STAT1 is a tumor suppressor in ALK+ ALCL. Phosphorylation and downregulation of STAT1 by NPM-ALK represent other mechanisms by which this oncogenic tyrosine kinase promotes tumorigenesis.

  14. ZEB1 Mediates Acquired Resistance to the Epidermal Growth Factor Receptor-Tyrosine Kinase Inhibitors in Non-Small Cell Lung Cancer.

    Directory of Open Access Journals (Sweden)

    Takeshi Yoshida

    Full Text Available Epithelial-mesenchymal transition (EMT is one mechanism of acquired resistance to inhibitors of the epidermal growth factor receptor-tyrosine kinases (EGFR-TKIs in non-small cell lung cancer (NSCLC. The precise mechanisms of EMT-related acquired resistance to EGFR-TKIs in NSCLC remain unclear. We generated erlotinib-resistant HCC4006 cells (HCC4006ER by chronic exposure of EGFR-mutant HCC4006 cells to increasing concentrations of erlotinib. HCC4006ER cells acquired an EMT phenotype and activation of the TGF-β/SMAD pathway, while lacking both T790M secondary EGFR mutation and MET gene amplification. We employed gene expression microarrays in HCC4006 and HCC4006ER cells to better understand the mechanism of acquired EGFR-TKI resistance with EMT. At the mRNA level, ZEB1 (TCF8, a known regulator of EMT, was >20-fold higher in HCC4006ER cells than in HCC4006 cells, and increased ZEB1 protein level was also detected. Furthermore, numerous ZEB1 responsive genes, such as CDH1 (E-cadherin, ST14, and vimentin, were coordinately regulated along with increased ZEB1 in HCC4006ER cells. We also identified ZEB1 overexpression and an EMT phenotype in several NSCLC cells and human NSCLC samples with acquired EGFR-TKI resistance. Short-interfering RNA against ZEB1 reversed the EMT phenotype and, importantly, restored erlotinib sensitivity in HCC4006ER cells. The level of micro-RNA-200c, which can negatively regulate ZEB1, was significantly reduced in HCC4006ER cells. Our results suggest that increased ZEB1 can drive EMT-related acquired resistance to EGFR-TKIs in NSCLC. Attempts should be made to explore targeting ZEB1 to resensitize TKI-resistant tumors.

  15. Receptor protein tyrosine kinase EphB4 is up-regulated in colon cancer

    Directory of Open Access Journals (Sweden)

    Hewett Peter J

    2001-12-01

    Full Text Available Abstract Background We have used commercially available cDNA arrays to identify EphB4 as a gene that is up-regulated in colon cancer tissue when compared with matched normal tissue from the same patient. Results Quantitative RT-PCR analysis of the expression of the EphB4 gene has shown that its expression is increased in 82% of tumour samples when compared with the matched normal tissue from the same patient. Using immunohistochemistry and Western analysis techniques with an EphB4-specific antibody, we also show that this receptor is expressed in the epithelial cells of the tumour tissue and either not at all, or in only low levels, in the normal tissue. Conclusion The results presented here supports the emerging idea that Eph receptors play a role in tumour formation and suggests that further elucidation of this signalling pathway may identify useful targets for cancer treatment therapies.

  16. Melatonin Sensitizes H1975 Non-Small-Cell Lung Cancer Cells Harboring a T790M-Targeted Epidermal Growth Factor Receptor Mutation to the Tyrosine Kinase Inhibitor Gefitinib

    Directory of Open Access Journals (Sweden)

    Miyong Yun

    2014-08-01

    Full Text Available Background/Aims: The use of tyrosine kinase inhibitors (TKIs to target active epidermal growth factor receptor (EGFR-harbouring mutations has been effective in patients with advanced non-small-cell lung cancer (NSCLC. However, the use of TKIs in NSCLS patients with somatic EGFR mutations, particularly T790M, causes drug resistance. Thus, in the present study, we investigated overcoming resistance against the TKI gefitinib by combination treatment with melatonin in H1975 NSCLC cells harbouring the T790M somatic mutation. Methods: H1975 and HCC827 cells were treated with melatonin in combination with gefitinib, and cell viability, cell cycle progression, apoptosis, and EGFR, AKT, p38, Bcl-2, Bcl-xL, caspase 3 and Bad protein levels were examined. Results: Treatment with melatonin dose-dependently decreased the viability of H1975 cells harbouring the T790M somatic mutation compared to HCC827 cells with an EGFR active mutation. Melatonin-mediated cell death resulted in decreased phosphorylation of EGFR and Akt, leading to attenuated expression of survival proteins, such as Bcl-2, Bcl-xL and survivin, and activated caspase 3 in H1975 cells, but not in HCC827 cells. However, we did not observe a significant change in expression of cell cycle proteins, such as cyclin D, cyclin A, p21 and CDK4 in H1975 cells. Surprisingly, co-treatment of gefitinib with melatonin effectively decreased the viability of H1975 cells, but not HCC827 cells. Moreover, co-treatment of H1975 cells caused consistent down-regulation of EGFR phosphorylation and induced apoptosis compared to treatment with gefitinib or melatonin alone. Conclusions: Our findings demonstrate that melatonin acts as a potent chemotherapeutic agent by sensitising to gefitinib TKI-resistant H1975 cells that harbour a EGFR T790M mutation.

  17. Proliferation of Ewing sarcoma cell lines is suppressed by the receptor tyrosine kinase inhibitors gefitinib and vandetanib

    Directory of Open Access Journals (Sweden)

    Åman Pierre

    2008-01-01

    Full Text Available Abstract Background Tyrosine kinase inhibitors (TKIs have gained much attention in recent years as targeted agents for the treatment of a wide range of human cancers. We have investigated the effect of the TKIs gefitinib and vandetanib on tumor cell lines derived from Ewing sarcoma, a highly malignant tumor affecting bone and soft tissue in children and young adults. Gefitinib is an inhibitor of epidermal growth factor receptor tyrosine kinase activity (EGFR and vandetanib selectively targets vascular endothelial growth factor receptor-2 (VEGFR-2 with additional activity against VEGFR-3, EGFR and RET kinase receptors. Results Two Ewing sarcoma cell lines investigated showed high levels of nuclear EGFR expression as well as moderate expression in plasma membrane and cytoplasm. When treated with concentrations of 5 μM and more of either gefitinib or vandetanib, we observed a significant decrease in cell proliferation. However, there were no detectable changes in p44/42 MAPK and Akt-1 phosphorylation, or in the expression of cyclin D1 or c-Myc following gefitinib or vandetanib treatment. Conclusion We conclude that Ewing sarcoma tumor cell proliferation is not highly sensitive to inhibition of EGFR signaling alone or the simultaneous inhibition of VEGFR receptors, EGFR and RET kinase. Decreased tumor cell proliferation could be achieved with gefitinib and vandetanib, but only at higher doses where non-specific effects of the compounds may be overriding. As Ewing tumor cells do not seem to depend on EGFR and VEGFR pathways for survival, other key factors in the cellular signaling of Ewing sarcoma should be targeted in order to obtain a potent therapeutic response.

  18. The conformational control inhibitor of tyrosine kinases DCC-2036 is effective for imatinib-resistant cells expressing T674I FIP1L1-PDGFRα.

    Directory of Open Access Journals (Sweden)

    Yingying Shen

    Full Text Available The cells expressing the T674I point mutant of FIP1-like-1-platelet-derived growth factor receptor alpha (FIP1L1-PDGFRα in hypereosinophilics syndrome (HES are resistant to imatinib and some second-generation tyrosine kinase inhibitors (TKIs. There is a desperate need to develop therapy to combat this acquired drug resistance. DCC-2036 has been synthesized as a third-generation TKI to combat especially the Bcr-Abl T315I mutant in chronic myeloid leukemia. This study evaluated the effect of DCC-2036 on FIP1L1-PDGFRα-positive cells, including the wild type (WT and the T674I mutant. The in vitro effects of DCC-2036 on the PDGFRα signal pathways, proliferation, cell cycling and apoptosis of FIP1L1-PDGFRα-positive cells were investigated, and a nude mouse xenograft model was employed to assess the in vivo antitumor activity. We found that DCC-2036 decreased the phosphorylated levels of PDGFRα and its downstream targets without apparent effects on total protein levels. DCC-2036 inhibited proliferation, and induced apoptosis with MEK-dependent up-regulation of the pro-apoptotic protein Bim in FIP1L1-PDGFRα-positive cells. DCC-2036 also exhibited in vivo antineoplastic activity against cells with T674I FIP1L1-PDGFRα. In summary, FIP1L1-PDGFRα-positive cells are sensitive to DCC-2036 regardless of their sensitivity to imatinib. DCC-2036 may be a potential compound to treat imatinib-resistant HES.

  19. Fully automated synthesis of (phosphopeptide arrays in microtiter plate wells provides efficient access to protein tyrosine kinase characterization

    Directory of Open Access Journals (Sweden)

    Goldstein David J

    2005-01-01

    Full Text Available Abstract Background Synthetic peptides have played a useful role in studies of protein kinase substrates and interaction domains. Synthetic peptide arrays and libraries, in particular, have accelerated the process. Several factors have hindered or limited the applicability of various techniques, such as the need for deconvolution of combinatorial libraries, the inability or impracticality of achieving full automation using two-dimensional or pin solid phases, the lack of convenient interfacing with standard analytical platforms, or the difficulty of compartmentalization of a planar surface when contact between assay components needs to be avoided. This paper describes a process for synthesis of peptides and phosphopeptides on microtiter plate wells that overcomes previous limitations and demonstrates utility in determination of the epitope of an autophosphorylation site phospho-motif antibody and utility in substrate utilization assays of the protein tyrosine kinase, p60c-src. Results The overall reproducibility of phospho-peptide synthesis and multiplexed EGF receptor (EGFR autophosphorylation site (pY1173 antibody ELISA (9H2 was within 5.5 to 8.0%. Mass spectrometric analyses of the released (phosphopeptides showed homogeneous peaks of the expected molecular weights. An overlapping peptide array of the complete EGFR cytoplasmic sequence revealed a high redundancy of 9H2 reactive sites. The eight reactive phospopeptides were structurally related and interestingly, the most conserved antibody reactive peptide motif coincided with a subset of other known EGFR autophosphorylation and SH2 binding motifs and an EGFR optimal substrate motif. Finally, peptides based on known substrate specificities of c-src and related enzymes were synthesized in microtiter plate array format and were phosphorylated by c-Src with the predicted specificities. The level of phosphorylation was proportional to c-Src concentration with sensitivities below 0.1 Units of

  20. Role of PAX8 in the regulation of MET and RON receptor tyrosine kinases in non-small cell lung cancer

    International Nuclear Information System (INIS)

    Non-small cell lung cancers (NSCLC) are highly heterogeneous at the molecular level and comprise 75% of all lung tumors. We have previously shown that the receptor tyrosine kinase (RTK) MET frequently suffers gain-of-function mutations that significantly promote lung tumorigenesis. Subsequent studies from our lab also revealed that PAX5 transcription factor is preferentially expressed in small cell lung cancer (SCLC) and promotes MET transcription. PAX8, however, is also expressed in NSCLC cell lines. We therefore investigated the role of PAX8 in NSCLC. Using IHC analysis, PAX8 protein expression was determined in archival NSCLC tumor tissues (n = 254). In order to study the effects of PAX8 knockdown on NSCLC cellular functions such as apoptosis and motility, siRNA against PAX8 was used. Confocal fluorescence microscopy was used to monitor the localization of MET, RON and PAX8. The combinatorial effect of PAX8 knockdown and MET inhibition using SU11274 was investigated in NSCLC cell viability assay. Relative levels of PAX8 protein were elevated (≥ + 2 on a scale of 0–3) in adenocarcinoma (58/94), large cell carcinoma (50/85), squamous cell carcinoma (28/47), and metastatic NSCLC (17/28; lymph node). Utilizing early progenitors isolated from NSCLC cell lines and fresh tumor tissues, we observed robust overexpression of PAX8, MET, and RON. PAX8 knockdown A549 cells revealed abrogated PAX8 expression with a concomitant loss in MET and the related RON kinase expression. A dramatic colocalization between the active form of MET (also RON) and PAX8 upon challenging A549 cells with HGF was visualized. A similar colocalization of MET and EGL5 (PAX8 ortholog) proteins was found in embryos of C. elegans. Most importantly, knockdown of PAX8 in A549 cells resulted in enhanced apoptosis (~6 fold) and decreased cell motility (~45%), thereby making PAX8 a potential therapeutic target. However, the combinatorial approach of PAX8 knockdown and treatment with MET inhibitor, SU

  1. Ultrasensitive electrochemical detection of protein tyrosine kinase-7 by gold nanoparticles and methylene blue assisted signal amplification.

    Science.gov (United States)

    Miao, Xiangmin; Li, Zongbing; Zhu, Aihua; Feng, Zhaozhong; Tian, Jun; Peng, Xue

    2016-09-15

    We present here an ultrasensitive and simple strategy for protein tyrosine kinase-7 (PTK7) detection based on the recognition-induced structure change of sgc8 aptamer, and the signal change of methylene blue (MB) that interacted with sandwiched DNA complex. To construct such a sensor, an homogeneous nano-surface was formed firstly on the glass carbon electrode (GCE) by using negatively charged Nafion (Nf) as the inner layer and positively charged gold nanoparticles ((+)AuNPs) as the outer layer, followed by the immobilization of sgc8 aptamer based on Au-S bond. In the presence of helper probe (HP), sandwiched DNA complex was formed between the sgc8 aptamer and the DNA modified gold nanoparticle probe (DNA-AuNPs). Then, a strong current signal was produced due to the capture of abundant MB molecules by both the sandwiched DNA complex and the multiple DNAs that modified on AuNPs surface. However, the specific binding of sgc8 aptamer with PNK7 would trigger a structure transition of it, and directly prevented the following formation of sandwiched structure and the capture of MB. Thus, PTK7 detection could be realized based on monitoring the signal reduction of MB upon incubation of sgc8 aptamer with PTK7. Under optimal conditions, a low detection limit of 372 fM was obtained for PNK7 detection. Due to the employment of sgc8 aptamer, the proposed biosensor exhibited high selectivity to PNK7. Moreover, satisfactory results were obtained when the proposed method was applied for PNK7 detection in cellular debris. PMID:27101533

  2. The catalytic role of aspartic acid-92 in a human dual-specific protein-tyrosine-phosphatase.

    Science.gov (United States)

    Denu, J M; Zhou, G; Guo, Y; Dixon, J E

    1995-03-14

    The mechanism of catalysis for the human dual-specific (vaccinia H1-related) protein-tyrosine-phosphatase was investigated. The pH dependence of the kcat value is bell-shaped when p-nitrophenyl phosphate was employed as a model substrate. The kcat/Km pH profile rises with a slope of 2 and decreases with a slope of -1, indicating that two groups must be unprotonated and one group must be protonated for activity. An amino acid residue with an apparent pKa value of 5.5 +/- 0.2 must be unprotonated and a residue with a pKa value of 5.7 must be unprotonated for activity. The pKa value of the catalytic cysteine-124 (C124) was 5.6 +/- 0.1. The aspartic acid-92-asparagine (D92N) mutant enzyme was 100-fold less active than the native enzyme and exhibited the loss of the basic limb in the pH profiles, suggesting that in the native enzyme D92 must be protonated for activity. The D92 residue is conserved throughout the entire family of dual-specific phosphatases. Mutants glutamic acid-6-glutamine, glutamic acid-32-glutamine, aspartic acid-14-asparagine, and aspartic acid-110-asparagine had less than a 2-fold effect on the kinetic parameters when compared to native enzyme. Based upon the lack of a "burst" in rapid reaction kinetics, formation of the intermediate is rate-limiting with both native and D92N mutant enzymes. In agreement with rate-limiting formation of the intermediate, the pKa value of 5.5 for the group which must be unprotonated for activity was assigned to C124.(ABSTRACT TRUNCATED AT 250 WORDS)

  3. 蛋白酪氨酸磷酸酶相互作用蛋白51的研究进展%Research progress on protein tyrosine phosphatase interacting protein 51

    Institute of Scientific and Technical Information of China (English)

    肖玉霞

    2012-01-01

    蛋白酪氨酸磷酸酶相互作用蛋白(PTPIP)51是一种进化保守的蛋白质,在人类和多种哺乳动物中具有同源性.PTPIP51蛋白表达十分广泛,在不同的组织可有不同的相对分子质量:在不同组织中可与相应的细胞因子结合,具有不同的功能;与多种受体复合物结合.触发和启动多种信号通路的级联反应,参与细胞的代谢、增殖和分化以及功能活动、程序性死亡和迁移等过程.本文就PTPIP51的生物学特性、表达及其调节、信号配体和生理功能以及与肿瘤的关系等研究进展作一综述.%The protein tyrosine phosphatase interacting protein 51 (PTPIP51) was a conserved protein whose amino acid sequence of human was found sequence homology with several other mammalian species. The expression profile of PTPIP51 protein were widely found in human and there were several different calculated molecular masses of the protein in a tissue-specific manner. The protein interacted with different signaling partners playing variety functions in human tissues. Interacting with its receptor complexes, signaling cascades of different pathways were activated and promoted mediating cells proliferation, differentiation, apoptosis and motility. In this paper, we will review the biological characteristics, and gene expression, regulation, signal ligands and the physiological functions and relationship with human tumors of PTPIP51.

  4. Cell-free H-cluster synthesis and [FeFe] hydrogenase activation: all five CO and CN⁻ ligands derive from tyrosine.

    Directory of Open Access Journals (Sweden)

    Jon M Kuchenreuther

    Full Text Available [FeFe] hydrogenases are promising catalysts for producing hydrogen as a sustainable fuel and chemical feedstock, and they also serve as paradigms for biomimetic hydrogen-evolving compounds. Hydrogen formation is catalyzed by the H-cluster, a unique iron-based cofactor requiring three carbon monoxide (CO and two cyanide (CN⁻ ligands as well as a dithiolate bridge. Three accessory proteins (HydE, HydF, and HydG are presumably responsible for assembling and installing the H-cluster, yet their precise roles and the biosynthetic pathway have yet to be fully defined. In this report, we describe effective cell-free methods for investigating H-cluster synthesis and [FeFe] hydrogenase activation. Combining isotopic labeling with FTIR spectroscopy, we conclusively show that each of the CO and CN⁻ ligands derive respectively from the carboxylate and amino substituents of tyrosine. Such in vitro systems with reconstituted pathways comprise a versatile approach for studying biosynthetic mechanisms, and this work marks a significant step towards an understanding of both the protein-protein interactions and complex reactions required for H-cluster assembly and hydrogenase maturation.

