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Sample records for bcr-abl-y177-mediated p27kip1 phosphorylation

  1. Stat1 phosphorylation determines Ras oncogenicity by regulating p27 kip1.

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    Shuo Wang

    Full Text Available Inactivation of p27 Kip1 is implicated in tumorigenesis and has both prognostic and treatment-predictive values for many types of human cancer. The transcription factor Stat1 is essential for innate immunity and tumor immunosurveillance through its ability to act downstream of interferons. Herein, we demonstrate that Stat1 functions as a suppressor of Ras transformation independently of an interferon response. Inhibition of Ras transformation and tumorigenesis requires the phosphorylation of Stat1 at tyrosine 701 but is independent of Stat1 phosphorylation at serine 727. Stat1 induces p27 Kip1 expression in Ras transformed cells at the transcriptional level through mechanisms that depend on Stat1 phosphorylation at tyrosine 701 and activation of Stat3. The tumor suppressor properties of Stat1 in Ras transformation are reversed by the inactivation of p27 Kip1. Our work reveals a novel functional link between Stat1 and p27 Kip1, which act in coordination to suppress the oncogenic properties of activated Ras. It also supports the notion that evaluation of Stat1 phosphorylation in human tumors may prove a reliable prognostic factor for patient outcome and a predictor of treatment response to anticancer therapies aimed at activating Stat1 and its downstream effectors.

  2. Phosphorylation and subcellular localization of p27Kip1 regulated by hydrogen peroxide modulation in cancer cells.

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    Irene L Ibañez

    Full Text Available The Cyclin-dependent kinase inhibitor 1B (p27Kip1 is a key protein in the decision between proliferation and cell cycle exit. Quiescent cells show nuclear p27Kip1, but this protein is exported to the cytoplasm in response to proliferating signals. We recently reported that catalase treatment increases the levels of p27Kip1 in vitro and in vivo in a murine model. In order to characterize and broaden these findings, we evaluated the regulation of p27Kip1 by hydrogen peroxide (H(2O(2 in human melanoma cells and melanocytes. We observed a high percentage of p27Kip1 positive nuclei in melanoma cells overexpressing or treated with exogenous catalase, while non-treated controls showed a cytoplasmic localization of p27Kip1. Then we studied the levels of p27Kip1 phosphorylated (p27p at serine 10 (S10 and at threonine 198 (T198 because phosphorylation at these sites enables nuclear exportation of this protein, leading to accumulation and stabilization of p27pT198 in the cytoplasm. We demonstrated by western blot a decrease in p27pS10 and p27pT198 levels in response to H(2O(2 removal in melanoma cells, associated with nuclear p27Kip1. Melanocytes also exhibited nuclear p27Kip1 and lower levels of p27pS10 and p27pT198 than melanoma cells, which showed cytoplasmic p27Kip1. We also showed that the addition of H(2O(2 (0.1 µM to melanoma cells arrested in G1 by serum starvation induces proliferation and increases the levels of p27pS10 and p27pT198 leading to cytoplasmic localization of p27Kip1. Nuclear localization and post-translational modifications of p27Kip1 were also demonstrated by catalase treatment of colorectal carcinoma and neuroblastoma cells, extending our findings to these other human cancer types. In conclusion, we showed in the present work that H(2O(2 scavenging prevents nuclear exportation of p27Kip1, allowing cell cycle arrest, suggesting that cancer cells take advantage of their intrinsic pro-oxidant state to favor cytoplasmic localization

  3. High-throughput screening reveals alsterpaullone, 2-cyanoethyl as a potent p27Kip1 transcriptional inhibitor.

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    Brandon J Walters

    Full Text Available p27Kip1 is a cell cycle inhibitor that prevents cyclin dependent kinase (CDK/cyclin complexes from phosphorylating their targets. p27Kip1 is a known tumor suppressor, as the germline loss of p27Kip1 results in sporadic pituitary formation in aged rodents, and its presence in human cancers is indicative of a poor prognosis. In addition to its role in cancer, loss of p27Kip1 results in regenerative phenotypes in some tissues and maintenance of stem cell pluripotency, suggesting that p27Kip1 inhibitors could be beneficial for tissue regeneration. Because p27Kip1 is an intrinsically disordered protein, identifying direct inhibitors of the p27Kip1 protein is difficult. Therefore, we pursued a high-throughput screening strategy to identify novel p27Kip1 transcriptional inhibitors. We utilized a luciferase reporter plasmid driven by the p27Kip1 promoter to transiently transfect HeLa cells and used cyclohexamide as a positive control for non-specific inhibition. We screened a "bioactive" library consisting of 8,904 (4,359 unique compounds, of which 830 are Food and Drug Administration (FDA approved. From this screen, we successfully identified 111 primary hits with inhibitory effect against the promoter of p27Kip1. These hits were further refined using a battery of secondary screens. Here we report four novel p27Kip1 transcriptional inhibitors, and further demonstrate that our most potent hit compound (IC50 = 200 nM Alsterpaullone 2-cyanoethyl, inhibits p27Kip1 transcription by preventing FoxO3a from binding to the p27Kip1 promoter. This screen represents one of the first attempts to identify inhibitors of p27Kip1 and may prove useful for future tissue regeneration studies.

  4. Synergistic effect of melatonin and ghrelin in preventing cisplatin-induced ovarian damage via regulation of FOXO3a phosphorylation and binding to the p27Kip1 promoter in primordial follicles.

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    Jang, Hoon; Na, Younghwa; Hong, Kwonho; Lee, Sangho; Moon, Sohyeon; Cho, Minha; Park, Miseon; Lee, Ok-Hee; Chang, Eun Mi; Lee, Dong Ryul; Ko, Jung Jae; Lee, Woo Sik; Choi, Youngsok

    2017-10-01

    Premature ovarian failure during chemotherapy is a serious problem for young women with cancer. To preserve the fertility of these patients, approaches to prevent chemotherapy-induced ovarian failure are needed. In a previous study, we reported that melatonin treatment prevents the depletion of the dormant follicle pool via repression of the simultaneous activation of dormant primordial follicles by cisplatin. However, melatonin's protective effect was only partial and thus insufficient. In this study, we found that the hormone ghrelin enhances the protective effect of melatonin against cisplatin-induced ovarian failure in mouse model. Co-administration of melatonin and ghrelin more effectively prevented cisplatin-induced follicle disruption. Simultaneous treatment with melatonin and ghrelin almost restored the number of primordial follicles and the corpus luteum in cisplatin-treated ovaries, compared with single administration. We found melatonin and ghrelin receptors on the cell membrane of premature oocytes of primordial follicles. In addition, melatonin and ghrelin co-administration inhibited the cisplatin-induced phosphorylation of PTEN and FOXO3a that induces cytoplasmic translocation of FOXO3a. Inhibition of FOXO3a phosphorylation by melatonin and ghrelin increased the binding affinity of FOXO3a for the p27 Kip1 promoter in primordial follicles. Co-administration of melatonin and ghrelin in cisplatin-treated ovaries restored the expression of p27 Kip1 , which is critical for retention of the dormant status of primordial follicles. In conclusion, these findings suggest that melatonin and ghrelin co-administration is suitable for use as a fertoprotective adjuvant therapy during cisplatin chemotherapy in young female cancer patients. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  5. p27Kip1 Is Required to Mediate a G1 Cell Cycle Arrest Downstream of ATM following Genotoxic Stress.

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    Erica K Cassimere

    Full Text Available The DNA damage response (DDR is a coordinated signaling network that ensures the maintenance of genome stability under DNA damaging stress. In response to DNA lesions, activation of the DDR leads to the establishment of cell cycle checkpoints that delay cell-cycle progression and allow repair of the defects. The tumor suppressor p27Kip1 is a cyclin-CDK inhibitor that plays an important role in regulating quiescence in a variety of tissues. Several studies have suggested that p27Kip1 also plays a role in the maintenance of genomic integrity. Here we demonstrate that p27Kip1 is essential for the establishment of a G1 checkpoint arrest after DNA damage. We also uncovered that ATM phosphorylates p27Kip1 on a previously uncharacterized residue (Ser-140, which leads to its stabilization after induction of DNA double-strand breaks. Inhibition of this stabilization by replacing endogenous p27Kip1 with a Ser-140 phospho-mutant (S140A significantly sensitized cells to IR treatments. Our findings reveal a novel role for p27Kip1 in the DNA damage response pathway and suggest that part of its tumor suppressing functions relies in its ability to mediate a G1 arrest after the induction of DNA double strand breaks.

  6. CRM-1 knockdown inhibits extrahepatic cholangiocarcinoma tumor growth by blocking the nuclear export of p27Kip1.

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    Luo, Jian; Chen, Yongjun; Li, Qiang; Wang, Bing; Zhou, Yanqiong; Lan, Hongzhen

    2016-08-01

    Cholangiocarcinoma is a deadly disease which responds poorly to surgery and conventional chemotherapy or radiotherapy. Early diagnosis is difficult due to the anatomical and biological characteristics of cholangiocarcinoma. Cyclin-dependent kinase inhibitor 1B (p27Kip1) is a cyclin‑dependent kinase inhibitor and in the present study, we found that p27Kip1 expression was suppressed in the nucleus and increased in the cytoplasm in 53 samples of cholangiocarcinoma from patients with highly malignant tumors (poorly-differentiated and tumor-node-metastsis (TNM) stage III-IV) compared with that in samples from 10 patients with chronic cholangitis. The expression of phosphorylated (p-)p27Kip1 (Ser10), one of the phosphorylated forms of p27Kip1, was increased in the patient samples with increasing malignancy and clinical stage. Coincidentally, chromosome region maintenance 1 (CRM-1; also referred to as exportin 1 or Xpo1), a critical protein responsible for protein translocation from the nucleus to the cytoplasm, was also overexpressed in the tumor samples which were poorly differentiated and of a higher clinical stage. Through specific short hairpin RNA (shRNA)-mediated knockdown of CRM-1 in the cholangiocarcinoma cell line QBC939, we identified an elevation of cytoplasmic p27Kip1 and a decrease of nuclear p27Kip1. Furthermore, the viability and colony formation ability of QBC939 cells was largely reduced with G1 arrest. Consistent with the findings of the in vitro experiments, in a xenograft mouse model, the tumors formed in the CRM-1 knockdown group were markedly smaller and weighed less than those in the control group in vivo. Taken together, these findings demonstrated that the interplay between CRM-1 and p27Kip1 may provide potentially potent biomarkers and functional targets for the development of future cholangiocarcinoma treatments.

  7. p27Kip1 Modulates Axonal Transport by Regulating α-Tubulin Acetyltransferase 1 Stability

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    Giovanni Morelli

    2018-05-01

    Full Text Available Summary: The protein p27Kip1 plays roles that extend beyond cell-cycle regulation during cerebral cortex development, such as the regulation of neuronal migration and neurite branching via signaling pathways that converge on the actin and microtubule cytoskeletons. Microtubule-dependent transport is essential for the maturation of neurons and the establishment of neuronal connectivity though synapse formation and maintenance. Here, we show that p27Kip1 controls the transport of vesicles and organelles along the axon of mice cortical projection neurons in vitro. Moreover, suppression of the p27Kip1 ortholog, dacapo, in Drosophila melanogaster disrupts axonal transport in vivo, leading to the reduction of locomotor activity in third instar larvae and adult flies. At the molecular level, p27Kip1 stabilizes the α-tubulin acetyltransferase 1, thereby promoting the acetylation of microtubules, a post-translational modification required for proper axonal transport. : Morelli et al. report that p27Kip1/Dacapo modulates the acetylation of microtubules in axons via stabilization of ATAT1, the main α-tubulin acetyltransferase. Its conditional loss leads to the reduction of bidirectional axonal transport of vesicles and mitochondria in vitro in mice and in vivo in Drosophila. Keywords: p27Kip1, dacapo, acetylation, axonal transport, ATAT1, alpha-tubulin, HDAC6, Drosophila, mouse, cerebral cortex

  8. Clinical significance of cyclin-dependent kinase inhibitor p27Kip1 expression and proliferation in non-Hodgkin's lymphoma

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    Møller, Michael Boe; Skjødt, Karsten; Mortensen, Leif Spange

    1999-01-01

    The cyclin-dependent kinase inhibitor p27Kip1 is a negative cell cycle regulator linking extracellular growth-regulatory signals to the cell cycle machinery in G1. We investigated the pattern and prognostic value of p27Kip1 expression in a population-based group of 203 non-Hodgkin's lymphoma (NHL...... between p27Kip1 and Ki-67 expression. Low expression of p27Kip1, defined as nuclear p27Kip1 expression in lymphomas behaved differently as those with low p27Kip1...... expression tended to do better. Likewise, a high proliferation rate (Ki-67 >40%) was associated with poor survival in indolent and aggressive lymphomas. Multivariate analysis using the proportional hazards model showed that only p27Kip1, and not Ki-67, maintained independent prognostic significance...

  9. Hes1 Directly Controls Cell Proliferation through the Transcriptional Repression of p27Kip1

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    Murata, Kaoru; Hattori, Masakazu; Hirai, Norihito; Shinozuka, Yoriko; Hirata, Hiromi; Kageyama, Ryoichiro; Sakai, Toshiyuki; Minato, Nagahiro

    2005-01-01

    A transcriptional regulator, Hes1, plays crucial roles in the control of differentiation and proliferation of neuronal, endocrine, and T-lymphocyte progenitors during development. Mechanisms for the regulation of cell proliferation by Hes1, however, remain to be verified. In embryonic carcinoma cells, endogenous Hes1 expression was repressed by retinoic acid in concord with enhanced p27Kip1 expression and cell cycle arrest. Conversely, conditional expression of a moderate but not maximal level of Hes1 in HeLa cells by a tetracycline-inducible system resulted in reduced p27Kip1 expression, which was attributed to decreased basal transcript rather than enhanced proteasomal degradation, with concomitant increases in the growth rate and saturation density. Hes1 induction repressed the promoter activity of a 5′ flanking basal enhancer region of p27Kip1 gene in a manner dependent on Hes1 expression levels, and this was mediated by its binding to class C sites in the promoter region. Finally, hypoplastic fetal thymi, as well as livers and brains of Hes1-deficient mice, showed significantly increased p27Kip1 transcripts compared with those of control littermates. These results have suggested that Hes1 directly contributes to the promotion of progenitor cell proliferation through transcriptional repression of a cyclin-dependent kinase inhibitor, p27Kip1. PMID:15870295

  10. Germline CDKN1B/p27Kip1 mutation in multiple endocrine neoplasia

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    Georgitsi, Marianthi; Raitila, Anniina; Karhu, Auli; van der Luijt, Rob B.; Aalfs, Cora M.; Sane, Timo; Vierimaa, Outi; Mäkinen, Markus J.; Tuppurainen, Karoliina; Paschke, Ralph; Gimm, Oliver; Koch, Christian A.; Gündogdu, Sadi; Lucassen, Anneke; Tischkowitz, Marc; Izatt, Louise; Aylwin, Simon; Bano, Gul; Hodgson, Shirley; de Menis, Ernesto; Launonen, Virpi; Vahteristo, Pia; Aaltonen, Lauri A.

    2007-01-01

    Germline mutations in the MEN1 gene predispose to multiple endocrine neoplasia type 1 (MEN1) syndrome, but in up to 20-25% of clinical MEN1 cases, no MEN1 mutations can be found. Recently, a germline mutation in the CDKN1B gene, encoding p27(Kip1), was reported in one suspected MEN1 family with two

  11. Resveratrol protects leukemic cells against cytotoxicity induced by proteasome inhibitors via induction of FOXO1 and p27Kip1

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    Niu, Xiao-Fang; Liu, Bao-Qin; Du, Zhen-Xian; Gao, Yan-Yan; Li, Chao; Li, Ning; Guan, Yifu; Wang, Hua-Qin

    2011-01-01

    It was reported recently that resveratrol could sensitize a number of cancer cells to the antitumoral effects of some conventional chemotherapy drugs. The current study was designed to investigate whether resveratrol could sensitize leukemic cells to proteasome inhibitors. Leukemic cells were treated with MG132 alone or in combination with resveratrol. Cell viability was investigated using MTT assay, and induction of apoptosis and cell cycle distribution was measured using flow cytometry. Western blot and real-time RT-PCR were used to investigate the expression of FOXO1 and p27 Kip1 . CHIP was performed to investigate the binding of FOXO1 to the p27 Kip1 promoter. Resveratrol strongly reduced cytotoxic activities of proteasome inhibitors against leukemic cells. MG132 in combination with resveratrol caused cell cycle blockade at G1/S transition via p27 Kip1 accumulation. Knockdown of p27 Kip1 using siRNA dramatically attenuated the protective effects of resveratrol on cytotoxic actions of proteasome inhibitors against leukemic cells. Resveratrol induced FOXO1 expression at the transcriptional level, while MG132 increased nuclear distribution of FOXO1. MG132 in combination with resveratrol caused synergistic induction of p27 Kip1 through increased recruitment of FOXO1 on the p27 Kip1 promoter. Resveratrol may have the potential to negate the cytotoxic effects of proteasome inhibitors via regulation of FOXO1 transcriptional activity and accumulation of p27 Kip1

  12. The human ubiquitin-conjugating enzyme Cdc34 controls cellular proliferation through regulation of p27Kip1 protein levels

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    Butz, Nicole; Ruetz, Stephan; Natt, Francois; Hall, Jonathan; Weiler, Jan; Mestan, Juergen; Ducarre, Monique; Grossenbacher, Rita; Hauser, Patrick; Kempf, Dominique; Hofmann, Francesco

    2005-01-01

    Ubiquitin-mediated degradation of the cyclin-dependent kinase inhibitor p27 Kip1 was shown to be required for the activation of key cyclin-dependent kinases, thereby triggering the onset of DNA replication and cell cycle progression. Although the SCF Skp2 ubiquitin ligase has been reported to mediate p27 Kip1 degradation, the nature of the human ubiquitin-conjugating enzyme involved in this process has not yet been determined at the cellular level. Here, we show that antisense oligonucleotides targeting the human ubiquitin-conjugating enzyme Cdc34 downregulate its expression, inhibit the degradation of p27 Kip1 , and prevent cellular proliferation. Elevation of p27 Kip1 protein level is found to be the sole requirement for the inhibition of cellular proliferation induced upon downregulation of Cdc34. Indeed, reducing the expression of p27 Kip1 with a specific antisense oligonucleotide is sufficient to reverse the anti-proliferative phenotype elicited by the Cdc34 antisense. Furthermore, downregulation of Cdc34 is found to specifically increase the abundance of the SCF Skp2 ubiquitin ligase substrate p27 Kip1 , but has no concomitant effect on the level of IkBα and β-catenin, which are known substrates of a closely related SCF ligase

  13. Histone Deacetylase Inhibitors Increase p27Kip1 by Affecting Its Ubiquitin-Dependent Degradation through Skp2 Downregulation

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    Adriana Borriello

    2016-01-01

    Full Text Available Histone deacetylase inhibitors (HDACIs represent an intriguing class of pharmacologically active compounds. Currently, some HDACIs are FDA approved for cancer therapy and many others are in clinical trials, showing important clinical activities at well tolerated doses. HDACIs also interfere with the aging process and are involved in the control of inflammation and oxidative stress. In vitro, HDACIs induce different cellular responses including growth arrest, differentiation, and apoptosis. Here, we evaluated the effects of HDACIs on p27Kip1, a key cyclin-dependent kinase inhibitor (CKI. We observed that HDACI-dependent antiproliferative activity is associated with p27Kip1 accumulation due to a reduced protein degradation. p27Kip1 removal requires a preliminary ubiquitination step due to the Skp2-SCF E3 ligase complex. We demonstrated that HDACIs increase p27Kip1 stability through downregulation of Skp2 protein levels. Skp2 decline is only partially due to a reduced Skp2 gene expression. Conversely, the protein decrease is more profound and enduring compared to the changes of Skp2 transcript. This argues for HDACIs effects on Skp2 protein posttranslational modifications and/or on its removal. In summary, we demonstrate that HDACIs increase p27Kip1 by hampering its nuclear ubiquitination/degradation. The findings might be of relevance in the phenotypic effects of these compounds, including their anticancer and aging-modulating activities.

  14. The inhibition of activated hepatic stellate cells proliferation by arctigenin through G0/G1 phase cell cycle arrest: persistent p27(Kip1) induction by interfering with PI3K/Akt/FOXO3a signaling pathway.

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    Li, Ao; Wang, Jun; Wu, Mingjun; Zhang, Xiaoxun; Zhang, Hongzhi

    2015-01-15

    Proliferation of hepatic stellate cells (HSCs) is vital for the development of fibrosis during liver injury. In this study, we describe that arctigenin (ATG), a major bioactive component of Fructus Arctii, exhibited selective cytotoxic activity via inhibiting platelet-derived growth factor-BB (PDGF-BB)-activated HSCs proliferation and arrested cell cycle at G0/G1 phase, which could not be observed in normal human hepatocytes in vitro. The cyclin-dependent kinase (CDK) 4/6 activities could be strongly inhibited by ATG through down-regulation of cyclin D1 and CDK4/6 expression in early G1 phase arrest. In the ATG-treated HSCs, the expression level of p27(Kip1) and the formation of CDK2-p27(Kip1) complex were also increased. p27(Kip1) silencing significantly attenuated the effect of ATG, including cell cycle arrest and suppression of proliferation in activated HSCs. We also found that ATG suppressed PDGF-BB-induced phosphorylation of Akt and its downstream transcription factor Forkhead box O 3a (FOXO3a), decreased binding of FOXO3a to 14-3-3 protein, and stimulated nuclear translocation of FOXO3a in activated HSCs. Furthermore, knockdown of FOXO3a expression by FOXO3a siRNA attenuated ATG-induced up-regulation of p27(Kip1) in activated HSCs. All the above findings suggested that ATG could increase the levels of p27(Kip1) protein through inhibition of Akt and improvement of FOXO3a activity, in turn inhibited the CDK2 kinase activity, and eventually caused an overall inhibition of HSCs proliferation. Copyright © 2014 Elsevier B.V. All rights reserved.

  15. MIB-1 (KI-67) proliferation index and cyclin-dependent kinase inhibitor p27(Kip1) protein expression in nephroblastoma

    NARCIS (Netherlands)

    M.A.I. Ghanem (Mazen); Th.H. van der Kwast (Theo); M.K. Sudaryo; R.B. Mathoera (Rejiv); M.M. van den Heuvel-Eibrink (Marry); A.A. Al-Doray; R.J.M. Nijman (Rien); G.J. van Steenbrugge (Gert Jan)

    2004-01-01

    textabstractPURPOSE: A number of studies have indicated that the tumor proliferation marker MIB-1 and cell cycle inhibitor p27(Kip1) expression are of prognostic importance in a variety of cancers. The present study was performed to evaluate the prognostic value of these

  16. Vitamin E δ-tocotrienol induces p27(Kip1-dependent cell-cycle arrest in pancreatic cancer cells via an E2F-1-dependent mechanism.

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    Pamela J Hodul

    Full Text Available Vitamin E δ-tocotrienol has been shown to have antitumor activity, but the precise molecular mechanism by which it inhibits the proliferation of cancer cells remains unclear. Here, we demonstrated that δ-tocotrienol exerted significant cell growth inhibition pancreatic ductal cancer (PDCA cells without affecting normal human pancreatic ductal epithelial cell growth. We also showed that δ-tocotrienol-induced growth inhibition occurred concomitantly with G(1 cell-cycle arrest and increased p27(Kip1 nuclear accumulation. This finding is significant considering that loss of nuclear p27(Kip1 expression is a well-established adverse prognostic factor in PDCA. Furthermore, δ-tocotrienol inactivated RAF-MEK-ERK signaling, a pathway known to suppress p27(Kip1 expression. To determine whether p27(Kip1 induction is required for δ-tocotrienol inhibition of PDCA cell proliferation, we stably silenced the CDKN1B gene, encoding p27(Kip1, in MIAPaCa-2 PDCA cells and demonstrated that p27(Kip1 silencing suppressed cell-cycle arrest induced by δ-tocotrienol. Furthermore, δ-tocotrienol induced p27(Kip1 mRNA expression but not its protein degradation. p27(Kip1 gene promoter activity was induced by δ-tocotrienol through the promoter's E2F-1 binding site, and this activity was attenuated by E2F-1 depletion using E2F-1 small interfering RNA. Finally, decreased proliferation, mediated by Ki67 and p27(Kip1 expression by δ-tocotrienol, was confirmed in vivo in a nude mouse xenograft pancreatic cancer model. Our findings reveal a new mechanism, dependent on p27(Kip1 induction, by which δ-tocotrienol can inhibit proliferation in PDCA cells, providing a new rationale for p27(Kip1 as a biomarker for δ-tocotrienol efficacy in pancreatic cancer prevention and therapy.

  17. oxLDL induces endothelial cell proliferation via Rho/ROCK/Akt/p27kip1 signaling: opposite effects of oxLDL and cholesterol loading.

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    Zhang, Chongxu; Adamos, Crystal; Oh, Myung-Jin; Baruah, Jugajyoti; Ayee, Manuela A A; Mehta, Dolly; Wary, Kishore K; Levitan, Irena

    2017-09-01

    Oxidized modifications of LDL (oxLDL) play a key role in the development of endothelial dysfunction and atherosclerosis. However, the underlying mechanisms of oxLDL-mediated cellular behavior are not completely understood. Here, we compared the effects of two major types of oxLDL, copper-oxidized LDL (Cu 2+ -oxLDL) and lipoxygenase-oxidized LDL (LPO-oxLDL), on proliferation of human aortic endothelial cells (HAECs). Cu 2+ -oxLDL enhanced HAECs' proliferation in a dose- and degree of oxidation-dependent manner. Similarly, LPO-oxLDL also enhanced HAEC proliferation. Mechanistically, both Cu 2+ -oxLDL and LPO-oxLDL enhance HAEC proliferation via activation of Rho, Akt phosphorylation, and a decrease in the expression of cyclin-dependent kinase inhibitor 1B (p27 kip1 ). Both Cu 2+ -oxLDL or LPO-oxLDL significantly increased Akt phosphorylation, whereas an Akt inhibitor, MK2206, blocked oxLDL-induced increase in HAEC proliferation. Blocking Rho with C3 or its downstream target ROCK with Y27632 significantly inhibited oxLDL-induced Akt phosphorylation and proliferation mediated by both Cu 2+ - and LPO-oxLDL. Activation of RhoA was blocked by Rho-GDI-1, which also abrogated oxLDL-induced Akt phosphorylation and HAEC proliferation. In contrast, blocking Rac1 in these cells had no effect on oxLDL-induced Akt phosphorylation or cell proliferation. Moreover, oxLDL-induced Rho/Akt signaling downregulated cell cycle inhibitor p27 kip1 Preloading these cells with cholesterol, however, prevented oxLDL-induced Akt phosphorylation and HAEC proliferation. These findings provide a new understanding of the effects of oxLDL on endothelial proliferation, which is essential for developing new treatments against neovascularization and progression of atherosclerosis. Copyright © 2017 the American Physiological Society.

  18. p27kip1 expression distinguishes papillary hyperplasia in Graves' disease from papillary thyroid carcinoma.

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    Erickson, L A; Yousef, O M; Jin, L; Lohse, C M; Pankratz, V S; Lloyd, R V

    2000-09-01

    In most cases, the histopathologic and cytologic distinction between Graves' disease and papillary thyroid carcinoma is relatively easy, but on occasion Graves' disease may simulate a thyroid papillary carcinoma. For example, papillary fronds with fibrovascular cores may be present in both Graves' disease and papillary carcinoma. p27kip1 (p27) is a cyclin-dependent kinase inhibitory protein that has been shown to be an independent prognostic factor in a variety of human tumors. Our previous studies of p27 expression in hyperplastic and neoplastic endocrine lesions showed that the level of p27 was quite different in these two conditions. To determine if this distinction could also be made between Graves' disease and papillary carcinoma, we analyzed expression of p27 and other cell cycle proteins in a series of cases of Graves' disease with papillary hyperplasia and a series of papillary thyroid carcinomas. Formalin-fixed paraffin-embedded tissues from 61 randomly selected patients with thyroid disease, including 29 cases of Graves' disease with papillary architectural features and 32 cases of papillary carcinoma, were analyzed for expression of p27, Ki-67, and DNA topoisomerase II alpha (topo II alpha) by immunostaining. The distribution of immunoreactivity was analyzed by quantifying the percentage of positive nuclei that was expressed as the labeling index (LI) plus or minus the standard error of the mean. The papillary hyperplasia of Graves' disease had a p27 LI of 68.2 +/- 3.1 (range, 24 to 88), whereas papillary carcinomas had a LI of 25.6 +/- 2.5 (range, 12 to 70) (P hyperplasia in Graves' disease and papillary carcinoma. These results indicate that p27 protein expression is significantly higher in papillary hyperplasia of Graves' disease compared to papillary carcinoma, which may be diagnostically useful in difficult cases.

  19. Accelerated turnover of taste bud cells in mice deficient for the cyclin-dependent kinase inhibitor p27Kip1

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    Perna Marla K

    2011-04-01

    Full Text Available Abstract Background Mammalian taste buds contain several specialized cell types that coordinately respond to tastants and communicate with sensory nerves. While it has long been appreciated that these cells undergo continual turnover, little is known concerning how adequate numbers of cells are generated and maintained. The cyclin-dependent kinase inhibitor p27Kip1 has been shown to influence cell number in several developing tissues, by coordinating cell cycle exit during cell differentiation. Here, we investigated its involvement in the control of taste cell replacement by examining adult mice with targeted ablation of the p27Kip1 gene. Results Histological and morphometric analyses of fungiform and circumvallate taste buds reveal no structural differences between wild-type and p27Kip1-null mice. However, when examined in functional assays, mutants show substantial proliferative changes. In BrdU incorporation experiments, more S-phase-labeled precursors appear within circumvallate taste buds at 1 day post-injection, the earliest time point examined. After 1 week, twice as many labeled intragemmal cells are present, but numbers return to wild-type levels by 2 weeks. Mutant taste buds also contain more TUNEL-labeled cells and 50% more apoptotic bodies than wild-type controls. In normal mice, p27 Kip1 is evident in a subset of receptor and presynaptic taste cells beginning about 3 days post-injection, correlating with the onset of taste cell maturation. Loss of gene function, however, does not alter the proportions of distinct immunohistochemically-identified cell types. Conclusions p27Kip1 participates in taste cell replacement by regulating the number of precursor cells available for entry into taste buds. This is consistent with a role for the protein in timing cell cycle withdrawal in progenitor cells. The equivalence of mutant and wild-type taste buds with regard to cell number, cell types and general structure contrasts with the hyperplasia

  20. Rp58 and p27kip1 coordinate cell cycle exit and neuronal migration within the embryonic mouse cerebral cortex.

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    Clément, Olivier; Hemming, Isabel Anne; Gladwyn-Ng, Ivan Enghian; Qu, Zhengdong; Li, Shan Shan; Piper, Michael; Heng, Julian Ik-Tsen

    2017-05-15

    During the development of the mammalian cerebral cortex, newborn postmitotic projection neurons are born from local neural stem cells and must undergo radial migration so as to position themselves appropriately to form functional neural circuits. The zinc finger transcriptional repressor Rp58 (also known as Znf238 or Zbtb18) is critical for coordinating corticogenesis, but its underlying molecular mechanism remains to be better characterised. Here, we demonstrate that the co-expression of Rp58 and the cyclin dependent kinase inhibitor (CDKI) p27 kip1 is important for E14.5-born cortical neurons to coordinate cell cycle exit and initiate their radial migration. Notably, we find that the impaired radial positioning of Rp58-deficient cortical neurons within the embryonic (E17.5) mouse cortex, as well as their multipolar to bipolar transition from the intermediate zone to the cortical plate can be restored by forced expression of p27 kip1 in concert with suppression of Rnd2, a downstream target gene of Rp58. Furthermore, the restorative effects of p27 kip1 and Rnd2 abrogation are reminiscent of suppressing RhoA signalling in Rp58-deficient cells. Our findings demonstrate functional interplay between a transcriptional regulator and a CDKI to mediate neuroprogenitor cell cycle exit, as well as to promote radial migration through a molecular mechanism consistent with suppression of RhoA signalling.

  1. Time and flow-dependent changes in the p27(kip1) gene network drive maladaptive vascular remodeling.

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    DeSart, Kenneth M; Butler, Khayree; O'Malley, Kerri A; Jiang, Zhihua; Berceli, Scott A

    2015-11-01

    Although clinical studies have identified that a single nucleotide polymorphism in the p27(kip1) gene is associated with success or failure after vein bypass grafting, the underlying mechanisms for this difference are not well defined. Using a high-throughput approach in a flow-dependent vein graft model, we explored the differences in p27(kip1)-related genes that drive the enhanced hyperplastic response under low-flow conditions. Bilateral rabbit carotid artery interposition grafts with jugular vein were placed with a unilateral distal outflow branch ligation to create differential flow states. Grafts were harvested at 2 hours and at 1, 3, 7, 14, and 28 days after implantation, measured for neointimal area, and assayed for cell proliferation. Whole-vessel messenger RNA was isolated and analyzed using an Affymetrix (Santa Clara, Calif) gene array platform. Ingenuity Pathway Analysis (Ingenuity, Redwood City, Calif) was used to identify the gene networks surrounding p27(kip1). This gene set was then analyzed for temporal expression changes after graft placement and for differential expression in the alternate flow conditions. Outflow branch ligation resulted in an eightfold difference in mean flow rates throughout the 28-day perfusion period (P Flow reduction led to a robust hyperplastic response, resulting in a significant increase in intimal area by 7 days (0.13 ± 0.04 mm(2) vs 0.014 ± 0.006 mm(2); P flow grafts demonstrated a burst of actively dividing intimal cells (36.4 ± 9.4 cells/mm(2) vs 11.5 ± 1.9 cells/mm(2); P = .04). Sixty-five unique genes within the microarray were identified as components of the p27(kip1) network. At a false discovery rate of 0.05, 26 genes demonstrated significant temporal changes, and two dominant patterns of expression were identified. Class comparison analysis identified differential expression of 11 genes at 2 hours and seven genes and 14 days between the high-flow and low-flow grafts (P flow and shear stress result in

  2. Prognostic implication of p27Kip1, Skp2 and Cks1 expression in renal cell carcinoma: a tissue microarray study

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    Wang Facheng

    2008-10-01

    Full Text Available Abstract Background p27Kip1 plays a major role as a negative regulator of the cell cycle. The regulation of p27Kip1 degradation is mediated by its specific ubiquitin ligase subunits S-phase kinase protein (Skp 2 and cyclin-dependent kinase subunit (Cks 1. However, little is known regarding the prognostic utility of p27Kip1, Skp2 and Cks1 expression in renal cell carcinoma. Methods Immunohistochemistry was performed for p27Kip1, Skp2 and Cks1 in tissue microarrays of 482 renal cell carcinomas with follow-up. The data were correlated with clinicopathological features. The univariate and multivariate survival analyses were also performed to determine their prognostic significance. Results Immunoreactivity of p27Kip1, Skp2 and Cks1 was noted in 357, 71 and 82 patients, respectively. Skp2 and Cks1 expression were not noted in chromophobe cancers. A strong correlation was found between Skp2 and Cks1 expression (P Kip1 levels (P = 0.006 and P Kip1 expression and Skp2 expression were correlated with larger tumor size and higher stage, as well as tumor necrosis. Cks1 expression was only correlated with tumor size. In univariate analysis, low p27Kip1 expression, Skp2 and Cks1 expression were all associated with a poor prognosis, while in multivariate analysis, only low p27Kip1 expression were independent prognostic factors for both cancer specific survival and recurrence-free survival in patients with RCC. Conclusion Our results suggest that immunohistochemical expression levels of p27Kip1, Skp2 and Cks1 may serve as markers with prognostic value in renal cell carcinoma.

  3. Ursodeoxycholic Acid Influences the Expression of p27kip1 but Not FoxO1 in Patients with Non-Cirrhotic Primary Biliary Cirrhosis

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    Malgorzata Milkiewicz

    2014-01-01

    Full Text Available Background. Enhanced expression of cell cycle inhibitor p27kip1 suppresses cell proliferation. Ursodeoxycholic acid (UDCA delays progression of primary biliary cirrhosis (PBC but its effect on p27kip1 expression is uncertain. Aims. To analyze the expression of p27kip1 and its transcription modulator FoxO1 in patients with PBC, and to assess the impact of UDCA on this pathway. Materials and Methods. The examined human tissue included explanted livers from patients with cirrhotic PBC (n=23, primary sclerosing cholangitis (PSC; n=9, alcoholic liver disease (ALD; n=9, and routine liver biopsies from patients with non-cirrhotic PBC (n=26. Healthy liver samples served as controls (n=19. Livers of FoxO-deficient mice were also studied. mRNA and protein expressions were analyzed by real-time PCR and Western blot. Results. p27kip1 expression was increased in cirrhotic and non-cirrhotic PBC. FoxO1 mRNA levels were increased in PBC (8.5-fold increase versus controls. FoxO1 protein expression in PBC was comparable to controls, but it was decreased in patients with PSC and ALD (63% and 70% reduction, respectively; both P<0.05 versus control. UDCA-treated non-cirrhotic patients with PBC showed decreased expression of p27kip1 mRNA. Conclusion. PBC progression is characterized by a FoxO1-independent increase of p27kip1 expression. In early PBC, UDCA may enhance liver regeneration via p27kip1-dependent mechanism.

  4. A syndrome of multiorgan hyperplasia with features of gigantism, tumorigenesis, and female sterility in p27(Kip1)-deficient mice.

    Science.gov (United States)

    Fero, M L; Rivkin, M; Tasch, M; Porter, P; Carow, C E; Firpo, E; Polyak, K; Tsai, L H; Broudy, V; Perlmutter, R M; Kaushansky, K; Roberts, J M

    1996-05-31

    Targeted disruption of the murine p27(Kip1) gene caused a gene dose-dependent increase in animal size without other gross morphologic abnormalities. All tissues were enlarged and contained more cells, although endocrine abnormalities were not evident. Thymic hyperplasia was associated with increased T lymphocyte proliferation, and T cells showed enhanced IL-2 responsiveness in vitro. Thus, p27 deficiency may cause a cell-autonomous defect resulting in enhanced proliferation in response to mitogens. In the spleen, the absence of p27 selectively enhanced proliferation of hematopoietic progenitor cells. p27 deletion, like deletion of the Rb gene, uniquely caused neoplastic growth of the pituitary pars intermedia, suggesting that p27 and Rb function in the same regulatory pathway. The absence of p27 also caused an ovulatory defect and female sterility. Maturation of secondary ovarian follicles into corpora lutea, which express high levels of p27, was markedly impaired.

  5. The RNA-binding protein Celf1 post-transcriptionally regulates p27Kip1 and Dnase2b to control fiber cell nuclear degradation in lens development.

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    Archana D Siddam

    2018-03-01

    Full Text Available Opacification of the ocular lens, termed cataract, is a common cause of blindness. To become transparent, lens fiber cells undergo degradation of their organelles, including their nuclei, presenting a fundamental question: does signaling/transcription sufficiently explain differentiation of cells progressing toward compromised transcriptional potential? We report that a conserved RNA-binding protein Celf1 post-transcriptionally controls key genes to regulate lens fiber cell differentiation. Celf1-targeted knockout mice and celf1-knockdown zebrafish and Xenopus morphants have severe eye defects/cataract. Celf1 spatiotemporally down-regulates the cyclin-dependent kinase (Cdk inhibitor p27Kip1 by interacting with its 5' UTR and mediating translation inhibition. Celf1 deficiency causes ectopic up-regulation of p21Cip1. Further, Celf1 directly binds to the mRNA of the nuclease Dnase2b to maintain its high levels. Together these events are necessary for Cdk1-mediated lamin A/C phosphorylation to initiate nuclear envelope breakdown and DNA degradation in fiber cells. Moreover, Celf1 controls alternative splicing of the membrane-organization factor beta-spectrin and regulates F-actin-crosslinking factor Actn2 mRNA levels, thereby controlling fiber cell morphology. Thus, we illustrate new Celf1-regulated molecular mechanisms in lens development, suggesting that post-transcriptional regulatory RNA-binding proteins have evolved conserved functions to control vertebrate oculogenesis.

  6. Inactivation of p27kip1 Promoted Nonspecific Inflammation by Enhancing Macrophage Proliferation in Islet Transplantation.

    Science.gov (United States)

    Li, Yang; Ding, Xiaoming; Fan, Ping; Guo, Jian; Tian, Xiaohui; Feng, Xinshun; Zheng, Jin; Tian, Puxun; Ding, Chenguang; Xue, Wujun

    2016-11-01

    Islet transplantation suffers from low efficiency caused by nonspecific inflammation-induced graft loss after transplantation. This study reports increased islet loss and enhanced inflammatory response in p27-deficient mice (p27-/-) and proposes a possible mechanism. Compared with wild type, p27-/- mice showed more severe functional injury of islet, with increased serum levels of inflammatory cytokines IL-1 and TNF-α, inducing macrophage proliferation. Furthermore, the increased number, proapoptotic proteins, and nuclear factor-kappa b (NF-κB) phosphorylation status of the infiltrating macrophages were accompanied by increased TNF-α mRNA level of islet graft site in p27-/- mice. Moreover, in vitro, we found that macrophages were still activated and cocultured with islet and promoted islet loss even blocking the direct effect of TNF-α on islets. Malondialdehyde (MDA, an end product of lipid peroxidation) in islet and media were increased after cocultured with macrophages. p27 deficiency also increased macrophage proliferation and islet injury. Therefore, p27 inactivation promotes injury islet graft loss via the elevation of proliferation and inflammatory cytokines secretion in infiltrating macrophages which induced nonspecific inflammation independent of TNF-α/nuclear factor-kappa b pathway. This potentially represents a promising therapeutic target in improving islet graft survival.

  7. Deficiency of cyclin-dependent kinase inhibitors p21Cip1 and p27Kip1 accelerates atherogenesis in apolipoprotein E-deficient mice

    International Nuclear Information System (INIS)

    Akyuerek, Levent M.; Boehm, Manfred; Olive, Michelle; Zhou, Alex-Xianghua; San, Hong; Nabel, Elizabeth G.

    2010-01-01

    Cyclin-dependent kinase inhibitors, p21 Cip1 and p27 Kip1 , are upregulated during vascular cell proliferation and negatively regulate growth of vascular cells. We hypothesized that absence of either p21 Cip1 or p27 Kip1 in apolipoprotein E (apoE)-deficiency may increase atherosclerotic plaque formation. Compared to apoE -/- aortae, both apoE -/- /p21 -/- and apoE -/- /p27 -/- aortae exhibited significantly more atherosclerotic plaque following a high-cholesterol regimen. This increase was particularly observed in the abdominal aortic regions. Deficiency of p27 Kip1 accelerated plaque formation significantly more than p21 -/- in apoE -/- mice. This increased plaque formation was in parallel with increased intima/media area ratios. Deficiency of p21 Cip1 and p27 Kip1 accelerates atherogenesis in apoE -/- mice. These findings have significant implications for our understanding of the molecular basis of atherosclerosis associated with excessive proliferation of vascular cells.

  8. miR-221 and miR-222 expression affects the proliferation potential of human prostate carcinoma cell lines by targeting p27Kip1.

    Science.gov (United States)

    Galardi, Silvia; Mercatelli, Neri; Giorda, Ezio; Massalini, Simone; Frajese, Giovanni Vanni; Ciafrè, Silvia Anna; Farace, Maria Giulia

    2007-08-10

    MicroRNAs are short regulatory RNAs that negatively modulate protein expression at a post-transcriptional level and are deeply involved in the pathogenesis of several types of cancers. Here we show that miR-221 and miR-222, encoded in tandem on chromosome X, are overexpressed in the PC3 cellular model of aggressive prostate carcinoma, as compared with LNCaP and 22Rv1 cell line models of slowly growing carcinomas. In all cell lines tested, we show an inverse relationship between the expression of miR-221 and miR-222 and the cell cycle inhibitor p27(Kip1). We recognize two target sites for the microRNAs in the 3' untranslated region of p27 mRNA, and we show that miR-221/222 ectopic overexpression directly results in p27 down-regulation in LNCaP cells. In those cells, we demonstrate that the ectopic overexpression of miR-221/222 strongly affects their growth potential by inducing a G(1) to S shift in the cell cycle and is sufficient to induce a powerful enhancement of their colony-forming potential in soft agar. Consistently, miR-221 and miR-222 knock-down through antisense LNA oligonucleotides increases p27(Kip1) in PC3 cells and strongly reduces their clonogenicity in vitro. Our results suggest that miR-221/222 can be regarded as a new family of oncogenes, directly targeting the tumor suppressor p27(Kip1), and that their overexpression might be one of the factors contributing to the oncogenesis and progression of prostate carcinoma through p27(Kip1) down-regulation.

  9. P27Kip1, regulated by glycogen synthase kinase-3β, results in HMBA-induced differentiation of human gastric cancer cells

    International Nuclear Information System (INIS)

    Wei, Min; Gu, Qinlong; Wang, Zhiwei; Yao, Hongliang; Yang, Zhongyin; Zhang, Qing; Liu, Bingya; Yu, Yingyan; Su, Liping; Zhu, Zhenggang

    2011-01-01

    Gastric cancer is the second most common cause of global cancer-related mortality. Although dedifferentiation predicts poor prognosis in gastric cancer, the molecular mechanism underlying dedifferentiation, which could provide fundamental insights into tumor development and progression, has yet to be elucidated. Furthermore, the molecular mechanism underlying the effects of hexamethylene bisacetamide (HMBA), a recently discovered differentiation inducer, requires investigation and there are no reported studies concerning the effect of HMBA on gastric cancer. Based on the results of FACS analysis, the levels of proteins involved in the cell cycle or apoptosis were determined using western blotting after single treatments and sequential combinations of HMBA and LiCl. GSK-3β and proton pump were investigated by western blotting after up-regulating Akt expression by Ad-Akt infection. To investigate the effects of HMBA on protein localization and the activities of GSK-3β, CDK2 and CDK4, kinase assays, immunoprecipitation and western blotting were performed. In addition, northern blotting and RNase protection assays were carried out to determine the functional concentration of HMBA. HMBA increased p27Kip1 expression and induced cell cycle arrest associated with gastric epithelial cell differentiation. In addition, treating gastric-derived cells with HMBA induced G0/G1 arrest and up-regulation of the proton pump, a marker of gastric cancer differentiation. Moreover, treatment with HMBA increased the expression and activity of GSK-3β in the nucleus but not the cytosol. HMBA decreased CDK2 activity and induced p27Kip1 expression, which could be rescued by inhibition of GSK-3β. Furthermore, HMBA increased p27Kip1 binding to CDK2, and this was abolished by GSK-3β inhibition. The results presented herein suggest that GSK-3β functions by regulating p27Kip1 assembly with CDK2, thereby playing a critical role in G0/G1 arrest associated with HMBA-induced gastric epithelial

  10. Insect peptide CopA3-induced protein degradation of p27Kip1 stimulates proliferation and protects neuronal cells from apoptosis

    International Nuclear Information System (INIS)

    Nam, Seung Taek; Kim, Dae Hong; Lee, Min Bum; Nam, Hyo Jung; Kang, Jin Ku; Park, Mi Jung; Lee, Ik Hwan; Seok, Heon; Lee, Dong Gun; Hwang, Jae Sam; Kim, Ho

    2013-01-01

    Highlights: •CopA3 peptide isolated from the Korean dung beetle has antimicrobial activity. •Our study reported that CopA3 has anticancer and immunosuppressive effects. •We here demonstrated that CopA3 has neurotropic and neuroprotective effects. •CopA3 degrades p27Kip1 protein and this mediates effects of CopA3 on neuronal cells. -- Abstract: We recently demonstrated that the antibacterial peptide, CopA3 (a D-type disulfide dimer peptide, LLCIALRKK), inhibits LPS-induced macrophage activation and also has anticancer activity in leukemia cells. Here, we examined whether CopA3 could affect neuronal cell proliferation. We found that CopA3 time-dependently increased cell proliferation by up to 31 ± 2% in human neuroblastoma SH-SY5Y cells, and up to 29 ± 2% in neural stem cells isolated from neonatal mouse brains. In both cell types, CopA3 also significantly inhibited the apoptosis and viability losses caused by 6-hydroxy dopamine (a Parkinson disease-mimicking agent) and okadaic acid (an Alzheimer’s disease-mimicking agent). Immunoblotting revealed that the p27Kip1 protein (a negative regulator of cell cycle progression) was markedly degraded in CopA3-treated SH-SY5Y cells. Conversely, an adenovirus expressing p27Kip1 significantly inhibited the antiapoptotic effects of CopA3 against 6-hydroxy dopamine- and okadaic acid-induced apoptosis, and decreased the neurotropic effects of CopA3. These results collectively suggest that CopA3-mediated protein degradation of p27Kip1 may be the main mechanism through which CopA3 exerts neuroprotective and neurotropic effects

  11. Insect peptide CopA3-induced protein degradation of p27Kip1 stimulates proliferation and protects neuronal cells from apoptosis

    Energy Technology Data Exchange (ETDEWEB)

    Nam, Seung Taek; Kim, Dae Hong; Lee, Min Bum; Nam, Hyo Jung; Kang, Jin Ku; Park, Mi Jung; Lee, Ik Hwan [Department of Life Science, College of Natural Science, Daejin University, Pocheon, Gyeonggido 487-711 (Korea, Republic of); Seok, Heon [Department of Biomedical Science, Jungwon University, Goesan, Chungcheongbukdo 367-700 (Korea, Republic of); Lee, Dong Gun [School of Life Sciences and Biotechnology, College of Natural Sciences, Kyungpook National University, Daegu 702-701 (Korea, Republic of); Hwang, Jae Sam [Department of Agricultural Biology, National Academy of Agricultural Science, RDA, Suwon 441-707 (Korea, Republic of); Kim, Ho, E-mail: hokim@daejin.ac.kr [Department of Life Science, College of Natural Science, Daejin University, Pocheon, Gyeonggido 487-711 (Korea, Republic of)

    2013-07-19

    Highlights: •CopA3 peptide isolated from the Korean dung beetle has antimicrobial activity. •Our study reported that CopA3 has anticancer and immunosuppressive effects. •We here demonstrated that CopA3 has neurotropic and neuroprotective effects. •CopA3 degrades p27Kip1 protein and this mediates effects of CopA3 on neuronal cells. -- Abstract: We recently demonstrated that the antibacterial peptide, CopA3 (a D-type disulfide dimer peptide, LLCIALRKK), inhibits LPS-induced macrophage activation and also has anticancer activity in leukemia cells. Here, we examined whether CopA3 could affect neuronal cell proliferation. We found that CopA3 time-dependently increased cell proliferation by up to 31 ± 2% in human neuroblastoma SH-SY5Y cells, and up to 29 ± 2% in neural stem cells isolated from neonatal mouse brains. In both cell types, CopA3 also significantly inhibited the apoptosis and viability losses caused by 6-hydroxy dopamine (a Parkinson disease-mimicking agent) and okadaic acid (an Alzheimer’s disease-mimicking agent). Immunoblotting revealed that the p27Kip1 protein (a negative regulator of cell cycle progression) was markedly degraded in CopA3-treated SH-SY5Y cells. Conversely, an adenovirus expressing p27Kip1 significantly inhibited the antiapoptotic effects of CopA3 against 6-hydroxy dopamine- and okadaic acid-induced apoptosis, and decreased the neurotropic effects of CopA3. These results collectively suggest that CopA3-mediated protein degradation of p27Kip1 may be the main mechanism through which CopA3 exerts neuroprotective and neurotropic effects.

  12. p27kip1 overexpression regulates IL-1β in the microenvironment of stem cells and eutopic endometriosis co-cultures.

    Science.gov (United States)

    Gonçalves, G A; Invitti, A L; Parreira, R M; Kopelman, A; Schor, E; Girão, M J B C

    2017-01-01

    Endometriosis is a gynecological benign chronic disease defined as the growth of endometrial glands and stroma in extra-uterine sites, most commonly implanted over visceral and peritoneal surfaces within the female pelvis causing inflammatory lesions. It affects around 10% of the female population and is often accompanied by chronic pelvic pain, adhesion formation and infertility. Therefore, endometriosis could be considered a "social disease", since it affects the quality of life, reproductivity and also has a socio-economic impact. The expression of cell cycle and inflammatory proteins is modified in the endometriotic tissues. Immunostaining of glandular and stromal cells in endometrial biopsies obtained from patients with endometriosis compared with those of healthy control demonstrated that endometriotic tissues have lower levels of p27 kip1 protein. Endometriosis endometrial cells cultures have also lower levels of p27 kip1 compared to health endometrial cells cultures and restore the cell cycle balance when transduced with an adenoviral vector carring the p27 kip1 coding gene (Adp27EGFP). The low levels of p27 kip1 are related to the S phase in the cell cycle, whereas higher levels lead to a G1 cell cycle arrest. The inflammatory cytokine IL-1β was recently identified as another key protein in the endometriosis proliferation. This cytokine has elevated levels during the proliferative and secretory phases of the menstrual cycle. In endometriosis endometrial cells cultures the IL-1β stimulates the production of IL-6 and IL-8, increasing the cell proliferation and reducing the apoptosis and Bax expression in these cells. According to these remarks, this work aims to evaluate the inflammatory effects in vitro, but more next to what happens in a woman's body, associating endometrial cells with stem cells, thus mimicking the endometrial microenvironment, with gene therapy using Adp27, notoriously known as controller cell cycle, apoptosis and potent modulator of

  13. SKP2 siRNA inhibits the degradation of P27kip1 and down-regulates the expression of MRP in HL-60/A cells.

    Science.gov (United States)

    Xiao, Jie; Yin, Songmei; Li, Yiqing; Xie, Shuangfeng; Nie, Danian; Ma, Liping; Wang, Xiuju; Wu, Yudan; Feng, Jianhong

    2009-08-01

    S-phase kinase-associated protein 2 (SKP2) gene is a tumor suppressor gene, and is involved in the ubiquitin-mediated degradation of P27kip1. SKP2 and P27kip1 affect the proceeding and prognosis of leukemia through regulating the proliferation, apoptosis and differentiation of leukemia cells. In this study, we explored the mechanism of reversing of HL-60/A drug resistance through SKP2 down-regulation. HL-60/A cells were nucleofected by Amaxa Nucleofector System with SKP2 siRNA. The gene and protein expression levels of Skp2, P27kip1, and multi-drug resistance associated protein (MRP) were determined by reverse transcription-polymerase chain reaction and western blot analysis, respectively. The cell cycle was analyzed by flow cytometry. The 50% inhibitory concentration value was calculated using cytotoxic analysis according to the death rate of these two kinds of cells under different concentrations of chemotherapeutics to compare the sensitivity of the cells. HL-60/A cells showed multi-drug resistance phenotype characteristic by cross-resistance to adriamycin, daunorubicin, and arabinosylcytosine, due to the expression of MRP. We found that the expression of SKP2 was higher in HL-60/A cells than in HL-60 cells, but the expression of P27kip1 was lower. The expression of SKP2 in HL-60/A cells nucleofected by SKP2 siRNA was down-regulated whereas the protein level of P27kip1 was up-regulated. Compared with the MRP expression level in the control group (nucleofected by control siRNA), the mRNA and protein expression levels of MRP in HL-60/A cells nucleofected by SKP2 siRNA were lower, and the latter cells were more sensitive to adriamycin, daunorubicin, and arabinosylcytosine. Down-regulating the SKP2 expression and arresting cells in the G0/G1 phase improve drug sensitivity of leukemia cells with down-regulated MRP expression.

  14. The retinoid X receptor agonist, 9-cis UAB30, inhibits cutaneous T-cell lymphoma proliferation through the SKP2-p27kip1 axis.

    Science.gov (United States)

    Chou, Chu-Fang; Hsieh, Yu-Hua; Grubbs, Clinton J; Atigadda, Venkatram R; Mobley, James A; Dummer, Reinhard; Muccio, Donald D; Eto, Isao; Elmets, Craig A; Garvey, W Timothy; Chang, Pi-Ling

    2018-06-01

    Bexarotene (Targretin ® ) is currently the only FDA approved retinoid X receptor (RXR) -selective agonist for the treatment of cutaneous T-cell lymphomas (CTCLs). The main side effects of bexarotene are hypothyroidism and elevation of serum triglycerides (TGs). The novel RXR ligand, 9-cis UAB30 (UAB30) does not elevate serum TGs or induce hypothyroidism in normal subjects. To assess preclinical efficacy and mechanism of action of UAB30 in the treatment of CTCLs and compare its action with bexarotene. With patient-derived CTCL cell lines, we evaluated UAB30 function in regulating growth, apoptosis, cell cycle check points, and cell cycle-related markers. Compared to bexarotene, UAB30 had lower half maximal inhibitory concentration (IC 50 ) values and was more effective in inhibiting the G1 cell cycle checkpoint. Both rexinoids increased the stability of the cell cycle inhibitor, p27kip1 protein, in part, through targeting components involved in the ubiquitination-proteasome system: 1) decreasing SKP2, a F-box protein that binds and targets p27kip1 for degradation by 26S proteasome and 2) suppressing 20S proteasome activity (cell line-dependent) through downregulation of PSMA7, a component of the 20S proteolytic complex in 26S proteasome. UAB30 and bexarotene induce both early cell apoptosis and suppress cell proliferation. Inhibition of the G1 to S cell cycle transition by rexinoids is mediated, in part, through downregulation of SKP2 and/or 20S proteasome activity, leading to increased p27kip1 protein stability. Because UAB30 has minimal effect in elevating serum TGs and inducing hypothyroidism, it is potentially a better alternative to bexarotene for the treatment of CTCLs. Copyright © 2018 Japanese Society for Investigative Dermatology. Published by Elsevier B.V. All rights reserved.

  15. In vivo regulation of colonic cell proliferation, differentiation, apoptosis, and P27Kip1 by dietary fish oil and butyrate in rats.

    Science.gov (United States)

    Hong, Mee Young; Turner, Nancy D; Murphy, Mary E; Carroll, Raymond J; Chapkin, Robert S; Lupton, Joanne R

    2015-11-01

    We have shown that dietary fish oil is protective against experimentally induced colon cancer, and the protective effect is enhanced by coadministration of pectin. However, the underlying mechanisms have not been fully elucidated. We hypothesized that fish oil with butyrate, a pectin fermentation product, protects against colon cancer initiation by decreasing cell proliferation and increasing differentiation and apoptosis through a p27(Kip1)-mediated mechanism. Rats were provided diets of corn or fish oil, with/without butyrate, and terminated 12, 24, or 48 hours after azoxymethane (AOM) injection. Proliferation (Ki-67), differentiation (Dolichos Biflorus Agglutinin), apoptosis (TUNEL), and p27(Kip1) (cell-cycle mediator) were measured in the same cell within crypts in order to examine the coordination of cell cycle as a function of diet. DNA damage (N(7)-methylguanine) was determined by quantitative IHC analysis. Dietary fish oil decreased DNA damage by 19% (P = 0.001) and proliferation by 50% (P = 0.003) and increased differentiation by 56% (P = 0.039) compared with corn oil. When combined with butyrate, fish oil enhanced apoptosis 24 hours after AOM injection compared with a corn oil/butyrate diet (P = 0.039). There was an inverse relationship between crypt height and apoptosis in the fish oil/butyrate group (r = -0.53, P = 0.040). The corn oil/butyrate group showed a positive correlation between p27(Kip1) expression and proliferation (r = 0.61, P = 0.035). These results indicate the in vivo effect of butyrate on apoptosis and proliferation is dependent on dietary lipid source. These results demonstrate the presence of an early coordinated colonocyte response by which fish oil and butyrate protects against colon tumorigenesis. ©2015 American Association for Cancer Research.

  16. Herbal composition of Cinnamomum cassia, Pinus densiflora, Curcuma longa and Glycyrrhiza glabra prevents atherosclerosis by upregulating p27 (Kip1) expression.

    Science.gov (United States)

    Lee, Jung-Jin; Lee, Ji-Hye; Cho, Won-Kyung; Han, Joo-Hui; Ma, Jin Yeul

    2016-07-28

    Kiom-18 is a novel composition of Cinnamomum cassia, Pinus densiflora, Curcuma longa and Glycyrrhiza glabra. Curcuma longa and Glycyrrhiza glabra, which are traditional medicines in Asia, have been reported to demonstrate preventive effects against atherosclerosis; however, they have not yet been developed into functional atherosclerosis treatments. We therefore studied the anti-atherosclerotic effects and possible molecular mechanisms of Kiom-18 using vascular smooth muscle cells (VSMCs). To assess the anti-proliferative effect of Kiom-18 in vitro, we performed thymidine incorporation, cell cycle progression, immunoblotting and immunofluorescence assays in VSMCs stimulated by platelet derived-growth factor (PDGF)-BB. In addition, we used LDLr knockout mice to identify the effects of Kiom-18 as a preliminary result in an atherosclerosis animal model. Kiom-18 inhibited platelet-derived growth factor (PDGF)-BB-stimulated-VSMC proliferation and DNA synthesis. Additionally, Kiom-18 arrested the cell cycle transition of G0/G1 stimulated by PDGF-BB and its cell cycle-related proteins. Correspondingly, the level of p27(kip1) expression was upregulated in the presence of the Kiom-18 extract. Moreover, in an atherosclerosis animal model of LDLr knockout mice, Kiom-18 extract showed a preventive effect for the formation of atherosclerotic plaque and suppressed body weight, fat weight, food treatment efficiency, neutrophil count, and triglyceride level. These results indicate that Kiom-18 exerts anti-atherosclerotic effects by inhibiting VSMC proliferation via G0/G1 arrest, which upregulates p27(Kip1) expression.

  17. Rare sugar D-allose induces specific up-regulation of TXNIP and subsequent G1 cell cycle arrest in hepatocellular carcinoma cells by stabilization of p27kip1.

    Science.gov (United States)

    Yamaguchi, Fuminori; Takata, Maki; Kamitori, Kazuyo; Nonaka, Machiko; Dong, Youyi; Sui, Li; Tokuda, Masaaki

    2008-02-01

    'Rare sugars' are defined as monosaccharides that exist in nature but are only present in limited quantities. The development of mass production method of rare sugars revealed some interesting physiological effects of these on animal cells, but the mechanisms have not been well studied. We examined the effect of D-allose on the proliferation of cancer cells and the underlying molecular mechanism of the action. The HuH-7 hepatocellular carcinoma cells were treated with various monosaccharides for 48 h and D-allose was shown to inhibit cell growth by 40% in a dose-dependent manner. D-allose induced G1 cell cycle arrest but not apoptosis. The microarray analysis revealed that D-allose significantly up-regulated thioredoxin interacting protein (TXNIP) gene expression, which is often suppressed in tumor cells and western blot analysis confirmed its increase at protein level. The overexpression of TXNIP also induced G1 cell cycle arrest. Analysis of cell cycle regulatory genes showed p27kip1, a key regulator of G1/S cell cycle transition, to be increased at the protein but not the transcriptional level. Protein interaction between TXNIP and jab1, and p27kip1 and jab1, was observed, suggesting stabilization of p27kip1 protein by the competitive inhibition of jab1-mediated nuclear export of p27kip1 by TXNIP. In addition, increased interaction and nuclear localization of TXNIP and p27kip1 were apparent after D-allose treatment. Our findings surprisingly suggest that D-allose, a simple monosaccharide, may act as a novel anticancer agent via unique TXNIP induction and p27kip1 protein stabilization.

  18. In Vivo Interplay between p27Kip1, GATA3, ATOH1, and POU4F3 Converts Non-sensory Cells to Hair Cells in Adult Mice

    Directory of Open Access Journals (Sweden)

    Bradley J. Walters

    2017-04-01

    Full Text Available Summary: Hearing loss is widespread and persistent because mature mammalian auditory hair cells (HCs are nonregenerative. In mice, the ability to regenerate HCs from surrounding supporting cells (SCs declines abruptly after postnatal maturation. We find that combining p27Kip1 deletion with ectopic ATOH1 expression surmounts this age-related decline, leading to conversion of SCs to HCs in mature mouse cochleae and after noise damage. p27Kip1 deletion, independent of canonical effects on Rb-family proteins, upregulated GATA3, a co-factor for ATOH1 that is lost from SCs with age. Co-activation of GATA3 or POU4F3 and ATOH1 promoted conversion of SCs to HCs in adult mice. Activation of POU4F3 alone also converted mature SCs to HCs in vivo. These data illuminate a genetic pathway that initiates auditory HC regeneration and suggest p27Kip1, GATA3, and POU4F3 as additional therapeutic targets for ATOH1-mediated HC regeneration. : Auditory hair cells are nonregenerative, resulting in persistent hearing loss upon damage. Walters et al. find that manipulating two genes, p27Kip1 and Atoh1, induces the conversion of nonsensory cells to hair cells in adult mice. This effect is mediated by GATA3 and POU4F3, where POU4F3 alone was found to convert nonsensory cells. Keywords: regeneration, aging, differentiation, proliferation, development, cancer, sensory, cochlea, hearing

  19. In vitro differentiation of HT-29 M6 mucus-secreting colon cancer cells involves a trychostatin A and p27(KIP1)-inducible transcriptional program of gene expression.

    Science.gov (United States)

    Mayo, Clara; Lloreta, Josep; Real, Francisco X; Mayol, Xavier

    2007-07-01

    Tumor cell dedifferentiation-such as the loss of cell-to-cell adhesion in epithelial tumors-is associated with tumor progression. To better understand the mechanisms that maintain carcinoma cells in a differentiated state, we have dissected in vitro differentiation pathways in the mucus-secretor HT-29 M6 colon cancer cell line, which spontaneously differentiates in postconfluent cultures. By lowering the extracellular calcium concentration to levels that prevent intercellular adhesion and epithelial polarization, our results reveal that differentiation is calcium-dependent and involves: (i) a process of cell cycle exit to G(0) and (ii) the induction of a transcriptional program of differentiation gene expression (i.e., mucins MUC1 and MUC5AC, and the apical membrane peptidase DPPIV). In calcium-deprived, non-differentiated postconfluent cultures, differentiation gene promoters are repressed by a trichostatin A (TSA)-sensitive mechanism, indicating that loss of gene expression by dedifferentiation is driven by histone deacetylases (HDAC). Since TSA treatment or extracellular calcium restoration allow gene promoter activation to similar levels, we suggest that induction of differentiation is one mechanism of HDAC inhibitor antitumor action. Moreover, transcriptional de-repression can also be induced in non-differentiating culture conditions by overexpressing the cyclin-dependent kinase inhibitor p27(KIP1), which is normally induced during spontaneous differentiation. Since p27(KIP1) downregulation in colon cancer is associated with poor prognosis independently of tumor cell division rates, we propose that p27 (KIP1) may prevent tumor progression by, at least in part, enhancing the expression of some differentiation genes. Therefore, the HT-29 M6 model allows the identification of some basic mechanisms of cancer cell differentiation control, so far revealing HDAC and p27(KIP1) as key regulatory factors of differentiation gene expression.