  5. Levels of circulating CD45dimCD34+VEGFR2+ progenitor cells correlate with outcome in metastatic renal cell carcinoma patients treated with tyrosine kinase inhibitors

    OpenAIRE

    Farace, F.; Gross-Goupil, M; Tournay, E; Taylor, M; Vimond, N.; Jacques, N; Billiot, F.; Mauguen, A.; Hill, C.; Escudier, B

    2011-01-01

    Background: Predicting the efficacy of antiangiogenic therapy would be of clinical value in patients (pts) with metastatic renal cell carcinoma (mRCC). We tested the hypothesis that circulating endothelial cell (CEC), bone marrow-derived CD45dimCD34+VEGFR2+ progenitor cell or plasma angiogenic factor levels are associated with clinical outcome in mRCC pts undergoing treatment with tyrosine kinase inhibitors (TKI). Methods: Fifty-five mRCC pts were prospectively monitored at baseline (day 1) a...

  6. Metabolic alterations and drug sensitivity of tyrosine kinase inhibitor resistant leukemia cells with a FLT3/ITD mutation.

    Science.gov (United States)

    Huang, Amin; Ju, Huai-Qiang; Liu, Kaiyan; Zhan, Guilian; Liu, Daolu; Wen, Shijun; Garcia-Manero, Guillermo; Huang, Peng; Hu, Yumin

    2016-07-28

    Internal tandem duplication (ITD) of the juxtamembrane region of FMS-like tyrosine kinase-3 (FLT3) receptor is a common type of mutation in adult acute myeloid leukemia (AML), and patient response to FLT3 inhibitors appears to be transient due to the emergence of drug resistance. We established two sorafenib-resistant cell lines carrying FLT3/ITD mutations, including the murine BaF3/ITD-R and human MV4-11-R cell lines. Gene expression profile analysis of the resistant and parental cells suggests that the highest ranked molecular and cellular functions of the differentially expressed genes are related to mitochondrial dysfunction. Both murine and human resistant cell lines display a longer doubling time, along with a significant inhibition of mitochondrial respiratory chain activity and substantial upregulation of glycolysis. The sorafenib-resistant cells exhibit increased expression of a majority of glycolytic enzymes, including hexokinase 2, which is also highly expressed in the mitochondrial fraction and is associated with resistance to apoptotic cell death. The sorafenib-resistant cells are collaterally sensitive to a number of glycolytic inhibitors including 2-deoxyglucose and 3-bromopyruvate propylester. Our study reveals a metabolic signature of sorafenib-resistant cells and suggests that glycolytic inhibition may override such resistance and warrant further clinical investigation. PMID:27132990

  7. Development of brain injury in mice by Angiostrongylus cantonensis infection is associated with the induction of transcription factor NF-kappaB, nuclear protooncogenes, and protein tyrosine phosphorylation.

    Science.gov (United States)

    Lee, H H; Shiow, S J; Chung, H C; Huang, C Y; Lin, C L; Hsu, J D; Shyu, L Y; Wang, C J

    2000-07-01

    Eosinophilic meningitis or meningoencephalitis caused by Angiostrongylus cantonensis is endemic to the Pacific area of Asia, especially Taiwan, Thailand, and Japan. Although eosinophilia is an important clinical manifestation of A. cantonensis infection, the role of eosinophils in the progress of the infection remains to be elucidated. In this experiment, we showed that A. cantonensis-caused eosinoplia and inflammation might lead to the induction of NF-kappaB and protooncogene expression via activation of the tyrosine phosphorylation signal pathway. After mice were infected daily with 30 third-stage larvae of A. cantonensis by oral adminstration for 6 weeks, no significant differences PKC-alpha, MEK-1, ERK-2, JNK, and p38 protein expression were found between the control and infected mice. However, the protein tyrosine phosphorylation levels, NF-kappaB, and iNOS protein products were significantly increased by 3.5-, 3.3-, and 6.3-fold, respectively, after 3 weeks of A. cantonensis infection. The same pattern was found for c-Myc, c-Jun, and c-Fos proteins, which were elevated by 3.2-, 2.3-, and 3.4-fold, respectively, compared to control animals after 3 weeks. The expression potency of these proteins started increasing in week 1, reaching maximal induction in week 3, and then declining in week 5 after A. cantonensis infection. Another consistent result was noted in the pathological observations, including eosinophilia, leukocyte infiltration, granulomatous reactions, and time responses in brain tissues of infected mice. These data suggest that the development of brain injury by eosinophlia of A. cantonensis infection is associated with NF-kappaB and/or nuclear protooncogenes expression, which is activated by the tyrosine phosphorylation pathway.

  8. Cell wall proteins in seedling cotyledons of Prosopis chilensis.

    Science.gov (United States)

    Rodríguez, J G; Cardemil, L

    1994-01-01

    Four cell wall proteins of cotyledons of Prosopis chilensis seedlings were characterized by PAGE and Western analyses using a polyclonal antibody, generated against soybean seed coat extensin. These proteins had M(r)s of 180,000, 126,000, 107,000 and 63,000, as determined by SDS-PAGE. The proteins exhibited a fluorescent positive reaction with dansylhydrazine suggesting that they are glycoproteins; they did not show peroxidase activity. The cell wall proteins were also characterized by their amino acid composition and by their amino-terminal sequence. These analyses revealed that there are two groups of related cell wall proteins in the cotyledons. The first group comprises the proteins of M(r)s 180,000, 126,000, 107,000 which are rich in glutamic acid/glutamine and aspartic acid/asparagine and they have almost identical NH2-terminal sequences. The second group comprises the M(r) 63,000 protein which is rich in proline, glycine, valine and tyrosine, with an NH2-terminal sequence which was very similar to that of soybean proline-rich proteins.

  9. Radiosensitivity of tumor cell lines after pretreatment with the EGFR tyrosine kinase inhibitor ZD1839 (Iressa {sup registered})

    Energy Technology Data Exchange (ETDEWEB)

    Burdak-Rothkamm, S. [Dept. of Radiotherapy, Saarland Univ. Hospital, Homburg/Saar (Germany); Gray Cancer Inst., Northwood, Middlesex (United Kingdom); Ruebe, C.E.; Nguyen, T.P.; Ludwig, D.; Ruebe, C. [Dept. of Radiotherapy, Saarland Univ. Hospital, Homburg/Saar (Germany); Feldmann, K. [AstraZeneca GmbH, Wedel (Germany); Wiegel, T. [Dept. of Radiotherapy, Univ. Hospital Benjamin Franklin, Berlin (Germany)

    2005-03-01

    Background and purpose: the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor ZD1839 (Iressa registered) reduces survival and augments radiation response of certain tumor cells. The aim of this study was to identify cellular events that are associated with the modulation of radiosensitivity by ZD1839. Material and methods: three tumor cell lines (A549, H596, FaDu) were exposed to ionizing radiation, treatment with ZD1839, and combined treatment. Clonogenic cell survival was determined by colony assays, EGFR and transforming growth factor-(TGF-){alpha} expression by quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR), cell cycle distribution and apoptosis by flow cytometry. Results: in A549 and H596 cells ZD1839 had little effect on clonogenic growth, but survival curves revealed a radiosensitizing effect of 5 {mu}M ZD1839 on A549 cells. Both cell lines expressed moderate amounts of EGFR mRNA and very low levels of TGF-{alpha} mRNA. FaDu cells expressed relatively high amounts of EGFR and TGF-{alpha} transcripts and showed marked inhibition of clonogenic growth, reduction of S-phase cells, and induction of apoptosis after treatment with 1 {mu}M ZD1839 and combined treatment. Surprisingly, the subpopulation of FaDu cells surviving ZD1839 pretreatment was more radioresistant. Exposure to ZD1839 caused a decrease in EGFR mRNA expression in A549 cells, no change in H596, and even an increase in FaDu cells. Conclusion: the sensitivity to ZD1839 correlated with the EGFR expression level, an inhibition of cell proliferation, and induction of apoptosis in the cell lines analyzed. A radiosensitizing effect of ZD1839 was associated with downregulation of EGFR mRNA expression. (orig.)

  10. A specific A/T polymorphism in Western tyrosine phosphorylation B-motifs regulates Helicobacter pylori CagA epithelial cell interactions.

    Science.gov (United States)

    Zhang, Xue-Song; Tegtmeyer, Nicole; Traube, Leah; Jindal, Shawn; Perez-Perez, Guillermo; Sticht, Heinrich; Backert, Steffen; Blaser, Martin J

    2015-02-01

    Helicobacter pylori persistently colonizes the human stomach, with mixed roles in human health. The CagA protein, a key host-interaction factor, is translocated by a type IV secretion system into host epithelial cells, where its EPIYA tyrosine phosphorylation motifs (TPMs) are recognized by host cell kinases, leading to multiple host cell signaling cascades. The CagA TPMs have been described as type A, B, C or D, each with a specific conserved amino acid sequence surrounding EPIYA. Database searching revealed strong non-random distribution of the B-motifs (including EPIYA and EPIYT) in Western H. pylori isolates. In silico analysis of Western H. pylori CagA sequences provided evidence that the EPIYT B-TPMs are significantly less associated with gastric cancer than the EPIYA B-TPMs. By generating and using a phosphorylated CagA B-TPM-specific antibody, we demonstrated the phosphorylated state of the CagA B-TPM EPIYT during H. pylori co-culture with host cells. We also showed that within host cells, CagA interaction with phosphoinositol 3-kinase (PI3-kinase) was B-TPM tyrosine-phosphorylation-dependent, and the recombinant CagA with EPIYT B-TPM had higher affinity to PI3-kinase and enhanced induction of AKT than the isogenic CagA with EPIYA B-TPM. Structural modeling of the CagA B-TPM motif bound to PI3-kinase indicated that the threonine residue at the pY+1 position forms a side-chain hydrogen bond to N-417 of PI3-kinase, which cannot be formed by alanine. During co-culture with AGS cells, an H. pylori strain with a CagA EPIYT B-TPM had significantly attenuated induction of interleukin-8 and hummingbird phenotype, compared to the isogenic strain with B-TPM EPIYA. These results suggest that the A/T polymorphisms could regulate CagA activity through interfering with host signaling pathways related to carcinogenesis, thus influencing cancer risk.

  11. A specific A/T polymorphism in Western tyrosine phosphorylation B-motifs regulates Helicobacter pylori CagA epithelial cell interactions.

    Directory of Open Access Journals (Sweden)

    Xue-Song Zhang

    2015-02-01

    Full Text Available Helicobacter pylori persistently colonizes the human stomach, with mixed roles in human health. The CagA protein, a key host-interaction factor, is translocated by a type IV secretion system into host epithelial cells, where its EPIYA tyrosine phosphorylation motifs (TPMs are recognized by host cell kinases, leading to multiple host cell signaling cascades. The CagA TPMs have been described as type A, B, C or D, each with a specific conserved amino acid sequence surrounding EPIYA. Database searching revealed strong non-random distribution of the B-motifs (including EPIYA and EPIYT in Western H. pylori isolates. In silico analysis of Western H. pylori CagA sequences provided evidence that the EPIYT B-TPMs are significantly less associated with gastric cancer than the EPIYA B-TPMs. By generating and using a phosphorylated CagA B-TPM-specific antibody, we demonstrated the phosphorylated state of the CagA B-TPM EPIYT during H. pylori co-culture with host cells. We also showed that within host cells, CagA interaction with phosphoinositol 3-kinase (PI3-kinase was B-TPM tyrosine-phosphorylation-dependent, and the recombinant CagA with EPIYT B-TPM had higher affinity to PI3-kinase and enhanced induction of AKT than the isogenic CagA with EPIYA B-TPM. Structural modeling of the CagA B-TPM motif bound to PI3-kinase indicated that the threonine residue at the pY+1 position forms a side-chain hydrogen bond to N-417 of PI3-kinase, which cannot be formed by alanine. During co-culture with AGS cells, an H. pylori strain with a CagA EPIYT B-TPM had significantly attenuated induction of interleukin-8 and hummingbird phenotype, compared to the isogenic strain with B-TPM EPIYA. These results suggest that the A/T polymorphisms could regulate CagA activity through interfering with host signaling pathways related to carcinogenesis, thus influencing cancer risk.

  12. Tyrosine kinase JAK1 is associated with the granulocyte-colony-stimulating factor receptor and both become tyrosine-phosphorylated after receptor activation.

    OpenAIRE

    Nicholson, S. E.; Oates, A. C.; Harpur, A G; Ziemiecki, A; Wilks, A F; Layton, J E

    1994-01-01

    Granulocyte-colony-stimulating factor (G-CSF) stimulates the proliferation and differentiation of cells of the neutrophil lineage by interaction with a specific receptor. Early signal transduction events following G-CSF receptor activation were studied. We detected tyrosine phosphorylation of both the G-CSF receptor and the protein tyrosine kinase JAK1 following G-CSF binding to the human G-CSF receptor. In vitro, the kinase activity of JAK1 was increased by G-CSF stimulation. Coimmunoprecipi...

  13. Novel irreversible EGFR tyrosine kinase inhibitor 324674 sensitizes human colon carcinoma HT29 and SW480 cells to apoptosis by blocking the EGFR pathway

    Energy Technology Data Exchange (ETDEWEB)

    Yu, Zhiwei; Cui, Binbin; Jin, Yinghu; Chen, Haipeng [Division of Colorectal Surgery, Third Affiliated Hospital of Harbin Medical University, Harbin (China); Wang, Xishan, E-mail: wxshan_oncologist@yahoo.com.cn [Division of Colorectal Surgery, Third Affiliated Hospital of Harbin Medical University, Harbin (China)

    2011-08-12

    Highlights: {yields} This article described the effects of the EGFR tyrosine kinase inhibitor on the cell proliferation and the apoptosis induction of the colon carcinoma cell lines. {yields} Demonstrated that 326474 is a more potent EGFR inhibitor on colon cancer cells than other three TKIs. {yields} It can be important when considering chemotherapy for colonic cancer patients. -- Abstract: Background: Epidermal growth factor receptor (EGFR) is widely expressed in multiple solid tumors including colorectal cancer by promoting cancer cell growth and proliferation. Therefore, the inhibition of EGFR activity may establish a clinical strategy of cancer therapy. Methods: In this study, using human colon adenocarcinoma HT29 and SW480 cells as research models, we compared the efficacy of four EGFR inhibitors in of EGFR-mediated pathways, including the novel irreversible inhibitor 324674, conventional reversible inhibitor AG1478, dual EGFR/HER2 inhibitor GW583340 and the pan-EGFR/ErbB2/ErbB4 inhibitor. Cell proliferation was assessed by MTT analysis, and apoptosis was evaluated by the Annexin-V binding assay. EGFR and its downstream signaling effectors were examined by western blotting analysis. Results: Among the four inhibitors, the irreversible EGFR inhibitor 324674 was more potent at inhibiting HT29 and SW480 cell proliferation and was able to efficiently induce apoptosis at lower concentrations. Western blotting analysis revealed that AG1478, GW583340 and pan-EGFR/ErbB2/ErbB4 inhibitors failed to suppress EGFR activation as well as the downstream mitogen-activated protein kinase (MAPK) and PI3K/AKT/mTOR (AKT) pathways. In contrast, 324674 inhibited EGFR activation and the downstream AKT signaling pathway in a dose-dependent manner. Conclusion: Our studies indicated that the novel irreversible EGFR inhibitor 324674 may have a therapeutic application in colon cancer therapy.

  14. Low HIP1R mRNA and protein expression are associated with worse survival in diffuse large B-cell lymphoma patients treated with R-CHOP

    DEFF Research Database (Denmark)

    Wong, K. K.; Ch'ng, E. S.; Loo, S. K.;

    2015-01-01

    Huntingtin-interacting protein 1-related (HIP1R) is an endocytic protein involved in receptor trafficking, including regulating cell surface expression of receptor tyrosine kinases. We have previously shown that low HIP1R protein expression was associated with poorer survival in diffuse large B-c...

  15. Down-regulation of estrogen receptor-alpha and rearranged during transfection tyrosine kinase is associated with withaferin a-induced apoptosis in MCF-7 breast cancer cells

    Directory of Open Access Journals (Sweden)

    Samadi Abbas K

    2011-10-01

    Full Text Available Abstract Background Withaferin A (WA, a naturally occurring withanolide, induces apoptosis in both estrogen-responsive MCF-7 and estrogen-independent MDA-MB-231 breast cancer cell lines with higher sensitivity in MCF-7 cells, but the underlying mechanisms are not well defined. The purpose of this study was to determine the anti-cancer effects of WA in MCF-7 breast cancer cells and explore alterations in estrogen receptor alpha (ERα and its associated molecules in vitro as novel mechanisms of WA action. Methods The effects of WA on MCF-7 viability and proliferation were evaluated by 3-(4, 5-dimethylthiazol-2-yl-5-(3-carboxymethoxyphenyl-2-(4-sulfophenyl-2H-tetrazolium (MTS assay and trypan blue exclusion assays. Apoptosis was evaluated by Annexin V-fluorescein isothiocyanate (FITC/propidium iodide (PI flow cytometry and Western blot analysis of poly (ADP-ribose polymerase (PARP cleavage. Cell cycle effects were analyzed by PI flow cytometry. Western blotting was also conducted to examine alterations in the expression of ERα and pathways that are associated with ERα function. Results WA resulted in growth inhibition and decreased viability in MCF-7 cells with an IC50 of 576 nM for 72 h. It also caused a dose- and time-dependent apoptosis and G2/M cell cycle arrest. WA-induced apoptosis was associated with down-regulation of ERα, REarranged during Transfection (RET tyrosine kinase, and heat shock factor-1 (HSF1, as well as up-regulation of phosphorylated p38 mitogen-activated protein kinase (phospho-p38 MAPK, p53 and p21 protein expression. Co-treatment with protein synthesis inhibitor cycloheximide or proteasome inhibitor MG132 revealed that depletion of ERα by WA is post-translational, due to proteasome-dependent ERα degradation. Conclusions Taken together, down-regulation of ERα, RET, HSF1 and up-regulation of phospho-p38 MAPK, p53, p21 are involved in the pro-apoptotic and growth-inhibitory effects of WA in MCF-7 breast cancer cells in

  16. Xanthium strumarium as an Inhibitor of α-Glucosidase, Protein Tyrosine Phosphatase 1β, Protein Glycation and ABTS⁺ for Diabetic and Its Complication.

    Science.gov (United States)

    Hwang, Seung Hwan; Wang, Zhiqiang; Yoon, Ha Na; Lim, Soon Sung

    2016-01-01

    Phytochemical investigation of the natural products from Xanthium strumarium led to the isolation of fourteen compounds including seven caffeoylquinic acid (CQA) derivatives. The individual compounds were screened for inhibition of α-glucosidase, protein tyrosine phosphatase 1β (PTP1β), advanced glycation end products (AGEs), and ABTS⁺ radical scavenging activity using in vitro assays. Among the isolated compounds, methyl-3,5-di-caffeoyquinic acid exhibited significant inhibitory activity against α-glucosidase (18.42 μM), PTP1β (1.88 μM), AGEs (82.79 μM), and ABTS⁺ (6.03 μM). This effect was marked compared to that of the positive controls (acarbose 584.79 μM, sumarin 5.51 μM, aminoguanidine 1410.00 μM, and trolox 29.72 μM respectively). In addition, 3,5-di-O-CQA (88.14 μM) and protocatechuic acid (32.93 μM) had a considerable inhibitory effect against α-glucosidase and ABTS⁺. Based on these findings, methyl-3,5-di-caffeoyquinic acid was assumed to be potentially responsible for the anti-diabetic actions of X. strumarium. PMID:27649132

  17. Coarse-grained molecular simulation of epidermal growth factor receptor protein tyrosine kinase multi-site self-phosphorylation.

    Directory of Open Access Journals (Sweden)

    John G Koland

    2014-01-01

    Full Text Available Upon the ligand-dependent dimerization of the epidermal growth factor receptor (EGFR, the intrinsic protein tyrosine kinase (PTK activity of one receptor monomer is activated, and the dimeric receptor undergoes self-phosphorylation at any of eight candidate phosphorylation sites (P-sites in either of the two C-terminal (CT domains. While the structures of the extracellular ligand binding and intracellular PTK domains are known, that of the ∼225-amino acid CT domain is not, presumably because it is disordered. Receptor phosphorylation on CT domain P-sites is critical in signaling because of the binding of specific signaling effector molecules to individual phosphorylated P-sites. To investigate how the combination of conventional substrate recognition and the unique topological factors involved in the CT domain self-phosphorylation reaction lead to selectivity in P-site phosphorylation, we performed coarse-grained molecular simulations of the P-site/catalytic site binding reactions that precede EGFR self-phosphorylation events. Our results indicate that self-phosphorylation of the dimeric EGFR, although generally believed to occur in trans, may well occur with a similar efficiency in cis, with the P-sites of both receptor monomers being phosphorylated to a similar extent. An exception was the case of the most kinase-proximal P-site-992, the catalytic site binding of which occurred exclusively in cis via an intramolecular reaction. We discovered that the in cis interaction of P-site-992 with the catalytic site was facilitated by a cleft between the N-terminal and C-terminal lobes of the PTK domain that allows the short CT domain sequence tethering P-site-992 to the PTK core to reach the catalytic site. Our work provides several new mechanistic insights into the EGFR self-phosphorylation reaction, and demonstrates the potential of coarse-grained molecular simulation approaches for investigating the complexities of self-phosphorylation in

  18. Protein tyrosine phosphatase 1B inhibitory activity of Indonesian herbal medicines and constituents of Cinnamomum burmannii and Zingiber aromaticum.

    Science.gov (United States)

    Saifudin, Azis; Kadota, Shigetoshi; Tezuka, Yasuhiro

    2013-04-01

    We screened water and methanol extracts of 28 Indonesian medicinal plants for their protein tyrosine phosphatase 1B (PTP1B) inhibitory activities. Nine water extracts, i.e., Alstonia scholaris leaf, Blumea balsamifera, Cinnamomum burmannii, Cymbopogon nardus, Melaleuca leucadendra, Phyllanthus niruri, Piper nigrum, Syzygium aromaticum, and Sy. polyanthum, exhibited ≥70 % inhibition at 25 μg/mL, whereas 11 methanol extracts, i.e., Als. scholaris, Andrographis paniculata, B. balsamifera, Ci. burmannii, Curcuma heyneana, Glycyrrhiza glabra, M. leucadendra, Punica granatum, Rheum palmatum, Sy. polyanthum, and Z. aromaticum, exhibited ≥70 % inhibition at 25 μg/mL. Water extracts of B. balsamifera (IC50, 2.26 μg/mL) and M. leucadendra (IC50, 2.05 μg/mL), and methanol extracts of Ci. burmannii (IC50, 2.47 μg/mL), Pu. granatum (IC50, 2.40 μg/mL), and Sy. polyanthum (IC50, 1.03 μg/mL) exhibited strong inhibitory activity, which was comparable with that of the positive control, RK-682 (IC50, 2.05 μg/mL). The PTP1B inhibitory activity of the constituents of Ci. burmannii and Z. aromaticum was then evaluated. 5'-Hydroxy-5-hydroxymethyl-4″,5″-methylenedioxy-1,2,3,4-dibenzo-1,3,5-cycloheptatriene (2; IC50, 29.7 μM) and trans-cinnamaldehyde (5; IC50, 57.6 μM) were the active constituents of Ci. burmannii, while humulatrien-5-ol-8-one (21; IC50, 27.7 μM), kaempferol-3,4'-di-O-methyl ether (32; IC50, 17.5 μM), and (S)-6-gingerol (33; IC50, 28.1 μM) were those of Z. aromaticum. These results suggest that these medicinal plants may contribute to the treatment and/or prevention of type II diabetes and/or obesity through PTP1B inhibition. PMID:22645080

  19. The anti-esophageal cancer cell activity by a novel tyrosine/phosphoinositide kinase inhibitor PP121

    Energy Technology Data Exchange (ETDEWEB)

    Peng, Yi; Zhou, Yajuan [Department of Radiation and Medical Oncology, Hubei Key Laboratory of Tumor Biological Behaviors, Zhongnan Hospital of Wuhan University, Wuhan 430071 (China); Department of Radiation Oncology, Hubei Cancer Hospital, Wuhan 430071 (China); Cheng, Long [Department of Interventional Radiology, the Second Affiliated Hospital of Soochow University, Soochow University, Suzhou 215001 (China); Hu, Desheng; Zhou, Xiaoyi; Wang, Zhaohua [Department of Radiation Oncology, Hubei Cancer Hospital, Wuhan 430071 (China); Xie, Conghua, E-mail: chxie_65@hotmail.com [Department of Radiation and Medical Oncology, Hubei Key Laboratory of Tumor Biological Behaviors, Zhongnan Hospital of Wuhan University, Wuhan 430071 (China); Zhou, Fuxiang, E-mail: ZhouFuxiangwuhan@126.com [Department of Radiation and Medical Oncology, Hubei Key Laboratory of Tumor Biological Behaviors, Zhongnan Hospital of Wuhan University, Wuhan 430071 (China)

    2015-09-11

    Here we explored the potential effect of PP121, a novel dual inhibitor of tyrosine and phosphoinositide kinases, against human esophageal cancer cells. We showed that PP121 exerted potent cytotoxic effect in primary (patient-derived) and established (Eca-109, TE-1 and TE-3 lines) esophageal cancer cells, possibly through activating caspase-3-dependnent apoptosis. PP121 was, however, non-cytotoxic to the normal human esophageal epithelial cells (EECs). At the molecular level, we showed that PP121 blocked Akt-mTOR (mammalian target of rapamycin) activation in esophageal cancer cells, which was restored by introducing a constitutively-active Akt (CA-Akt). Yet, CA-Akt only partly inhibited cytotoxicity by PP121 in Eca-109 cells. Importantly, we showed that PP121 inhibited nuclear factor kappa B (NFκB) signaling activation in esophageal cancer cells, which appeared independent of Akt-mTOR blockage. In vivo, oral administration of PP121 remarkably inhibited Eca-109 xenograft growth in nude mice, and significantly improved mice survival. Further, the immunohistochemistry (IHC) and Western blot assays analyzing xenografted tumors showed that PP121 inhibited Akt-mTOR and NFκB activations in vivo. Together, we demonstrate that PP121 potently inhibits esophageal cancer cells in vitro and in vivo, possibly through concurrently inhibiting Akt-mTOR and NFκB signalings. - Highlights: • PP121 is cytotoxic against primary and established esophageal cancer cells. • PP121 induces caspase-3-dependnent apoptosis in esophageal cancer cells. • PP121 blocks Akt-mTOR activation in esophageal cancer cells. • PP121 inhibits NFκB activation, independent of Akt-mTOR blockage. • PP121 inhibits Eca-109 xenograft growth and Akt-mTOR/NFκB activation in vivo.

  20. Combined treatment of tyrosine kinase inhibitor labeled gold nanorod encapsulated albumin with laser thermal ablation in a renal cell carcinoma model

    Science.gov (United States)

    This manuscript served to characterize and evaluate Human Serum Albumin-encapsulated Nanoparticles (NPs) for drug delivery of a tyrosine kinase inhibitor combined with induction of photothermal ablation (PTA) combination therapy of Renal Cell Carcinoma (RCC). RCC is the most common type of kidney c...

  1. Signaling mechanisms coupled to tyrosines in the granulocyte colony-stimulating factor receptor orchestrate G-CSF-induced expansion of myeloid progenitor cells

    NARCIS (Netherlands)

    M.H. Hermans; C. Heijmans-Antonissen (Claudia); J. Gits (Judith); D. van Leeuwen (Daphne); A.C. Ward (Alister); I.P. Touw (Ivo); G.J.M. van de Geijn (Gert-Jan)

    2003-01-01

    textabstractGranulocyte colony-stimulating factor (G-CSF) is the major regulator of neutrophil production. Studies in cell lines have established that conserved tyrosines Tyr704, Tyr729, Tyr744, Tyr764 within the cytoplasmic domain of G-CSF receptor (G-CSF-R) contribute significant

  2. The Bruton tyrosine kinase inhibitor PCI-32765 thwarts chronic lymphocytic leukemia cell survival and tissue homing in vitro and in vivo

    OpenAIRE

    Ponader, Sabine; Chen, Shih-Shih; Buggy, Joseph J.; Balakrishnan, Kumudha; Gandhi, Varsha; William G Wierda; Michael J Keating; O'Brien, Susan; Chiorazzi, Nicholas; Burger, Jan A.

    2012-01-01

    B-cell receptor (BCR) signaling is a critical pathway in the pathogenesis of several B-cell malignancies, including chronic lymphocytic leukemia (CLL), and can be targeted by inhibitors of BCR-associated kinases, such as Bruton tyrosine kinase (Btk). PCI-32765, a selective, irreversible Btk inhibitor, is a novel, molecularly targeted agent for patients with B-cell malignancies, and is particularly active in patients with CLL. In this study, we analyzed the mechanism of action of PCI-32765 in ...

  3. Expression profiling of receptor tyrosine kinases in high-grade neuroendocrine carcinoma of the lung: a comparative analysis with adenocarcinoma and squamous cell carcinoma

    OpenAIRE

    MATSUMURA, YUKI; Umemura, Shigeki; Ishii, Genichiro; Tsuta, Koji; Matsumoto, Shingo; Aokage, Keiju; Hishida, Tomoyuki; Yoshida, Junji; Ohe, Yuichiro; Suzuki, Hiroyuki; Ochiai, Atsushi; Goto, Koichi; Nagai, Kanji; Tsuchihara, Katsuya

    2015-01-01

    Background As the comprehensive genomic analysis of small cell lung cancer (SCLC) progresses, novel treatments for this disease need to be explored. With attention to the direct connection between the receptor tyrosine kinases (RTKs) of tumor cells and the pharmacological effects of specific inhibitors, we systematically assessed the RTK expressions of high-grade neuroendocrine carcinomas of the lung [HGNECs, including SCLC and large cell neuroendocrine carcinoma (LCNEC)]. Patients and method...

  4. Activity of Bruton's tyrosine-kinase inhibitor ibrutinib in patients with CD117-positive acute myeloid leukaemia: a mechanistic study using patient-derived blast cells

    OpenAIRE

    Rushworth, Stuart; Pillinger, Genevra; Abdul-Aziz, Amina; Piddock, Rachel; Shafat, Manar S.; Murray, Megan Y; Zaitseva, Lyubov; Lawes, Matthew J.; MacEwan, David J.; Bowles, Kristian M.

    2015-01-01

    Summary Background Roughly 80% of patients with acute myeloid leukaemia have high activity of Bruton's tyrosine-kinase (BTK) in their blast cells compared with normal haemopoietic cells, rendering the cells sensitive to the oral BTK inhibitor ibrutinib in vitro. We aimed to develop the biological understanding of the BTK pathway in acute myeloid leukaemia to identify clinically relevant diagnostic information that might define a subset of patients that should respond to ibrutinib treatment. M...

  5. A selective Seoul-Fluor-based bioprobe, SfBP, for vaccinia H1-related phosphatase--a dual-specific protein tyrosine phosphatase.

    Science.gov (United States)

    Jeong, Myeong Seon; Kim, Eunha; Kang, Hyo Jin; Choi, Eun Joung; Cho, Alvin R; Chung, Sang J; Park, Seung Bum

    2012-07-01

    We report a Seoul-Fluor-based bioprobe, SfBP, for selective monitoring of protein tyrosine phosphatases (PTPs). A rational design based on the structures at the active site of dual-specific PTPs can enable SfBP to selectively monitor the activity of these PTPs with a 93-fold change in brightness. Moreover, screening results of SfBP against 30 classical PTPs and 35 dual-specific PTPs show that it is selective toward vaccinia H1-related (VHR) phosphatase, a dual-specific PTP (DUSP-3).

  6. SILAC-based quantification of changes in protein tyrosine phosphorylation induced by Interleukin-2 (IL-2) and IL-15 in T-lymphocytes

    DEFF Research Database (Denmark)

    Osinalde, Nerea; Sánchez-Quiles, Virginia; Akimov, Vyacheslav;

    2015-01-01

    This data article presents the first large-scale quantitative phosphoproteomics dataset generated to decipher the signaling networks initiated by IL-2 and IL-15 in T-lymphocytes. Data was collected by combining immunoprecipitation of tyrosine phosphorylated proteins and TiO2-based phosphopeptide....... The data supplied in this article is related to the research work entitled "Simultaneous dissection and comparison of IL-2 and IL-15 signaling pathways by global quantitative phosphoproteomics" [1]. All mass spectrometry data have been deposited in the ProteomeXchange with the identifier PXD001129....

  7. The V protein of canine distemper virus is required for virus replication in human epithelial cells.

    Directory of Open Access Journals (Sweden)

    Noriyuki Otsuki

    Full Text Available Canine distemper virus (CDV becomes able to use human receptors through a single amino acid substitution in the H protein. In addition, CDV strains possessing an intact C protein replicate well in human epithelial H358 cells. The present study showed that CDV strain 007Lm, which was isolated from lymph node tissue of a dog with distemper, failed to replicate in H358 cells, although it possessed an intact C protein. Sequence analyses suggested that a cysteine-to-tyrosine substitution at position 267 of the V protein caused this growth defect. Analyses using H358 cells constitutively expressing the CDV V protein showed that the V protein with a cysteine, but not that with a tyrosine, at this position effectively blocked the interferon-stimulated signal transduction pathway, and supported virus replication of 007Lm in H358 cells. Thus, the V protein as well as the C protein appears to be functional and essential for CDV replication in human epithelial cells.