  20. ER, PgR, Ki67, p27Kip1, and histological grade as predictors of pathological complete response in patients with HER2-positive breast cancer receiving neoadjuvant chemotherapy using taxanes followed by fluorouracil, epirubicin, and cyclophosphamide concomitant with trastuzumab

    International Nuclear Information System (INIS)

    Kurozumi, Sasagu; Inoue, Kenichi; Takei, Hiroyuki; Matsumoto, Hiroshi; Kurosumi, Masafumi; Horiguchi, Jun; Takeyoshi, Izumi; Oyama, Tetsunari

    2015-01-01

    Neoadjuvant chemotherapy (NAC) with taxanes followed by fluorouracil, epirubicin, and cyclophosphamide (FEC), and concurrent trastuzumab is a potent regimen for HER2 over-expressing breast cancer. A high pathological complete response (pCR) rate has been achieved using this regimen; however, the predictive factors and prognostic effects of pCR currently remain unclear. In the present study, we determined whether pCR was related to histological grade (HG) and several biological factors including p27 Kip1 . We also assessed the prognosis of the pCR and non-pCR groups, and expected differences between those positive and negative for lymph node metastasis after chemotherapy. A total of 129 Japanese women with HER2-positive invasive breast cancer received either paclitaxel or docetaxel followed by FEC, with the concomitant administration of trastuzumab. The statuses of HG, ER, PgR, Ki67, and p27 Kip1 were evaluated to determine their relationship with pCR. Relapse-free survival (RFS) and overall survival (OS) were also analyzed for their relationship with pCR and pathological nodal involvement. pCR was obtained in 84 out of 129 patients and the pCR rate was 65.1 %. The pCR rates related to 5 factors were as follows: HG (grade 3, 70.0 % vs. grades 1–2, 36.8 %), ER (negative, 78.6 % vs. positive, 40.0 %), PgR (negative, 75.3 % vs. positive, 38.9 %), Ki67 (high, 72.0 % vs. low, 47.2 %), and p27 Kip1 (low, 71.0 % vs. high, 50.0 %). RFS was significantly better in the pCR group than in the non-pCR group (p = 0.018). Patients with remaining nodal disease in the pCR group had worse OS (p = 0.0002). High-HG, low-ER, low-PgR, high-Ki67, and low-p27 Kip1 were identified as predictive factors of pCR in NAC with trastuzumab, while pCR and negative nodes were predictive of better survivals. The online version of this article (doi:10.1186/s12885-015-1641-y) contains supplementary material, which is available to authorized users

  1. The Rho GTPase Effector ROCK Regulates Cyclin A, Cyclin D1, and p27Kip1 Levels by Distinct Mechanisms

    OpenAIRE

    Croft, Daniel R.; Olson, Michael F.

    2006-01-01

    The members of the Rho GTPase family are well known for their regulation of actin cytoskeletal structures. In addition, they influence progression through the cell cycle. The RhoA and RhoC proteins regulate numerous effector proteins, with a central and vital signaling role mediated by the ROCK I and ROCK II serine/threonine kinases. The requirement for ROCK function in the proliferation of numerous cell types has been revealed by studies utilizing ROCK-selective inhibitors such as Y-27632. H...

  2. The Cell Cycle Inhibitor p27KIP1: A Key of G1 Arrest by Androgen Ablation and by Vitamin D3 Analog

    National Research Council Canada - National Science Library

    Slingerland, Joyce

    2001-01-01

    .... Effects of androgens and vDR activation by EB 1089 on p27 function were assayed. We demonstrated that physiologic concentrations of DHT and EB 1089 have synergistic effects to upregulate p27 and inhibit growth of prostate cancer cells...

  3. VEGF-A promotes lymphoma tumour growth by activation of STAT proteins and inhibition of p27(KIP1) via paracrine mechanisms

    NARCIS (Netherlands)

    Roorda, Berber D.; ter Elst, Arja; Scherpen, Frank J. G.; Meemusen-de Boer, Tiny G. J.; Kamps, Willem A.; de Bont, Eveline S. J. M.

    Increased levels of circulating VEGF-A have been demonstrated in patients with non-Hodgkin lymphoma (NHL) and are associated with progressive disease and poor clinical outcome. We investigated the role of VEGF-A in lymphoma tumour growth on a molecular level in order to identify the mechanism of

  4. PLD2 has both enzymatic and cell proliferation-inducing capabilities, that are differentially regulated by phosphorylation and dephosphorylation

    International Nuclear Information System (INIS)

    Henkels, Karen M.; Short, Stephen; Peng, Hong-Juan; Fulvio, Mauricio Di; Gomez-Cambronero, Julian

    2009-01-01

    Phospholipase D2 (PLD2) overexpression in mammalian cells results in cell transformation. We have hypothesized that this is due to an increase of de novo DNA synthesis. We show here that overexpression of PLD2-WT leads to an increased DNA synthesis, as measured by the expression levels of the proliferation markers PCNA, p27 KIP1 and phospho-histone-3. The enhancing effect was even higher with phosphorylation-deficient PLD2-Y179F and PLD2-Y511F mutants. The mechanism for this did not involve the enzymatic activity of the lipase, but, rather, the presence of the protein tyrosine phosphatase CD45, as silencing with siRNA for CD45 abrogated the effect. The two Y→F mutants had in common a YxN consensus site that, in the phosphorylated counterparts, could be recognized by SH2-bearing proteins, such as Grb2. Even though Y179F and Y511F cannot bind Grb2, they could still find other protein partners, one of which, we have reasoned, could be CD45 itself. Affinity purified PLD2 is indeed activated by Grb2 and deactivated by CD45 in vitro. We concluded that phosphorylated PLD2, aided by Grb2, mediates lipase activity, whereas dephosphorylated PLD2 mediates an induction of cell proliferation, and the specific residues involved in this newly discovered regulation of PLD2 are Y 179 and Y 511 .

  5. CDK2 phosphorylation of Smad2 disrupts TGF-beta transcriptional regulation in resistant primary bone marrow myeloma cells.

    Science.gov (United States)

    Baughn, Linda B; Di Liberto, Maurizio; Niesvizky, Ruben; Cho, Hearn J; Jayabalan, David; Lane, Joseph; Liu, Fang; Chen-Kiang, Selina

    2009-02-15

    Resistance to growth suppression by TGF-beta1 is common in cancer; however, mutations in this pathway are rare in hematopoietic malignancies. In multiple myeloma, a fatal cancer of plasma cells, malignant cells accumulate in the TGF-beta-rich bone marrow due to loss of both cell cycle and apoptotic controls. Herein we show that TGF-beta activates Smad2 but fails to induce cell cycle arrest or apoptosis in primary bone marrow myeloma and human myeloma cell lines due to its inability to activate G(1) cyclin-dependent kinase (CDK) inhibitors (p15(INK4b), p21(CIP1/WAF1), p27(KIP1), p57(KIP2)) or to repress c-myc and Bcl-2 transcription. Correlating with aberrant activation of CDKs, CDK-dependent phosphorylation of Smad2 on Thr(8) (pT8), a modification linked to impaired Smad activity, is elevated in primary bone marrow myeloma cells, even in asymptomatic monoclonal gammopathy of undetermined significance. Moreover, CDK2 is the predominant CDK that phosphorylates Smad2 on T8 in myeloma cells, leading to inhibition of Smad2-Smad4 association that precludes transcriptional regulation by Smad2. Our findings provide the first direct evidence that pT8 Smad2 couples dysregulation of CDK2 to TGF-beta resistance in primary cancer cells, and they suggest that disruption of Smad2 function by CDK2 phosphorylation acts as a mechanism for TGF-beta resistance in multiple myeloma.

  6. Cytokine-mediated FOXO3a phosphorylation suppresses FasL expression in hemopoietic cell lines: investigations of the role of Fas in apoptosis due to cytokine starvation.

    Science.gov (United States)

    Behzad, Hayedeh; Jamil, Sarwat; Denny, Trisha A; Duronio, Vincent

    2007-05-01

    We have investigated phosphatidylinositol 3-kinase (PI3K)-dependent survival signalling pathways using several cytokines in three different hemopoietic cell lines, MC/9, FDC-P1, and TF-1. Cytokines caused PI3K- and PKB-dependent phosphorylation of FOXO3a (previously known as FKHRL1) at three distinct sites. Following cytokine withdrawal or PI3K inhibition, both of which are known to lead to apoptosis, there was a loss of FOXO3a phosphorylation, and a resulting increase in forkhead transcriptional activity, along with increased expression of Fas Ligand (FasL), which could be detected at the cell surface. Concurrently, an increase in cell surface expression of Fas was also detected. Despite the presence of both FasL and Fas, there was no detectable evidence that activation of Fas-mediated apoptotic events was contributing to apoptosis resulting from cytokine starvation or inhibition of PI3K activity. Thus, inhibition of FOXO3a activity is mediated by the PI3K-PKB pathway, but regulation of FasL is not the primary means by which cell survival is regulated in cytokine-dependent hemopoietic cells. We were also able to confirm increased expression of known FOXO3a targets, Bim and p27kip1. Together, these results support the conclusion that mitochondrial-mediated signals play the major role in apoptosis of hemopoietic cells due to loss of cytokine signalling.

  7. Antitumor activity of intratumoral injection of pcDNA3.1-p27Kip1mt followed by in vivo electroporation in a malignant Burkitt’s lymphoma cell xenograft

    OpenAIRE

    Supriatno Supriatno; Sartari Entin Yuletnawati

    2012-01-01

    Background: Human malignant Burkitt’s lymphomas are an uncommon type of Non-Hodgkin Lymphoma commonly affects in children. It is a highly aggressive type of B-cell lymphoma. Treatment for this malignant are still limited. However, a new strategy for refractory cancer, gene therapy is watched with keen interest. Recently, a novel method for high-efficiency and region-controlled in vivo gene transfer was developed by combining in vivo electroporation and plasmid cDNA. In the present study, a no...

  8. Insertional mutagenesis in mice deficient for p15Ink4b, p16Ink4a, p21Cip1, and p27Kip1 reveals cancer gene interactions and correlations with tumor phenotypes

    DEFF Research Database (Denmark)

    Kool, Jaap; Uren, Anthony G; Martins, Carla P

    2010-01-01

    -throughput murine leukemia virus insertional mutagenesis screens in mice that are deficient for one or two CDK inhibitors. We retrieved 9,117 retroviral insertions from 476 lymphomas to define hundreds of loci that are mutated more frequently than expected by chance. Many of these loci are skewed toward a specific...... revealed a significant overlap between the datasets. Together, our findings highlight the importance of genetic context within large-scale mutation detection studies, and they show a novel use for insertional mutagenesis data in prioritizing disease-associated genes that emerge from genome-wide association...

  9. Caffeic acid phenethyl ester induced cell cycle arrest and growth inhibition in androgen-independent prostate cancer cells via regulation of Skp2, p53, p21Cip1 and p27Kip1

    OpenAIRE

    Lin, Hui-Ping; Lin, Ching-Yu; Huo, Chieh; Hsiao, Ping-Hsuan; Su, Liang-Cheng; Jiang, Shih Sheng; Chan, Tzu-Min; Chang, Chung-Ho; Chen, Li-Tzong; Kung, Hsing-Jien; Wang, Horng-Dar; Chuu, Chih-Pin

    2015-01-01

    Prostate cancer (PCa) patients receiving the androgen ablation therapy ultimately develop recurrent castration-resistant prostate cancer (CRPC) within 1?3 years. Treatment with caffeic acid phenethyl ester (CAPE) suppressed cell survival and proliferation via induction of G1 or G2/M cell cycle arrest in LNCaP 104-R1, DU-145, 22Rv1, and C4?2 CRPC cells. CAPE treatment also inhibited soft agar colony formation and retarded nude mice xenograft growth of LNCaP 104-R1 cells. We identified that CAP...

  10. Synemin promotes AKT-dependent glioblastoma cell proliferation by antagonizing PP2A

    OpenAIRE

    Pitre, Aaron; Davis, Nathan; Paul, Madhumita; Orr, A Wayne; Skalli, Omar

    2012-01-01

    The intermediate filament protein synemin is present in astrocyte progenitors and glioblastoma cells but not in mature astrocytes. Here we demonstrate a role for synemin in enhancing glioblastoma cell proliferation and clonogenic survival, as synemin RNA interference decreased both behaviors by inducing G1 arrest along with Rb hypophosphorylation and increased protein levels of the G1/S inhibitors p21Cip1 and p27Kip1. Akt involvement was demonstrated by decreased phosphorylation of its substr...

  11. Prolonged mechanical ventilation induces cell cycle arrest in newborn rat lung.

    Directory of Open Access Journals (Sweden)

    Andreas A Kroon

    Full Text Available RATIONALE: The molecular mechanism(s by which mechanical ventilation disrupts alveolar development, a hallmark of bronchopulmonary dysplasia, is unknown. OBJECTIVE: To determine the effect of 24 h of mechanical ventilation on lung cell cycle regulators, cell proliferation and alveolar formation in newborn rats. METHODS: Seven-day old rats were ventilated with room air for 8, 12 and 24 h using relatively moderate tidal volumes (8.5 mL.kg⁻¹. MEASUREMENT AND MAIN RESULTS: Ventilation for 24 h (h decreased the number of elastin-positive secondary crests and increased the mean linear intercept, indicating arrest of alveolar development. Proliferation (assessed by BrdU incorporation was halved after 12 h of ventilation and completely arrested after 24 h. Cyclin D1 and E1 mRNA and protein levels were decreased after 8-24 h of ventilation, while that of p27(Kip1 was significantly increased. Mechanical ventilation for 24 h also increased levels of p57(Kip2, decreased that of p16(INK4a, while the levels of p21(Waf/Cip1 and p15(INK4b were unchanged. Increased p27(Kip1 expression coincided with reduced phosphorylation of p27(Kip1 at Thr¹⁵⁷, Thr¹⁸⁷ and Thr¹⁹⁸ (p<0.05, thereby promoting its nuclear localization. Similar -but more rapid- changes in cell cycle regulators were noted when 7-day rats were ventilated with high tidal volume (40 mL.kg⁻¹ and when fetal lung epithelial cells were subjected to a continuous (17% elongation cyclic stretch. CONCLUSION: This is the first demonstration that prolonged (24 h of mechanical ventilation causes cell cycle arrest in newborn rat lungs; the arrest occurs in G₁ and is caused by increased expression and nuclear localization of Cdk inhibitor proteins (p27(Kip1, p57(Kip2 from the Kip family.

  12. Oxidative phosphorylation revisited

    DEFF Research Database (Denmark)

    Nath, Sunil; Villadsen, John

    2015-01-01

    The fundamentals of oxidative phosphorylation and photophosphorylation are revisited. New experimental data on the involvement of succinate and malate anions respectively in oxidative phosphorylation and photophosphorylation are presented. These new data offer a novel molecular mechanistic...

  13. Lycopene and Beta-Carotene Induce Growth Inhibition and Proapoptotic Effects on ACTH-Secreting Pituitary Adenoma Cells

    Science.gov (United States)

    Leite de Oliveira, Felipe; Soares, Nathália; de Mattos, Rômulo Medina; Hecht, Fábio; Dezonne, Rômulo Sperduto; Vairo, Leandro; Goldenberg, Regina Coeli dos Santos; Gomes, Flávia Carvalho Alcântara; de Carvalho, Denise Pires; Gadelha, Mônica R.; Nasciutti, Luiz Eurico; Miranda-Alves, Leandro

    2013-01-01

    Pituitary adenomas comprise approximately 10–15% of intracranial tumors and result in morbidity associated with altered hormonal patterns, therapy and compression of adjacent sella turcica structures. The use of functional foods containing carotenoids contributes to reduce the risk of chronic diseases such as cancer and vascular disorders. In this study, we evaluated the influence of different concentrations of beta-carotene and lycopene on cell viability, colony formation, cell cycle, apoptosis, hormone secretion, intercellular communication and expression of connexin 43, Skp2 and p27kip1 in ACTH-secreting pituitary adenoma cells, the AtT20 cells, incubated for 48 and 96 h with these carotenoids. We observed a decrease in cell viability caused by the lycopene and beta-carotene treatments; in these conditions, the clonogenic ability of the cells was also significantly decreased. Cell cycle analysis revealed that beta-carotene induced an increase of the cells in S and G2/M phases; furthermore, lycopene increased the proportion of these cells in G0/G1 while decreasing the S and G2/M phases. Also, carotenoids induced apoptosis after 96 h. Lycopene and beta-carotene decreased the secretion of ACTH in AtT20 cells in a dose-dependent manner. Carotenoids blocked the gap junction intercellular communication. In addition, the treatments increased the expression of phosphorylated connexin43. Finally, we also demonstrate decreased expression of S-phase kinase-associated protein 2 (Skp2) and increased expression of p27kip1 in carotenoid-treated cells. These results show that lycopene and beta-carotene were able to negatively modulate events related to the malignant phenotype of AtT-20 cells, through a mechanism that could involve changes in the expression of connexin 43, Skp2 and p27kip1; and suggest that these compounds might provide a novel pharmacological approach to the treatment of Cushing’s disease. PMID:23667519

  14. Lycopene and beta-carotene induce growth inhibition and proapoptotic effects on ACTH-secreting pituitary adenoma cells.

    Directory of Open Access Journals (Sweden)

    Natália F Haddad

    Full Text Available Pituitary adenomas comprise approximately 10-15% of intracranial tumors and result in morbidity associated with altered hormonal patterns, therapy and compression of adjacent sella turcica structures. The use of functional foods containing carotenoids contributes to reduce the risk of chronic diseases such as cancer and vascular disorders. In this study, we evaluated the influence of different concentrations of beta-carotene and lycopene on cell viability, colony formation, cell cycle, apoptosis, hormone secretion, intercellular communication and expression of connexin 43, Skp2 and p27(kip1 in ACTH-secreting pituitary adenoma cells, the AtT20 cells, incubated for 48 and 96 h with these carotenoids. We observed a decrease in cell viability caused by the lycopene and beta-carotene treatments; in these conditions, the clonogenic ability of the cells was also significantly decreased. Cell cycle analysis revealed that beta-carotene induced an increase of the cells in S and G2/M phases; furthermore, lycopene increased the proportion of these cells in G0/G1 while decreasing the S and G2/M phases. Also, carotenoids induced apoptosis after 96 h. Lycopene and beta-carotene decreased the secretion of ACTH in AtT20 cells in a dose-dependent manner. Carotenoids blocked the gap junction intercellular communication. In addition, the treatments increased the expression of phosphorylated connexin43. Finally, we also demonstrate decreased expression of S-phase kinase-associated protein 2 (Skp2 and increased expression of p27(kip1 in carotenoid-treated cells. These results show that lycopene and beta-carotene were able to negatively modulate events related to the malignant phenotype of AtT-20 cells, through a mechanism that could involve changes in the expression of connexin 43, Skp2 and p27(kip1; and suggest that these compounds might provide a novel pharmacological approach to the treatment of Cushing's disease.

  15. About phosphorylation of lappaconitine

    International Nuclear Information System (INIS)

    Burdelnaya, E.V.; Turmukhambetov, A.Zh.

    2005-01-01

    In the article chemical modifications of alkaloid lappaconitine are investigated. It was shown that synthesis of the phosphorylated derivatives are the ways to create new biologically active compounds. Interaction of lappaconitine with phosphorus pentachloride was used to obtain new phosphoric derivatives of alkaloid. The composition and structure of the new phosphorus-containing compounds were confirmed by elemental analysis: IR, UV and 13 C, 1 H, 31 P NMR -spectroscopy

  16. Gambogic Acid Lysinate Induces Apoptosis in Breast Cancer MCF-7 Cells by Increasing Reactive Oxygen Species

    Directory of Open Access Journals (Sweden)

    Yong-Zhan Zhen

    2015-01-01

    Full Text Available Gambogic acid (GA inhibits the proliferation of various human cancer cells. However, because of its water insolubility, the antitumor efficacy of GA is limited. Objectives. To investigate the antitumor activity of gambogic acid lysinate (GAL and its mechanism. Methods. Inhibition of cell proliferation was determined by MTT assay; intracellular ROS level was detected by staining cells with DCFH-DA; cell apoptosis was determined by flow cytometer and the mechanism of GAL was investigated by Western blot. Results. GAL inhibited the proliferation of MCF-7 cells with IC50 values 1.46 μmol/L comparable with GA (IC50, 1.16 μmol/L. GAL promoted the production of ROS; however NAC could remove ROS and block the effect of GAL. GAL inhibited the expression of SIRT1 but increased the phosphorylation of FOXO3a and the expression of p27Kip1. At knockdown of FOXO3a, cell apoptosis induced by GAL can be partly blocked. In addition it also enhanced the cleavage of caspase-3. Conclusions. GAL inhibited MCF-7 cell proliferation and induced MCF-7 cell apoptosis by increasing ROS level which could induce cell apoptosis by both SIRT1/FOXO3a/p27Kip1 and caspase-3 signal pathway. These results suggested that GAL might be useful as a modulation agent in cancer chemotherapy.

  17. Properties of phosphorylated thymidylate synthase

    DEFF Research Database (Denmark)

    Frączyk, Tomasz; Ruman, Tomasz; Wilk, Piotr

    2015-01-01

    by (31)P NMR to be modified only on histidine residues, like potassium phosphoramidate (KPA)-phosphorylated TS proteins. NanoLC-MS/MS, enabling the use of CID and ETD peptide fragmentation methods, identified several phosphohistidine residues, but certain phosphoserine and phosphothreonine residues were...... also implicated. Molecular dynamics studies, based on the mouse TS crystal structure, allowed one to assess potential of several phosphorylated histidine residues to affect catalytic activity, the effect being phosphorylation site dependent....

  18. Phosphorylation of chicken growth hormone

    International Nuclear Information System (INIS)

    Aramburo, C.; Montiel, J.L.; Donoghue, D.; Scanes, C.G.; Berghman, L.R.

    1990-01-01

    The possibility that chicken growth hormone (cGH) can be phosphorylated has been examined. Both native and biosynthetic cGH were phosphorylated by cAMP-dependent protein kinase (and γ- 32 P-ATP). The extent of phosphorylation was however less than that observed with ovine prolactin. Under the conditions employed, glycosylated cGH was not phosphorylated. Chicken anterior pituitary cells in primary culture were incubated in the presence of 32 P-phosphate. Radioactive phosphate was incorporated in vitro into the fraction immunoprecipitable with antisera against cGH. Incorporation was increased with cell number and time of incubation. The presence of GH releasing factor (GRF) increased the release of 32 P-phosphate labeled immunoprecipitable GH into the incubation media but not content of immunoprecipitable GH in the cells. The molecular weight of the phosphorylated immunoreactive cGH in the cells corresponded to cGH dimer

  19. Glycogen phosphorylation and Lafora disease.

    Science.gov (United States)

    Roach, Peter J

    2015-12-01

    Covalent phosphorylation of glycogen, first described 35 years ago, was put on firm ground through the work of the Whelan laboratory in the 1990s. But glycogen phosphorylation lay fallow until interest was rekindled in the mid 2000s by the finding that it could be removed by a glycogen-binding phosphatase, laforin, and that mutations in laforin cause a fatal teenage-onset epilepsy, called Lafora disease. Glycogen phosphorylation is due to phosphomonoesters at C2, C3 and C6 of glucose residues. Phosphate is rare, ranging from 1:500 to 1:5000 phosphates/glucose depending on the glycogen source. The mechanisms of glycogen phosphorylation remain under investigation but one hypothesis to explain C2 and perhaps C3 phosphate is that it results from a rare side reaction of the normal synthetic enzyme glycogen synthase. Lafora disease is likely caused by over-accumulation of abnormal glycogen in insoluble deposits termed Lafora bodies in neurons. The abnormality in the glycogen correlates with elevated phosphorylation (at C2, C3 and C6), reduced branching, insolubility and an enhanced tendency to aggregate and become insoluble. Hyperphosphorylation of glycogen is emerging as an important feature of this deadly childhood disease. Copyright © 2015 Elsevier Ltd. All rights reserved.

  20. Genistein inhibits proliferation of colon cancer cells by attenuating a negative effect of epidermal growth factor on tumor suppressor FOXO3 activity

    International Nuclear Information System (INIS)

    Qi, Wentao; Weber, Christopher R; Wasland, Kaarin; Savkovic, Suzana D

    2011-01-01

    Soy consumption is associated with a lower incidence of colon cancer which is believed to be mediated by one of its of components, genistein. Genistein may inhibit cancer progression by inducing apoptosis or inhibiting proliferation, but mechanisms are not well understood. Epidermal growth factor (EGF)-induced proliferation of colon cancer cells plays an important role in colon cancer progression and is mediated by loss of tumor suppressor FOXO3 activity. The aim of this study was to assess if genistein exerts anti-proliferative properties by attenuating the negative effect of EGF on FOXO3 activity. The effect of genistein on proliferation stimulated by EGF-mediated loss of FOXO3 was examined in human colonic cancer HT-29 cells. EGF-induced FOXO3 phosphorylation and translocation were assessed in the presence of genistein. EGF-mediated loss of FOXO3 interactions with p53 (co-immunoprecipitation) and promoter of p27kip1 (ChIP assay) were examined in presence of genistein in cells with mutated p53 (HT-29) and wild type p53 (HCT116). Silencing of p53 determined activity of FOXO3 when it is bound to p53. Genistein inhibited EGF-induced proliferation, while favoring dephosphorylation and nuclear retention of FOXO3 (active state) in colon cancer cells. Upstream of FOXO3, genistein acts via the PI3K/Akt pathway to inhibit EGF-stimulated FOXO3 phosphorylation (i.e. favors active state). Downstream, EGF-induced disassociation of FOXO3 from mutated tumor suppressor p53, but not wild type p53, is inhibited by genistein favoring FOXO3-p53(mut) interactions with the promoter of the cell cycle inhibitor p27kip1 in colon cancer cells. Thus, the FOXO3-p53(mut) complex leads to elevated p27kip1 expression and promotes cell cycle arrest. These novel anti-proliferative mechanisms of genistein suggest a possible role of combining genistein with other chemoreceptive agents for the treatment of colon cancer

  1. Propofol directly increases tau phosphorylation.

    Directory of Open Access Journals (Sweden)

    Robert A Whittington

    2011-01-01

    Full Text Available In Alzheimer's disease (AD and other tauopathies, the microtubule-associated protein tau can undergo aberrant hyperphosphorylation potentially leading to the development of neurofibrillary pathology. Anesthetics have been previously shown to induce tau hyperphosphorylation through a mechanism involving hypothermia-induced inhibition of protein phosphatase 2A (PP2A activity. However, the effects of propofol, a common clinically used intravenous anesthetic, on tau phosphorylation under normothermic conditions are unknown. We investigated the effects of a general anesthetic dose of propofol on levels of phosphorylated tau in the mouse hippocampus and cortex under normothermic conditions. Thirty min following the administration of propofol 250 mg/kg i.p., significant increases in tau phosphorylation were observed at the AT8, CP13, and PHF-1 phosphoepitopes in the hippocampus, as well as at AT8, PHF-1, MC6, pS262, and pS422 epitopes in the cortex. However, we did not detect somatodendritic relocalization of tau. In both brain regions, tau hyperphosphorylation persisted at the AT8 epitope 2 h following propofol, although the sedative effects of the drug were no longer evident at this time point. By 6 h following propofol, levels of phosphorylated tau at AT8 returned to control levels. An initial decrease in the activity and expression of PP2A were observed, suggesting that PP2A inhibition is at least partly responsible for the hyperphosphorylation of tau at multiple sites following 30 min of propofol exposure. We also examined tau phosphorylation in SH-SY5Y cells transfected to overexpress human tau. A 1 h exposure to a clinically relevant concentration of propofol in vitro was also associated with tau hyperphosphorylation. These findings suggest that propofol increases tau phosphorylation both in vivo and in vitro under normothermic conditions, and further studies are warranted to determine the impact of this anesthetic on the acceleration of

  2. Mel-18 negatively regulates INK4a/ARF-independent cell cycle progression via Akt inactivation in breast cancer.

    Science.gov (United States)

    Lee, Jeong-Yeon; Jang, Ki-Seok; Shin, Dong-Hui; Oh, Mi-Yun; Kim, Hyun-Jun; Kim, Yongseok; Kong, Gu

    2008-06-01

    Mel-18, a polycomb group (PcG) protein, has been suggested as a tumor suppressor in human breast cancer. Previously, we reported that Mel-18 has antiproliferative activity in breast cancer cells. However, its functional mechanism has not been fully elucidated. Here, we investigated the role of Mel-18 in human breast cancer. We saw an inverse correlation between Mel-18 and phospho-Akt, which were expressed at low and high levels, respectively, in primary breast tumor tissues from 40 breast cancer patients. The effect of Mel-18 on cell growth was examined in two breast cancer cell lines, SK-BR-3 and T-47D, which express relatively low and high levels of endogenous Mel-18, respectively. On Mel-18 overexpression in SK-BR-3 cells, cell growth was attenuated and G(1) arrest was observed. Likewise, suppression of Mel-18 by antisense expression in T-47D cells led to enhanced cell growth and accelerated G(1)-S phase transition. In these cells, cyclin-dependent kinase (Cdk)-4 and Cdk2 activities were affected by Mel-18, which were mediated by changes in cyclin D1 expression and p27(Kip1) phosphorylation at Thr(157), but not by INK4a/ARF genes. The changes were both dependent on the phosphatidylinositol 3-kinase/Akt signaling pathway. Akt phosphorylation at Ser(473) was reduced by Mel-18 overexpression in SK-BR-3 cells and enhanced by Mel-18 suppression in T-47D cells. Akt-mediated cytoplasmic localization of p27(Kip1) was inhibited by Mel-18 in SK-BR-3 cells. Moreover, Mel-18 overexpression showed reduced glycogen synthase kinase-3beta phosphorylation, beta-catenin nuclear localization, T-cell factor/lymphoid enhancer factor promoter activity, and cyclin D1 mRNA level. Taken together, we established a linear relationship between Mel-18-->Akt-->G(1) phase regulators.

  3. SYMPOSIUM ON PLANT PROTEIN PHOSPHORYLATION

    Energy Technology Data Exchange (ETDEWEB)

    JOHN C WALKER

    2011-11-01

    Protein phosphorylation and dephosphorylation play key roles in many aspects of plant biology, including control of cell division, pathways of carbon and nitrogen metabolism, pattern formation, hormonal responses, and abiotic and biotic responses to environmental signals. A Symposium on Plant Protein Phosphorylation was hosted on the Columbia campus of the University of Missouri from May 26-28, 2010. The symposium provided an interdisciplinary venue at which scholars studying protein modification, as it relates to a broad range of biological questions and using a variety of plant species, presented their research. It also provided a forum where current international challenges in studies related to protein phosphorylation could be examined. The symposium also stimulated research collaborations through interactions and networking among those in the research community and engaged students and early career investigators in studying issues in plant biology from an interdisciplinary perspective. The proposed symposium, which drew 165 researchers from 13 countries and 21 States, facilitated a rapid dissemination of acquired knowledge and technical expertise regarding protein phosphorylation in plants to a broad range of plant biologists worldwide.

  4. Tyrosine phosphorylation in human lymphomas

    NARCIS (Netherlands)

    Haralambieva, E; Jones, M.; Roncador, GM; Cerroni, L; Lamant, L; Ott, G; Rosenwald, A; Sherman, C; Thorner, P; Kusec, R; Wood, KM; Campo, E; Falini, B; Ramsay, A; Marafioti, T; Stein, H; Kluin, PM; Pulford, K; Mason, DY

    2002-01-01

    In a previous study, we showed that the high level of protein tyrosine phosphorylation present in lymphomas containing an anaplastic lymphoma kinase (ALK) can be demonstrated in routinely processed paraffin tissue sections using immunolabelling techniques. In the present study we investigated

  5. Heterogenic final cell cycle by chicken retinal Lim1 horizontal progenitor cells leads to heteroploid cells with a remaining replicated genome.

    Directory of Open Access Journals (Sweden)

    Shahrzad Shirazi Fard

    Full Text Available Retinal progenitor cells undergo apical mitoses during the process of interkinetic nuclear migration and newly generated post-mitotic neurons migrate to their prospective retinal layer. Whereas this is valid for most types of retinal neurons, chicken horizontal cells are generated by delayed non-apical mitoses from dedicated progenitors. The regulation of such final cell cycle is not well understood and we have studied how Lim1 expressing horizontal progenitor cells (HPCs exit the cell cycle. We have used markers for S- and G2/M-phase in combination with markers for cell cycle regulators Rb1, cyclin B1, cdc25C and p27Kip1 to characterise the final cell cycle of HPCs. The results show that Lim1+ HPCs are heterogenic with regards to when and during what phase they leave the final cell cycle. Not all horizontal cells were generated by a non-apical (basal mitosis; instead, the HPCs exhibited three different behaviours during the final cell cycle. Thirty-five percent of the Lim1+ horizontal cells was estimated to be generated by non-apical mitoses. The other horizontal cells were either generated by an interkinetic nuclear migration with an apical mitosis or by a cell cycle with an S-phase that was not followed by any mitosis. Such cells remain with replicated DNA and may be regarded as somatic heteroploids. The observed heterogeneity of the final cell cycle was also seen in the expression of Rb1, cyclin B1, cdc25C and p27Kip1. Phosphorylated Rb1-Ser608 was restricted to the Lim1+ cells that entered S-phase while cyclin B1 and cdc25C were exclusively expressed in HPCs having a basal mitosis. Only HPCs that leave the cell cycle after an apical mitosis expressed p27Kip1. We speculate that the cell cycle heterogeneity with formation of heteroploid cells may present a cellular context that contributes to the suggested propensity of these cells to generate cancer when the retinoblastoma gene is mutated.

  6. Tyrosine phosphorylation of WW proteins

    Science.gov (United States)

    Reuven, Nina; Shanzer, Matan

    2015-01-01

    A number of key regulatory proteins contain one or two copies of the WW domain known to mediate protein–protein interaction via proline-rich motifs, such as PPxY. The Hippo pathway components take advantage of this module to transduce tumor suppressor signaling. It is becoming evident that tyrosine phosphorylation is a critical regulator of the WW proteins. Here, we review the current knowledge on the involved tyrosine kinases and their roles in regulating the WW proteins. PMID:25627656

  7. Phosphorylation regulates SIRT1 function.

    Directory of Open Access Journals (Sweden)

    Tsutomu Sasaki

    Full Text Available BACKGROUND: SIR2 is an NAD(+-dependent deacetylase [1]-[3] implicated in the regulation of lifespan in species as diverse as yeast [4], worms [5], and flies [6]. We previously reported that the level of SIRT1, the mammalian homologue of SIR2 [7], [8], is coupled to the level of mitotic activity in cells both in vitro and in vivo[9]. Cells from long-lived mice maintained SIRT1 levels of young mice in tissues that undergo continuous cell replacement by proliferating stem cells. Changes in SIRT1 protein level were not associated with changes in mRNA level, suggesting that SIRT1 could be regulated post-transcriptionally. However, other than a recent report on sumoylation [10] and identification of SIRT1 as a nuclear phospho-protein by mass spectrometry [11], post-translational modifications of this important protein have not been reported. METHODOLOGY/PRINCIPAL FINDINGS: We identified 13 residues in SIRT1 that are phosphorylated in vivo using mass spectrometry. Dephosphorylation by phosphatases in vitro resulted in decreased NAD(+-dependent deacetylase activity. We identified cyclinB/Cdk1 as a cell cycle-dependent kinase that forms a complex with and phosphorylates SIRT1. Mutation of two residues phosphorylated by Cyclin B/Cdk1 (threonine 530 and serine 540 disturbs normal cell cycle progression and fails to rescue proliferation defects in SIRT1-deficient cells [12], [13]. CONCLUSIONS/SIGNIFICANCE: Pharmacological manipulation of SIRT1 activity is currently being tested as a means of extending lifespan in mammals. Treatment of obese mice with resveratrol, a pharmacological activator of SIRT1, modestly but significantly improved longevity and, perhaps more importantly, offered some protection against the development of type 2 diabetes mellitus and metabolic syndrome [14]-[16]. Understanding the endogenous mechanisms that regulate the level and activity of SIRT1, therefore, has obvious relevance to human health and disease. Our results identify

  8. Phosphorylated nano-diamond/ Polyimide Nanocomposites

    International Nuclear Information System (INIS)

    Beyler-Çiǧil, Asli; Çakmakçi, Emrah; Kahraman, Memet Vezir

    2014-01-01

    In this study, a novel route to synthesize polyimide (PI)/phosphorylated nanodiamond films with improved thermal and mechanical properties was developed. Surface phosphorylation of nano-diamond was performed in dichloromethane. Phosphorylation dramatically enhanced the thermal stability of nano-diamond. Poly(amic acid) (PAA), which is the precursor of PI, was successfully synthesized with 3,3',4,4'-Benzophenonetetracarboxylic dianhydride (BTDA) and 4,4'-oxydianiline (4,4'-ODA) in the solution of N,N- dimethylformamide (DMF). Pure BTDA-ODA polyimide films and phosphorylated nanodiamond containing BTDA-ODA PI films were prepared. The PAA displayed good compatibility with phosphorylated nano-diamond. The morphology of the polyimide (PI)/phosphorylated nano-diamond was characterized by scanning electron microscopy (SEM). Chemical structure of polyimide and polyimide (PI)/phosphorylated nano-diamond was characterized by FTIR. SEM and FTIR results showed that the phosphorylated nano-diamond was successfully prepared. Thermal properties of the polyimide (PI)/phosphorylated nanodiamond was characterized by thermogravimetric analysis (TGA). TGA results showed that the thermal stability of (PI)/phosphorylated nano-diamond film was increased

  9. Tyrosine phosphorylation in signal transduction

    International Nuclear Information System (INIS)

    Roberts, T.M.; Kaplan, D.; Morgan, W.; Keller, T.; Mamon, H.; Piwnica-Worms, H.; Druker, B.; Whitman, M.; Morrison, D.; Cohen, B.; Schaffhausen, B.; Cantley, L.; Rapp, U.

    1988-01-01

    Recent work has focused on the elucidation of the mechanisms by which membrane-bound tyrosine kinases transmit signals within the cell. To examine the role of tyrosine phosphorylation the authors have employed the following strategy. First, they have utilized antibodies to phosphotyrosine (anti-P.Tyr) to identify candidate substrates of various tyrosine kinases, such as pp60 c-src , the CSF- receptor, or the platelet-derived growth factor (PDGF) receptor. Second, they have attempted to characterize the biochemical properties of the putative substrates and to determine in what manner these properties are modified by phosphorylation on tyrosine residues. In this endeavor, they are recapitulating the classic biochemical analysis used to study the effect of kinases on metabolism. The final portion of our work consists of using modern molecular biological strategies to clone the genes or cDNAs for the substrates and overproduce the relevant proteins for studies in vitro in defined systems. This paper describes the first and second aspects of this strategy, the identification and characterization of novel substrate molecules

  10. Conformational Clusters of Phosphorylated Tyrosine.

    Science.gov (United States)

    Abdelrasoul, Maha; Ponniah, Komala; Mao, Alice; Warden, Meghan S; Elhefnawy, Wessam; Li, Yaohang; Pascal, Steven M

    2017-12-06

    Tyrosine phosphorylation plays an important role in many cellular and intercellular processes including signal transduction, subcellular localization, and regulation of enzymatic activity. In 1999, Blom et al., using the limited number of protein data bank (PDB) structures available at that time, reported that the side chain structures of phosphorylated tyrosine (pY) are partitioned into two conserved conformational clusters ( Blom, N.; Gammeltoft, S.; Brunak, S. J. Mol. Biol. 1999 , 294 , 1351 - 1362 ). We have used the spectral clustering algorithm to cluster the increasingly growing number of protein structures with pY sites, and have found that the pY residues cluster into three distinct side chain conformations. Two of these pY conformational clusters associate strongly with a narrow range of tyrosine backbone conformation. The novel cluster also highly correlates with the identity of the n + 1 residue, and is strongly associated with a sequential pYpY conformation which places two adjacent pY side chains in a specific relative orientation. Further analysis shows that the three pY clusters are associated with distinct distributions of cognate protein kinases.

  11. Threonine phosphorylation of rat liver glycogen synthase

    International Nuclear Information System (INIS)

    Arino, J.; Arro, M.; Guinovart, J.J.

    1985-01-01

    32 P-labeled glycogen synthase specifically immunoprecipitated from 32 P-phosphate incubated rat hepatocytes contains, in addition to [ 32 P] phosphoserine, significant levels of [ 32 P] phosphothreonine. When the 32 P-immunoprecipitate was cleaved with CNBr, the [ 32 P] phosphothreonine was recovered in the large CNBr fragment (CB-2, Mapp 28 Kd). Homogeneous rat liver glycogen synthase was phosphorylated by all the protein kinases able to phosphorylate CB-2 in vitro. After analysis of the immunoprecipitated enzyme for phosphoaminoacids, it was observed that only casein kinase II was able to phosphorylate on threonine and 32 P-phosphate was only found in CB-2. These results demonstrate that rat liver glycogen synthase is phosphorylated at threonine site(s) contained in CB-2 and strongly indicate that casein kinase II may play a role in the ''in vivo'' phosphorylation of liver glycogen synthase. This is the first protein kinase reported to phosphorylate threonine residues in liver glycogen synthase

  12. Mapping of p140Cap phosphorylation sites

    DEFF Research Database (Denmark)

    Repetto, Daniele; Aramu, Simona; Boeri Erba, Elisabetta

    2013-01-01

    phosphorylation and tunes its interactions with other regulatory molecules via post-translation modification. In this work, using mass spectrometry, we found that p140Cap is in vivo phosphorylated on tyrosine (Y) within the peptide GEGLpYADPYGLLHEGR (from now on referred to as EGLYA) as well as on three serine...... residues. Consistently, EGLYA has the highest score of in silico prediction of p140Cap phosphorylation. To further investigate the p140Cap function, we performed site specific mutagenesis on tyrosines inserted in EGLYA and EPLYA, a second sequence with the same highest score of phosphorylation. The mutant...

  13. Phosphorylation of human link proteins

    International Nuclear Information System (INIS)

    Oester, D.A.; Caterson, B.; Schwartz, E.R.

    1986-01-01

    Three link proteins of 48, 44 and 40 kDa were purified from human articular cartilage and identified with monoclonal anti-link protein antibody 8-A-4. Two sets of lower molecular weight proteins of 30-31 kDa and 24-26 kDa also contained link protein epitopes recognized by the monoclonal antibody and were most likely degradative products of the intact link proteins. The link proteins of 48 and 40 kDa were identified as phosphoproteins while the 44 kDa link protein did not contain 32 P. The phosphorylated 48 and 40 kDa link proteins contained approximately 2 moles PO 4 /mole link protein

  14. SIMAC - A phosphoproteomic strategy for the rapid separation of mono-phosphorylated from multiply phosphorylated peptides

    DEFF Research Database (Denmark)

    Thingholm, Tine E; Jensen, Ole N; Robinson, Phillip J

    2008-01-01

    spectrometric analysis, such as immobilized metal affinity chromatography or titanium dioxide the coverage of the phosphoproteome of a given sample is limited. Here we report a simple and rapid strategy - SIMAC - for sequential separation of mono-phosphorylated peptides and multiply phosphorylated peptides from...... and an optimized titanium dioxide chromatographic method. More than double the total number of identified phosphorylation sites was obtained with SIMAC, primarily from a three-fold increase in recovery of multiply phosphorylated peptides....

  15. Membrane phosphorylation and nerve cell function

    International Nuclear Information System (INIS)

    Baer, P.R.

    1982-01-01

    This thesis deals with the phosphorylation of membrane components. In part I a series of experiments is described using the hippocampal slice as a model system. In part II a different model system - cultured hybrid cells - is used to study protein and lipid phosphorylation, influenced by incubation with neuropeptides. In part III in vivo and in vitro studies are combined to study protein phosphorylation after neuroanatomical lesions. In a section of part II (Page 81-90) labelling experiments of the membrane inositol-phospholipids are described. 32 P-ATP was used to label phospholipids in intact hybrid cells, and short incubations were found to be the most favourable. (C.F.)

  16. Protein-Tyrosine Phosphorylation in Bacillus subtilis

    DEFF Research Database (Denmark)

    Mijakovic, Ivan; Petranovic, Dina; Bottini, N.

    2005-01-01

    phosphorylation, indicating that this post-translational modifi cation could regulate physiological processes ranging from stress response and exopolysaccharide synthesis to DNA metabolism. Some interesting work in this fi eld was done in Bacillus subtilis , and we here present the current state of knowledge...... on protein-tyrosine phosphorylation in this gram-positive model organism. With its two kinases, two kinase modulators, three phosphatases and at least four different tyrosine-phosphorylated substrates, B. subtilis is the bacterium with the highest number of presently known participants in the global network...

  17. Insulin treatment promotes tyrosine phosphorylation of PKR and inhibits polyIC induced PKR threonine phosphorylation.

    Science.gov (United States)

    Swetha, Medchalmi; Ramaiah, Kolluru V A

    2015-11-01

    Tyrosine phosphorylation of insulin receptor beta (IRβ) in insulin treated HepG2 cells is inversely correlated to ser(51) phosphorylation in the alpha-subunit of eukaryotic initiation factor 2 (eIF2α) that regulates protein synthesis. Insulin stimulates interaction between IRβ and PKR, double stranded RNA-dependent protein kinase, also known as EIF2AK2, and phosphorylation of tyrosine residues in PKR, as analyzed by immunoprecipitation and pull down assays using anti-IRβ and anti-phosphotyrosine antibodies, recombinant IRβ and immunopurified PKR. Further polyIC or synthetic double stranded RNA-induced threonine phosphorylation or activation of immunopurified and cellular PKR is suppressed in the presence of insulin treated purified IRβ and cell extracts. Acute, but not chronic, insulin treatment enhances tyrosine phosphorylation of IRβ, its interaction with PKR and tyrosine phosphorylation of PKR. In contrast, lipopolysaccharide that stimulates threonine phosphorylation of PKR and eIF2α phosphorylation and AG 1024, an inhibitor of the tyrosine kinase activity of IRβ, reduces PKR association with the receptor, IRβ in HepG2 cells. These findings therefore may suggest that tyrosine phosphorylated PKR plays a role in the regulation of insulin induced protein synthesis and in maintaining insulin sensitivity, whereas, suppression of polyIC-mediated threonine phosphorylation of PKR by insulin compromises its ability to fight against virus infection in host cells. Copyright © 2015 Elsevier Inc. All rights reserved.

  18. Protein phosphorylation in bcterial signaling and regulation

    KAUST Repository

    Mijakovic, Ivan

    2016-01-01

    . Evolutionary studies based on genome comparison indicate that BY-kinases exist only in bacteria. They are non-essential (present in about 40% bacterial genomes), and their knockouts lead to pleiotropic phenotypes, since they phosphorylate many substrates

  19. Fibronectin phosphorylation by ecto-protein kinase

    International Nuclear Information System (INIS)

    Imada, Sumi; Sugiyama, Yayoi; Imada, Masaru

    1988-01-01

    The presence of membrane-associated, extracellular protein kinase (ecto-protein kinase) and its substrate proteins was examined with serum-free cultures of Swiss 3T3 fibroblast. When cells were incubated with [γ- 32 ]ATP for 10 min at 37 degree C, four proteins with apparent molecular weights between 150 and 220 kDa were prominently phosphorylated. These proteins were also radiolabeled by lactoperoxidase catalyzed iodination and were sensitive to mild tryptic digestion, suggesting that they localized on the cell surface or in the extracellular matrix. Phosphorylation of extracellular proteins with [γ- 32 P]ATP in intact cell culture is consistent with the existence of ecto-protein kinase. Anti-fibronectin antibody immunoprecipitated one of the phosphoproteins which comigrated with a monomer and a dimer form of fibronectin under reducing and nonreducing conditions of electrophoresis, respectively. The protein had affinity for gelatin as demonstrated by retention with gelatin-conjugated agarose. This protein substrate of ecto-protein kinase was thus concluded to be fibronectin. The sites of phosphorylation by ecto-protein kinase were compared with those of intracellularly phosphorylated fibronectin by the analysis of radiolabeled amino acids and peptides. Ecto-protein kinase phosphorylated fibronectin at serine and threonine residues which were distinct from the sites of intracellular fibronectin phosphorylation

  20. Phosphorylation of human skeletal muscle myosin

    International Nuclear Information System (INIS)

    Houston, M.E.; Lingley, M.D.; Stuart, D.S.; Hoffman-Goetz, L.

    1986-01-01

    Phosphorylation of the P-light chains (phosphorylatable light chains) in human skeletal muscle myosin was studied in vitro and in vivo under resting an d contracted conditions. biopsy samples from rested vastus lateralis muscle of male and female subjects were incubated in oxygenated physiological solution at 30 0 C. Samples frozen following a quiescent period showed the presence of only unphosphorylated P-light chains designated LC2f (light chain two of fast myosin) CL2s and LC2s'(light chains two of slow myosin). Treatment with caffeine (10 mM) or direct electrical stimulation resulted in the appearance of three additional bands which were identified as the phosphorylated forms of the P-light chains i.e. LC2f-P, LC2s-P and LC2s'-P. The presence of phosphate was confirmed by prior incubation with ( 30 P) orthophosphate. Muscle samples rapidly frozen from resting vastus lateralis muscle revealed the presence of unphosphorylated and phosphorylated P-light chains in approximately equal ratios. Muscle samples rapidly frozen following a maximal 10 second isometric contraction showed virtually only phosphorylated fast and slow P-light chains. These results reveal that the P-light chains in human fast and slow myosin may be rapidly phosphorylated, but the basal level of phosphorylation in rested human muscle considerably exceeds that observed in animal muscles studied in vitro or in situ

  1. Protein phosphorylation during coconut zygotic embryo development

    International Nuclear Information System (INIS)

    Islas-Flores, I.; Oropeza, C.; Hernandez-Sotomayor, S.M.T.

    1998-01-01

    Evidence was obtained on the occurrence of protein threonine, serine, and tyrosine (Tyr) kinases in developing coconut (Cocos nucifera L.) zygotic embryos, based on in vitro phosphorylation of proteins in the presence of [gamma-32P]ATP, alkaline treatment, and thin-layer chromatography analysis, which showed the presence of [32P]phosphoserine, [32P]phosphothreonine, and [32P]phosphotyrosine in [32P]-labeled protein hydrolyzates. Tyr kinase activity was further confirmed in extracts of embryos at different stages of development using antiphosphotyrosine monoclonal antibodies and the synthetic peptide derived from the amino acid sequence surrounding the phosphorylation site in pp60src (RR-SRC), which is specific for Tyr kinases. Anti-phosphotyrosine western blotting revealed a changing profile of Tyr-phosphorylated proteins during embryo development. Tyr kinase activity, as assayed using RR-SRC, also changed during embryo development, showing two peaks of activity, one during early and another during late embryo development. In addition, the use of genistein, a Tyr kinase inhibitor, diminished the ability of extracts to phosphorylate RR-SRC. Results presented here show the occurrence of threonine, serine, and Tyr kinases in developing coconut zygotic embryos, and suggest that protein phosphorylation, and the possible inference of Tyr phosphorylation in particular, may play a role in the coordination of the development of embryos in this species

  2. PKA regulates calcineurin function through the phosphorylation of RCAN1: Identification of a novel phosphorylation site

    International Nuclear Information System (INIS)

    Kim, Seon Sook; Lee, Eun Hye; Lee, Kooyeon; Jo, Su-Hyun; Seo, Su Ryeon

    2015-01-01

    Calcineurin is a calcium/calmodulin-dependent phosphatase that has been implicated in T cell activation through the induction of nuclear factors of activated T cells (NFAT). We have previously suggested that endogenous regulator of calcineurin (RCAN1, also known as DSCR1) is targeted by protein kinase A (PKA) for the control of calcineurin activity. In the present study, we characterized the PKA-mediated phosphorylation site in RCAN1 by mass spectrometric analysis and revealed that PKA directly phosphorylated RCAN1 at the Ser 93. PKA-induced phosphorylation and the increase in the half-life of the RCAN1 protein were prevented by the substitution of Ser 93 with Ala (S93A). Furthermore, the PKA-mediated phosphorylation of RCAN1 at Ser 93 potentiated the inhibition of calcineurin-dependent pro-inflammatory cytokine gene expression by RCAN1. Our results suggest the presence of a novel phosphorylation site in RCAN1 and that its phosphorylation influences calcineurin-dependent inflammatory target gene expression. - Highlights: • We identify novel phosphorylation sites in RCAN1 by LC-MS/MS analysis. • PKA-dependent phosphorylation of RCAN1 at Ser 93 inhibits calcineurin-mediated intracellular signaling. • We show the immunosuppressive function of RCAN1 phosphorylation at Ser 93 in suppressing cytokine expression

  3. Protein phosphorylation systems in postmortem human brain

    International Nuclear Information System (INIS)

    Walaas, S.I.; Perdahl-Wallace, E.; Winblad, B.; Greengard, P.

    1989-01-01

    Protein phosphorylation systems regulated by cyclic adenosine 3',5'-monophosphate (cyclic AMP), or calcium in conjunction with calmodulin or phospholipid/diacylglycerol, have been studied by phosphorylation in vitro of particulate and soluble fractions from human postmortem brain samples. One-dimensional or two-dimensional gel electrophoretic protein separations were used for analysis. Protein phosphorylation catalyzed by cyclic AMP-dependent protein kinase was found to be highly active in both particulate and soluble preparations throughout the human CNS, with groups of both widely distributed and region-specific substrates being observed in different brain nuclei. Dopamine-innervated parts of the basal ganglia and cerebral cortex contained the phosphoproteins previously observed in rodent basal ganglia. In contrast, calcium/phospholipid-dependent and calcium/calmodulin-dependent protein phosphorylation systems were less prominent in human postmortem brain than in rodent brain, and only a few widely distributed substrates for these protein kinases were found. Protein staining indicated that postmortem proteolysis, particularly of high-molecular-mass proteins, was prominent in deeply located, subcortical regions in the human brain. Our results indicate that it is feasible to use human postmortem brain samples, when obtained under carefully controlled conditions, for qualitative studies on brain protein phosphorylation. Such studies should be of value in studies on human neurological and/or psychiatric disorders

  4. Src kinase regulation by phosphorylation and dephosphorylation

    International Nuclear Information System (INIS)

    Roskoski, Robert

    2005-01-01

    Src and Src-family protein-tyrosine kinases are regulatory proteins that play key roles in cell differentiation, motility, proliferation, and survival. The initially described phosphorylation sites of Src include an activating phosphotyrosine 416 that results from autophosphorylation, and an inhibiting phosphotyrosine 527 that results from phosphorylation by C-terminal Src kinase (Csk) and Csk homologous kinase. Dephosphorylation of phosphotyrosine 527 increases Src kinase activity. Candidate phosphotyrosine 527 phosphatases include cytoplasmic PTP1B, Shp1 and Shp2, and transmembrane enzymes include CD45, PTPα, PTPε, and PTPλ. Dephosphorylation of phosphotyrosine 416 decreases Src kinase activity. Thus far PTP-BL, the mouse homologue of human PTP-BAS, has been shown to dephosphorylate phosphotyrosine 416 in a regulatory fashion. The platelet-derived growth factor receptor protein-tyrosine kinase mediates the phosphorylation of Src Tyr138; this phosphorylation has no direct effect on Src kinase activity. The platelet-derived growth factor receptor and the ErbB2/HER2 growth factor receptor protein-tyrosine kinases mediate the phosphorylation of Src Tyr213 and activation of Src kinase activity. Src kinase is also a substrate for protein-serine/threonine kinases including protein kinase C (Ser12), protein kinase A (Ser17), and CDK1/cdc2 (Thr34, Thr46, and Ser72). Of the three protein-serine/threonine kinases, only phosphorylation by CDK1/cdc2 has been demonstrated to increase Src kinase activity. Although considerable information on the phosphoprotein phosphatases that catalyze the hydrolysis of Src phosphotyrosine 527 is at hand, the nature of the phosphatases that mediate the hydrolysis of phosphotyrosine 138 and 213, and phosphoserine and phosphothreonine residues has not been determined

  5. Proteasome phosphorylation regulates cocaine-induced sensitization.

    Science.gov (United States)

    Gonzales, Frankie R; Howell, Kristin K; Dozier, Lara E; Anagnostaras, Stephan G; Patrick, Gentry N

    2018-04-01

    Repeated exposure to cocaine produces structural and functional modifications at synapses from neurons in several brain regions including the nucleus accumbens. These changes are thought to underlie cocaine-induced sensitization. The ubiquitin proteasome system plays a crucial role in the remodeling of synapses and has recently been implicated in addiction-related behavior. The ATPase Rpt6 subunit of the 26S proteasome is phosphorylated by Ca 2+ /calmodulin-dependent protein kinases II alpha at ser120 which is thought to regulate proteasome activity and distribution in neurons. Here, we demonstrate that Rpt6 phosphorylation is involved in cocaine-induced locomotor sensitization. Cocaine concomitantly increases proteasome activity and Rpt6 S120 phosphorylation in cultured neurons and in various brain regions of wild type mice including the nucleus accumbens and prefrontal cortex. In contrast, cocaine does not increase proteasome activity in Rpt6 phospho-mimetic (ser120Asp) mice. Strikingly, we found a complete absence of cocaine-induced locomotor sensitization in the Rpt6 ser120Asp mice. Together, these findings suggest a critical role for Rpt6 phosphorylation and proteasome function in the regulation cocaine-induced behavioral plasticity. Copyright © 2017 Elsevier Inc. All rights reserved.

  6. Regulation of protein phosphorylation in oat mitochondria

    International Nuclear Information System (INIS)

    Pike, C.; Kopeck, K.; Sceppa, E.

    1989-01-01

    We sought to identify phosphorylated proteins in isolated oat mitocchondria and to characterize the enzymatic and regulatory properties of the protein kinase(s). Mitochondria from oats (Avena sativa L. cv. Garry) were purified on Percoll gradients. Mitochondria were incubated with 32 P-γ-ATP; proteins were separated by SDS-PAGE. A small number of bands was detected on autoradiograms, most prominently at 70 kD and 42 kD; the latter band has been tentatively identified as a subunit of the pyruvate dehydrogenase complex, a well-known phosphoprotein. The protein kinase(s) could also phosphorylate casein, but not histone. Spermine enhanced the phosphorylation of casein and inhibited the phosphorylation of the 42 kD band. These studies were carried out on both intact and burst mitochondria. Control by calcium and other ions was investigated. The question of the action of regulators on protein kinase or protein phosphatase was studied by the use of 35 S-adenosine thiotriphosphate

  7. Tyrosine phosphorylation switching of a G protein.

    Science.gov (United States)

    Li, Bo; Tunc-Ozdemir, Meral; Urano, Daisuke; Jia, Haiyan; Werth, Emily G; Mowrey, David D; Hicks, Leslie M; Dokholyan, Nikolay V; Torres, Matthew P; Jones, Alan M

    2018-03-30

    Heterotrimeric G protein complexes are molecular switches relaying extracellular signals sensed by G protein-coupled receptors (GPCRs) to downstream targets in the cytoplasm, which effect cellular responses. In the plant heterotrimeric GTPase cycle, GTP hydrolysis, rather than nucleotide exchange, is the rate-limiting reaction and is accelerated by a receptor-like regulator of G signaling (RGS) protein. We hypothesized that posttranslational modification of the Gα subunit in the G protein complex regulates the RGS-dependent GTPase cycle. Our structural analyses identified an invariant phosphorylated tyrosine residue (Tyr 166 in the Arabidopsis Gα subunit AtGPA1) located in the intramolecular domain interface where nucleotide binding and hydrolysis occur. We also identified a receptor-like kinase that phosphorylates AtGPA1 in a Tyr 166 -dependent manner. Discrete molecular dynamics simulations predicted that phosphorylated Tyr 166 forms a salt bridge in this interface and potentially affects the RGS protein-accelerated GTPase cycle. Using a Tyr 166 phosphomimetic substitution, we found that the cognate RGS protein binds more tightly to the GDP-bound Gα substrate, consequently reducing its ability to accelerate GTPase activity. In conclusion, we propose that phosphorylation of Tyr 166 in AtGPA1 changes the binding pattern with AtRGS1 and thereby attenuates the steady-state rate of the GTPase cycle. We coin this newly identified mechanism "substrate phosphoswitching." © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

  8. Protein phosphorylation in isolated human adipocytes - Adrenergic control of the phosphorylation of hormone-sensitive lipase

    International Nuclear Information System (INIS)

    Smiley, R.M.; Paul, S.; Browning, M.D.; Leibel, R.L.; Hirsch, J.

    1990-01-01

    The effect of adrenergic agents on protein phosphorylation in human adipocytes was examined. Freshly isolated human fat cells were incubated with 32 PO 4 in order to label intracellular ATP, then treated with a variety of adrenergic and other pharmacologic agents. Treatment with the β-adrenergic agonist isoproterenol led to a significant increase in phosphate content of at least five protein bands (M r 52, 53, 63, 67, 84 kDa). The increase in phosphorylation was partially inhibited by the α-2 agonist clonidine. Epinephrine, a combined α and β agonist, was less effective at increasing phosphate content of the proteins than was isoproterenol. Neither insulin nor the α-1 agonist phenylephrine had any discernible effect on the pattern of protein phosphorylation. The 84 kDa phosphorylated peptide band appears to contain hormone-sensitive lipase, a key enzyme in the lipolytic pathway which is activated by phosphorylation. These results are somewhat different than previously reported results for rat adipocytes, and represent the first report of overall pattern and adrenergic modulation of protein phosphorylation in human adipocytes

  9. Ponatinib promotes a G1 cell-cycle arrest of merlin/NF2-deficient human schwann cells.

    Science.gov (United States)

    Petrilli, Alejandra M; Garcia, Jeanine; Bott, Marga; Klingeman Plati, Stephani; Dinh, Christine T; Bracho, Olena R; Yan, Denise; Zou, Bing; Mittal, Rahul; Telischi, Fred F; Liu, Xue-Zhong; Chang, Long-Sheng; Welling, D Bradley; Copik, Alicja J; Fernández-Valle, Cristina

    2017-05-09

    Neurofibromatosis type 2 (NF2) is a genetic syndrome that predisposes individuals to multiple benign tumors of the central and peripheral nervous systems, including vestibular schwannomas. Currently, there are no FDA approved drug therapies for NF2. Loss of function of merlin encoded by the NF2 tumor suppressor gene leads to activation of multiple mitogenic signaling cascades, including platelet-derived growth factor receptor (PDGFR) and SRC in Schwann cells. The goal of this study was to determine whether ponatinib, an FDA-approved ABL/SRC inhibitor, reduced proliferation and/or survival of merlin-deficient human Schwann cells (HSC). Merlin-deficient HSC had higher levels of phosphorylated PDGFRα/β, and SRC than merlin-expressing HSC. A similar phosphorylation pattern was observed in phospho-protein arrays of human vestibular schwannoma samples compared to normal HSC. Ponatinib reduced merlin-deficient HSC viability in a dose-dependent manner by decreasing phosphorylation of PDGFRα/β, AKT, p70S6K, MEK1/2, ERK1/2 and STAT3. These changes were associated with decreased cyclin D1 and increased p27Kip1levels, leading to a G1 cell-cycle arrest as assessed by Western blotting and flow cytometry. Ponatinib did not modulate ABL, SRC, focal adhesion kinase (FAK), or paxillin phosphorylation levels. These results suggest that ponatinib is a potential therapeutic agent for NF2-associated schwannomas and warrants further in vivo investigation.

  10. Flux control through protein phosphorylation in yeast

    DEFF Research Database (Denmark)

    Chen, Yu; Nielsen, Jens

    2016-01-01

    Protein phosphorylation is one of the most important mechanisms regulating metabolism as it can directly modify metabolic enzymes by the addition of phosphate groups. Attributed to such a rapid and reversible mechanism, cells can adjust metabolism rapidly in response to temporal changes. The yeast...... as well as identify mechanisms underlying human metabolic diseases. Here we collect functional phosphorylation events of 41 enzymes involved in yeast metabolism and demonstrate functional mechanisms and the application of this information in metabolic engineering. From a systems biology perspective, we...... describe the development of phosphoproteomics in yeast as well as approaches to analysing the phosphoproteomics data. Finally, we focus on integrated analyses with other omics data sets and genome-scale metabolic models. Despite the advances, future studies improving both experimental technologies...

  11. Solid polymer electrolyte from phosphorylated chitosan

    Energy Technology Data Exchange (ETDEWEB)

    Fauzi, Iqbal, E-mail: arcana@chem.itb.ac.id; Arcana, I Made, E-mail: arcana@chem.itb.ac.id [Inorganic and Physical Chemistry Research Groups, Faculty of Mathematics and Natural Sciences, Institut Teknologi Bandung, Jl. Ganesha 10, Bandung 40132 (Indonesia)

    2014-03-24

    Recently, the need of secondary battery application continues to increase. The secondary battery which using a liquid electrolyte was indicated had some weakness. A solid polymer electrolyte is an alternative electrolytes membrane which developed in order to replace the liquid electrolyte type. In the present study, the effect of phosphorylation on to polymer electrolyte membrane which synthesized from chitosan and lithium perchlorate salts was investigated. The effect of the component’s composition respectively on the properties of polymer electrolyte, was carried out by analyzed of it’s characterization such as functional groups, ion conductivity, and thermal properties. The mechanical properties i.e tensile resistance and the morphology structure of membrane surface were determined. The phosphorylation processing of polymer electrolyte membrane of chitosan and lithium perchlorate was conducted by immersing with phosphoric acid for 2 hours, and then irradiated on a microwave for 60 seconds. The degree of deacetylation of chitosan derived from shrimp shells was obtained around 75.4%. Relative molecular mass of chitosan was obtained by viscometry method is 796,792 g/mol. The ionic conductivity of chitosan membrane was increase from 6.33 × 10{sup −6} S/cm up to 6.01 × 10{sup −4} S/cm after adding by 15 % solution of lithium perchlorate. After phosphorylation, the ionic conductivity of phosphorylated lithium chitosan membrane was observed 1.37 × 10{sup −3} S/cm, while the tensile resistance of 40.2 MPa with a better thermal resistance. On the strength of electrolyte membrane properties, this polymer electrolyte membrane was suggested had one potential used for polymer electrolyte in field of lithium battery applications.

  12. Protein phosphorylation in bcterial signaling and regulation

    KAUST Repository

    Mijakovic, Ivan

    2016-01-26

    In 2003, it was demonstrated for the first time that bacteria possess protein-tyrosine kinases (BY-kinases), capable of phosphorylating other cellular proteins and regulating their activity. It soon became apparent that these kinases phosphorylate a number of protein substrates, involved in different cellular processes. More recently, we found out that BY-kinases can be activated by several distinct protein interactants, and are capable of engaging in cross-phosphorylation with other kinases. Evolutionary studies based on genome comparison indicate that BY-kinases exist only in bacteria. They are non-essential (present in about 40% bacterial genomes), and their knockouts lead to pleiotropic phenotypes, since they phosphorylate many substrates. Surprisingly, BY-kinase genes accumulate mutations at an increased rate (non-synonymous substitution rate significantly higher than other bacterial genes). One direct consequence of this phenomenon is no detectable co-evolution between kinases and their substrates. Their promiscuity towards substrates thus seems to be “hard-wired”, but why would bacteria maintain such promiscuous regulatory devices? One explanation is the maintenance of BY-kinases as rapidly evolving regulators, which can readily adopt new substrates when environmental changes impose selective pressure for quick evolution of new regulatory modules. Their role is clearly not to act as master regulators, dedicated to triggering a single response, but they might rather be employed to contribute to fine-tuning and improving robustness of various cellular responses. This unique feature makes BY-kinases a potentially useful tool in synthetic biology. While other bacterial kinases are very specific and their signaling pathways insulated, BY-kinase can relatively easily be engineered to adopt new substrates and control new biosynthetic processes. Since they are absent in humans, and regulate some key functions in pathogenic bacteria, they are also very promising

  13. Peroxides and radiation impairment of oxidative phosphorylation

    Energy Technology Data Exchange (ETDEWEB)

    Dovgii, I E; Akoev, I G

    1975-09-01

    An increase in the peroxidase activity of the mitochondria and a simultaneous rise in the amount of peroxide compounds, which are half lipid-like substances, are detected within the first 10 minutes after irradiation (1000 r). A mechanism of radiation impairment of oxidative phosphorylation is connected with the penetration of its inhibitors to the mitochondria due to the disturbed permeability of membranes affected by peroxides.

  14. Phosphorylation of proteins in Clostridium thermohydrosulfuricum

    International Nuclear Information System (INIS)

    Londesborough, J.

    1986-01-01

    Cell extracts of the thermophile Clostridium thermohydrosulfuricum catalyzed the phosphorylation by (γ- 32 P)ATP of several endogenous proteins with M/sub r/s between 13,000 and 100,000. Serine and tyrosine were the main acceptors. Distinct substrate proteins were found in the soluble (e.g., proteins p66, p63, and p53 of M/sub r/s 66,000, 63,000, and 53,000, respectively) and particulate (p76 and p30) fractions, both of which contained protein kinase and phosphatase activity. The soluble fraction suppressed the phosphorylation of particulate proteins and contained a protein kinase inhibitor. Phosphorylation of p53 was promoted by 10μM fructose 1,6-bisphosphate or glucose 1,6-bisphosphate and suppressed by hexose monophosphates, whereas p30 and p13 were suppressed by 5 μM brain (but not spinach) calmodulin. Polyamines, including the odd polyamines characteristic of thermophiles, modulated the labeling of most of the phosphoproteins. Apart from p66, all the proteins labeled in vitro were also rapidly labeled in intact cells by 32 P/sub i/. Several proteins strongly labeled in vivo were labeled slowly or not at all in vitro

  15. Resveratrol induces growth arrest and apoptosis through activation of FOXO transcription factors in prostate cancer cells.