  8. Hypoxia increases rate of transcription and stability of tyrosine hydroxylase mRNA in pheochromocytoma (PC12) cells.

    Science.gov (United States)

    Czyzyk-Krzeska, M F; Furnari, B A; Lawson, E E; Millhorn, D E

    1994-01-01

    Reduced arterial oxygen tension (i.e. hypoxia) is a powerful physiological stimulus that induces synthesis and release of dopamine from O2-sensitive (type I) cells in the mammalian carotid bodies. We reported recently that hypoxia stimulates gene expression for tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine synthesis in type I cells of the carotid body. Efforts to identify the mechanisms regulating TH gene expression in O2-sensitive cells during hypoxia have been hampered by the lack of an appropriate model cell culture system. Here we report that TH gene expression in the rat pheochromocytoma cell line (PC12) is regulated during hypoxia in a manner similar to that measured in carotid body type I cells. PC12 cells might therefore be useful as an experimental model for identifying the molecular mechanisms that regulate TH gene expression during hypoxia. Nuclear runoff assays revealed that transcription of the wild type TH gene was enhanced during exposures to hypoxia lasting 12 h. Chloramphenicol acetyltransferase assays with constructs that contained different fragments of TH promoter revealed that the regulatory sequences that mediate the hypoxia-induced increase in transcription are located between bases -272 and +27 of the TH gene. Findings from experiments in which transcription was inhibited either with actinomycin D or 5,6-dichloro-1-D-ribofuranosylbenzimidazole, as well as pulse-chase experiments using 4-thiouridine showed that the half-life of TH mRNA was substantially increased during hypoxia. Thus, in the present paper we show that TH gene expression in PC12 cells during hypoxia is regulated by increases in both the rate of TH gene transcription and TH mRNA stability. PMID:7903970

  9. Phosphorylation at tyrosine 114 of Proliferating Cell Nuclear Antigen (PCNA) is required for adipogenesis in response to high fat diet

    Energy Technology Data Exchange (ETDEWEB)

    Lo, Yuan-Hung; Ho, Po-Chun; Chen, Min-Shan; Hugo, Eric; Ben-Jonathan, Nira [Department of Cancer Biology, University of Cincinnati College of Medicine, 3125 Eden Avenue, Cincinnati, OH 45267-0521 (United States); Department of Environmental Health, University of Cincinnati College of Medicine, 3223 Eden Avenue, Kettering Laboratory, Cincinnati, OH 45267-0056 (United States); Wang, Shao-Chun, E-mail: shao-chun.wang@uc.edu [Department of Cancer Biology, University of Cincinnati College of Medicine, 3125 Eden Avenue, Cincinnati, OH 45267-0521 (United States); Department of Environmental Health, University of Cincinnati College of Medicine, 3223 Eden Avenue, Kettering Laboratory, Cincinnati, OH 45267-0056 (United States)

    2013-01-04

    Highlights: Black-Right-Pointing-Pointer Proliferating Cell Nuclear Antigen (PCNA) is phosphorylated at Y114. Black-Right-Pointing-Pointer Phospho-Y114 of PCNA is not required for cell proliferation for normal growth. Black-Right-Pointing-Pointer MCE during adipogenesis is abolished in the lack of the phosphorylation. Black-Right-Pointing-Pointer Homozygous Y114F mice are resistant to high fat diet induced obesity. Black-Right-Pointing-Pointer Our results shed light on the interface between proliferation and differentiation. -- Abstract: Clonal proliferation is an obligatory component of adipogenesis. Although several cell cycle regulators are known to participate in the transition between pre-adipocyte proliferation and terminal adipocyte differentiation, how the core DNA synthesis machinery is coordinately regulated in adipogenesis remains elusive. PCNA (Proliferating Cell Nuclear Antigen) is an indispensable component for DNA synthesis during proliferation. Here we show that PCNA is subject to phosphorylation at the highly conserved tyrosine residue 114 (Y114). Replacing the Y114 residue with phenylalanine (Y114F), which is structurally similar to tyrosine but cannot be phosphorylated, does not affect normal animal development. However, when challenged with high fat diet, mice carrying homozygous Y114F alleles (PCNA{sup F/F}) are resistant to adipose tissue enlargement in comparison to wild-type (WT) mice. Mouse embryonic fibroblasts (MEFs) harboring WT or Y114F mutant PCNA proliferate at similar rates. However, when subjected to adipogenesis induction in culture, PCNA{sup F/F} MEFs are not able to re-enter the cell cycle and fail to form mature adipocytes, while WT MEFs undergo mitotic clonal expansion in response to the adipogenic stimulation, accompanied by enhanced Y114 phosphorylation of PCNA, and differentiate to mature adipocytes. Consistent with the function of Y114 phosphorylation in clonal proliferation in adipogenesis, fat tissues isolated from WT

  10. SILAC-based quantification of changes in protein tyrosine phosphorylation induced by Interleukin-2 (IL-2 and IL-15 in T-lymphocytes

    Directory of Open Access Journals (Sweden)

    Nerea Osinalde

    2015-12-01

    Full Text Available This data article presents the first large-scale quantitative phosphoproteomics dataset generated to decipher the signaling networks initiated by IL-2 and IL-15 in T-lymphocytes. Data was collected by combining immunoprecipitation of tyrosine phosphorylated proteins and TiO2-based phosphopeptide enrichment with SILAC-based quantitative mass spectrometry. We report all the proteins and phosphotyrosine-containing peptides identified and quantified in IL-2- and IL-15-stimulated T-lymphocytes. The gene ontology analysis of IL-2 and IL-15 effector proteins detected in the present work is also included. The data supplied in this article is related to the research work entitled “Simultaneous dissection and comparison of IL-2 and IL-15 signaling pathways by global quantitative phosphoproteomics” [1]. All mass spectrometry data have been deposited in the ProteomeXchange with the identifier PXD001129.

  11. Activation of cardiac chloride conductance by the tyrosine kinase inhibitor, genistein.

    OpenAIRE

    Shuba, L. M.; Asai, T.; Pelzer, S.; McDonald, T. F.

    1996-01-01

    1. Genistein (GST), an inhibitor of protein tyrosine kinase (PTK), Na3VO4 (VO4), an inhibitor of phosphotyrosine phosphatase (PTPase), and forskolin (FSK), an activator of the cyclic AMP-dependent, cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel, were applied to guinea-pig ventricular myocytes to probe for a possible role of tyrosine phosphorylation in the regulation of cardiac Cl- channels. 2. Myocytes in the standard whole-cell configuration were pulsed to various pot...

  12. The V Protein of Canine Distemper Virus Is Required for Virus Replication in Human Epithelial Cells

    OpenAIRE

    Noriyuki Otsuki; Yuichiro Nakatsu; Toru Kubota; Tsuyoshi Sekizuka; Fumio Seki; Kouji Sakai; Makoto Kuroda; Ryoji Yamaguchi; Makoto Takeda

    2013-01-01

    Canine distemper virus (CDV) becomes able to use human receptors through a single amino acid substitution in the H protein. In addition, CDV strains possessing an intact C protein replicate well in human epithelial H358 cells. The present study showed that CDV strain 007Lm, which was isolated from lymph node tissue of a dog with distemper, failed to replicate in H358 cells, although it possessed an intact C protein. Sequence analyses suggested that a cysteine-to-tyrosine substitution at posit...

  13. X-box-binding protein 1-modified neural stem cells for treatment of Parkinson's disease.

    Science.gov (United States)

    Si, Lihui; Xu, Tianmin; Wang, Fengzhang; Liu, Qun; Cui, Manhua

    2012-04-01

    X-box-binding protein 1-transfected neural stem cells were transplanted into the right lateral ventricles of rats with rotenone-induced Parkinson's disease. The survival capacities and differentiation rates of cells expressing the dopaminergic marker tyrosine hydroxylase were higher in X-box-binding protein 1-transfected neural stem cells compared to non-transfected cells. Moreover, dopamine and 3,4-dihydroxyphenylacetic acid levels in the substantia nigra were significantly increased, α-synuclein expression was decreased, and neurological behaviors were significantly ameliorated in rats following transplantation of X-box-binding protein 1-transfected neural stem cells. These results indicate that transplantation of X-box-binding protein 1-transfected neural stem cells can promote stem cell survival and differentiation into dopaminergic neurons, increase dopamine and 3,4-dihydroxyphenylacetic acid levels, reduce α-synuclein aggregation in the substantia nigra, and improve the symptoms of Parkinson's disease in rats.

  14. X-box-binding protein 1-modified neural stem cells for treatment of Parkinson's disease

    Institute of Scientific and Technical Information of China (English)

    Lihui Si; Tianmin Xu; Fengzhang Wang; Qun Liu; Manhua Cui

    2012-01-01

    X-box-binding protein 1-transfected neural stem cells were transplanted into the right lateral ventricles of rats with rotenone-induced Parkinson's disease. The survival capacities and differentiation rates of cells expressing the dopaminergic marker tyrosine hydroxylase were higher in X-box-binding protein 1-transfected neural stem cells compared to non-transfected cells. Moreover, dopamine and 3,4-dihydroxyphenylacetic acid levels in the substantia nigra were significantly increased, α-synuclein expression was decreased, and neurological behaviors were significantly ameliorated in rats following transplantation of X-box-binding protein 1-transfected neural stem cells. These results indicate that transplantation of X-box-binding protein 1-transfected neural stem cells can promote stem cell survival and differentiation into dopaminergic neurons, increase dopamine and 3,4-dihydroxyphenylacetic acid levels, reduce α-synuclein aggregation in the substantia nigra, and improve the symptoms of Parkinson's disease in rats.

  15. QSAR AND PHARMACOPHORE MODELING BASED DRUG DESIGNING FOR SPLEEN TYROSINE KINASE (SYK PROTEIN FOR HUMAN USING ACCELRYS DISCOVERY STUDIO SOFTWARE IN LINUX SERVER

    Directory of Open Access Journals (Sweden)

    Kuldeep Sahu

    2013-11-01

    Full Text Available From this current research, Syk (spleen tyrosine kinase protein and gene information is analyzed by different genomics, proteomics tools & databases. One crystal ligand 4DFL was collected from protein data bank (pdb. From different literature review 131 syk protein inhibitors were collected. Molecular modeling of these 131 molecules was done through Accelrys discovery studio (ADS. Choose appropriate force-field & minimization (Smart, Stephent Descent, and Conjugate Gradient according to selected molecules. Then collected crystal ligand is purified by protein purification method and used appropriate conformation (BEST, FAST, and CAESAR. Docking methods were analyzed with protein, crystal ligand and similar inhibitors to know the best protein-ligand interaction. Pharmacophore research is done through HIPHOP and HYPOGEN method. Protein with final compound docking method is done after completion of virtual screening method. Pharmacophore research with final molecule was done. Quantitative structure activity relationship (qsar method is analyzed to know the correlation between the above selective structures. From virtual screening method, best and final compound is analyzed. So, final molecule can be a drug molecule for SYK protein abnormality diseases. However, the scope for fine tuning and optimizing this potent class of syK inhibitors could lead to the generation of new therapeutic agents.

  16. p66Shc longevity protein regulates the proliferation of human ovarian cancer cells.

    Science.gov (United States)

    Muniyan, Sakthivel; Chou, Yu-Wei; Tsai, Te-Jung; Thomes, Paul; Veeramani, Suresh; Benigno, Benedict B; Walker, L DeEtte; McDonald, John F; Khan, Shafiq A; Lin, Fen-Fen; Lele, Subodh M; Lin, Ming-Fong

    2015-08-01

    p66Shc functions as a longevity protein in murine and exhibits oxidase activity in regulating diverse biological activities. In this study, we investigated the role of p66Shc protein in regulating ovarian cancer (OCa) cell proliferation. Among three cell lines examined, the slowest growing OVCAR-3 cells have the lowest level of p66Shc protein. Transient transfection with p66Shc cDNA expression vector in OVCAR-3 cells increases cell proliferation. Conversely, knock-down of p66Shc by shRNA in rapidly growing SKOV-3 cells results in decreased cell growth. In estrogen (E2)-treated CaOV-3 cells, elevated p66Shc protein level correlates with ROS level, ErbB-2 and ERK/MAPK activation, and cell proliferation. Further, the E2-stimulated proliferation of CaOV-3 cells was blocked by antioxidants and ErbB-2 inhibitor. Additionally, in E2-stimulated cells, the tartrate-sensitive, but not the tartrate-resistant, phosphatase activity decreases; concurrently, the tyrosine phosphorylation of ErbB-2 increases. Conversely, inhibition of phosphatase activity by L(+)-tartrate treatment increases p66Shc protein level, ErbB-2 tyrosine phosphorylation, ERK/MAPK activation, and cell growth. Further, inhibition of the ERK/MAPK pathway by PD98059 blocks E2-induced ERK/MAPK activation and cell proliferation in CaOV-3 cells. Moreover, immunohistochemical analyses showed that the p66Shc protein level was significantly higher in cancerous cells than in noncancerous cells in archival OCa tissues (n = 76; P = 0.00037). These data collectively indicate that p66Shc protein plays a critical role in up-regulating OCa progression. PMID:24395385

  17. Therapeutic potential and challenges of targeting receptor tyrosine kinase ROR1 with monoclonal antibodies in B-cell malignancies.

    Directory of Open Access Journals (Sweden)

    Jiahui Yang

    Full Text Available BACKGROUND: Based on its selective cell surface expression in chronic lymphocytic leukemia (CLL and mantle cell lymphoma (MCL, receptor tyrosine kinase ROR1 has recently emerged as a promising target for therapeutic monoclonal antibodies (mAbs. To further assess the suitability of ROR1 for targeted therapy of CLL and MCL, a panel of mAbs was generated and its therapeutic utility was investigated. METHODOLOGY AND PRINCIPAL FINDINGS: A chimeric rabbit/human Fab library was generated from immunized rabbits and selected by phage display. Chimeric rabbit/human Fab and IgG1 were investigated for their capability to bind to human and mouse ROR1, to mediate antibody-dependent cellular cytotoxicity (ADCC, complement-dependent cytotoxicity (CDC, and internalization, and to agonize or antagonize apoptosis using primary CLL cells from untreated patients as well as MCL cell lines. A panel of mAbs demonstrated high affinity and specificity for a diverse set of epitopes that involve all three extracellular domains of ROR1, are accessible on the cell surface, and mediate internalization. The mAb with the highest affinity and slowest rate of internalization was found to be the only mAb that mediated significant, albeit weak, ADCC. None of the mAbs mediated CDC. Alone, they did not enhance or inhibit apoptosis. CONCLUSIONS AND SIGNIFICANCE: Owing to its relatively low cell surface density, ROR1 may be a preferred target for armed rather than naked mAbs. Provided is a panel of fully sequenced and thoroughly characterized anti-ROR1 mAbs suitable for conversion to antibody-drug conjugates, immunotoxins, chimeric antigen receptors, and other armed mAb entities for preclinical and clinical studies.

  18. Change of glutamic acid decarboxylase antibody and protein tyrosine phosphatase antibody in Chinese patients with acute-onset type 1 diabetes mellitus

    Institute of Scientific and Technical Information of China (English)

    CHAO Chen; HUANG Gan; LI Xia; YANG Lin; LIN Jian; JIN Ping; LUO Shuo-ming

    2013-01-01

    Background Glutamic acid decarboxylase antibody (GADA) and protein tyrosine phosphatase antibody (IA-2A) are two major autoantibodies,which exert important roles in the process of type 1 diabetes mellitus (T1D).Our study aimed to investigate the changes in positivity and titers of GADA and IA-2A during the course of Chinese acute-onset T1D patients and their relationships with clinical features.Methods Two hundreds and forty-seven Chinese newly diagnosed acute-onset T1D patients were consecutively recruited.GADA and IA-2A were detected at the time of diagnosis,one year later,3-5 years later after diagnosis during the follow-up; all the clinical data were recorded and analyzed as well.Results During the course of acute-onset T1D,the majority of patients remained stable for GADA or IA-2A,however,a few patients changed from positivity to negativity and fewer patients converted from negativity to positivity.The prevalence of GADA was 56.3% at diagnosis,decreasing to 50.5% one year later,and 43.3% 3-5 years later while the corresponding prevalence of IA-2A were 32.8%,31.0% and 23.3%,respectively.The median GADA titers were 0.0825 at diagnosis,declining to 0.0585 one year later and 0.0383 3-5 years later (P <0.001),while the corresponding median titers were 0.0016,0.0010,0.0014 for IA-2A,respectively.Fasting C-peptide (FCP) and postprandial C-peptide 2 hours (PCP2h)levels of GADA or IA-2A negativity persistence patients were higher than those of positivity persistence and negativity conversion patients (P <0.05) which indicated GADA or IA-2A negativity persistence T1D patients had a less loss of β cell function.Conclusion Our data suggest that repeated detection of GADA and IA-2A are necessary for differential diagnosis of autoimmune diabetes and the indirect prediction of the β cell function in Chinese patients.

  19. Phenylalanine and tyrosine levels are rate-limiting factors in production of health promoting metabolites in Vitis vinifera cv. Gamay Red cell suspension.

    Science.gov (United States)

    Manela, Neta; Oliva, Moran; Ovadia, Rinat; Sikron-Persi, Noga; Ayenew, Biruk; Fait, Aaron; Galili, Gad; Perl, Avichai; Weiss, David; Oren-Shamir, Michal

    2015-01-01

    Environmental stresses such as high light intensity and temperature cause induction of the shikimate pathway, aromatic amino acids (AAA) pathways, and of pathways downstream from AAAs. The induction leads to production of specialized metabolites that protect the cells from oxidative damage. The regulation of the diverse AAA derived pathways is still not well understood. To gain insight on that regulation, we increased AAA production in red grape Vitis vinifera cv. Gamay Red cell suspension, without inducing external stress on the cells, and characterized the metabolic effect of this induction. Increased AAA production was achieved by expressing a feedback-insensitive bacterial form of 3-deoxy- D-arabino-heptulosonate 7-phosphate synthase enzyme (AroG (*)) of the shikimate pathway under a constitutive promoter. The presence of AroG(*) protein led to elevated levels of primary metabolites in the shikimate and AAA pathways including phenylalanine and tyrosine, and to a dramatic increase in phenylpropanoids. The AroG (*) transformed lines accumulated up to 20 and 150 fold higher levels of resveratrol and dihydroquercetin, respectively. Quercetin, formed from dihydroquercetin, and resveratrol, are health promoting metabolites that are induced due to environmental stresses. Testing the expression level of key genes along the stilbenoids, benzenoids, and phenylpropanoid pathways showed that transcription was not affected by AroG (*). This suggests that concentrations of AAAs, and of phenylalanine in particular, are rate-limiting in production of these metabolites. In contrast, increased phenylalanine production did not lead to elevated concentrations of anthocyanins, even though they are also phenylpropanoid metabolites. This suggests a control mechanism of this pathway that is independent of AAA concentration. Interestingly, total anthocyanin concentrations were slightly lower in AroG(*) cells, and the relative frequencies of the different anthocyanins changed as well.