    Directory of Open Access Journals (Sweden)

    Qinghe Chen

    2010-12-01

    Full Text Available Resveratrol, a naturally occurring phytopolyphenol compound, has attracted extensive interest in recent years because of its diverse pharmacological characteristics. Although resveratrol possesses chemopreventive properties against several cancers, the molecular mechanisms by which it inhibits cell growth and induces apoptosis have not been clearly understood. The present study was carried out to examine whether PI3K/AKT/FOXO pathway mediates the biological effects of resveratrol.Resveratrol inhibited the phosphorylation of PI3K, AKT and mTOR. Resveratrol, PI3K inhibitors (LY294002 and Wortmannin and AKT inhibitor alone slightly induced apoptosis in LNCaP cells. These inhibitors further enhanced the apoptosis-inducing potential of resveratrol. Overexpression of wild-type PTEN slightly induced apoptosis. Wild type PTEN and PTEN-G129E enhanced resveratrol-induced apoptosis, whereas PTEN-G129R had no effect on proapoptotic effects of resveratrol. Furthermore, apoptosis-inducing potential of resveratrol was enhanced by dominant negative AKT, and inhibited by wild-type AKT and constitutively active AKT. Resveratrol has no effect on the expression of FKHR, FKHRL1 and AFX genes. The inhibition of FOXO phosphorylation by resveratrol resulted in its nuclear translocation, DNA binding and transcriptional activity. The inhibition of PI3K/AKT pathway induced FOXO transcriptional activity resulting in induction of Bim, TRAIL, p27/KIP1, DR4 and DR5, and inhibition of cyclin D1. Similarly, resveratrol-induced FOXO transcriptional activity was further enhanced when activation of PI3K/AKT pathway was blocked. Over-expression of phosphorylation deficient mutants of FOXO proteins (FOXO1-TM, FOXO3A-TM and FOXO4-TM induced FOXO transcriptional activity, which was further enhanced by resveratrol. Inhibition of FOXO transcription factors by shRNA blocked resveratrol-induced upregulation of Bim, TRAIL, DR4, DR5, p27/KIP1 and apoptosis, and inhibition of cyclin D1 by

  16. Unraveling a phosphorylation event in a folded protein by NMR spectroscopy: phosphorylation of the Pin1 WW domain by PKA

    Energy Technology Data Exchange (ETDEWEB)

    Smet-Nocca, Caroline, E-mail: caroline.smet@univ-lille1.fr; Launay, Helene; Wieruszeski, Jean-Michel; Lippens, Guy; Landrieu, Isabelle, E-mail: isabelle.landrieu@univ-lille1.fr [Universite de Lille-Nord de France, Institut Federatif de Recherches 147, CNRS UMR 8576 (France)

    2013-04-15

    The Pin1 protein plays a critical role in the functional regulation of the hyperphosphorylated neuronal Tau protein in Alzheimer's disease and is by itself regulated by phosphorylation. We have used Nuclear Magnetic Resonance (NMR) spectroscopy to both identify the PKA phosphorylation site in the Pin1 WW domain and investigate the functional consequences of this phosphorylation. Detection and identification of phosphorylation on serine/threonine residues in a globular protein, while mostly occurring in solvent-exposed flexible loops, does not lead to chemical shift changes as obvious as in disordered proteins and hence does not necessarily shift the resonances outside the spectrum of the folded protein. Other complications were encountered to characterize the extent of the phosphorylation, as part of the {sup 1}H,{sup 15}N amide resonances around the phosphorylation site are specifically broadened in the unphosphorylated state. Despite these obstacles, NMR spectroscopy was an efficient tool to confirm phosphorylation on S16 of the WW domain and to quantify the level of phosphorylation. Based on this analytical characterization, we show that WW phosphorylation on S16 abolishes its binding capacity to a phosphorylated Tau peptide. A reduced conformational heterogeneity and flexibility of the phospho-binding loop upon S16 phosphorylation could account for part of the decreased affinity for its phosphorylated partner. Additionally, a structural model of the phospho-WW obtained by molecular dynamics simulation and energy minimization suggests that the phosphate moiety of phospho-S16 could compete with the phospho-substrate.

  17. Unraveling a phosphorylation event in a folded protein by NMR spectroscopy: phosphorylation of the Pin1 WW domain by PKA

    International Nuclear Information System (INIS)

    Smet-Nocca, Caroline; Launay, Hélène; Wieruszeski, Jean-Michel; Lippens, Guy; Landrieu, Isabelle

    2013-01-01

    The Pin1 protein plays a critical role in the functional regulation of the hyperphosphorylated neuronal Tau protein in Alzheimer’s disease and is by itself regulated by phosphorylation. We have used Nuclear Magnetic Resonance (NMR) spectroscopy to both identify the PKA phosphorylation site in the Pin1 WW domain and investigate the functional consequences of this phosphorylation. Detection and identification of phosphorylation on serine/threonine residues in a globular protein, while mostly occurring in solvent-exposed flexible loops, does not lead to chemical shift changes as obvious as in disordered proteins and hence does not necessarily shift the resonances outside the spectrum of the folded protein. Other complications were encountered to characterize the extent of the phosphorylation, as part of the 1 H, 15 N amide resonances around the phosphorylation site are specifically broadened in the unphosphorylated state. Despite these obstacles, NMR spectroscopy was an efficient tool to confirm phosphorylation on S16 of the WW domain and to quantify the level of phosphorylation. Based on this analytical characterization, we show that WW phosphorylation on S16 abolishes its binding capacity to a phosphorylated Tau peptide. A reduced conformational heterogeneity and flexibility of the phospho-binding loop upon S16 phosphorylation could account for part of the decreased affinity for its phosphorylated partner. Additionally, a structural model of the phospho-WW obtained by molecular dynamics simulation and energy minimization suggests that the phosphate moiety of phospho-S16 could compete with the phospho-substrate.

  18. Identification of the protein kinase C phosphorylation site in neuromodulin

    International Nuclear Information System (INIS)

    Apel, E.D.; Byford, M.F.; Au, D.; Walsh, K.A.; Storm, D.R.

    1990-01-01

    Neuromodulin (P-57, GAP-43, B-50, F-1) is a neurospecific calmodulin binding protein that is phosphorylated by protein kinase C. Phosphorylation by protein kinase C has been shown to abolish the affinity of neuromodulin for calmodulin and the authors have proposed that the concentration of free CaM in neurons may be regulated by phosphorylation and dephosphorylation of neuromodulin. The purpose of this study was to identify the protein kinase C phosphorylation site(s) in neuromodulin using recombinant neuromodulin as a substrate. Toward this end, it was demonstrated that recombinant neuromodulin purified from Escherichia coli and bovine neuromodulin were phosphorylated with similar K m values and stoichiometries and that protein kinase C mediated phosphorylation of both proteins abolished binding to calmodulin-Sepharose. Recombinant neuromodulin was phosphorylated by using protein kinase C and [γ- 32 P]ATP and digested with trypsin, and the resulting peptides were separated by HPLC. Only one 32 P-labeled tryptic peptide was generated from phosphorylated neuromodulin. They conclude that serine-41 is the protein kinase C phosphorylation site of neuromodulin and that phosphorylation of this amino acid residue blocks binding of calmoculin to neuromodulin. The proximity of serine-41 to the calmodulin binding domain in neuromodulin very likely explains the effect of phosphorylation on the affinity of neuromodulin for calmodulin

  19. Protein Kinase C alpha (PKCα) dependent signaling mediates endometrial cancer cell growth and tumorigenesis

    Science.gov (United States)

    Haughian, James M.; Reno, Elaine M.; Thorne, Alicia M.; Bradford, Andrew P.

    2009-01-01

    Endometrial cancer is the most common invasive gynecologic malignancy, yet molecular mechanisms and signaling pathways underlying its etiology and pathophysiology remain poorly characterized. We sought to define a functional role for the protein kinase C (PKC) isoform, PKCα, in an established cell model of endometrial adenocarcinoma. Ishikawa cells depleted of PKCα protein grew slower, formed fewer colonies in anchorage-independent growth assays and exhibited impaired xenograft tumor formation in nude mice. Consistent with impaired growth, PKCα knockdown increased levels of the cyclin dependent kinase (CDK) inhibitors p21Cip1/WAF1 (p21) and p27Kip1 (p27). Despite the absence of functional phosphatase and tensin homologue (PTEN) protein in Ishikawa cells, PKCα knockdown reduced Akt phosphorylation at serine 473 and concomitantly inhibited phosphorylation of the Akt target, glycogen synthase kinase-3β (GSK-3β). PKCα knockdown also resulted in decreased basal ERK phosphorylation and attenuated ERK activation following EGF stimulation. p21 and p27 expression was not increased by treatment of Ishikawa cells with ERK and Akt inhibitors, suggesting PKCα regulates CDK expression independently of Akt and ERK. Immunohistochemical analysis of grade 1 endometrioid adenocarcinoma revealed aberrant PKCα expression, with foci of elevated PKCα staining, not observed in normal endometrium. These studies demonstrate a critical role for PKCα signaling in endometrial tumorigenesis by regulating expression of CDK inhibitors p21 and p27 and activation of Akt and ERK dependent proliferative pathways. Thus, targeting PKCα may provide novel therapeutic options in endometrial tumors. PMID:19672862

  20. Synemin promotes AKT-dependent glioblastoma cell proliferation by antagonizing PP2A.

    Science.gov (United States)

    Pitre, Aaron; Davis, Nathan; Paul, Madhumita; Orr, A Wayne; Skalli, Omar

    2012-04-01

    The intermediate filament protein synemin is present in astrocyte progenitors and glioblastoma cells but not in mature astrocytes. Here we demonstrate a role for synemin in enhancing glioblastoma cell proliferation and clonogenic survival, as synemin RNA interference decreased both behaviors by inducing G1 arrest along with Rb hypophosphorylation and increased protein levels of the G1/S inhibitors p21(Cip1) and p27(Kip1). Akt involvement was demonstrated by decreased phosphorylation of its substrate, p21(Cip1), and reduced Akt catalytic activity and phosphorylation at essential activation sites. Synemin silencing, however, did not affect the activities of PDPK1 and mTOR complex 2, which directly phosphorylate Akt activation sites, but instead enhanced the activity of the major regulator of Akt dephosphorylation, protein phosphatase type 2A (PP2A). This was accompanied by changes in PP2A subcellular distribution resulting in increased physical interactions between PP2A and Akt, as shown by proximity ligation assays (PLAs). PLAs and immunoprecipitation experiments further revealed that synemin and PP2A form a protein complex. In addition, treatment of synemin-silenced cells with the PP2A inhibitor cantharidic acid resulted in proliferation and pAkt and pRb levels similar to those of controls. Collectively these results indicate that synemin positively regulates glioblastoma cell proliferation by helping sequester PP2A away from Akt, thereby favoring Akt activation.

  1. Phosphoryl functionalized mesoporous silica for uranium adsorption

    International Nuclear Information System (INIS)

    Xue, Guo; Yurun, Feng; Li, Ma; Dezhi, Gao; Jie, Jing; Jincheng, Yu; Haibin, Sun; Hongyu, Gong; Yujun, Zhang

    2017-01-01

    Highlights: • Phosphoryl functionalized mesoporous silica (TBP-SBA-15) is synthesized. • The amino and phosphoryl groups are successfully grafted on SBA-15. • TBP-SBA-15 has high and rapid uranium adsorption capacity in broad pH range. • The U(VI) adsorption of TBP-SBA-15 is spontaneous and belongs to chemical adsorption. - Abstract: Phosphoryl functionalized mesoporous silica (TBP-SBA-15) was synthesized by modified mesoporous silica with γ-amino propyl triethoxy silane and tributyl phosphate. The obtained samples were characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM), small angle X-ray diffraction (SAXRD), thermo-gravimetric/differential thermalanalyzer (TG/DTA), N_2 adsorption–desorption (BET) and Fourier transform infrared spectroscopy (FT-IR) techniques. Results showed that TBP-SBA-15 had large surface areas with ordered channel structure. Moreover, the effects of adsorption time, sorbent dose, solution pH, initial uranium concentration and temperature on the uranium adsorption behaviors were investigated. TBP-SBA-15 showed a high uranium adsorption capacity in a broad range of pH values. The U(VI) adsorption rate of TBP-SBA-15 was fast and nearly achieved completion in 10 min with the sorbent dose of 1 g/L. The U(VI) adsorption of TBP-SBA-15 followed the pseudo-second-order kinetic model and Freundlich isotherm model, indicating that the process was belonged to chemical adsorption. Furthermore, the thermodynamic parameters (ΔG"0, ΔH"0 and ΔS"0) confirmed that the adsorption process was endothermic and spontaneous.

  2. Phosphoryl functionalized mesoporous silica for uranium adsorption

    Energy Technology Data Exchange (ETDEWEB)

    Xue, Guo; Yurun, Feng; Li, Ma; Dezhi, Gao; Jie, Jing; Jincheng, Yu; Haibin, Sun [Key Laboratory for Liquid-Solid Structural Evolution & Processing of Materials of Ministry of Education, Shandong University, Jinan 250061 (China); Key Laboratory of Special Functional Aggregated Materials, Ministry of Education, Shandong University, Jinan 250061 (China); Hongyu, Gong, E-mail: gong_hongyu@163.com [Key Laboratory for Liquid-Solid Structural Evolution & Processing of Materials of Ministry of Education, Shandong University, Jinan 250061 (China); Key Laboratory of Special Functional Aggregated Materials, Ministry of Education, Shandong University, Jinan 250061 (China); Yujun, Zhang, E-mail: yujunzhangcn@163.com [Key Laboratory for Liquid-Solid Structural Evolution & Processing of Materials of Ministry of Education, Shandong University, Jinan 250061 (China); Key Laboratory of Special Functional Aggregated Materials, Ministry of Education, Shandong University, Jinan 250061 (China)

    2017-04-30

    Highlights: • Phosphoryl functionalized mesoporous silica (TBP-SBA-15) is synthesized. • The amino and phosphoryl groups are successfully grafted on SBA-15. • TBP-SBA-15 has high and rapid uranium adsorption capacity in broad pH range. • The U(VI) adsorption of TBP-SBA-15 is spontaneous and belongs to chemical adsorption. - Abstract: Phosphoryl functionalized mesoporous silica (TBP-SBA-15) was synthesized by modified mesoporous silica with γ-amino propyl triethoxy silane and tributyl phosphate. The obtained samples were characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM), small angle X-ray diffraction (SAXRD), thermo-gravimetric/differential thermalanalyzer (TG/DTA), N{sub 2} adsorption–desorption (BET) and Fourier transform infrared spectroscopy (FT-IR) techniques. Results showed that TBP-SBA-15 had large surface areas with ordered channel structure. Moreover, the effects of adsorption time, sorbent dose, solution pH, initial uranium concentration and temperature on the uranium adsorption behaviors were investigated. TBP-SBA-15 showed a high uranium adsorption capacity in a broad range of pH values. The U(VI) adsorption rate of TBP-SBA-15 was fast and nearly achieved completion in 10 min with the sorbent dose of 1 g/L. The U(VI) adsorption of TBP-SBA-15 followed the pseudo-second-order kinetic model and Freundlich isotherm model, indicating that the process was belonged to chemical adsorption. Furthermore, the thermodynamic parameters (ΔG{sup 0}, ΔH{sup 0} and ΔS{sup 0}) confirmed that the adsorption process was endothermic and spontaneous.

  3. Monitoring HPV-16 E7 phosphorylation events

    Energy Technology Data Exchange (ETDEWEB)

    Nogueira, Marcela O.; Hošek, Tomáš; Calçada, Eduardo O.; Castiglia, Francesca [Magnetic Resonance Center (CERM) and Department of Chemistry “Ugo Schiff”, University of Florence, via Luigi Sacconi 6, Sesto Fiorentino (Italy); Massimi, Paola; Banks, Lawrence [International Centre for Genetic Engineering and Biotechnology (ICGEB), Padriciano 99, Trieste (Italy); Felli, Isabella C., E-mail: felli@cerm.unifi.it [Magnetic Resonance Center (CERM) and Department of Chemistry “Ugo Schiff”, University of Florence, via Luigi Sacconi 6, Sesto Fiorentino (Italy); Pierattelli, Roberta, E-mail: pierattelli@cerm.unifi.it [Magnetic Resonance Center (CERM) and Department of Chemistry “Ugo Schiff”, University of Florence, via Luigi Sacconi 6, Sesto Fiorentino (Italy)

    2017-03-15

    HPV-16 E7 is one of the key proteins that, by interfering with the host metabolism through many protein-protein interactions, hijacks cell regulation and contributes to malignancy. Here we report the high resolution investigation of the CR3 region of HPV-16 E7, both as an isolated domain and in the full-length protein. This opens the way to the atomic level study of the many interactions in which HPV-16 E7 is involved. Along these lines we show here the effect of one of the key post-translational modifications of HPV-16 E7, the phosphorylation by casein kinase II.

  4. SH3 domain tyrosine phosphorylation--sites, role and evolution.

    Directory of Open Access Journals (Sweden)

    Zuzana Tatárová

    Full Text Available BACKGROUND: SH3 domains are eukaryotic protein domains that participate in a plethora of cellular processes including signal transduction, proliferation, and cellular movement. Several studies indicate that tyrosine phosphorylation could play a significant role in the regulation of SH3 domains. RESULTS: To explore the incidence of the tyrosine phosphorylation within SH3 domains we queried the PhosphoSite Plus database of phosphorylation sites. Over 100 tyrosine phosphorylations occurring on 20 different SH3 domain positions were identified. The tyrosine corresponding to c-Src Tyr-90 was by far the most frequently identified SH3 domain phosphorylation site. A comparison of sequences around this tyrosine led to delineation of a preferred sequence motif ALYD(Y/F. This motif is present in about 15% of human SH3 domains and is structurally well conserved. We further observed that tyrosine phosphorylation is more abundant than serine or threonine phosphorylation within SH3 domains and other adaptor domains, such as SH2 or WW domains. Tyrosine phosphorylation could represent an important regulatory mechanism of adaptor domains. CONCLUSIONS: While tyrosine phosphorylation typically promotes signaling protein interactions via SH2 or PTB domains, its role in SH3 domains is the opposite - it blocks or prevents interactions. The regulatory function of tyrosine phosphorylation is most likely achieved by the phosphate moiety and its charge interfering with binding of polyproline helices of SH3 domain interacting partners.

  5. Systematic inference of functional phosphorylation events in yeast metabolism.

    Science.gov (United States)

    Chen, Yu; Wang, Yonghong; Nielsen, Jens

    2017-07-01

    Protein phosphorylation is a post-translational modification that affects proteins by changing their structure and conformation in a rapid and reversible way, and it is an important mechanism for metabolic regulation in cells. Phosphoproteomics enables high-throughput identification of phosphorylation events on metabolic enzymes, but identifying functional phosphorylation events still requires more detailed biochemical characterization. Therefore, development of computational methods for investigating unknown functions of a large number of phosphorylation events identified by phosphoproteomics has received increased attention. We developed a mathematical framework that describes the relationship between phosphorylation level of a metabolic enzyme and the corresponding flux through the enzyme. Using this framework, it is possible to quantitatively estimate contribution of phosphorylation events to flux changes. We showed that phosphorylation regulation analysis, combined with a systematic workflow and correlation analysis, can be used for inference of functional phosphorylation events in steady and dynamic conditions, respectively. Using this analysis, we assigned functionality to phosphorylation events of 17 metabolic enzymes in the yeast Saccharomyces cerevisiae , among which 10 are novel. Phosphorylation regulation analysis cannot only be extended for inference of other functional post-translational modifications but also be a promising scaffold for multi-omics data integration in systems biology. Matlab codes for flux balance analysis in this study are available in Supplementary material. yhwang@ecust.edu.cn or nielsenj@chalmers.se. Supplementary data are available at Bioinformatics online. © The Author 2017. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com

  6. Phenobarbital Meets Phosphorylation of Nuclear Receptors.

    Science.gov (United States)

    Negishi, Masahiko

    2017-05-01

    Phenobarbital was the first therapeutic drug to be characterized for its induction of hepatic drug metabolism. Essentially at the same time, cytochrome P450, an enzyme that metabolizes drugs, was discovered. After nearly 50 years of investigation, the molecular target of phenobarbital induction has now been delineated to phosphorylation at threonine 38 of the constitutive androstane receptor (NR1I3), a member of the nuclear receptor superfamily. Determining this mechanism has provided us with the molecular basis to understand drug induction of drug metabolism and disposition. Threonine 38 is conserved as a phosphorylation motif in the majority of both mouse and human nuclear receptors, providing us with an opportunity to integrate diverse functions of nuclear receptors. Here, I review the works and accomplishments of my laboratory at the National Institutes of Health National Institute of Environmental Health Sciences and the future research directions of where our study of the constitutive androstane receptor might take us. U.S. Government work not protected by U.S. copyright.

  7. Regulation of cardiac C-protein phosphorylation

    International Nuclear Information System (INIS)

    Titus, F.L.

    1985-01-01

    Molecular mechanisms of cardiac sympathetic and parasympathetic responses were addressed by studying subcellular changes in protein phosphorylation, cAMP-dependent protein kinase activity and protein phosphatase activity in frog hearts. B-adrenergic agonists increased and muscarinic cholinergic agonists decreased [ 32 P]phosphate incorporation into C-protein, a thick filament component. Regulation of protein phosphatase activity by Iso and methacholine (MCh) was assayed using extracts of drug treated frog hearts and [ 32 P]phospho-C-protein as substrate. Total phosphatase activity decreased 21% in extracts from hearts perfused with 0.1 μM Iso and 17% in hearts exposed to Iso plus 1 μM methacholine. This decrease reflected decreased phosphatase-2A activity. No changes in total phosphatase activity were measurable in broken cells treated with Iso or MCh. The results suggest adrenergic stimulation changes contractile activity in frog hearts by activating cAMP-dependent protein kinase associated with particulate cellular elements and inactivating soluble protein phosphatase-2A. This is the first demonstration of coordinated regulation of these enzymes by B-adrenergic agonists favoring phosphorylation of effector proteins. Coordinated regulation by methacholine in the presence of Iso was not observed

  8. Modelling the Krebs cycle and oxidative phosphorylation.

    Science.gov (United States)

    Korla, Kalyani; Mitra, Chanchal K

    2014-01-01

    The Krebs cycle and oxidative phosphorylation are the two most important sets of reactions in a eukaryotic cell that meet the major part of the total energy demands of a cell. In this paper, we present a computer simulation of the coupled reactions using open source tools for simulation. We also show that it is possible to model the Krebs cycle with a simple black box with a few inputs and outputs. However, the kinetics of the internal processes has been modelled using numerical tools. We also show that the Krebs cycle and oxidative phosphorylation together can be combined in a similar fashion - a black box with a few inputs and outputs. The Octave script is flexible and customisable for any chosen set-up for this model. In several cases, we had no explicit idea of the underlying reaction mechanism and the rate determining steps involved, and we have used the stoichiometric equations that can be easily changed as and when more detailed information is obtained. The script includes the feedback regulation of the various enzymes of the Krebs cycle. For the electron transport chain, the pH gradient across the membrane is an essential regulator of the kinetics and this has been modelled empirically but fully consistent with experimental results. The initial conditions can be very easily changed and the simulation is potentially very useful in a number of cases of clinical importance.

  9. Chemoselective synthesis and analysis of naturally occurring phosphorylated cysteine peptides

    Science.gov (United States)

    Bertran-Vicente, Jordi; Penkert, Martin; Nieto-Garcia, Olaia; Jeckelmann, Jean-Marc; Schmieder, Peter; Krause, Eberhard; Hackenberger, Christian P. R.

    2016-09-01

    In contrast to protein O-phosphorylation, studying the function of the less frequent N- and S-phosphorylation events have lagged behind because they have chemical features that prevent their manipulation through standard synthetic and analytical methods. Here we report on the development of a chemoselective synthetic method to phosphorylate Cys side-chains in unprotected peptides. This approach makes use of a reaction between nucleophilic phosphites and electrophilic disulfides accessible by standard methods. We achieve the stereochemically defined phosphorylation of a Cys residue and verify the modification using electron-transfer higher-energy dissociation (EThcD) mass spectrometry. To demonstrate the use of the approach in resolving biological questions, we identify an endogenous Cys phosphorylation site in IICBGlc, which is known to be involved in the carbohydrate uptake from the bacterial phosphotransferase system (PTS). This new chemical and analytical approach finally allows further investigating the functions and significance of Cys phosphorylation in a wide range of crucial cellular processes.

  10. Phosphorylation of mouse serine racemase regulates D-serine synthesis

    DEFF Research Database (Denmark)

    Foltyn, Veronika N; Zehl, Martin; Dikopoltsev, Elena

    2010-01-01

    Serine racemase (SR) catalyses the synthesis of the transmitter/neuromodulator D-serine, which plays a major role in synaptic plasticity and N-methyl D-aspartate receptor neurotoxicity. We now report that SR is phosphorylated at Thr71 and Thr227 as revealed by mass spectrometric analysis and in v...... with a phosphorylation-deficient SR mutant indicate that Thr71 phosphorylation increases SR activity, suggesting a novel mechanism for regulating D-serine production....

  11. The in vivo phosphorylation sites of rat brain dynamin I

    DEFF Research Database (Denmark)

    Graham, Mark E; Anggono, Victor; Bache, Nicolai

    2007-01-01

    -824). To resolve the discrepancy and to better understand the biological roles of dynI phosphorylation, we undertook a systematic identification of all phosphorylation sites in rat brain nerve terminal dynI. Using phosphoamino acid analysis, exclusively phospho-serine residues were found. Thr(780) phosphorylation...... of their relative abundance and relative responses to depolarization. The multiple phospho-sites suggest subtle regulation of synaptic vesicle endocytosis by new protein kinases and new protein-protein interactions. The homologous dynI and dynIII phosphorylation indicates a high mechanistic similarity. The results...

  12. Altered phosphorylation of rhodopsin in retinal dystrophic Irish Setters

    International Nuclear Information System (INIS)

    Cunnick, J.; Takemoto, D.J.; Takemoto, L.J.

    1986-01-01

    The carboxyl-terminus of rhodopsin in retinal dystrophic (rd) Irish Setters is altered near a possible phosphorylation site. To determine if this alteration affects ATP-mediated phosphorylation they compared the phosphorylation of rhodopsin from rd affected Irish Setters and normal unaffected dogs. Retinas from 8-week-old Irish Setters were phosphorylated with γ- 32 P-ATP and separated on SDS-PAGE. Compared to unaffected normal retinas, equalized for rhodopsin content, phosphorylation of rd rhodopsin was drastically reduced. When rd retinas were mixed with normal dog retinas, phosphorylation of the latter was inhibited. Inhibition also occurred when bovine retinas were mixed with rd retinas. The rd-mediated inhibition of phosphorylation was prevented by including 1mM NaF in the reaction mixture. Likewise, 1mM NaF restored phosphorylation of rd rhodopsin to normal levels. Phosphopeptide maps of rd and normal rhodopsin were identical and indicated 5 phosphopeptides present in each. Results suggest that one cause of the depressed rd rhodopsin phosphorylation is an increased phosphatase activity

  13. Cisplatinum and Taxol Induce Different Patterns of p53 Phosphorylation

    Directory of Open Access Journals (Sweden)

    Giovanna Damia

    2001-01-01

    Full Text Available Posttranslational modifications of p53 induced by two widely used anticancer agents, cisplatinum (DDP and taxol were investigated in two human cancer cell lines. Although both drugs were able to induce phosphorylation at serine 20 (Ser20, only DDP treatment induced p53 phosphorylation at serine 15 (Ser15. Moreover, both drug treatments were able to increase p53 levels and consequently the transcription of waf1 and mdm-2 genes, although DDP treatment resulted in a stronger inducer of both genes. Using two ataxia telangiectasia mutated (ATM cell lines, the role of ATM in druginduced p53 phosphorylations was investigated. No differences in drug-induced p53 phosphorylation could be observed, indicating that ATM is not the kinase involved in these phosphorylation events. In addition, inhibition of DNA-dependent protein kinase activity by wortmannin did not abolish p53 phosphorylation at Ser15 and Ser20, again indicating that DNA-PK is unlikely to be the kinase involved. After both taxol and DDP treatments, an activation of hCHK2 was found and this is likely to be responsible for phosphorylation at Ser20. In contrast, only DDP was able to activate ATR, which is the candidate kinase for phosphorylation of Ser15 by this drug. This data clearly suggests that differential mechanisms are involved in phosphorylation and activation of p53 depending on the drug type.

  14. Tyrosine phosphorylation of Grb14 by Tie2

    Directory of Open Access Journals (Sweden)

    Dumont Daniel J

    2010-10-01

    Full Text Available Abstract Background Growth factor receptor bound (Grb proteins 7, 10 and 14 are a family of structurally related multi-domain adaptor proteins involved in a variety of biological processes. Grb7, 10 and 14 are known to become serine and/or threonine phosphorylated in response to growth factor (GF stimulation. Grb7 and 10 have also been shown to become tyrosine phosphorylated under certain conditions. Under experimental conditions Grb7 is tyrosine phosphorylated by the Tie2/Tie-2/Tek angiogenic receptor tyrosine kinase (RTK. Furthermore, Grb14 has also been shown to interact with Tie2, however tyrosine phosphorylation of this Grb family member has yet to be reported. Results Here we report for the first time tyrosine phosphorylation of Grb14. This phosphorylation requires a kinase competent Tie2 as well as intact tyrosines 1100 and 1106 (Y1100 and Y1106 on the receptor. Furthermore, a complete SH2 domain on Grb14 is required for Grb14 tyrosine phosphorylation by Tie2. Grb14 was also able to become tyrosine phosphorylated in primary endothelial cells when treated with a soluble and potent variant of the Tie2 ligand, cartilage oligomeric matrix protein (COMP Ang1. Conclusion Our results show that Grb14, like its family members Grb7 and Grb10, is able to be tyrosine phosphorylated. Furthermore, our data indicate a role for Grb14 in endothelial signaling downstream of the Tie2 receptor.

  15. Parkinson's disease associated with impaired oxidative phosphorylation

    International Nuclear Information System (INIS)

    Finsterer, J.; Jarius, C.; Baumgartner, M.

    2001-01-01

    Parkinson's disease may be due to primary or secondary oxidative phosphorylation (OXPHOS) defects. In a 76-year-old man with Parkinson's disease since 1992, slightly but recurrently elevated creatine phosphokinase, recurrently elevated blood glucose, thickening of the left ventricular myocardium, bifascicular block and hypacusis were found. Cerebral MRI showed atrophy, periventricular demyelination, multiple, disseminated, supra- and infratentorial lacunas, and haemosiderin deposits in both posterior horns. Muscle biopsy showed typical features of an OXPHOS defect. Whether the association of Parkinson's disease and impaired OXPHOS was causative or coincidental remains unknown. Possibly, the mitochondrial defect acted as an additional risk factor for Parkinson's disease or the OXPHOS defect worsened the preexisting neurological impairments by a cumulative or synergistic mechanism. In conclusion, this case shows that Parkinson's disease may be associated with a mitochondrially or nuclearly encoded OXPHOS defect, manifesting as hypacusis, myopathy, axonal polyneuropathy, cardiomyopathy and recurrent subclinical ischaemic strokes and haemorrhages. (orig.)

  16. ATM regulates Cdt1 stability during the unperturbed S phase to prevent re-replication

    Science.gov (United States)

    Iwahori, Satoko; Kohmon, Daisuke; Kobayashi, Junya; Tani, Yuhei; Yugawa, Takashi; Komatsu, Kenshi; Kiyono, Tohru; Sugimoto, Nozomi; Fujita, Masatoshi

    2014-01-01

    Ataxia-telangiectasia mutated (ATM) plays crucial roles in DNA damage responses, especially with regard to DNA double-strand breaks (DSBs). However, it appears that ATM can be activated not only by DSB, but also by some changes in chromatin architecture, suggesting potential ATM function in cell cycle control. Here, we found that ATM is involved in timely degradation of Cdt1, a critical replication licensing factor, during the unperturbed S phase. At least in certain cell types, degradation of p27Kip1 was also impaired by ATM inhibition. The novel ATM function for Cdt1 regulation was dependent on its kinase activity and NBS1. Indeed, we found that ATM is moderately phosphorylated at Ser1981 during the S phase. ATM silencing induced partial reduction in levels of Skp2, a component of SCFSkp2 ubiquitin ligase that controls Cdt1 degradation. Furthermore, Skp2 silencing resulted in Cdt1 stabilization like ATM inhibition. In addition, as reported previously, ATM silencing partially prevented Akt phosphorylation at Ser473, indicative of its activation, and Akt inhibition led to modest stabilization of Cdt1. Therefore, the ATM-Akt-SCFSkp2 pathway may partly contribute to the novel ATM function. Finally, ATM inhibition rendered cells hypersensitive to induction of re-replication, indicating importance for maintenance of genome stability. PMID:24280901

  17. A grammar inference approach for predicting kinase specific phosphorylation sites.

    Science.gov (United States)

    Datta, Sutapa; Mukhopadhyay, Subhasis

    2015-01-01

    Kinase mediated phosphorylation site detection is the key mechanism of post translational mechanism that plays an important role in regulating various cellular processes and phenotypes. Many diseases, like cancer are related with the signaling defects which are associated with protein phosphorylation. Characterizing the protein kinases and their substrates enhances our ability to understand the mechanism of protein phosphorylation and extends our knowledge of signaling network; thereby helping us to treat such diseases. Experimental methods for predicting phosphorylation sites are labour intensive and expensive. Also, manifold increase of protein sequences in the databanks over the years necessitates the improvement of high speed and accurate computational methods for predicting phosphorylation sites in protein sequences. Till date, a number of computational methods have been proposed by various researchers in predicting phosphorylation sites, but there remains much scope of improvement. In this communication, we present a simple and novel method based on Grammatical Inference (GI) approach to automate the prediction of kinase specific phosphorylation sites. In this regard, we have used a popular GI algorithm Alergia to infer Deterministic Stochastic Finite State Automata (DSFA) which equally represents the regular grammar corresponding to the phosphorylation sites. Extensive experiments on several datasets generated by us reveal that, our inferred grammar successfully predicts phosphorylation sites in a kinase specific manner. It performs significantly better when compared with the other existing phosphorylation site prediction methods. We have also compared our inferred DSFA with two other GI inference algorithms. The DSFA generated by our method performs superior which indicates that our method is robust and has a potential for predicting the phosphorylation sites in a kinase specific manner.

  18. A Grammar Inference Approach for Predicting Kinase Specific Phosphorylation Sites

    Science.gov (United States)

    Datta, Sutapa; Mukhopadhyay, Subhasis

    2015-01-01

    Kinase mediated phosphorylation site detection is the key mechanism of post translational mechanism that plays an important role in regulating various cellular processes and phenotypes. Many diseases, like cancer are related with the signaling defects which are associated with protein phosphorylation. Characterizing the protein kinases and their substrates enhances our ability to understand the mechanism of protein phosphorylation and extends our knowledge of signaling network; thereby helping us to treat such diseases. Experimental methods for predicting phosphorylation sites are labour intensive and expensive. Also, manifold increase of protein sequences in the databanks over the years necessitates the improvement of high speed and accurate computational methods for predicting phosphorylation sites in protein sequences. Till date, a number of computational methods have been proposed by various researchers in predicting phosphorylation sites, but there remains much scope of improvement. In this communication, we present a simple and novel method based on Grammatical Inference (GI) approach to automate the prediction of kinase specific phosphorylation sites. In this regard, we have used a popular GI algorithm Alergia to infer Deterministic Stochastic Finite State Automata (DSFA) which equally represents the regular grammar corresponding to the phosphorylation sites. Extensive experiments on several datasets generated by us reveal that, our inferred grammar successfully predicts phosphorylation sites in a kinase specific manner. It performs significantly better when compared with the other existing phosphorylation site prediction methods. We have also compared our inferred DSFA with two other GI inference algorithms. The DSFA generated by our method performs superior which indicates that our method is robust and has a potential for predicting the phosphorylation sites in a kinase specific manner. PMID:25886273

  19. PAK6 Phosphorylates 14-3-3γ to Regulate Steady State Phosphorylation of LRRK2

    Directory of Open Access Journals (Sweden)

    Laura Civiero

    2017-12-01

    Full Text Available Mutations in Leucine-rich repeat kinase 2 (LRRK2 are associated with Parkinson's disease (PD and, as such, LRRK2 is considered a promising therapeutic target for age-related neurodegeneration. Although the cellular functions of LRRK2 in health and disease are incompletely understood, robust evidence indicates that PD-associated mutations alter LRRK2 kinase and GTPase activities with consequent deregulation of the downstream signaling pathways. We have previously demonstrated that one LRRK2 binding partner is P21 (RAC1 Activated Kinase 6 (PAK6. Here, we interrogate the PAK6 interactome and find that PAK6 binds a subset of 14-3-3 proteins in a kinase dependent manner. Furthermore, PAK6 efficiently phosphorylates 14-3-3γ at Ser59 and this phosphorylation serves as a switch to dissociate the chaperone from client proteins including LRRK2, a well-established 14-3-3 binding partner. We found that 14-3-3γ phosphorylated by PAK6 is no longer competent to bind LRRK2 at phospho-Ser935, causing LRRK2 dephosphorylation. To address whether these interactions are relevant in a neuronal context, we demonstrate that a constitutively active form of PAK6 rescues the G2019S LRRK2-associated neurite shortening through phosphorylation of 14-3-3γ. Our results identify PAK6 as the kinase for 14-3-3γ and reveal a novel regulatory mechanism of 14-3-3/LRRK2 complex in the brain.

  20. Phosphorylated benzimedazoles. 8. Synthesis of phosphorylated with /sup 32/P benzimidazoles

    Energy Technology Data Exchange (ETDEWEB)

    Makarov, A M; Matevosyan, G L; Zavlin, P M [Leningradskij Sel' skokhozyajstvennyj Inst. (USSR)

    1983-03-01

    Accessible methods of synthesis and identification of phosphorylated benzimidazoles with specific activity close to the maximum permissible with labelled /sup 32/P are developed. These methods permit to determine the permissible residual amounts of the above preparations in nutrition products and the maximum permissible amounts of growth regulators in different objects of the environment, because it is impossible to detect, for example, tri(1-benzimidazolido)phosphate with other physico-chemical methods with the existing concentration of 10/sup -9/%.

  1. Separation Options for Phosphorylated Osteopontin from Transgenic Microalgae Chlamydomonas reinhardtii

    Directory of Open Access Journals (Sweden)

    Ayswarya Ravi

    2018-02-01

    Full Text Available Correct folding and post-translational modifications are vital for therapeutic proteins to elicit their biological functions. Osteopontin (OPN, a bone regenerative protein present in a range of mammalian cells, is an acidic phosphoprotein with multiple potential phosphorylation sites. In this study, the ability of unicellular microalgae, Chlamydomonas reinhardtii, to produce phosphorylated recombinant OPN in its chloroplast is investigated. This study further explores the impact of phosphorylation and expression from a “plant-like” algae on separation of OPN. Chromatography resins ceramic hydroxyapatite (CHT and Gallium-immobilized metal affinity chromatography (Ga-IMAC were assessed for their binding specificity to phosphoproteins. Non-phosphorylated recombinant OPN expressed in E. coli was used to compare the specificity of interaction of the resins to phosphorylated OPN. We observed that CHT binds OPN by multimodal interactions and was better able to distinguish phosphorylated proteins in the presence of 250 mM NaCl. Ga-IMAC interaction with OPN was not selective to phosphorylation, irrespective of salt, as the resin bound OPN from both algal and bacterial sources. Anion exchange chromatography proved an efficient capture method to partially separate major phosphorylated host cell protein impurities such as Rubisco from OPN.

  2. Distribution pattern of histone H3 phosphorylation at serine 10

    Indian Academy of Sciences (India)

    We evaluated the pattern of H3 phosphorylation using immunodetection during mitosis and meiosis in both diploid and tetraploid genotypes of Brachiaria species. Results revealed differences in chromosome distribution of H3S10ph when mitosis and meiosis were compared. Whole chromosomes were phosphorylated ...

  3. Phosphorylation of eukaryotic aminoacyl-tRNA synthetases

    International Nuclear Information System (INIS)

    Pendergast, A.M.

    1986-01-01

    The phosphorylation of the highly purified aminoacyl-tRNA synthetase complex from rabbit reticulocytes was examined. The synthetase complex contained, in addition to eight aminoacyl-tRNA synthetases, three unidentified proteins and was free of endogenous protein kinase activity. Incubation of the complex with casein kinase I in the presence of ATP resulted in the phosphorylation of four synthetases, the glutamyl-, isoleucyl-, methionyl-, and lysyl-tRNA synthetases. Phosphorylation by casein kinase I altered binding to tRNA-Sepharose such that the phosphorylated complex eluted at 190 mM NaCl instead of the 275 mM salt observed for the nonphosphorylated form. Phosphorylation by casein kinase I resulted in a significant inhibition of aminoacylation with the four synthetases; the activities of the nonphosphorylated synthetases were unchanged. One of the unidentified proteins in the complex (M/sub r/ 37,000) was also an excellent substrate for casein kinase I. A comparison of the properties and two-dimensional phosphopeptide pattern of this protein with that of casein kinase I suggest that the 37,000 dalton protein in the synthetase complex is an inactive form of casein kinase I. Two other protein kinases were shown to phosphorylate aminoacyl-tRNA synthetases in the complex. The phosphorylation of threonyl-tRNA synthetase was also investigated. Five aminoacyl-tRNA synthetases in the high molecular weight complex were shown to be phosphorylated in rabbit reticulocytes following labeling with ( 32 P)orthophosphate

  4. Quantitative phosphoproteomics reveals widespread full phosphorylation site occupancy during mitosis

    DEFF Research Database (Denmark)

    Miller, Martin Lee; Brunak, Søren; Olsen, JV

    2010-01-01

    and phosphorylation sites were grouped according to their cell cycle kinetics and compared to publicly available messenger RNA microarray data. Most detected phosphorylation sites and more than 20% of all quantified proteins showed substantial regulation, mainly in mitotic cells. Kinase-motif analysis revealed global...

  5. Myosin light chain kinase phosphorylation in tracheal smooth muscle

    International Nuclear Information System (INIS)

    Stull, J.T.; Hsu, L.C.; Tansey, M.G.; Kamm, K.E.

    1990-01-01

    Purified myosin light chain kinase from smooth muscle is phosphorylated by cyclic AMP-dependent protein kinase, protein kinase C, and the multifunctional calmodulin-dependent protein kinase II. Because phosphorylation in a specific site (site A) by any one of these kinases desensitizes myosin light chain kinase to activation by Ca2+/calmodulin, kinase phosphorylation could play an important role in regulating smooth muscle contractility. This possibility was investigated in 32 P-labeled bovine tracheal smooth muscle. Treatment of tissues with carbachol, KCl, isoproterenol, or phorbol 12,13-dibutyrate increased the extent of kinase phosphorylation. Six primary phosphopeptides (A-F) of myosin light chain kinase were identified. Site A was phosphorylated to an appreciable extent only with carbachol or KCl, agents which contract tracheal smooth muscle. The extent of site A phosphorylation correlated to increases in the concentration of Ca2+/calmodulin required for activation. These results show that cyclic AMP-dependent protein kinase and protein kinase C do not affect smooth muscle contractility by phosphorylating site A in myosin light chain kinase. It is proposed that phosphorylation of myosin light chain kinase in site A in contracting tracheal smooth muscle may play a role in the reported desensitization of contractile elements to activation by Ca2+

  6. Novel Role of Src in Priming Pyk2 Phosphorylation.

    Directory of Open Access Journals (Sweden)

    Ming Zhao

    Full Text Available Proline-rich tyrosine kinase 2 (Pyk2 is a member of the focal adhesion kinase (FAK family of non-receptor tyrosine kinases and plays an important role in diverse cellular events downstream of the integrin-family of receptors, including cell migration, proliferation and survival. Here, we have identified a novel role for Src kinase in priming Pyk2 phosphorylation and subsequent activation upon cell attachment on the integrin-ligand fibronectin. By using complementary methods, we show that Src activity is indispensable for the initial Pyk2 phosphorylation on the Y402 site observed in response to cell attachment. In contrast, the initial fibronectin-induced autophosphorylation of FAK in the homologous Y397 site occurs in a Src-independent manner. We demonstrate that the SH2-domain of Src is required for Src binding to Pyk2 and for Pyk2 phosphorylation at sites Y402 and Y579. Moreover, Y402 phosphorylation is a prerequisite for the subsequent Y579 phosphorylation. While this initial phosphorylation of Pyk2 by Src is independent of Pyk2 kinase activity, subsequent autophosphorylation of Pyk2 in trans is required for full Pyk2 phosphorylation and activation. Collectively, our studies reveal a novel function of Src in priming Pyk2 (but not FAK phosphorylation and subsequent activation downstream of integrins, and shed light on the signaling events that regulate the function of Pyk2.

  7. Effects of protein phosphorylation on color stability of ground meat.

    Science.gov (United States)

    Li, Meng; Li, Xin; Xin, Jianzeng; Li, Zheng; Li, Guixia; Zhang, Yan; Du, Manting; Shen, Qingwu W; Zhang, Dequan

    2017-03-15

    The influence of protein phosphorylation on meat color stability was investigated in this study. Phosphatase and protein kinase inhibitors were added to minced ovine Longissimus thoracis et lumborum (LTL) muscle to manipulate the global phosphorylation of sarcoplasmic proteins. The data obtained show that the rate and extent of pH decline, along with lactate accumulation in postmortem muscle, were related to protein phosphorylation. Analysis of meat color and the relative content of myoglobin redox forms revealed that meat color stability was inversely related to the phosphorylation of sarcoplasmic proteins. Thus, this study suggests that protein phosphorylation may be involved in meat color development by regulating glycolysis and the redox stability of myoglobin. Copyright © 2016 Elsevier Ltd. All rights reserved.

  8. Interaction of butylated hydroxyanisole with mitochondrial oxidative phosphorylation.

    Science.gov (United States)

    Fusi, F; Sgaragli, G; Murphy, M P

    1992-03-17

    The antioxidant, butylated hydroxyanisole (BHA), has a number of effects on mitochondrial oxidative phosphorylation. In this study we apply the novel approach developed by Brand (Brand MD, Biochim Biophys Acta 1018: 128-133, 1990) to investigate the site of action of BHA on oxidative phosphorylation in rat liver mitochondria. Using this approach we show that BHA increases the proton leak through the mitochondrial inner membrane and that it also inhibits the delta p (proton motive force across the mitochondrial inner membrane) generating system, but has no effect on the phosphorylation system. This demonstrates that compounds having pleiotypic effects on mitochondrial oxidative phosphorylation in vitro can be analysed and their many effects distinguished. This approach is of general use in analysing many other compounds of pharmacological interest which interact with mitochondria. The implications of these results for the mechanism of interaction of BHA with mitochondrial oxidative phosphorylation are discussed.

  9. Importance of tyrosine phosphorylation in receptor kinase complexes.

    Science.gov (United States)

    Macho, Alberto P; Lozano-Durán, Rosa; Zipfel, Cyril

    2015-05-01

    Tyrosine phosphorylation is an important post-translational modification that is known to regulate receptor kinase (RK)-mediated signaling in animals. Plant RKs are annotated as serine/threonine kinases, but recent work has revealed that tyrosine phosphorylation is also crucial for the activation of RK-mediated signaling in plants. These initial observations have paved the way for subsequent detailed studies on the mechanism of activation of plant RKs and the biological relevance of tyrosine phosphorylation for plant growth and immunity. In this Opinion article we review recent reports on the contribution of RK tyrosine phosphorylation in plant growth and immunity; we propose that tyrosine phosphorylation plays a major regulatory role in the initiation and transduction of RK-mediated signaling in plants. Copyright © 2015 Elsevier Ltd. All rights reserved.

  10. Phosphorylation of the Epstein-Barr virus nuclear antigen 2

    DEFF Research Database (Denmark)

    Grässer, F A; Göttel, S; Haiss, P

    1992-01-01

    A major in vivo phosphorylation site of the Epstein-Barr virus nuclear antigen 2 (EBNA-2) was found to be localized at the C-terminus of the protein. In vitro phosphorylation studies using casein kinase 1 (CK-1) and casein kinase 2 (CK-2) revealed that EBNA-2 is a substrate for CK-2, but not for CK......-1. The CK-2 specific phosphorylation site was localized in the 140 C-terminal amino acids using a recombinant trpE-C-terminal fusion protein. In a similar experiment, the 58 N-terminal amino acids expressed as a recombinant trpE-fusion protein were not phosphorylated. Phosphorylation of a synthetic...

  11. Sequential phosphorylation of GRASP65 during mitotic Golgi disassembly

    Directory of Open Access Journals (Sweden)

    Danming Tang

    2012-09-01

    GRASP65 phosphorylation during mitosis and dephosphorylation after mitosis are required for Golgi disassembly and reassembly during the cell cycle. At least eight phosphorylation sites on GRASP65 have been identified, but whether they are modified in a coordinated fashion during mitosis is so far unknown. In this study, we raised phospho-specific antibodies that recognize phosphorylated T220/T224, S277 and S376 residues of GRASP65, respectively. Biochemical analysis showed that cdc2 phosphorylates all three sites, while plk1 enhances the phosphorylation. Microscopic studies using these antibodies for double and triple labeling demonstrate sequential phosphorylation and dephosphorylation during the cell cycle. S277 and S376 are phosphorylated from late G2 phase through metaphase until telophase when the new Golgi is reassembled. T220/224 is not modified until prophase, but is highly modified from prometaphase to anaphase. In metaphase, phospho-T220/224 signal localizes on both Golgi haze and mitotic Golgi clusters that represent dispersed Golgi vesicles and Golgi remnants, respectively, while phospho-S277 and S376 labeling is more concentrated on mitotic Golgi clusters. Expression of a phosphorylation-resistant GRASP65 mutant T220A/T224A inhibited mitotic Golgi fragmentation to a much larger extent than the expression of the S277A and S376A mutants. In cytokinesis, T220/224 dephosphorylation occurs prior to that of S277, but after S376. This study provides evidence that GRASP65 is sequentially phosphorylated and dephosphorylated during mitosis at different sites to orchestrate Golgi disassembly and reassembly during cell division, with phosphorylation of the T220/224 site being most critical in the process.

  12. Measuring brain glucose phosphorylation with labeled glucose

    International Nuclear Information System (INIS)

    Brondsted, H.E.; Gjedde, A.

    1988-01-01

    This study tested whether glucose labeled at the C-6 position generates metabolites that leave brain so rapidly that C-6-labeled glucose cannot be used to measure brain glucose phosphorylation (CMRGlc). In pentobarbital-anesthetized rats, the parietal cortex uptake of [ 14 C]glucose labeled in the C-6 position was followed for times ranging from 10 s to 60 min. We subtracted the observed radioactivity from the radioactivity expected with no loss of labeled metabolites from brain by extrapolation of glucose uptake in an initial period when loss was negligible. The observed radioactivity was a monoexponentially declining function of the total radioactivity expected in the absence of metabolite loss. The constant of decline was 0.0077.min-1 for parietal cortex. Metabolites were lost from the beginning of the experiment. However, with correction for the loss of labeled metabolites, it was possible to determine an average CMRGlc between 4 and 60 min of circulation of 64 +/- 4 (SE; n = 49) mumol.hg-1.min-1

  13. Phosphorylation site on yeast pyruvate dehydrogenase complex

    International Nuclear Information System (INIS)

    Uhlinger, D.J.

    1986-01-01

    The pyruvate dehydrogenase complex was purified to homogeneity from baker's yeast (Saccharomyces cerevisiae). Yeast cells were disrupted in a Manton-Gaulin laboratory homogenizer. The pyruvate dehydrogenase complex was purified by fractionation with polyethylene glycol, isoelectric precipitation, ultracentrifugation and chromatography on hydroxylapatite. Final purification of the yeast pyruvate dehydrogenase complex was achieved by cation-exchange high pressure liquid chromatography (HPLC). No endogenous pyruvate dehydrogenase kinase activity was detected during the purification. However, the yeast pyruvate dehydrogenase complex was phosphorylated and inactivated with purified pyruvate dehydrogenase kinase from bovine kidney. Tryptic digestion of the 32 P-labeled complex yielded a single phosphopeptide which was purified to homogeniety. The tryptic digest was subjected to chromatography on a C-18 reverse phase HPLC column with a linear gradient of acetonitrile. Radioactive fractions were pooled, concentrated, and subjected to anion-exchange HPLC. The column was developed with a linear gradient of ammonium acetate. Final purification of the phosphopeptide was achieved by chromatography on a C-18 reverse phase HPLC column developed with a linear gradient of acetonitrile. The amino acid sequence of the homogeneous peptide was determined by manual modified Edman degradation

  14. Phosphorylation sites of Arabidopsis MAP Kinase Substrate 1 (MKS1)

    DEFF Research Database (Denmark)

    Caspersen, M.B.; Qiu, J.-L.; Zhang, X.

    2007-01-01

    The Arabidopsis MAP kinase 4 (MPK4) substrate MKS1 was expressed in Escherichia coli and purified, full-length, 6x histidine (His)-tagged MKS1 was phosphorylated in vitro by hemagglutinin (HA)-tagged MPK4 immuno-precipitated from plants. MKS1 phosphorylation was initially verified by electrophore......The Arabidopsis MAP kinase 4 (MPK4) substrate MKS1 was expressed in Escherichia coli and purified, full-length, 6x histidine (His)-tagged MKS1 was phosphorylated in vitro by hemagglutinin (HA)-tagged MPK4 immuno-precipitated from plants. MKS1 phosphorylation was initially verified...... phosphopeptide detection. As MAP kinases generally phosphorylate serine or threonine followed by proline (Ser/Thr-Pro), theoretical masses of potentially phosphorylated peptides were calculated and mass spectrometric peaks matching these masses were fragmented and searched for a neutral-loss signal...... at approximately 98 Da indicative of phosphorylation. Additionally, mass spectrometric peaks present in the MPK4-treated MKS1, but not in the control peptide map of untreated MKS1, were fragmented. Fragmentation spectra were subjected to a MASCOT database search which identified three of the twelve Ser-Pro serine...

  15. Cytochrome C is tyrosine 97 phosphorylated by neuroprotective insulin treatment.

    Directory of Open Access Journals (Sweden)

    Thomas H Sanderson

    Full Text Available Recent advancements in isolation techniques for cytochrome c (Cytc have allowed us to discover post-translational modifications of this protein. We previously identified two distinct tyrosine phosphorylated residues on Cytc in mammalian liver and heart that alter its electron transfer kinetics and the ability to induce apoptosis. Here we investigated the phosphorylation status of Cytc in ischemic brain and sought to determine if insulin-induced neuroprotection and inhibition of Cytc release was associated with phosphorylation of Cytc. Using an animal model of global brain ischemia, we found a ∼50% decrease in neuronal death in the CA1 hippocampal region with post-ischemic insulin administration. This insulin-mediated increase in neuronal survival was associated with inhibition of Cytc release at 24 hours of reperfusion. To investigate possible changes in the phosphorylation state of Cytc we first isolated the protein from ischemic pig brain and brain that was treated with insulin. Ischemic brains demonstrated no detectable tyrosine phosphorylation. In contrast Cytc isolated from brains treated with insulin showed robust phosphorylation of Cytc, and the phosphorylation site was unambiguously identified as Tyr97 by immobilized metal affinity chromatography/nano-liquid chromatography/electrospray ionization mass spectrometry. We next confirmed these results in rats by in vivo application of insulin in the absence or presence of global brain ischemia and determined that Cytc Tyr97-phosphorylation is strongly induced under both conditions but cannot be detected in untreated controls. These data suggest a mechanism whereby Cytc is targeted for phosphorylation by insulin signaling, which may prevent its release from the mitochondria and the induction of apoptosis.

  16. Amino acid chirality breaking by N-phosphorylation

    International Nuclear Information System (INIS)

    Zhao Yufen; Yan Qingjin.

    1995-01-01

    The chirality breaking of amino acid is a focus issue in the origin of life. For chemists, there are some interesting chemical approaches to solve the symmetry breaking problem. Our previous experiments indicated that when amino acids were phosphorylated, there were many bio-mimic reactions happened. In this paper, it was found that there had significant difference between the N-phosphoryl L- and D- amino acids such as serine and threonine. The optical rotation tracing experiments of the racemic N-phosphoamino acids also showed the similar results. The chirality breaking of amino acids by N-phosphorylation was a novel phenomena. (author). 3 refs, 1 fig. Abstract only

  17. Rat1p maintains RNA polymerase II CTD phosphorylation balance

    DEFF Research Database (Denmark)

    Jimeno-González, Silvia; Schmid, Manfred; Malagon, Francisco

    2014-01-01

    . Here we describe a function of Rat1p in regulating phosphorylation levels of the C-terminal domain (CTD) of the largest RNAPII subunit, Rpb1p, during transcription elongation. The rat1-1 mutant exhibits highly elevated levels of CTD phosphorylation as well as RNAPII distribution and transcription...... termination defects. These phenotypes are all rescued by overexpression of the CTD phosphatase Fcp1p, suggesting a functional relationship between the absence of Rat1p activity, elevated CTD phosphorylation, and transcription defects. We also demonstrate that rat1-1 cells display increased RNAPII...

  18. Rosamines targeting the cancer oxidative phosphorylation pathway.

    Directory of Open Access Journals (Sweden)

    Siang Hui Lim

    Full Text Available Reprogramming of energy metabolism is pivotal to cancer, so mitochondria are potential targets for anticancer therapy. A prior study has demonstrated the anti-proliferative activity of a new class of mitochondria-targeting rosamines. This present study describes in vitro cytotoxicity of second-generation rosamine analogs, their mode of action, and their in vivo efficacies in a tumor allografted mouse model. Here, we showed that these compounds exhibited potent cytotoxicity (average IC50<0.5 µM, inhibited Complex II and ATP synthase activities of the mitochondrial oxidative phosphorylation pathway and induced loss of mitochondrial transmembrane potential. A NCI-60 cell lines screen further indicated that rosamine analogs 4 and 5 exhibited potent antiproliferative effects with Log10GI50 = -7 (GI50 = 0.1 µM and were more effective against a colorectal cancer sub-panel than other cell lines. Preliminary in vivo studies on 4T1 murine breast cancer-bearing female BALB/c mice indicated that treatment with analog 5 in a single dosing of 5 mg/kg or a schedule dosing of 3 mg/kg once every 2 days for 6 times (q2d×6 exhibited only minimal induction of tumor growth delay. Our results suggest that rosamine analogs may be further developed as mitochondrial targeting agents. Without a doubt proper strategies need to be devised to enhance tumor uptake of rosamines, i.e. by integration to carrier molecules for better therapeutic outcome.

  19. Growth-Inhibitory and Antiangiogenic Activity of the MEK Inhibitor PD0325901 in Malignant Melanoma with or without BRAF Mutations

    Directory of Open Access Journals (Sweden)

    Ludovica Ciuffreda

    2009-08-01

    Full Text Available The Raf/MEK/ERK pathway is an importantmediator of tumor cell proliferation and angiogenesis. Here, weinvestigated the growth-inhibitory and antiangiogenic properties of PD0325901, a novel MEK inhibitor, in human melanoma cells. PD0325901 effects were determined in a panel of melanoma cell lines with different genetic aberrations. PD0325901 markedly inhibited ERK phosphorylation and growth of both BRAF mutant and wild-type melanoma cell lines, with IC50 in the nanomolar range even in the least responsive models. Growth inhibition was observed both in vitro and in vivo in xenograft models, regardless of BRAF mutation status, and was due to G1-phase cell cycle arrest and subsequent induction of apoptosis. Cell cycle (cyclin D1, c-Myc, and p27KIP1 and apoptosis (Bcl-2 and survivin regulators were modulated by PD0325901 at the protein level. Gene expression profiling revealed profound modulation of several genes involved in the negative control of MAPK signaling and melanoma cell differentiation, suggesting alternative, potentially relevant mechanisms of action. Finally, PD0325901 inhibited the production of the proangiogenic factors vascular endothelial growth factor and interleukin 8 at a transcriptional level. In conclusion, PD0325901 exerts potent growth-inhibitory, proapoptotic, and antiangiogenic activity in melanoma lines, regardless of their BRAF mutation status. Deeper understanding of the molecular mechanisms of action of MEK inhibitors will likely translate into more effective treatment strategies for patients experiencing malignant melanoma.

  20. Long-term insulin-like growth factor-I expression in skeletal muscles attenuates the enhanced in vitro proliferation ability of the resident satellite cells in transgenic mice

    Science.gov (United States)

    Chakravarthy, M. V.; Fiorotto, M. L.; Schwartz, R. J.; Booth, F. W.

    2001-01-01

    Insulin-like growth factor-I (IGF-I) overexpression for 1-month in mouse skeletal muscle increases satellite cell proliferation potential. However, it is unknown whether this beneficial enhancement by IGF-I expression would persist over a longer-term duration in aged mice. This is an important issue to address if a prolonged course of IGF-I is to be used clinically in muscle-wasting conditions where satellite cells may become limiting. Using the IGF-I transgenic (IGF-I Tg) mouse that selectively expresses the IGF-I transgene in striated muscles, we found that 18-months of continuous IGF-I overexpression led to a loss in the enhanced in vitro proliferative capacity of satellite cells from Tg skeletal muscles. Also 18-month-old IGF-I Tg satellite cells lost the enhanced BrdU incorporation, greater pRb and Akt phosphorylations, and decreased p27(Kip1) levels initially observed in cells from 1-month-old IGF-I Tg mice. The levels of those biochemical markers reverted to similar values seen in the 18-months WT littermates. These findings, therefore, suggest that there is no further beneficial effect on enhancing satellite cell proliferation ability with persistent long-term expression of IGF-I in skeletal muscles of these transgenic mice.

  1. Phosphoinositide-3-kinases p110alpha and p110beta mediate S phase entry in astroglial cells in the marginal zone of rat neocortex

    Directory of Open Access Journals (Sweden)

    Rabea eMüller

    2013-03-01

    Full Text Available In cells cultured from neocortex of newborn rats, phosphoinositide-3-kinases of class I regulate the DNA synthesis in a subgroup of astroglial cells. We have studied the location of these cells as well as the kinase isoforms which facilitate the S phase entry. Using dominant negative isoforms as well as selective pharmacological inhibitors we quantified S phase entry by nuclear labeling with bromodeoxyuridine. Only in astroglial cells harvested from the marginal zone of the neocortex inhibition of phosphoinositide-3-kinases reduced the nuclear labeling with bromodeoxyuridine, indicating that neocortical astroglial cells differ in the regulation of proliferation. The two kinase isoforms p110 and p110were essential for S phase entry. p110 diminished the level of the p27Kip1 which inactivates the complex of cyclin E and CDK2 necessary for entry into the S phase. p110phosphorylated and inhibited glycogen synthase kinase-3which can prevent S-phase entry. Taken together, both isoforms mediated S phase in a subgroup of neocortical astroglial cells and acted via distinct pathways.

  2. Age-specific functional epigenetic changes in p21 and p16 in injury-activated satellite cells

    Science.gov (United States)

    Li, Ju; Han, Suhyoun; Cousin, Wendy; Conboy, Irina M.

    2014-01-01

    The regenerative capacity of muscle dramatically decreases with age because old muscle stem cells fail to proliferate in response to tissue damage. Here we uncover key age-specific differences underlying this proliferative decline: namely, the genetic loci of CDK inhibitors (CDKI) p21 and p16 are more epigenetically silenced in young muscle stem cells, as compared to old, both in quiescent cells and those responding to tissue injury. Interestingly, phosphorylated ERK (pERK) induced in these cells by ectopic FGF-2 is found in association with p21 and p16 promoters, and moreover, only in the old cells. Importantly, in the old satellite cells FGF-2/pERK silences p21 epigenetically and transcriptionally, which leads to reduced p21 protein levels and enhanced cell proliferation. In agreement with the epigenetic silencing of the loci, young muscle stem cells do not depend as much as old on ectopic FGF/pERK for their myogenic proliferation. In addition, other CDKIs, such asp15INK4B and p27KIP1, become elevated in satellite cells with age, confirming and explaining the profound regenerative defect of old muscle. This work enhances our understanding of tissue aging, promoting strategies for combating age-imposed tissue degeneration. PMID:25447026

  3. Cytochrome c Is Tyrosine 97 Phosphorylated by Neuroprotective Insulin Treatment

    Czech Academy of Sciences Publication Activity Database

    Sanderson, T. H.; Mahapatra, G.; Pecina, Petr; Ji, Q.; Yu, K.; Sinkler, Ch.; Varughese, A.; Kumar, R.; Bukowski, M. J.; Tousignant, R. N.; Salomon, A. R.; Lee, I.; Hüttemann, M.

    2013-01-01

    Roč. 8, č. 11 (2013), e78627 E-ISSN 1932-6203 Institutional support: RVO:67985823 Keywords : cytochrome c * tyrosine phosphorylation * brain ischemia * insulin Subject RIV: ED - Physiology Impact factor: 3.534, year: 2013

  4. Bad phosphorylation as a target of inhibition in oncology.

    Science.gov (United States)

    Bui, Ngoc-Linh-Chi; Pandey, Vijay; Zhu, Tao; Ma, Lan; Basappa; Lobie, Peter E

    2018-02-28

    Bcl-2 agonist of cell death (BAD) is a BH3-only member of the Bcl-2 family which possesses important regulatory function in apoptosis. BAD has also been shown to possess many non-apoptotic functions closely linked to cancer including regulation of glycolysis, autophagy, cell cycle progression and immune system development. Interestingly, BAD can be either pro-apoptotic or pro-survival depending on the phosphorylation state of three specific serine residues (human S75, S99 and S118). Expression of BAD and BAD phosphorylation patterns have been shown to influence tumor initiation and progression and play a predictive role in disease prognosis, drug response and chemosensitivity in various cancers. This review aims to summarize the current evidence on the functional role of BAD phosphorylation in human cancer and evaluate the potential utility of modulating BAD phosphorylation in cancer. Copyright © 2017 Elsevier B.V. All rights reserved.

  5. Genetics Home Reference: combined oxidative phosphorylation deficiency 1

    Science.gov (United States)

    ... a severe condition that primarily impairs neurological and liver function. Most people with combined oxidative phosphorylation deficiency 1 have severe brain dysfunction (encephalopathy) that worsens over time; they also have difficulty ...

  6. In vivo phosphorylation of a peptide tag for protein purification.

    Science.gov (United States)

    Goux, Marine; Fateh, Amina; Defontaine, Alain; Cinier, Mathieu; Tellier, Charles

    2016-05-01

    To design a new system for the in vivo phosphorylation of proteins in Escherichia coli using the co-expression of the α-subunit of casein kinase II (CKIIα) and a target protein, (Nanofitin) fused with a phosphorylatable tag. The level of the co-expressed CKIIα was controlled by the arabinose promoter and optimal phosphorylation was obtained with 2 % (w/v) arabinose as inductor. The effectiveness of the phosphorylation system was demonstrated by electrophoretic mobility shift assay (NUT-PAGE) and staining with a specific phosphoprotein-staining gel. The resulting phosphorylated tag was also used to purify the phosphoprotein by immobilized metal affinity chromatography, which relies on the specific interaction of phosphate moieties with Fe(III). The use of a single tag for both the purification and protein array anchoring provides a simple and straightforward system for protein analysis.