  20. Phenylalanine and tyrosine levels are rate-limiting factors in production of health promoting metabolites in Vitis vinifera cv. Gamay Red cell suspension

    Directory of Open Access Journals (Sweden)

    Neta eManela

    2015-07-01

    Full Text Available Environmental stresses such as high light intensity and temperature cause induction of the shikimate pathway, aromatic amino acids (AAA pathways, and of pathways downstream from AAAs. The induction leads to production of specialized metabolites that protect the cells from oxidative damage. The regulation of the diverse AAA derived pathways is still not well understood. To gain insight on that regulation, we increased AAA production in red grape Vitis vinifera cv. Gamay Red cell suspension, without inducing external stress on the cells, and characterized the metabolic effect of this induction. Increased AAA production was achieved by expressing a feedback-insensitive bacterial form of 3-deoxy- D-arabino-heptulosonate 7-phosphate synthase enzyme (AroG* of the shikimate pathway under a constitutive promoter. The presence of AroG* protein led to elevated levels of primary metabolites in the shikimate and AAA pathways including phenylalanine and tyrosine, and to a dramatic increase in phenylpropanoids. The AroG* transformed lines accumulated up to 20 and 150 fold higher levels of resveratrol and dihydroquercetin, respectively. Quercetin, formed from dihydroquercetin, and resveratrol, are health promoting metabolites that are induced due to environmental stresses. Testing the expression level of key genes along the stilbenoids, benzenoids and phenylpropanoid pathways showed that transcription was not affected by AroG*. This suggests that concentrations of AAAs, and of phenylalanine in particular, are rate-limiting in production of these metabolites. In contrast, increased phenylalanine production did not lead to elevated concentrations of anthocyanins, even though they are also phenylpropanoid metabolites. This suggests a control mechanism of this pathway that is independent of AAA concentration. Interestingly, total anthocyanin concentrations were slightly lower in AroG* cells, and the relative frequencies of the different anthocyanins changed as

  1. Neuromedin B receptors regulate EGF receptor tyrosine phosphorylation in lung cancer cells

    OpenAIRE

    Moody, Terry W.; Berna, Marc J.; Mantey, Samuel; Sancho, Veronica; Ridnour, Lisa; Wink, David A.; Chan, Daniel; Giaccone, Giuseppe; Jensen, Robert T.

    2010-01-01

    Neuromedin B (NMB), a member of the bombesin family of peptides, is an autocrine growth factor for many lung cancer cells. The present study investigated the ability of NMB to cause transactivation of the epidermal growth factor (EGF) receptor in lung cancer cells. By Western blot, addition of NMB or related peptides to NCI-H1299 human non-small cell lung cancer (NSCLC) cells, caused phosphorylation of Tyr1068 of the EGF receptor. The signal was amplified using NCI-H1299 cells stably transect...

  2. Effects of Tyrosine Kinase inhibitor Imatinib (Glivec) on PDGFR-positive primary and metastatic melanoma cells

    International Nuclear Information System (INIS)

    In summary these preliminary results indicate that Imatinib is able to induce apoptosis in metastatic cells and to sensitize these cells to pro-apoptotic agents commonly used in melanoma therapy, e.g. radiation or Cisplatin. Conversely, primary melanoma cells seem to be intrinsically resistant either to Imatinib given alone or in combination with Cisplatin or radiation. By contrast, these cells underwent autophagy and replicative senescence boostering their survival. Interestingly, the use of Imatinib in combination with anti-CD95/Fas antibodies sensitizes primary melanoma cells to apoptosis

  3. Heteromeric MAPPIT: a novel strategy to study modification-dependent protein-protein interactions in mammalian cells.

    Science.gov (United States)

    Lemmens, Irma; Eyckerman, Sven; Zabeau, Lennart; Catteeuw, Dominiek; Vertenten, Els; Verschueren, Kristin; Huylebroeck, Danny; Vandekerckhove, Joël; Tavernier, Jan

    2003-07-15

    We recently reported a two-hybrid trap for detecting protein-protein interactions in intact mammalian cells (MAPPIT). The bait protein was fused to a STAT recruitment-deficient, homodimeric cytokine receptor and the prey protein to functional STAT recruitment sites. In such a configuration, STAT-dependent responses can be used to monitor a given bait-prey interaction. Using this system, we were able to demonstrate both modification-independent and tyrosine phosphorylation- dependent interactions. Protein modification in this approach is, however, strictly dependent on the receptor-associated JAK tyrosine kinases. We have now extended this concept by using extracellular domains of the heteromeric granulocyte/macrophage colony-stimulating factor receptor (GM-CSFR). Herein, the bait was fused to the (beta)c chain and its modifying enzyme to the GM-CSFRalpha chain (or vice versa). We demonstrate several serine phosphorylation-dependent interactions in the TGFbeta/Smad pathway using the catalytic domains of the ALK4 or ALK6 serine/threonine kinase receptors. In all cases tested, STAT-dependent signaling was completely abolished when mutant baits were used wherein critical serine residues were replaced by alanines. This approach operates both in transient and stable expression systems and may not be limited to serine phosphorylation but has the potential for studying various different types of protein modification-dependent interactions in intact cells. PMID:12853652

  4. Imaging the intracellular distribution of tyrosine kinase inhibitors in living cells with quantitative hyperspectral stimulated Raman scattering

    Science.gov (United States)

    Fu, Dan; Zhou, Jing; Zhu, Wenjing Suzanne; Manley, Paul W.; Wang, Y. Karen; Hood, Tami; Wylie, Andrew; Xie, X. Sunney

    2014-07-01

    ABL1 tyrosine-kinase inhibitors (TKI) are front-line therapy for chronic myelogenous leukaemia and are among the best-known examples of targeted cancer therapeutics. However, the dynamic uptake into cells of TKIs of low molecular weight and their intracellular behaviour is unknown because of the difficulty of observing non-fluorescent small molecules at subcellular resolution. Here we report the direct label-free visualization and quantification of two TKI drugs (imatinib and nilotinib) inside living cells using hyperspectral stimulated Raman scattering imaging. Concentrations of both drugs were enriched over 1,000-fold in lysosomes as a result of their lysosomotropic properties. In addition, low solubility appeared to contribute significantly to the surprisingly large accumulation of nilotinib. We further show that the lysosomal trapping of imatinib was reduced more than tenfold when chloroquine is used simultaneously, which suggests that chloroquine may increase the efficacy of TKIs through lysosome-mediated drug-drug interaction in addition to the commonly proposed autophagy-inhibition mechanism.

  5. Molecular Mechanisms Contributing to Resistance to Tyrosine Kinase-Targeted Therapy for Non-Small Cell Lung Cancer

    International Nuclear Information System (INIS)

    One of the most important pathways in non-small cell lung cancer (NSCLC) is the epidermal growth factor receptor (EGFR) pathway. This pathway affects several crucial processes in tumor development and progression, including tumor cell proliferation, apoptosis regulation, angiogenesis, and metastatic invasion. Targeting EGFR is currently being intensely explored. We are witnessing the development of a number of potential molecular-inhibiting treatments for application in clinical oncology. In the last decade, the tyrosine kinase (TK) domain of the EGFR was identified in NSCLC patients, and it has responded very well with a dramatic clinical improvement to TK inhibitors such are gefitinib and erlotinib. Unfortunately, there were primary and/or secondary resistance to these treatments, as shown by clinical trials. Subsequent molecular biology studies provided some explanations for the drug resistance phenomenon. The molecular mechanisms of resistance need to be clarified. An in-depth understanding of these targeted-therapy resistance may help us explore new strategies for overcoming or reversing the resistance to these inhibitors for the future of NSCLC treatment

  6. Enediyne lidamycin enhances the effect of epidermal growth factor receptor tyrosine kinase inhibitor, gefitinib, in epidermoid carcinoma A431 cells and lung carcinoma H460 cells.

    Science.gov (United States)

    Liu, Hong; Li, Liang; Li, Xing-Qi; Liu, Xiu-Jun; Zhen, Yong-Su

    2009-01-01

    Gefitinib, a low-molecular-weight epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, is effective in a wide variety of tumor types. Preclinical studies have shown potentiated antitumor efficacies of this agent in combination with chemotherapy or radiotherapy. The antitumor antibiotic lidamycin (LDM) showed extremely potent cytotoxicity in vitro and marked therapeutic effect in vivo. In this report, the cytotoxic and biochemical activity of LDM and gefitinib on human epidermoid carcinoma A431 cells and human large cell lung cancer H460 cells as a single agent or in combination has been evaluated. In the MTT assay, LDM showed much more potent cytotoxicity than gefitinib to both cell lines. A431 cells with a highly EGFR-expressing level were more sensitive to gefitinib than H460 cells, which expressed EGFR at an intermediate level. LDM plus gefitinib showed potentiation of antiproliferative activity and apoptosis induction, which were associated with downregulation of EGFR signaling pathway and nuclear factor-kappa B expression, and the increase of cleaved poly (adenosine diphosphate-ribose) polymerase in the two cell lines, although to a lesser degree in H460 cells. Combined treatment induced G1 phase arrest similar to that of gefitinib alone in A431 cells and intensified G2/M phase accumulation in H460 cells. The above results indicate that LDM potentiates the effects of gefitinib in both gefitinib sensitive and less sensitive cells in association with enhanced inhibition of EGFR-dependent signaling. PMID:19342999

  7. The Mertk Receptor Tyrosine Kinase Promotes T-B interaction Stimulated by IgD B-cell Receptor Cross-linking

    OpenAIRE

    Shao, Wen-Hai; Zhen, Yuxuan; Finkelman, Fred D.; Cohen, Philip L.

    2014-01-01

    The Mertk receptor tyrosine kinase facilitates macrophage and DC apoptotic-cell clearance and regulates immune tolerance. Mertk may also contribute to B-cell activation, because Mertk-KO mice fail to develop autoantibodies when allo-activated by T cells. We investigated this possibility with a well-characterized model in which injection of mice with goat anti-IgD antibody causes membrane IgD cross-linking that induces T-independent B cell activation and antigen presentation to T cells. Goat a...

  8. Cell cycle-dependent phosphorylation of pRb-like protein in root meristem cells of Vicia faba.

    Science.gov (United States)

    Polit, Justyna Teresa; Kaźmierczak, Andrzej; Walczak-Drzewiecka, Aurelia

    2012-01-01

    The retinoblastoma tumor suppressor protein (pRb) regulates cell cycle progression by controlling the G1-to-S phase transition. As evidenced in mammals, pRb has three functionally distinct binding domains and interacts with a number of proteins including the E2F family of transcription factors, proteins with a conserved LxCxE motif (D-type cyclin), and c-Abl tyrosine kinase. CDK-mediated phosphorylation of pRb inhibits its ability to bind target proteins, thus enabling further progression of the cell cycle. As yet, the roles of pRb and pRb-binding factors have not been well characterized in plants. By using antibody which specifically recognizes phosphorylated serines (S807/811) in the c-Abl tyrosine kinase binding C-domain of human pRb, we provide evidence for the cell cycle-dependent changes in pRb-like proteins in root meristems cells of Vicia faba. An increased phosphorylation of this protein has been found correlated with the G1-to-S phase transition.

  9. Interleukins 2, 4, 7, and 15 stimulate tyrosine phosphorylation of insulin receptor substrates 1 and 2 in T cells. Potential role of JAK kinases.

    Science.gov (United States)

    Johnston, J A; Wang, L M; Hanson, E P; Sun, X J; White, M F; Oakes, S A; Pierce, J H; O'Shea, J J

    1995-12-01

    The signaling molecules insulin receptor substrate (IRS)-1 and the newly described IRS-2 (4PS) molecule are major insulin and interleukin 4 (IL-4)-dependent phosphoproteins. We report here that IL-2, IL-7, and IL-15, as well as IL-4, rapidly stimulate the tyrosine phosphorylation of IRS-1 and IRS-2 in human peripheral blood T cells, NK cells, and in lymphoid cell lines. In addition, we show that the Janus kinases, JAK1 and JAK3, associate with IRS-1 and IRS-2 in T cells. Coexpression studies demonstrate that these kinases can tyrosine-phosphorylate IRS-2, suggesting a possible mechanism by which cytokine receptors may induce the tyrosine phosphorylation of IRS-1 and IRS-2. We further demonstrate that the p85 subunit of phosphoinositol 3-kinase associates with IRS-1 in response to IL-2 and IL-4 in T cells. Therefore, these data indicate that IRS-1 and IRS-2 may have important roles in T lymphocyte activation not only in response to IL-4, but also in response to IL-2, IL-7, and IL-15.

  10. Activation of protein tyrosine phosphatase non-receptor type 2 by spermidine exerts anti-inflammatory effects in human THP-1 monocytes and in a mouse model of acute colitis.

    Directory of Open Access Journals (Sweden)

    Belén Morón

    Full Text Available BACKGROUND: Spermidine is a dietary polyamine that is able to activate protein tyrosine phosphatase non-receptor type 2 (PTPN2. As PTPN2 is known to be a negative regulator of interferon-gamma (IFN-γ-induced responses, and IFN-γ stimulation of immune cells is a critical process in the immunopathology of inflammatory bowel disease (IBD, we wished to explore the potential of spermidine for reducing pro-inflammatory effects in vitro and in vivo. METHODS: Human THP-1 monocytes were treated with IFN-γ and/or spermidine. Protein expression and phosphorylation were analyzed by Western blot, cytokine expression by quantitative-PCR, and cytokine secretion by ELISA. Colitis was induced in mice by dextran sodium sulfate (DSS administration. Disease severity was assessed by recording body weight, colonoscopy and histology. RESULTS: Spermidine increased expression and activity of PTPN2 in THP-1 monocytes and reduced IFN-γ-induced phosphorylation of signal transducer and activator of transcription (STAT 1 and 3, as well as p38 mitogen-activated protein kinase (MAPK in a PTPN2 dependent manner. Subsequently, IFN-γ-induced expression/secretion of intracellular cell adhesion molecule (ICAM-1 mRNA, monocyte chemoattractant protein (MCP-1, and interleukin (IL-6 was reduced in spermidine-treated cells. The latter effects were absent in PTPN2-knockdown cells. In mice with DSS-induced colitis, spermidine treatment resulted in ameliorated weight loss and decreased mucosal damage indicating reduced disease severity. CONCLUSIONS: Activation of PTPN2 by spermidine ameliorates IFN-γ-induced inflammatory responses in THP-1 cells. Furthermore, spermidine treatment significantly reduces disease severity in mice with DSS-induced colitis; hence, spermidine supplementation and subsequent PTPN2 activation may be helpful in the treatment of chronic intestinal inflammation such as IBD.

  11. Tyrosine kinase inhibitors influence ABCG2 expression in EGFR-positive MDCK BCRP cells via the PI3K/Akt signaling pathway.

    Science.gov (United States)

    Pick, Anne; Wiese, Michael

    2012-04-01

    Multidrug resistance observed in cancer chemotherapy is commonly attributed to overexpression of efflux transporter proteins. These proteins act as ATP-dependent drug efflux pumps, actively extruding chemotherapeutic agents from cells and causing a decrease in intracellular drug accumulation. Besides the well-recognized role of P-glycoprotein (P-gp, ABCB1), the breast cancer resistance protein (BCRP, ABCG2) is becoming increasingly accepted as playing an important role in multidrug resistance. In contrast to P-glycoprotein, only a few inhibitors of ABCG2 are known. According to the literature, tyrosine kinase inhibitors (TKIs) can be considered to be broad-spectrum inhibitors, interacting with ABCB1, ABCC1 and ABCG2. Here, we investigated seven different TKIs, gefitinib, erlotinib, AG1478, PD158780, PD153035, nilotinib and imatinib, for their potential to restore ABCG2 sensitivity to cells. Furthermore, we analyzed the alteration of ABCG2 expression caused by TKIs and demonstrated that EGFR inhibitors such as gefitinib and PD158780 reduced both total and surface expression of ABCG2 in EGRF-positive MDCK BCRP cells by interaction with the PI3K/Akt signaling pathway. The reduced ABCG2 content led to an increased effect of XR9577, a well-known ABCG2 modulator, lowering the concentration required for half maximal inhibition. On the other hand, BCR-ABL inhibitors had no influence on ABCG2 expression and modulator activity. Interestingly, a combination of an EGFR inhibitor with the PI3K/Akt inhibitor LY294002 led to a significant reduction of ABCG2 expression at low concentrations of the drugs. Based on our results, we assume that EGFR exerts a post-transcriptional enhancing effect on ABCG2 expression via the PI3K/Akt signaling pathway, which can be attenuated by EGFR inhibitors. Blocking the key signaling pathway regulating ABCG2 expression with EGFR inhibitors, combined with the inhibition of ABCG2 with potent modulators might be a promising approach to circumvent MDR

  12. Dopaminergic Receptors and Tyrosine Hydroxylase Expression in Peripheral Blood Mononuclear Cells: A Distinct Pattern in Central Obesity

    Science.gov (United States)

    Leite, Fernanda; Lima, Margarida; Marino, Franca; Cosentino, Marco; Ribeiro, Laura

    2016-01-01

    Background Dopamine (DA) may be involved in central obesity (CO), an inflammatory condition, through its role in the central nervous system and in periphery, where it may affect immune cell function through five different DA receptors (DR). Whether dopaminergic pathways in peripheral immune cells are implicated in the inflammatory condition linked to CO is however unknown. Methods In a cohort of blood donors with and without CO, categorized by waist circumference (WC) (CO: WC ≥0.80 m in women and ≥0.94 m in men), we studied the expression of DR and tyrosine hydroxylase (TH), the rate-limiting enzyme in the synthesis of DA, in peripheral blood mononuclear cells (PBMCs) and their relation with anthropometric and metabolic/endocrine and inflammatory parameters. DR D1-5 and TH expression was assessed by semi quantitative real-time PCR. As inflammatory markers we investigated the immunophenotype of monocyte subsets by flow cytometry, staining for CD14, CD16, CD11b and CD36. Results CO individuals showed higher plasma levels of leptin and higher inflammatory pattern of monocytes compared with non-CO. PBMC expression of DR D2, DR D4 and DR D5 as well as of TH were lower in CO in comparison with non-CO. DR D2, and DR D5 expression correlated with lower WC and weight, and with lower inflammatory pattern of monocytes, and TH expression correlated with lower WC. DR D4 expression correlated with lower plasma levels of glycosylated hemoglobin, and DR D2 expression correlated with lower CO. Conclusions Results show that CO is associated with peripheral inflammation and downregulation of dopaminergic pathways in PBMCs, possibly suggesting DR expressed on immune cells as pharmacological targets in obesity for better metabolic outcome. PMID:26808524

  13. H pylori stimulates proliferation of gastric cancer cells through activating mitogen-activated protein kinase cascade

    Institute of Scientific and Technical Information of China (English)

    Yong-Chang Chen; Ying Wang; Jing-Yan Li; Wen-Rong Xu; You-Li Zhang

    2006-01-01

    AIM: To explore the mechanism by which H pylori causes activation of gastric epithelial cells.METHODS: A VacA (+) and CagA (+) standard Hpyloriline NCTC 11637 and a human gastric adenocarcinoma derived gastric epithelial cell line BGC-823 were applied in the study. MTT assay and 3H-TdR incorporation test were used to detect the proliferation of BGC-823 cells and Western blotting was used to detect the activity and existence of related proteins.RESULTS: Incubation with Hpylori extract increased the proliferation of gastric epithelial cells, reflected by both live cell number and DNA synthesis rate. The activity of extracellular signal-regulated protein kinase (ERK) signal transduction cascade increased within 20 min after incubation with Hpylori extract and appeared to be a sustained event. MAPK/ERK kinase (MEK) inhibitor PD98059abolished the action of H pylori extract on both ERK activity and cell proliferation. Incubation with H pyloriextract increased c-Fos expression and SRE-dependentgene expression. H pylori extract caused phosphorylation of several proteins including a protein with molecular size of 97.4 kDa and tyrosine kinase inhibitor genistein inhibited the activation of ERK and the proliferation of cells caused by H pylori extract.CONCLUSION: Biologically active elements in H pylori extract cause proliferation of gastric epithelial cells through activating tyrosine kinase and ERK signal transduction cascade.

  14. The myeloperoxidase-derived oxidant hypothiocyanous acid inhibits protein tyrosine phosphatases via oxidation of key cysteine residues

    DEFF Research Database (Denmark)

    Cook, Naomi L.; Moeke, Cassidy H.; Fantoni, Luca I.;

    2016-01-01

    , can modulate the activity of multiple members of the PTP superfamily via oxidation of Cys residues to sulfenic acids. This alteration of the balance of PTP/kinase activity may perturb protein phosphorylation and disrupt cell signaling with subsequent induction of apoptosis at sites of inflammation.......-) oxidation by H2O2 to form hypothiocyanous acid (HOSCN), an oxidant that targets Cys residues. Dysregulated phosphorylation and elevated MPO levels have been associated with chronic inflammatory diseases where HOSCN can be generated. Previous studies have shown that HOSCN inhibits isolated PTP1B and induces......-PTP; CD45 and Src homology phosphatase-1, Shp-1) by targeting Cys residues. Isolated PTP activity, and activity in lysates of human monocyte-derived macrophages (HMDM) was inhibited by 0-100 μM HOSCN with this being accompanied by reversible oxidation of Cys residues, formation of sulfenic acids...

  15. The novel tyrosine kinase inhibitor AKN-028 has significant antileukemic activity in cell lines and primary cultures of acute myeloid leukemia

    International Nuclear Information System (INIS)

    Aberrantly expressed tyrosine kinases have emerged as promising targets for drug development in acute myeloid leukemia (AML). We report that AKN-028, a novel tyrosine kinase inhibitor (TKI), is a potent FMS-like receptor tyrosine kinase 3 (FLT3) inhibitor (IC50=6 nℳ), causing dose-dependent inhibition of FLT3 autophosphorylation. Inhibition of KIT autophosphorylation was shown in a human megakaryoblastic leukemia cell line overexpressing KIT. In a panel of 17 cell lines, AKN-028 showed cytotoxic activity in all five AML cell lines included. AKN-028 triggered apoptosis in MV4-11 by activation of caspase 3. In primary AML samples (n=15), AKN-028 induced a clear dose-dependent cytotoxic response (mean IC50 1 μℳ). However, no correlation between antileukemic activity and FLT3 mutation status, or to the quantitative expression of FLT3, was observed. Combination studies showed synergistic activity when cytarabine or daunorubicin was added simultaneously or 24 h before AKN-028. In mice, AKN-028 demonstrated high oral bioavailability and antileukemic effect in primary AML and MV4-11 cells, with no major toxicity observed in the experiment. In conclusion, AKN-028 is a novel TKI with significant preclinical antileukemic activity in AML. Possible sequence-dependent synergy with standard AML drugs and good oral bioavailability has made it a candidate drug for clinical trials (ongoing)

  16. The adaptor molecule signaling lymphocytic activation molecule (SLAM)-associated protein (SAP) is essential in mechanisms involving the Fyn tyrosine kinase for induction and progression of collagen-induced arthritis.

    Science.gov (United States)

    Zhong, Ming-Chao; Veillette, André

    2013-11-01

    Signaling lymphocytic activation molecule-associated protein (SAP) is an Src homology 2 domain-only adaptor involved in multiple immune cell functions. It has also been linked to immunodeficiencies and autoimmune diseases, such as systemic lupus erythematosus. Here, we examined the role and mechanism of action of SAP in autoimmunity using a mouse model of autoimmune arthritis, collagen-induced arthritis (CIA). We found that SAP was essential for development of CIA in response to collagen immunization. It was also required for production of collagen-specific antibodies, which play a key role in disease pathogenesis. These effects required SAP expression in T cells, not in B cells. In mice immunized with a high dose of collagen, the activity of SAP was nearly independent of its ability to bind the protein tyrosine kinase Fyn and correlated with the capacity of SAP to promote full differentiation of follicular T helper (TFH) cells. However, with a lower dose of collagen, the role of SAP was more dependent on Fyn binding, suggesting that additional mechanisms other than TFH cell differentiation were involved. Further studies suggested that this might be due to a role of the SAP-Fyn interaction in natural killer T cell development through the ability of SAP-Fyn to promote Vav-1 activation. We also found that removal of SAP expression during progression of CIA attenuated disease severity. However, it had no effect on disease when CIA was clinically established. Together, these results indicate that SAP plays an essential role in CIA because of Fyn-independent and Fyn-dependent effects on TFH cells and, possibly, other T cell types.

  17. Receptor tyrosine kinase targeting in multicellular spheroids.

    Science.gov (United States)

    Breslin, Susan; O'Driscoll, Lorraine

    2015-01-01

    While growing cells as a monolayer is the traditional method for cell culture, the incorporation of multicellular spheroids into experimental design is becoming increasingly popular. This is due to the understanding that cells grown as spheroids tend to replicate the in vivo situation more reliably than monolayer cells. Thus, the use of multicellular spheroids may be more clinically relevant than monolayer cell cultures. Here, we describe methods for multicellular 3D spheroid generation that may be used to provide samples for receptor tyrosine kinase (and other protein) detection. Methods described include the forced-floating poly-HEMA method, the hanging-drop method, and the use of ECM to form multicellular 3D spheroids. PMID:25319898

  18. Tau protein function in living cells

    OpenAIRE

    1986-01-01

    Tau protein from mammalian brain promotes microtubule polymerization in vitro and is induced during nerve cell differentiation. However, the effects of tau or any other microtubule-associated protein on tubulin assembly within cells are presently unknown. We have tested tau protein activity in vivo by microinjection into a cell type that has no endogenous tau protein. Immunofluorescence shows that tau protein microinjected into fibroblast cells associates specifically with microtubules. The i...

  19. Reprogramming cells with synthetic proteins

    Directory of Open Access Journals (Sweden)

    Xiaoxiao Yang

    2015-06-01

    Full Text Available Conversion of one cell type into another cell type by forcibly expressing specific cocktails of transcription factors (TFs has demonstrated that cell fates are not fixed and that cellular differentiation can be a two-way street with many intersections. These experiments also illustrated the sweeping potential of TFs to "read" genetically hardwired regulatory information even in cells where they are not normally expressed and to access and open up tightly packed chromatin to execute gene expression programs. Cellular reprogramming enables the modeling of diseases in a dish, to test the efficacy and toxicity of drugs in patient-derived cells and ultimately, could enable cell-based therapies to cure degenerative diseases. Yet, producing terminally differentiated cells that fully resemble their in vivocounterparts in sufficient quantities is still an unmet clinical need. While efforts are being made to reprogram cells nongenetically by using drug-like molecules, defined TF cocktails still dominate reprogramming protocols. Therefore, the optimization of TFs by protein engineering has emerged as a strategy to enhance reprogramming to produce functional, stable and safe cells for regenerative biomedicine. Engineering approaches focused on Oct4, MyoD, Sox17, Nanog and Mef2c and range from chimeric TFs with added transactivation domains, designer transcription activator-like effectors to activate endogenous TFs to reprogramming TFs with rationally engineered DNA recognition principles. Possibly, applying the complete toolkit of protein design to cellular reprogramming can help to remove the hurdles that, thus far, impeded the clinical use of cells derived from reprogramming technologies.

  20. Photooxidation of Tryptophan and Tyrosine Residues in Human Serum Albumin Sensitized by Pterin: A Model for Globular Protein Photodamage in Skin.

    Science.gov (United States)

    Reid, Lara O; Roman, Ernesto A; Thomas, Andrés H; Dántola, M Laura

    2016-08-30

    Human serum albumin (HSA) is the most abundant protein in the circulatory system. Oxidized albumin was identified in the skin of patients suffering from vitiligo, a depigmentation disorder in which the protection against ultraviolet (UV) radiation fails because of the lack of melanin. Oxidized pterins, efficient photosensitizers under UV-A irradiation, accumulate in the skin affected by vitiligo. In this work, we have investigated the ability of pterin (Ptr), the parent compound of oxidized pterins, to induce structural and chemical changes in HSA under UV-A irradiation. Our results showed that Ptr is able to photoinduce oxidation of the protein in at least two amino acid residues: tryptophan (Trp) and tyrosine (Tyr). HSA undergoes oligomerization, yielding protein structures whose molecular weight increases with irradiation time. The protein cross-linking, due to the formation of dimers of Tyr, does not significantly affect the secondary and tertiary structures of HSA. Trp is consumed in the photosensitized process, and N-formylkynurenine was identified as one of its oxidation products. The photosensitization of HSA takes place via a purely dynamic process, which involves the triplet excited state of Ptr. The results presented in this work suggest that protein photodamage mediated by endogenous photosensitizers can significantly contribute to the harmful effects of UV-A radiation on the human skin. PMID:27500308

  1. Nomogram Predicting Clinical Outcomes in Non-small Cell Lung Cancer Patients Treated with Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors

    OpenAIRE

    Keam, Bhumsuk; Kim, Dong-Wan; Park, Jin Hyun; Lee, Jeong-Ok; Kim, Tae Min; Lee, Se-Hoon; Chung, Doo Hyun; Heo, Dae Seog

    2014-01-01

    Purpose The aim of this study was to develop a pragmatic nomogram for prediction of progressionfree survival (PFS) for the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) in EGFR mutant non-small cell lung cancer (NSCLC). Materials and Methods A total of 306 recurred or metastatic NSCLC patients with EGFR mutation, who received EGFR TKIs, were enrolled in this study. We developed the nomogram, using a Cox proportional hazard regression model for PFS. Results The median...

  2. Effects of angiopoietins-1 and -2 on the receptor tyrosine kinase Tie2 are differentially regulated at the endothelial cell surface

    OpenAIRE

    Hansen, Tania M.; Singh, Harprit; Tahir, Tariq A.; Nicholas P J Brindle

    2010-01-01

    Angiopoietin-1 (Ang1) and Ang2 are ligands for the receptor tyrosine kinase Tie2. Structural data suggest that the two ligands bind Tie2 similarly. However, in endothelial cells Ang1 activates Tie2 whereas Ang2 can act as an apparent antagonist. In addition, each ligand exhibits distinct kinetics of release following binding. These observations suggest that additional factors influence function and binding of angiopoietins with receptors in the cellular context. Previous work has shown that A...

  3. Role of SRC-like adaptor protein (SLAP) in immune and malignant cell signaling.

    Science.gov (United States)

    Kazi, Julhash U; Kabir, Nuzhat N; Rönnstrand, Lars

    2015-07-01

    SRC-like adaptor protein (SLAP) is an adaptor protein structurally similar to the SRC family protein kinases. Like SRC, SLAP contains an SH3 domain followed by an SH2 domain but the kinase domain has been replaced by a unique C-terminal region. SLAP is expressed in a variety of cell types. Current studies suggest that it regulates signaling of various cell surface receptors including the B cell receptor, the T cell receptor, cytokine receptors and receptor tyrosine kinases which are important regulator of immune and cancer cell signaling. SLAP targets receptors, or its associated components, by recruiting the ubiquitin machinery and thereby destabilizing signaling. SLAP directs receptors to ubiquitination-mediated degradation and controls receptors turnover as well as signaling. Thus, SLAP appears to be an important component in regulating signal transduction required for immune and malignant cells.

  4. Cell-Surface Receptors Transactivation Mediated by G Protein-Coupled Receptors

    Directory of Open Access Journals (Sweden)

    Fabio Cattaneo

    2014-10-01

    Full Text Available G protein-coupled receptors (GPCRs are seven transmembrane-spanning proteins belonging to a large family of cell-surface receptors involved in many intracellular signaling cascades. Despite GPCRs lack intrinsic tyrosine kinase activity, tyrosine phosphorylation of a tyrosine kinase receptor (RTK occurs in response to binding of specific agonists of several such receptors, triggering intracellular mitogenic cascades. This suggests that the notion that GPCRs are associated with the regulation of post-mitotic cell functions is no longer believable. Crosstalk between GPCR and RTK may occur by different molecular mechanism such as the activation of metalloproteases, which can induce the metalloprotease-dependent release of RTK ligands, or in a ligand-independent manner involving membrane associated non-receptor tyrosine kinases, such as c-Src. Reactive oxygen species (ROS are also implicated as signaling intermediates in RTKs transactivation. Intracellular concentration of ROS increases transiently in cells stimulated with GPCR agonists and their deliberated and regulated generation is mainly catalyzed by enzymes that belong to nicotinamide adenine dinucleotide phosphate (NADPH oxidase family. Oxidation and/or reduction of cysteine sulfhydryl groups of phosphatases tightly controls the activity of RTKs and ROS-mediated inhibition of cellular phosphatases results in an equilibrium shift from the non-phosphorylated to the phosphorylated state of RTKs. Many GPCR agonists activate phospholipase C, which catalyze the hydrolysis of phosphatidylinositol 4,5-bis-phosphate to produce inositol 1,4,5-triphosphate and diacylglicerol. The consequent mobilization of Ca2+ from endoplasmic reticulum leads to the activation of protein kinase C (PKC isoforms. PKCα mediates feedback inhibition of RTK transactivation during GPCR stimulation. Recent data have expanded the coverage of transactivation to include Serine/Threonine kinase receptors and Toll-like receptors

  5. Fluorogenic Tagging of Peptide and Protein 3-Nitrotyrosine with 4-(Aminomethyl)-benzenesulfonic Acid for Quantitative Analysis of Protein Tyrosine Nitration.

    Science.gov (United States)

    Sharov, Victor S; Dremina, Elena S; Galeva, Nadezhda A; Gerstenecker, Gary S; Li, Xiaobao; Dobrowsky, Rick T; Stobaugh, John F; Schöneich, Christian

    2010-01-01

    Protein 3-nitrotyrosine (3-NT) has been recognized as an important biomarker of nitroxidative stress associated with inflammatory and degenerative diseases, and biological aging. Analysis of protein-bound 3-NT continues to represent a challenge since in vivo it frequently does not accumulate on proteins in amounts detectable by quantitative analytical methods. Here, we describe a novel approach of fluorescent tagging and quantitation of peptide-bound 3-NT residues based on the selective reduction to 3-AT followed by reaction with 4-(amino-methyl)benzenesulfonic acid (ABS) in the presence of K(3)Fe(CN)(6) to form a highly fluorescent 2-phenylbenzoxazole product. Synthetic 3-NT peptide (0.005-1 μM) upon reduction with 10 mM sodium dithionite and tagging with 2 mM ABS and 5 μM K(3)Fe(CN)(6) in 0.1 M Na(2)HPO(4) buffer (pH 9.0) was converted with yields >95% to a single fluorescent product incorporating two ABS molecules per 3-NT residue, with fluorescence excitation and emission maxima at 360 ± 2 and 490 ± 2 nm, respectively, and a quantum yield of 0.77 ± 0.08, based on reverse-phase LC with UV and fluorescence detection, fluorescence spectroscopy and LC-MS-MS analysis. This protocol was successfully tested for quantitative analysis of in vitro Tyr nitration in a model protein, rabbit muscle phosphorylase b, and in a complex mixture of proteins from C2C12 cultured cells exposed to peroxynitrite, with a detection limit of ca. 1 pmol 3-NT by fluorescence spectrometry, and an apparent LOD of 12 and 40 pmol for nitropeptides alone or in the presence of 100 μg digested cell proteins, respectively. LC-MS-MS analysis of ABS tagged peptides revealed that the fluorescent derivatives undergo efficient backbone fragmentations, allowing for sequence-specific characterization of protein Tyr nitration in proteomic studies. Fluorogenic tagging with ABS also can be instrumental for detection and visualization of protein 3-NT in LC and gel-based protein separations. PMID

  6. SHP1 tyrosine phosphatase negatively regulates NPM-ALK tyrosine kinase signaling.

    Science.gov (United States)

    Honorat, Jean-François; Ragab, Ashraf; Lamant, Laurence; Delsol, Georges; Ragab-Thomas, Jeannie

    2006-05-15

    Anaplastic large-cell lymphoma (ALCL) is frequently associated with the 2;5 translocation and expresses the NPM-ALK fusion protein, which possesses a constitutive tyrosine kinase activity. We analyzed SHP1 tyrosine phosphatase expression and activity in 3 ALK-positive ALCL cell lines (Karpas 299, Cost, and SU-DHL1) and in lymph node biopsies (n = 40). We found an inverse correlation between the level of NPM-ALK phosphorylation and SHP1 phosphatase activity. Pull-down and coimmunoprecipitation experiments demonstrated a SHP1/NPM-ALK association. Furthermore, confocal microscopy performed on ALCL cell lines and biopsy specimens showed the colocalization of the 2 proteins in cytoplasmic bodies containing Y664-phosphorylated NPM-ALK. Dephosphorylation of NPM-ALK by SHP1 demonstrated that NPM-ALK was a SHP1 substrate. Downregulation of SHP1 expression by RNAi in Karpas cells led to hyperphosphorylation of NPM-ALK, STAT3 activation, and increase in cell proliferation. Furthermore, SHP1 overexpression in 3T3 fibroblasts stably expressing NPM-ALK led to the decrease of NPM-ALK phosphorylation, lower cell proliferation, and tumor progression in nude mice. These findings show that SHP1 is a negative regulator of NPM-ALK signaling. The use of tissue microarrays revealed that 50% of ALK-positive ALCLs were positive for SHP1. Our results suggest that SHP1 could be a critical enzyme in ALCL biology and a potential therapeutic target.