  7. PhosphoBase: a database of phosphorylation sites

    DEFF Research Database (Denmark)

    Blom, Nikolaj; Kreegipuu, Andres; Brunak, Søren

    1998-01-01

    PhosphoBase is a database of experimentally verified phosphorylation sites. Version 1.0 contains 156 entries and 398 experimentally determined phosphorylation sites. Entries are compiled and revised from the literature and from major protein sequence databases such as SwissProt and PIR. The entries...... provide information about the phosphoprotein and the exact position of its phosphorylation sites. Furthermore, part of the entries contain information about kinetic data obtained from enzyme assays on specific peptides. To illustrate the use of data extracted from PhosphoBase we present a sequence logo...... displaying the overall conservation of positions around serines phosphorylated by protein kinase A (PKA). PhosphoBase is available on the WWW at http://www.cbs.dtu.dk/databases/PhosphoBase/....

  8. Annealing properties of potato starches with different degrees of phosphorylation

    DEFF Research Database (Denmark)

    Muhrbeck, Per; Svensson, E

    1996-01-01

    Changes in the gelatinization temperature interval and gelatinization enthalpy with annealing time at 50 degrees C were followed for a number of potato starch samples, with different degrees of phosphorylation, using differential scanning calorimetry. The gelatinization temperature increased...

  9. Exploring the diversity of protein modifications: special bacterial phosphorylation systems

    DEFF Research Database (Denmark)

    Mijakovic, Ivan; Grangeasse, Christophe; Turgay, Kürşad

    2016-01-01

    Protein modifications not only affect protein homeostasis but can also establish new cellular protein functions and are important components of complex cellular signal sensing and transduction networks. Among these post-translational modifications, protein phosphorylation represents the one that ...

  10. CAPS Activity in Priming Vesicle Exocytosis Requires CK2 Phosphorylation*

    OpenAIRE

    Nojiri, Mari; Loyet, Kelly M.; Klenchin, Vadim A.; Kabachinski, Gregory; Martin, Thomas F. J.

    2009-01-01

    CAPS (Ca2+-dependent activator protein for secretion) functions in priming Ca2+-dependent vesicle exocytosis, but the regulation of CAPS activity has not been characterized. Here we show that phosphorylation by protein kinase CK2 is required for CAPS activity. Dephosphorylation eliminated CAPS activity in reconstituting Ca2+-dependent vesicle exocytosis in permeable and intact PC12 cells. Ser-5, -6, and -7 and Ser-1281 were identified by mass spectrometry as the major phosphorylation sites in...

  11. LRRK2 mediated Rab8a phosphorylation promotes lipid storage.

    Science.gov (United States)

    Yu, Miao; Arshad, Muhammad; Wang, Wenmin; Zhao, Dongyu; Xu, Li; Zhou, Linkang

    2018-02-27

    Several mutations in leucine rich repeat kinase 2 (LRRK2) gene have been associated with pathogenesis of Parkinson's disease (PD), a neurodegenerative disorder marked by resting tremors, and rigidity, leading to Postural instability. It has been revealed that mutations that lead to an increase of kinase activity of LRRK2 protein are significantly associated with PD pathogenesis. Recent studies have shown that some Rab GTPases, especially Rab8, serve as substrates of LRRK2 and undergo phosphorylation in its switch II domain upon interaction. Current study was performed in order to find out the effects of the phosphorylation of Rab8 and its mutants on lipid metabolism and lipid droplets growth. The phosphorylation status of Rab8a was checked by phos-tag gel. Point mutant construct were generated to investigate the function of Rab8a. 3T3L1 cells were transfected with indicated plasmids and the lipid droplets were stained with Bodipy. Fluorescent microscopy experiments were performed to examine the sizes of lipid droplets. The interactions between Rab8a and Optineurin were determined by immunoprecipitation and western blot. Our assays demonstrated that Rab8a was phosphorylated by mutated LRRK2 that exhibits high kinase activity. Phosphorylation of Rab8a on amino acid residue T72 promoted the formation of large lipid droplets. T72D mutant of Rab8a had higher activity to promote the formation of large lipid droplets compared with wild type Rab8a, with increase in average diameter of lipid droplets from 2.10 μm to 2.46 μm. Moreover, phosphorylation of Rab8a weakened the interaction with its effector Optineurin. Y1699C mutated LRRK2 was able to phosphorylate Rab8a and phosphorylation of Rab8a on site 72 plays important role in the fusion and enlargement of lipid droplets. Taken together, our study suggests an indirect relationship between enhanced lipid storage capacity and PD pathogenesis.

  12. Cortactin Tyrosine Phosphorylation Promotes Its Deacetylation and Inhibits Cell Spreading

    Science.gov (United States)

    Meiler, Eugenia; Nieto-Pelegrín, Elvira; Martinez-Quiles, Narcisa

    2012-01-01

    Background Cortactin is a classical Src kinase substrate that participates in actin cytoskeletal dynamics by activating the Arp2/3 complex and interacting with other regulatory proteins, including FAK. Cortactin has various domains that may contribute to the assembly of different protein platforms to achieve process specificity. Though the protein is known to be regulated by post-translational modifications such as phosphorylation and acetylation, how tyrosine phosphorylation regulates cortactin activity is poorly understood. Since the basal level of tyrosine phosphorylation is low, this question must be studied using stimulated cell cultures, which are physiologically relevant but unreliable and difficult to work with. In fact, their unreliability may be the cause of some contradictory findings about the dynamics of tyrosine phosphorylation of cortactin in different processes. Methodology/Principal Findings In the present study, we try to overcome these problems by using a Functional Interaction Trap (FIT) system, which involves cotransfecting cells with a kinase (Src) and a target protein (cortactin), both of which are fused to complementary leucine-zipper domains. The FIT system allowed us to control precisely the tyrosine phosphorylation of cortactin and explore its relationship with cortactin acetylation. Conclusions/Significance Using this system, we provide definitive evidence that a competition exists between acetylation and tyrosine phosphorylation of cortactin and that phosphorylation inhibits cell spreading. We confirmed the results from the FIT system by examining endogenous cortactin in different cell types. Furthermore, we demonstrate that cell spreading promotes the association of cortactin and FAK and that tyrosine phosphorylation of cortactin disrupts this interaction, which may explain how it inhibits cell spreading. PMID:22479425

  13. Protein phosphorylation in isolated hepatocytes of septic and endotoxemic rats

    International Nuclear Information System (INIS)

    Deaciuc, I.V.; Spitzer, J.A.

    1989-01-01

    The purpose of this study was to investigate possible alterations induced by sepsis and endotoxicosis in the late phase of Ca2+-dependent signaling in rat liver. Hepatocytes isolated from septic or chronically endotoxin (ET)-treated rats were labeled with [32P]H3PO4 and stimulated with various agents. Proteins were resolved by one-dimensional polyacrylamide gel electrophoresis and autoradiographed. Vasopressin (VP)- and phenylephrine (PE)-induced responses were attenuated in both septic and ET-treated rats for cytosolic and membrane proteins compared with their respective controls. Glucagon and 12-O-myristate phorbol-13-acetate (TPA) affected only the phosphorylation of membrane proteins. Glucagon-induced changes in the phosphorylation of membrane proteins were affected by both sepsis and endotoxicosis, whereas TPA-stimulated phosphorylation was lowered only in endotoxicosis. Response to the Ca2+ ionophore A23187 was depressed in septic rats for cytosolic proteins. The phosphorylation of two cytosolic proteins, i.e., 93 and 61 kDa (previously identified as glycogen phosphorylase and pyruvate kinase, respectively), in response to VP, PE, and A23187 was severely impaired by endotoxicosis and sepsis. TPA did not affect the phosphorylation state of these two proteins. The results show that sepsis and endotoxicosis produce perturbations of the phosphorylation step in Ca2+ transmembrane signaling. Such changes can explain alterations of glycogenolysis and gluconeogenesis associated with sepsis and endotoxicosis

  14. Characterisation and properties of homo- and heterogenously phosphorylated nanocellulose.

    Science.gov (United States)

    Kokol, Vanja; Božič, Mojca; Vogrinčič, Robert; Mathew, Aji P

    2015-07-10

    Nano-sized cellulose ester derivatives having phosphoryl side groups were synthesised by phosphorylation of nanofibrilated cellulose (NFC) and nanocrystaline cellulose (NCC), using different heterogeneous (in water) and homogeneous (in molten urea) processes with phosphoric acid as phosphoryl donor. The phosphorylation mechanism, efficacy, stability, as well as its influence on the NC crystallinity and thermal properties, were evaluated using ATR-FTIR and (13)C NMR spectroscopies, potentiometric titration, capillary electrophoresis, X-ray diffraction, colorimetry, thermogravimmetry and SEM. Phosphorylation under both processes created dibasic phosphate and monobasic tautomeric phosphite groups at C6 and C3 positioned hydroxyls of cellulose, yielded 60-fold (∼1,173 mmol/kg) and 2-fold (∼1.038 mmol/kg) higher surface charge density for p-NFC and p-NCC, respectively, under homogenous conditions. None of the phosphorylations affected neither the NC crystallinity degree nor the structure, and noticeably preventing the derivatives from weight loss during the pyrolysis process. The p-NC showed high hydrolytic stability to water at all pH mediums. Reusing of the treatment bath was examined after the heterogeneous process. Copyright © 2015 Elsevier Ltd. All rights reserved.

  15. Protein phosphorylation and its role in archaeal signal transduction

    Science.gov (United States)

    Esser, Dominik; Hoffmann, Lena; Pham, Trong Khoa; Bräsen, Christopher; Qiu, Wen; Wright, Phillip C.; Albers, Sonja-Verena; Siebers, Bettina

    2016-01-01

    Reversible protein phosphorylation is the main mechanism of signal transduction that enables cells to rapidly respond to environmental changes by controlling the functional properties of proteins in response to external stimuli. However, whereas signal transduction is well studied in Eukaryotes and Bacteria, the knowledge in Archaea is still rather scarce. Archaea are special with regard to protein phosphorylation, due to the fact that the two best studied phyla, the Euryarchaeota and Crenarchaeaota, seem to exhibit fundamental differences in regulatory systems. Euryarchaeota (e.g. halophiles, methanogens, thermophiles), like Bacteria and Eukaryotes, rely on bacterial-type two-component signal transduction systems (phosphorylation on His and Asp), as well as on the protein phosphorylation on Ser, Thr and Tyr by Hanks-type protein kinases. Instead, Crenarchaeota (e.g. acidophiles and (hyper)thermophiles) only depend on Hanks-type protein phosphorylation. In this review, the current knowledge of reversible protein phosphorylation in Archaea is presented. It combines results from identified phosphoproteins, biochemical characterization of protein kinases and protein phosphatases as well as target enzymes and first insights into archaeal signal transduction by biochemical, genetic and polyomic studies. PMID:27476079

  16. ZDHHC3 Tyrosine Phosphorylation Regulates Neural Cell Adhesion Molecule Palmitoylation

    Science.gov (United States)

    Lievens, Patricia Marie-Jeanne; Kuznetsova, Tatiana; Kochlamazashvili, Gaga; Cesca, Fabrizia; Gorinski, Natalya; Galil, Dalia Abdel; Cherkas, Volodimir; Ronkina, Natalia; Lafera, Juri; Gaestel, Matthias

    2016-01-01

    The neural cell adhesion molecule (NCAM) mediates cell-cell and cell-matrix adhesion. It is broadly expressed in the nervous system and regulates neurite outgrowth, synaptogenesis, and synaptic plasticity. Previous in vitro studies revealed that palmitoylation of NCAM is required for fibroblast growth factor 2 (FGF2)-stimulated neurite outgrowth and identified the zinc finger DHHC (Asp-His-His-Cys)-containing proteins ZDHHC3 and ZDHHC7 as specific NCAM-palmitoylating enzymes. Here, we verified that FGF2 controlled NCAM palmitoylation in vivo and investigated molecular mechanisms regulating NCAM palmitoylation by ZDHHC3. Experiments with overexpression and pharmacological inhibition of FGF receptor (FGFR) and Src revealed that these kinases control tyrosine phosphorylation of ZDHHC3 and that ZDHHC3 is phosphorylated by endogenously expressed FGFR and Src proteins. By site-directed mutagenesis, we found that Tyr18 is an FGFR1-specific ZDHHC3 phosphorylation site, while Tyr295 and Tyr297 are specifically phosphorylated by Src kinase in cell-based and cell-free assays. Abrogation of tyrosine phosphorylation increased ZDHHC3 autopalmitoylation, enhanced interaction with NCAM, and upregulated NCAM palmitoylation. Expression of ZDHHC3 with tyrosine mutated in cultured hippocampal neurons promoted neurite outgrowth. Our findings for the first time highlight that FGFR- and Src-mediated tyrosine phosphorylation of ZDHHC3 modulates ZDHHC3 enzymatic activity and plays a role in neuronal morphogenesis. PMID:27247265

  17. Cholinergic regulation of protein phosphorylation in bovine adrenal chromaffin cells

    International Nuclear Information System (INIS)

    Haycock, J.W.; Browning, M.D.; Greengard, P.

    1988-01-01

    Chromaffin cells were isolated from bovine adrenal medullae and maintained in primary culture. After prelabeling with 32 PO 4 , exposure of the chromaffin cells to acetylcholine increased the phosphorylation of a M/sub r/ ≅ 100,000 protein and a M/sub r/ ≅ 60,000 protein (tyrosine hydroxylase), visualized after separation of total cellular proteins in NaDodSO 4 /polyacrylamide gels. Immunoprecipitation with antibodies to three known phosphoproteins (100-kDa, 87-kDa, and protein III) revealed an acetylcholine-dependent phosphorylation of these proteins. These three proteins were also shown to be present in bovine adrenal chromaffin cells by immunolabeling techniques. 100-kDa is a M/sub r/ ≅ 100,000 protein selectively phosphorylated by calcium/calmodulin-dependent protein kinase III, 87-kDa is a M/sub r/ ≅ 87,000 protein selectively phosphorylated by protein kinase C, and protein III is a phosphoprotein doublet of M/sub r/ ≅ 74,000 (IIIa) and M/sub r/ ≅ 55,000 (IIIb) phosphorylated by cAMP-dependent protein kinase and calcium/calmodulin-dependent protein kinase I. The data demonstrate that cholinergic activation of chromaffin cells increases the phosphorylation of several proteins and that several protein kinase systems may be involved in these effects

  18. Raptor is phosphorylated by cdc2 during mitosis.

    Directory of Open Access Journals (Sweden)

    Dana M Gwinn

    2010-02-01

    Full Text Available The appropriate control of mitotic entry and exit is reliant on a series of interlocking signaling events that coordinately drive the biological processes required for accurate cell division. Overlaid onto these signals that promote orchestrated cell division are checkpoints that ensure appropriate mitotic spindle formation, a lack of DNA damage, kinetochore attachment, and that each daughter cell has the appropriate complement of DNA. We recently discovered that AMP-activated protein kinase (AMPK modulates the G2/M phase of cell cycle progression in part through its suppression of mammalian target of rapamycin (mTOR signaling. AMPK directly phosphorylates the critical mTOR binding partner raptor inhibiting mTORC1 (mTOR-raptor rapamycin sensitive mTOR kinase complex 1. As mTOR has been previously tied to mitotic control, we examined further how raptor may contribute to this process.We have discovered that raptor becomes highly phosphorylated in cells in mitosis. Utilizing tandem mass spectrometry, we identified a number of novel phosphorylation sites in raptor, and using phospho-specific antibodies demonstrated that raptor becomes phosphorylated on phospho-serine/threonine-proline sites in mitosis. A combination of site-directed mutagenesis in a tagged raptor cDNA and analysis with a series of new phospho-specific antibodies generated against different sites in raptor revealed that Serine 696 and Threonine 706 represent two key sites in raptor phosphorylated in mitosis. We demonstrate that the mitotic cyclin-dependent kinase cdc2/CDK1 is the kinase responsible for phosphorylating these sites, and its mitotic partner Cyclin B efficiently coimmunoprecipitates with raptor in mitotic cells.This study demonstrates that the key mTOR binding partner raptor is directly phosphorylated during mitosis by cdc2. This reinforces previous studies suggesting that mTOR activity is highly regulated and important for mitotic progression, and points to a direct

  19. Phosphorylation of the Yeast Choline Kinase by Protein Kinase C

    Science.gov (United States)

    Choi, Mal-Gi; Kurnov, Vladlen; Kersting, Michael C.; Sreenivas, Avula; Carman, George M.

    2005-01-01

    The Saccharomyces cerevisiae CKI1-encoded choline kinase catalyzes the committed step in phosphatidylcholine synthesis via the Kennedy pathway. The enzyme is phosphorylated on multiple serine residues, and some of this phosphorylation is mediated by protein kinase A. In this work, we examined the hypothesis that choline kinase is also phosphorylated by protein kinase C. Using choline kinase as a substrate, protein kinase C activity was dose- and time-dependent, and dependent on the concentrations of choline kinase (Km = 27 μg/ml) and ATP (Km = 15 μM). This phosphorylation, which occurred on a serine residue, was accompanied by a 1.6-fold stimulation of choline kinase activity. The synthetic peptide SRSSS25QRRHS (Vmax/Km = 17.5 mM-1 μmol min-1 mg-1) that contains the protein kinase C motif for Ser25 was a substrate for protein kinase C. A Ser25 to Ala (S25A) mutation in choline kinase resulted in a 60% decrease in protein kinase C phosphorylation of the enzyme. Phosphopeptide mapping analysis of the S25A mutant enzyme confirmed that Ser25 was a protein kinase C target site. In vivo, the S25A mutation correlated with a decrease (55%) in phosphatidylcholine synthesis via the Kennedy pathway whereas an S25D phosphorylation site mimic correlated with an increase (44%) in phosphatidylcholine synthesis. Whereas the S25A (protein kinase C site) mutation did not affect the phosphorylation of choline kinase by protein kinase A, the S30A (protein kinase A site) mutation caused a 46% reduction in enzyme phosphorylation by protein kinase C. A choline kinase synthetic peptide (SQRRHS30LTRQ) containing Ser30 was a substrate (Vmax/Km = 3.0 mM−1 μmol min−1 mg−1) for protein kinase C. Comparison of phosphopeptide maps of the wild type and S30A mutant choline kinase enzymes phosphorylated by protein kinase C confirmed that Ser30 was also a target site for protein kinase C. PMID:15919656

  20. Neurofilament subunit (NFL) head domain phosphorylation regulates axonal transport of neurofilaments.

    LENUS (Irish Health Repository)

    Yates, Darran M

    2009-04-01

    Neurofilaments are the intermediate filaments of neurons and are synthesised in neuronal cell bodies and then transported through axons. Neurofilament light chain (NFL) is a principal component of neurofilaments, and phosphorylation of NFL head domain is believed to regulate the assembly of neurofilaments. However, the role that NFL phosphorylation has on transport of neurofilaments is poorly understood. To address this issue, we monitored axonal transport of phosphorylation mutants of NFL. We mutated four known phosphorylation sites in NFL head domain to either preclude phosphorylation, or mimic permanent phosphorylation. Mutation to preclude phosphorylation had no effect on transport but mutation of three sites to mimic permanent phosphorylation inhibited transport. Mutation of all four sites together to mimic permanent phosphorylation proved especially potent at inhibiting transport and also disrupted neurofilament assembly. Our results suggest that NFL head domain phosphorylation is a regulator of neurofilament axonal transport.

  1. beta2-adaptin is constitutively de-phosphorylated by serine/threonine protein phosphatase PP2A and phosphorylated by a staurosporine-sensitive kinase

    DEFF Research Database (Denmark)

    Lauritsen, Jens Peter Holst; Menné, C; Kastrup, J

    2000-01-01

    Clathrin-mediated endocytosis includes cycles of assembly and disassembly of the clathrin-coated vesicle constituents. How these cycles are regulated is still not fully known but previous studies have indicated that phosphorylation of coat subunits may play a role. Here we describe that beta2-ada...... the hypothesis that phosphorylation/de-phosphorylation of coat proteins plays a regulatory role in the assembly/disassembly cycle of clathrin-coated vesicles.......Clathrin-mediated endocytosis includes cycles of assembly and disassembly of the clathrin-coated vesicle constituents. How these cycles are regulated is still not fully known but previous studies have indicated that phosphorylation of coat subunits may play a role. Here we describe that beta2......-adaptin undergoes cycles of phosphorylation/de-phosphorylation in intact cells. Thus, beta2-adaptin was constitutively de-phosphorylated by serine/threonine protein phosphatase 2A and phosphorylated by a staurosporine-sensitive kinase in vivo. Confocal laser scanning microscopy demonstrated...

  2. Inhibition of peptide aggregation by means of enzymatic phosphorylation

    Directory of Open Access Journals (Sweden)

    Kristin Folmert

    2016-11-01

    Full Text Available As is the case in numerous natural processes, enzymatic phosphorylation can be used in the laboratory to influence the conformational populations of proteins. In nature, this information is used for signal transduction or energy transfer, but has also been shown to play an important role in many diseases like tauopathies or diabetes. With the goal of determining the effect of phosphorylation on amyloid fibril formation, we designed a model peptide which combines structural characteristics of α-helical coiled-coils and β-sheets in one sequence. This peptide undergoes a conformational transition from soluble structures into insoluble amyloid fibrils over time and under physiological conditions and contains a recognition motif for PKA (cAMP-dependent protein kinase that enables enzymatic phosphorylation. We have analyzed the pathway of amyloid formation and the influence of enzymatic phosphorylation on the different states along the conformational transition from random-coil to β-sheet-rich oligomers to protofilaments and on to insoluble amyloid fibrils, and we found a remarkable directing effect from β-sheet-rich structures to unfolded structures in the initial growth phase, in which small oligomers and protofilaments prevail if the peptide is phosphorylated.

  3. Stimulation of glucose phosphorylation by fructose in isolated rat hepatocytes.

    Science.gov (United States)

    Van Schaftingen, E; Vandercammen, A

    1989-01-15

    The phosphorylation of glucose was measured by the formation of [3H]H2O from [2-3H]glucose in suspensions of freshly isolated rat hepatocytes. Fructose (0.2 mM) stimulated 2-4-fold the rate of phosphorylation of 5 mM glucose although not of 40 mM glucose, thus increasing the apparent affinity of the glucose phosphorylating system. A half-maximal stimulatory effect was observed at about 50 microM fructose. Stimulation was maximal 5 min after addition of the ketose and was stable for at least 40 min, during which period 60% of the fructose was consumed. The effect of fructose was reversible upon removal of the ketose. Sorbitol and tagatose were as potent as fructose in stimulating the phosphorylation of 5 mM glucose. D-Glyceraldehyde also had a stimulatory effect but at tenfold higher concentrations. In contrast, dihydroxyacetone had no significant effect and glycerol inhibited the detritiation of glucose. Oleate did not affect the phosphorylation of glucose, even in the presence of fructose, although it stimulated the formation of ketone bodies severalfold, indicating that it was converted to its acyl-CoA derivative. These results allow the conclusion that fructose stimulates glucokinase in the intact hepatocyte. They also suggest that this effect is mediated through the formation of fructose 1-phosphate, which presumably interacts with a competitive inhibitor of glucokinase other than long-chain acyl-CoAs.

  4. Tyrosine Phosphorylation in Toll-Like Receptor Signaling

    Science.gov (United States)

    Chattopadhyay, Saurabh; Sen, Ganes C.

    2014-01-01

    There is a wealth of knowledge about how different Ser/Thr protein kinases participate in Toll-like receptor (TLR) signaling. In many cases, we know the identities of the Ser/Thr residues of various components of the TLR-signaling pathways that are phosphorylated, the functional consequences of the phosphorylation and the responsible protein kinases. In contrast, the analysis of Tyr-phosphorylation of TLRs and their signaling proteins is currently incomplete, because several existing analyses are not systematic or they do not rely on robust experimental data. Nevertheless, it is clear that many TLRs require, for signaling, ligand-dependent phosphorylation of specific Tyr residues in their cytoplasmic domains; the list includes TLR2, TLR3, TLR4, TLR5, TLR8 and TLR9. In this article, we discuss the current status of knowledge on the effect of Tyr-phosphorylation of TLRs and their signaling proteins on their biochemical and biological functions, the possible identities of the relevant protein tyrosine kinases (PTKs) and the nature of regulations of PTK-mediated activation of TLR signaling pathways. PMID:25022196

  5. Phosphorylation of Large T Antigen Regulates Merkel Cell Polyomavirus Replication

    International Nuclear Information System (INIS)

    Diaz, Jason; Wang, Xin; Tsang, Sabrina H.; Jiao, Jing; You, Jianxin

    2014-01-01

    Merkel Cell Polyomavirus (MCPyV) was recently discovered as a novel human polyomavirus that is associated with ~80% of Merkel Cell Carcinomas. The Large Tumor antigen (LT) is an early viral protein which has a variety of functions, including manipulation of the cell cycle and initiating viral DNA replication. Phosphorylation plays a critical regulatory role for polyomavirus LT proteins, but no investigation of MCPyV LT phosphorylation has been performed to date. In this report mass spectrometry analysis reveals three unique phosphorylation sites: T271, T297 and T299. In vivo replication assays confirm that phosphorylation of T271 does not play a role in viral replication, while modification at T297 and T299 have dramatic and opposing effects on LT’s ability to initiate replication from the viral origin. We test these mutants for their ability to bind, unwind, and act as a functional helicase at the viral origin. These studies provide a framework for understanding how phosphorylation of LT may dynamically regulate viral replication. Although the natural host cell of MCPyV has not yet been established, this work provides a foundation for understanding how LT activity is regulated and provides tools for better exploring this regulation in both natural host cells and Merkel cells

  6. Phosphorylation of Large T Antigen Regulates Merkel Cell Polyomavirus Replication

    Energy Technology Data Exchange (ETDEWEB)

    Diaz, Jason; Wang, Xin; Tsang, Sabrina H. [Department of Microbiology, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104 (United States); Jiao, Jing [Department of Pathology and Laboratory Medicine, Children’s Hospital of Philadelphia, Philadelphia, PA 19104 (United States); You, Jianxin, E-mail: jianyou@mail.med.upenn.edu [Department of Microbiology, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104 (United States)

    2014-07-08

    Merkel Cell Polyomavirus (MCPyV) was recently discovered as a novel human polyomavirus that is associated with ~80% of Merkel Cell Carcinomas. The Large Tumor antigen (LT) is an early viral protein which has a variety of functions, including manipulation of the cell cycle and initiating viral DNA replication. Phosphorylation plays a critical regulatory role for polyomavirus LT proteins, but no investigation of MCPyV LT phosphorylation has been performed to date. In this report mass spectrometry analysis reveals three unique phosphorylation sites: T271, T297 and T299. In vivo replication assays confirm that phosphorylation of T271 does not play a role in viral replication, while modification at T297 and T299 have dramatic and opposing effects on LT’s ability to initiate replication from the viral origin. We test these mutants for their ability to bind, unwind, and act as a functional helicase at the viral origin. These studies provide a framework for understanding how phosphorylation of LT may dynamically regulate viral replication. Although the natural host cell of MCPyV has not yet been established, this work provides a foundation for understanding how LT activity is regulated and provides tools for better exploring this regulation in both natural host cells and Merkel cells.

  7. Identification and quantitation of signal molecule-dependent protein phosphorylation

    KAUST Repository

    Groen, Arnoud J.

    2013-09-03

    Phosphoproteomics is a fast-growing field that aims at characterizing phosphorylated proteins in a cell or a tissue at a given time. Phosphorylation of proteins is an important regulatory mechanism in many cellular processes. Gel-free phosphoproteome technique involving enrichment of phosphopeptide coupled with mass spectrometry has proven to be invaluable to detect and characterize phosphorylated proteins. In this chapter, a gel-free quantitative approach involving 15N metabolic labelling in combination with phosphopeptide enrichment by titanium dioxide (TiO2) and their identification by MS is described. This workflow can be used to gain insights into the role of signalling molecules such as cyclic nucleotides on regulatory networks through the identification and quantification of responsive phospho(proteins). © Springer Science+Business Media New York 2013.

  8. Study of ATM Phosphorylation by Cdk5 in Neuronal Cells.

    Science.gov (United States)

    She, Hua; Mao, Zixu

    2017-01-01

    The phosphatidylinositol-3-kinase-like kinase ATM (ataxia-telangiectasia mutated) plays a central role in coordinating the DNA damage responses including cell cycle checkpoint control, DNA repair, and apoptosis. Mutations of ATM cause a spectrum of defects ranging from neurodegeneration to cancer predisposition. We previously showed that Cdk5 (cyclin-dependent kinase 5) is activated by DNA damage and directly phosphorylates ATM at serine 794 in postmitotic neurons. Phosphorylation at serine 794 precedes and is required for ATM autophosphorylation at serine 1981, and activates ATM kinase activity. Cdk5-ATM pathway plays a crucial role in DNA damage-induced neuronal injury. This chapter describes protocols used in analyzing ATM phosphorylation by Cdk5 in CGNs (cerebellar granule neurons) and its effects on neuronal survival.

  9. Common extraction of Tc, Pd and Eu by phosphorylated calixarenes

    International Nuclear Information System (INIS)

    Babain, V.; Smirnov, I.; Kvasnitskiy, I.; Karavan, M.; Boiko, V.; Miroshnichenko, V.; Klimchuk, O.; Kalchenko, V.

    2003-01-01

    The present work is aimed at studying the extraction systems based on neutral organophosphorus extractants - phosphorylated calixarenes for recovery of Pd and Tc together with Am and Cm from high-level radioactive wastes. Extraction of Pd, Tc and Eu (Am) was studied for phosphorylated calixarenes in meta-nitrobenzotrifluoride (NBTF). Main results are presented in Table. On the basis of available data one can suggest that type and position of phosphor-organic substituents are not so important for extraction of Tc and Pd, as for Eu and Am extraction. The phosphorylated at upper rim calix[4]arenas with small alkyl substituents at phosphorus atom are of prime interest for joint recovery of europium, americium, technetium and palladium. (authors)

  10. Phosphorylation site dynamics of early T-cell receptor signaling

    DEFF Research Database (Denmark)

    Chylek, Lily A; Akimov, Vyacheslav; Dengjel, Jörn

    2014-01-01

    In adaptive immune responses, T-cell receptor (TCR) signaling impacts multiple cellular processes and results in T-cell differentiation, proliferation, and cytokine production. Although individual protein-protein interactions and phosphorylation events have been studied extensively, we lack...... that diverse dynamic patterns emerge within seconds. We detected phosphorylation dynamics as early as 5 s and observed widespread regulation of key TCR signaling proteins by 30 s. Development of a computational model pointed to the presence of novel regulatory mechanisms controlling phosphorylation of sites...... a systems-level understanding of how these components cooperate to control signaling dynamics, especially during the crucial first seconds of stimulation. Here, we used quantitative proteomics to characterize reshaping of the T-cell phosphoproteome in response to TCR/CD28 co-stimulation, and found...

  11. Histones and their phosphorylation during germination of rice seeds

    International Nuclear Information System (INIS)

    Iqbal Ahmed, C.M.; Padayatti, J.D.

    1980-01-01

    Histones from nuclei of rice embryos were identified by their mobilities on 15% acid-urea polyacrylamide gel electrophoreogram, incorporation of ( 3 H)lysine and ( 14 C) arginine and lack of incorporation of ( 3 H)tryptophan. The ratio of histone to DNA in ungerminated embryos was 2.7 which decreased during germination reaching unity by 48 hr. There was preferential phosphorylation of lysine-rich histones, which paralleled the synthesis of DNA. In the presence of cytosine arabinoside and mitomycin-C, which inhibited the synthesis of DNA to the extend of 75-80% the phosphorylation of lysine-rich histone was reduced by 50-60% suggesting the dependence of phosphorylation on the ongoing synthesis of DNA. (auth.)

  12. Characterization and Prediction of Protein Phosphorylation Hotspots in Arabidopsis thaliana.

    Science.gov (United States)

    Christian, Jan-Ole; Braginets, Rostyslav; Schulze, Waltraud X; Walther, Dirk

    2012-01-01

    The regulation of protein function by modulating the surface charge status via sequence-locally enriched phosphorylation sites (P-sites) in so called phosphorylation "hotspots" has gained increased attention in recent years. We set out to identify P-hotspots in the model plant Arabidopsis thaliana. We analyzed the spacing of experimentally detected P-sites within peptide-covered regions along Arabidopsis protein sequences as available from the PhosPhAt database. Confirming earlier reports (Schweiger and Linial, 2010), we found that, indeed, P-sites tend to cluster and that distributions between serine and threonine P-sites to their respected closest next P-site differ significantly from those for tyrosine P-sites. The ability to predict P-hotspots by applying available computational P-site prediction programs that focus on identifying single P-sites was observed to be severely compromised by the inevitable interference of nearby P-sites. We devised a new approach, named HotSPotter, for the prediction of phosphorylation hotspots. HotSPotter is based primarily on local amino acid compositional preferences rather than sequence position-specific motifs and uses support vector machines as the underlying classification engine. HotSPotter correctly identified experimentally determined phosphorylation hotspots in A. thaliana with high accuracy. Applied to the Arabidopsis proteome, HotSPotter-predicted 13,677 candidate P-hotspots in 9,599 proteins corresponding to 7,847 unique genes. Hotspot containing proteins are involved predominantly in signaling processes confirming the surmised modulating role of hotspots in signaling and interaction events. Our study provides new bioinformatics means to identify phosphorylation hotspots and lays the basis for further investigating novel candidate P-hotspots. All phosphorylation hotspot annotations and predictions have been made available as part of the PhosPhAt database at http://phosphat.mpimp-golm.mpg.de.

  13. Mechanism of APC/CCDC20 activation by mitotic phosphorylation.

    Science.gov (United States)

    Qiao, Renping; Weissmann, Florian; Yamaguchi, Masaya; Brown, Nicholas G; VanderLinden, Ryan; Imre, Richard; Jarvis, Marc A; Brunner, Michael R; Davidson, Iain F; Litos, Gabriele; Haselbach, David; Mechtler, Karl; Stark, Holger; Schulman, Brenda A; Peters, Jan-Michael

    2016-05-10

    Chromosome segregation and mitotic exit are initiated by the 1.2-MDa ubiquitin ligase APC/C (anaphase-promoting complex/cyclosome) and its coactivator CDC20 (cell division cycle 20). To avoid chromosome missegregation, APC/C(CDC20) activation is tightly controlled. CDC20 only associates with APC/C in mitosis when APC/C has become phosphorylated and is further inhibited by a mitotic checkpoint complex until all chromosomes are bioriented on the spindle. APC/C contains 14 different types of subunits, most of which are phosphorylated in mitosis on multiple sites. However, it is unknown which of these phospho-sites enable APC/C(CDC20) activation and by which mechanism. Here we have identified 68 evolutionarily conserved mitotic phospho-sites on human APC/C bound to CDC20 and have used the biGBac technique to generate 47 APC/C mutants in which either all 68 sites or subsets of them were replaced by nonphosphorylatable or phospho-mimicking residues. The characterization of these complexes in substrate ubiquitination and degradation assays indicates that phosphorylation of an N-terminal loop region in APC1 is sufficient for binding and activation of APC/C by CDC20. Deletion of the N-terminal APC1 loop enables APC/C(CDC20) activation in the absence of mitotic phosphorylation or phospho-mimicking mutations. These results indicate that binding of CDC20 to APC/C is normally prevented by an autoinhibitory loop in APC1 and that its mitotic phosphorylation relieves this inhibition. The predicted location of the N-terminal APC1 loop implies that this loop controls interactions between the N-terminal domain of CDC20 and APC1 and APC8. These results reveal how APC/C phosphorylation enables CDC20 to bind and activate the APC/C in mitosis.

  14. Potential Role of Inorganic Confined Environments in Prebiotic Phosphorylation.

    Science.gov (United States)

    Dass, Avinash Vicholous; Jaber, Maguy; Brack, André; Foucher, Frédéric; Kee, Terence P; Georgelin, Thomas; Westall, Frances

    2018-03-05

    A concise outlook on the potential role of confinement in phosphorylation and phosphate condensation pertaining to prebiotic chemistry is presented. Inorganic confinement is a relatively uncharted domain in studies concerning prebiotic chemistry, and even more so in terms of experimentation. However, molecular crowding within confined dimensions is central to the functioning of contemporary biology. There are numerous advantages to confined environments and an attempt to highlight this fact, within this article, has been undertaken, keeping in context the limitations of aqueous phase chemistry in phosphorylation and, to a certain extent, traditional approaches in prebiotic chemistry.

  15. Training-induced adaptation of oxidative phosphorylation in skeletal muscles.

    Science.gov (United States)

    Korzeniewski, Bernard; Zoladz, Jerzy A

    2003-08-15

    Muscle training/conditioning improves the adaptation of oxidative phosphorylation in skeletal muscles to physical exercise. However, the mechanisms underlying this adaptation are still not understood fully. By quantitative analysis of the existing experimental results, we show that training-induced acceleration of oxygen-uptake kinetics at the onset of exercise and improvement of ATP/ADP stability due to physical training are mainly caused by an increase in the amount of mitochondrial proteins and by an intensification of the parallel activation of ATP usage and ATP supply (increase in direct stimulation of oxidative phosphorylation complexes accompanying stimulation of ATP consumption) during exercise.

  16. Tau Phosphorylation by GSK3 in Different Conditions

    Directory of Open Access Journals (Sweden)

    Jesús Avila

    2012-01-01

    Full Text Available Almost a 20% of the residues of tau protein are phosphorylatable amino acids: serine, threonine, and tyrosine. In this paper we comment on the consequences for tau of being a phosphoprotein. We will focus on serine/threonine phosphorylation. It will be discussed that, depending on the modified residue in tau molecule, phosphorylation could be protective, in processes like hibernation, or toxic like in development of those diseases known as tauopathies, which are characterized by an hyperphosphorylation and aggregation of tau.

  17. The occurrence of a phosphorylated glycosphingolipid in Aspergillus niger.

    Science.gov (United States)

    Brennan, P J; Roe, J

    1975-01-01

    A novel type of water-soluble phosphorylated glycosphingolipid was isolated from Aspergillus niger by a simple procedure involving precipitation, DEAE-cellulose chromatography and preparative t.l.c. Besides ceramide and phosphorus it contains inositol, galactose, mannose and small amounts of glucosamine. Images PLATE 1 PMID:1156383

  18. A mathematical model of phosphorylation AKT in Acute Myeloid Leukemia

    Science.gov (United States)

    Adi, Y. A.; Kusumo, F. A.; Aryati, L.; Hardianti, M. S.

    2016-04-01

    In this paper we consider a mathematical model of PI3K/AKT signaling pathways in phosphorylation AKT. PI3K/AKT pathway is an important mediator of cytokine signaling implicated in regulation of hematopoiesis. Constitutive activation of PI3K/AKT signaling pathway has been observed in Acute Meyloid Leukemia (AML) it caused by the mutation of Fms-like Tyrosine Kinase 3 in internal tandem duplication (FLT3-ITD), the most common molecular abnormality associated with AML. Depending upon its phosphorylation status, protein interaction, substrate availability, and localization, AKT can phosphorylate or inhibite numerous substrates in its downstream pathways that promote protein synthesis, survival, proliferation, and metabolism. Firstly, we present a mass action ordinary differential equation model describing AKT double phosphorylation (AKTpp) in a system with 11 equations. Finally, under the asumtion enzyme catalyst constant and steady state equilibrium, we reduce the system in 4 equation included Michaelis Menten constant. Simulation result suggested that a high concentration of PI3K and/or a low concentration of phospatase increased AKTpp activation. This result also indicates that PI3K is a potential target theraphy in AML.

  19. Activation of purified calcium channels by stoichiometric protein phosphorylation

    Energy Technology Data Exchange (ETDEWEB)

    Nunoki, K.; Florio, V.; Catterall, W.A. (Univ. of Washington, Seattle (USA))

    1989-09-01

    Purified dihydropyridine-sensitive calcium channels from rabbit skeletal muscle were reconstituted into phosphatidylcholine vesicles to evaluate the effect of phosphorylation by cyclic AMP-dependent protein kinase (PK-A) on their function. Both the rate and extent of {sup 45}Ca{sup 2+} uptake into vesicles containing reconstituted calcium channels were increased severalfold after incubation with ATP and PK-A. The degree of stimulation of {sup 45}Ca{sup 2+} uptake was linearly proportional to the extent of phosphorylation of the alpha 1 and beta subunits of the calcium channel up to a stoichiometry of approximately 1 mol of phosphate incorporated into each subunit. The calcium channels activated by phosphorylation were determined to be incorporated into the reconstituted vesicles in the inside-out orientation and were completely inhibited by low concentrations of dihydropyridines, phenylalkylamines, Cd{sup 2+}, Ni{sup 2+}, and Mg{sup 2+}. The results demonstrate a direct relationship between PK-A-catalyzed phosphorylation of the alpha 1 and beta subunits of the purified calcium channel and activation of the ion conductance activity of the dihydropyridine-sensitive calcium channels.

  20. Activation of purified calcium channels by stoichiometric protein phosphorylation

    International Nuclear Information System (INIS)

    Nunoki, K.; Florio, V.; Catterall, W.A.

    1989-01-01

    Purified dihydropyridine-sensitive calcium channels from rabbit skeletal muscle were reconstituted into phosphatidylcholine vesicles to evaluate the effect of phosphorylation by cyclic AMP-dependent protein kinase (PK-A) on their function. Both the rate and extent of 45 Ca 2+ uptake into vesicles containing reconstituted calcium channels were increased severalfold after incubation with ATP and PK-A. The degree of stimulation of 45 Ca 2+ uptake was linearly proportional to the extent of phosphorylation of the alpha 1 and beta subunits of the calcium channel up to a stoichiometry of approximately 1 mol of phosphate incorporated into each subunit. The calcium channels activated by phosphorylation were determined to be incorporated into the reconstituted vesicles in the inside-out orientation and were completely inhibited by low concentrations of dihydropyridines, phenylalkylamines, Cd 2+ , Ni 2+ , and Mg 2+ . The results demonstrate a direct relationship between PK-A-catalyzed phosphorylation of the alpha 1 and beta subunits of the purified calcium channel and activation of the ion conductance activity of the dihydropyridine-sensitive calcium channels

  1. Extraction of rhenium(VII) by phosphorylated podands

    International Nuclear Information System (INIS)

    Turanov, A.N.; Karandashev, V.K.; Baulin, V.E.

    2006-01-01

    Interphase distribution of ReO 4 - between aqueous solutions of H 2 SO 4 and solutions of phosphoryl-containing podands in organic solvents is studied. Stoichiometry of the complexes extracted is determined. Effect of extractant structure and nature of organic solvent on efficiency of rhenium extraction into organic phase is determined [ru

  2. Linear motif atlas for phosphorylation-dependent signaling

    DEFF Research Database (Denmark)

    Miller, Martin Lee; Jensen, LJ; Diella, F

    2008-01-01

    bind to them remains a challenge. NetPhorest is an atlas of consensus sequence motifs that covers 179 kinases and 104 phosphorylation-dependent binding domains [Src homology 2 (SH2), phosphotyrosine binding (PTB), BRCA1 C-terminal (BRCT), WW, and 14-3-3]. The atlas reveals new aspects of signaling...

  3. Genetic defects in the oxidative phosphorylation (OXPHOS) system.

    NARCIS (Netherlands)

    Janssen, R.J.R.J.; Heuvel, L.P.W.J. van den; Smeitink, J.A.M.

    2004-01-01

    The oxidative phosphorylation (OXPHOS) system consists of five multiprotein complexes and two mobile electron carriers embedded in the lipid bilayer of the mitochondrial inner membrane. With the exception of complex II and the mobile carriers, the other parts of the OXPHOS system are under dual

  4. Phosphorylation of intact erythrocytes in human muscular dystrophy

    International Nuclear Information System (INIS)

    Johnson, R.M.; Nigro, M.

    1986-01-01

    The uptake of exogenous 32 Pi into the membrane proteins of intact erythrocytes was measured in 8 patients with Duchenne muscular dystrophy. No abnormalities were noted after autoradiographic analysis. This contrasts with earlier results obtained when isolated membranes were phosphorylated with gamma-[ 32 P]ATP, and suggests a possible reinterpretation of those experiments

  5. Phosphorylation and disassembly of intermediate filaments in mitotic cells

    International Nuclear Information System (INIS)

    Chou, Yinghao; Rosevear, E.; Goldman, R.D.

    1989-01-01

    As baby hamster kidney (BHK-21) cells enter mitosis, networks of intermediate filaments (IFs) are transformed into cytoplasmic aggregates of protofilaments. Coincident with this morphological change, the phosphate content of vimentin increases from 0.3 mol of P i per mol of protein in interphase to 1.9 mol of P i per mol of protein in mitosis. A similar increase in phosphate content is observed with desmin, from 0.5 mol of P i per mol of protein to 1.5 mol of P i per mol of protein. Fractionation of mitotic cell lysates by hydroxylapatite column chromatography reveals the presence of two IF protein kinase activities, designated as IF protein kinase I and IF protein kinase II. Comparison of two-dimensional 32 P-labeled phosphopeptide maps of vimentin and desmin phosphorylated in vivo in mitosis, and in vitro using partially purified kinase fractions, reveals extensive similarity in the two sets of phosphorylation sites. Phosphorylation of in vitro polymerized IFs by IF protein kinase II induces complete disassembly as determined by negative-stain electron microscopy. The results support the idea that the disassembly of IFs in mitosis is regulated by the phosphorylation of its subunit proteins

  6. Phosphorylation of formate dehydrogenase in potato tuber mitochondria

    DEFF Research Database (Denmark)

    Bykova, N.V.; Stensballe, A.; Egsgaard, H.

    2003-01-01

    Two highly phosphorylated proteins were detected after two-dimensional (blue native/SDS-PAGE) gel electrophoretic separation of the matrix fraction isolated from potato tuber mitochondria. These two phosphoproteins were identified by mass spectrometry as formate dehydrogenase (FDH) and the E1alpha...

  7. Syndecan-4 Phosphorylation Is a Control Point for Integrin Recycling

    Science.gov (United States)

    Morgan, Mark R.; Hamidi, Hellyeh; Bass, Mark D.; Warwood, Stacey; Ballestrem, Christoph; Humphries, Martin J.

    2013-01-01

    Summary Precise spatiotemporal coordination of integrin adhesion complex dynamics is essential for efficient cell migration. For cells adherent to fibronectin, differential engagement of α5β1 and αVβ3 integrins is used to elicit changes in adhesion complex stability, mechanosensation, matrix assembly, and migration, but the mechanisms responsible for receptor regulation have remained largely obscure. We identify phosphorylation of the membrane-intercalated proteoglycan syndecan-4 as an essential switch controlling integrin recycling. Src phosphorylates syndecan-4 and, by driving syntenin binding, leads to suppression of Arf6 activity and recycling of αVβ3 to the plasma membrane at the expense of α5β1. The resultant elevation in αVβ3 engagement promotes stabilization of focal adhesions. Conversely, abrogation of syndecan-4 phosphorylation drives surface expression of α5β1, destabilizes adhesion complexes, and disrupts cell migration. These data identify the dynamic spatiotemporal regulation of Src-mediated syndecan-4 phosphorylation as an essential switch controlling integrin trafficking and adhesion dynamics to promote efficient cell migration. PMID:23453597

  8. Systematic inference of functional phosphorylation events in yeast metabolism

    DEFF Research Database (Denmark)

    Chen, Yu; Wang, Yonghong; Nielsen, Jens

    2017-01-01

    Motivation: Protein phosphorylation is a post-translational modification that affects proteins by changing their structure and conformation in a rapid and reversible way, and it is an important mechanism for metabolic regulation in cells. Phosphoproteomics enables high-throughput identification o...

  9. Stress induces pain transition by potentiation of AMPA receptor phosphorylation.

    Science.gov (United States)

    Li, Changsheng; Yang, Ya; Liu, Sufang; Fang, Huaqiang; Zhang, Yong; Furmanski, Orion; Skinner, John; Xing, Ying; Johns, Roger A; Huganir, Richard L; Tao, Feng

    2014-10-08

    Chronic postsurgical pain is a serious issue in clinical practice. After surgery, patients experience ongoing pain or become sensitive to incident, normally nonpainful stimulation. The intensity and duration of postsurgical pain vary. However, it is unclear how the transition from acute to chronic pain occurs. Here we showed that social defeat stress enhanced plantar incision-induced AMPA receptor GluA1 phosphorylation at the Ser831 site in the spinal cord and greatly prolonged plantar incision-induced pain. Interestingly, targeted mutation of the GluA1 phosphorylation site Ser831 significantly inhibited stress-induced prolongation of incisional pain. In addition, stress hormones enhanced GluA1 phosphorylation and AMPA receptor-mediated electrical activity in the spinal cord. Subthreshold stimulation induced spinal long-term potentiation in GluA1 phosphomimetic mutant mice, but not in wild-type mice. Therefore, spinal AMPA receptor phosphorylation contributes to the mechanisms underlying stress-induced pain transition. Copyright © 2014 the authors 0270-6474/14/3413737-10$15.00/0.

  10. Changes in protein composition and protein phosphorylation during ...

    African Journals Online (AJOL)

    Changes in protein profiles and protein phosphorylation were studied in various stages of germinating somatic and zygotic embryos. Many proteins, which were expressed in cotyledonary stage somatic embryos, were also present in the zygotic embryos obtained from mature dry seed. The intensity of 22 kDa protein was ...

  11. A mathematical model of phosphorylation AKT in Acute Myeloid Leukemia

    Energy Technology Data Exchange (ETDEWEB)

    Adi, Y. A., E-mail: yudi.adi@math.uad.ac.id [Department of Mathematic Faculty of MIPA Universitas Ahmad Dahlan (Indonesia); Department of Mathematic Faculty of MIPA Universitas Gadjah Mada (Indonesia); Kusumo, F. A.; Aryati, L. [Department of Mathematic Faculty of MIPA Universitas Gadjah Mada (Indonesia); Hardianti, M. S. [Department of Internal Medicine, Faculty of Medicine, Universitas Gadjah Mada (Indonesia)

    2016-04-06

    In this paper we consider a mathematical model of PI3K/AKT signaling pathways in phosphorylation AKT. PI3K/AKT pathway is an important mediator of cytokine signaling implicated in regulation of hematopoiesis. Constitutive activation of PI3K/AKT signaling pathway has been observed in Acute Meyloid Leukemia (AML) it caused by the mutation of Fms-like Tyrosine Kinase 3 in internal tandem duplication (FLT3-ITD), the most common molecular abnormality associated with AML. Depending upon its phosphorylation status, protein interaction, substrate availability, and localization, AKT can phosphorylate or inhibite numerous substrates in its downstream pathways that promote protein synthesis, survival, proliferation, and metabolism. Firstly, we present a mass action ordinary differential equation model describing AKT double phosphorylation (AKTpp) in a system with 11 equations. Finally, under the asumtion enzyme catalyst constant and steady state equilibrium, we reduce the system in 4 equation included Michaelis Menten constant. Simulation result suggested that a high concentration of PI3K and/or a low concentration of phospatase increased AKTpp activation. This result also indicates that PI3K is a potential target theraphy in AML.

  12. Topographical distribution of phosphorylation sites of phosvitins by mass spectrometry.

    Science.gov (United States)

    Czernick, Drew; Liu, Jess; Serge, Dibart; Salih, Erdjan

    2013-05-27

    Phosvitin, derived from the vitellogenin II gene protein, is a highly phosphorylated protein found in egg yolk. A second hypothetical protein has been predicted based on the vitellogenin I gene, but has not been defined at the protein level. Mass spectrometric analysis was used to identify the phosphopeptide sequences and the precise sites of phosphorylation of two phosvitins, phosvitin 1 and phosvitin 2 derived from vitellogenins I and II, respectively. Samples of native phosvitin were subjected to tryptic digestion followed by mass spectrometric analysis: (i) native phosvitin peptides, (ii) after treatment with NaOH, and (iii) after chemical derivatization of P-Ser/P-Thr residues by dithiothreitol under base-catalyzed conditions. A combination of these approaches led to the identification of 68 and 35 phosphopeptides with 89 (81 P-Ser and 8 P-Thr residues) and 62 (57 P-Ser and 5 P-Thr residues) phosphorylation sites of phosvitin 1 and phosvitin 2, respectively. These data for the first time documented on a large scale the major states and sites of phosphorylation of phosvitins with a total of 151 phosphorylation sites. Importantly, the present work also provided the first direct de novo protein amino-acid sequence data for phosvitin 1 protein and evidence for the full expression of vitellogenin I gene. We have for the first time generated a large number of phosphopeptides (~100) and identified 151 phosphorylation sites of phosvitin 1 and phosvitin 2, respectively. Importantly, this study also led to the discovery of a novel phosvitin 1 and provided the first direct de novo protein amino-acid sequence data for the full expression of vitellogenin I gene. There is considerable interest in naturally occurring phosphopeptides/phosphoproteins and their application in biomedical fields and in the food industry because of their molecular characteristics and non-toxic nature, hence, our work opens new avenues to pursue such endeavors. In addition, the results provide

  13. Phosphorylation of acidic ribosomal proteins from rabbit reticulocytes by a ribosome-associated casein kinase

    DEFF Research Database (Denmark)

    Issinger, O G

    1977-01-01

    Two acidic proteins from 80-S ribosomes were isolated and purified to homogeneity. The purified acidic proteins could be phosphorylated by casein kinase using [gamma-32P]ATP and [gamma-32P]GTP as a phosphoryl donor. The proteins became phosphorylated in situ, too. Sodium dodecyl sulfate polyacryl......Two acidic proteins from 80-S ribosomes were isolated and purified to homogeneity. The purified acidic proteins could be phosphorylated by casein kinase using [gamma-32P]ATP and [gamma-32P]GTP as a phosphoryl donor. The proteins became phosphorylated in situ, too. Sodium dodecyl sulfate...

  14. Proteomic analysis of tyrosine phosphorylation during human liver transplantation

    Directory of Open Access Journals (Sweden)

    Boutros Tarek

    2007-01-01

    Full Text Available Abstract Background Ischemia-reperfusion (I/R causes a dramatic reprogramming of cell metabolism during liver transplantation and can be linked to an alteration of the phosphorylation level of several cellular proteins. Over the past two decades, it became clear that tyrosine phosphorylation plays a pivotal role in a variety of important signalling pathways and was linked to a wide spectrum of diseases. Functional profiling of the tyrosine phosphoproteome during liver transplantation is therefore of great biological significance and is likely to lead to the identification of novel targets for drug discovery and provide a basis for novel therapeutic strategies. Results Using liver biopsies collected during the early phases of organ procurement and transplantation, we aimed at characterizing the global patterns of tyrosine phosphorylation during hepatic I/R. A proteomic approach, based on the purification of tyrosine phosphorylated proteins followed by their identification using mass spectrometry, allowed us to identify Nck-1, a SH2/SH3 adaptor, as a potential regulator of I/R injury. Using immunoblot, cell fractionation and immunohistochemistry, we demonstrate that Nck-1 phosphorylation, expression and localization were affected in liver tissue upon I/R. In addition, mass spectrometry identification of Nck-1 binding partners during the course of the transplantation also suggested a dynamic interaction between Nck-1 and actin during I/R. Conclusion Taken together, our data suggest that Nck-1 may play a role in I/R-induced actin reorganization, which was previously reported to be detrimental for the hepatocytes of the transplanted graft. Nck-1 could therefore represent a target of choice for the design of new organ preservation strategies, which could consequently help to reduce post-reperfusion liver damages and improve transplantation outcomes.

  15. Discrimination between acid and alkali-labile phosphorylated residues on Immobilon: phosphorylation studies of nucleoside diphosphate kinase

    DEFF Research Database (Denmark)

    Biondi, R M; Walz, K; Issinger, O G

    1996-01-01

    of phosphoserine after strong acid hydrolysis of the histidine autophosphorylated enzyme is in fact a nonenzymatic transphosphorylation from phosphohistidine due to the harsh acid treatment. This methodology was also applied to in vivo phosphorylation studies of C. albicans NDP kinase. We believe...

  16. Haloperidol Regulates the State of Phosphorylation of Ribosomal Protein S6 via Activation of PKA and Phosphorylation of DARPP-32

    Science.gov (United States)

    Valjent, Emmanuel; Bertran-Gonzalez, Jesus; Bowling, Heather; Lopez, Sébastien; Santini, Emanuela; Matamales, Miriam; Bonito-Oliva, Alessandra; Hervé, Denis; Hoeffer, Charles; Klann, Eric; Girault, Jean-Antoine; Fisone, Gilberto

    2011-01-01

    Administration of typical antipsychotic drugs, such as haloperidol, promotes cAMP-dependent signaling in the medium spiny neurons (MSNs) of the striatum. In this study, we have examined the effect of haloperidol on the state of phosphorylation of the ribosomal protein S6 (rpS6), a component of the small 40S ribosomal subunit. We found that haloperidol increases the phosphorylation of rpS6 at the dual site Ser235/236, which is involved in the regulation of mRNA translation. This effect was exerted in the MSNs of the indirect pathway, which express specifically dopamine D2 receptors (D2Rs) and adenosine A2 receptors (A2ARs). The effect of haloperidol was decreased by blockade of A2ARs or by genetic attenuation of the Gαolf protein, which couples A2ARs to activation of adenylyl cyclase. Moreover, stimulation of cAMP-dependent protein kinase A (PKA) increased Ser235/236 phosphorylation in cultured striatal neurons. The ability of haloperidol to promote rpS6 phosphorylation was abolished in knock-in mice deficient for PKA activation of the protein phosphatase-1 inhibitor, dopamine- and cAMP-regulated phosphoprotein of 32 kDa. In contrast, pharmacological or genetic inactivation of p70 rpS6 kinase 1, or extracellular signal-regulated kinases did not affect haloperidol-induced rpS6 phosphorylation. These results identify PKA as a major rpS6 kinase in neuronal cells and suggest that regulation of protein synthesis through rpS6 may be a potential target of antipsychotic drugs. PMID:21814187

  17. HSP20 phosphorylation and airway smooth muscle relaxation

    Directory of Open Access Journals (Sweden)

    Mariam Ba

    2009-06-01

    Full Text Available Mariam Ba1, Cherie A Singer1, Manoj Tyagi2, Colleen Brophy3, Josh E Baker4, Christine Cremo4, Andrew Halayko5, William T Gerthoffer21Department of Pharmacology, University of Nevada School of Medicine, Reno, NV, USA; 2Department of Biochemistry and Molecular Biology, University of South Alabama, Mobile, AL, USA; 3Harrington Department of Biochemistry, Arizona State University, Tempe, AZ, USA; 4Department of Biochemistry and Molecular Biology, University of Nevada, Reno, NV, USA; 5Departments of Physiology and Internal Medicine, University of Manitoba, Winnipeg, MB, CanadaAbstract: HSP20 (HSPB6 is a small heat shock protein expressed in smooth muscles that is hypothesized to inhibit contraction when phosphorylated by cAMP-dependent protein kinase. To investigate this hypothesis in airway smooth muscle (ASM we showed that HSP20 was constitutively expressed as well as being inducible in cultured hASM cells by treatment with 1 µM isoproterenol or 10 µM salmeterol. In contrast, a mixture of proinflammatory mediators (interleukin-1β, tumor necrosis factor α, and interferon γ inhibited expression of HSP20 by about 50% in 48 hours. To determine whether phosphorylation of HSP20 is sufficient to induce relaxation, canine tracheal smooth muscle was treated with a cell permeant phosphopeptide that mimics the phosphorylation of HSP20. The HSP20 phosphopeptide antagonized carbacholinduced contraction by 60% with no change in myosin light chain phosphorylation. Recombinant full length HSP20 inhibited skeletal actin binding to smooth muscle myosin subfragment 1 (S1, and recombinant cell permeant TAT-HSP20 S16D mutant reduced F-actin filaments in cultured hASM cells. Carbachol stimulation of canine tracheal smooth muscle tissue caused redistribution of HSP20 from large macromolecular complexes (200–500 kDa to smaller complexes (<60 kDa. The results are consistent with HSP20 expression and macromolecular structure being dynamically regulated in airway

  18. Molecular mechanism of APC/C activation by mitotic phosphorylation.

    Science.gov (United States)

    Zhang, Suyang; Chang, Leifu; Alfieri, Claudio; Zhang, Ziguo; Yang, Jing; Maslen, Sarah; Skehel, Mark; Barford, David

    2016-05-12

    In eukaryotes, the anaphase-promoting complex (APC/C, also known as the cyclosome) regulates the ubiquitin-dependent proteolysis of specific cell-cycle proteins to coordinate chromosome segregation in mitosis and entry into the G1 phase. The catalytic activity of the APC/C and its ability to specify the destruction of particular proteins at different phases of the cell cycle are controlled by its interaction with two structurally related coactivator subunits, Cdc20 and Cdh1. Coactivators recognize substrate degrons, and enhance the affinity of the APC/C for its cognate E2 (refs 4-6). During mitosis, cyclin-dependent kinase (Cdk) and polo-like kinase (Plk) control Cdc20- and Cdh1-mediated activation of the APC/C. Hyperphosphorylation of APC/C subunits, notably Apc1 and Apc3, is required for Cdc20 to activate the APC/C, whereas phosphorylation of Cdh1 prevents its association with the APC/C. Since both coactivators associate with the APC/C through their common C-box and Ile-Arg tail motifs, the mechanism underlying this differential regulation is unclear, as is the role of specific APC/C phosphorylation sites. Here, using cryo-electron microscopy and biochemical analysis, we define the molecular basis of how phosphorylation of human APC/C allows for its control by Cdc20. An auto-inhibitory segment of Apc1 acts as a molecular switch that in apo unphosphorylated APC/C interacts with the C-box binding site and obstructs engagement of Cdc20. Phosphorylation of the auto-inhibitory segment displaces it from the C-box-binding site. Efficient phosphorylation of the auto-inhibitory segment, and thus relief of auto-inhibition, requires the recruitment of Cdk-cyclin in complex with a Cdk regulatory subunit (Cks) to a hyperphosphorylated loop of Apc3. We also find that the small-molecule inhibitor, tosyl-l-arginine methyl ester, preferentially suppresses APC/C(Cdc20) rather than APC/C(Cdh1), and interacts with the binding sites of both the C-box and Ile-Arg tail motifs. Our

  19. Large-scale analysis of phosphorylation site occupancy in eukaryotic proteins

    DEFF Research Database (Denmark)

    Rao, R Shyama Prasad; Møller, Ian Max

    2012-01-01

    in proteins is currently lacking. We have therefore analyzed the occurrence and occupancy of phosphorylated sites (~ 100,281) in a large set of eukaryotic proteins (~ 22,995). Phosphorylation probability was found to be much higher in both the  termini of protein sequences and this is much pronounced...... maximum randomness. An analysis of phosphorylation motifs indicated that just 40 motifs and a much lower number of associated kinases might account for nearly 50% of the known phosphorylations in eukaryotic proteins. Our results provide a broad picture of the phosphorylation sites in eukaryotic proteins.......Many recent high throughput technologies have enabled large-scale discoveries of new phosphorylation sites and phosphoproteins. Although they have provided a number of insights into protein phosphorylation and the related processes, an inclusive analysis on the nature of phosphorylated sites...

  20. Contraction regulates site-specific phosphorylation of TBC1D1 in skeletal muscle.

    Science.gov (United States)

    Vichaiwong, Kanokwan; Purohit, Suneet; An, Ding; Toyoda, Taro; Jessen, Niels; Hirshman, Michael F; Goodyear, Laurie J

    2010-10-15

    TBC1D1 (tre-2/USP6, BUB2, cdc16 domain family member 1) is a Rab-GAP (GTPase-activating protein) that is highly expressed in skeletal muscle, but little is known about TBC1D1 regulation and function. We studied TBC1D1 phosphorylation on three predicted AMPK (AMP-activated protein kinase) phosphorylation sites (Ser231, Ser660 and Ser700) and one predicted Akt phosphorylation site (Thr590) in control mice, AMPKα2 inactive transgenic mice (AMPKα2i TG) and Akt2-knockout mice (Akt2 KO). Muscle contraction significantly increased TBC1D1 phosphorylation on Ser231 and Ser660, tended to increase Ser700 phosphorylation, but had no effect on Thr590. AICAR (5-aminoimidazole-4-carboxyamide ribonucleoside) also increased phosphorylation on Ser231, Ser660 and Ser700, but not Thr590, whereas insulin only increased Thr590 phosphorylation. Basal and contraction-stimulated TBC1D1 Ser231, Ser660 and Ser700 phosphorylation were greatly reduced in AMPKα2i TG mice, although contraction still elicited a small increase in phosphorylation. Akt2 KO mice had blunted insulin-stimulated TBC1D1 Thr590 phosphorylation. Contraction-stimulated TBC1D1 Ser231 and Ser660 phosphorylation were normal in high-fat-fed mice. Glucose uptake in vivo was significantly decreased in tibialis anterior muscles overexpressing TBC1D1 mutated on four predicted AMPK phosphorylation sites. In conclusion, contraction causes site-specific phosphorylation of TBC1D1 in skeletal muscle, and TBC1D1 phosphorylation on AMPK sites regulates contraction-stimulated glucose uptake. AMPK and Akt regulate TBC1D1 phosphorylation, but there must be additional upstream kinases that mediate TBC1D1 phosphorylation in skeletal muscle.

  1. Caffeine inhibits cell proliferation by G0/G1 phase arrest in JB6 cells.

    Science.gov (United States)

    Hashimoto, Takashi; He, Zhiwei; Ma, Wei-Ya; Schmid, Patricia C; Bode, Ann M; Yang, Chung S; Dong, Zigang

    2004-05-01

    Caffeine is a major biologically active constituent in coffee and tea. Because caffeine has been reported to inhibit carcinogenesis in UVB-exposed mice, the cancer-preventing effect of caffeine has attracted considerable attention. In the present study, the effect of caffeine in quiescent (G0 phase) cells was investigated. Pretreatment with caffeine suppressed cell proliferation in a dose-dependent manner 36 h after addition of fetal bovine serum as a cell growth stimulator. Analysis by flow cytometry showed that caffeine suppressed cell cycle progression at the G0/G1 phase, i.e., 18 h after addition of fetal bovine serum, the percentages of cells in G0/G1 phase in 1 mM caffeine-treated cells and in caffeine-untreated cells were 61.7 and 29.0, respectively. The percentage of cells in G0/G1 phase at 0 h was 75.5. Caffeine inhibited phosphorylation of retinoblastoma protein at Ser780 and Ser807/Ser811, the sites where retinoblastoma protein has been reported to be phosphorylated by cyclin-dependent kinase 4 (cdk4). Furthermore, caffeine inhibited the activation of the cyclin D1-cdk4 complex in a dose-dependent manner. However this compound did not directly inhibit the activity of this complex. In addition, caffeine did not affect p16INK4 or p27Kip1 protein levels, but inhibited the phosphorylation of protein kinase B (Akt) and glycogen synthase kinase 3beta. Our results showed that caffeine suppressed the progression of quiescent cells into the cell cycle. The inhibitory mechanism may be due to the inhibition of cell growth signal-induced activation of cdk4, which may be involved in the inhibition of carcinogenesis in vivo.

  2. Cucurbitacin IIb exhibits anti-inflammatory activity through modulating multiple cellular behaviors of mouse lymphocytes.

    Directory of Open Access Journals (Sweden)

    Yao Wang

    Full Text Available Cucurbitacin IIb (CuIIb is one of the major active compounds in Hemsleyadine tablets which have been used for clinical treatment of bacillary dysentery, enteritis and acute tonsilitis. However, its action mechanism has not been completely understood. This study aimed to explore the anti-inflammatory activity of CuIIb and its underlying mechanism in mitogen-activated lymphocytes isolated from mouse mesenteric lymph nodes. The results showed that CuIIb inhibited the proliferation of concanavalin A (Con A-activated lymphocytes in a time- and dose-dependent manner. CuIIb treatment arrested their cell cycle in S and G2/M phases probably due to the disruption of the actin cytoskeleton and the modulation of p27(Kip1 and cyclin levels. Moreover, the surface expression of activation markers CD69 and CD25 on Con A-activated CD3(+ T lymphocytes was suppressed by CuIIb treatment. Both Con A- and phorbol ester plus ionomycin-induced expression of TNF-α, IFN-γ and IL-6 proteins was attenuated upon exposure to CuIIb. Mechanistically, CuIIb treatment suppressed the phosphorylation of JNK and Erk1/2 but not p38 in Con A-activated lymphocytes. Although CuIIb unexpectedly enhanced the phosphorylation of IκB and NF-κB (p65, it blocked the nuclear translocation of NF-κB (p65. In support of this, CuIIb significantly decreased the mRNA levels of IκBα and TNF-α, two target genes of NF-κB, in Con A-activated lymphocytes. In addition, CuIIb downregulated Con A-induced STAT3 phosphorylation and increased cell apoptosis. Collectively, these results suggest that CuIIb exhibits its anti-inflammatory activity through modulating multiple cellular behaviors and signaling pathways, leading to the suppression of the adaptive immune response.