  7. Irradiation and various cytotoxic drugs enhance tyrosine phosphorylation and {beta}{sub 1}-integrin clustering in human A549 lung cancer cells in a substratum-dependent manner in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Cordes, N.; Beinke, C.; Beuningen, D. van [Inst. of Radiobiology, German Armed Forces, Munich (Germany); Plasswilm, L. [Dept. of Radiation Oncology, Univ. Hospital Basel (Swaziland)

    2004-03-01

    Background and purpose: interactions of cells with a substratum, especially extracellular matrix proteins, initiate clustering of integrin receptors in the cell membrane. This process represents the initial step for the activation of signaling pathways regulating survival, proliferation, differentiation, adhesion, and migration, and could, furthermore, be important for cellular resistance-mediating mechanisms against radiation or cytotoxic drugs. The lack of data elucidating the impact of irradiation or cytotoxic drugs on this important phenomenon led to this study on human A549 lung cancer cells in vitro. Material and methods: the human lung carcinoma cell line A549 grown on polystyrene or fibronectin (FN) was irradiated with 0-8 Gy or treated with cisplatin (0.1-50 {mu}M), paclitaxel (0.1-50 nM), or mitomycin (0.1-50 {mu}M). Colony formation assays, immunofluorescence staining in combination with activation of integrin clustering using anti-{beta}{sub 1}-integrin antibodies (K20), and Western blotting for tyrosine phosphorylation under treatment of cells with the IC{sub 50} for irradiation (2 Gy; IC{sub 50} = 2.2 Gy), cisplatin (2 {mu}M), paclitaxel (5 nM), or mitomycin (7 {mu}M) were performed. Results: attachment of cells to FN resulted in a significantly reduced radio- and chemosensitivity compared to polystyrene. The clustering of {beta}{sub 1}-integrins examined by immunofluorescence staining was only stimulated by irradiation, cisplatin, paclitaxel, or mitomycin in case of cell attachment to FN. By contrast, tyrosine phosphorylation, as one of the major events following {beta}{sub 1}-integrin clustering, showed a 3.7-fold, FN-related enhancement, and treatment of cells with the IC{sub 50} of radiation, cisplatin, paclitaxel, or mitomycin showed a substratum-dependent induction. Conclusion: for the first time, a strong influence of irradiation and a variety of cytotoxic drugs on the clustering of {beta}{sub 1}-integrins could be shown. This event is a

  8. Interleukin-2 induces tyrosine phosphorylation and nuclear translocation of stat3 in human T lymphocytes

    DEFF Research Database (Denmark)

    Nielsen, M; Svejgaard, A; Skov, S;

    1994-01-01

    stimulation through the IL-2R induced tyrosine phosphorylation and subsequent nuclear translocation of stat3, a newly identified member of the signal transducers and activators of transcription (STAT) family of proteins. In contrast, stat1 proteins were not tyrosine phosphorylated after IL-2 ligation, whereas...... an apparent molecular mass of 84 kDa and was not recognized by stat3 or stat1 mAb or antisera. Since IL-2 induced nuclear translocation of the 84 kDa protein and stat3 followed identical kinetics, p84 is a candidate for a new, yet undefined, member of the STAT family. Taken together, we report that...... IL-2 induces tyrosine phosphorylation and subsequent nuclear translocation of stat3 and an as yet undefined 84-kDa protein in antigen-specific human T cell lines....

  9. Oxidation of Tyrosine-Phosphopeptides by Titanium Dioxide Photocatalysis.

    Science.gov (United States)

    Ruokolainen, Miina; Ollikainen, Elisa; Sikanen, Tiina; Kotiaho, Tapio; Kostiainen, Risto

    2016-06-22

    Protein phosphorylation has a key role in cell regulation. Oxidation of proteins, in turn, is related to many diseases and to aging, but the effects of phosphorylation on the oxidation of proteins and peptides have been rarely studied. The aim of this study was to examine the mechanistic effect of phosphorylation on peptide oxidation induced by titanium dioxide photocatalysis. The effect of phosphorylation was compared between nonphosphorylated and tyrosine phosphorylated peptides using electrospray tandem mass spectrometry. We observed that tyrosine was the most preferentially oxidized amino acid, but the oxidation reaction was significantly inhibited by its phosphorylation. The study also shows that titanium dioxide photocatalysis provides a fast and easy method to study oxidation reactions of biomolecules, such as peptides.

  10. Two homologous putative protein tyrosine phosphatases, OsPFA-DSP2 and AtPFA-DSP4, negatively regulate the pathogen response in transgenic plants.

    Directory of Open Access Journals (Sweden)

    Hanjie He

    Full Text Available Protein phosphatases, together with protein kinases, regulate protein phosphorylation and dephosphorylation, and play critical roles in plant growth and biotic stress responses. However, little is known about the biological functions of plant protein tyrosine dual-specificity phosphatase (PFA-DSP in biotic stresses. Here, we found that OsPFA-DSP2 was mainly expressed in calli, seedlings, roots, and young panicles, and localized in cytoplasm and nucleus. Ectopic overexpression of OsPFA-DSP2 in rice increased sensitivity to Magnaporthe grisea (M. grisea Z1 strain, inhibited the accumulation of hydrogen peroxide (H(2O(2 and suppressed the expression of pathogenesis-related (PR genes after fungal infection. Interestingly, transgenic Arabidopsis plants overexpressing AtPFA-DSP4, which is homologous to OsPFA-DSP2, also exhibited sensitivity to Pseudomonas syringae pv. tomato DC3000 (Pst DC3000, reduced accumulation of H(2O(2 and decreased photosynthesic capacity after infection compared with Col-0. These results indicate that OsPFA-DSP2 and AtPFA-DSP4 act as negative regulators of the pathogen response in transgenic plants.

  11. Cloning and characterization of R-PTP-kappa, a new member of the receptor protein tyrosine phosphatase family with a proteolytically cleaved cellular adhesion molecule-like extracellular region

    DEFF Research Database (Denmark)

    Jiang, Y P; Wang, H; D'Eustachio, P;

    1993-01-01

    We describe a new member of the receptor protein tyrosine phosphatase family, R-PTP-kappa, cDNA cloning predicts that R-PTP-kappa is synthesized from a precursor protein of 1,457 amino acids. Its intracellular domain displays the classical tandemly repeated protein tyrosine phosphatase homology......, separated from the transmembrane segment by an uncharacteristically large juxta-membrane region. The extracellular domain of the R-PTP-kappa precursor protein contains an immunoglobulin-like domain and four fibronectin type III-like repeats, preceded by a signal peptide and a region of about 150 amino acids...... with similarity to the Xenopus A5 antigen, a putative neuronal recognition molecule (S. Takagi, T. Hsrata, K. Agata, M. Mochii, G. Eguchi, and H. Fujisawa, Neuron 7:295-307, 1991). Antibodies directed against the intra- and extracellular domains reveal that the R-PTP-kappa precursor protein undergoes proteolytic...

  12. Investigation of the vitamin D receptor gene (VDR) and its interaction with protein tyrosine phosphatase, non-receptor type 2 gene (PTPN2) on risk of islet autoimmunity and type 1 diabetes : The Diabetes Autoimmunity Study in the Young (DAISY)

    NARCIS (Netherlands)

    Frederiksen, B.; Liu, E.; Romanos, J.; Steck, A. K.; Yin, X.; Kroehl, M.; Fingerlin, T. E.; Erlich, H.; Eisenbarth, G. S.; Rewers, M.; Norris, J. M.

    2013-01-01

    The present study investigated the association between variants in the vitamin D receptor gene (VDR) and protein tyrosine phosphatase, non-receptor type 2 gene (PTPN2), as well as an interaction between VDR and PTPN2 and the risk of islet autoimmunity (IA) and progression to type 1 diabetes (T1D). T

  13. Salmonella induces SRC protein tyrosine kinase, c-Jun N-terminal kinase (JNK), and NF-kappaBp65 signaling pathways in commercial and wild-type turkey leukocytes

    Science.gov (United States)

    Previous studies comparing signaling in wild-type turkey (WT) leukocytes and commercial turkey (CT) leukocytes found that the activity of protein tyrosine kinases and MAP kinases, ERK 1/2 and p38, were significantly higher in WT leukocytes compared to CT lines upon exposure to both SE and OPSE on d...

  14. The monoclonal antibody Zt/f2 targeting RON receptor tyrosine kinase as potential therapeutics against tumor growth-mediated by colon cancer cells

    Directory of Open Access Journals (Sweden)

    Zhang Rui-Wen

    2011-07-01

    Full Text Available Abstract Background Overexpression of the RON receptor tyrosine kinase contributes to epithelial cell transformation, malignant progression, and acquired drug resistance. RON also has been considered as a potential target for therapeutic intervention. This study determines biochemical features and inhibitory activity of a mouse monoclonal antibody (mAb Zt/f2 in experimental cancer therapy. Results Zt/f2 is a mouse IgG2a mAb that is highly specific and sensitive to human RON and its oncogenic variants such as RON160 (ED50 = 2.3 nmol/L. Receptor binding studies revealed that Zt/f2 interacts with an epitope(s located in a 49 amino acid sequence coded by exon 11 in the RON β-chain extracellular sequences. This sequence is critical in regulating RON maturation and phosphorylation. Zt/f2 did not compete with ligand macrophage-stimulating protein for binding to RON; however, its engagement effectively induced RON internalization, which diminishes RON expression and impairs downstream signaling activation. These biochemical features provide the cellular basis for the use of Zt/f2 to inhibit tumor growth in animal model. Repeated administration of Zt/f2 as a single agent into Balb/c mice results in partial inhibition of tumor growth caused by transformed NIH-3T3 cells expressing oncogenic RON160. Colon cancer HT-29 cell-mediated tumor growth in athymic nude mice also was attenuated following Zt/f2 treatment. In both cases, ~50% inhibition of tumor growth as measured by tumor volume was achieved. Moreover, Zt/f2 in combination with 5-fluorouracil showed an enhanced inhibition effect of ~80% on HT-29 cell-mediated tumor growth in vivo. Conclusions Zt/f2 is a potential therapeutic mAb capable of inhibiting RON-mediated oncogenesis by colon cancer cells in animal models. The inhibitory effect of Zt/f2 in vivo in combination with chemoagent 5-fluorouracil could represent a novel strategy for future colon cancer therapy.

  15. Protein Kinase G facilitates EGFR-mediated cell death in MDA-MB-468 cells.

    Science.gov (United States)

    Jackson, Nicole M; Ceresa, Brian P

    2016-08-15

    The Epidermal Growth Factor Receptor (EGFR) is a transmembrane receptor tyrosine kinase with critical implications in cell proliferation, migration, wound healing and the regulation of apoptosis. However, the EGFR has been shown to be hyper-expressed in a number of human malignancies. The MDA-MB-468 metastatic breast cell line is one example of this. This particular cell line hyper-expresses the EGFR and undergoes EGFR-mediated apoptosis in response to EGF ligand. The goal of this study was to identify the kinases that could be potential intermediates for the EGFR-mediated induction of apoptosis intracellularly. After identifying Cyclic GMP-dependent Protein Kinase G (PKG) as a plausible intermediate, we wanted to determine the temporal relationship of these two proteins in the induction of apoptosis. We observed a dose-dependent decrease in MDA-MB-468 cell viability, which was co-incident with increased PKG activity as measured by VASPSer239 phosphorylation. In addition, we observed a dose dependent decrease in cell viability, as well as an increase in apoptosis, in response to two different PKG agonists, 8-Bromo-cGMP and 8-pCPT-cGMP. MDA-MB-468 cells with reduced PKG activity had attenuated EGFR-mediated apoptosis. These findings indicate that PKG does not induce cell death via transphosphorylation of the EGFR. Instead, PKG activity occurs following EGFR activation. Together, these data indicate PKG as an intermediary in EGFR-mediated cell death, likely via apoptotic pathway. PMID:27381222

  16. Tyrosine 402 phosphorylation of Pyk2 is involved in ionomycin-induced neurotransmitter release.

    Directory of Open Access Journals (Sweden)

    Zhao Zhang

    Full Text Available Protein tyrosine kinases, which are highly expressed in the central nervous system, are implicated in many neural processes. However, the relationship between protein tyrosine kinases and neurotransmitter release remains unknown. In this study, we found that ionomycin, a Ca²⁺ ionophore, concurrently induced asynchronous neurotransmitter release and phosphorylation of a non-receptor protein tyrosine kinase, proline-rich tyrosine kinase 2 (Pyk2, in clonal rat pheochromocytoma PC12 cells and cerebellar granule cells, whereas introduction of Pyk2 siRNA dramatically suppressed ionomycin-induced neurotransmitter release. Further study indicated that Tyr-402 (Y402 in Pyk2, instead of other tyrosine sites, underwent rapid phosphorylation after ionomycin induction in 1 min to 2 min. We demonstrated that the mutant of Pyk2 Y402 could abolish ionomycin-induced dopamine (DA release by transfecting cells with recombinant Pyk2 and its mutants (Y402F, Y579F, Y580F, and Y881F. In addition, Src inhibition could prolong phosphorylation of Pyk2 Y402 and increase DA release. These findings suggested that Pyk2 was involved in ionomycin-induced neurotransmitter release through phosphorylation of Y402.

  17. Endocytosis of the ASGP receptor H1 is reduced by mutation of tyrosine-5 but still occurs via coated pits

    OpenAIRE

    Fuhrer, C; Geffen, I; Spiess, M.

    1991-01-01

    The clustering of plasma membrane receptors in clathrin-coated pits depends on determinants within their cytoplasmic domains. In several cases, individual tyrosine residues were shown to be necessary for rapid internalization. We have mutated the single tyrosine at position 5 in the cytoplasmic domain of the major subunit H1 of the asialoglycoprotein receptor to alanine. Expressed in fibroblasts cells, the mutant protein was accumulated in the plasma membrane, and its rate of internalization ...

  18. The pan-ErbB tyrosine kinase inhibitor canertinib induces caspase-mediated cell death in human T-cell leukemia (Jurkat) cells

    Energy Technology Data Exchange (ETDEWEB)

    Trinks, Cecilia, E-mail: Cecilia.trinks@liu.se [Division of Oncology, Department of Clinical and Experimental Medicine, Faculty of Health Sciences, Linkoeping University, Linkoeping (Sweden); Severinsson, Emelie A., E-mail: Emelie.severinsson@liu.se [Division of Oncology, Department of Clinical and Experimental Medicine, Faculty of Health Sciences, Linkoeping University, Linkoeping (Sweden); Holmlund, Birgitta, E-mail: Birgitta.holmlund@lio.se [Department of Oncology, County Council of Ostergoetland, Linkoeping (Sweden); Green, Anna, E-mail: Anna.green@liu.se [Division of Cell Biology, Department of Clinical and Experimental Medicine, Faculty of Health Sciences, Linkoeping University, Linkoeping (Sweden); Green, Henrik, E-mail: Henrik.green@liu.se [Clinical Pharmacology, Division of Drug Research, Department of Medical and Health Sciences, Faculty of Health Sciences, Linkoeping University, Linkoeping (Sweden); Joensson, Jan-Ingvar, E-mail: Jan-ingvar.jonsson@liu.se [Experimental Hematology Unit, Department of Clinical and Experimental Medicine, Faculty of Health Sciences, Linkoeping University, Linkoeping (Sweden); Hallbeck, Anna-Lotta, E-mail: Anna-Lotta.Hallbeck@lio.se [Division of Oncology, Department of Clinical and Experimental Medicine, Faculty of Health Sciences, Linkoeping University, Linkoeping (Sweden); Department of Oncology, County Council of Ostergoetland, Linkoeping (Sweden); Walz, Thomas M., E-mail: Thomas.Walz@lio.se [Division of Oncology, Department of Clinical and Experimental Medicine, Faculty of Health Sciences, Linkoeping University, Linkoeping (Sweden); Department of Oncology, County Council of Ostergoetland, Linkoeping (Sweden)

    2011-07-08

    Highlights: {yields} Canertinib induces caspase-mediated apoptosis in T-cell leukemia cells in vitro. {yields} Canertinib mediates activation of the intrinsic apoptotic pathway. {yields} Canertinib induces apoptosis in an ErbB receptor independent manner. {yields} Lymphocyte specific proteins as well as survival kinases are inhibited. {yields} Canertinib may act as a multi-kinase inhibiting drug in human T-cell malignancies. -- Abstract: Canertinib is a novel ErbB-receptor inhibitor currently in clinical development for the treatment of solid tumors overexpressing ErbB-receptors. We have recently demonstrated that canertinib displays anti-proliferative and pro-apoptotic effects in human myeloid leukemia cells devoid of ErbB-receptors. The mechanism mediating these effects are however unknown. In this study, we show that canertinib is able to act as a multi-kinase inhibitor by inhibition of several intracellular kinases involved in T-cell signaling such as Akt, Erk1/2 and Zap-70, and reduced Lck protein expression in the human T-cell leukemia cell line Jurkat. Treatment with canertinib at a concentration of 2 {mu}M caused accumulation of Jurkat cells in the G{sub 1} cell cycle phase and increased doses induced apoptosis in a time-dependent manner. Apoptotic signs of treated cells were detected by Annexin V staining and cleavage of PARP, caspase-3, -8, -9, -10 and Bid. A subset of the pro-apoptotic signals mediated by canertinib could be significantly reduced by specific caspase inhibitors. Taken together, these results demonstrate the dual ability of canertinib to downregulate important signaling pathways and to activate caspase-mediated intrinsic apoptosis pathway in human T-cell leukemia cells.