  3. Identification of ATM Protein Kinase Phosphorylation Sites by Mass Spectrometry.

    Science.gov (United States)

    Graham, Mark E; Lavin, Martin F; Kozlov, Sergei V

    2017-01-01

    ATM (ataxia-telangiectasia mutated) protein kinase is a key regulator of cellular responses to DNA damage and oxidative stress. DNA damage triggers complex cascade of signaling events leading to numerous posttranslational modification on multitude of proteins. Understanding the regulation of ATM kinase is therefore critical not only for understanding the human genetic disorder ataxia-telangiectasia and potential treatment strategies, but essential for deciphering physiological responses of cells to stress. These responses play an important role in carcinogenesis, neurodegeneration, and aging. We focus here on the identification of DNA damage inducible ATM phosphorylation sites to understand the importance of autophosphorylation in the mechanism of ATM kinase activation. We demonstrate the utility of using immunoprecipitated ATM in quantitative LC-MS/MS workflow with stable isotope dimethyl labeling of ATM peptides for identification of phosphorylation sites.

  4. Tyrosine 370 phosphorylation of ATM positively regulates DNA damage response

    Science.gov (United States)

    Lee, Hong-Jen; Lan, Li; Peng, Guang; Chang, Wei-Chao; Hsu, Ming-Chuan; Wang, Ying-Nai; Cheng, Chien-Chia; Wei, Leizhen; Nakajima, Satoshi; Chang, Shih-Shin; Liao, Hsin-Wei; Chen, Chung-Hsuan; Lavin, Martin; Ang, K Kian; Lin, Shiaw-Yih; Hung, Mien-Chie

    2015-01-01

    Ataxia telangiectasia mutated (ATM) mediates DNA damage response by controling irradiation-induced foci formation, cell cycle checkpoint, and apoptosis. However, how upstream signaling regulates ATM is not completely understood. Here, we show that upon irradiation stimulation, ATM associates with and is phosphorylated by epidermal growth factor receptor (EGFR) at Tyr370 (Y370) at the site of DNA double-strand breaks. Depletion of endogenous EGFR impairs ATM-mediated foci formation, homologous recombination, and DNA repair. Moreover, pretreatment with an EGFR kinase inhibitor, gefitinib, blocks EGFR and ATM association, hinders CHK2 activation and subsequent foci formation, and increases radiosensitivity. Thus, we reveal a critical mechanism by which EGFR directly regulates ATM activation in DNA damage response, and our results suggest that the status of ATM Y370 phosphorylation has the potential to serve as a biomarker to stratify patients for either radiotherapy alone or in combination with EGFR inhibition. PMID:25601159

  5. Reactions of α-phosphorylated carbonyl compounds with amino alcohols

    International Nuclear Information System (INIS)

    Moskva, V.V.; Sitdikova, T.Sh.; Zykova, T.V.; Alparova, M.V.; Shagvaleev, F.Sh.

    1986-01-01

    2-Aminoethanol reacts with carbonyl compounds with the formation, depending on the structure of the latter, either of a mixture of azomethines and oxazolidines, or of only azomethines. In the development of investigations on the reactivity of α-phosphorylated carbonyl compounds the authors studied the reactions of a number of amino alcohols with phosphorylated acetaldehyde and acetone. In both cases they observed the formation of compounds of enamine structure, oxazolidines and azomethines were not observed. By means of NMR spectroscopy they established clearly the formation of the E-isomeric products. The 1 H, 31 P, and 13 C NMR spectra were recorded on a WP-80 spectrometer. Chemical shifts of protons and 13 C nuclei are given relative to TMS, and phosphorus nuclei relative to orthophosphoric acid

  6. GABAB receptor phosphorylation regulates KCTD12-induced K+ current desensitization

    DEFF Research Database (Denmark)

    Adelfinger, L; Turecek, R; Ivankova, K

    2014-01-01

    released from the G-protein. Receptor-activated K+ currents desensitize in the sustained presence of agonist to avoid excessive effects on neuronal activity. Desensitization of K+ currents integrates distinct mechanistic underpinnings. GABAB receptor activity reduces protein kinase-A activity, which...... reduces phosphorylation of serine-892 in GABAB2 and promotes receptor degradation. This form of desensitization operates on the time scale of several minutes to hours. A faster form of desensitization is induced by the auxiliary subunit KCTD12, which interferes with channel activation by binding to the G......-protein βγ subunits. Here we show that the two mechanisms of desensitization influence each other. Serine-892 phosphorylation in heterologous cells rearranges KCTD12 at the receptor and slows KCTD12-induced desensitization. Likewise, protein kinase-A activation in hippocampal neurons slows fast...

  7. Modulation of repulsive forces between neurofilaments by sidearm phosphorylation

    International Nuclear Information System (INIS)

    Kumar, Sanjay; Hoh, Jan H.

    2004-01-01

    Recent studies have advanced the notion that the axonal organization of neurofilaments (NFs) is based on mutual steric repulsion between the unstructured 'sidearm' domains of adjacent NFs. Here, we present experimental evidence that these repulsive forces are modulated by the degree of sidearm phosphorylation. When NFs are sedimented into a gelatinous pellet, pellet volume falls with increasing ionic strength and enzymatic dephosphorylation; sedimentation of phosphorylated NFs in the presence of divalent cations also dramatically reduces pellet volume. Further, atomic force microscopy imaging of isolated mammalian NFs reveals robust exclusion of colloidal particles from the NF backbone that is reduced at high ionic strength and attenuated when the filaments are enzymatically dephosphorylated. Phosphate-phosphate repulsion on the NF sidearm appears to modulate NF excluded volume in a graded fashion, thereby controlling axonal NF organization through interfilament forces

  8. Investigating quantitation of phosphorylation using MALDI-TOF mass spectrometry.

    Science.gov (United States)

    Parker, Laurie; Engel-Hall, Aaron; Drew, Kevin; Steinhardt, George; Helseth, Donald L; Jabon, David; McMurry, Timothy; Angulo, David S; Kron, Stephen J

    2008-04-01

    Despite advances in methods and instrumentation for analysis of phosphopeptides using mass spectrometry, it is still difficult to quantify the extent of phosphorylation of a substrate because of physiochemical differences between unphosphorylated and phosphorylated peptides. Here we report experiments to investigate those differences using MALDI-TOF mass spectrometry for a set of synthetic peptides by creating calibration curves of known input ratios of peptides/phosphopeptides and analyzing their resulting signal intensity ratios. These calibration curves reveal subtleties in sequence-dependent differences for relative desorption/ionization efficiencies that cannot be seen from single-point calibrations. We found that the behaviors were reproducible with a variability of 5-10% for observed phosphopeptide signal. Although these data allow us to begin addressing the issues related to modeling these properties and predicting relative signal strengths for other peptide sequences, it is clear that this behavior is highly complex and needs to be further explored. John Wiley & Sons, Ltd

  9. ERK phosphorylation regulates sleep and plasticity in Drosophila.

    Directory of Open Access Journals (Sweden)

    William M Vanderheyden

    Full Text Available Given the relationship between sleep and plasticity, we examined the role of Extracellular signal-regulated kinase (ERK in regulating baseline sleep, and modulating the response to waking experience. Both sleep deprivation and social enrichment increase ERK phosphorylation in wild-type flies. The effects of both sleep deprivation and social enrichment on structural plasticity in the LNvs can be recapitulated by expressing an active version of ERK (UAS-ERK(SEM pan-neuronally in the adult fly using GeneSwitch (Gsw Gsw-elav-GAL4. Conversely, disrupting ERK reduces sleep and prevents both the behavioral and structural plasticity normally induced by social enrichment. Finally, using transgenic flies carrying a cAMP response Element (CRE-luciferase reporter we show that activating ERK enhances CRE-Luc activity while disrupting ERK reduces it. These data suggest that ERK phosphorylation is an important mediator in transducing waking experience into sleep.

  10. Novel tyrosine phosphorylation sites in rat skeletal muscle revealed by phosphopeptide enrichment and HPLC-ESI-MS/MS

    DEFF Research Database (Denmark)

    Zhang, Xiangmin; Højlund, Kurt; Luo, Moulun

    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 (...

  11. Phosphorylation and function of DGAT1 in skeletal muscle cells

    OpenAIRE

    Yu, Jinhai; Li, Yiran; Zou, Fei; Xu, Shimeng; Liu, Pingsheng

    2015-01-01

    Aberrant intramuscular triacylglycerol (TAG) storage in human skeletal muscle is closely related to insulin insensitivity. Excessive lipid storage can induce insulin resistance of skeletal muscle, and under severe conditions, lead to type 2 diabetes. The balance of interconversion between diacylglycerol and TAG greatly influences lipid storage and utilization. Diacylglycerol O-acyltransferase 1 (DGAT1) plays a key role in this process, but its activation and phosphorylation requires further d...

  12. Modulation of P1798 lymphosarcoma proliferation by protein phosphorylation

    International Nuclear Information System (INIS)

    Michnoff, C.A.H.

    1983-01-01

    The role of protein kinases in modulating cell proliferation was examined. Studies characterized the regulation of cell proliferation by adenosine 3':5'-monophosphate-dependent protein kinase (cA-Pk). Calcium/calmodulin-dependent myosin light chain kinase (MLCK) was isolated and examined as a potential substrate regulated by cA-PK in the rapidly proliferating P1798 lymphosarcoma. Modulation of cell proliferation by cA-PK was characterized by quantitating cell division by [methyl- 3 H] thymidine ([ 3 H]-dT) incorporation into DNA, cAMP accumulations, and activation of cA-PK using P1798 lymphosarcoma cells. Epinephrine and prostaglandin E 1 (PGE 1 ) were demonstrated to suppress [ 3 H]-dT incorporation into DNA, to stimulate cAMP accumulation, and to activate cA-PK with dose-dependency. Calcium/calmodulin-dependent MLCK was partially purified from P1798 lymphosarcoma. P1798 MLCK phosphorylated myosin regulatory light chains (P-LC) from thymus, cardiac and skeletal muscles. One mol [ 32 Pi] was transferred into one mol cardiac or skeletal P-LC by P1798 MLCK. Apparent Km values of 65 μM and 51 μM were determined for ATP and cardiac P-LC, respectively. The apparent molecular weight of P1798 MLCK was 135,000. P1798 MLCK was phosphorylated by cA-PK. Phosphorylated MLCK showed a 41% decrease in calcium-dependent activity. Two additional protein kinases from P1798 lymphosarcoma phosphorylated cardiac and skeletal light chains

  13. Phosphorylation Modulates Ameloblastin Self-assembly and Ca2+ Binding

    Czech Academy of Sciences Publication Activity Database

    Stakkestad, O.; Lyngstadaas, S. P.; Thiede, B.; Vondrášek, Jiří; Skalhegg, B. S.; Reseland, J. E.

    2017-01-01

    Roč. 8, Jul 27 (2017), č. článku 531. ISSN 1664-042X Institutional support: RVO:61388963 Keywords : ameloblastin * phosphorylation * self-assembly * Ca2+-binding * enamel * intrinsically disordered proteins Subject RIV: CE - Biochemistry OBOR OECD: Biochemistry and molecular biology Impact factor: 4.134, year: 2016 http://journal.frontiersin.org/article/10.3389/fphys.2017.00531/full

  14. Training-induced adaptation of oxidative phosphorylation in skeletal muscles.

    OpenAIRE

    Korzeniewski, Bernard; Zoladz, Jerzy A

    2003-01-01

    Muscle training/conditioning improves the adaptation of oxidative phosphorylation in skeletal muscles to physical exercise. However, the mechanisms underlying this adaptation are still not understood fully. By quantitative analysis of the existing experimental results, we show that training-induced acceleration of oxygen-uptake kinetics at the onset of exercise and improvement of ATP/ADP stability due to physical training are mainly caused by an increase in the amount of mitochondrial protein...

  15. Urokinase receptor expression involves tyrosine phosphorylation of phosphoglycerate kinase.

    Science.gov (United States)

    Shetty, Praveenkumar; Velusamy, Thirunavukkarasu; Bhandary, Yashodhar P; Liu, Ming C; Shetty, Sreerama

    2010-02-01

    The interaction of urokinase-type plasminogen activator (uPA) with its receptor, uPAR, plays a central role in several pathophysiological processes, including cancer. uPA induces its own cell surface receptor expression through stabilization of uPAR mRNA. The mechanism involves binding of a 51 nt uPAR mRNA coding sequence with phosphoglycerate kinase (PGK) to down regulate cell surface uPAR expression. Tyrosine phosphorylation of PGK mediated by uPA treatment enhances uPAR mRNA stabilization. In contrast, inhibition of tyrosine phosphorylation augments PGK binding to uPAR mRNA and attenuates uPA-induced uPAR expression. Mapping the specific peptide region of PGK indicated that its first quarter (amino acids 1-100) interacts with uPAR mRNA. To determine if uPAR expression by uPA is regulated through activation of tyrosine residues of PGK, we mutated the specific tyrosine residue and tested mutant PGK for its ability to interfere with uPAR expression. Inhibition of tyrosine phosphorylation by mutating Y76 residue abolished uPAR expression induced by uPA treatment. These findings collectively demonstrate that Y76 residue present in the first quarter of the PGK molecule is involved in lung epithelial cell surface uPAR expression. This region can effectively mimic the function of a whole PGK molecule in inhibiting tumor cell growth.

  16. The Regulation of NF-κB Subunits by Phosphorylation

    Directory of Open Access Journals (Sweden)

    Frank Christian

    2016-03-01

    Full Text Available The NF-κB transcription factor is the master regulator of the inflammatory response and is essential for the homeostasis of the immune system. NF-κB regulates the transcription of genes that control inflammation, immune cell development, cell cycle, proliferation, and cell death. The fundamental role that NF-κB plays in key physiological processes makes it an important factor in determining health and disease. The importance of NF-κB in tissue homeostasis and immunity has frustrated therapeutic approaches aimed at inhibiting NF-κB activation. However, significant research efforts have revealed the crucial contribution of NF-κB phosphorylation to controlling NF-κB directed transactivation. Importantly, NF-κB phosphorylation controls transcription in a gene-specific manner, offering new opportunities to selectively target NF-κB for therapeutic benefit. This review will focus on the phosphorylation of the NF-κB subunits and the impact on NF-κB function.

  17. Coilin phosphorylation mediates interaction with SMN and SmB′

    Science.gov (United States)

    Toyota, Cory G.; Davis, Misty D.; Cosman, Angela M.; Hebert, Michael D.

    2010-01-01

    Cajal bodies (CBs) are subnuclear domains that participate in spliceosomal small nuclear ribonucleoprotein (snRNP) biogenesis and play a part in the assembly of the spliceosomal complex. The CB marker protein, coilin, interacts with survival of motor neuron (SMN) and Sm proteins. Several coilin phosphoresidues have been identified by mass spectrometric analysis. Phosphorylation of coilin affects its self-interaction and localization in the nucleus. We hypothesize that coilin phosphorylation also impacts its binding to SMN and Sm proteins. In vitro binding studies with a C-terminal fragment of coilin and corresponding phosphomimics show that SMN binds preferentially to dephosphorylated analogs and that SmB′ binds preferentially to phosphomimetic constructs. Bacterially expressed full-length coilin binds more SMN and SmB′ than does the C-terminal fragment. Co-immunoprecipitation and phosphatase experiments show that SMN also binds dephosphorylated coilin in vivo. These data show that phosphorylation of coilin influences interaction with its target proteins and, thus, may be significant in managing the flow of snRNPs through the CB. PMID:19997741

  18. Coilin phosphorylation mediates interaction with SMN and SmB'.

    Science.gov (United States)

    Toyota, Cory G; Davis, Misty D; Cosman, Angela M; Hebert, Michael D

    2010-04-01

    Cajal bodies (CBs) are subnuclear domains that participate in spliceosomal small nuclear ribonucleoprotein (snRNP) biogenesis and play a part in the assembly of the spliceosomal complex. The CB marker protein, coilin, interacts with survival of motor neuron (SMN) and Sm proteins. Several coilin phosphoresidues have been identified by mass spectrometric analysis. Phosphorylation of coilin affects its self-interaction and localization in the nucleus. We hypothesize that coilin phosphorylation also impacts its binding to SMN and Sm proteins. In vitro binding studies with a C-terminal fragment of coilin and corresponding phosphomimics show that SMN binds preferentially to dephosphorylated analogs and that SmB' binds preferentially to phosphomimetic constructs. Bacterially expressed full-length coilin binds more SMN and SmB' than does the C-terminal fragment. Co-immunoprecipitation and phosphatase experiments show that SMN also binds dephosphorylated coilin in vivo. These data show that phosphorylation of coilin influences interaction with its target proteins and, thus, may be significant in managing the flow of snRNPs through the CB.

  19. Phosphorylation and antiaging activity of polysaccharide from Trichosanthes peel

    Directory of Open Access Journals (Sweden)

    Min Zhang

    2017-10-01

    Full Text Available Polysaccharides from Trichosanthes peel (TPP were obtained by ultrasound-assisted extraction. TPP-1 was separated from the TPP by Sephadex G-100 column chromatography. Phosphorylation of TPP-1 was carried out and phosphorylated TPP-1 was named as PTTP-1. The results of infrared spectra, 13C nuclear magnetic resonance spectra and 31P nuclear magnetic resonance spectra showed that the main structure of PTPP-1 was similar to that of TPP-1 and -H2PO3 groups which were conjugated to C-6 of →4-α-D-Manp-(1→, C-4 of →6-α-D-Galp-(1→, C-2 and C-3 of →1-α-L-Araf, C-2 of →1-α-L-Araf-(3→, and C-6 and C-3 of →1-α-D-Glcp. In vivo antiaging activity results proved that TTP-1 and PTTP-1 could both significantly improve the body weight, spleen index, and thymus index of the D-galactose-induced aging mice, increase the levels of superoxide dismutase, catalase, glutathione peroxidase, and reduce malondialdehyde contents in the liver, brain, and serum of aging mice. These results indicated that both TPP-1 and PTTP-1 presented significant antiaging activity. Moreover, PTTP-1 showed stronger antiaging effects in aging mice, indicating that phosphorylation improved antiaging effect.

  20. Regulation of the autophagy protein LC3 by phosphorylation

    Science.gov (United States)

    Cherra, Salvatore J.; Kulich, Scott M.; Uechi, Guy; Balasubramani, Manimalha; Mountzouris, John; Day, Billy W.

    2010-01-01

    Macroautophagy is a major catabolic pathway that impacts cell survival, differentiation, tumorigenesis, and neurodegeneration. Although bulk degradation sustains carbon sources during starvation, autophagy contributes to shrinkage of differentiated neuronal processes. Identification of autophagy-related genes has spurred rapid advances in understanding the recruitment of microtubule-associated protein 1 light chain 3 (LC3) in autophagy induction, although braking mechanisms remain less understood. Using mass spectrometry, we identified a direct protein kinase A (PKA) phosphorylation site on LC3 that regulates its participation in autophagy. Both metabolic (rapamycin) and pathological (MPP+) inducers of autophagy caused dephosphorylation of endogenous LC3. The pseudophosphorylated LC3 mutant showed reduced recruitment to autophagosomes, whereas the nonphosphorylatable mutant exhibited enhanced puncta formation. Finally, autophagy-dependent neurite shortening induced by expression of a Parkinson disease–associated G2019S mutation in leucine-rich repeat kinase 2 was inhibited by dibutyryl–cyclic adenosine monophosphate, cytoplasmic expression of the PKA catalytic subunit, or the LC3 phosphorylation mimic. These data demonstrate a role for phosphorylation in regulating LC3 activity. PMID:20713600

  1. ATM-mediated Snail Serine 100 phosphorylation regulates cellular radiosensitivity

    International Nuclear Information System (INIS)

    Boohaker, Rebecca J.; Cui, Xiaoli; Stackhouse, Murray; Xu, Bo

    2013-01-01

    Purpose: Activation of the DNA damage responsive protein kinase ATM is a critical step for cellular survival in response to ionizing irradiation (IR). Direct targets of ATM regulating radiosensitivity remain to be fully investigated. We have recently reported that ATM phosphorylates the transcriptional repressor Snail on Serine 100. We aimed to further study the functional significance of ATM-mediated Snail phosphorylation in response to IR. Material and methods: We transfected vector-only, wild-type, the Serine 100 to alanine (S100A) or to glutamic acid (S100E) substitution of Snail into various cell lines. We assessed colony formation, γ-H2AX focus formation and the invasion index in the cells treated with or without IR. Results: We found that over-expression of the S100A mutant Snail in HeLa cells significantly increased radiosensitivity. Meanwhile the expression of S100E, a phospho-mimicking mutation, resulted in enhanced radio-resistance. Interestingly, S100E could rescue the radiosensitive phenotype in ATM-deficient cells. We also found that expression of S100E increased γ-H2AX focus formation and compromised inhibition of invasion in response to IR independent of cell survival. Conclusion: ATM-mediated Snail Serine 100 phosphorylation in response to IR plays an important part in the regulation of radiosensitivity

  2. Synthesis of rigid polyurethane foams from phosphorylated biopolyols.

    Science.gov (United States)

    de Haro, Juan Carlos; López-Pedrajas, Daniel; Pérez, Ángel; Rodríguez, Juan Francisco; Carmona, Manuel

    2017-08-18

    Renewable resources are playing a key role on the synthesis of biodegradable polyols. Moreover, the incorporation of covalently linked additives is increasing in importance in the polyurethane (PU) market. In this work, previously epoxidized grape seed oil and methyl oleate were transformed into phosphorylated biopolyols through an acid-catalyzed ring-opening hydrolysis in the presence of H 3 PO 4 . The formation of phosphate polyesters was confirmed by FT-IR and 31 P-NMR. However, the synthesis of a high-quality PU rigid foam was not possible using exclusively these polyols attending to their low hydroxyl value. In that way, different rigid PU foams were prepared from the phosphorylated biopolyols and the commercial polyol Alcupol R4520. It was observed that phosphorylated biopolyols can be incorporated up to a 57 wt.% in the PU synthesis without significant structural changes with respect to the commercial foam. Finally, thermogravimetric and EDAX analyses revealed an improvement of thermal stability by the formation of a protective phosphorocarbonaceous char layer.

  3. Determination of sites of U50,488H-promoted phosphorylation of the mouse κ opioid receptor (KOPR): disconnect between KOPR phosphorylation and internalization.

    Science.gov (United States)

    Chen, Chongguang; Chiu, Yi-Ting; Wu, Wenman; Huang, Peng; Mann, Anika; Schulz, Stefan; Liu-Chen, Lee-Yuan

    2016-02-15

    Phosphorylation sites of KOPR (κ opioid receptor) following treatment with the selective agonist U50,488H {(-)(trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidiny)cyclo-hexyl]benzeneacetamide} were identified after affinity purification, SDS/PAGE, in-gel digestion with Glu-C and HPLC-MS/MS. Single- and double-phosphorylated peptides were identified containing phosphorylated Ser(356), Thr(357), Thr(363) and Ser(369) in the C-terminal domain. Antibodies were generated against three phosphopeptides containing pSer(356)/pThr(357), pThr(363) and pSer(369) respectively, and affinity-purified antibodies were found to be highly specific for phospho-KOPR. U50,488H markedly enhanced staining of the KOPR by pThr(363)-, pSer(369)- and pSer(356)/pThr(357)-specific antibodies in immunoblotting, which was blocked by the selective KOPR antagonist norbinaltorphimine. Ser(369) phosphorylation affected Thr(363) phosphorylation and vice versa, and Thr(363) or Ser(369) phosphorylation was important for Ser(356)/Thr(357) phosphorylation, revealing a phosphorylation hierarchy. U50,488H, but not etorphine, promoted robust KOPR internalization, although both were full agonists. U50,488H induced higher degrees of phosphorylation than etorphine at Ser(356)/Thr(357), Thr(363) and Ser(369) as determined by immunoblotting. Using SILAC (stable isotope labelling by amino acids in cell culture) and HPLC-MS/MS, we found that, compared with control (C), U50,488H (U) and etorphine (E) KOPR promoted single phosphorylation primarily at Thr(363) and Ser(369) with U/E ratios of 2.5 and 2 respectively. Both induced double phosphorylation at Thr(363)+Ser(369) and Thr(357)+Ser(369) with U/E ratios of 3.3 and 3.4 respectively. Only U50,488H induced triple phosphorylation at Ser(356)+Thr(357)+Ser(369). An unphosphorylated KOPR-(354-372) fragment containing all of the phosphorylation sites was detected with a C/E/U ratio of 1/0.7/0.4, indicating that ∼60% and ∼30% of the mouse KOPR are phosphorylated

  4. Phototropism and Protein Phosphorylation in Higher Plants: Unilateral Blue Light Irradiation Generates a Directional Gradient of Protein Phosphorylation Across the Oat Coleoptile

    International Nuclear Information System (INIS)

    Salomon, M.; Zacherl, M.; Rüdiger, W.

    1997-01-01

    Blue light induces the phosphorylation of a 116 kDa oat protein found in plasma membrane preparations from coleoptile tips. We developed a very sensitive in vitro method that allowed us to determine the tissue distribution of protein phosphorylation after applying unilateral and bilateral blue light pulses in vivo. We found that following unilateral in vivo irradiation the degree in phosphorylation of the 116 kDa protein is significantly higher at the irradiated than at the shaded side of the coleoptile tip. This asymmetry can be considered as previously missing criterion that protein phosphorylation represents an early event within the transduction chain for phototropism. (author)

  5. Phosphorylation of αB-crystallin: Role in stress, aging and patho-physiological conditions.

    Science.gov (United States)

    Bakthisaran, Raman; Akula, Kranthi Kiran; Tangirala, Ramakrishna; Rao, Ch Mohan

    2016-01-01

    αB-crystallin, once thought to be a lenticular protein, is ubiquitous and has critical roles in several cellular processes that are modulated by phosphorylation. Serine residues 19, 45 and 59 of αB-crystallin undergo phosphorylation. Phosphorylation of S45 is mediated by p44/42 MAP kinase, whereas S59 phosphorylation is mediated by MAPKAP kinase-2. Pathway involved in S19 phosphorylation is not known. The review highlights the role of phosphorylation in (i) oligomeric structure, stability and chaperone activity, (ii) cellular processes such as apoptosis, myogenic differentiation, cell cycle regulation and angiogenesis, and (iii) aging, stress, cardiomyopathy-causing αB-crystallin mutants, and in other diseases. Depending on the context and extent of phosphorylation, αB-crystallin seems to confer beneficial or deleterious effects. Phosphorylation alters structure, stability, size distribution and dynamics of the oligomeric assembly, thus modulating chaperone activity and various cellular processes. Phosphorylated αB-crystallin has a tendency to partition to the cytoskeleton and hence to the insoluble fraction. Low levels of phosphorylation appear to be protective, while hyperphosphorylation has negative implications. Mutations in αB-crystallin, such as R120G, Q151X and 464delCT, associated with inherited myofibrillar myopathy lead to hyperphosphorylation and intracellular inclusions. An ongoing study in our laboratory with phosphorylation-mimicking mutants indicates that phosphorylation of R120GαB-crystallin increases its propensity to aggregate. Phosphorylation of αB-crystallin has dual role that manifests either beneficial or deleterious consequences depending on the extent of phosphorylation and interaction with cytoskeleton. Considering that disease-causing mutants of αB-crystallin are hyperphosphorylated, moderation of phosphorylation may be a useful strategy in disease management. This article is part of a Special Issue entitled Crystallin

  6. PPARγ1 phosphorylation enhances proliferation and drug resistance in human fibrosarcoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Pang, Xiaojuan; Shu, Yuxin; Niu, Zhiyuan; Zheng, Wei; Wu, Haochen [State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing (China); Lu, Yan, E-mail: luyan@nju.edu.cn [State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing (China); Shen, Pingping, E-mail: ppshen@nju.edu.cn [State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing (China); Model Animal Research Center (MARC), Nanjing University, Nanjing (China)

    2014-03-10

    Post-translational regulation plays a critical role in the control of cell growth and proliferation. The phosphorylation of peroxisome proliferator-activated receptor γ (PPARγ) is the most important post-translational modification. The function of PPARγ phosphorylation has been studied extensively in the past. However, the relationship between phosphorylated PPARγ1 and tumors remains unclear. Here we investigated the role of PPARγ1 phosphorylation in human fibrosarcoma HT1080 cell line. Using the nonphosphorylation (Ser84 to alanine, S84A) and phosphorylation (Ser84 to aspartic acid, S84D) mutant of PPARγ1, the results suggested that phosphorylation attenuated PPARγ1 transcriptional activity. Meanwhile, we demonstrated that phosphorylated PPARγ1 promoted HT1080 cell proliferation and this effect was dependent on the regulation of cell cycle arrest. The mRNA levels of cyclin-dependent kinase inhibitor (CKI) p21{sup Waf1/Cip1} and p27{sup Kip1} descended in PPARγ1{sup S84D} stable HT1080 cell, whereas the expression of p18{sup INK4C} was not changed. Moreover, compared to the PPARγ1{sup S84A}, PPARγ1{sup S84D} up-regulated the expression levels of cyclin D1 and cyclin A. Finally, PPARγ1 phosphorylation reduced sensitivity to agonist rosiglitazone and increased resistance to anticancer drug 5-fluorouracil (5-FU) in HT1080 cell. Our findings establish PPARγ1 phosphorylation as a critical event in human fibrosarcoma growth. These findings raise the possibility that chemical compounds that prevent the phosphorylation of PPARγ1 could act as anticancer drugs. - Highlights: • Phosphorylation attenuates PPARγ1 transcriptional activity. • Phosphorylated PPARγ1 promotes HT1080 cells proliferation. • PPARγ1 phosphorylation regulates cell cycle by mediating expression of cell cycle regulators. • PPARγ1 phosphorylation reduces sensitivity to agonist and anticancer drug. • Our findings establish PPARγ1 phosphorylation as a critical event in HT1080

  7. Mcm2 phosphorylation and the response to replicative stress

    Directory of Open Access Journals (Sweden)

    Stead Brent E

    2012-05-01

    Full Text Available Abstract Background The replicative helicase in eukaryotic cells is comprised of minichromosome maintenance (Mcm proteins 2 through 7 (Mcm2-7 and is a key target for regulation of cell proliferation. In addition, it is regulated in response to replicative stress. One of the protein kinases that targets Mcm2-7 is the Dbf4-dependent kinase Cdc7 (DDK. In a previous study, we showed that alanine mutations of the DDK phosphorylation sites at S164 and S170 in Saccharomyces cerevisiae Mcm2 result in sensitivity to caffeine and methyl methanesulfonate (MMS leading us to suggest that DDK phosphorylation of Mcm2 is required in response to replicative stress. Results We show here that a strain with the mcm2 allele lacking DDK phosphorylation sites (mcm2AA is also sensitive to the ribonucleotide reductase inhibitor, hydroxyurea (HU and to the base analogue 5-fluorouracil (5-FU but not the radiomimetic drug, phleomycin. We screened the budding yeast non-essential deletion collection for synthetic lethal interactions with mcm2AA and isolated deletions that include genes involved in the control of genome integrity and oxidative stress. In addition, the spontaneous mutation rate, as measured by mutations in CAN1, was increased in the mcm2AA strain compared to wild type, whereas with a phosphomimetic allele (mcm2EE the mutation rate was decreased. These results led to the idea that the mcm2AA strain is unable to respond properly to DNA damage. We examined this by screening the deletion collection for suppressors of the caffeine sensitivity of mcm2AA. Deletions that decrease spontaneous DNA damage, increase homologous recombination or slow replication forks were isolated. Many of the suppressors of caffeine sensitivity suppressed other phenotypes of mcm2AA including sensitivity to genotoxic drugs, the increased frequency of cells with RPA foci and the increased mutation rate. Conclusions Together these observations point to a role for DDK-mediated phosphorylation

  8. The upper and lower limits of the mechanistic stoichiometry of mitochondrial oxidative phosphorylation. Stoichiometry of oxidative phosphorylation.

    Science.gov (United States)

    Beavis, A D; Lehninger, A L

    1986-07-15

    Determination of the intrinsic or mechanistic P/O ratio of oxidative phosphorylation is difficult because of the unknown magnitude of leak fluxes. Applying a new approach developed to overcome this problem (see our preceding paper in this journal), the relationships between the rate of O2 uptake [( Jo)3], the net rate of phosphorylation (Jp), the P/O ratio, and the respiratory control ratio (RCR) have been determined in rat liver mitochondria when the rate of phosphorylation was systematically varied by three specific means. (a) When phosphorylation is titrated with carboxyatractyloside, linear relationships are observed between Jp and (Jo)3. These data indicate that the upper limit of the mechanistic P/O ratio is 1.80 for succinate and 2.90 for 3-hydroxybutyrate oxidation. (b) Titration with malonate or antimycin yields linear relationships between Jp and (Jo)3. These data give the lower limit of the mechanistic P/O ratio of 1.63 for succinate and 2.66 for 3-hydroxybutyrate oxidation. (c) Titration with a protonophore yields linear relationships between Jp, (Jo)3, and (Jo)4 and between P/O and 1/RCR. Extrapolation of the P/O ratio to 1/RCR = 0 yields P/O ratios of 1.75 for succinate and 2.73 for 3-hydroxybutyrate oxidation which must be equal to or greater than the mechanistic stoichiometry. When published values for the H+/O and H+/ATP ejection ratios are taken into consideration, these measurements suggest that the mechanistic P/O ratio is 1.75 for succinate oxidation and 2.75 for NADH oxidation.

  9. In cellulo phosphorylation of XRCC4 Ser320 by DNA-PK induced by DNA damage

    International Nuclear Information System (INIS)

    Sharma, Mukesh Kumar; Imamichi, Shoji; Fukuchi, Mikoto; Samarth, Ravindra Mahadeo; Tomita, Masanori; Matsumoto, Yoshihisa

    2016-01-01

    XRCC4 is a protein associated with DNA Ligase IV, which is thought to join two DNA ends at the final step of DNA double-strand break repair through non-homologous end joining. In response to treatment with ionizing radiation or DNA damaging agents, XRCC4 undergoes DNA-PK-dependent phosphorylation. Furthermore, Ser260 and Ser320 (or Ser318 in alternatively spliced form) of XRCC4 were identified as the major phosphorylation sites by purified DNA-PK in vitro through mass spectrometry. However, it has not been clear whether these sites are phosphorylated in vivo in response to DNA damage. In the present study, we generated an antibody that reacts with XRCC4 phosphorylated at Ser320 and examined in cellulo phosphorylation status of XRCC4 Ser320. The phosphorylation of XRCC4 Ser320 was induced by γ-ray irradiation and treatment with Zeocin. The phosphorylation of XRCC4 Ser320 was detected even after 1 Gy irradiation and increased in a manner dependent on radiation dose. The phosphorylation was observed immediately after irradiation and remained mostly unchanged for up to 4 h. The phosphorylation was inhibited by DNA-PK inhibitor NU7441 and was undetectable in DNA-PKcs-deficient cells, indicating that the phosphorylation was mainly mediated by DNA-PK. These results suggested potential usefulness of the phosphorylation status of XRCC4 Ser320 as an indicator of DNA-PK functionality in living cells

  10. 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.

  11. Identification of Mitosis-Specific Phosphorylation in Mitotic Chromosome-Associated Proteins.

    Science.gov (United States)

    Ohta, Shinya; Kimura, Michiko; Takagi, Shunsuke; Toramoto, Iyo; Ishihama, Yasushi

    2016-09-02

    During mitosis, phosphorylation of chromosome-associated proteins is a key regulatory mechanism. Mass spectrometry has been successfully applied to determine the complete protein composition of mitotic chromosomes, but not to identify post-translational modifications. Here, we quantitatively compared the phosphoproteome of isolated mitotic chromosomes with that of chromosomes in nonsynchronized cells. We identified 4274 total phosphorylation sites and 350 mitosis-specific phosphorylation sites in mitotic chromosome-associated proteins. Significant mitosis-specific phosphorylation in centromere/kinetochore proteins was detected, although the chromosomal association of these proteins did not change throughout the cell cycle. This mitosis-specific phosphorylation might play a key role in regulation of mitosis. Further analysis revealed strong dependency of phosphorylation dynamics on kinase consensus patterns, thus linking the identified phosphorylation sites to known key mitotic kinases. Remarkably, chromosomal axial proteins such as non-SMC subunits of condensin, TopoIIα, and Kif4A, together with the chromosomal periphery protein Ki67 involved in the establishment of the mitotic chromosomal structure, demonstrated high phosphorylation during mitosis. These findings suggest a novel mechanism for regulation of chromosome restructuring in mitosis via protein phosphorylation. Our study generated a large quantitative database on protein phosphorylation in mitotic and nonmitotic chromosomes, thus providing insights into the dynamics of chromatin protein phosphorylation at mitosis onset.

  12. Effect of phosphorylation on antioxidant activities of pumpkin (Cucurbita pepo, Lady godiva) polysaccharide.

    Science.gov (United States)

    Song, Yi; Ni, Yuanying; Hu, Xiaosong; Li, Quanhong

    2015-11-01

    Phosphorylated derivatives of pumpkin polysaccharide with different degree of substitution were synthesized using POCl3 and pyridine. Antioxidant activities and cytoprotective effects of unmodified polysaccharide and phosphorylated derivatives were investigated employing various in vitro systems. Results showed that high ratio of POCl3/pyridine could increase the degree of substitution and no remarkable degradation occurred in the phosphorylation process. Characteristic absorption of phosphorylation appeared both in the IR and (31)P NMR spectrum. The df values between 2.27 and 2.55 indicated the relatively expanded conformation of the phosphorylated derivatives. All the phosphorylated polysaccharides exhibited higher antioxidant activities. H2O2-induced oxidative damages on rat thymic lymphocyte were also prevented by the derivatives. In general, phosphorylation could improve the antioxidant activities of pumpkin polysaccharide both in vitro and in a cell system. Copyright © 2015 Elsevier B.V. All rights reserved.

  13. Characterization of mitosis-specific phosphorylation of tumor-associated microtubule-associated protein.

    Science.gov (United States)

    Hong, Kyung Uk; Kim, Hyun-Jun; Bae, Chang-Dae; Park, Joobae

    2009-11-30

    Tumor-associated microtubule-associated protein (TMAP), also known as cytoskeleton associated protein 2 (CKAP2), has been recently shown to be involved in the assembly and maintenance of mitotic spindle and also plays an essential role in maintaining the fidelity of chromosome segregation during mitosis. We have previously reported that TMAP is phosphorylated at multiple residues specifically during mitosis, and characterized the mechanism and functional importance of phosphorylation at one of the mitosis-specific phosphorylation residues (i.e., Thr-622). However, the phosphorylation events at the remaining mitotic phosphorylation sites of TMAP have not been fully characterized in detail. Here, we report on generation and characterization of phosphorylated Thr-578- and phosphorylated Thr-596-specific antibodies. Using the antibodies, we show that phosphorylation of TMAP at Thr-578 and Thr-596 indeed occurs specifically during mitosis. Immunofluorescent staining using the antibodies shows that these residues become phosphorylated starting at prophase and then become rapidly dephosphorylated soon after initiation of anaphase. Subtle differences in the kinetics of phosphorylation between Thr-578 and Thr-596 imply that they may be under different mechanisms of phosphorylation during mitosis. Unlike the phosphorylation-deficient mutant form for Thr-622, the mutant in which both Thr-578 and Thr-596 had been mutated to alanines did not induce significant delay in progression of mitosis. These results show that the majority of mitosis-specific phosphorylation of TMAP is limited to pre-anaphase stages and suggest that the multiple phosphorylation may not act in concert but serve diverse functions.

  14. Thyroid states regulate subcellular glucose phosphorylation activity in male mice

    Directory of Open Access Journals (Sweden)

    Flavia Letícia Martins Peçanha

    2017-07-01

    Full Text Available The thyroid hormones (THs, triiodothyronine (T3 and thyroxine (T4, are very important in organism metabolism and regulate glucose utilization. Hexokinase (HK is responsible for the first step of glycolysis, catalyzing the conversion of glucose to glucose 6-phosphate. HK has been found in different cellular compartments, and new functions have been attributed to this enzyme. The effects of hyperthyroidism on subcellular glucose phosphorylation in mouse tissues were examined. Tissues were removed, subcellular fractions were isolated from eu- and hyperthyroid (T3, 0.25 μg/g, i.p. during 21 days mice and HK activity was assayed. Glucose phosphorylation was increased in the particulate fraction in soleus (312.4% ± 67.1, n = 10, gastrocnemius (369.2% ± 112.4, n = 10 and heart (142.2% ± 13.6, n = 10 muscle in the hyperthyroid group compared to the control group. Hexokinase activity was not affected in brain or liver. No relevant changes were observed in HK activity in the soluble fraction for all tissues investigated. Acute T3 administration (single dose of T3, 1.25 μg/g, i.p. did not modulate HK activity. Interestingly, HK mRNA levels remained unchanged and HK bound to mitochondria was increased by T3 treatment, suggesting a posttranscriptional mechanism. Analysis of the AKT pathway showed a 2.5-fold increase in AKT and GSK3B phosphorylation in the gastrocnemius muscle in the hyperthyroid group compared to the euthyroid group. Taken together, we show for the first time that THs modulate HK activity specifically in particulate fractions and that this action seems to be under the control of the AKT and GSK3B pathways.

  15. Trans10,cis12 conjugated linoleic acid inhibits proliferation and migration of ovarian cancer cells by inducing ER stress, autophagy, and modulation of Src.

    Directory of Open Access Journals (Sweden)

    Mian M K Shahzad

    Full Text Available The goal of this study was to investigate the anti-cancer effects of Trans10,cis12 conjugated linoleic acid (t10,c12 CLA. MTT assays and QCM™ chemotaxis 96-wells were used to test the effect of t10,c12 CLA on the proliferation and migration and invasion of cancer cells. qPCR and Western Blotting were used to determine the expression of specific factors. RNA sequencing was conducted using the Illumina platform and apoptosis was measured using a flow cytometry assay. t10,c12 CLA (IC50, 7 μM inhibited proliferation of ovarian cancer cell lines SKOV-3 and A2780. c9,t11 CLA did not attenuate the proliferation of these cells. Transcription of 165 genes was significantly repressed and 28 genes were elevated. Genes related to ER stress, ATF4, CHOP, and GADD34 were overexpressed whereas EDEM2 and Hsp90, genes required for proteasomal degradation of misfolded proteins, were downregulated upon treatment. While apoptosis was not detected, t10,c12 CLA treatment led to 9-fold increase in autophagolysosomes and higher levels of LC3-II. G1 cell cycle arrest in treated cells was correlated with phosphorylation of GSK3β and loss of β-catenin. microRNA miR184 and miR215 were upregulated. miR184 likely contributed to G1 arrest by downregulating E2F1. miR215 upregulation was correlated with increased expression of p27/Kip-1. t10,c12 CLA-mediated inhibition of invasion and migration correlated with decreased expression of PTP1b and decreased Src activation by inhibiting phosphorylation at Tyr416. Due to its ability to inhibit proliferation and migration, t10,c12 CLA should be considered for treatment of ovarian cancer.

  16. Trans10,cis12 conjugated linoleic acid inhibits proliferation and migration of ovarian cancer cells by inducing ER stress, autophagy, and modulation of Src.

    Science.gov (United States)

    Shahzad, Mian M K; Felder, Mildred; Ludwig, Kai; Van Galder, Hannah R; Anderson, Matthew L; Kim, Jong; Cook, Mark E; Kapur, Arvinder K; Patankar, Manish S

    2018-01-01

    The goal of this study was to investigate the anti-cancer effects of Trans10,cis12 conjugated linoleic acid (t10,c12 CLA). MTT assays and QCM™ chemotaxis 96-wells were used to test the effect of t10,c12 CLA on the proliferation and migration and invasion of cancer cells. qPCR and Western Blotting were used to determine the expression of specific factors. RNA sequencing was conducted using the Illumina platform and apoptosis was measured using a flow cytometry assay. t10,c12 CLA (IC50, 7 μM) inhibited proliferation of ovarian cancer cell lines SKOV-3 and A2780. c9,t11 CLA did not attenuate the proliferation of these cells. Transcription of 165 genes was significantly repressed and 28 genes were elevated. Genes related to ER stress, ATF4, CHOP, and GADD34 were overexpressed whereas EDEM2 and Hsp90, genes required for proteasomal degradation of misfolded proteins, were downregulated upon treatment. While apoptosis was not detected, t10,c12 CLA treatment led to 9-fold increase in autophagolysosomes and higher levels of LC3-II. G1 cell cycle arrest in treated cells was correlated with phosphorylation of GSK3β and loss of β-catenin. microRNA miR184 and miR215 were upregulated. miR184 likely contributed to G1 arrest by downregulating E2F1. miR215 upregulation was correlated with increased expression of p27/Kip-1. t10,c12 CLA-mediated inhibition of invasion and migration correlated with decreased expression of PTP1b and decreased Src activation by inhibiting phosphorylation at Tyr416. Due to its ability to inhibit proliferation and migration, t10,c12 CLA should be considered for treatment of ovarian cancer.

  17. Identification of Phosphorylated Proteins on a Global Scale.

    Science.gov (United States)

    Iliuk, Anton

    2018-05-31

    Liquid chromatography (LC) coupled with tandem mass spectrometry (MS/MS) has enabled researchers to analyze complex biological samples with unprecedented depth. It facilitates the identification and quantification of modifications within thousands of proteins in a single large-scale proteomic experiment. Analysis of phosphorylation, one of the most common and important post-translational modifications, has particularly benefited from such progress in the field. Here, detailed protocols are provided for a few well-regarded, common sample preparation methods for an effective phosphoproteomic experiment. © 2018 by John Wiley & Sons, Inc. Copyright © 2018 John Wiley & Sons, Inc.

  18. Injectable hydrogels derived from phosphorylated alginic acid calcium complexes

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Han-Sem; Song, Minsoo, E-mail: minsoosong00@gmail.com; Lee, Eun-Jung; Shin, Ueon Sang, E-mail: usshin12@dankook.ac.kr

    2015-06-01

    Phosphorylation of sodium alginate salt (NaAlg) was carried out using H{sub 3}PO{sub 4}/P{sub 2}O{sub 5}/Et{sub 3}PO{sub 4} followed by acid–base reaction with Ca(OAc){sub 2} to give phosphorylated alginic acid calcium complexes (CaPAlg), as a water dispersible alginic acid derivative. The modified alginate derivatives including phosphorylated alginic acid (PAlg) and CaPAlg were characterized by nuclear magnetic resonance spectroscopy for {sup 1}H, and {sup 31}P nuclei, high resolution inductively coupled plasma optical emission spectroscopy, Fourier transform infrared spectroscopy, and thermogravimetric analysis. CaPAlg hydrogels were prepared simply by mixing CaPAlg solution (2 w/v%) with NaAlg solution (2 w/v%) in various ratios (2:8, 4:6, 6:4, 8:2) of volume. No additional calcium salts such as CaSO{sub 4} or CaCl{sub 2} were added externally. The gelation was completed within about 3–40 min indicating a high potential of hydrogel delivery by injection in vivo. Their mechanical properties were tested to be ≤ 6.7 kPa for compressive strength at break and about 8.4 kPa/mm for elastic modulus. SEM analysis of the CaPAlg hydrogels showed highly porous morphology with interconnected pores of width in the range of 100–800 μm. Cell culture results showed that the injectable hydrogels exhibited comparable properties to the pure alginate hydrogel in terms of cytotoxicity and 3D encapsulation of cells for a short time period. The developed injectable hydrogels showed suitable physicochemical and mechanical properties for injection in vivo, and could therefore be beneficial for the field of soft tissue engineering. - Highlights: • Preparation of water-soluble alginic acid complexes with calcium phosphate • Self-assembly of the phosphorylated alginic acid calcium complexes with sodium alginate • Preparation of injectable hydrogels with diverse gelation times within about 3–40 min.

  19. Regulation of PCNA Function by Tyrosine Phosphorylation in Prostate Cancer

    Science.gov (United States)

    2012-10-01

    ylated wild-type sequence did not bind to any of the functional domains. In contrast, incubation with the phosphorylated peptide identified the SH2 domain...Recently, He et al. reported that c-Abl interacted with PCNA through a putative PCNA-binding motif in the SH2 domain of c- Abl [22]. This proposed motif...motif of c-Abl may play a role in anti-apoptosis, interaction between Abl/ SH2 with PCNA/phospho-Y211 can confer a signaling for growth advantage in

  20. Protein Ser/Thr/Tyr phosphorylation in the Archaea.

    Science.gov (United States)

    Kennelly, Peter J

    2014-04-04

    The third domain of life, the Archaea (formerly Archaebacteria), is populated by a physiologically diverse set of microorganisms, many of which reside at the ecological extremes of our global environment. Although ostensibly prokaryotic in morphology, the Archaea share much closer evolutionary ties with the Eukarya than with the superficially more similar Bacteria. Initial genomic, proteomic, and biochemical analyses have revealed the presence of "eukaryotic" protein kinases and phosphatases and an intriguing set of serine-, threonine-, and tyrosine-phosphorylated proteins in the Archaea that may offer new insights into this important regulatory mechanism.

  1. Constitutive phosphorylation of Shc proteins in human tumors

    DEFF Research Database (Denmark)

    Pelicci, G; Lanfrancone, L; Salcini, A E

    1995-01-01

    The Shc gene encodes three overlapping proteins which all contain a carboxy-terminal SH2 domain. Shc proteins are ubiquitously expressed and are downstream targets and effectors of activated tyrosine kinases (TK). We investigated tyrosine-phosphorylation of Shc proteins in normal and transformed...... of the Shc-associated phosphoproteins (EGFR, PDGFR, erbB-2, Met, bcr-abl, H4-ret) bound both the Shc- and Grb2-SH2 domains in vitro; others (p175; p70-p80) only the Shc-SH2 domain and yet others (p140) only the Grb2-SH3 domains. These results indicate that Shc proteins are common substrates of constitutively...

  2. Phosphorylation prevents C/EBPβ from the calpain-dependent degradation

    International Nuclear Information System (INIS)

    Zhang, Yuan-yuan; Li, Shu-fen; Qian, Shu-wen; Zhang, You-you; Liu, Yuan; Tang, Qi-Qun; Li, Xi

    2012-01-01

    Highlights: ► Phosphorylation protected C/EBPβ from μ-calpain-mediated proteolysis in vitro. ► Phosphorylation mimic C/EBPβ was insensitive to calpain accelerator and inhibitor. ► Phosphorylation on Thr 188 contributed more to the stabilization of C/EBPβ. -- Abstract: CCAAT/enhancer-binding protein (C/EBP) β plays an important role in proliferation and differentiation of 3T3-L1 preadipocytes. C/EBPβ is sequentially phosphorylated during the 3T3-L1 adipocyte differentiation program, first by MAPK/Cyclin A/cdk2 on Thr 188 and subsequently by GSK3β on Ser 184 or Thr 179 . Dual phosphorylation is critical for the gain of DNA binding activity of C/EBPβ. In this manuscript, we found that phosphorylation also contributed to the stability of C/EBPβ. Both ex vivo and in vitro experiments showed that phosphorylation by MAPK/Cyclin A/cdk2 and GSK3β protected C/EBPβ from μ-calpain-mediated proteolysis, while phosphorylation on Thr 188 by MAPK/Cyclin A/cdk2 contributed more to the stabilization of C/EBPβ, Further studies indicated that phosphorylation mimic C/EBPβ was insensitive to both calpain accelerator and calpain inhibitor. Thus, phosphorylation might contribute to the stability as well as the gain of DNA binding activity of C/EBPβ.

  3. Phosphorylation of Mycobacterium tuberculosis Ser/Thr phosphatase by PknA and PknB.

    Directory of Open Access Journals (Sweden)

    Andaleeb Sajid

    2011-03-01

    Full Text Available The integrated functions of 11 Ser/Thr protein kinases (STPKs and one phosphatase manipulate the phosphorylation levels of critical proteins in Mycobacterium tuberculosis. In this study, we show that the lone Ser/Thr phosphatase (PstP is regulated through phosphorylation by STPKs.PstP is phosphorylated by PknA and PknB and phosphorylation is influenced by the presence of Zn(2+-ions and inorganic phosphate (Pi. PstP is differentially phosphorylated on the cytosolic domain with Thr(137, Thr(141, Thr(174 and Thr(290 being the target residues of PknB while Thr(137 and Thr(174 are phosphorylated by PknA. The Mn(2+-ion binding residues Asp(38 and Asp(229 are critical for the optimal activity of PstP and substitution of these residues affects its phosphorylation status. Native PstP and its phosphatase deficient mutant PstP(c (D38G are phosphorylated by PknA and PknB in E. coli and addition of Zn(2+/Pi in the culture conditions affect the phosphorylation level of PstP. Interestingly, the phosphorylated phosphatase is more active than its unphosphorylated equivalent.This study establishes the novel mechanisms for regulation of mycobacterial Ser/Thr phosphatase. The results indicate that STPKs and PstP may regulate the signaling through mutually dependent mechanisms. Consequently, PstP phosphorylation may play a critical role in regulating its own activity. Since, the equilibrium between phosphorylated and non-phosphorylated states of mycobacterial proteins is still unexplained, understanding the regulation of PstP may help in deciphering the signal transduction pathways mediated by STPKs and the reversibility of the phenomena.

  4. A Mass Spectrometry-Based Predictive Strategy Reveals ADAP1 is Phosphorylated at Tyrosine 364

    Energy Technology Data Exchange (ETDEWEB)

    Littrell, BobbiJo R [National Renewable Energy Laboratory (NREL), Golden, CO (United States)

    2018-04-16

    The goal of this work was to identify phosphorylation sites within the amino acid sequence of human ADAP1. Using traditional mass spectrometry-based techniques we were unable to produce interpretable spectra demonstrating modification by phosphorylation. This prompted us to employ a strategy in which phosphorylated peptides were first predicted using peptide mapping followed by targeted MS/MS acquisition. ADAP1 was immunoprecipitated from extracts of HEK293 cells stably-transfected with ADAP1 cDNA. Immunoprecipitated ADAP1 was digested with proteolytic enzymes and analyzed by LC-MS in MS1 mode by high-resolution quadrupole time-of-flight mass spectrometry (QTOF-MS). Peptide molecular features were extracted using an untargeted data mining algorithm. Extracted peptide neutral masses were matched against the ADAP1 amino acid sequence with phosphorylation included as a predicted modification. Peptides with predicted phosphorylation sites were analyzed by targeted LC-MS2. Acquired MS2 spectra were then analyzed using database search engines to confirm phosphorylation. Spectra of phosphorylated peptides were validated by manual interpretation. Further confirmation was performed by manipulating phospho-peptide abundance using calf intestinal phosphatase (CIP) and the phorbol ester, phorbol 12-myristate 13-acetate (PMA). Of five predicted phosphopeptides, one, comprised of the sequence AVDRPMLPQEYAVEAHFK, was confirmed to be phosphorylated on a Tyrosine at position 364. Pre-treatment of cells with PMA prior to immunoprecipitation increased the ratio of phosphorylated to unphosphorylated peptide as determined by area counts of extracted ion chromatograms (EIC). Addition of CIP to immunoprecipitation reactions eliminated the phosphorylated form. A novel phosphorylation site was identified at Tyrosine 364. Phosphorylation at this site is increased by treatment with PMA. PMA promotes membrane translocation and activation of protein kinase C (PKC), indicating that Tyrosine 364

  5. Metal ion interaction with phosphorylated tyrosine analogue monolayers on gold.

    Science.gov (United States)

    Petoral, Rodrigo M; Björefors, Fredrik; Uvdal, Kajsa

    2006-11-23

    Phosphorylated tyrosine analogue molecules (pTyr-PT) were assembled onto gold substrates, and the resulting monolayers were used for metal ion interaction studies. The monolayers were characterized by X-ray photoelectron spectroscopy (XPS), infrared reflection-absorption spectroscopy (IRAS), cyclic voltammetry (CV), and electrochemical impedance spectroscopy (EIS), both prior to and after exposure to metal ions. XPS verified the elemental composition of the molecular adsorbate and the presence of metal ions coordinated to the phosphate groups. Both the angle-dependent XPS and IRAS results were consistent with the change in the structural orientation of the pTyr-PT monolayer upon exposure to metal ions. The differential capacitance of the monolayers upon coordination of the metal ions was evaluated using EIS. These metal ions were found to significantly change the capacitance of the pTyr-PT monolayers in contrast to the nonphosphorylated tyrosine analogue (TPT). CV results showed reduced electrochemical blocking capabilities of the phosphorylated analogue monolayer when exposed to metal ions, supporting the change in the structure of the monolayer observed by XPS and IRAS. The largest change in the structure and interfacial capacitance was observed for aluminum ions, compared to calcium, magnesium, and chromium ions. This type of monolayer shows an excellent capability to coordinate metal ions and has a high potential for use as sensing layers in biochip applications to monitor the presence of metal ions.

  6. Immunohistochemistry of colorectal cancer biomarker phosphorylation requires controlled tissue fixation.

    Directory of Open Access Journals (Sweden)

    Abbey P Theiss

    Full Text Available Phosphorylated signaling molecules are biomarkers of cancer pathophysiology and resistance to therapy, but because phosphoprotein analytes are often labile, poorly controlled clinical laboratory practices could prevent translation of research findings in this area from the bench to the bedside. We therefore compared multiple biomarker and phosphoprotein immunohistochemistry (IHC results in 23 clinical colorectal carcinoma samples after either a novel, rapid tissue fixation protocol or a standard tissue fixation protocol employed by clinical laboratories, and we also investigated the effect of a defined post-operative "cold" ischemia period on these IHC results. We found that a one-hour cold ischemia interval, allowed by ASCO/CAP guidelines for certain cancer biomarker assays, is highly deleterious to certain phosphoprotein analytes, specifically the phosphorylated epidermal growth factor receptor (pEGFR, but shorter ischemic intervals (less than 17 minutes facilitate preservation of phosphoproteins. Second, we found that a rapid 4-hour, two temperature, formalin fixation yielded superior staining in several cases with select markers (pEGFR, pBAD, pAKT compared to a standard overnight room temperature fixation protocol, despite taking less time. These findings indicate that the future research and clinical utilities of phosphoprotein IHC for assessing colorectal carcinoma pathophysiology absolutely depend upon attention to preanalytical factors and rigorously controlled tissue fixation protocols.

  7. Regulation of Endothelial Adherens Junctions by Tyrosine Phosphorylation

    Science.gov (United States)

    Adam, Alejandro Pablo

    2015-01-01

    Endothelial cells form a semipermeable, regulated barrier that limits the passage of fluid, small molecules, and leukocytes between the bloodstream and the surrounding tissues. The adherens junction, a major mechanism of intercellular adhesion, is comprised of transmembrane cadherins forming homotypic interactions between adjacent cells and associated cytoplasmic catenins linking the cadherins to the cytoskeleton. Inflammatory conditions promote the disassembly of the adherens junction and a loss of intercellular adhesion, creating openings or gaps in the endothelium through which small molecules diffuse and leukocytes transmigrate. Tyrosine kinase signaling has emerged as a central regulator of the inflammatory response, partly through direct phosphorylation and dephosphorylation of the adherens junction components. This review discusses the findings that support and those that argue against a direct effect of cadherin and catenin phosphorylation in the disassembly of the adherens junction. Recent findings indicate a complex interaction between kinases, phosphatases, and the adherens junction components that allow a fine regulation of the endothelial permeability to small molecules, leukocyte migration, and barrier resealing. PMID:26556953

  8. Solid-phase assay for the phosphorylation of proteins blotted on nitrocellulose membrane filters

    International Nuclear Information System (INIS)

    Valtorta, F.; Schiebler, W.; Jahn, R.; Ceccarelli, B.; Greengard, P.

    1986-01-01

    A new procedure for the phosphorylation and assay of phosphoproteins is described. Proteins are solubilized from tissue samples, separated by polyacrylamide gel electrophoresis, transferred onto nitrocellulose membrane filters, and the blotted polypeptides are phyosphorylated with the catalytic subunit of cyclic AMP (adenosine 3':5'-monophosphate)-dependent protein kinase. The method was developed for the assay of dephosphosynapsin I, but it has also proven suitable for the phosphorylation of other proteins. The patterns of phosphorylation of tissue samples phosphorylated using the new method are similar to those obtained using the conventional test tube assay. Once phosphorylated, the adsorbed proteins can be digested with proteases and subjected to phosphopeptide mapping. The phosphorylated blotted proteins can also be analyzed by overlay techniques for the immunological detection of polypeptides

  9. Synthesis of Isomeric Phosphoubiquitin Chains Reveals that Phosphorylation Controls Deubiquitinase Activity and Specificity

    Directory of Open Access Journals (Sweden)

    Nicolas Huguenin-Dezot

    2016-07-01

    Full Text Available Ubiquitin is post-translationally modified by phosphorylation at several sites, but the consequences of these modifications are largely unknown. Here, we synthesize multi-milligram quantities of ubiquitin phosphorylated at serine 20, serine 57, and serine 65 via genetic code expansion. We use these phosphoubiquitins for the enzymatic assembly of 20 isomeric phosphoubiquitin dimers, with different sites of isopeptide linkage and/or phosphorylation. We discover that phosphorylation of serine 20 on ubiquitin converts UBE3C from a dual-specificity E3 ligase into a ligase that primarily synthesizes K48 chains. We profile the activity of 31 deubiquitinases on the isomeric phosphoubiquitin dimers in 837 reactions, and we discover that phosphorylation at distinct sites in ubiquitin can activate or repress cleavage of a particular linkage by deubiquitinases and that phosphorylation at a single site in ubiquitin can control the specificity of deubiquitinases for distinct ubiquitin linkages.

  10. Akt phosphorylation is essential for nuclear translocation and retention in NGF-stimulated PC12 cells

    International Nuclear Information System (INIS)

    Truong Le Xuan Nguyen; Choi, Joung Woo; Lee, Sang Bae; Ye, Keqiang; Woo, Soo-Dong; Lee, Kyung-Hoon; Ahn, Jee-Yin

    2006-01-01

    Nerve growth factor (NGF) elicits Akt translocation into the nucleus, where it phosphorylates nuclear targets. Here, we describe that Akt phosphorylation can promote the nuclear translocation of Akt and is necessary for its nuclear retention. Overexpression of Akt-K179A, T308A, S473A-mutant failed to show either nuclear translocation or nuclear Akt phosphorylation, whereas expression of wild-type counterpart elicited profound Akt phosphorylation and induced nuclear translocation under NGF stimulation. Employing the PI3K inhibitor and a variety of mutants PI3K, we showed that nuclear translocation of Akt was mediated by activation of PI3K, and Akt phosphorylation status in the nucleus required PI3K activity. Thus the activity of PI3K might contribute to the nuclear translocation of Akt, and that Akt phosphorylation is essential for its nuclear retention under NGF stimulation conditions

  11. A CK2 site is reversibly phosphorylated in the photosystem II subunit CP29.

    Science.gov (United States)

    Testi, M G; Croce, R; Polverino-De Laureto, P; Bassi, R

    1996-12-16

    Protein phosphorylation is a major mechanism in the regulation of protein function. In chloroplast thylakoids several photosystem II subunits, including the major antenna light-harvesting complex II and several core complex components, are reversibly phosphorylated depending on the redox state of the electron carriers. A previously unknown reversible phosphorylation event has recently been described on the CP29 subunit which leads to conformational changes and protection from cold stress (Bergantino, E., Dainese, P., Cerovic, Z. Sechi, S. and Bassi, R. (1995) J. Biol Chem. 270, 8474-8481). In this study, we have identified the phosphorylation site on the N-terminal, stroma-exposed domain, showing that it is located in a sequence not homologous to the other members of the Lhc family. The phosphorylated sequence is unique in chloroplast membranes since it meets the requirements for CK2 (casein kinase II) kinases. The possibility that this phosphorylation is involved in a signal transduction pathway is discussed.

  12. Dynamic phosphorylation of Ebola virus VP30 in NP-induced inclusion bodies.

    Science.gov (United States)

    Lier, Clemens; Becker, Stephan; Biedenkopf, Nadine

    2017-12-01

    Zaire Ebolavirus (EBOV) causes a severe feverish disease with high case fatality rates. Transcription of EBOV is dependent on the activity of the nucleocapsid protein VP30 which represents an essential viral transcription factor. Activity of VP30 is regulated via phosphorylation at six N-terminal serine residues. Recent data demonstrated that dynamic phosphorylation and dephosphorylation of serine residue 29 is essential for transcriptional support activity of VP30. To analyze the spatio/temporal dynamics of VP30 phosphorylation, we generated a peptide antibody recognizing specifically VP30 phosphorylated at serine 29. Using this antibody we could demonstrate that (i) the majority of VP30 molecules in EBOV-infected cells is dephosphorylated at the crucial position serine 29, (ii) both, VP30 phosphorylation and dephosphorylation take place in viral inclusion bodies that are induced by the nucleoprotein NP and (iii) NP influences the phosphorylation state of VP30. Copyright © 2017 Elsevier Inc. All rights reserved.

  13. Muscarinic agonists and phorbol esters increase tyrosine phosphorylation of a 40-kilodalton protein in hippocampal slices

    International Nuclear Information System (INIS)

    Stratton, K.R.; Worley, P.F.; Huganir, R.L.; Baraban, J.M.

    1989-01-01

    The authors have used the hippocampal slice preparation to investigate the regulation of protein tyrosine phosphorylation in brain. After pharmacological treatment of intact slices, proteins were separated by electrophoresis, and levels of protein tyrosine phosphorylation were assessed by immunoblotting with specific anti-phosphotyrosine antibodies. Phorbol esters, activators of the serine- and threonine-phosphorylating enzyme protein kinase C, selectively increase tyrosine phosphorylation of a soluble protein with an apparent molecular mass of approximately 40 kilodaltons. Muscarinic agonists such as carbachol and oxotremorine M that strongly activate the inositol phospholipid system also increase tyrosine phosphorylation of this protein. Neurotransmitter activation of the inositol phospholipid system and protein kinase C appears to trigger a cascade leading to increased tyrosine phosphorylation

  14. Tyrosine phosphorylation of Eps15 is required for ligand-regulated, but not constitutive, endocytosis

    DEFF Research Database (Denmark)

    Confalonieri, S; Salcini, A E; Puri, C

    2000-01-01

    for endocytosis of the epidermal growth factor receptor (EGFR), the prototypical ligand-inducible receptor, but not of the transferrin receptor (TfR), the prototypical constitutively internalized receptor. Eps15, an endocytic protein that is tyrosine phosphorylated by EGFR, is a candidate for such a function....... Here, we show that tyrosine phosphorylation of Eps15 is necessary for internalization of the EGFR, but not of the TfR. We mapped Tyr 850 as the major in vivo tyrosine phosphorylation site of Eps15. A phosphorylation-negative mutant of Eps15 acted as a dominant negative on the internalization...... of the EGFR, but not of the TfR. A phosphopeptide, corresponding to the phosphorylated sequence of Eps15, inhibited EGFR endocytosis, suggesting that phosphotyrosine in Eps15 serves as a docking site for a phosphotyrosine binding protein. Thus, tyrosine phosphorylation of Eps15 represents the first molecular...

  15. Changes in phosphorylation of myofibrillar proteins during postmortem development of porcine muscle

    DEFF Research Database (Denmark)

    Huang, Honggang; Larsen, Martin Røssel; Lametsch, Rene

    2012-01-01

    A gel-based phosphoproteomic study was performed to investigate the postmortem (PM) changes in protein phosphorylation of the myofibrillar proteins in three groups of pigs with different pH decline rates, from PM 1 h to 24 h. The global phosphorylation level in the group with a fast pH decline rate...... was higher than that in the slow and intermediate groups at early PM time, but became the lowest at 24 h. The protein phosphorylation level of seven individual protein bands was only significantly (ppH...... phosphorylated protein bands with the highest scores. The results indicate that the phosphorylation pattern of myofibrillar proteins in PM muscle is mainly changed with PM time, but only to a minor extent influenced by the rate of pH decline, suggesting that the phosphorylation of myofibrillar proteins may...

  16. Replication Protein A (RPA) Phosphorylation Prevents RPA Association with Replication Centers

    OpenAIRE

    Vassin, Vitaly M.; Wold, Marc S.; Borowiec, James A.

    2004-01-01

    Mammalian replication protein A (RPA) undergoes DNA damage-dependent phosphorylation at numerous sites on the N terminus of the RPA2 subunit. To understand the functional significance of RPA phosphorylation, we expressed RPA2 variants in which the phosphorylation sites were converted to aspartate (RPA2D) or alanine (RPA2A). Although RPA2D was incorporated into RPA heterotrimers and supported simian virus 40 DNA replication in vitro, the RPA2D mutant was selectively unable to associate with re...

  17. Emerging roles for protein histidine phosphorylation in cellular signal transduction: lessons from the islet ?-cell

    OpenAIRE

    Kowluru, Anjaneyulu

    2008-01-01

    Protein phosphorylation represents one of the key regulatory events in physiological insulin secretion from the islet ?-cell. In this context, several classes of protein kinases (e.g. calcium-, cyclic nucleotide- and phospholipid-dependent protein kinases and tyrosine kinases) have been characterized in the ?-cell. The majority of phosphorylated amino acids identified include phosphoserine, phosphothreonine and phosphotyrosine. Protein histidine phosphorylation has been implicated in the prok...

  18. Protein kinases responsible for the phosphorylation of the nuclear egress core complex of human cytomegalovirus.

    Science.gov (United States)

    Sonntag, Eric; Milbradt, Jens; Svrlanska, Adriana; Strojan, Hanife; Häge, Sigrun; Kraut, Alexandra; Hesse, Anne-Marie; Amin, Bushra; Sonnewald, Uwe; Couté, Yohann; Marschall, Manfred

    2017-10-01

    Nuclear egress of herpesvirus capsids is mediated by a multi-component nuclear egress complex (NEC) assembled by a heterodimer of two essential viral core egress proteins. In the case of human cytomegalovirus (HCMV), this core NEC is defined by the interaction between the membrane-anchored pUL50 and its nuclear cofactor, pUL53. NEC protein phosphorylation is considered to be an important regulatory step, so this study focused on the respective role of viral and cellular protein kinases. Multiply phosphorylated pUL50 varieties were detected by Western blot and Phos-tag analyses as resulting from both viral and cellular kinase activities. In vitro kinase analyses demonstrated that pUL50 is a substrate of both PKCα and CDK1, while pUL53 can also be moderately phosphorylated by CDK1. The use of kinase inhibitors further illustrated the importance of distinct kinases for core NEC phosphorylation. Importantly, mass spectrometry-based proteomic analyses identified five major and nine minor sites of pUL50 phosphorylation. The functional relevance of core NEC phosphorylation was confirmed by various experimental settings, including kinase knock-down/knock-out and confocal imaging, in which it was found that (i) HCMV core NEC proteins are not phosphorylated solely by viral pUL97, but also by cellular kinases; (ii) both PKC and CDK1 phosphorylation are detectable for pUL50; (iii) no impact of PKC phosphorylation on NEC functionality has been identified so far; (iv) nonetheless, CDK1-specific phosphorylation appears to be required for functional core NEC interaction. In summary, our findings provide the first evidence that the HCMV core NEC is phosphorylated by cellular kinases, and that the complex pattern of NEC phosphorylation has functional relevance.

  19. Musite, a tool for global prediction of general and kinase-specific phosphorylation sites.

    Science.gov (United States)

    Gao, Jianjiong; Thelen, Jay J; Dunker, A Keith; Xu, Dong

    2010-12-01

    Reversible protein phosphorylation is one of the most pervasive post-translational modifications, regulating diverse cellular processes in various organisms. High throughput experimental studies using mass spectrometry have identified many phosphorylation sites, primarily from eukaryotes. However, the vast majority of phosphorylation sites remain undiscovered, even in well studied systems. Because mass spectrometry-based experimental approaches for identifying phosphorylation events are costly, time-consuming, and biased toward abundant proteins and proteotypic peptides, in silico prediction of phosphorylation sites is potentially a useful alternative strategy for whole proteome annotation. Because of various limitations, current phosphorylation site prediction tools were not well designed for comprehensive assessment of proteomes. Here, we present a novel software tool, Musite, specifically designed for large scale predictions of both general and kinase-specific phosphorylation sites. We collected phosphoproteomics data in multiple organisms from several reliable sources and used them to train prediction models by a comprehensive machine-learning approach that integrates local sequence similarities to known phosphorylation sites, protein disorder scores, and amino acid frequencies. Application of Musite on several proteomes yielded tens of thousands of phosphorylation site predictions at a high stringency level. Cross-validation tests show that Musite achieves some improvement over existing tools in predicting general phosphorylation sites, and it is at least comparable with those for predicting kinase-specific phosphorylation sites. In Musite V1.0, we have trained general prediction models for six organisms and kinase-specific prediction models for 13 kinases or kinase families. Although the current pretrained models were not correlated with any particular cellular conditions, Musite provides a unique functionality for training customized prediction models

  20. Formaldehyde-induced histone H3 phosphorylation via JNK and the expression of proto-oncogenes

    International Nuclear Information System (INIS)

    Yoshida, Ikuma; Ibuki, Yuko

    2014-01-01

    Graphical abstract: - Highlights: • Formaldehyde modified histones. • The phosphorylation of H3S10 was increased at the promoter regions of proto-oncogenes. • The phosphorylation of H2AXS139 was attributed to FA-induced DNA damage. • The FA-induced initiation and promotion of cancer could be judged by these modifications. - Abstract: Formaldehyde (FA) is a very reactive compound that forms DNA adducts and DNA-protein crosslinks, which are known to contribute to FA-induced mutations and carcinogenesis. Post-translational modifications to histones have recently attracted attention due to their link with cancer. In the present study, we examined histone modifications following a treatment with FA. FA significantly phosphorylated histone H3 at serine 10 (H3S10), and at serine 28 (H3S28), the time-course of which was similar to the phosphorylation of H2AX at serine 139 (γ-H2AX), a marker of DNA double strand breaks. The temporal deacetylation of H3 was observed due to the reaction of FA with the lysine residues of histones. The phosphorylation mechanism was then analyzed by focusing on H3S10. The nuclear distribution of the phosphorylation of H3S10 and γ-H2AX did not overlap, and the phosphorylation of H3S10 could not be suppressed with an inhibitor of ATM/ATR, suggesting that the phosphorylation of H3S10 was independent of the DNA damage response. ERK and JNK in the MAPK pathways were phosphorylated by the treatment with FA, in which the JNK pathway was the main target for phosphorylation. The phosphorylation of H3S10 increased at the promoter regions of c-fos and c-jun, indicating a relationship between FA-induced tumor promotion activity and phosphorylation of H3S10. These results suggested that FA both initiates and promotes cancer, as judged by an analysis of histone modifications

  1. Formaldehyde-induced histone H3 phosphorylation via JNK and the expression of proto-oncogenes

    Energy Technology Data Exchange (ETDEWEB)

    Yoshida, Ikuma; Ibuki, Yuko, E-mail: ibuki@u-shizuoka-ken.ac.jp

    2014-12-15

    Graphical abstract: - Highlights: • Formaldehyde modified histones. • The phosphorylation of H3S10 was increased at the promoter regions of proto-oncogenes. • The phosphorylation of H2AXS139 was attributed to FA-induced DNA damage. • The FA-induced initiation and promotion of cancer could be judged by these modifications. - Abstract: Formaldehyde (FA) is a very reactive compound that forms DNA adducts and DNA-protein crosslinks, which are known to contribute to FA-induced mutations and carcinogenesis. Post-translational modifications to histones have recently attracted attention due to their link with cancer. In the present study, we examined histone modifications following a treatment with FA. FA significantly phosphorylated histone H3 at serine 10 (H3S10), and at serine 28 (H3S28), the time-course of which was similar to the phosphorylation of H2AX at serine 139 (γ-H2AX), a marker of DNA double strand breaks. The temporal deacetylation of H3 was observed due to the reaction of FA with the lysine residues of histones. The phosphorylation mechanism was then analyzed by focusing on H3S10. The nuclear distribution of the phosphorylation of H3S10 and γ-H2AX did not overlap, and the phosphorylation of H3S10 could not be suppressed with an inhibitor of ATM/ATR, suggesting that the phosphorylation of H3S10 was independent of the DNA damage response. ERK and JNK in the MAPK pathways were phosphorylated by the treatment with FA, in which the JNK pathway was the main target for phosphorylation. The phosphorylation of H3S10 increased at the promoter regions of c-fos and c-jun, indicating a relationship between FA-induced tumor promotion activity and phosphorylation of H3S10. These results suggested that FA both initiates and promotes cancer, as judged by an analysis of histone modifications.

  2. The Staphylococcus aureus autoinducer-2 synthase LuxS is regulated by Ser/Thr phosphorylation.

    Science.gov (United States)

    Cluzel, Marie-Eve; Zanella-Cléon, Isabelle; Cozzone, Alain J; Fütterer, Klaus; Duclos, Bertrand; Molle, Virginie

    2010-12-01

    The Staphylococcus aureus autoinducer-2 (AI-2) producer protein LuxS is phosphorylated by the Ser/Thr kinase Stk1 at a unique position, Thr14. The enzymatic activity of the phosphorylated isoform of LuxS was abrogated compared to that of nonphosphorylated LuxS, thus providing the first evidence of an AI-2-producing enzyme regulated by phosphorylation and demonstrating that S. aureus possesses an original and specific system for controlling AI-2 synthesis.

  3. Tyrosine Phosphorylation of Jak2 in the JH2 Domain Inhibits Cytokine Signaling

    OpenAIRE

    Feener, Edward P.; Rosario, Felicia; Dunn, Sarah L.; Stancheva, Zlatina; Myers, Martin G.

    2004-01-01

    Jak family tyrosine kinases mediate signaling by cytokine receptors to regulate diverse biological processes. Although Jak2 and other Jak kinase family members are phosphorylated on numerous sites during cytokine signaling, the identity and function of most of these sites remains unknown. Using tandem mass spectroscopic analysis of activated Jak2 protein from intact cells, we identified Tyr221 and Tyr570 as novel sites of Jak2 phosphorylation. Phosphorylation of both sites was stimulated by c...

  4. Protein Kinase B/Akt Binds and Phosphorylates PED/PEA-15, Stabilizing Its Antiapoptotic Action

    OpenAIRE

    Trencia, Alessandra; Perfetti, Anna; Cassese, Angela; Vigliotta, Giovanni; Miele, Claudia; Oriente, Francesco; Santopietro, Stefania; Giacco, Ferdinando; Condorelli, Gerolama; Formisano, Pietro; Beguinot, Francesco

    2003-01-01

    The antiapoptotic protein PED/PEA-15 features an Akt phosphorylation motif upstream from Ser116. In vitro, recombinant PED/PEA-15 was phosphorylated by Akt with a stoichiometry close to 1. Based on Western blotting with specific phospho-Ser116 PED/PEA-15 antibodies, Akt phosphorylation of PED/PEA-15 occurred mainly at Ser116. In addition, a mutant of PED/PEA-15 featuring the substitution of Ser116→Gly (PEDS116→G) showed 10-fold-decreased phosphorylation by Akt. In intact 293 cells, Akt also i...

  5. Phosphorylation of p37 is important for Golgi disassembly at mitosis

    International Nuclear Information System (INIS)

    Kaneko, Yayoi; Tamura, Kaori; Totsukawa, Go; Kondo, Hisao

    2010-01-01

    Research highlights: → p37 is phosphorylated on Serine-56 and Threonine-59 by Cdc2 at mitosis. → Phosphorylated p37 does not bind to Golgi membranes. → p37 phosphorylation inhibits p97/p37-mediated Golgi membrane fusion. -- Abstract: In mammals, the Golgi apparatus is disassembled at early mitosis and reassembled at the end of mitosis. For Golgi disassembly, membrane fusion needs to be blocked. Golgi biogenesis requires two distinct p97ATPase-mediated membrane fusion, the p97/p47 and p97/p37 pathways. We previously reported that p47 phosphorylation on Serine-140 by Cdc2 results in mitotic inhibition of the p97/p47 pathway . In this study, we demonstrate that p37 is phosphorylated on Serine-56 and Threonine-59 by Cdc2 at mitosis, and this phosphorylated p37 does not bind to Golgi membranes. Using an in vitro Golgi reassembly assay, we show that mutated p37(S56D, T59D), which mimics mitotic phosphorylation, does not cause any cisternal regrowth, indicating that p37 phosphorylation inhibits the p97/p37 pathway. Our results demonstrate that p37 phosphorylation on Serine-56 and Threonine-59 is important for Golgi disassembly at mitosis.

  6. Mitotic phosphorylation of VCIP135 blocks p97ATPase-mediated Golgi membrane fusion

    Energy Technology Data Exchange (ETDEWEB)

    Totsukawa, Go; Matsuo, Ayaka; Kubota, Ayano; Taguchi, Yuya; Kondo, Hisao, E-mail: hk228@med.kyushu-u.ac.jp

    2013-04-05

    Highlights: •VCIP135 is mitotically phosphorylated on Threonine-760 and Serine-767 by Cdc2. •Phosphorylated VCIP135 does not bind to p97ATPase. •The phosphorylation of VCIP135 inhibits p97ATPase-mediated Golgi membrane fusion. -- Abstract: In mammals, the Golgi apparatus is disassembled early mitosis and reassembled at the end of mitosis. For Golgi disassembly, membrane fusion needs to be blocked. Golgi biogenesis requires two distinct p97ATPase-mediated membrane fusion, the p97/p47 and p97/p37 pathways. We previously reported that p47 phosphorylation on Serine-140 and p37 phosphorylation on Serine-56 and Threonine-59 result in mitotic inhibition of the p97/p47 and the p97/p37 pathways, respectively [11,14]. In this study, we show another mechanism of mitotic inhibition of p97-mediated Golgi membrane fusion. We clarified that VCIP135, an essential factor in both p97 membrane fusion pathways, is phosphorylated on Threonine-760 and Serine-767 by Cdc2 at mitosis and that this phosphorylated VCIP135 does not bind to p97. An in vitro Golgi reassembly assay revealed that VCIP135(T760E, S767E), which mimics mitotic phosphorylation, caused no cisternal regrowth. Our results indicate that the phosphorylation of VCIP135 on Threonine-760 and Serine-767 inhibits p97-mediated Golgi membrane fusion at mitosis.

  7. Tampering with springs: phosphorylation of titin affecting the mechanical function of cardiomyocytes.

    Science.gov (United States)

    Hamdani, Nazha; Herwig, Melissa; Linke, Wolfgang A

    2017-06-01

    Reversible post-translational modifications of various cardiac proteins regulate the mechanical properties of the cardiomyocytes and thus modulate the contractile performance of the heart. The giant protein titin forms a continuous filament network in the sarcomeres of striated muscle cells, where it determines passive tension development and modulates active contraction. These mechanical properties of titin are altered through post-translational modifications, particularly phosphorylation. Titin contains hundreds of potential phosphorylation sites, the functional relevance of which is only beginning to emerge. Here, we provide a state-of-the-art summary of the phosphorylation sites in titin, with a particular focus on the elastic titin spring segment. We discuss how phosphorylation at specific amino acids can reduce or increase the stretch-induced spring force of titin, depending on where the spring region is phosphorylated. We also review which protein kinases phosphorylate titin and how this phosphorylation affects titin-based passive tension in cardiomyocytes. A comprehensive overview is provided of studies that have measured altered titin phosphorylation and titin-based passive tension in myocardial samples from human heart failure patients and animal models of heart disease. As our understanding of the broader implications of phosphorylation in titin progresses, this knowledge could be used to design targeted interventions aimed at reducing pathologically increased titin stiffness in patients with stiff hearts.

  8. PKCδ-mediated IRS-1 Ser24 phosphorylation negatively regulates IRS-1 function

    International Nuclear Information System (INIS)

    Greene, Michael W.; Ruhoff, Mary S.; Roth, Richard A.; Kim, Jeong-a; Quon, Michael J.; Krause, Jean A.

    2006-01-01

    The IRS-1 PH and PTB domains are essential for insulin-stimulated IRS-1 Tyr phosphorylation and insulin signaling, while Ser/Thr phosphorylation of IRS-1 disrupts these signaling events. To investigate consensus PKC phosphorylation sites in the PH-PTB domains of human IRS-1, we changed Ser24, Ser58, and Thr191 to Ala (3A) or Glu (3E), to block or mimic phosphorylation, respectively. The 3A mutant abrogated the inhibitory effect of PKCδ on insulin-stimulated IRS-1 Tyr phosphorylation, while reductions in insulin-stimulated IRS-1 Tyr phosphorylation, cellular proliferation, and Akt activation were observed with the 3E mutant. When single Glu mutants were tested, the Ser24 to Glu mutant had the greatest inhibitory effect on insulin-stimulated IRS-1 Tyr phosphorylation. PKCδ-mediated IRS-1 Ser24 phosphorylation was confirmed in cells with PKCδ catalytic domain mutants and by an RNAi method. Mechanistic studies revealed that IRS-1 with Ala and Glu point mutations at Ser24 impaired phosphatidylinositol-4,5-bisphosphate binding. In summary, our data are consistent with the hypothesis that Ser24 is a negative regulatory phosphorylation site in IRS-1

  9. Phosphorylation of rat thymus histones, its control and the effects thereon of γ-irradiation

    International Nuclear Information System (INIS)

    Fonagy, A.; Ord, M.G.; Stocken, L.A.

    1977-01-01

    The phosphate content of rat thymus histones was determined. As expected for a replicating tissue, histones 1 and 2B were more phosphorylated and had higher 32 P uptakes than did histones from resting liver nuclei; the other histones all showed 32 P uptake, but the phosphate content and uptake of histone 2A was about half that for liver histone 2A. When thymus nuclei were incubated in a slightly hypo-osmotic medium, non-histone proteins and phosphorylated histones were released into solution; this was enhanced if ATP was present in the medium. [γ- 32 P]ATP was incorporated into non-histone proteins, including Pl, and into the ADP-ribosylated form of histone 1; negligible 32 P was incorporated into the other, bound, histones. Histones 1 and 2B added to the incubation medium were extensively, and histones 2A and 4 slightly, phosphorylated. Histones released by increasing the ionic strength of the medium were phosphorylated. Added lysozyme and cytochrome c were neither bound nor phosphorylated, but added non-histone protein Pl was phosphorylated, causing other histones to be released from the nuclei, especially histones 2A and 3. The released histones were phosphorylated. γ-irradiation decreased 32 P uptake into the non-ADP-ribosylated histones 1 and 4; phosphorylation of histone 1 in vitro was unaffected. The importance of non-histone proteins, ATP availability and nuclear protein kinases to the control of histone phosphorylation in vivo is discussed. (author)

  10. Synthesis of tritium labelled nucleoside triphosphates by enzymatic phosphorylation

    International Nuclear Information System (INIS)

    Shen Decun; Ji Linzhen; Liao Sha

    1986-01-01

    [5- 3 H]UMP, [5- 3 H]CMP, [8- 3 H]AMP and [8- 3 H]GMP were prepared from 5BrUMP 5BrCMP 8BrAMP and 8BrGMP by catalytic halogentritium replacement at the same time. [5- 3 H]UTP, [5- 3 H]CTP, [8- 3 H]ATP and [8- 3 H]GTP were subsequently synthesized from [5- 3 H]UMP, [5- 3 H]CMP, [8- 3 H]AMP and [8- 3 H]GMP by enzymatic phosphorylation with the crude enzyme prepared from brewer's yeasts and purified by paper chromatography simultaneously. In addition, four kinds of tritium labelled nucleoside monophosphates and four kinds of tritium labelled nucleoside diphosphates were obtained as the by-products. The specific activity of these products is between 14-19 Ci/mmol and the radiochemical purity is more than 98%

  11. Ultrasensitivity in phosphorylation-dephosphorylation cycles with little substrate.

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    Bruno M C Martins

    Full Text Available Cellular decision-making is driven by dynamic behaviours, such as the preparations for sunrise enabled by circadian rhythms and the choice of cell fates enabled by positive feedback. Such behaviours are often built upon ultrasensitive responses where a linear change in input generates a sigmoidal change in output. Phosphorylation-dephosphorylation cycles are one means to generate ultrasensitivity. Using bioinformatics, we show that in vivo levels of kinases and phosphatases frequently exceed the levels of their corresponding substrates in budding yeast. This result is in contrast to the conditions often required by zero-order ultrasensitivity, perhaps the most well known means for how such cycles become ultrasensitive. We therefore introduce a mechanism to generate ultrasensitivity when numbers of enzymes are higher than numbers of substrates. Our model combines distributive and non-distributive actions of the enzymes with two-stage binding and concerted allosteric transitions of the substrate. We use analytical and numerical methods to calculate the Hill number of the response. For a substrate with [Formula: see text] phosphosites, we find an upper bound of the Hill number of [Formula: see text], and so even systems with a single phosphosite can be ultrasensitive. Two-stage binding, where an enzyme must first bind to a binding site on the substrate before it can access the substrate's phosphosites, allows the enzymes to sequester the substrate. Such sequestration combined with competition for each phosphosite provides an intuitive explanation for the sigmoidal shifts in levels of phosphorylated substrate. Additionally, we find cases for which the response is not monotonic, but shows instead a peak at intermediate levels of input. Given its generality, we expect the mechanism described by our model to often underlay decision-making circuits in eukaryotic cells.

  12. Characterization of osteopontin-uranyl interaction: role of multiple phosphorylations

    International Nuclear Information System (INIS)

    Qi, Lei

    2014-01-01

    While some metals are essential for Life, other ones are only toxicants for living organisms, tolerated below well-definite concentrations. This is the case for uranium, a natural element which has no known biological function. It is a low α emitter and its chemical toxicity rather than its radiological toxicity is a subject of concern. Once in the body, this metal reaches the blood and accumulates in the bones under the action of unknown mechanisms. Uranium mainly exists in form of uranyl ion (UO 2 2+ ) in aqueous media and particularly reacts with carboxylates, phenolates and phosphates of the proteins. Previous studies have highlighted that UO 2 2+ modulates the SPP1 expression, a gene which codes for osteopontin (OPN). This highly phosphorylated glycoprotein plays an important role in bone homeostasis. This role and its biochemical properties led us to hypothesize that OPN might be a potential target of UO 2 2+ and involved in its accumulation in bones. A simple and original purification process was optimized to produce very highly purified OPN starting from human and bovine milk. Various biophysical approaches were set up and confirmed that both bovine and human OPN display very high affinity for UO 2 2+ . Moreover, the formation of stable UO 2 -protein complexes originating from structural changes was evidenced. The major role of phosphorylations, both on the OPN's affinity for UO 2 2+ and the stability of the UO 2 -protein complexes, was confirmed. These results demonstrate that OPN presents all the characteristics to be a major UO 2 2+ binding-protein in vitro, and they open new insights in the understanding of the UO 2 2+ mineralization process mechanisms. (author) [fr

  13. Phosphorylated aminosugars: Synthesis, properties, and reactivity in enzymatic reactions

    International Nuclear Information System (INIS)

    Sem, D.S.; Cleland, W.W.

    1991-01-01

    A number of phosphorylated aminosugars have been prepared and tested as substrates for metabolic reactions. 6-Aminoglucose is a slow substrate for yeast hexokinase with a V max that is only 0.012% that of glucose. While V max is pH independent, V/K decreases below the pK of 9.0 of the amino group. 6-Aminoglucose is a competitive inhibitor vs glucose with a K i value increasing below the pK of 9 but leveling off at 33 mM below pH 7.16. Thus, protonation decreases binding affinity by 2.4 kcal/mol and only the neutral amine is catalytically competent. 6-Aminoglucose-6-P was synthesized enzymatically with hexokinase. Its pK's determined by 31 P NMR were 2.46 and 8.02 (α anomer) and 2.34 and 7.85 (β anomer), with a β:α ratio of 3.0. It is most stable at pH 12, while as a monoanion its half-life is 3 h. The 31 P NMR chemical shifts of the analogues are 8-8.5 ppm at pH 9.5. Their relative stability is 6-aminogluconate-6-P > 3-aminoglyceraldehyde-3-P > 6-aminoglucose-6-P > 6-aminofructose-1,6-bis-P≅6-aminofructose-6-P > 5-aminoribulose-5-P. These analogues were tested as substrates for their respective enzymes. Phosphorylated aminosugars are thus excellent isosteric analogues of normal metabolic intermediates, except for reactions catalyzed by kinases

  14. Molecular dynamics simulation of phosphorylated KID post-translational modification.

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    Hai-Feng Chen

    2009-08-01

    Full Text Available Kinase-inducible domain (KID as transcriptional activator can stimulate target gene expression in signal transduction by associating with KID interacting domain (KIX. NMR spectra suggest that apo-KID is an unstructured protein. After post-translational modification by phosphorylation, KID undergoes a transition from disordered to well folded protein upon binding to KIX. However, the mechanism of folding coupled to binding is poorly understood.To get an insight into the mechanism, we have performed ten trajectories of explicit-solvent molecular dynamics (MD for both bound and apo phosphorylated KID (pKID. Ten MD simulations are sufficient to capture the average properties in the protein folding and unfolding.Room-temperature MD simulations suggest that pKID becomes more rigid and stable upon the KIX-binding. Kinetic analysis of high-temperature MD simulations shows that bound pKID and apo-pKID unfold via a three-state and a two-state process, respectively. Both kinetics and free energy landscape analyses indicate that bound pKID folds in the order of KIX access, initiation of pKID tertiary folding, folding of helix alpha(B, folding of helix alpha(A, completion of pKID tertiary folding, and finalization of pKID-KIX binding. Our data show that the folding pathways of apo-pKID are different from the bound state: the foldings of helices alpha(A and alpha(B are swapped. Here we also show that Asn139, Asp140 and Leu141 with large Phi-values are key residues in the folding of bound pKID. Our results are in good agreement with NMR experimental observations and provide significant insight into the general mechanisms of binding induced protein folding and other conformational adjustment in post-translational modification.

  15. Stimulation of JNK Phosphorylation by the PTTH in Prothoracic Glands of the Silkworm, Bombyx mori

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    Shi-Hong Gu

    2018-02-01

    Full Text Available In this study, phosphorylation of c-Jun N-terminal kinase (JNK by the prothoracicotropic hormone (PTTH was investigated in prothoracic glands (PGs of the silkworm, Bombyx mori. Results showed that JNK phosphorylation was stimulated by the PTTH in time- and dose-dependent manners. In vitro activation of JNK phosphorylation in PGs by the PTTH was also confirmed in an in vivo experiment, in which a PTTH injection greatly increased JNK phosphorylation in PGs of day-6 last instar larvae. JNK phosphorylation caused by PTTH stimulation was greatly inhibited by U73122, a potent and specific inhibitor of phospholipase C (PLC and an increase in JNK phosphorylation was also detected when PGs were treated with agents (either A23187 or thapsigargin that directly elevated the intracellular Ca2+ concentration, thereby indicating involvement of PLC and Ca2+. Pretreatment with an inhibitor (U0126 of mitogen-activated protein kinase (MAPK/extracellular signal-regulated kinase (ERK kinase (MEK and an inhibitor (LY294002 of phosphoinositide 3-kinase (PI3K failed to significantly inhibit PTTH-stimulated JNK phosphorylation, indicating that ERK and PI3K were not related to JNK. We further investigated the effect of modulation of the redox state on JNK phosphorylation. In the presence of either an antioxidant (N-acetylcysteine, NAC or diphenylene iodonium (DPI, PTTH-stimulated JNK phosphorylation was blocked. The JNK kinase inhibitor, SP600125, markedly inhibited PTTH-stimulated JNK phosphorylation and ecdysteroid synthesis. The kinase assay of JNK in PGs confirmed its stimulation by PTTH and inhibition by SP600125. Moreover, PTTH treatment did not affect JNK or Jun mRNA expressions. Based on these findings, we concluded that PTTH stimulates JNK phosphorylation in Ca2+- and PLC-dependent manners and that the redox-regulated JNK signaling pathway is involved in PTTH-stimulated ecdysteroid synthesis in B. mori PGs.

  16. Cdc15 Phosphorylates the C-terminal Domain of RNA Polymerase II for Transcription during Mitosis.

    Science.gov (United States)

    Singh, Amit Kumar; Rastogi, Shivangi; Shukla, Harish; Asalam, Mohd; Rath, Srikanta Kumar; Akhtar, Md Sohail

    2017-03-31

    In eukaryotes, the basal transcription in interphase is orchestrated through the regulation by kinases (Kin28, Bur1, and Ctk1) and phosphatases (Ssu72, Rtr1, and Fcp1), which act through the post-translational modification of the C-terminal domain (CTD) of the largest subunit of RNA polymerase II. The CTD comprises the repeated Tyr-Ser-Pro-Thr-Ser-Pro-Ser motif with potential epigenetic modification sites. Despite the observation of transcription and periodic expression of genes during mitosis with entailing CTD phosphorylation and dephosphorylation, the associated CTD specific kinase(s) and its role in transcription remains unknown. Here we have identified Cdc15 as a potential kinase phosphorylating Ser-2 and Ser-5 of CTD for transcription during mitosis in the budding yeast. The phosphorylation of CTD by Cdc15 is independent of any prior Ser phosphorylation(s). The inactivation of Cdc15 causes reduction of global CTD phosphorylation during mitosis and affects the expression of genes whose transcript levels peak during mitosis. Cdc15 also influences the complete transcription of clb2 gene and phosphorylates Ser-5 at the promoter and Ser-2 toward the 3' end of the gene. The observation that Cdc15 could phosphorylate Ser-5, as well as Ser-2, during transcription in mitosis is in contrast to the phosphorylation marks put by the kinases in interphase (G 1 , S, and G 2 ), where Cdck7/Kin28 phosphorylates Ser-5 at promoter and Bur1/Ctk1 phosphorylates Ser-2 at the 3' end of the genes. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  17. Petri net-based prediction of therapeutic targets that recover abnormally phosphorylated proteins in muscle atrophy.

    Science.gov (United States)

    Jung, Jinmyung; Kwon, Mijin; Bae, Sunghwa; Yim, Soorin; Lee, Doheon

    2018-03-05

    Muscle atrophy, an involuntary loss of muscle mass, is involved in various diseases and sometimes leads to mortality. However, therapeutics for muscle atrophy thus far have had limited effects. Here, we present a new approach for therapeutic target prediction using Petri net simulation of the status of phosphorylation, with a reasonable assumption that the recovery of abnormally phosphorylated proteins can be a treatment for muscle atrophy. The Petri net model was employed to simulate phosphorylation status in three states, i.e. reference, atrophic and each gene-inhibited state based on the myocyte-specific phosphorylation network. Here, we newly devised a phosphorylation specific Petri net that involves two types of transitions (phosphorylation or de-phosphorylation) and two types of places (activation with or without phosphorylation). Before predicting therapeutic targets, the simulation results in reference and atrophic states were validated by Western blotting experiments detecting five marker proteins, i.e. RELA, SMAD2, SMAD3, FOXO1 and FOXO3. Finally, we determined 37 potential therapeutic targets whose inhibition recovers the phosphorylation status from an atrophic state as indicated by the five validated marker proteins. In the evaluation, we confirmed that the 37 potential targets were enriched for muscle atrophy-related terms such as actin and muscle contraction processes, and they were also significantly overlapping with the genes associated with muscle atrophy reported in the Comparative Toxicogenomics Database (p-value net. We generated a list of the potential therapeutic targets whose inhibition recovers abnormally phosphorylated proteins in an atrophic state. They were evaluated by various approaches, such as Western blotting, GO terms, literature, known muscle atrophy-related genes and shortest path analysis. We expect the new proposed strategy to provide an understanding of phosphorylation status in muscle atrophy and to provide assistance towards

  18. O-GlcNAc modification: why so intimately associated with phosphorylation?

    Directory of Open Access Journals (Sweden)

    Ande Sudharsana R

    2011-01-01

    Full Text Available Abstract Post-translational modification of proteins at serine and threonine side chains by β-N-acetylglucosamine (O-GlcNAc mediated by the enzyme β-N-acetylglucosamine transferase has been emerging as a fundamental regulatory mechanism encompassing a wide range of proteins involved in cell division, metabolism, transcription and cell signaling. Furthermore, an extensive interplay between O-GlcNAc modification and serine/threonine phosphorylation in a variety of proteins has been reported to exist. However, our understanding of the regulatory mechanisms involved in O-GlcNAc modification and its interplay with serine/threonine phosphorylation in proteins is still elusive. Recent success in the mapping of O-GlcNAc modification sites in proteins as a result of technological advancement in mass spectrometry have revealed two important clues which may be inherently connected to the regulation of O-GlcNAc modification and its interplay with phosphorylation in proteins. First, almost all O-GlcNAc modified proteins are known phospho proteins. Second, the prevalence of tyrosine phosphorylation among O-GlcNAc modified proteins is exceptionally higher (~68% than its normal occurrence (~2% alone. We hypothesize that phosphorylation may be a requisite for O-GlcNAc modification and tyrosine phosphorylation plays a role in the interplay between O-GlcNAc modification and serine/threonine phosphorylation in proteins. In other words, the interplay between O-GlcNAc modification and phosphorylation is not limited to serine/threonine phosphorylation but also includes tyrosine phosphorylation. Our hypothesis provides an opportunity to understand the underlying mechanism involved in O-GlcNAc modification and its interplay with serine/threonine phosphorylation in proteins. Furthermore, implication of our hypothesis extends to tyrosine kinase signaling.

  19. Smad, PI3K/Akt, and Wnt-dependent signaling pathways are involved in BMP-4-induced ESC self-renewal.

    Science.gov (United States)

    Lee, Min Young; Lim, Hyun Woo; Lee, Sang Hun; Han, Ho Jae

    2009-08-01

    It is known that bone morphogenetic protein 4 (BMP-4) has a diverse effect on ESCs. However, its precise mechanism in mouse ESCs is not fully understood. We evaluated the effect of BMP-4 on ESC proliferation and its related signal cascades in this study. BMP-4 significantly increased the level of [(3)H]-thymidine incorporation in time- (> or =8 hours) and dose- (> or =10 ng/ml) dependent manners. Additionally, BMP-4 increased cyclin D1 and decreased p27(kip1) expression values in a time-dependent manner. The increases in BMP-4-induced [(3)H]-thymidine incorporation and cyclin D1 expression were inhibited by the BMP-4 receptor antagonist noggin. BMP-4 increased Wnt1 expression. Wnt1 expression was attenuated by Smad4 small interfering RNA (siRNA), and BMP-4-induced cyclin D1 expression was inhibited by Smad4 and Wnt1 siRNAs. BMP-4 also activated beta-catenin, which was blocked by Smad4 and Wnt1 siRNAs. In addition, BMP-4 induced Akt phosphorylation. BMP-4-induced beta-catenin activation and cyclin D1 expression were attenuated by phosphatidyl inositol 3-kinase (PI3K) siRNA and Akt inhibitor. Additionally, downregulation of Smad4, Wnt1, and PI3K expression by siRNA decreased the levels of pluripotency marker mRNAs of ESCs, including Oct4, Sox2, and FoxD3. Our results suggested that BMP-4-induced [(3)H]-thymidine incorporation was significantly attenuated by Smad4, Wnt1, and PI3K knockdown. In conclusion, BMP-4 contributed to the maintenance of cell proliferation and the pluripotent state by Smad, PI3K/Akt, and Wnt1/beta-catenin in mouse ESCs.

  20. Silencing of reversion-inducing cysteine-rich protein with Kazal motifs stimulates hyperplastic phenotypes through activation of epidermal growth factor receptor and hypoxia-inducible factor-2α.

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    You Mie Lee

    Full Text Available Reversion-inducing cysteine-rich protein with Kazal motifs (RECK, a tumor suppressor is down-regulated by the oncogenic signals and hypoxia, but the biological function of RECK in early tumorigenic hyperplastic phenotypes is largely unknown. Knockdown of RECK by small interfering RNA (siRECK or hypoxia significantly promoted cell proliferation in various normal epithelial cells. Hypoxia as well as knockdown of RECK by siRNA increased the cell cycle progression, the levels of cyclin D1 and c-Myc, and the phosphorylation of Rb protein (p-pRb, but decreased the expression of p21(cip1, p27(kip1, and p16(ink4A. HIF-2α was upregulated by knockdown of RECK, indicating HIF-2α is a downstream target of RECK. As knockdown of RECK induced the activation of epidermal growth factor receptor (EGFR and treatment of an EGFR kinase inhibitor, gefitinib, suppressed HIF-2α expression induced by the silencing of RECK, we can suggest that the RECK silenicng-EGFR-HIF-2α axis might be a key molecular mechanism to induce hyperplastic phenotype of epithelial cells. It was also found that shRNA of RECK induced larger and more numerous colonies than control cells in an anchorage-independent colony formation assay. Using a xenograft assay, epithelial cells with stably transfected with shRNA of RECK formed a solid mass earlier and larger than those with control cells in nude mice. In conclusion, the suppression of RECK may promote the development of early tumorigenic hyperplastic characteristics in hypoxic stress.

  1. Anoxia-responsive regulation of the FoxO transcription factors in freshwater turtles, Trachemys scripta elegans.

    Science.gov (United States)

    Krivoruchko, Anastasia; Storey, Kenneth B

    2013-11-01

    The forkhead class O (FoxO) transcription factors are important regulators of multiple aspects of cellular metabolism. We hypothesized that activation of these transcription factors could play crucial roles in low oxygen survival in the anoxia-tolerant turtle, Trachemys scripta elegans. Two FoxOs, FoxO1 and FoxO3, were examined in turtle tissues in response to 5 and 20h of anoxic submergence using techniques of RT-PCR, western immunoblotting and DNA-binding assays to assess activation. Transcript levels of FoxO-responsive genes were also quantified using RT-PCR. FoxO1 was anoxia-responsive in the liver, with increases in transcript levels, protein levels, nuclear levels and DNA-binding of 1.7-4.8fold in response to anoxia. Levels of phosphorylated FoxO1 also decreased to 57% of control values in response to 5h of anoxia, indicating activation. FoxO3 was activated in the heart, kidney and liver in response to anoxia, with nuclear levels increasing by 1.5-3.7fold and DNA-binding activity increasing by 1.3-2.9fold. Transcript levels of two FoxO-target genes, p27kip1 and catalase, also rose by 2.4-2.5fold in the turtle liver under anoxia. The results suggest that the FoxO transcription factors are activated in response to anoxia in T. scripta elegans, potentially contributing to the regulation of stress resistance and metabolic depression. This study provides the first demonstration of activation of FoxOs in a natural model for vertebrate anoxia tolerance, further improving understanding of how tissues can survive without oxygen. © 2013.

  2. The goya mouse mutant reveals distinct newly identified roles for MAP3K1 in the development and survival of cochlear sensory hair cells

    Directory of Open Access Journals (Sweden)

    Andrew Parker

    2015-12-01

    Full Text Available Mitogen-activated protein kinase, MAP3K1, plays an important role in a number of cellular processes, including epithelial migration during eye organogenesis. In addition, studies in keratinocytes indicate that MAP3K1 signalling through JNK is important for actin stress fibre formation and cell migration. However, MAP3K1 can also act independently of JNK in the regulation of cell proliferation and apoptosis. We have identified a mouse mutant, goya, which exhibits the eyes-open-at-birth and microphthalmia phenotypes. In addition, these mice also have hearing loss. The goya mice carry a splice site mutation in the Map3k1 gene. We show that goya and kinase-deficient Map3k1 homozygotes initially develop supernumerary cochlear outer hair cells (OHCs that subsequently degenerate, and a progressive profound hearing loss is observed by 9 weeks of age. Heterozygote mice also develop supernumerary OHCs, but no cellular degeneration or hearing loss is observed. MAP3K1 is expressed in a number of inner-ear cell types, including outer and inner hair cells, stria vascularis and spiral ganglion. Investigation of targets downstream of MAP3K1 identified an increase in p38 phosphorylation (Thr180/Tyr182 in multiple cochlear tissues. We also show that the extra OHCs do not arise from aberrant control of proliferation via p27KIP1. The identification of the goya mutant reveals a signalling molecule involved with hair-cell development and survival. Mammalian hair cells do not have the ability to regenerate after damage, which can lead to irreversible sensorineural hearing loss. Given the observed goya phenotype, and the many diverse cellular processes that MAP3K1 is known to act upon, further investigation of this model might help to elaborate upon the mechanisms underlying sensory hair cell specification, and pathways important for their survival. In addition, MAP3K1 is revealed as a new candidate gene for human sensorineural hearing loss.

  3. The cytoplasmic C-terminus of polycystin-1 increases cell proliferation in kidney epithelial cells through serum-activated and Ca2+-dependent pathway(s)

    International Nuclear Information System (INIS)

    Manzati, Elisa; Aguiari, Gianluca; Banzi, Manuela; Manzati, Michele; Selvatici, Rita; Falzarano, Sofia; Maestri, Iva; Pinton, Paolo; Rizzuto, Rosario; Senno, Laura del

    2005-01-01

    Polycystin-1 (PC1) is a large transmembrane protein important in renal differentiation and defective in most cases of autosomal dominant polycystic kidney disease (ADPKD), a common cause of renal failure in adults. Although the genetic basis of ADPKD has been elucidated, molecular and cellular mechanisms responsible for the dysregulation of epithelial cell growth in ADPKD cysts are still not well defined. We approached this issue by investigating the role of the carboxyl cytoplasmic domain of PC1 involved in signal transduction on the control of kidney cell proliferation. Therefore, we generated human HEK293 cells stably expressing the PC1 cytoplasmic tail as a membrane targeted TrkA-PC1 chimeric receptor protein (TrkPC1). We found that TrkPC1 increased cell proliferation through an increase in cytoplasmic Ca 2+ levels and activation of PKCα, thereby upregulating D1 and D3 cyclin, downregulating p21 waf1 and p27 kip1 cyclin inhibitors, and thus inducing cell cycle progression from G0/G1 to the S phase. Interestingly, TrkPC1-dependent Ca 2+ increase and PKCα activation are not constitutive, but require serum factor(s) as parallel component. In agreement with this observation, a significant increase in ERK1/2 phosphorylation was observed. Consistently, inhibitors specifically blocking either PKCα or ERK1/2 prevented the TrkPC1-dependent proliferation increase. NGF, the TrkA ligand, blocked this increase. We propose that in kidney epithelial cells the overexpression of PC1 C-terminus upregulates serum-evoked intracellular Ca 2+ by counteracting the growth-suppression activity of endogenous PC1 and leading to an increase in cell proliferation

  4. ANGPTL3 is a novel biomarker as it activates ERK/MAPK pathway in oral cancer

    International Nuclear Information System (INIS)

    Koyama, Tomoyoshi; Ogawara, Katsunori; Kasamatsu, Atsushi; Okamoto, Atsushi; Kasama, Hiroki; Minakawa, Yasuyuki; Shimada, Ken; Yokoe, Hidetaka; Shiiba, Masashi; Tanzawa, Hideki; Uzawa, Katsuhiro

    2015-01-01

    Angiopoietin-like 3 (ANGPTL3), which is involved in new blood vessel growth and stimulation of mitogen-activated protein kinase (MAPK), is expressed aberrantly in several types of human cancers. However, little is known about the relevance of ANGPTL3 in the behavior of oral squamous cell carcinoma (OSCC). In this study, we evaluated ANGPTL3 mRNA and protein in OSCC-derived cell lines (n = 8) and primary OSCCs (n = 109) and assessed the effect of ANGPTL3 on the biology and function of OSCCs in vitro and in vivo. Significant (P < 0.05) ANGPTL3 upregulation was detected in the cell lines and most primary OSCCs (60%) compared with the normal counterparts. The ANGPTL3 expression level was correlated closely (P < 0.05) with tumoral size. In patients with T3/T4 tumors, the overall survival rate with an ANGPTL3-positive tumor was significantly (P < 0.05) lower than that of ANGPTL3-negative cases. In vitro, cellular growth in ANGPTL3 knockdown cells significantly (P < 0.05) decreased with inactivated extracellular regulated kinase (ERK) and cell-cycle arrest at the G1 phase resulting from upregulation of the cyclin-dependent kinase inhibitors, including p21 Cip1 and p27 Kip1 . We also observed a marked (P < 0.05) reduction in the growth in ANGPTL3 knockdown-cell xenografts with decreased levels of phosphorylated ERK relative to control-cell xenografts. The current data indicated that ANGPTL3 may play a role in OSCCs via MAPK signaling cascades, making it a potentially useful diagnostic/therapeutic target for use in patients with OSCC

  5. Identification of Ser-543 as the major regulatory phosphorylation site in spinach leaf nitrate reductase

    Science.gov (United States)

    Bachmann, M.; Shiraishi, N.; Campbell, W. H.; Yoo, B. C.; Harmon, A. C.; Huber, S. C.; Davies, E. (Principal Investigator)

    1996-01-01

    Spinach leaf NADH:nitrate reductase (NR) responds to light/dark signals and photosynthetic activity in part as a result of rapid regulation by reversible protein phosphorylation. We have identified the major regulatory phosphorylation site as Ser-543, which is located in the hinge 1 region connecting the cytochrome b domain with the molybdenum-pterin cofactor binding domain of NR, using recombinant NR fragments containing or lacking the phosphorylation site sequence. Studies with NR partial reactions indicated that the block in electron flow caused by phosphorylation also could be localized to the hinge 1 region. A synthetic peptide (NR6) based on the phosphorylation site sequence was phosphorylated readily by NR kinase (NRk) in vitro. NR6 kinase activity tracked the ATP-dependent inactivation of NR during several chromatographic steps and completely inhibited inactivation/phosphorylation of native NR in vitro. Two forms of NRk were resolved by using anion exchange chromatography. Studies with synthetic peptide analogs indicated that both forms of NRk had similar specificity determinants, requiring a basic residue at P-3 (i.e., three amino acids N-terminal to the phosphorylated serine) and a hydrophobic residue at P-5. Both forms are strictly calcium dependent but belong to distinct families of protein kinases because they are distinct immunochemically.

  6. Calcium-regulated in vivo protein phosphorylation in Zea mays L. root tips

    Science.gov (United States)

    Raghothama, K. G.; Reddy, A. S.; Friedmann, M.; Poovaiah, B. W.

    1987-01-01

    Calcium dependent protein phosphorylation was studied in corn (Zea mays L.) root tips. Prior to in vivo protein phosphorylation experiments, the effect of calcium, ethyleneglycol-bis-(beta-aminoethyl ether)-N-N' -tetraacetic acid (EGTA) and calcium ionophore (A-23187) on phosphorus uptake was studied. Calcium increased phosphorus uptake, whereas EGTA and A-23187 decreased it. Consequently, phosphorus concentration in the media was adjusted so as to attain similar uptake in different treatments. Phosphoproteins were analyzed by two-dimensional gel electrophoresis. Distinct changes in phosphorylation were observed following altered calcium levels. Calcium depletion in root tips with EGTA and A-23187 decreased protein phosphorylation. However, replenishment of calcium following EGTA and ionophore pretreatment enhanced phosphorylation of proteins. Preloading of the root tips with 32P in the presence of EGTA and A-23187 followed by a ten minute calcium treatment, resulted in increased phosphorylation indicating the involvement of calcium, calcium and calmodulin-dependent kinases. Calmodulin antagonist W-7 was effective in inhibiting calcium-promoted phosphorylation. These studies suggest a physiological role for calcium-dependent phosphorylation in calcium-mediated processes in plants.

  7. Pea DNA topoisomerase I is phosphorylated and stimulated by casein kinase 2 and protein kinase C.

    Science.gov (United States)

    Tuteja, Narendra; Reddy, Malireddy Kodandarami; Mudgil, Yashwanti; Yadav, Badam Singh; Chandok, Meena Rani; Sopory, Sudhir Kumar

    2003-08-01

    DNA topoisomerase I catalyzes the relaxation of superhelical DNA tension and is vital for DNA metabolism; therefore, it is essential for growth and development of plants. Here, we have studied the phosphorylation-dependent regulation of topoisomerase I from pea (Pisum sativum). The purified enzyme did not show autophosphorylation but was phosphorylated in an Mg(2+)-dependent manner by endogenous protein kinases present in pea nuclear extracts. This phosphorylation was abolished with calf intestinal alkaline phosphatase and lambda phosphatase. It was also phosphorylated by exogenous casein kinase 2 (CK2), protein kinase C (PKC; from animal sources), and an endogenous pea protein, which was purified using a novel phorbol myristate acetate affinity chromatography method. All of these phosphorylations were inhibited by heparin (inhibitor of CK2) and calphostin (inhibitor of PKC), suggesting that pea topoisomerase I is a bona fide substrate for these kinases. Spermine and spermidine had no effect on the CK2-mediated phosphorylation, suggesting that it is polyamine independent. Phospho-amino acid analysis showed that only serine residues were phosphorylated, which was further confirmed using antiphosphoserine antibody. The topoisomerase I activity increased after phosphorylation with exogenous CK2 and PKC. This study shows that these kinases may contribute to the physiological regulation of DNA topoisomerase I activity and overall DNA metabolism in plants.

  8. Involvement of Phosphorylated "Apis Mellifera" CREB in Gating a Honeybee's Behavioral Response to an External Stimulus

    Science.gov (United States)

    Gehring, Katrin B.; Heufelder, Karin; Feige, Janina; Bauer, Paul; Dyck, Yan; Ehrhardt, Lea; Kühnemund, Johannes; Bergmann, Anja; Göbel, Josefine; Isecke, Marlene; Eisenhardt, Dorothea

    2016-01-01

    The transcription factor cAMP-response element-binding protein (CREB) is involved in neuronal plasticity. Phosphorylation activates CREB and an increased level of phosphorylated CREB is regarded as an indicator of CREB-dependent transcriptional activation. In honeybees ("Apis mellifera") we recently demonstrated a particular high…

  9. Detection of phosphorylation states by intermolecular sensitization of lanthanide-peptide conjugates.

    Science.gov (United States)

    Pazos, Elena; Goličnik, Marko; Mascareñas, José L; Vázquez, M Eugenio

    2012-10-04

    The luminescence of a designed peptide equipped with a coordinatively-unsaturated lanthanide complex is modulated by the phosphorylation state of a serine residue in the sequence. While the phosphorylated state is weakly emissive, even in the presence of an external antenna, removal of the phosphate allows coordination of the sensitizer to the metal, yielding a highly emissive supramolecular complex.

  10. Model and simulation of Na+/K+ pump phosphorylation in the presence of palytoxin.

    Science.gov (United States)

    Rodrigues, Antônio M; Almeida, Antônio-Carlos G; Infantosi, Antonio F C; Teixeira, Hewerson Z; Duarte, Mario A

    2008-02-01

    The ATP hydrolysis reactions responsible for the Na(+)/K(+)-ATPase phosphorylation, according to recent experimental evidences, also occur for the PTX-Na(+)/K(+) pump complex. Moreover, it has been demonstrated that PTX interferes with the enzymes phosphorylation status. However, the reactions involved in the PTX-Na(+)/K(+) pump complex phosphorylation are not very well established yet. This work aims at proposing a reaction model for PTX-Na(+)/K(+) pump complex, with similar structure to the Albers-Post model, to contribute to elucidate the PTX effect over Na(+)/K(+)-ATPase phosphorylation and dephosphorylation. Computational simulations with the proposed model support several hypotheses and also suggest: (i) phosphorylation promotes an increase of the open probability of induced channels; (ii) PTX reduces the Na(+)/K(+) pump phosphorylation rate; (iii) PTX may cause conformational changes to substates where the Na(+)/K(+)-ATPase may not be phosphorylated; (iv) PTX can bind to substates of the two principal states E1 and E2, with highest affinity to phosphorylated enzymes and with ATP bound to its low-affinity sites. The proposed model also allows previewing the behavior of the PTX-pump complex substates for different levels of intracellular ATP concentrations.

  11. High-accuracy identification and bioinformatic analysis of in vivo protein phosphorylation sites in yeast

    DEFF Research Database (Denmark)

    Gnad, Florian; de Godoy, Lyris M F; Cox, Jürgen

    2009-01-01

    Protein phosphorylation is a fundamental regulatory mechanism that affects many cell signaling processes. Using high-accuracy MS and stable isotope labeling in cell culture-labeling, we provide a global view of the Saccharomyces cerevisiae phosphoproteome, containing 3620 phosphorylation sites ma...

  12. ERK5 pathway regulates the phosphorylation of tumour suppressor hDlg during mitosis

    Energy Technology Data Exchange (ETDEWEB)

    Inesta-Vaquera, Francisco A. [Departamento de Inmunologia y Oncologia, Centro Nacional de Biotecnologia-CSIC, Campus de Cantoblanco-UAM, 28049 Madrid (Spain); Campbell, David G.; Arthur, J. Simon C. [MRC Protein Phosphorylation Unit, Sir James Black Building, School of Life Sciences, University of Dundee, Dundee DD1 5EH (United Kingdom); Cuenda, Ana, E-mail: acuenda@cnb.csic.es [Departamento de Inmunologia y Oncologia, Centro Nacional de Biotecnologia-CSIC, Campus de Cantoblanco-UAM, 28049 Madrid (Spain)

    2010-08-13

    Research highlights: {yields} hDlg is phosphorylated during mitosis in multiple residues. {yields} Prospho-hDlg is excluded from the midbody during mitosis. {yields} hDlg is not phosphorylated by p38{gamma} or JNK1/2 during mitosis. {yields} ERK5 pathway mediates hDlg phosphorylation in mitosis. -- Abstract: Human disc-large (hDlg) is a scaffold protein critical for the maintenance of cell polarity and adhesion. hDlg is thought to be a tumour suppressor that regulates the cell cycle and proliferation. However, the mechanism and pathways involved in hDlg regulation during these processes is still unclear. Here we report that hDlg is phosphorylated during mitosis, and we establish the identity of at least three residues phosphorylated in hDlg; some are previously unreported. Phosphorylation affects hDlg localisation excluding it from the contact point between the two daughter cells. Our results reveal a previously unreported pathway for hDlg phosphorylation in mitosis and show that ERK5 pathway mediates hDlg cell cycle dependent phosphorylation. This is likely to have important implications in the correct timely mitotic entry and mitosis progression.

  13. NetPhosYeast: prediction of protein phosphorylation sites in yeast

    DEFF Research Database (Denmark)

    Ingrell, C.R.; Miller, Martin Lee; Jensen, O.N.

    2007-01-01

    sites compared to those in humans, suggesting the need for an yeast-specific phosphorylation site predictor. NetPhosYeast achieves a correlation coefficient close to 0.75 with a sensitivity of 0.84 and specificity of 0.90 and outperforms existing predictors in the identification of phosphorylation sites...

  14. Distinct phosphorylation events regulate p130- and p107-mediated repression of E2F-4

    DEFF Research Database (Denmark)

    Farkas, Thomas; Hansen, Klaus; Holm, Karin

    2002-01-01

    The "pocket proteins" pRb (retinoblastoma tumor suppressor protein), p107, and p130 regulate cell proliferation via phosphorylation-sensitive interactions with E2F transcription factors and other proteins. We previously identified 22 in vivo phosphorylation sites in human p130, including three...

  15. A CK2 site is reversibly phosphorylated in the photosystem II subunit CP29

    NARCIS (Netherlands)

    Testi, Maria Grazia; Croce, Roberta; Polverino-De Laureto, Patrizia; Bassi, Roberto

    1996-01-01

    Protein phosphorylation is a major mechanism in the regulation of protein function. In chloroplast thylakoids several photosystem II subunits, including the major antenna light-harvesting complex II and several core complex components, are reversibly phosphorylated depending on the redox state of

  16. Phosphorylation of nm23/nucleoside diphosphate kinase by casein kinase 2 in vitro

    DEFF Research Database (Denmark)

    Engel, M; Issinger, O G; Lascu, I

    1994-01-01

    We have investigated phosphorylation of human nucleoside diphosphate kinase (NDPK) and of homologous NDPK from different species by human casein kinase 2 (CK-2). The human NDPK isotypes A and B were phosphorylated by CK-2 in vitro both when the purified proteins and total lysate of HL-60 leukemia...

  17. ERK5 pathway regulates the phosphorylation of tumour suppressor hDlg during mitosis

    International Nuclear Information System (INIS)

    Inesta-Vaquera, Francisco A.; Campbell, David G.; Arthur, J. Simon C.; Cuenda, Ana

    2010-01-01

    Research highlights: → hDlg is phosphorylated during mitosis in multiple residues. → Prospho-hDlg is excluded from the midbody during mitosis. → hDlg is not phosphorylated by p38γ or JNK1/2 during mitosis. → ERK5 pathway mediates hDlg phosphorylation in mitosis. -- Abstract: Human disc-large (hDlg) is a scaffold protein critical for the maintenance of cell polarity and adhesion. hDlg is thought to be a tumour suppressor that regulates the cell cycle and proliferation. However, the mechanism and pathways involved in hDlg regulation during these processes is still unclear. Here we report that hDlg is phosphorylated during mitosis, and we establish the identity of at least three residues phosphorylated in hDlg; some are previously unreported. Phosphorylation affects hDlg localisation excluding it from the contact point between the two daughter cells. Our results reveal a previously unreported pathway for hDlg phosphorylation in mitosis and show that ERK5 pathway mediates hDlg cell cycle dependent phosphorylation. This is likely to have important implications in the correct timely mitotic entry and mitosis progression.

  18. Phospho.ELM: A database of experimentally verified phosphorylation sites in eukaryotic proteins

    DEFF Research Database (Denmark)

    Diella, F.; Cameron, S.; Gemund, C.

    2004-01-01

    Background: Post-translational phosphorylation is one of the most common protein modifications. Phosphoserine, threonine and tyrosine residues play critical roles in the regulation of many cellular processes. The fast growing number of research reports on protein phosphorylation points to a gener...

  19. Monitoring the native phosphorylation state of plasma membrane proteins from a single mouse cerebellum

    DEFF Research Database (Denmark)

    Schindler, J.; Ye, J. Y.; Jensen, Ole Nørregaard

    2013-01-01

    Neuronal processing in the cerebellum involves the phosphorylation and dephosphorylation of various plasma membrane proteins such as AMPA or NMDA receptors. Despite the importance of changes in phosphorylation pattern, no global phospho-proteome analysis has yet been performed. As plasma membrane...

  20. Vasopressin induces phosphorylation of the thiazide-sensitive sodium chloride cotransporter in the distal convoluted tubule

    DEFF Research Database (Denmark)

    Pedersen, Nis Borbye; Hofmeister, Marlene Vind; Rosenbaek, Lena L

    2010-01-01

    The thiazide-sensitive Na(+)-Cl(-) cotransporter (NCC) is important for renal electrolyte balance and its phosphorylation causes an increase in its transport activity and cellular localization. Here, we generated phospho-specific antibodies against two conserved N-terminal phosphorylation sites...

  1. Phosphorylated lignin as a halogen-free flame retardant additive for epoxy composites

    Science.gov (United States)

    Gamini P. Mendis; Sydney G. Weiss; Matthew Korey; Charles R. Boardman; Mark Dietenberger; Jeffrey P. Youngblood; John A. Howarter

    2016-01-01

    Sustainable, non-halogenated flame retardants are desired for a variety of industry applications. Lignin, as an industrially processed wood derivative, has been examined as a potential sustainable flame retardant additive to polymer systems. Here, the lignin is phosphorylated using a pyridine-catalysed esterification reaction with diphenyl phosphoryl chloride to...

  2. Identification of phosphorylation sites in protein kinase A substrates using artificial neural networks and mass spectrometry

    DEFF Research Database (Denmark)

    Hjerrild, M.; Stensballe, A.; Rasmussen, T.E.

    2004-01-01

    Protein phosphorylation plays a key role in cell regulation and identification of phosphorylation sites is important for understanding their functional significance. Here, we present an artificial neural network algorithm: NetPhosK (http://www.cbs.dtu.dk/services/NetPhosK/) that predicts protein...

  3. Serine/threonine/tyrosine phosphorylation regulates DNA binding of bacterial transcriptional regulators

    DEFF Research Database (Denmark)

    Kalantari, Aida; Derouiche, Abderahmane; Shi, Lei

    2015-01-01

    Reversible phosphorylation of bacterial transcriptional regulators (TRs) belonging to the family of two-component systems (TCSs) is a well-established mechanism for regulating gene expression. Recent evidence points to the fact that reversible phosphorylation of bacterial TRs on other types...

  4. In vitro phosphorylation of the movement protein of tomato mosaic tobamovirus by a cellular kinase.

    Science.gov (United States)

    Matsushita, Y; Hanazawa, K; Yoshioka, K; Oguchi, T; Kawakami, S; Watanabe, Y; Nishiguchi, M; Nyunoya, H

    2000-08-01

    The movement protein (MP) of tomato mosaic virus (ToMV) was produced in E. coli as a soluble fusion protein with glutathione S-transferase. When immobilized on glutathione affinity beads, the recombinant protein was phosphorylated in vitro by incubating with cell extracts of Nicotiana tabacum and tobacco suspension culture cells (BY-2) in the presence of [gamma-(32)P]ATP. Phosphorylation occurred even after washing the beads with a detergent-containing buffer, indicating that the recombinant MP formed a stable complex with some protein kinase(s) during incubation with the cell extract. Phosphoamino acid analysis revealed that the MP was phosphorylated on serine and threonine residues. Phosphorylation of the MP was decreased by addition of kinase inhibitors such as heparin, suramin and quercetin, which are known to be effective for casein kinase II (CK II). The phosphorylation level was not changed by other types of inhibitor. In addition, as shown for animal and plant CK II, [gamma-(32)P]GTP was efficiently used as a phosphoryl donor. Phosphorylation was not affected by amino acid replacements at serine-37 and serine-238, but was completely inhibited by deletion of the carboxy-terminal 9 amino acids, including threonine-256, serine-257, serine-261 and serine-263. These results suggest that the MP of ToMV could be phosphorylated in plant cells by a host protein kinase that is closely related to CK II.

  5. Tousled-like kinases phosphorylate Asf1 to promote histone supply during DNA replication

    DEFF Research Database (Denmark)

    Klimovskaia, Ilnaz M; Young, Clifford; Strømme, Caroline B

    2014-01-01

    During DNA replication, nucleosomes are rapidly assembled on newly synthesized DNA to restore chromatin organization. Asf1, a key histone H3-H4 chaperone required for this process, is phosphorylated by Tousled-like kinases (TLKs). Here, we identify TLK phosphorylation sites by mass spectrometry...

  6. FAK tyrosine phosphorylation is regulated by AMPK and controls metabolism in human skeletal muscle

    DEFF Research Database (Denmark)

    Lassiter, David G; Nylén, Carolina; Sjögren, Rasmus J O

    2018-01-01

    the FAK gene, PTK2. RESULTS: AMPK activation reduced tyrosine phosphorylation of FAK in skeletal muscle. AICAR reduced p-FAKY397in isolated human skeletal muscle and cultured myotubes. Insulin stimulation did not alter FAK phosphorylation. Serum starvation increased AMPK activation, as demonstrated...

  7. Phosphorylation variation during the cell cycle scales with structural propensities of proteins.

    Directory of Open Access Journals (Sweden)

    Stefka Tyanova

    Full Text Available Phosphorylation at specific residues can activate a protein, lead to its localization to particular compartments, be a trigger for protein degradation and fulfill many other biological functions. Protein phosphorylation is increasingly being studied at a large scale and in a quantitative manner that includes a temporal dimension. By contrast, structural properties of identified phosphorylation sites have so far been investigated in a static, non-quantitative way. Here we combine for the first time dynamic properties of the phosphoproteome with protein structural features. At six time points of the cell division cycle we investigate how the variation of the amount of phosphorylation correlates with the protein structure in the vicinity of the modified site. We find two distinct phosphorylation site groups: intrinsically disordered regions tend to contain sites with dynamically varying levels, whereas regions with predominantly regular secondary structures retain more constant phosphorylation levels. The two groups show preferences for different amino acids in their kinase recognition motifs - proline and other disorder-associated residues are enriched in the former group and charged residues in the latter. Furthermore, these preferences scale with the degree of disorderedness, from regular to irregular and to disordered structures. Our results suggest that the structural organization of the region in which a phosphorylation site resides may serve as an additional control mechanism. They also imply that phosphorylation sites are associated with different time scales that serve different functional needs.

  8. Phosphorylation Variation during the Cell Cycle Scales with Structural Propensities of Proteins

    DEFF Research Database (Denmark)

    Tyanova, S.; Frishman, D.; Cox, J.

    2013-01-01

    of the cell division cycle we investigate how the variation of the amount of phosphorylation correlates with the protein structure in the vicinity of the modified site. We find two distinct phosphorylation site groups: intrinsically disordered regions tend to contain sites with dynamically varying levels...

  9. Identification of phosphorylation sites in protein kinase A substrates using artificial neural networks and mass spectrometry

    DEFF Research Database (Denmark)

    Hjerrild, Majbrit; Stensballe, Allan; Rasmussen, Thomas E

    2011-01-01

    Protein phosphorylation plays a key role in cell regulation and identification of phosphorylation sites is important for understanding their functional significance. Here, we present an artificial neural network algorithm: NetPhosK (http://www.cbs.dtu.dk/services/NetPhosK/) that predicts protein...

  10. The physiological link between metabolic rate depression and tau phosphorylation in mammalian hibernation.

    Directory of Open Access Journals (Sweden)

    Jens T Stieler

    Full Text Available Abnormal phosphorylation and aggregation of tau protein are hallmarks of a variety of neurological disorders, including Alzheimer's disease (AD. Increased tau phosphorylation is assumed to represent an early event in pathogenesis and a pivotal aspect for aggregation and formation of neurofibrillary tangles. However, the regulation of tau phosphorylation in vivo and the causes for its increased stage of phosphorylation in AD are still not well understood, a fact that is primarily based on the lack of adequate animal models. Recently we described the reversible formation of highly phosphorylated tau protein in hibernating European ground squirrels. Hence, mammalian hibernation represents a model system very well suited to study molecular mechanisms of both tau phosphorylation and dephosphorylation under in vivo physiological conditions. Here, we analysed the extent and kinetics of hibernation-state dependent tau phosphorylation in various brain regions of three species of hibernating mammals: arctic ground squirrels, Syrian hamsters and black bears. Overall, tau protein was highly phosphorylated in torpor states and phosphorylation levels decreased after arousal in all species. Differences between brain regions, hibernation-states and phosphosites were observed with respect to degree and kinetics of tau phosphorylation. Furthermore, we tested the phosphate net turnover of tau protein to analyse potential alterations in kinase and/or phosphatase activities during hibernation. Our results demonstrate that the hibernation-state dependent phosphorylation of tau protein is specifically regulated but involves, in addition, passive, temperature driven regulatory mechanisms. By determining the activity-state profile for key enzymes of tau phosphorylation we could identify kinases potentially involved in the differentially regulated, reversible tau phosphorylation that occurs during hibernation. We show that in black bears hibernation is associated with

  11. Differential regulation of the transcriptional activity of the glucocorticoid receptor through site-specific phosphorylation

    Directory of Open Access Journals (Sweden)

    Raj Kumar

    2008-08-01

    Full Text Available Raj Kumar1, William J Calhoun21Division of Gastroenterology; 2Division of Allergy, Pulmonary, Immunology, Critical Care, and Sleep (APICS, Department of Internal Medicine, University of Texas Medical Branch, Galveston, TX, USAAbstract: Post-translational modifications such as phosphorylation are known to play an important role in the gene regulation by the transcription factors including the nuclear hormone receptor superfamily of which the glucocorticoid receptor (GR is a member. Protein phosphorylation often switches cellular activity from one state to another. Like many other transcription factors, the GR is a phosphoprotein, and phosphorylation plays an important role in the regulation of GR activity. Cell signaling pathways that regulate phosphorylation of the GR and its associated proteins are important determinants of GR function under various physiological conditions. While the role of many phosphorylation sites in the GR is still not fully understood, the role of others is clearer. Several aspects of transcription factor function, including DNA binding affinity, interaction of transactivation domains with the transcription initiation complex, and shuttling between the cytoplasmic compartments, have all been linked to site-specific phosphorylation. All major phosphorylation sites in the human GR are located in the N-terminal domain including the major transactivation domain, AF1. Available literature clearly indicates that many of these potential phosphorylation sites are substrates for multiple kinases, suggesting the potential for a very complex regulatory network. Phosphorylated GR interacts favorably with critical coregulatory proteins and subsequently enhances transcriptional activity. In addition, the activities and specificities of coregulators may be subject to similar regulation by phosphorylation. Regulation of the GR activity due to phosphorylation appears to be site-specific and dependent upon specific cell signaling cascade

  12. Postmortem Changes in Pork Muscle Protein Phosphorylation in Relation to the RN Genotype

    DEFF Research Database (Denmark)

    Lametsch, René; Larsen, Martin Røssel; Essén-Gustavsson, Birgitta

    2011-01-01

    Postmortem changes in pork muscle protein phosphorylation in relation to the RN(-) genotype were investigated using one-dimensional gel electrophoresis and a phosphor specific staining. The phosphorylation levels of several protein bands were found to be affected by the RN(-) genotype and to change...... of phosphorylation of these key enzymes during the postmortem metabolism. The results illustrate that the protein phosphorylation level of the muscle proteins could be interpreted as a global metabolic fingerprint containing information about the activity status of the enzymes in the postmortem metabolism....... during postmortem development. Glycogen phosphorylase, phosphofructokinase, and pyruvate kinase were found in protein bands affected by the RN(-) genotype, and the phosphorylation profile indicates that part of the increased rate and extended pH decline of the RN(-) genotype could be a consequence...