  19. Novel Tyrosine Phosphorylation Sites in Rat Skeletal Muscle Revealed by Phosphopeptide Enrichment and HPLC-ESI-MS/MS

    OpenAIRE

    Zhang, Xiangmin; Højlund, Kurt; Luo, Moulun; Meyer, Christian; Thangiah, Geetha; Yi, Zhengping

    2012-01-01

    Tyrosine phosphorylation plays a fundamental role in many cellular processes including differentiation, growth and insulin signaling. In insulin resistant muscle, aberrant tyrosine phosphorylation of several proteins has been detected. However, due to the low abundance of tyrosine phosphorylation (

  20. Inhibitory Activities of Epidermal Growth Factor Receptor Tyrosine Kinase-Targeted Dihydroxyisoflavone and Trihydroxydeoxybenzoin Derivatives on Sarcocystis neurona, Neospora caninum, and Cryptosporidium parvum Development

    OpenAIRE

    Gargala, G.; Baishanbo, A.; Favennec, L; François, A; Ballet, J J; Rossignol, J.-F.

    2005-01-01

    Several gene sequences of parasitic protozoa belonging to protein kinase gene families and epidermal growth factor (EGF)-like peptides, which act via binding to receptor tyrosine kinases of the EGF receptor (EGFR) family, appear to mediate host-protozoan interactions. As a clue to EGFR protein tyrosine kinase (PTK) mediation and a novel approach for identifying anticoccidial agents, activities against Sarcocystis neurona, Neospora caninum, and Cryptosporidium parvum grown in BM and HCT-8 cell...

  1. A redundant role of the CD3 gamma-immunoreceptor tyrosine-based activation motif in mature T cell function

    NARCIS (Netherlands)

    Haks, MC; Cordaro, TA; van den Brakel, JHN; Haanen, JBAG; de Vries, EFR; Borst, J; Krimpenfort, P; Kruisbeek, AM

    2001-01-01

    At least four different CD3 polypeptide chains are contained within the mature TCR complex, each encompassing one (CD3 gamma, CD3 delta, and CD3 epsilon) or three (CD3 zeta) immunoreceptof tyrosine-based activation motifs (ITAMs) within their cytoplasmic domains. Why so many ITAMs are required is un

  2. L-3-[I-123]iodo-alpha-methyl-tyrosine SPECT in non-small cell lung cancer : Preliminary observations

    NARCIS (Netherlands)

    Jager, PL; Groen, HJM; van der Leest, A; van Putten, JWG; Pieterman, RM; de Vries, EGE; Piers, DA

    2001-01-01

    L-3-[(123)]iodo-alpha -methyl-tyrosine (IMT) is a modified amino acid that is avidly taken up by many tumors. Uptake is based on the increased transmembrane transport of amino acids in malignancies. IMT is the only amino acid tracer suitable for SPECT. The aim of this study was to determine the feas

  3. The Tyrosine Aminomutase TAM1 Is Required for β-Tyrosine Biosynthesis in Rice

    Science.gov (United States)

    Yan, Jian; Aboshi, Takako; Teraishi, Masayoshi; Strickler, Susan R.; Spindel, Jennifer E.; Tung, Chih-Wei; Takata, Ryo; Matsumoto, Fuka; Maesaka, Yoshihiro; McCouch, Susan R.; Okumoto, Yutaka; Mori, Naoki; Jander, Georg

    2015-01-01

    Non-protein amino acids, often isomers of the standard 20 protein amino acids, have defense-related functions in many plant species. A targeted search for jasmonate-induced metabolites in cultivated rice (Oryza sativa) identified (R)-β-tyrosine, an isomer of the common amino acid (S)-α-tyrosine in the seeds, leaves, roots, and root exudates of the Nipponbare cultivar. Assays with 119 diverse cultivars showed a distinct presence/absence polymorphism, with β-tyrosine being most prevalent in temperate japonica cultivars. Genetic mapping identified a candidate gene on chromosome 12, which was confirmed to encode a tyrosine aminomutase (TAM1) by transient expression in Nicotiana benthamiana and in vitro enzyme assays. A point mutation in TAM1 eliminated β-tyrosine production in Nipponbare. Rice cultivars that do not produce β-tyrosine have a chromosome 12 deletion that encompasses TAM1. Although β-tyrosine accumulation was induced by the plant defense signaling molecule jasmonic acid, bioassays with hemipteran and lepidopteran herbivores showed no negative effects at physiologically relevant β-tyrosine concentrations. In contrast, root growth of Arabidopsis thaliana and other tested dicot plants was inhibited by concentrations as low as 1 μM. As β-tyrosine is exuded into hydroponic medium at higher concentrations, it may contribute to the allelopathic potential of rice. PMID:25901084

  4. Proline-rich tyrosine kinase 2 (Pyk2 promotes cell motility of hepatocellular carcinoma through induction of epithelial to mesenchymal transition.

    Directory of Open Access Journals (Sweden)

    Chris K Sun

    Full Text Available AIMS: Proline-rich tyrosine kinase 2 (Pyk2, a non-receptor tyrosine kinase of the focal adhesion kinase (FAK family, is up-regulated in more than 60% of the tumors of hepatocellular carcinoma (HCC patients. Forced overexpression of Pyk2 can promote the proliferation and invasion of HCC cells. In this study, we aimed to explore the underlying molecular mechanism of Pyk2-mediated cell migration of HCC cells. METHODOLOGY/PRINCIPAL FINDINGS: We demonstrated that Pyk2 transformed the epithelial HCC cell line Hep3B into a mesenchymal phenotype via the induction of epithelial to mesenchymal transition (EMT, signified by the up-regulation of membrane ruffle formation, activation of Rac/Rho GTPases, down-regulation of epithelial genes E-cadherin and cytokeratin as well as promotion of cell motility in presence of lysophosphatidic acid (LPA. Suppression of Pyk2 by overexpression of dominant negative PRNK domain in the metastatic HCC cell line MHCC97L transformed its fibroblastoid phenotype to an epithelial phenotype with up-regulation of epithelial genes, down-regulation of mesenchymal genes N-cadherin and STAT5b, and reduction of LPA-induced membrane ruffle formation and cell motility. Moreover, overexpression of Pyk2 in Hep3B cells promoted the phosphorylation and localization of mesenchymal gene Hic-5 onto cell membrane while suppression of Pyk2 in MHCC97L cells attenuated its phosphorylation and localization. CONCLUSION: These data provided new evidence of the underlying mechanism of Pyk2 in controlling cell motility of HCC cells through regulation of genes associated with EMT.

  5. Cadherin-mediated cell-cell adhesion is perturbed by v-src tyrosine phosphorylation in metastatic fibroblasts

    OpenAIRE

    1992-01-01

    Rat 3Y1 cells acquire metastatic potential when transformed with v-src, and this potential is enhanced by double transformation with v-src and v-fos (Taniguchi, S., T. Kawano, T. Mitsudomi, G. Kimura, and T. Baba. 1986. Jpn. J. Cancer Res. 77:1193-1197). We compared the activity of cadherin cell adhesion molecules of normal 3Y1 cells with that of v-src transformed (SR3Y1) and v-src and v-fos double transformed (fosSR3Y1) 3Y1 cells. These cells expressed similar amounts of P-cadherin, and show...

  6. Non-small-cell lung cancer cells combat epidermal growth factor receptor tyrosine kinase inhibition through immediate adhesion-related responses

    Directory of Open Access Journals (Sweden)

    Wang HY

    2016-05-01

    Full Text Available Hsian-Yu Wang,1,2 Min-Kung Hsu,3,4 Kai-Hsuan Wang,1 Ching-Ping Tseng,2,4 Feng-Chi Chen,3,4 John T-A Hsu1,4 1Institute of Biotechnology and Pharmaceutical Research, National Health Research Institutes (NHRI, Zhunan, Miaoli County, 2Institute of Molecular Medicine and Bioengineering, National Chiao Tung University (NCTU, Hsinchu, 3Division of Biostatistics and Bioinformatics, Institute of Population Health Sciences, National Health Research Institutes (NHRI, Zhunan, Miaoli County, 4Department of Biological Science and Technology, National Chiao Tung University (NCTU, Hsinchu, Taiwan, Republic of China Background: Epidermal growth factor receptor (EGFR tyrosine kinase inhibitors (TKIs, such as gefitinib, erlotinib, and afatinib, have greatly improved treatment efficacy in non-small cell lung cancer (NSCLC patients with drug-sensitive EGFR mutations. However, in some TKI responders, the benefits of such targeted therapies are limited by the rapid development of resistance, and strategies to overcome this resistance are urgently needed. Studies of drug resistance in cancer cells typically involve long term in vitro induction to obtain stably acquired drug-resistant cells followed by elucidation of resistance mechanisms, but the immediate responses of cancer cells upon drug treatment have been ignored. The aim of this study was to investigate the immediate responses of NSCLC cells upon treatment with EGFR TKIs.Results: Both NSCLC cells, ie, PC9 and H1975, showed immediate enhanced adhesion-related responses as an apoptosis-countering mechanism upon first-time TKI treatment. By gene expression and pathway analysis, adhesion-related pathways were enriched in gefitinib-treated PC9 cells. Pathway inhibition by small-hairpin RNAs or small-molecule drugs revealed that within hours of EGFR TKI treatment, NSCLC cells used adhesion-related responses to combat the drugs. Importantly, we show here that the Src family inhibitor, dasatinib, dramatically inhibits

  7. PTP1B-dependent regulation of receptor tyrosine kinase signaling by the actin-binding protein Mena

    NARCIS (Netherlands)

    Hughes, Shannon K; Oudin, Madeleine J; Tadros, Jenny; Neil, Jason; Del Rosario, Amanda; Joughin, Brian A; Ritsma, Laila; Wyckoff, Jeff; Vasile, Eliza; Eddy, Robert; Philippar, Ulrike; Lussiez, Alisha; Condeelis, John S; van Rheenen, Jacco; White, Forest; Lauffenburger, Douglas A; Gertler, Frank B

    2015-01-01

    During breast cancer progression, alternative mRNA splicing produces functionally distinct isoforms of Mena, an actin regulator with roles in cell migration and metastasis. Aggressive tumor cell subpopulations express Mena(INV), which promotes tumor cell invasion by potentiating EGF responses. Howev

  8. Roles of tyrosine-rich precursor glycoproteins and dityrosine- and 3,4-dihydroxyphenylalanine-mediated protein cross-linking in development of the oocyst wall in the coccidian parasite Eimeria maxima

    DEFF Research Database (Denmark)

    Belli, Sabina I; Wallach, Michael G; Luxford, Catherine;

    2003-01-01

    infection by several organisms of medical and veterinary importance such as Eimeria, Plasmodium, Toxoplasma, Cyclospora, and Neospora could be developed. Here, we show that two tyrosine-rich precursor glycoproteins, gam56 and gam82, found in specialized organelles (wall-forming bodies) in the sexual stage...... cuticles, yeast cell walls, mussel byssal threads, and sea urchin fertilization membranes....

  9. Targeting of nucleotide-binding proteins by HAMLET--a conserved tumor cell death mechanism.

    Science.gov (United States)

    Ho, J C S; Nadeem, A; Rydström, A; Puthia, M; Svanborg, C

    2016-02-18

    HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) kills tumor cells broadly suggesting that conserved survival pathways are perturbed. We now identify nucleotide-binding proteins as HAMLET binding partners, accounting for about 35% of all HAMLET targets in a protein microarray comprising 8000 human proteins. Target kinases were present in all branches of the Kinome tree, including 26 tyrosine kinases, 10 tyrosine kinase-like kinases, 13 homologs of yeast sterile kinases, 4 casein kinase 1 kinases, 15 containing PKA, PKG, PKC family kinases, 15 calcium/calmodulin-dependent protein kinase kinases and 13 kinases from CDK, MAPK, GSK3, CLK families. HAMLET acted as a broad kinase inhibitor in vitro, as defined in a screen of 347 wild-type, 93 mutant, 19 atypical and 17 lipid kinases. Inhibition of phosphorylation was also detected in extracts from HAMLET-treated lung carcinoma cells. In addition, HAMLET recognized 24 Ras family proteins and bound to Ras, RasL11B and Rap1B on the cytoplasmic face of the plasma membrane. Direct cellular interactions between HAMLET and activated Ras family members including Braf were confirmed by co-immunoprecipitation. As a consequence, oncogenic Ras and Braf activity was inhibited and HAMLET and Braf inhibitors synergistically increased tumor cell death in response to HAMLET. Unlike most small molecule kinase inhibitors, HAMLET showed selectivity for tumor cells in vitro and in vivo. The results identify nucleotide-binding proteins as HAMLET targets and suggest that dysregulation of the ATPase/kinase/GTPase machinery contributes to cell death, following the initial, selective recognition of HAMLET by tumor cells. The findings thus provide a molecular basis for the conserved tumoricidal effect of HAMLET, through dysregulation of kinases and oncogenic GTPases, to which tumor cells are addicted. PMID:26028028

  10. Targeting of nucleotide-binding proteins by HAMLET--a conserved tumor cell death mechanism.

    Science.gov (United States)

    Ho, J C S; Nadeem, A; Rydström, A; Puthia, M; Svanborg, C

    2016-02-18

    HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) kills tumor cells broadly suggesting that conserved survival pathways are perturbed. We now identify nucleotide-binding proteins as HAMLET binding partners, accounting for about 35% of all HAMLET targets in a protein microarray comprising 8000 human proteins. Target kinases were present in all branches of the Kinome tree, including 26 tyrosine kinases, 10 tyrosine kinase-like kinases, 13 homologs of yeast sterile kinases, 4 casein kinase 1 kinases, 15 containing PKA, PKG, PKC family kinases, 15 calcium/calmodulin-dependent protein kinase kinases and 13 kinases from CDK, MAPK, GSK3, CLK families. HAMLET acted as a broad kinase inhibitor in vitro, as defined in a screen of 347 wild-type, 93 mutant, 19 atypical and 17 lipid kinases. Inhibition of phosphorylation was also detected in extracts from HAMLET-treated lung carcinoma cells. In addition, HAMLET recognized 24 Ras family proteins and bound to Ras, RasL11B and Rap1B on the cytoplasmic face of the plasma membrane. Direct cellular interactions between HAMLET and activated Ras family members including Braf were confirmed by co-immunoprecipitation. As a consequence, oncogenic Ras and Braf activity was inhibited and HAMLET and Braf inhibitors synergistically increased tumor cell death in response to HAMLET. Unlike most small molecule kinase inhibitors, HAMLET showed selectivity for tumor cells in vitro and in vivo. The results identify nucleotide-binding proteins as HAMLET targets and suggest that dysregulation of the ATPase/kinase/GTPase machinery contributes to cell death, following the initial, selective recognition of HAMLET by tumor cells. The findings thus provide a molecular basis for the conserved tumoricidal effect of HAMLET, through dysregulation of kinases and oncogenic GTPases, to which tumor cells are addicted.

  11. Phenylketonuria : tyrosine supplementation in phenylalanine-restricted diets

    NARCIS (Netherlands)

    van Spronsen, FJ; van Rijn, M; Bekhof, J; Koch, R; Smit, PGA

    2001-01-01

    Treatment of phenylketonuria (PKU) consists of restriction of natural protein and provision of a protein substitute that lacks phenylalanine but is enriched in tyrosine. Large and unexplained differences exist, however, in the tyrosine enrichment of the protein substitutes. Furthermore, some investi

  12. Detecting protein-protein interactions in living cells

    DEFF Research Database (Denmark)

    Gottschalk, Marie; Bach, Anders; Hansen, Jakob Lerche;

    2009-01-01

    to the endogenous C-terminal peptide of the NMDA receptor, as evaluated by a cell-free protein-protein interaction assay. However, it is important to address both membrane permeability and effect in living cells. Therefore a bioluminescence resonance energy transfer (BRET) assay was established, where the C...

  13. The Mertk receptor tyrosine kinase promotes T-B interaction stimulated by IgD B-cell receptor cross-linking.

    Science.gov (United States)

    Shao, Wen-Hai; Zhen, Yuxuan; Finkelman, Fred D; Cohen, Philip L

    2014-09-01

    The Mertk receptor tyrosine kinase facilitates macrophage and DC apoptotic-cell clearance and regulates immune tolerance. Mertk may also contribute to B-cell activation, because Mertk-KO mice fail to develop autoantibodies when allo-activated by T cells. We investigated this possibility with a well-characterized model in which injection of mice with goat anti-IgD antibody causes membrane IgD cross-linking that induces T-independent B cell activation and antigen presentation to T cells. Goat anti-mouse IgD antibody-injected C57BL/6 Mertk-KO mice had normal initial B cell activation and proliferation, but significantly lower T cell activation and proliferation, as well as lower IgE and IgG anti-goat IgG responses, as compared to C57BL/6 WT controls. B cell antigen processing, analyzed by evaluating B cell fluorescence following injection of monoclonal anti-IgD antibody labeled with biotin or FITC, was comparable between Mertk-KO mice and WT mice. IgD Ab primed B cells from Mertk-KO mice exhibited significantly lower ability in activating memory T cells isolated from WT mice injected with the same antigen 10 days before. These observations suggest that Mertk expression is required for optimal B-cell antigen presentation, which is, in turn, required in this model for optimal T cell activation and subsequent T cell-dependent B cell differentiation. PMID:24768065

  14. Antitumor activity of F90,an epidermal growth factor receptor tyrosine kinase inhibitor,on glioblastoma cell line SHG-44

    Institute of Scientific and Technical Information of China (English)

    LIU Fang-jun; GUI Song-bai; LI Chu-zhong; SUN Ze-lin; ZHANG Ya-zhuo

    2008-01-01

    Background Over-expression of epidermal growth factor receptor (EGFR) is thought to be related to cell proliferation,invasion,metastasis,resistance to chemoradiotherapy and poor prognosis of various human cancers.Forty percent to fifty percent of glioblastoma multiforme (GBM) possess deregulated EGFR,which may contribute to the aggressive and refractory course of GBM.Therefore,blockade of EGFR signal transduction may be a promising treatment strategy for GBM.Methods MIT assay,cell growth curve assay and tumor xenograft model were used to evaluate the antitumor activity of F90 against SHG-44 in vitro and in vivo.Western blot assay was applied to evaluate the expression of p-EGFR,p-ERK1,p-JNK,p-P38,Bcl2 and P53 proteins.Results F90