  13. Phosphorylation of plant plasma membrane H+-ATPase by the heterologous host S. cerevisiae

    DEFF Research Database (Denmark)

    Rudashevskaya, Elena; Ye, Juanying; Young, Clifford

     It is known, that phosphorylation of both plant and yeast plasma membrane H+-ATPase results in enzyme activation or inhibition. Several sites at the regulatory C-terminus of the enzyme have been found to undergo phosphorylation in vivo in both plant and yeast. The C-termini of plant H...... of heterologous system of yeast cells, expressing plant proton pump. Therefore identification of possible regulatory effects by phosphorylation events in plant H+-ATPase in the system is significant. A number of putative phosphorylation sites at regulatory C-domain of H+-ATPase (AHA2) have been point...... functioning of the residues and suggests, that plant H+-ATPase could be regulated by phosphorylation at several sites being in yeast cells. Plant H+-ATPase purified from yeast cells by his-tag affinity chromatography was subjected to IMAC and TiO2 for enrichment of phosphopeptides. The phosphopeptides were...

  14. Characterization of a novel phosphorylation site in the sodium-chloride cotransporter, NCC

    DEFF Research Database (Denmark)

    Rosenbaek, L L; Assentoft, M; Pedersen, N B

    2012-01-01

    The sodium-chloride cotransporter, NCC, is essential for renal electrolyte balance. NCC function can be modulated by protein phosphorylation. In this study, we characterized the role and physiological regulation of a novel phosphorylation site in NCC at Ser124 (S124). Novel phospho-specific antib......The sodium-chloride cotransporter, NCC, is essential for renal electrolyte balance. NCC function can be modulated by protein phosphorylation. In this study, we characterized the role and physiological regulation of a novel phosphorylation site in NCC at Ser124 (S124). Novel phospho......-related proline-alanine-rich kinase and oxidative stress-response kinases (SPAK and OSR1) were not able to phosphorylate NCC at S124. Protein kinase arrays identified multiple kinases that were able to bind to the region surrounding S124. Four of these kinases (IRAK2, CDK6/Cyclin D1, NLK and m...

  15. Phosphorylated SAP155, the spliceosomal component, is localized to chromatin in postnatal mouse testes

    Energy Technology Data Exchange (ETDEWEB)

    Eto, Ko, E-mail: etoko@gpo.kumamoto-u.ac.jp [Department of Biological Sciences, Graduate School of Science and Technology, Kumamoto University, Kumamoto 860-8555 (Japan); Sonoda, Yoshiyuki [Department of Biological Sciences, Graduate School of Science and Technology, Kumamoto University, Kumamoto 860-8555 (Japan); Jin, Yuji [School of Basic Medicine, Jilin Medical College, Jilin 132013 (China); Abe, Shin-ichi [Department of Biological Sciences, Graduate School of Science and Technology, Kumamoto University, Kumamoto 860-8555 (Japan)

    2010-03-19

    SAP155 is an essential component of the spliceosome and its phosphorylation is required for splicing catalysis, but little is known concerning its expression and regulation during spermatogenesis in postnatal mouse testes. We report that SAP155 is ubiquitously expressed in nuclei of germ and Sertoli cells within the seminiferous tubules of 6- and 35-day postpartum (dpp) testes. Analyses by fractionation of testes revealed that (1) phosphorylated SAP155 was found in the fraction containing nuclear structures at 6 dpp in amounts much larger than that at other ages; (2) non-phosphorylated SAP155 was detected in the fraction containing nucleoplasm; and (3) phosphorylated SAP155 was preferentially associated with chromatin. Our findings suggest that the active spliceosome, containing phosphorylated SAP155, performs pre-mRNA splicing on chromatin concomitant with transcription during testicular development.

  16. Axin-mediated CKI phosphorylation of beta-catenin at Ser 45

    DEFF Research Database (Denmark)

    Amit, Sharon; Hatzubai, Ada; Birman, Yaara

    2002-01-01

    The Wnt pathway controls numerous developmental processes via the beta-catenin-TCF/LEF transcription complex. Deregulation of the pathway results in the aberrant accumulation of beta-catenin in the nucleus, often leading to cancer. Normally, cytoplasmic beta-catenin associates with APC and axin...... and is continuously phosphorylated by GSK-3beta, marking it for proteasomal degradation. Wnt signaling is considered to prevent GSK-3beta from phosphorylating beta-catenin, thus causing its stabilization. However, the Wnt mechanism of action has not been resolved. Here we study the regulation of beta......-catenin phosphorylation and degradation by the Wnt pathway. Using mass spectrometry and phosphopeptide-specific antibodies, we show that a complex of axin and casein kinase I (CKI) induces beta-catenin phosphorylation at a single site: serine 45 (S45). Immunopurified axin and recombinant CKI phosphorylate beta...

  17. Phosphorylation-dependent regulation of plant chromatin and chromatin-associated proteins

    KAUST Repository

    Bigeard, Jean; Rayapuram, Naganand; Pflieger, Delphine; Hirt, Heribert

    2014-01-01

    In eukaryotes, most of the DNA is located in the nucleus where it is organized with histone proteins in a higher order structure as chromatin. Chromatin and chromatin-associated proteins contribute to DNA-related processes such as replication and transcription as well as epigenetic regulation. Protein functions are often regulated by PTMs among which phosphorylation is one of the most abundant PTM. Phosphorylation of proteins affects important properties, such as enzyme activity, protein stability, or subcellular localization. We here describe the main specificities of protein phosphorylation in plants and review the current knowledge on phosphorylation-dependent regulation of plant chromatin and chromatin-associated proteins. We also outline some future challenges to further elucidate protein phosphorylation and chromatin regulation.

  18. Phosphorylation-dependent regulation of plant chromatin and chromatin-associated proteins

    KAUST Repository

    Bigeard, Jean

    2014-07-10

    In eukaryotes, most of the DNA is located in the nucleus where it is organized with histone proteins in a higher order structure as chromatin. Chromatin and chromatin-associated proteins contribute to DNA-related processes such as replication and transcription as well as epigenetic regulation. Protein functions are often regulated by PTMs among which phosphorylation is one of the most abundant PTM. Phosphorylation of proteins affects important properties, such as enzyme activity, protein stability, or subcellular localization. We here describe the main specificities of protein phosphorylation in plants and review the current knowledge on phosphorylation-dependent regulation of plant chromatin and chromatin-associated proteins. We also outline some future challenges to further elucidate protein phosphorylation and chromatin regulation.

  19. A novel post-translational modification in nerve terminals: O-linked N-acetylglucosamine phosphorylation

    DEFF Research Database (Denmark)

    Graham, Mark E; Thaysen-Andersen, Morten; Bache, Nicolai

    2011-01-01

    Protein phosphorylation and glycosylation are the most common post-translational modifications observed in biology, frequently on the same protein. Assembly protein AP180 is a synapse-specific phosphoprotein and O-linked beta-N-acetylglucosamine (O-GlcNAc) modified glycoprotein. AP180 is involved......NAc-P to a Thr residue was confirmed by electron transfer dissociation MS. A second AP180 tryptic peptide was also glycosyl phosphorylated, but the site of modification was not assigned. Sequence similarities suggest there may be a common motif within AP180 involving glycosyl phosphorylation and dual flanking...... phosphorylation sites within 4 amino acid residues. This novel type of protein glycosyl phosphorylation adds a new signaling mechanism to the regulation of neurotransmission and more complexity to the study of O-GlcNAc modification....

  20. Temperature controls oxidative phosphorylation and reactive oxygen species production through uncoupling in rat skeletal muscle mitochondria.

    Science.gov (United States)

    Jarmuszkiewicz, Wieslawa; Woyda-Ploszczyca, Andrzej; Koziel, Agnieszka; Majerczak, Joanna; Zoladz, Jerzy A

    2015-06-01

    Mitochondrial respiratory and phosphorylation activities, mitochondrial uncoupling, and hydrogen peroxide formation were studied in isolated rat skeletal muscle mitochondria during experimentally induced hypothermia (25 °C) and hyperthermia (42 °C) compared to the physiological temperature of resting muscle (35 °C). For nonphosphorylating mitochondria, increasing the temperature from 25 to 42 °C led to a decrease in membrane potential, hydrogen peroxide production, and quinone reduction levels. For phosphorylating mitochondria, no temperature-dependent changes in these mitochondrial functions were observed. However, the efficiency of oxidative phosphorylation decreased, whereas the oxidation and phosphorylation rates and oxidative capacities of the mitochondria increased, with increasing assay temperature. An increase in proton leak, including uncoupling protein-mediated proton leak, was observed with increasing assay temperature, which could explain the reduced oxidative phosphorylation efficiency and reactive oxygen species production. Copyright © 2015 Elsevier Inc. All rights reserved.

  1. Phosphorylation of dynamin I on Ser-795 by protein kinase C blocks its association with phospholipids

    DEFF Research Database (Denmark)

    Powell, K A; Valova, V A; Malladi, C S

    2000-01-01

    Dynamin I is phosphorylated in nerve terminals exclusively in the cytosolic compartment and in vitro by protein kinase C (PKC). Dephosphorylation is required for synaptic vesicle retrieval, suggesting that its phosphorylation affects its subcellular localization. An in vitro phospholipid binding ...... assay was established that prevents lipid vesiculation and dynamin lipid insertion into the lipid. Dynamin I bound the phospholipid in a concentration-dependent and saturable manner, with an apparent affinity of 230 +/- 51 nM. Optimal binding occurred with mixtures of phosphatidylserine...... the phosphorylation site in PKCalpha-phosphorylated dynamin I as a single site at Ser-795, located near a binding site for the SH3 domain of p85, the regulatory subunit of phosphatidylinositol 3-kinase. However, phosphorylation had no effect on dynamin binding to a bacterially expressed p85-SH3 domain. Thus...

  2. Histone phosphorylation during radiation-induced mitotic delay in synchronous plasmodia of Physarum polycephalum

    International Nuclear Information System (INIS)

    Brewer, E.N.; Oleinick, N.L.

    1980-01-01

    Using the nearly perfect synchrony of the mitotic stages in Physarum plasmodia, and making use of 32 P as a tracer, studies were made to define the time course of histone phosphorylation during the late G2 and prophase and the alterations in that time course accompanying radiation-induced mitotic delay. Histone H1 was phosphorylated throughout the last 2-3 hours of the mitotic cycle coincident with the early stages of chromosome condensation. H1 phosphorylation appeared to be reduced in irradiated plasmodia. It is postulated that a longer time period, i.e. the mitotic delay, may be required to obtain the same eventual level of H1-phosphate. In normal cultures, nucleosome core histones were phosphorylated late in G2 and prophase, the peak corresponding closely with the γ-transition point. In irradiated plasmodia, phosphorylation of the core histones had an extended time course similar to H1. (U.K.)

  3. Mediator phosphorylation prevents stress response transcription during non-stress conditions.

    Science.gov (United States)

    Miller, Christian; Matic, Ivan; Maier, Kerstin C; Schwalb, Björn; Roether, Susanne; Strässer, Katja; Tresch, Achim; Mann, Matthias; Cramer, Patrick

    2012-12-28

    The multiprotein complex Mediator is a coactivator of RNA polymerase (Pol) II transcription that is required for the regulated expression of protein-coding genes. Mediator serves as an end point of signaling pathways and regulates Pol II transcription, but the mechanisms it uses are not well understood. Here, we used mass spectrometry and dynamic transcriptome analysis to investigate a functional role of Mediator phosphorylation in gene expression. Affinity purification and mass spectrometry revealed that Mediator from the yeast Saccharomyces cerevisiae is phosphorylated at multiple sites of 17 of its 25 subunits. Mediator phosphorylation levels change upon an external stimulus set by exposure of cells to high salt concentrations. Phosphorylated sites in the Mediator tail subunit Med15 are required for suppression of stress-induced changes in gene expression under non-stress conditions. Thus dynamic and differential Mediator phosphorylation contributes to gene regulation in eukaryotic cells.

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

    DEFF Research Database (Denmark)

    Mijakovic, Ivan; Petranovic, Dina; Macek, B

    2006-01-01

    for phosphotyrosine-containing proteins in Streptomyces griseus by immunoaffinity chromatography identified bacterial SSBs as a novel target of bacterial tyrosine kinases. Since genes encoding protein-tyrosine kinases (PTKs) have not been recognized in streptomycetes, and SSBs from Streptomyces coelicolor (Sc......SSB) and Bacillus subtilis (BsSSB) share 38.7% identity, we used a B.subtilis protein-tyrosine kinase YwqD to phosphorylate two cognate SSBs (BsSSB and YwpH) in vitro. We demonstrate that in vivo phosphorylation of B.subtilis SSB occurs on tyrosine residue 82, and this reaction is affected antagonistically...... 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...

  5. Pinpointing Phosphorylation Sites: Quantitative Filtering and a Novel Site-specific x-Ion Fragment

    DEFF Research Database (Denmark)

    Kelstrup, Christian D; Hekmat, Omid; Francavilla, Chiara

    2011-01-01

    Phosphoproteomics deals with the identification and quantification of thousands of phosphopeptides. Localizing the phosphorylation site is however much more difficult than establishing the identity of a phosphorylated peptide. Further, recent findings have raised doubts of the validity of the site......-phase phosphate rearrangement reactions during collision-induced dissociation (CID) and used these spectra to devise a quantitative filter that by comparing signal intensities of putative phosphorylated fragment ions with their nonphosphorylated counterparts allowed us to accurately pinpoint which fragment ions...... contain a phosphorylated residue and which ones do not. We also evaluated higher-energy collisional dissociation (HCD) and found this to be an accurate method for correct phosphorylation site localization with no gas-phase rearrangements observed above noise level. Analyzing a large set of HCD spectra...

  6. Phosphorylation of p300 by ATM controls the stability of NBS1

    International Nuclear Information System (INIS)

    Jang, Eun Ryoung; Choi, Jae Duk; Jeong, Gajin; Lee, Jong-Soo

    2010-01-01

    Acetyltransferase, p300 is a transcriptional cofactor of signal-responsive transcriptional regulation. The surveillance kinase ataxia-telangiectasia mutated (ATM) plays a central role in regulation of a wide range of cellular DNA damage responses. Here, we investigated whether and how ATM mediates phosphorylation of p300 in response to DNA damage and how p300 phosphorylation is functionally linked to DNA damage. ATM-phosphorylated p300 in vitro and in vivo, in response to DNA damage. Phosphorylation of p300 proteins was observed upon γ-irradiation in ATM + cells but not ATM - cells. Importantly, expression of nonphosphorylatable serine to alanine form of p300 (S106A) destabilized both p300 and NBS1 proteins, after DNA damage. These data demonstrate that ATM transduces a DNA damage signal to p300, and that ATM-dependent phosphorylation of p300 is required for stabilization of NBS1 proteins in response to DNA damage.

  7. Smooth muscle myosin light chain kinase efficiently phosphorylates serine 15 of cardiac myosin regulatory light chain

    International Nuclear Information System (INIS)

    Josephson, Matthew P.; Sikkink, Laura A.; Penheiter, Alan R.; Burghardt, Thomas P.; Ajtai, Katalin

    2011-01-01

    Highlights: ► Cardiac myosin regulatory light chain (MYL2) is phosphorylated at S15. ► Smooth muscle myosin light chain kinase (smMLCK) is a ubiquitous kinase. ► It is a widely believed that MYL2 is a poor substrate for smMLCK. ► In fact, smMLCK efficiently and rapidly phosphorylates S15 in MYL2. ► Phosphorylation kinetics measured by novel fluorescence method without radioactivity. -- Abstract: Specific phosphorylation of the human ventricular cardiac myosin regulatory light chain (MYL2) modifies the protein at S15. This modification affects MYL2 secondary structure and modulates the Ca 2+ sensitivity of contraction in cardiac tissue. Smooth muscle myosin light chain kinase (smMLCK) is a ubiquitous kinase prevalent in uterus and present in other contracting tissues including cardiac muscle. The recombinant 130 kDa (short) smMLCK phosphorylated S15 in MYL2 in vitro. Specific modification of S15 was verified using the direct detection of the phospho group on S15 with mass spectrometry. SmMLCK also specifically phosphorylated myosin regulatory light chain S15 in porcine ventricular myosin and chicken gizzard smooth muscle myosin (S20 in smooth muscle) but failed to phosphorylate the myosin regulatory light chain in rabbit skeletal myosin. Phosphorylation kinetics, measured using a novel fluorescence method eliminating the use of radioactive isotopes, indicates similar Michaelis–Menten V max and K M for regulatory light chain S15 phosphorylation rates in MYL2, porcine ventricular myosin, and chicken gizzard myosin. These data demonstrate that smMLCK is a specific and efficient kinase for the in vitro phosphorylation of MYL2, cardiac, and smooth muscle myosin. Whether smMLCK plays a role in cardiac muscle regulation or response to a disease causing stimulus is unclear but it should be considered a potentially significant kinase in cardiac tissue on the basis of its specificity, kinetics, and tissue expression.

  8. Phosphorylation coexists with O-GlcNAcylation in a plant virus protein and influences viral infection.

    Science.gov (United States)

    Martínez-Turiño, Sandra; Pérez, José De Jesús; Hervás, Marta; Navajas, Rosana; Ciordia, Sergio; Udeshi, Namrata D; Shabanowitz, Jeffrey; Hunt, Donald F; García, Juan Antonio

    2018-06-01

    Phosphorylation and O-GlcNAcylation are two widespread post-translational modifications (PTMs), often affecting the same eukaryotic target protein. Plum pox virus (PPV) is a member of the genus Potyvirus which infects a wide range of plant species. O-GlcNAcylation of the capsid protein (CP) of PPV has been studied extensively, and some evidence of CP phosphorylation has also been reported. Here, we use proteomics analyses to demonstrate that PPV CP is phosphorylated in vivo at the N-terminus and the beginning of the core region. In contrast with the 'yin-yang' mechanism that applies to some mammalian proteins, PPV CP phosphorylation affects residues different from those that are O-GlcNAcylated (serines Ser-25, Ser-81, Ser-101 and Ser-118). Our findings show that PPV CP can be concurrently phosphorylated and O-GlcNAcylated at nearby residues. However, an analysis using a differential proteomics strategy based on iTRAQ (isobaric tags for relative and absolute quantitation) showed a significant enhancement of phosphorylation at Ser-25 in virions recovered from O-GlcNAcylation-deficient plants, suggesting that crosstalk between O-GlcNAcylation and phosphorylation in PPV CP takes place. Although the preclusion of phosphorylation at the four identified phosphotarget sites only had a limited impact on viral infection, the mimicking of phosphorylation prevents PPV infection in Prunus persica and weakens infection in Nicotiana benthamiana and other herbaceous hosts, prompting the emergence of potentially compensatory second mutations. We postulate that the joint action of phosphorylation and O-GlcNAcylation in the N-proximal segment of CP allows a fine-tuning of protein stability, providing the amount of CP required in each step of viral infection. © 2017 BSPP AND JOHN WILEY & SONS LTD.

  9. Phosphorylation prevents C/EBP{beta} from the calpain-dependent degradation

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Yuan-yuan; Li, Shu-fen; Qian, Shu-wen; Zhang, You-you; Liu, Yuan; Tang, Qi-Qun; Li, Xi, E-mail: lixi@shmu.edu.cn

    2012-03-16

    Highlights: Black-Right-Pointing-Pointer Phosphorylation protected C/EBP{beta} from {mu}-calpain-mediated proteolysis in vitro. Black-Right-Pointing-Pointer Phosphorylation mimic C/EBP{beta} was insensitive to calpain accelerator and inhibitor. Black-Right-Pointing-Pointer Phosphorylation on Thr{sub 188} contributed more to the stabilization of C/EBP{beta}. -- Abstract: CCAAT/enhancer-binding protein (C/EBP) {beta} plays an important role in proliferation and differentiation of 3T3-L1 preadipocytes. C/EBP{beta} is sequentially phosphorylated during the 3T3-L1 adipocyte differentiation program, first by MAPK/Cyclin A/cdk2 on Thr{sub 188} and subsequently by GSK3{beta} on Ser{sub 184} or Thr{sub 179}. Dual phosphorylation is critical for the gain of DNA binding activity of C/EBP{beta}. In this manuscript, we found that phosphorylation also contributed to the stability of C/EBP{beta}. Both ex vivo and in vitro experiments showed that phosphorylation by MAPK/Cyclin A/cdk2 and GSK3{beta} protected C/EBP{beta} from {mu}-calpain-mediated proteolysis, while phosphorylation on Thr{sub 188} by MAPK/Cyclin A/cdk2 contributed more to the stabilization of C/EBP{beta}, Further studies indicated that phosphorylation mimic C/EBP{beta} was insensitive to both calpain accelerator and calpain inhibitor. Thus, phosphorylation might contribute to the stability as well as the gain of DNA binding activity of C/EBP{beta}.

  10. Characterization and validation of new tools for measuring site-specific cardiac troponin I phosphorylation.

    Science.gov (United States)

    Thoemmes, Stephen F; Stutzke, Crystal A; Du, Yanmei; Browning, Michael D; Buttrick, Peter M; Walker, Lori A

    2014-01-31

    Phosphorylation of cardiac troponin I is a well established mechanism by which cardiac contractility is modulated. However, there are a number of phosphorylation sites on TnI which contribute singly or in combination to influence cardiac function. Accordingly, methods for accurately measuring site-specific TnI phosphorylation are needed. Currently, two strategies are employed: mass spectrometry, which is costly, difficult and has a low throughput; and Western blotting using phospho-specific antibodies, which is limited by the availability of reagents. In this report, we describe a cohort of new site-specific TnI phosphoantibodies, generated against physiologically relevant phosphorylation sites, that are superior to the current commercially available antibodies: to phospho-serine 22/23 which shows a >5-fold phospho-specificity for phosphorylated TnI; to phospho-serine 43, which has >3-fold phospho-specificity for phosphorylated TnI; and phospho-serine 150 which has >2-fold phospho-specificity for phosphorylated TnI. These new antibodies demonstrated greater sensitivity and specificity for the phosphorylated TnI than the most widely used commercially available reagents. For example, at a protein load of 20 μg of total cardiac extract, a commercially available antibody recognized both phosphorylated and dephosphorylated TnI to the same degree. At the same protein load our phospho-serine 22/23 antibody exhibited no cross-reactivity with dephosphorylated TnI. These new tools should allow a more accurate assessment and a better understanding of the role of TnI phosphorylation in the response of the heart to pathologic stress. Copyright © 2013 Elsevier B.V. All rights reserved.

  11. PER1 Phosphorylation Specifies Feeding Rhythm in Mice

    Directory of Open Access Journals (Sweden)

    Zhiwei Liu

    2014-06-01

    Full Text Available Organization of circadian behavior, physiology, and metabolism is important for human health. An S662G mutation in hPER2 has been linked to familial advanced sleep-phase syndrome (FASPS. Although the paralogous phosphorylation site S714 in PER1 is conserved in mice, its specific function in circadian organization remains unknown. Here, we find that the PER1S714G mutation accelerates the molecular feedback loop. Furthermore, hPER1S714G mice, but not hPER2S662G mice, exhibit peak time of food intake that is several hours before daily energy expenditure peaks. Both the advanced feeding behavior and the accelerated clock disrupt the phase of expression of several key metabolic regulators in the liver and adipose tissue. Consequently, hPER1S714G mice rapidly develop obesity on a high-fat diet. Our studies demonstrate that PER1 and PER2 are linked to different downstream pathways and that PER1 maintains coherence between the circadian clock and energy metabolism.

  12. Importance of glycolysis and oxidative phosphorylation in advanced melanoma

    Directory of Open Access Journals (Sweden)

    Ho Jonhan

    2012-10-01

    Full Text Available Abstract Serum lactate dehydrogenase (LDH is a prognostic factor for patients with stage IV melanoma. To gain insights into the biology underlying this prognostic factor, we analyzed total serum LDH, serum LDH isoenzymes, and serum lactate in up to 49 patients with metastatic melanoma. Our data demonstrate that high serum LDH is associated with a significant increase in LDH isoenzymes 3 and 4, and a decrease in LDH isoenzymes 1 and 2. Since LDH isoenzymes play a role in both glycolysis and oxidative phosphorylation (OXPHOS, we subsequently determined using tissue microarray (TMA analysis that the levels of proteins associated with mitochondrial function, lactate metabolism, and regulators of glycolysis were all elevated in advanced melanomas compared with nevic melanocytes. To investigate whether in advanced melanoma, the glycolysis and OXPHOS pathways might be linked, we determined expression of the monocarboxylate transporters (MCT 1 and 4. Analysis of a nevus-to-melanoma progression TMA revealed that MCT4, and to a lesser extend MCT1, were elevated with progression to advanced melanoma. Further analysis of human melanoma specimens using the Seahorse XF24 extracellular flux analyzer indicated that metastatic melanoma tumors derived a large fraction of energy from OXPHOS. Taken together, these findings suggest that in stage IV melanomas with normal serum LDH, glycolysis and OXPHOS may provide metabolic symbiosis within the same tumor, whereas in stage IV melanomas with high serum LDH glycolysis is the principle source of energy.

  13. Efficient, crosswise catalytic promiscuity among enzymes that catalyze phosphoryl transfer.

    Science.gov (United States)

    Mohamed, Mark F; Hollfelder, Florian

    2013-01-01

    The observation that one enzyme can accelerate several chemically distinct reactions was at one time surprising because the enormous efficiency of catalysis was often seen as inextricably linked to specialization for one reaction. Originally underreported, and considered a quirk rather than a fundamental property, enzyme promiscuity is now understood to be important as a springboard for adaptive evolution. Owing to the large number of promiscuous enzymes that have been identified over the last decade, and the increased appreciation for promiscuity's evolutionary importance, the focus of research has shifted to developing a better understanding of the mechanistic basis for promiscuity and the origins of tolerant or restrictive specificity. We review the evidence for widespread crosswise promiscuity amongst enzymes that catalyze phosphoryl transfer, including several members of the alkaline phosphatase superfamily, where large rate accelerations between 10(6) and 10(17) are observed for both native and multiple promiscuous reactions. This article is part of a Special Issue entitled: Chemistry and mechanism of phosphatases, diesterases and triesterases. Copyright © 2012 Elsevier B.V. All rights reserved.

  14. Oxidative Phosphorylation System in Gastric Carcinomas and Gastritis

    Science.gov (United States)

    Skaria, Tom; Wessler, Silja; Cover, Timothy L.; Posselt, Gernot; Sperl, Wolfgang; Kofler, Barbara

    2017-01-01

    Switching of cellular energy production from oxidative phosphorylation (OXPHOS) by mitochondria to aerobic glycolysis occurs in many types of tumors. However, the significance of this switching for the development of gastric carcinoma and what connection it may have to Helicobacter pylori infection of the gut, a primary cause of gastric cancer, are poorly understood. Therefore, we investigated the expression of OXPHOS complexes in two types of human gastric carcinomas (“intestinal” and “diffuse”), bacterial gastritis with and without metaplasia, and chemically induced gastritis by using immunohistochemistry. Furthermore, we analyzed the effect of HP infection on several key mitochondrial proteins. Complex I expression was significantly reduced in intestinal type (but not diffuse) gastric carcinomas compared to adjacent control tissue, and the reduction was independent of HP infection. Significantly, higher complex I and complex II expression was present in large tumors. Furthermore, higher complex II and complex III protein levels were also obvious in grade 3 versus grade 2. No differences of OXPHOS complexes and markers of mitochondrial biogenesis were found between bacterially caused and chemically induced gastritis. Thus, intestinal gastric carcinomas, but not precancerous stages, are frequently characterized by loss of complex I, and this pathophysiology occurs independently of HP infection. PMID:28744336

  15. Oxidative Phosphorylation System in Gastric Carcinomas and Gastritis.

    Science.gov (United States)

    Feichtinger, René G; Neureiter, Daniel; Skaria, Tom; Wessler, Silja; Cover, Timothy L; Mayr, Johannes A; Zimmermann, Franz A; Posselt, Gernot; Sperl, Wolfgang; Kofler, Barbara

    2017-01-01

    Switching of cellular energy production from oxidative phosphorylation (OXPHOS) by mitochondria to aerobic glycolysis occurs in many types of tumors. However, the significance of this switching for the development of gastric carcinoma and what connection it may have to Helicobacter pylori infection of the gut, a primary cause of gastric cancer, are poorly understood. Therefore, we investigated the expression of OXPHOS complexes in two types of human gastric carcinomas ("intestinal" and "diffuse"), bacterial gastritis with and without metaplasia, and chemically induced gastritis by using immunohistochemistry. Furthermore, we analyzed the effect of HP infection on several key mitochondrial proteins. Complex I expression was significantly reduced in intestinal type (but not diffuse) gastric carcinomas compared to adjacent control tissue, and the reduction was independent of HP infection. Significantly, higher complex I and complex II expression was present in large tumors. Furthermore, higher complex II and complex III protein levels were also obvious in grade 3 versus grade 2. No differences of OXPHOS complexes and markers of mitochondrial biogenesis were found between bacterially caused and chemically induced gastritis. Thus, intestinal gastric carcinomas, but not precancerous stages, are frequently characterized by loss of complex I, and this pathophysiology occurs independently of HP infection.

  16. Homeostasis of the protonmotive force in phosphorylating mitochondria.

    Science.gov (United States)

    Duszyński, J; Bogucka, K; Wojtczak, L

    1984-12-18

    The relationship between the respiration rate and the magnitude of the electrochemical proton potential (delta mu H+) in rat liver mitochondria was investigated. (1) Under the active-state conditions, the action of inhibitors of either phosphorylation (oligomycin) or respiration (rotenone, malonate) on the respiration and delta mu H+ was measured. Both inhibitors diminished the respiration, whereas rotenone resulted in a decrease of delta mu H+, and oligomycin produced an increase of this potential. The effect of the inhibitors was much more pronounced on the respiration rate than on delta mu H+; for example, the excess of oligomycin produced a 90% inhibition of the respiration while delta mu H+ was changed only by 9%. (2) Under the resting-state conditions, small concentrations of the uncoupler stimulated the respiration while changing delta mu H+ to a relatively small extent. The uncoupler concentrations which doubled and tripled the respiration rate produced only 5 and 9% decrease of delta mu H+, respectively. (3) The present results enabled us to propose a model describing the interrelationship between respiration and delta mu H+.

  17. The Phosphorylation of Ribosomal Protein in Lemna minor

    Science.gov (United States)

    Trewavas, A.

    1973-01-01

    Sterile cultures of Lemna minor have been labeled with 32P1, and the ribosomal proteins have been examined for radioactivity. In relatively short term labeling a radioactive protein was found which ran as a single component in both urea/acetic acid and sodium lauryl sulfate gel electrophoresis. Acid hydrolysis of the labeled protein permitted the isolation of serine phosphate. After labeling to equilibrium with 32P1, calculation indicated only 0.6 to 0.75 atom of this protein phosphorus per ribosome. The phosphorylated protein is found in both polysomes and “derived” monomers and appears to be located in the ribosomal small subunit. Its apparent molecular weight is 42,000. Addition of growth-inhibiting concentrations of abscisic acid does not alter the apparent degree of labeling of this protein in 5 hours, but after 24 hours of treatment the total protein phosphorus was reduced from 0.75 atom of phosphorus per ribosome to 0.36 atom of phosphorus per ribosome. PMID:16658405

  18. BAG3 controls angiogenesis through regulation of ERK phosphorylation.

    Science.gov (United States)

    Falco, A; Festa, M; Basile, A; Rosati, A; Pascale, M; Florenzano, F; Nori, S L; Nicolin, V; Di Benedetto, M; Vecchione, M L; Arra, C; Barbieri, A; De Laurenzi, V; Turco, M C

    2012-12-13

    BAG3 is a co-chaperone of the heat shock protein (Hsp) 70, is expressed in many cell types upon cell stress, however, its expression is constitutive in many tumours. We and others have previously shown that in neoplastic cells BAG3 exerts an anti-apoptotic function thus favoring tumour progression. As a consequence we have proposed BAG3 as a target of antineoplastic therapies. Here we identify a novel role for BAG3 in regulation of neo-angiogenesis and show that its downregulation results in reduced angiogenesis therefore expanding the role of BAG3 as a therapeutical target. In brief we show that BAG3 is expressed in endothelial cells and is essential for the interaction between ERK and its phosphatase DUSP6, as a consequence its removal results in reduced binding of DUSP6 to ERK and sustained ERK phosphorylation that in turn determines increased levels of p21 and p15 and cell-cycle arrest in the G1 phase.

  19. Detection and characterization of 3D-signature phosphorylation site motifs and their contribution towards improved phosphorylation site prediction in proteins

    Directory of Open Access Journals (Sweden)

    Selbig Joachim

    2009-04-01

    Full Text Available Abstract Background Phosphorylation of proteins plays a crucial role in the regulation and activation of metabolic and signaling pathways and constitutes an important target for pharmaceutical intervention. Central to the phosphorylation process is the recognition of specific target sites by protein kinases followed by the covalent attachment of phosphate groups to the amino acids serine, threonine, or tyrosine. The experimental identification as well as computational prediction of phosphorylation sites (P-sites has proved to be a challenging problem. Computational methods have focused primarily on extracting predictive features from the local, one-dimensional sequence information surrounding phosphorylation sites. Results We characterized the spatial context of phosphorylation sites and assessed its usability for improved phosphorylation site predictions. We identified 750 non-redundant, experimentally verified sites with three-dimensional (3D structural information available in the protein data bank (PDB and grouped them according to their respective kinase family. We studied the spatial distribution of amino acids around phosphorserines, phosphothreonines, and phosphotyrosines to extract signature 3D-profiles. Characteristic spatial distributions of amino acid residue types around phosphorylation sites were indeed discernable, especially when kinase-family-specific target sites were analyzed. To test the added value of using spatial information for the computational prediction of phosphorylation sites, Support Vector Machines were applied using both sequence as well as structural information. When compared to sequence-only based prediction methods, a small but consistent performance improvement was obtained when the prediction was informed by 3D-context information. Conclusion While local one-dimensional amino acid sequence information was observed to harbor most of the discriminatory power, spatial context information was identified as

  20. Insulin increase in MAP kinase phosphorylation is shifted to early time-points by overexpressing APS, while Akt phosphorylation is not influenced.

    Science.gov (United States)

    Onnockx, Sheela; Xie, Jingwei; Degraef, Chantal; Erneux, Christophe; Pirson, Isabelle

    2009-09-10

    Upon insulin stimulation, the adaptor protein APS is recruited to the insulin receptor and tyrosine phosphorylated. APS initiates the insulin-induced TC10 cascade which participates to GLUT4 translocation to the plasma membrane. Nevertheless, the molecular mechanism that governs APS and its SH2 and PH domains action on the insulin transduction cascade is not yet fully understood. Here, we show that APS co-immunoprecipitates with the class I PI 3-kinase regulatory subunit p85, through its SH2 domain but that APS does not modulate neither PtdIns(3,4,5)P3 levels nor Akt phosphorylation provoked by insulin. We have confirmed a previously described positive effect of APS overexpression on insulin-induced MAPK phosphorylation upregulation. Consequently, we analyzed the role of SH2 and PH domains of APS in the APS increased MAPK phosphorylation observed upon insulin stimulation and correlated this with the membrane localization of the protein. The effect observed on MAPK phosphorylation requires the intact PH binding domain of APS as well as its SH2 domain.

  1. Phosphorylated RPA recruits PALB2 to stalled DNA replication forks to facilitate fork recovery.

    Science.gov (United States)

    Murphy, Anar K; Fitzgerald, Michael; Ro, Teresa; Kim, Jee Hyun; Rabinowitsch, Ariana I; Chowdhury, Dipanjan; Schildkraut, Carl L; Borowiec, James A

    2014-08-18

    Phosphorylation of replication protein A (RPA) by Cdk2 and the checkpoint kinase ATR (ATM and Rad3 related) during replication fork stalling stabilizes the replisome, but how these modifications safeguard the fork is not understood. To address this question, we used single-molecule fiber analysis in cells expressing a phosphorylation-defective RPA2 subunit or lacking phosphatase activity toward RPA2. Deregulation of RPA phosphorylation reduced synthesis at forks both during replication stress and recovery from stress. The ability of phosphorylated RPA to stimulate fork recovery is mediated through the PALB2 tumor suppressor protein. RPA phosphorylation increased localization of PALB2 and BRCA2 to RPA-bound nuclear foci in cells experiencing replication stress. Phosphorylated RPA also stimulated recruitment of PALB2 to single-strand deoxyribonucleic acid (DNA) in a cell-free system. Expression of mutant RPA2 or loss of PALB2 expression led to significant DNA damage after replication stress, a defect accentuated by poly-ADP (adenosine diphosphate) ribose polymerase inhibitors. These data demonstrate that phosphorylated RPA recruits repair factors to stalled forks, thereby enhancing fork integrity during replication stress. © 2014 Murphy et al.

  2. Plk1 phosphorylation of IRS2 prevents premature mitotic exit via AKT inactivation

    Science.gov (United States)

    Chen, Long; Li, Zhiguo; Ahmad, Nihal; Liu, Xiaoqi

    2016-01-01

    Insulin receptor substrate (IRS) proteins play important roles by acting as a platform in transducing signals from transmembrane receptors upon growth factor stimulation. Although tyrosine phosphorylation on IRS proteins plays critical roles in signal transduction, phosphorylation of IRS proteins on serine/threonine residues are believed to play various regulatory roles on IRS protein function. However, studies on serine/threonine phosphorylation of IRS proteins are very limited, especially for insulin receptor substrate 2 (IRS2), one member of the IRS protein family. In this study, we identify Polo-like kinase 1 (Plk1) as the responsible kinase for phosphorylation of IRS2 on two serine residues, Ser 556 and Ser 1098. Phosphorylation of IRS2 on these two serine residues by Plk1 prevents the activation of the PI3K pathway upon growth factor stimulation by inhibiting the binding between IRS2 and the PI3K pathway components and increasing IRS2 protein degradation. Of significance, we show that IRS2 phosphorylation is cell cycle regulated and that Plk1 phosphorylation of IRS2 prevents premature mitotic exit via AKT inactivation. PMID:25830382

  3. PhosphoRice: a meta-predictor of rice-specific phosphorylation sites

    Directory of Open Access Journals (Sweden)

    Que Shufu

    2012-02-01

    Full Text Available Abstract Background As a result of the growing body of protein phosphorylation sites data, the number of phosphoprotein databases is constantly increasing, and dozens of tools are available for predicting protein phosphorylation sites to achieve fast automatic results. However, none of the existing tools has been developed to predict protein phosphorylation sites in rice. Results In this paper, the phosphorylation site predictors, NetPhos 2.0, NetPhosK, Kinasephos, Scansite, Disphos and Predphosphos, were integrated to construct meta-predictors of rice-specific phosphorylation sites using several methods, including unweighted voting, unreduced weighted voting, reduced unweighted voting and weighted voting strategies. PhosphoRice, the meta-predictor produced by using weighted voting strategy with parameters selected by restricted grid search and conditional random search, performed the best at predicting phosphorylation sites in rice. Its Matthew's Correlation Coefficient (MCC and Accuracy (ACC reached to 0.474 and 73.8%, respectively. Compared to the best individual element predictor (Disphos_default, PhosphoRice archieved a significant increase in MCC of 0.071 (P Conclusions PhosphoRice is a powerful tool for predicting unidentified phosphorylation sites in rice. Compared to the existing methods, we found that our tool showed greater robustness in ACC and MCC. PhosphoRice is available to the public at http://bioinformatics.fafu.edu.cn/PhosphoRice.

  4. Obesity does not Lead to Imbalance Between Myocardial Phospholamban Phosphorylation and Dephosphorylation

    Energy Technology Data Exchange (ETDEWEB)

    Freire, Paula Paccielli, E-mail: freirepp@hotmail.com; Alves, Carlos Augusto Barnabe; Deus, Adriana Fernandes de [Departamento de Clínica Médica - Faculdade de Medicina de Botucatu - Universidade Estadual Paulista, Botucatu, SP (Brazil); Leopoldo, Ana Paula Lima; Leopoldo, André Soares [Centro de Educação Física e Desportos - Universidade Federal do Espírito Santo, Vitória, ES (Brazil); Silva, Danielle Cristina Tomaz da; Tomasi, Loreta Casquel de; Campos, Dijon Henrique Salomé; Cicogna, Antonio Carlos [Departamento de Clínica Médica - Faculdade de Medicina de Botucatu - Universidade Estadual Paulista, Botucatu, SP (Brazil)

    2014-07-15

    The activation of the beta-adrenergic system promotes G protein stimulation that, via cyclic adenosine monophosphate (cAMP), alters the structure of protein kinase A (PKA) and leads to phospholamban (PLB) phosphorylation. This protein participates in the system that controls intracellular calcium in muscle cells, and it is the primary regulator of sarcoplasmic reticulum calcium pump activity. In obesity, the beta-adrenergic system is activated by the influence of increased leptin, therefore, resulting in higher myocardial phospholamban phosphorylation via cAMP-PKA. To investigate the involvement of proteins which regulate the degree of PLB phosphorylation due to beta-adrenergic activation in obesity. In the present study, we hypothesized that there is an imbalance between phospholamban phosphorylation and dephosphorylation, with prevalence of protein phosphorylation. Male Wistar rats were randomly distributed into two groups: control (n = 14), fed with normocaloric diet; and obese (n = 13), fed with a cycle of four unsaturated high-fat diets. Obesity was determined by the adiposity index, and protein expressions of phosphatase 1 (PP-1), PKA, PLB, phosphorylated phospholamban at serine16 (PPLB-Ser16) were assessed by Western blot. Obesity caused glucose intolerance, hyperinsulinemia, hypertriglyceridemia, hyperleptinemia and did not alter the protein expression of PKA, PP-1, PLB, PPLB-Ser16. Obesity does not promote an imbalance between myocardial PLB phosphorylation and dephosphorylation via beta-adrenergic system.

  5. Obesity does not Lead to Imbalance Between Myocardial Phospholamban Phosphorylation and Dephosphorylation

    International Nuclear Information System (INIS)

    Freire, Paula Paccielli; Alves, Carlos Augusto Barnabe; Deus, Adriana Fernandes de; Leopoldo, Ana Paula Lima; Leopoldo, André Soares; Silva, Danielle Cristina Tomaz da; Tomasi, Loreta Casquel de; Campos, Dijon Henrique Salomé; Cicogna, Antonio Carlos

    2014-01-01

    The activation of the beta-adrenergic system promotes G protein stimulation that, via cyclic adenosine monophosphate (cAMP), alters the structure of protein kinase A (PKA) and leads to phospholamban (PLB) phosphorylation. This protein participates in the system that controls intracellular calcium in muscle cells, and it is the primary regulator of sarcoplasmic reticulum calcium pump activity. In obesity, the beta-adrenergic system is activated by the influence of increased leptin, therefore, resulting in higher myocardial phospholamban phosphorylation via cAMP-PKA. To investigate the involvement of proteins which regulate the degree of PLB phosphorylation due to beta-adrenergic activation in obesity. In the present study, we hypothesized that there is an imbalance between phospholamban phosphorylation and dephosphorylation, with prevalence of protein phosphorylation. Male Wistar rats were randomly distributed into two groups: control (n = 14), fed with normocaloric diet; and obese (n = 13), fed with a cycle of four unsaturated high-fat diets. Obesity was determined by the adiposity index, and protein expressions of phosphatase 1 (PP-1), PKA, PLB, phosphorylated phospholamban at serine16 (PPLB-Ser16) were assessed by Western blot. Obesity caused glucose intolerance, hyperinsulinemia, hypertriglyceridemia, hyperleptinemia and did not alter the protein expression of PKA, PP-1, PLB, PPLB-Ser16. Obesity does not promote an imbalance between myocardial PLB phosphorylation and dephosphorylation via beta-adrenergic system

  6. Phosphorylated alpha(1 leads to 4) glucans as substrate for potato starch-branching enzyme I

    International Nuclear Information System (INIS)

    Vikso-Nielsen, A.; Blennow, A.; Nielsen, T.H.; Moller, B.L.

    1998-01-01

    The possible involvement of potato (Solanum tuberosum L.) starch-branching enzyme I (PSBE-I) in the in vivo synthesis of phosphorylated amylopectin was investigated in in vitro experiments with isolated PSBE-I using 33P-labeled phosphorylated and 3H end-labeled nonphosphorylated alpha(1 leads to 4) glucans as the substrates. From these radiolabeled substrates PSBE-I was shown to catalyze the formation of dual-labeled (3H/33P) phosphorylated branched polysaccharides with an average degree of polymerization of 80 to 85. The relatively high molecular mass indicated that the product was the result of multiple chain-transfer reactions. The presence of alpha(1 leads to 6) branch points was documented by isoamylase treatment and anion-exchange chromatography. Although the initial steps of the in vivo mechanism responsible for phosphorylation of potato starch remains elusive, the present study demonstrates that the enzyme machinery available in potato has the ability to incorporate phosphorylated alpha(1 leads to 4) glucans into neutral polysaccharides in an interchain catalytic reaction. Potato mini tubers synthesized phosphorylated starch from exogenously supplied 33PO4(3-) and [U-14C]Glc at rates 4 times higher than those previously obtained using tubers from fully grown potato plants. This system was more reproducible compared with soil-grown tubers and was therefore used for preparation of 33P-labeled phosphorylated alpha(1 leads to 4) glucan chains

  7. dbPAF: an integrative database of protein phosphorylation in animals and fungi.

    Science.gov (United States)

    Ullah, Shahid; Lin, Shaofeng; Xu, Yang; Deng, Wankun; Ma, Lili; Zhang, Ying; Liu, Zexian; Xue, Yu

    2016-03-24

    Protein phosphorylation is one of the most important post-translational modifications (PTMs) and regulates a broad spectrum of biological processes. Recent progresses in phosphoproteomic identifications have generated a flood of phosphorylation sites, while the integration of these sites is an urgent need. In this work, we developed a curated database of dbPAF, containing known phosphorylation sites in H. sapiens, M. musculus, R. norvegicus, D. melanogaster, C. elegans, S. pombe and S. cerevisiae. From the scientific literature and public databases, we totally collected and integrated 54,148 phosphoproteins with 483,001 phosphorylation sites. Multiple options were provided for accessing the data, while original references and other annotations were also present for each phosphoprotein. Based on the new data set, we computationally detected significantly over-represented sequence motifs around phosphorylation sites, predicted potential kinases that are responsible for the modification of collected phospho-sites, and evolutionarily analyzed phosphorylation conservation states across different species. Besides to be largely consistent with previous reports, our results also proposed new features of phospho-regulation. Taken together, our database can be useful for further analyses of protein phosphorylation in human and other model organisms. The dbPAF database was implemented in PHP + MySQL and freely available at http://dbpaf.biocuckoo.org.

  8. Tyrosine phosphorylation of AAV2 vectors and its consequences on viral intracellular trafficking and transgene expression

    Science.gov (United States)

    Zhong, Li; Li, Baozheng; Jayandharan, Giridhararao; Mah, Cathryn S.; Govindasamy, Lakshmanan; Agbandje-McKenna, Mavis; Herzog, Roland W.; Weigel-Van Aken, Kirsten A.; Hobbs, Jacqueline A.; Zolotukhin, Sergei; Muzyczka, Nicholas; Srivastava, Arun

    2008-01-01

    We have documented that epidermal growth factor receptor protein tyrosine kinase (EGFR-PTK) signaling negatively affects intracellular trafficking and transduction efficiency of recombinant adeno-associated virus 2 (AAV2) vectors. Specifically, inhibition of EGFR-PTK signaling leads to decreased ubiquitination of AAV2 capsid proteins, which in turn, facilitates viral nuclear transport by limiting proteasome-mediated degradation of AAV2 vectors. In the present studies, we observed that AAV capsids can indeed be phosphorylated at tyrosine residues by EGFR-PTK in in vitro phosphorylation assays and that phosphorylated AAV capsids retain their structural integrity. However, although phosphorylated AAV vectors enter cells as efficiently as their unphosphorylated counterparts, their transduction efficiency is significantly reduced. This reduction is not due to impaired viral second-strand DNA synthesis since transduction efficiency of both single-stranded AAV (ssAAV) and self-complementary AAV (scAAV) vectors is decreased by ~68% and ~74%, respectively. We also observed that intracellular trafficking of tyrosine-phosphorylated AAV vectors from cytoplasm to nucleus is significantly decreased, which leads to ubiquitination of AAV capsids followed by proteasome-mediated degradation, although downstream consequences of capsid ubiquitination may also be affected by tyrosine-phosphorylation. These studies provide new insights into the role of tyrosine-phosphorylation of AAV capsids in various steps in the virus life cycle, which has implications in the optimal use of recombinant AAV vectors in human gene therapy. PMID:18834608

  9. Insulin stimulates the tyrosine phosphorylation of a Mr = 160,000 glycoprotein in adipocyte plasma membranes

    International Nuclear Information System (INIS)

    Yu, K.T.; Khalaf, N.; Czech, M.P.

    1986-01-01

    In an attempt to identify putative substrates for the insulin receptor kinase, adipocyte plasma membranes were incubated with [γ- 32 P]ATP in the presence and absence of insulin. Insulin stimulates the tyrosine phosphorylation of its receptor β subunit but does not detectably alter the phosphorylation of other membrane proteins. In contrast, when plasma membranes from insulin-treated adipocytes are phosphorylated, the 32 P-labeling of a Mr=160,000 species (p160) and insulin receptor β subunit are markedly increased when compared to controls. p160 exhibits a rapid response (max. at 1 min) and high sensitivity (ED 50 = 2 x 10 -10 M) to insulin. The stimulatory effect of insulin on the phosphorylation of p160 is rapidly reversed following the addition of anti-insulin serum. Cold chase experiments indicate that insulin promotes the phosphorylation of p160 rather than inhibiting its dephosphorylation. p160 is a glycoprotein as evidenced by its adsorption to immobilized lectins and does not represent the insulin receptor precursor. The action of insulin on p160 tyrosine phosphorylation is mimicked by concanavalin A but not by EGF and other insulin-like agents such as hydrogen peroxide and vanadate. These results suggest that p160 tyrosine phosphorylation is an insulin receptor-mediated event and may participate in signalling by the insulin receptor

  10. Impact of SNPs on Protein Phosphorylation Status in Rice (Oryza sativa L.

    Directory of Open Access Journals (Sweden)

    Shoukai Lin

    2016-11-01

    Full Text Available Single nucleotide polymorphisms (SNPs are widely used in functional genomics and genetics research work. The high-quality sequence of rice genome has provided a genome-wide SNP and proteome resource. However, the impact of SNPs on protein phosphorylation status in rice is not fully understood. In this paper, we firstly updated rice SNP resource based on the new rice genome Ver. 7.0, then systematically analyzed the potential impact of Non-synonymous SNPs (nsSNPs on the protein phosphorylation status. There were 3,897,312 SNPs in Ver. 7.0 rice genome, among which 9.9% was nsSNPs. Whilst, a total 2,508,261 phosphorylated sites were predicted in rice proteome. Interestingly, we observed that 150,197 (39.1% nsSNPs could influence protein phosphorylation status, among which 52.2% might induce changes of protein kinase (PK types for adjacent phosphorylation sites. We constructed a database, SNP_rice, to deposit the updated rice SNP resource and phosSNPs information. It was freely available to academic researchers at http://bioinformatics.fafu.edu.cn. As a case study, we detected five nsSNPs that potentially influenced heterotrimeric G proteins phosphorylation status in rice, indicating that genetic polymorphisms showed impact on the signal transduction by influencing the phosphorylation status of heterotrimeric G proteins. The results in this work could be a useful resource for future experimental identification and provide interesting information for better rice breeding.

  11. 3-phosphorylated and -thiophosphorylated 2-thiazolidine- and 2-oxazolidine-thiones

    International Nuclear Information System (INIS)

    Vorob'eva, N.N.; Razvodovskaya, L.V.; Negrebetskii, V.V.; Grapov, A.F.; Mel'nikov, N.N.

    1987-01-01

    We investigated the phosphorylation and thiophosphorylation of 2-thiazolidine- and 2-oxazolidine-thiones. The presence in the heterocycle of the ambident triad HN-C=S can also lead to two series of phosphorylation products formed at the nitrogen and at the sulfur atom. It was therefore of interest to determine the dependence of the site of the phosphorylation on the structures of the heterocycle and off the phosphorylating agent. The formation of the N-phosphorylation products is confirmed by the 1 H NMR spectra, in which the signals of protons of the methylene group of the heteroring (C 4 H 2 ) are split on account of interaction with the phosphorus atom ( 3 JPH 0.5-2.3 Hz). We observed analogous values of 3 JPH constants for 2-aminothiazolines phosphorylated on the endocyclic nitrogen atom. In the 13 C NMR spectra of these compounds there are also coupling constants for the interaction of the carbon atoms C 4 and C 5 of the heterocycle with the phosphorus atom. The existence of the compounds as N-phosphorylated heterocycles is evidenced also by the 31 P chemical shifts

  12. Phosphorylated form of adrenocorticotropin and corticotropin-like intermediary lobe peptide in human tumors

    International Nuclear Information System (INIS)

    Massias, J.F.; Hardouin, S.; Vieau, D.; Lenne, F.; Bertagna, X.

    1994-01-01

    Many peptides contribute to the heterogeneity of immunoreactive adrenocorticotropin (ACTH) in man. The use of a radioimmunoassay (RIA) specifically directed against the C-terminal end of ACTH allowed the precise study of the following four peptides: ACTH itself, corticotropin-like intermediary lobe peptide (CLIP) or ACTH and their phosphorylated forms on SeR 31 . The authors have set up a high-performance liquid chromatography system that separates these four molecules in a single run, to establish their relative distributions in tumors responsible for Cushing's disease or for the ectopic ACTH syndrome, and to evaluate the possible interference of phospho-Ser 31 on various RIA or immuno-radiometric assay (IRMA) recognition systems for ACTH. In this system, alkaline phosphatase treatment shifted the retention time of the phosphorylated peptides to that of their non-phosphorylated counterparts. In three tumors responsible for the ectopic ACTH syndrome, CLIP peptides were predominant in two and phosphorylated molecules represented between 22% and 50% of immuno-reactive materials. In five pituitary tumors responsible for Cushing's disease, ACTH peptides were predominant and the phosphorylated molecules varied between 35% and 75% in four of them. In the same tumor the ratios of phosphorylated to non-phosphorylated CLIP or ACTH were identical. The presence of phospho-Ser 31 did not affect the recognition ability of two mid-ACTH and two C-terminal ACTH RIA's, nor of the ACTH IRMA. 15 refs., 5 figs., 2 tabs

  13. Identification and functional analysis of novel phosphorylation sites in the RNA surveillance protein Upf1.

    Science.gov (United States)

    Lasalde, Clarivel; Rivera, Andrea V; León, Alfredo J; González-Feliciano, José A; Estrella, Luis A; Rodríguez-Cruz, Eva N; Correa, María E; Cajigas, Iván J; Bracho, Dina P; Vega, Irving E; Wilkinson, Miles F; González, Carlos I

    2014-02-01

    One third of inherited genetic diseases are caused by mRNAs harboring premature termination codons as a result of nonsense mutations. These aberrant mRNAs are degraded by the Nonsense-Mediated mRNA Decay (NMD) pathway. A central component of the NMD pathway is Upf1, an RNA-dependent ATPase and helicase. Upf1 is a known phosphorylated protein, but only portions of this large protein have been examined for phosphorylation sites and the functional relevance of its phosphorylation has not been elucidated in Saccharomyces cerevisiae. Using tandem mass spectrometry analyses, we report the identification of 11 putative phosphorylated sites in S. cerevisiae Upf1. Five of these phosphorylated residues are located within the ATPase and helicase domains and are conserved in higher eukaryotes, suggesting a biological significance for their phosphorylation. Indeed, functional analysis demonstrated that a small carboxy-terminal motif harboring at least three phosphorylated amino acids is important for three Upf1 functions: ATPase activity, NMD activity and the ability to promote translation termination efficiency. We provide evidence that two tyrosines within this phospho-motif (Y-738 and Y-742) act redundantly to promote ATP hydrolysis, NMD efficiency and translation termination fidelity.

  14. Monitoring protein phosphorylation by acrylamide pendant Phos-Tag™ in various plants

    Directory of Open Access Journals (Sweden)

    Slavka eBekesova

    2015-05-01

    Full Text Available The aim of the present study is to rationalize acrylamide pendant Phos-Tag™ in-gel discrimination of phosphorylated and non-phosphorylated plant protein species with standard immunoblot analysis, and optimize sample preparation, efficient electrophoretic separation and transfer. We tested variants of the method including extraction buffers suitable for preservation of phosphorylated protein species in crude extracts from plants and we addressed the importance of the cation (Mn2+ or Zn2+ used in the gel recipe for efficient transfer to PVDF membranes for further immunoblot analysis. We demonstrate the monitoring of Medicago sativa stress-induced mitogen activated protein kinase (SIMK in stress-treated wild type plants and transgenic SIMKK RNAi line. We further show the hyperosmotically-induced phosphorylation of the previously uncharacterized HvMPK4 of barley. The method is validated using inducible phosphorylation of barley and wheat α-tubulin and of Arabidopsis MPK6. Acrylamide pendant Phos-Tag™ offers a flexible tool for studying protein phosphorylation in crops and Arabidopsis circumventing radioactive labeling and the use of phosphorylation specific antibodies.

  15. Rapid changes in plasma membrane protein phosphorylation during initiation of cell wall digestion

    International Nuclear Information System (INIS)

    Blowers, D.P.; Boss, W.F.; Trewavas, A.J.

    1988-01-01

    Plasma membrane vesicles from wild carrot cells grown in suspension culture were isolated by aqueous two-phase partitioning, and ATP-dependent phosphorylation was measured with [γ- 32 P]ATP in the presence and absence of calcium. Treatment of the carrot cells with the cell wall digestion enzymes, driselase, in a sorbitol osmoticum for 1.5 min altered the protein phosphorylation pattern compared to that of cells treated with sorbitol alone. Driselase treatment resulted in decreased phosphorylation of a band of M r 80,000 which showed almost complete calcium dependence in the osmoticum treated cells; decreased phosphorylation of a band of M r 15,000 which showed little calcium activation, and appearance of a new band of calcium-dependent phosphorylation at M r 22,000. However, protein phosphorylation was decreased. Adding driselase to the in vitro reaction mixture caused a general decrease in the membrane protein phosphorylation either in the presence or absence of calcium which did not mimic the in vivo response. Cells labeled in vivo with inorganic 32 P also showed a response to the Driselase treatment. An enzymically active driselas preparation was required for the observed responses

  16. An unusual protein kinase phosphorylates the chemotactic receptor of Dictystelium discoideum

    International Nuclear Information System (INIS)

    Meier, K.; Klein, C.

    1988-01-01

    The authors report the cAMP-dependent phosphorylation of the chemotactic receptor of Dictyostelium discoideum in partially purified plasma membranes. The protein kinase responsible for receptor phosphorylation is associated with this fraction and preferentially phosphorylates the ligand-occupied form of the receptor. 8-Azido[ 32 P]cAMP labeling of the cell surface has shown that the cAMP receptor exists in two forms. A 45-kDa protein is predominant on unstimulated cells. cAMP stimulation results in an increased receptor phosphorylation such that the receptor migrates on NaDodSO 4 /PAGE as a 47-kDa protein. Phosphorylation of the chemotactic receptor is not detected in membrane preparations unless cAMP is added to the incubation mixture. Only under those conditions is the phosphorylated 47-kDa form observed. The requirement for cAMP reflects the fact that the kinase involved preferentially uses the ligand-occupied receptor as a substrate. In vitro phosphorylation of the receptor does not involve tyrosine residues. The enzyme does not appear to be a cAMP- or cGMP-dependent protein kinase nor is it sensitive to guanine nucleotides, Ca 2+ /calmodulin, Ca 2+ /phospholipid, or EGTA. Similarities with the β-adrenergic receptor protein kinase are discussed

  17. Obesity does not Lead to Imbalance Between Myocardial Phospholamban Phosphorylation and Dephosphorylation

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    Paula Paccielli Freire

    2014-07-01

    Full Text Available Background: The activation of the beta-adrenergic system promotes G protein stimulation that, via cyclic adenosine monophosphate (cAMP, alters the structure of protein kinase A (PKA and leads to phospholamban (PLB phosphorylation. This protein participates in the system that controls intracellular calcium in muscle cells, and it is the primary regulator of sarcoplasmic reticulum calcium pump activity. In obesity, the beta-adrenergic system is activated by the influence of increased leptin, therefore, resulting in higher myocardial phospholamban phosphorylation via cAMP-PKA. Objective: To investigate the involvement of proteins which regulate the degree of PLB phosphorylation due to beta-adrenergic activation in obesity. In the present study, we hypothesized that there is an imbalance between phospholamban phosphorylation and dephosphorylation, with prevalence of protein phosphorylation. Methods: Male Wistar rats were randomly distributed into two groups: control (n = 14, fed with normocaloric diet; and obese (n = 13, fed with a cycle of four unsaturated high-fat diets. Obesity was determined by the adiposity index, and protein expressions of phosphatase 1 (PP-1, PKA, PLB, phosphorylated phospholamban at serine16 (PPLB-Ser16 were assessed by Western blot. Results: Obesity caused glucose intolerance, hyperinsulinemia, hypertriglyceridemia, hyperleptinemia and did not alter the protein expression of PKA, PP-1, PLB, PPLB-Ser16. Conclusion: Obesity does not promote an imbalance between myocardial PLB phosphorylation and dephosphorylation via beta-adrenergic system.

  18. Histone H1 phosphorylation is associated with transcription by RNA polymerases I and II

    Science.gov (United States)

    Zheng, Yupeng; John, Sam; Pesavento, James J.; Schultz-Norton, Jennifer R.; Schiltz, R. Louis; Baek, Sonjoon; Nardulli, Ann M.; Hager, Gordon L.; Kelleher, Neil L.

    2010-01-01

    Histone H1 phosphorylation affects chromatin condensation and function, but little is known about how specific phosphorylations impact the function of H1 variants in higher eukaryotes. In this study, we show that specific sites in H1.2 and H1.4 of human cells are phosphorylated only during mitosis or during both mitosis and interphase. Antisera generated to individual H1.2/H1.4 interphase phosphorylations reveal that they are distributed throughout nuclei and enriched in nucleoli. Moreover, interphase phosphorylated H1.4 is enriched at active 45S preribosomal RNA gene promoters and is rapidly induced at steroid hormone response elements by hormone treatment. Our results imply that site-specific interphase H1 phosphorylation facilitates transcription by RNA polymerases I and II and has an unanticipated function in ribosome biogenesis and control of cell growth. Differences in the numbers, structure, and locations of interphase phosphorylation sites may contribute to the functional diversity of H1 variants. PMID:20439994

  19. Impaired degradation of WNK by Akt and PKA phosphorylation of KLHL3.

    Science.gov (United States)

    Yoshizaki, Yuki; Mori, Yutaro; Tsuzaki, Yoshihito; Mori, Takayasu; Nomura, Naohiro; Wakabayashi, Mai; Takahashi, Daiei; Zeniya, Moko; Kikuchi, Eriko; Araki, Yuya; Ando, Fumiaki; Isobe, Kiyoshi; Nishida, Hidenori; Ohta, Akihito; Susa, Koichiro; Inoue, Yuichi; Chiga, Motoko; Rai, Tatemitsu; Sasaki, Sei; Uchida, Shinichi; Sohara, Eisei

    2015-11-13

    Mutations in with-no-lysine kinase (WNK) 1, WNK4, Kelch-like 3 (KLHL3), and Cullin3 result in an inherited hypertensive disease, pseudohypoaldosteronism type II. WNK activates the Na-Cl cotransporter (NCC), increasing sodium reabsorption in the kidney. Further, KLHL3, an adapter protein of Cullin3-based E3 ubiquitin ligase, has been recently found to bind to WNK, thereby degrading them. Insulin and vasopressin have been identified as powerful activators of WNK signaling. In this study, we investigated effects of Akt and PKA, key downstream substrates of insulin and vasopressin signaling, respectively, on KLHL3. Mass spectrometry analysis revealed that KLHL3 phosphorylation at S433. Phospho-specific antibody demonstrated defective binding between phosphorylated KLHL3 and WNK4. Consistent with the fact that S433 is a component of Akt and PKA phosphorylation motifs, in vitro kinase assay demonstrated that Akt and PKA can phosphorylate KLHL3 at S433, that was previously reported to be phosphorylated by PKC. Further, forskolin, a representative PKA stimulator, increased phosphorylation of KLHL3 at S433 and WNK4 protein expression in HEK293 cells by inhibiting the KLHL3 effect that leads to WNK4 degradation. Insulin also increased phosphorylation of KLHL3 at S433 in cultured cells. In conclusion, we found that Akt and PKA phosphorylated KLHL3 at S433, and phosphorylation of KLHL3 by PKA inhibited WNK4 degradation. This could be a novel mechanism on how insulin and vasopressin physiologically activate the WNK signal. Copyright © 2015 Elsevier Inc. All rights reserved.

  20. The role of glucocorticoid receptor phosphorylation in Mcl-1 and NOXA gene expression

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    Demonacos Constantinos

    2010-02-01

    Full Text Available Abstract Background The cyclin-dependent kinase (CDK and mitogen-activated protein kinase (MAPK mediated phosphorylation of glucocorticoid receptor (GR exerts opposite effects on GR transcriptional activity and affects other posttranslational modifications within this protein. The major phosphorylation site of human GR targeted by MAPK family is the serine 226 and multiple kinase complexes phosphorylate receptor at the serine 211 residue. We hypothesize that GR posttranslational modifications are involved in the determination of the cellular fate in human lymphoblastic leukemia cells. We investigated whether UV signalling through alternative GR phosphorylation determined the cell type specificity of glucocorticoids (GCs mediated apoptosis. Results We have identified putative Glucocorticoid Response Elements (GREs within the promoter regulatory regions of the Bcl-2 family members NOXA and Mcl-1 indicating that they are direct GR transcriptional targets. These genes were differentially regulated in CEM-C7-14, CEM-C1-15 and A549 cells by glucocorticoids and JNK pathway. In addition, our results revealed that the S211 phosphorylation was dominant in CEM-C7-14, whereas the opposite was the case in CEM-C1-15 where prevalence of S226 GR phosphorylation was observed. Furthermore, multiple GR isoforms with cell line specific patterns were identified in CEM-C7-14 cells compared to CEM-C1-15 and A549 cell lines with the same antibodies. Conclusions GR phosphorylation status kinetics, and site specificity as well as isoform variability differ in CEM-C7-14, CEM-C1-15, and A549 cells. The positive or negative response to GCs induced apoptosis in these cell lines is a consequence of the variable equilibrium of NOXA and Mcl-1 gene expression potentially mediated by alternatively phosphorylated GR, as well as the balance of MAPK/CDK pathways controlling GR phosphorylation pattern. Our results provide molecular base and valuable knowledge for improving the GC

  1. Phosphorylation of Nanog is Essential to Regulate Bmi1 and Promote Tumorigenesis

    Science.gov (United States)

    Xie, Xiujie; Piao, Longzhu; Cavey, Greg S.; Old, Matthew; Teknos, Theodoros N.; Mapp, Anna K; Pan, Quintin

    2014-01-01

    Emerging evidence indicates that Nanog is intimately involved in tumorigenesis in part through regulation of the cancer initiating cell population. However, the regulation and role of Nanog in tumorigenesis are still poorly understood. In this study, human Nanog was identified to be phosphorylated by human PKCε at multiple residues including T200 and T280. Our work indicated that phosphorylation at T200 and T280 modulates Nanog function through several regulatory mechanisms. Results with phosphorylation-insensitive and phosphorylation-mimetic mutant Nanog revealed that phosphorylation at T200 and T280 enhance Nanog protein stability. Moreover, phosphorylation-insensitive T200A and T280A mutant Nanog had a dominant-negative function to inhibit endogenous Nanog transcriptional activity. Inactivation of Nanog was due to impaired homodimerization, DNA binding, promoter occupancy, and p300, a transcriptional co-activator, recruitment resulting in a defect in target gene promoter activation. Ectopic expression of phosphorylation-insensitive T200A or T280A mutant Nanog reduced cell proliferation, colony formation, invasion, migration, and the cancer initiating cell population in head and neck squamous cell carcinoma (HNSCC) cells. The in vivo cancer initiating ability was severely compromised in HNSCC cells expressing phosphorylation-insensitive T200A or T280A mutant Nanog; 87.5% (14/16), 12.5% (1/8), and 0% (0/8) for control, T200A, and T280A, respectively. Nanog occupied the Bmi1 promoter to directly transactivate and regulate Bmi1. Genetic ablation and rescue experiments demonstrated that Bmi1 is a critical downstream signaling node for the pleiotropic, pro-oncogenic effects of Nanog. Taken together, our study revealed, for the first time, that post-translational phosphorylation of Nanog is essential to regulate Bmi1 and promote tumorigenesis. PMID:23708658

  2. Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay.

    Science.gov (United States)

    Cui, Heying; Loftus, Kyle M; Noell, Crystal R; Solmaz, Sozanne R

    2018-05-03

    Cyclin-dependent kinase 1 (Cdk1) is a master controller for the cell cycle in all eukaryotes and phosphorylates an estimated 8 - 13% of the proteome; however, the number of identified targets for Cdk1, particularly in human cells is still low. The identification of Cdk1-specific phosphorylation sites is important, as they provide mechanistic insights into how Cdk1 controls the cell cycle. Cell cycle regulation is critical for faithful chromosome segregation, and defects in this complicated process lead to chromosomal aberrations and cancer. Here, we describe an in vitro kinase assay that is used to identify Cdk1-specific phosphorylation sites. In this assay, a purified protein is phosphorylated in vitro by commercially available human Cdk1/cyclin B. Successful phosphorylation is confirmed by SDS-PAGE, and phosphorylation sites are subsequently identified by mass spectrometry. We also describe purification protocols that yield highly pure and homogeneous protein preparations suitable for the kinase assay, and a binding assay for the functional verification of the identified phosphorylation sites, which probes the interaction between a classical nuclear localization signal (cNLS) and its nuclear transport receptor karyopherin α. To aid with experimental design, we review approaches for the prediction of Cdk1-specific phosphorylation sites from protein sequences. Together these protocols present a very powerful approach that yields Cdk1-specific phosphorylation sites and enables mechanistic studies into how Cdk1 controls the cell cycle. Since this method relies on purified proteins, it can be applied to any model organism and yields reliable results, especially when combined with cell functional studies.

  3. Identification of membrane-type 1 matrix metalloproteinase tyrosine phosphorylation in association with neuroblastoma progression

    International Nuclear Information System (INIS)

    Nyalendo, Carine; Sartelet, Hervé; Barrette, Stéphane; Ohta, Shigeru; Gingras, Denis; Béliveau, Richard

    2009-01-01

    Neuroblastoma is a pediatric tumor of neural crest cells that is clinically characterized by its variable evolution, from spontaneous regression to malignancy. Despite many advances in neuroblastoma research, 60% of neuroblastoma, which are essentially metastatic cases, are associated with poor clinical outcome due to the lack of effectiveness of current therapeutic strategies. Membrane-type 1 matrix metalloproteinase (MT1-MMP, MMP-14), an enzyme involved in several steps in tumor progression, has previously been shown to be associated with poor clinical outcome for neuroblastoma. Based on our recent demonstration that MT1-MMP phosphorylation is involved in the growth of fibrosarcoma tumors, we examined the potential role of phosphorylated MT1-MMP in neuroblastoma progression. Tyrosine phosphorylated MT1-MMP was immunostained on tissue microarray samples from 55 patients with neuroblastoma detected by mass screening (known to be predominantly associated with favourable outcome), and from 234 patients with standard diagnosed neuroblastoma. In addition, the effects of a non phosphorylable version of MT1-MMP on neuroblastoma cell migration and proliferation were investigated within three-dimensional collagen matrices. Although there is no correlation between the extent of tyrosine phosphorylation of MT1-MMP (pMT1-MMP) and MYCN amplification or clinical stage, we observed greater phosphorylation of pMT1-MMP in standard neuroblastoma, while it is less evident in neuroblastoma from mass screening samples (P = 0.0006) or in neuroblastoma samples from patients younger than one year (P = 0.0002). In vitro experiments showed that overexpression of a non-phosphorylable version of MT1-MMP reduced MT1-MMP-mediated neuroblastoma cell migration and proliferation within a three-dimensional type I collagen matrix, suggesting a role for the phosphorylated enzyme in the invasive properties of neuroblastoma cells. Overall, these results suggest that tyrosine phosphorylated MT1-MMP

  4. Pro-Tumorigenic Phosphorylation of p120 Catenin in Renal and Breast Cancer.

    Directory of Open Access Journals (Sweden)

    Antonis Kourtidis

    Full Text Available Altered protein expression and phosphorylation are common events during malignant transformation. These perturbations have been widely explored in the context of E-cadherin cell-cell adhesion complexes, which are central in the maintenance of the normal epithelial phenotype. A major component of these complexes is p120 catenin (p120, which binds and stabilizes E-cadherin to promote its adhesive and tumor suppressing function. However, p120 is also an essential mediator of pro-tumorigenic signals driven by oncogenes, such as Src, and can be phosphorylated at multiple sites. Although alterations in p120 expression have been extensively studied by immunohistochemistry (IHC in the context of tumor progression, little is known about the status and role of p120 phosphorylation in cancer. Here we show that tyrosine and threonine phosphorylation of p120 in two sites, Y228 and T916, is elevated in renal and breast tumor tissue samples. We also show that tyrosine phosphorylation of p120 at its N-terminus, including at the Y228 site is required for its pro-tumorigenic potential. In contrast, phosphorylation of p120 at T916 does not affect this p120 function. However, phosphorylation of p120 at T916 interferes with epitope recognition of the most commonly used p120 antibody, namely pp120. As a result, this antibody selectively underrepresents p120 levels in tumor tissues, where p120 is phosphorylated. Overall, our data support a role of p120 phosphorylation as a marker and mediator of tumor transformation. Importantly, they also argue that the level and localization of p120 in human cancer tissues immunostained with pp120 needs to be re-evaluated.

  5. TNNI3K is a novel mediator of myofilament function and phosphorylates cardiac troponin I

    International Nuclear Information System (INIS)

    Wang, Hui; Wang, Lin; Song, Li; Zhang, Yan-Wan; Ye, Jue; Xu, Rui-Xia; Shi, Na; Meng, Xian-Min

    2013-01-01

    The phosphorylation of cardiac troponin I (cTnI) plays an important role in the contractile dysfunction associated with heart failure. Human cardiac troponin I-interacting kinase (TNNI3K) is a novel cardiac-specific functional kinase that can bind to cTnI in a yeast two-hybrid screen. The purpose of this study was to investigate whether TNNI3K can phosphorylate cTnI at specific sites and to examine whether the phosphorylation of cTnI caused by TNNI3K can regulate cardiac myofilament contractile function. Co-immunoprecipitation was performed to confirm that TNNI3K could interact with cTnI. Kinase assays further indicated that TNNI3K did not phosphorylate cTnI at Ser23/24 and Ser44, but directly phosphorylated Ser43 and Thr143 in vitro. The results obtained for adult rat cardiomyocytes also indicated that enhanced phosphorylation of cTnI at Ser43 and Thr143 correlated with rTNNI3K (rat TNNI3K) overexpression, and phosphorylation was reduced when rTNNI3K was knocked down. To determine the contractile function modulated by TNNI3K-mediated phosphorylation of cTnI, cardiomyocyte contraction was studied in adult rat ventricular myocytes. The contraction of cardiomyocytes increased with rTNNI3K overexpression and decreased with rTNNI3K knockdown. We conclude that TNNI3K may be a novel mediator of cTnI phosphorylation and contribute to the regulation of cardiac myofilament contraction function

  6. Regulation of gap junction conductance by calcineurin through Cx43 phosphorylation: implications for action potential conduction.

    Science.gov (United States)

    Jabr, Rita I; Hatch, Fiona S; Salvage, Samantha C; Orlowski, Alejandro; Lampe, Paul D; Fry, Christopher H

    2016-11-01

    Cardiac arrhythmias are associated with raised intracellular [Ca 2+ ] and slowed action potential conduction caused by reduced gap junction (GJ) electrical conductance (Gj). Ventricular GJs are composed of connexin proteins (Cx43), with Gj determined by Cx43 phosphorylation status. Connexin phosphorylation is an interplay between protein kinases and phosphatases but the precise pathways are unknown. We aimed to identify key Ca 2+ -dependent phosphorylation sites on Cx43 that regulate cardiac gap junction conductance and action potential conduction velocity. We investigated the role of the Ca 2+ -dependent phosphatase, calcineurin. Intracellular [Ca 2+ ] was raised in guinea-pig myocardium by a low-Na solution or increased stimulation. Conduction velocity and Gj were measured in multicellular strips. Phosphorylation of Cx43 serine residues (S365 and S368) and of the intermediary regulator I1 at threonine35 was measured by Western blot. Measurements were made in the presence and absence of inhibitors to calcineurin, I1 or protein phosphatase-1 and phosphatase-2.Raised [Ca 2 + ] i decreased Gj, reduced Cx43 phosphorylation at S365 and increased it at S368; these changes were reversed by calcineurin inhibitors. Cx43-S368 phosphorylation was reversed by the protein kinase C inhibitor chelerythrine. Raised [Ca 2+ ] i also decreased I1 phosphorylation, also prevented by calcineurin inhibitors, to increase activity of the Ca 2+ -independent phosphatase, PPI. The PP1 inhibitor, tautomycin, prevented Cx43-365 dephosphorylation, Cx43-S368 phosphorylation and Gj reduction in raised [Ca 2+ ] i . PP2A had no role. Conduction velocity was reduced by raised [Ca 2+ ] i and reversed by calcineurin inhibitors. Reduced action potential conduction and Gj in raised [Ca 2+ ] are regulated by calcineurin-dependent Cx43-S365 phosphorylation, leading to Cx43-S368 dephosphorylation. The calcineurin action is indirect, via I1 dephosphorylation and subsequent activation of PP1.

  7. Role of Protein Phosphorylation in the Regulation of Cell Cycle and DNA-Related Processes in Bacteria

    DEFF Research Database (Denmark)

    Garcia-Garcia, Transito; Poncet, Sandrine; Derouiche, Abderahmane

    2016-01-01

    In all living organisms, the phosphorylation of proteins modulates various aspects of their functionalities. In eukaryotes, protein phosphorylation plays a key role in cell signaling, gene expression, and differentiation. Protein phosphorylation is also involved in the global control of DNA repli...

  8. Multiplexed Imaging of Protein Phosphorylation on Membranes Based on Ti(IV) Functionalized Nanopolymers.

    Science.gov (United States)

    Iliuk, Anton; Li, Li; Melesse, Michael; Hall, Mark C; Tao, W Andy

    2016-05-17

    Accurate protein phosphorylation analysis reveals dynamic cellular signaling events not evident from protein expression levels. The most dominant biochemical assay, western blotting, suffers from the inadequate availability and poor quality of phospho-specific antibodies for phosphorylated proteins. Furthermore, multiplexed assays based on antibodies are limited by steric interference between the antibodies. Here we introduce a multifunctionalized nanopolymer for the universal detection of phosphoproteins that, in combination with regular antibodies, allows multiplexed imaging and accurate determination of protein phosphorylation on membranes. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. Evidence that phosphorylation by the mitotic kinase Cdk1 promotes ICER monoubiquitination and nuclear delocalization

    Energy Technology Data Exchange (ETDEWEB)

    Memin, Elisabeth, E-mail: molinac@mail.montclair.edu [Department of Biochemistry and Molecular Biology, University of Medicine and Dentistry of New Jersey-New Jersey Medical School, Newark, NJ 07103 (United States); Genzale, Megan [Department of Biochemistry and Molecular Biology, University of Medicine and Dentistry of New Jersey-New Jersey Medical School, Newark, NJ 07103 (United States); Crow, Marni; Molina, Carlos A. [Department of Biology and Molecular Biology, Montclair State University, Montclair, NJ, 07043 (United States)

    2011-10-15

    In contrast to normal prostatic cells, the transcriptional repressor Inducible cAMP Early Repressor (ICER) is undetected in the nuclei of prostate cancer cells. The molecular mechanisms for ICER abnormal expression in prostate cancer cells remained largely unknown. In this report data is presented demonstrating that ICER is phosphorylated by the mitotic kinase cdk1. Phosphorylation of ICER on a discrete residue targeted ICER to be monoubiquitinated. Different from unphosphorylated, phosphorylated and polyubiquitinated ICER, monoubiquitinated ICER was found to be cytosolic. Taken together, these results hinted on a mechanism for the observed abnormal subcellular localization of ICER in human prostate tumors.

  10. Cereal bioengineering: Amylopectin-free and hyper-phosphorylated barley starch

    DEFF Research Database (Denmark)

    Carciofi, Massimiliano; Shaik, Shahnoor Sultana; Jensen, Susanne Langgård

    Barley lines producing grains with either amylopectin-free or hyper-phosphorylated starches were made by transgenic methods. Cereals producing these kind of starches have not been reported before. Amylopectin-free barley was generated by simultaneously silencing the three genes encoding the starch...... and T1) of transgenic grains was tenfold higher than from vector control and wild type grains. Amylose content was not affected in hyper-phosphorylated grains. Hyper-phosphorylated starch granules had several pores on the surfaces, similar to pores seen on enzymatically semi-degraded granules...

  11. Cereal bioengineering: Amylopectin-free and hyper-phosphorylated barley starch

    DEFF Research Database (Denmark)

    Carciofi, Massimiliano; Shaik, Shahnoor Sultana; Jensen, Susanne Langgård

    2011-01-01

    Barley lines producing grains with either amylopectin-free or hyper-phosphorylated starches were made by transgenic methods. Cereals producing these kind of starches have not been reported before. Amylopectin-free barley was generated by simultaneously silencing the three genes encoding the starch...... and T1) of transgenic grains was tenfold higher than from vector control and wild type grains. Amylose content was not affected in hyper-phosphorylated grains. Hyper-phosphorylated starch granules had several pores on the surfaces, similar to pores seen on enzymatically semi-degraded granules...

  12. Studies on the effect of phosphorylation on the dipeptides actions by radiation chemistry

    Energy Technology Data Exchange (ETDEWEB)

    Zhang Manwei; Wang Zhiyong; Chu Gaosheng; Zhang Zhicheng [Univ. of Science and Technology of China, Hefei (China)

    2000-03-01

    The electron transfer within several dipeptides and their corresponding phosphorylated dipeptides was studied by electron pulse radiolysis, laser photolysis and electron spin resonance. The electron transfer rate constants were calculated by data modeling and kinetic analysis. It is found that the phosphoryl group in peptides participates the electron transfer process, and reduces the electron transfer rate in all cases. These are very important in life science since every biological process refers to the phosphorylation and nonphosphorylation of protein. It may be concerned in personalities and individualities of the personae. (author)

  13. Cell cycle-specific UNG2 phosphorylations regulate protein turnover, activity and association with RPA

    DEFF Research Database (Denmark)

    Hagen, Lars; Kavli, Bodil; Sousa, Mirta M L

    2008-01-01

    -catalytic domain that confer distinct functional properties to UNG2. These are apparently generated by cyclin-dependent kinases through stepwise phosphorylation of S23, T60 and S64 in the cell cycle. Phosphorylation of S23 in late G1/early S confers increased association with replication protein A (RPA......) and replicating chromatin and markedly increases the catalytic turnover of UNG2. Conversely, progressive phosphorylation of T60 and S64 throughout S phase mediates reduced binding to RPA and flag UNG2 for breakdown in G2 by forming a cyclin E/c-myc-like phosphodegron. The enhanced catalytic turnover of UNG2 p-S23...

  14. Effect of binding in cyclic phosphorylation-dephosphorylation process and in energy transformation.

    Science.gov (United States)

    Sarkar, A; Beard, D A; Franza, B R

    2006-07-01

    The effects of binding on the phosphorylation-dephosphorylation cycle (PDPC) - one of the key components of the signal transduction processes - is analyzed based on a mathematical model. The model shows that binding of proteins, forming a complex, diminishes the ultrasensitivity of the PDPC to the differences in activity between kinase and phosphatase in the cycle. It is also found that signal amplification depends upon the strength of the binding affinity of the protein (phosphorylated or dephosphorylated) to other proteins . It is also observed that the amplification of signal is not only dependent on phosphorylation potential but also on binding properties and resulting adjustments in binding energies.

  15. Thermodynamic study of the native and phosphorylated regulatory domain of the CFTR

    Energy Technology Data Exchange (ETDEWEB)

    Marasini, Carlotta, E-mail: marasini@ge.ibf.cnr.it [Istituto di Biofisica, Consiglio Nazionale delle Ricerche, Via De Marini 6, 16149 Genova (Italy); Galeno, Lauretta; Moran, Oscar [Istituto di Biofisica, Consiglio Nazionale delle Ricerche, Via De Marini 6, 16149 Genova (Italy)

    2012-07-06

    Highlights: Black-Right-Pointing-Pointer CFTR mutations produce cystic fibrosis. Black-Right-Pointing-Pointer Chloride transport depends on the regulatory domain phosphorylation. Black-Right-Pointing-Pointer Regulatory domain is intrinsically disordered. Black-Right-Pointing-Pointer Secondary structure and protein stability change upon phosphorylation. -- Abstract: The regulatory domain (RD) of the cystic fibrosis transmembrane conductance regulator (CFTR), the defective protein in cystic fibrosis, is the region of the channel that regulates the CFTR activity with multiple phosphorylation sites. This domain is an intrinsically disordered protein, characterized by lack of stable or unique tertiary structure. The disordered character of a protein is directly correlated with its function. The flexibility of RD may be important for its regulatory role: the continuous conformational change may be necessary for the progressive phosphorylation, and thus activation, of the channel. However, the lack of a defined and stable structure results in a considerable limitation when trying to in build a unique molecular model for the RD. Moreover, several evidences indicate significant structural differences between the native, non-phosphorylated state, and the multiple phosphorylated state of the protein. The aim of our work is to provide data to describe the conformations and the thermodynamic properties in these two functional states of RD. We have done the circular dichroism (CD) spectra in samples with a different degree of phosphorylation, from the non-phosphorylated state to a bona fide completely phosphorylated state. Analysis of CD spectra showed that the random coil and {beta}-sheets secondary structure decreased with the polypeptide phosphorylation, at expenses of an increase of {alpha}-helix. This observation lead to interpret phosphorylation as a mechanism favoring a more structured state. We also studied the thermal denaturation curves of the protein in the two

  16. Studies on the effect of phosphorylation on the dipeptides actions by radiation chemistry

    International Nuclear Information System (INIS)

    Zhang Manwei; Wang Zhiyong; Chu Gaosheng; Zhang Zhicheng

    2000-01-01

    The electron transfer within several dipeptides and their corresponding phosphorylated dipeptides was studied by electron pulse radiolysis, laser photolysis and electron spin resonance. The electron transfer rate constants were calculated by data modeling and kinetic analysis. It is found that the phosphoryl group in peptides participates the electron transfer process, and reduces the electron transfer rate in all cases. These are very important in life science since every biological process refers to the phosphorylation and nonphosphorylation of protein. It may be concerned in personalities and individualities of the personae. (author)

  17. Thermodynamic study of the native and phosphorylated regulatory domain of the CFTR

    International Nuclear Information System (INIS)

    Marasini, Carlotta; Galeno, Lauretta; Moran, Oscar

    2012-01-01

    Highlights: ► CFTR mutations produce cystic fibrosis. ► Chloride transport depends on the regulatory domain phosphorylation. ► Regulatory domain is intrinsically disordered. ► Secondary structure and protein stability change upon phosphorylation. -- Abstract: The regulatory domain (RD) of the cystic fibrosis transmembrane conductance regulator (CFTR), the defective protein in cystic fibrosis, is the region of the channel that regulates the CFTR activity with multiple phosphorylation sites. This domain is an intrinsically disordered protein, characterized by lack of stable or unique tertiary structure. The disordered character of a protein is directly correlated with its function. The flexibility of RD may be important for its regulatory role: the continuous conformational change may be necessary for the progressive phosphorylation, and thus activation, of the channel. However, the lack of a defined and stable structure results in a considerable limitation when trying to in build a unique molecular model for the RD. Moreover, several evidences indicate significant structural differences between the native, non-phosphorylated state, and the multiple phosphorylated state of the protein. The aim of our work is to provide data to describe the conformations and the thermodynamic properties in these two functional states of RD. We have done the circular dichroism (CD) spectra in samples with a different degree of phosphorylation, from the non-phosphorylated state to a bona fide completely phosphorylated state. Analysis of CD spectra showed that the random coil and β-sheets secondary structure decreased with the polypeptide phosphorylation, at expenses of an increase of α-helix. This observation lead to interpret phosphorylation as a mechanism favoring a more structured state. We also studied the thermal denaturation curves of the protein in the two conditions, monitoring the changes of the mean residue ellipticity measured at 222 nm as a function of temperature

  18. Phosphorylation of both nucleoplasmin domains is required for activation of its chromatin decondensation activity

    DEFF Research Database (Denmark)

    Bañuelos, Sonia; Omaetxebarria, Miren J; Ramos, Isbaal

    2007-01-01

    Nucleoplasmin (NP) is a histone chaperone involved in nucleosome assembly, chromatin decondensation at fertilization, and apoptosis. To carry out these activities NP has to interact with different types of histones, an interaction that is regulated by phosphorylation. Here we have identified...... are found at the tail domain, flanking the nuclear localization signal. Phosphorylation-mimicking mutations render a recombinant protein as active in chromatin decondensation as hyperphosphorylated NP isolated from Xenopus laevis eggs. Comparison of mutants in which the core and tail domains of the protein...... were independently or simultaneously "activated" indicates that activation or phosphorylation of both protein domains is required for NP to efficiently extract linker-type histones from chromatin....

  19. Site-specific mapping of the human SUMO proteome reveals co-modification with phosphorylation

    DEFF Research Database (Denmark)

    Hendriks, Ivo A; Lyon, David; Young, Clifford

    2017-01-01

    that were co-modified by ubiquitylation, acetylation and methylation. Notably, 9% of the identified SUMOylome occurred proximal to phosphorylation, and numerous SUMOylation sites were found to be fully dependent on prior phosphorylation events. SUMO-proximal phosphorylation occurred primarily in a proline......-directed manner, and inhibition of cyclin-dependent kinases dynamically affected co-modification. Collectively, we present a comprehensive analysis of the SUMOylated proteome, uncovering the structural preferences for SUMO and providing system-wide evidence for a remarkable degree of cross-talk between...

  20. Identifying protein phosphorylation sites with kinase substrate specificity on human viruses.

    Directory of Open Access Journals (Sweden)

    Neil Arvin Bretaña

    Full Text Available Viruses infect humans and progress inside the body leading to various diseases and complications. The phosphorylation of viral proteins catalyzed by host kinases plays crucial regulatory roles in enhancing replication and inhibition of normal host-cell functions. Due to its biological importance, there is a desire to identify the protein phosphorylation sites on human viruses. However, the use of mass spectrometry-based experiments is proven to be expensive and labor-intensive. Furthermore, previous studies which have identified phosphorylation sites in human viruses do not include the investigation of the responsible kinases. Thus, we are motivated to propose a new method to identify protein phosphorylation sites with its kinase substrate specificity on human viruses. The experimentally verified phosphorylation data were extracted from virPTM--a database containing 301 experimentally verified phosphorylation data on 104 human kinase-phosphorylated virus proteins. In an attempt to investigate kinase substrate specificities in viral protein phosphorylation sites, maximal dependence decomposition (MDD is employed to cluster a large set of phosphorylation data into subgroups containing significantly conserved motifs. The experimental human phosphorylation sites are collected from Phospho.ELM, grouped according to its kinase annotation, and compared with the virus MDD clusters. This investigation identifies human kinases such as CK2, PKB, CDK, and MAPK as potential kinases for catalyzing virus protein substrates as confirmed by published literature. Profile hidden Markov model is then applied to learn a predictive model for each subgroup. A five-fold cross validation evaluation on the MDD-clustered HMMs yields an average accuracy of 84.93% for Serine, and 78.05% for Threonine. Furthermore, an independent testing data collected from UniProtKB and Phospho.ELM is used to make a comparison of predictive performance on three popular kinase

  1. Identifying protein phosphorylation sites with kinase substrate specificity on human viruses.

    Science.gov (United States)

    Bretaña, Neil Arvin; Lu, Cheng-Tsung; Chiang, Chiu-Yun; Su, Min-Gang; Huang, Kai-Yao; Lee, Tzong-Yi; Weng, Shun-Long

    2012-01-01

    Viruses infect humans and progress inside the body leading to various diseases and complications. The phosphorylation of viral proteins catalyzed by host kinases plays crucial regulatory roles in enhancing replication and inhibition of normal host-cell functions. Due to its biological importance, there is a desire to identify the protein phosphorylation sites on human viruses. However, the use of mass spectrometry-based experiments is proven to be expensive and labor-intensive. Furthermore, previous studies which have identified phosphorylation sites in human viruses do not include the investigation of the responsible kinases. Thus, we are motivated to propose a new method to identify protein phosphorylation sites with its kinase substrate specificity on human viruses. The experimentally verified phosphorylation data were extracted from virPTM--a database containing 301 experimentally verified phosphorylation data on 104 human kinase-phosphorylated virus proteins. In an attempt to investigate kinase substrate specificities in viral protein phosphorylation sites, maximal dependence decomposition (MDD) is employed to cluster a large set of phosphorylation data into subgroups containing significantly conserved motifs. The experimental human phosphorylation sites are collected from Phospho.ELM, grouped according to its kinase annotation, and compared with the virus MDD clusters. This investigation identifies human kinases such as CK2, PKB, CDK, and MAPK as potential kinases for catalyzing virus protein substrates as confirmed by published literature. Profile hidden Markov model is then applied to learn a predictive model for each subgroup. A five-fold cross validation evaluation on the MDD-clustered HMMs yields an average accuracy of 84.93% for Serine, and 78.05% for Threonine. Furthermore, an independent testing data collected from UniProtKB and Phospho.ELM is used to make a comparison of predictive performance on three popular kinase-specific phosphorylation site

  2. Phosphorylation of Human Metapneumovirus M2-1 Protein Upregulates Viral Replication and Pathogenesis.

    Science.gov (United States)

    Cai, Hui; Zhang, Yu; Lu, Mijia; Liang, Xueya; Jennings, Ryan; Niewiesk, Stefan; Li, Jianrong

    2016-08-15

    Human metapneumovirus (hMPV) is a major causative agent of upper- and lower-respiratory-tract infections in infants, the elderly, and immunocompromised individuals worldwide. Like all pneumoviruses, hMPV encodes the zinc binding protein M2-1, which plays important regulatory roles in RNA synthesis. The M2-1 protein is phosphorylated, but the specific role(s) of the phosphorylation in viral replication and pathogenesis remains unknown. In this study, we found that hMPV M2-1 is phosphorylated at amino acid residues S57 and S60. Subsequent mutagenesis found that phosphorylation is not essential for zinc binding activity and oligomerization, whereas inhibition of zinc binding activity abolished the phosphorylation and oligomerization of the M2-1 protein. Using a reverse genetics system, recombinant hMPVs (rhMPVs) lacking either one or both phosphorylation sites in the M2-1 protein were recovered. These recombinant viruses had a significant decrease in both genomic RNA replication and mRNA transcription. In addition, these recombinant viruses were highly attenuated in cell culture and cotton rats. Importantly, rhMPVs lacking phosphorylation in the M2-1 protein triggered high levels of neutralizing antibody and provided complete protection against challenge with wild-type hMPV. Collectively, these data demonstrated that phosphorylation of the M2-1 protein upregulates hMPV RNA synthesis, replication, and pathogenesis in vivo The pneumoviruses include many important human and animal pathogens, such as human respiratory syncytial virus (hRSV), hMPV, bovine RSV, and avian metapneumovirus (aMPV). Among these viruses, hRSV and hMPV are the leading causes of acute respiratory tract infection in infants and children. Currently, there is no antiviral or vaccine to combat these diseases. All known pneumoviruses encode a zinc binding protein, M2-1, which is a transcriptional antitermination factor. In this work, we found that phosphorylation of M2-1 is essential for virus

  3. The thermodynamic efficiency of ATP synthesis in oxidative phosphorylation.

    Science.gov (United States)

    Nath, Sunil

    2016-12-01

    As the chief energy source of eukaryotic cells, it is important to determine the thermodynamic efficiency of ATP synthesis in oxidative phosphorylation (OX PHOS). Previous estimates of the thermodynamic efficiency of this vital process have ranged from Lehninger's original back-of-the-envelope calculation of 38% to the often quoted value of 55-60% in current textbooks of biochemistry, to high values of 90% from recent information theoretic considerations, and reports of realizations of close to ideal 100% efficiencies by single molecule experiments. Hence this problem has been reinvestigated from first principles. The overall thermodynamic efficiency of ATP synthesis in the mitochondrial energy transduction OX PHOS process has been found to lie between 40 and 41% from four different approaches based on a) estimation using structural and biochemical data, b) fundamental nonequilibrium thermodynamic analysis, c) novel insights arising from Nath's torsional mechanism of energy transduction and ATP synthesis, and d) the overall balance of cellular energetics. The torsional mechanism also offers an explanation for the observation of a thermodynamic efficiency approaching 100% in some experiments. Applications of the unique, molecular machine mode of functioning of F 1 F O -ATP synthase involving direct inter-conversion of chemical and mechanical energies in the design and fabrication of novel, man-made mechanochemical devices have been envisaged, and some new ways to exorcise Maxwell's demon have been proposed. It is hoped that analysis of the fundamental problem of energy transduction in OX PHOS from a fresh perspective will catalyze new avenues of research in this interdisciplinary field. Copyright © 2016 Elsevier B.V. All rights reserved.

  4. Analysis of cardiac myosin binding protein-C phosphorylation in human heart muscle.

    Science.gov (United States)

    Copeland, O'Neal; Sadayappan, Sakthivel; Messer, Andrew E; Steinen, Ger J M; van der Velden, Jolanda; Marston, Steven B

    2010-12-01

    A unique feature of MyBP-C in cardiac muscle is that it has multiple phosphorylation sites. MyBP-C phosphorylation, predominantly by PKA, plays an essential role in modulating contractility as part of the cellular response to β-adrenergic stimulation. In vitro studies indicate MyBP-C can be phosphorylated at Serine 273, 282, 302 and 307 (mouse sequence) but little is known about the level of MyBP-C phosphorylation or the sites phosphorylated in heart muscle. Since current methodologies are limited in specificity and are not quantitative we have investigated the use of phosphate affinity SDS-PAGE together with a total anti MyBP-C antibody and a range of phosphorylation site-specific antibodies for the main sites (Ser-273, -282 and -302). With these newly developed methods we have been able to make a detailed quantitative analysis of MyBP-C phosphorylation in heart tissue in situ. We have found that MyBP-C is highly phosphorylated in non-failing human (donor) heart or mouse heart; tris and tetra-phosphorylated species predominate and less than 10% of MyBP-C is unphosphorylated (0, 9.3 ± 1%: 1P, 13.4 ± 2.7%: 2P, 10.5 ± 3.3%: 3P, 28.7 ± 3.7%: 4P, 36.4 ± 2.7%, n=21). Total phosphorylation was 2.7 ± 0.07 mol Pi/mol MyBP-C. In contrast in failing heart and in myectomy samples from HCM patients the majority of MyBP-C was unphosphorylated. Total phosphorylation levels were 23% of normal in failing heart myofibrils (0, 60.1 ± 2.8%: 1P, 27.8 ± 2.8%: 2P, 4.8 ± 2.0%: 3P, 3.7 ± 1.2%: 4P, 2.8 ± 1.3%, n=19) and 39% of normal in myectomy samples. The site-specific antibodies showed a distinctive distribution pattern of phosphorylation sites in the multiple phosphorylation level species. We found that phosphorylated Ser-273, Ser-282 and Ser-302 were all present in the 4P band of MyBP-C but none of them were significant in the 1P band, indicating that there must be at least one other site of MyBP-C phosphorylation in human heart. The pattern of phosphorylation at the

  5. Phosphorylation of ribosomal proteins induced by auxins in maize embryonic tissues

    International Nuclear Information System (INIS)

    Perez, L.; Aguilar, R.; Mendez, A.P.; de Jimenez, E.S.

    1990-01-01

    The effect of auxin on ribosomal protein phosphorylation of germinating maize (Zea mays) tissues was investigated. Two-dimensional gel electrophoresis and autoradiography of [ 32 P] ribosomal protein patterns for natural and synthetic auxin-treated tissues were performed. Both the rate of 32 P incorporation and the electrophoretic patterns were dependent on 32 P pulse length, suggesting that active protein phosphorylation-dephosphorylation occurred in small and large subunit proteins, in control as well as in auxin-treated tissues. The effect of ribosomal protein phosphorylation on in vitro translation was tested. Measurements of poly(U) translation rates as a function of ribosome concentration provided apparent K m values significantly different for auxin-treated and nontreated tissues. These findings suggest that auxin might exert some kind of translational control by regulating the phosphorylated status of ribosomal proteins

  6. Highly selective enrichment of phosphorylated peptides from peptide mixtures using titanium dioxide microcolumns

    DEFF Research Database (Denmark)

    Larsen, Martin Røssel; Thingholm, Tine E; Jensen, Ole N

    2005-01-01

    based on TiO2microcolumns and peptide loading in 2,5-dihydroxybenzoic acid (DHB). The effect of DHB was a very efficient reduction in the binding of nonphosphorylated peptides to TiO2 while retaining its high binding affinity for phosphorylated peptides. Thus, inclusion of DHB dramatically increased...... the selectivity of the enrichment of phosphorylated peptides by TiO2. We demonstrated that this new procedure was more selective for binding phosphorylated peptides than IMAC using MALDI mass spectrometry. In addition, we showed that LC-ESI-MSMS was biased toward monophosphorylated peptides, whereas MALDI MS...... was not. Other substituted aromatic carboxylic acids were also capable of specifically reducing binding of nonphosphorylated peptides, whereas phosphoric acid reduced binding of both phosphorylated and nonphosphorylated peptides. A putative mechanism for this intriguing effect is presented....

  7. Single-well monitoring of protein-protein interaction and phosphorylation-dephosphorylation events.

    Science.gov (United States)

    Arcand, Mathieu; Roby, Philippe; Bossé, Roger; Lipari, Francesco; Padrós, Jaime; Beaudet, Lucille; Marcil, Alexandre; Dahan, Sophie

    2010-04-20

    We combined oxygen channeling assays with two distinct chemiluminescent beads to detect simultaneously protein phosphorylation and interaction events that are usually monitored separately. This novel method was tested in the ERK1/2 MAP kinase pathway. It was first used to directly monitor dissociation of MAP kinase ERK2 from MEK1 upon phosphorylation and to evaluate MAP kinase phosphatase (MKP) selectivity and mechanism of action. In addition, MEK1 and ERK2 were probed with an ATP competitor and an allosteric MEK1 inhibitor, which generated distinct phosphorylation-interaction patterns. Simultaneous monitoring of protein-protein interactions and substrate phosphorylation can provide significant mechanistic insight into enzyme activity and small molecule action.

  8. Developing CNS mitochondria oxidative phosphorylation P/O/ADP/O index for rats

    International Nuclear Information System (INIS)

    Egana, E.; Diaz, G.

    1975-01-01

    The effect of whole-body-gamma irradiation on developing CNS mitochondria oxidative phosphorylation was studied through the P/O/ADP/O index; three irradiation doses (5, 50 and 500 R) were employed at neonatal stage and both 'prompt' (10 min approx,) and 'delayed' (7 days for 500 R exposure, 21 days for 5 and 50 R) effects were observed. In the 'prompt' effects investigated after 500 R exposure, the oxidative phosphorylation diminished; the same occurred at 7 days with this dose ('delayed' effect). With doses of 5 and 50 R there was no alteration of oxidative phosphorylation as a 'prompt' effect, but it diminished at 21 days post irradiation. The uncoupling between respiration and oxidative phosphorylation should explain - at least, in part -these results. (author)

  9. Insulin increases phosphorylation of mitochondrial proteins in human skeletal muscle in vivo

    DEFF Research Database (Denmark)

    Zhao, Xiaolu; Bak, Steffen; Pedersen, Andreas James Thestrup

    2014-01-01

    , we investigated the effect of insulin on the phosphorylation of mitochondrial proteins in human skeletal muscle in vivo. Using a combination of TiO2 phosphopeptide-enrichment, HILIC fractionation, and LC−MS/MS, we compared the phosphoproteomes of isolated mitochondria from skeletal muscle samples...... obtained from healthy individuals before and after 4 h of insulin infusion. In total, we identified 207 phosphorylation sites in 95 mitochondrial proteins. Of these phosphorylation sites, 45% were identified in both basal and insulin-stimulated samples. Insulin caused a 2-fold increase in the number...... of different mitochondrial phosphopeptides (87 ± 7 vs 40 ± 7, p = 0.015) and phosphoproteins (46 ± 2 vs 26 ± 3, p = 0.005) identified in each mitochondrial preparation. Almost half of the mitochondrial phosphorylation sites (n = 94) were exclusively identified in the insulin-stimulated state and included...

  10. Understanding Alzheimer's disease by global quantification of protein phosphorylation and sialylated N-linked glycosylation profiles

    DEFF Research Database (Denmark)

    Lassen, Pernille S.; Thygesen, Camilla; Larsen, Martin R.

    2017-01-01

    elucidated them in neurodegenerative diseases such as Alzheimer's disease. Here, we comprehensively review Alzheimer's pathology in relation to protein phosphorylation and glycosylation on synaptic plasticity from neuroproteomics data. Moreover, we highlight several mass spectrometry-based sample processing...

  11. Role of Phosphorylated Neurofilament H as a diagnostic and prognostic marker in traumatic brain injury

    Directory of Open Access Journals (Sweden)

    Moh Omar Ghonemi

    2013-09-01

    Conclusion: Phosphorylated Neurofilament H can be used as a diagnostic and prognostic marker in patients with TBI as seen by the presence of significant correlations between the marker levels and different clinical and radiological tools.

  12. Megacomplex organization of the oxidative phosphorylation system by structural analysis of respiratory supercomplexes from potato

    NARCIS (Netherlands)

    Bultema, Jelle B.; Braun, Hans-Peter; Boekema, Egbert J.; Kouřil, Roman

    The individual protein complexes of the oxidative phosphorylation system (OXPHOS complexes 1 to V) specifically interact and form defined supramolecular structures, the so-called "respiratory supercomplexes". Some supercomplexes appear to associate into larger structures, or megacomplexes, such as a

  13. Methods for the Analysis of Protein Phosphorylation-Mediated Cellular Signaling Networks

    Science.gov (United States)

    White, Forest M.; Wolf-Yadlin, Alejandro

    2016-06-01

    Protein phosphorylation-mediated cellular signaling networks regulate almost all aspects of cell biology, including the responses to cellular stimulation and environmental alterations. These networks are highly complex and comprise hundreds of proteins and potentially thousands of phosphorylation sites. Multiple analytical methods have been developed over the past several decades to identify proteins and protein phosphorylation sites regulating cellular signaling, and to quantify the dynamic response of these sites to different cellular stimulation. Here we provide an overview of these methods, including the fundamental principles governing each method, their relative strengths and weaknesses, and some examples of how each method has been applied to the analysis of complex signaling networks. When applied correctly, each of these techniques can provide insight into the topology, dynamics, and regulation of protein phosphorylation signaling networks.

  14. Phosphorylation of ARD1 by IKKβ contributes to its destabilization and degradation

    International Nuclear Information System (INIS)

    Kuo, Hsu-Ping; Lee, Dung-Fang; Xia, Weiya; Lai, Chien-Chen; Li, Long-Yuan; Hung, Mien-Chie

    2009-01-01

    IκB kinase β (IKKβ), a major kinase downstream of various proinflammatory signals, mediates multiple cellular functions through phosphorylation and regulation of its substrates. On the basis of protein sequence analysis, we identified arrest-defective protein 1 (ARD1), a protein involved in apoptosis and cell proliferation processes in many human cancer cells, as a new IKKβ substrate. We provided evidence showing that ARD1 is indeed a bona fide substrate of IKKβ. IKKβ physically associated with ARD1 and phosphorylated it at Ser209. Phosphorylation by IKKβ destabilized ARD1 and induced its proteasome-mediated degradation. Impaired growth suppression was observed in ARD1 phosphorylation-mimic mutant (S209E)-transfected cells as compared with ARD1 non-phosphorylatable mutant (S209A)-transfected cells. Our findings of molecular interactions between ARD1 and IKKβ may enable further understanding of the upstream regulation mechanisms of ARD1 and of the diverse functions of IKKβ.

  15. Phosphorylation of myelin basic proteins and its relevance to myelin biogenesis

    Energy Technology Data Exchange (ETDEWEB)

    Ulmer, J.B.

    1985-01-01

    Age-related differences in the in vivo incorporation of (32-P) into mouse myelin basic proteins (MBPs) of the central nervous system were observed. The resulting specific radioactivity (S.A.) of the MBPs appeared to be related to the S.A. of the acid-soluble pool of phosphates of myelin. In development, MBPs were phosphorylated in vivo prior to the onset of myelination in the brain, indicating that MBPs are phosphorylated prior to their deposition in the myelin sheath. The incorporation of (32-P) into MBPs and the turnover rates of MBP phosphates were studied in vivo in developmentally-related myelin compartments. The results suggest that there are two separate events in MBP phosphorylation and that the turnover rates of the MBP phosphates derived from these two events are different. A model for MBP phosphorylation, that could explain in these observations, is postulated and discussed in the light of existing information.

  16. Developing CNS mitochondria oxidative phosphorylation P/O/ADP/O index for rats

    Energy Technology Data Exchange (ETDEWEB)

    Egana, E; Diaz, G [Institute of Experimental Medicine, Santiago (Chile). Lab. of Neurochemistry

    1975-11-01

    The effect of whole-body-gamma irradiation on developing CNS mitochondria oxidative phosphorylation was studied through the P/O/ADP/O index; three irradiation doses (5, 50 and 500 R) were employed at neonatal stage and both 'prompt' (10 min approx,) and 'delayed' (7 days for 500 R exposure, 21 days for 5 and 50 R) effects were observed. In the 'prompt' effects investigated after 500 R exposure, the oxidative phosphorylation diminished; the same occurred at 7 days with this dose ('delayed' effect). With doses of 5 and 50 R there was no alteration of oxidative phosphorylation as a 'prompt' effect, but it diminished at 21 days post irradiation. The uncoupling between respiration and oxidative phosphorylation should explain - at least, in part -these results.

  17. Quantitation of multisite EGF receptor phosphorylation using mass spectrometry and a novel normalization approach

    DEFF Research Database (Denmark)

    Erba, Elisabetta Boeri; Matthiesen, Rune; Bunkenborg, Jakob

    2007-01-01

    Using stable isotope labeling and mass spectrometry, we performed a sensitive, quantitative analysis of multiple phosphorylation sites of the epidermal growth factor (EGF) receptor. Phosphopeptide detection efficiency was significantly improved by using the tyrosine phosphatase inhibitor sodium p...

  18. Probing the Tyrosine Phosphorylation State in Breast Cancer by Src Homology 2 Domain Binding

    National Research Council Canada - National Science Library

    Mayer, Bruce J

    2006-01-01

    .... The overall goal of this project was to develop a novel molecular diagnostic method, termed SH2 profiling, that can classify cell samples based on their global protein tyrosine phosphorylation state...

  19. Probing the Tyrosine Phosphorylation State in Breast Cancer by Src Homology 2 Domain Binding

    National Research Council Canada - National Science Library

    Mayer, Bruce

    2004-01-01

    .... The overall goal of this project is to develop a novel molecular diagnostic method, termed SH2 profiling, that can classify cell samples based on their global protein tyrosine phosphorylation state...

  20. Cryopreservation of common carp (Cyprinus carpio L.) sperm induces protein phosphorylation in tyrosine and threonine residues

    Czech Academy of Sciences Publication Activity Database

    Li, P.; Hulák, M.; Li, Z.; H.; Šulc, Miroslav; Pšenička, M.; Rodina, M.; Gela, D.; Linhart, O.

    2013-01-01

    Roč. 80, č. 2 (2013), s. 84-89 ISSN 0093-691X Institutional support: RVO:61388971 Keywords : Cryopreservation * Sperm * Phosphorylation Subject RIV: CE - Biochemistry Impact factor: 1.845, year: 2013

  1. Effect of some organic solvents on oxidative phosphorylation in rat liver mitochondria

    DEFF Research Database (Denmark)

    Syed, Muzeeb; Skonberg, Christian; Hansen, Steen Honoré

    2013-01-01

    The effect of acetone, acetonitrile, dimethyl sulfoxide (DMSO), ethanol and methanol on oxidative phosphorylation (ATP synthesis) in rat liver mitochondria has been studied. All the organic solvents inhibited the oxidative phosphorylation in a concentration dependent manner, but with differences...... in potencies. Among the tested organic solvents, acetonitrile and acetone were more potent than ethanol, methanol, and DMSO. There was no significant difference in oxidative phosphorylation, compared to controls, when the concentrations of acetone was below 1% (v/v), of acetonitrile below 2% (v/v), of DMSO...... below 10% (v/v), of ethanol below 5% or of methanol below 2%, respectively. There was complete inhibition of oxidative phosphorylation at 50% (v/v) of acetone, acetonitrile and ethanol. But in the case of DMSO and methanol there were some residual activities observed at the 50% concentration level. DMSO...

  2. EFFECT OF PHOSPHOLIPASE-A2 ON SYK-MEDIATED PHOSPHORYLATION OF HUMAN ERYTHROCYTE MEMBRANE

    Directory of Open Access Journals (Sweden)

    Lucian Bordin

    2006-08-01

    , lacking in p36 isoform, are responsible of the lower ability of kinase of catalysing band 3 Tyr-phosphorylation, probably due either to steric bulk, or to particular sequestering of the holoenzyme into membrane compartment.

  3. Exercise increases TBC1D1 phosphorylation in human skeletal muscle

    Science.gov (United States)

    Jessen, Niels; An, Ding; Lihn, Aina S.; Nygren, Jonas; Hirshman, Michael F.; Thorell, Anders

    2011-01-01

    Exercise and weight loss are cornerstones in the treatment and prevention of type 2 diabetes, and both interventions function to increase insulin sensitivity and glucose uptake into skeletal muscle. Studies in rodents demonstrate that the underlying mechanism for glucose uptake in muscle involves site-specific phosphorylation of the Rab-GTPase-activating proteins AS160 (TBC1D4) and TBC1D1. Multiple kinases, including Akt and AMPK, phosphorylate TBC1D1 and AS160 on distinct residues, regulating their activity and allowing for GLUT4 translocation. In contrast to extensive rodent-based studies, the regulation of AS160 and TBC1D1 in human skeletal muscle is not well understood. In this study, we determined the effects of dietary intervention and a single bout of exercise on TBC1D1 and AS160 site-specific phosphorylation in human skeletal muscle. Ten obese (BMI 33.4 ± 2.4, M-value 4.3 ± 0.5) subjects were studied at baseline and after a 2-wk dietary intervention. Muscle biopsies were obtained from the subjects in the resting (basal) state and immediately following a 30-min exercise bout (70% V̇o2 max). Muscle lysates were analyzed for AMPK activity and Akt phosphorylation and for TBC1D1 and AS160 phosphorylation on known or putative AMPK and Akt sites as follows: AS160 Ser711 (AMPK), TBC1D1 Ser231 (AMPK), TBC1D1 Ser660 (AMPK), TBC1D1 Ser700 (AMPK), and TBC1D1 Thr590 (Akt). The diet intervention that consisted of a major shift in the macronutrient composition resulted in a 4.2 ± 0.4 kg weight loss (P < 0.001) and a significant increase in insulin sensitivity (M value 5.6 ± 0.6), but surprisingly, there was no effect on expression or phosphorylation of any of the muscle-signaling proteins. Exercise increased muscle AMPKα2 activity but did not increase Akt phosphorylation. Exercise increased phosphorylation on AS160 Ser711, TBC1D1 Ser231, and TBC1D1 Ser660 but had no effect on TBC1D1 Ser700. Exercise did not increase TBC1D1 Thr590 phosphorylation or TBC1D1/AS160 PAS

  4. Platelet-derived growth factor induces phosphorylation of a 64-kDa nuclear protein

    International Nuclear Information System (INIS)

    Shawver, L.K.; Pierce, G.F.; Kawahara, R.S.; Deuel, T.F.

    1989-01-01

    The platelet-derived growth factor (PDGF) stimulated the phosphorylation of a nuclear protein of 64 kDa (pp64) in nuclei of nontransformed normal rat kidney (NRK) cells. Low levels of phosphorylation of pp64 were observed in nuclei of serum-starved NRK cells. Fetal calf serum (FCS), PDGF, and homodimeric v-sis and PDGF A-chain protein enhanced the incorporation of 32P into pp64 over 4-fold within 30 min and over 8-fold within 2 h of exposure of NRK cells to the growth factors. In contrast, constitutive phosphorylation of 32P-labeled pp64 in nuclei of NRK cells transformed by the simian sarcoma virus (SSV) was high and only minimally stimulated by PDGF and FCS. 32P-Labeled pp64 was isolated from nuclei of PDGF-stimulated nontransformed NRK cells; the 32P of pp64 was labile in 1 M KOH, and pp64 was not significantly recognized by anti-phosphotyrosine antisera, suggesting that the PDGF-induced phosphorylation of pp64 occurred on serine or on threonine residues. However, pp64 from SSV-transformed NRK cell nuclei was significantly stable to base hydrolysis and was immunoprecipitated with anti-phosphotyrosine antisera, suggesting that pp64 from SSV-transformed cell nuclei is phosphorylated also on tyrosine. FCS, PDGF, and PDGF A- and B-chain homodimers thus stimulate the rapid time-dependent phosphorylation of a 64-kDa nuclear protein shortly after stimulation of responsive cells. The growth factor-stimulated phosphorylation of pp64 and the constitutive high levels of pp64 phosphorylation in cells transformed by SSV suggest important roles for pp64 and perhaps regulated nuclear protein kinases and phosphatases in cell division and proliferation

  5. Phosphorylation regulates human T-cell leukemia virus type 1 Rex function

    Directory of Open Access Journals (Sweden)

    Ward Michael

    2009-11-01

    Full Text Available Abstract Background Human T-cell leukemia virus type 1 (HTLV-1 is a pathogenic complex deltaretrovirus, which is the causative agent of adult T-cell leukemia/lymphoma (ATL and HTLV-1-associated myelopathy/tropical spastic paraparesis. In addition to the structural and enzymatic viral gene products, HTLV-1 encodes the positive regulatory proteins Tax and Rex along with viral accessory proteins. Tax and Rex proteins orchestrate the timely expression of viral genes important in viral replication and cellular transformation. Rex is a nucleolar-localizing shuttling protein that acts post-transcriptionally by binding and facilitating the export of the unspliced and incompletely spliced viral mRNAs from the nucleus to the cytoplasm. HTLV-1 Rex (Rex-1 is a phosphoprotein and general protein kinase inhibition correlates with reduced function. Therefore, it has been proposed that Rex-1 function may be regulated through site-specific phosphorylation. Results We conducted a phosphoryl mapping of Rex-1 over-expressed in transfected 293 T cells using a combination of affinity purification and liquid chromatography tandem mass spectrometry. We achieved 100% physical coverage of the Rex-1 polypeptide and identified five novel phosphorylation sites at Thr-22, Ser-36, Thr-37, Ser-97, and Ser-106. We also confirmed evidence of two previously identified residues, Ser-70 and Thr-174, but found no evidence of phosphorylation at Ser-177. The functional significance of these phosphorylation events was evaluated using a Rex reporter assay and site-directed mutational analysis. Our results indicate that phosphorylation at Ser-97 and Thr-174 is critical for Rex-1 function. Conclusion We have mapped completely the site-specific phosphorylation of Rex-1 identifying a total of seven residues; Thr-22, Ser-36, Thr-37, Ser-70, Ser-97, Ser-106, and Thr-174. Overall, this work is the first to completely map the phosphorylation sites in Rex-1 and provides important insight into

  6. Lithium potentiates GSK-3β activity by inhibiting phosphoinositide 3-kinase-mediated Akt phosphorylation

    International Nuclear Information System (INIS)

    Tian, Nie; Kanno, Takeshi; Jin, Yu; Nishizaki, Tomoyuki

    2014-01-01

    Highlights: • Lithium suppresses Akt activity by reducing PI3K-mediated Akt phosphorylation. • Lithium enhances GSK-3β activity by reducing Akt-mediated GSK-3β phosphorylation. • Lithium suppresses GSK-3β activity through its direct inhibition. - Abstract: Accumulating evidence has pointed to the direct inhibitory action of lithium, an anti-depressant, on GSK-3β. The present study investigated further insight into lithium signaling pathways. In the cell-free assay Li 2 CO 3 significantly inhibited phosphoinositide 3-kinase (PI3K)-mediated phosphorylation of Akt1 at Ser473, but Li 2 CO 3 did not affect PI3K-mediated PI(3,4,5)P 3 production and 3-phosphoinositide-dependent protein kinase 1 (PDK1)-mediated phosphorylation of Akt1 at Thr308. This indicates that lithium could enhance GSK-3β activity by suppressing Akt-mediated Ser9 phosphorylation of GSK-3β in association with inhibition of PI3K-mediated Akt activation. There was no direct effect of Li 2 CO 3 on Akt1-induced phosphorylation of GSK-3β at Ser9, but otherwise Li 2 CO 3 significantly reduced GSK-3β-mediated phosphorylation of β-catenin at Ser33/37 and Thr41. This indicates that lithium directly inhibits GSK-3β in an Akt-independent manner. In rat hippocampal slices Li 2 CO 3 significantly inhibited phosphorylation of Akt1/2 at Ser473/474, GSK-3β at Ser9, and β-catenin at Ser33/37 and Thr41. Taken together, these results indicate that lithium exerts its potentiating and inhibiting bidirectional actions on GSK-3β activity

  7. Specific mixing facilitates the comparative quantification of phosphorylation sites with significant dysregulations

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Jing [Key Laboratory of Separation Sciences for Analytical Chemistry, National Chromatographic R& A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences (CAS), Dalian 116023 (China); University of Chinese Academy of Sciences, Beijing 100049 (China); Xu, Bo [Key Laboratory of Separation Sciences for Analytical Chemistry, National Chromatographic R& A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences (CAS), Dalian 116023 (China); Liu, Zheyi; Dong, Mingming; Mao, Jiawei; Zhou, Ye; Chen, Jin [Key Laboratory of Separation Sciences for Analytical Chemistry, National Chromatographic R& A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences (CAS), Dalian 116023 (China); University of Chinese Academy of Sciences, Beijing 100049 (China); Wang, Fangjun, E-mail: wangfj@dicp.ac.cn [Key Laboratory of Separation Sciences for Analytical Chemistry, National Chromatographic R& A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences (CAS), Dalian 116023 (China); Zou, Hanfa [Key Laboratory of Separation Sciences for Analytical Chemistry, National Chromatographic R& A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences (CAS), Dalian 116023 (China)

    2017-01-15

    Mass spectrometry (MS) based quantitative analyses of proteome and proteome post-translational modifications (PTMs) play more and more important roles in biological, pharmaceutical and clinical studies. However, it is still a big challenge to accurately quantify the proteins or proteins PTM sites with extreme relative abundances in comparative protein samples, such as the significantly dysregulated ones. Herein, a novel quantification strategy, Mixing at Specific Ratio (MaSR) before isotope labeling, had been developed to improve the quantification accuracy and coverage of extreme proteins and protein phosphorylation sites. Briefly, the comparative protein samples were firstly mixed together at specific ratios of 9:1 and 1:9 (w/w), followed with mass differentiate light and heavy isotope labeling, respectively. The extreme proteins and protein phosphorylation sites, even if the newly expressed or disappeared ones, could be accurately quantified due to all of the proteins' relative abundances had been adjusted to 2 orders of magnitude (1/9-9) by this strategy. The number of quantified phosphorylation sites with more than 20 folds changes was improved about 10 times in comparative quantification of pervanadate stimulated phosphoproteome of HeLa cells, and 134 newly generated and 21 disappeared phosphorylation sites were solely quantified by the MaSR strategy. The significantly up-regulated phosphorylation sites were mainly involved in the key phosphoproteins regulating the insulin-related pathways, such as PI3K-AKT and RAS-MAPK pathways. Therefore, the MaSR strategy exhibits as a promising way in elucidating the biological processes with significant dysregulations. - Highlights: • All the proteins' relative abundances were adjusted into 2 orders of magnitude (1/9-9). • The quantification accuracy and coverage of extreme proteins and protein phosphorylation sites had been improved. • The newly expressed or disappeared proteins and protein

  8. Myosin Light Chain Kinase and the Role of Myosin Light Chain Phosphorylation in Skeletal Muscle

    Science.gov (United States)

    Stull, James T.; Kamm, Kristine E.; Vandenboom, Rene

    2011-01-01

    Skeletal muscle myosin light chain kinase (skMLCK) is a dedicated Ca2+/calmodulin-dependent serine-threonine protein kinase that phosphorylates the regulatory light chain (RLC) of sarcomeric myosin. It is expressed from the MYLK2 gene specifically in skeletal muscle fibers with most abundance in fast contracting muscles. Biochemically, activation occurs with Ca2+ binding to calmodulin forming a (Ca2+)4•calmodulin complex sufficient for activation with a diffusion limited, stoichiometic binding and displacement of a regulatory segment from skMLCK catalytic core. The N-terminal sequence of RLC then extends through the exposed catalytic cleft for Ser15 phosphorylation. Removal of Ca2+ results in the slow dissociation of calmodulin and inactivation of skMLCK. Combined biochemical properties provide unique features for the physiological responsiveness of RLC phosphorylation, including (1) rapid activation of MLCK by Ca2+/calmodulin, (2) limiting kinase activity so phosphorylation is slower than contraction, (3) slow MLCK inactivation after relaxation and (4) much greater kinase activity relative to myosin light chain phosphatase (MLCP). SkMLCK phosphorylation of myosin RLC modulates mechanical aspects of vertebrate skeletal muscle function. In permeabilized skeletal muscle fibers, phosphorylation-mediated alterations in myosin structure increase the rate of force-generation by myosin cross bridges to increase Ca2+-sensitivity of the contractile apparatus. Stimulation-induced increases in RLC phosphorylation in intact muscle produces isometric and concentric force potentiation to enhance dynamic aspects of muscle work and power in unfatigued or fatigued muscle. Moreover, RLC phosphorylation-mediated enhancements may interact with neural strategies for human skeletal muscle activation to ameliorate either central or peripheral aspects of fatigue. PMID:21284933

  9. Phosphorylation of histone H3 at threonine 11 establishes a novel chromatin mark for transcriptional regulation

    OpenAIRE

    Metzger, Eric; Yin, Na; Wissmann, Melanie; Kunowska, Natalia; Fischer, Kristin; Friedrichs, Nicolaus; Patnaik, Debasis; Higgins, Jonathan M.G.; Potier, Noelle; Scheidtmann, Karl-Heinz; Buettner, Reinhard; Schüle, Roland

    2007-01-01

    Posttranslational modifications of histones such as methylation, acetylation, and phosphorylation regulate chromatin structure and gene expression. Here we show that protein kinase C-related kinase 1 (PRK1) phosphorylates histone H3 at threonine 11 (H3T11) upon ligand-dependent recruitment to androgen receptor (AR) target genes. PRK1 is pivotal to AR function since PRK1 knockdown or inhibition impedes AR-dependent transcription. Blocking PRK1 function abrogates androgen-induced H3T11 phosphor...

  10. Phosphorylation of AIB1 at Mitosis Is Regulated by CDK1/CYCLIN B

    Science.gov (United States)

    Ferrero, Macarena; Ferragud, Juan; Orlando, Leonardo; Valero, Luz; Sánchez del Pino, Manuel; Farràs, Rosa; Font de Mora, Jaime

    2011-01-01

    Background Although the AIB1 oncogene has an important role during the early phase of the cell cycle as a coactivator of E2F1, little is known about its function during mitosis. Methodology/Principal Findings Mitotic cells isolated by nocodazole treatment as well as by shake-off revealed a post-translational modification occurring in AIB1 specifically during mitosis. This modification was sensitive to the treatment with phosphatase, suggesting its modification by phosphorylation. Using specific inhibitors and in vitro kinase assays we demonstrate that AIB1 is phosphorylated on Ser728 and Ser867 by Cdk1/cyclin B at the onset of mitosis and remains phosphorylated until exit from M phase. Differences in the sensitivity to phosphatase inhibitors suggest that PP1 mediates dephosphorylation of AIB1 at the end of mitosis. The phosphorylation of AIB1 during mitosis was not associated with ubiquitylation or degradation, as confirmed by western blotting and flow cytometry analysis. In addition, luciferase reporter assays showed that this phosphorylation did not alter the transcriptional properties of AIB1. Importantly, fluorescence microscopy and sub-cellular fractionation showed that AIB1 phosphorylation correlated with the exclusion from the condensed chromatin, thus preventing access to the promoters of AIB1-dependent genes. Phospho-specific antibodies developed against Ser728 further demonstrated the presence of phosphorylated AIB1 only in mitotic cells where it was localized preferentially in the periphery of the cell. Conclusions Collectively, our results describe a new mechanism for the regulation of AIB1 during mitosis, whereby phosphorylation of AIB1 by Cdk1 correlates with the subcellular redistribution of AIB1 from a chromatin-associated state in interphase to a more peripheral localization during mitosis. At the exit of mitosis, AIB1 is dephosphorylated, presumably by PP1. This exclusion from chromatin during mitosis may represent a mechanism for governing the

  11. Phosphorylation of Tropomyosin Extends Cooperative Binding of Myosin Beyond a Single Regulatory Unit

    OpenAIRE

    Rao, Vijay S.; Marongelli, Ellisha N.; Guilford, William H.

    2009-01-01

    Tropomyosin (Tm) is one of the major phosphoproteins comprising the thin filament of muscle. However, the specific role of Tm phosphorylation in modulating the mechanics of actomyosin interaction has not been determined. Here we show that Tm phosphorylation is necessary for long-range cooperative activation of myosin binding. We used a novel optical trapping assay to measure the isometric stall force of an ensemble of myosin molecules moving actin filaments reconstituted with either natively ...

  12. CD44 regulates cell migration in human colon cancer cells via Lyn kinase and AKT phosphorylation.

    Science.gov (United States)

    Subramaniam, Venkateswaran; Vincent, Isabella R; Gardner, Helena; Chan, Emily; Dhamko, Helena; Jothy, Serge

    2007-10-01

    Colon cancer is among the leading causes of cancer death in North America. CD44, an adhesion and antiapoptotic molecule is overexpressed in colon cancer. Cofilin is involved in the directional motility of cells. In the present study, we looked at how CD44 might modulate cell migration in human colon cancer via cofilin. We used a human colon cancer cell line, HT29, which expresses CD44, HT29 where CD44 expression was knocked down by siRNA, SW620, a human colon cancer cell line which does not express CD44, stably transfected exons of CD44 in SW620 cells and the colon from CD44 knockout and wild-type mouse. Western blot analysis of siRNA CD44 lysates showed increased level of AKT phosphorylation and decreased level of cofilin expression. Similar results were also observed with SW620 cells and CD44 knockout mouse colon lysates. Experiments using the AKT phosphorylation inhibitor LY294002 indicate that AKT phosphorylation downregulates cofilin. Immunoprecipitation studies showed CD44 complex formation with Lyn, providing an essential link between CD44 and AKT phosphorylation. LY294002 also stabilized Lyn from phosphorylated AKT, suggesting an interaction between Lyn and AKT phosphorylation. Immunocytochemistry showed that cofilin and Lyn expression were downregulated in siRNA CD44 cells and CD44 knockout mouse colon. siRNA CD44 cells had significantly less migration compared to HT29 vector. Given the well-defined roles of CD44, phosphorylated AKT in apoptosis and cancer, these results indicate that CD44-induced cell migration is dependent on its complex formation with Lyn and its consequent regulation of AKT phosphorylation and cofilin expression.

  13. Phosphorylation of CPAP by Aurora-A Maintains Spindle Pole Integrity during Mitosis

    OpenAIRE

    En-Ju Chou; Liang-Yi Hung; Chieh-Ju C. Tang; Wen-Bin Hsu; Hsin-Yi Wu; Pao-Chi Liao; Tang K. Tang

    2016-01-01

    CPAP is required for centriole elongation during S/G2 phase, but the role of CPAP in mitosis is incompletely understood. Here, we show that CPAP maintains spindle pole integrity through its phosphorylation by Aurora-A during mitosis. Depletion of CPAP induced a prolonged delay in mitosis, pericentriolar material (PCM) dispersion, and multiple mitotic abnormalities. Further studies demonstrated that CPAP directly interacts with and is phosphorylated by Aurora-A at serine 467 during mitosis. In...

  14. Characterization of mitosis-specific phosphorylation of tumor-associated microtubule-associated protein

    OpenAIRE

    Hong, Kyung Uk; Kim, Hyun-Jun; Bae, Chang-Dae; Park, Joobae

    2009-01-01

    Tumor-associated microtubule-associated protein (TMAP), also known as cytoskeleton associated protein 2 (CKAP2), has been recently shown to be involved in the assembly and maintenance of mitotic spindle and also plays an essential role in maintaining the fidelity of chromosome segregation during mitosis. We have previously reported that TMAP is phosphorylated at multiple residues specifically during mitosis, and characterized the mechanism and functional importance of phosphorylation at one o...

  15. Tyrosine phosphorylation in T cells is regulated by phosphatase activity: studies with phenylarsine oxide.

    OpenAIRE

    Garcia-Morales, P; Minami, Y; Luong, E; Klausner, R D; Samelson, L E

    1990-01-01

    Activation of T cells induces rapid tyrosine phosphorylation on the T-cell receptor zeta chain and other substrates. These phosphorylations can be regulated by a number of protein-tyrosine kinases (ATP: protein-tyrosine O-phosphotransferase, EC 2.7.1.112) and protein-tyrosine-phosphatases (protein-tyrosine-phosphate phosphohydrolase, EC 3.1.3.48). In this study, we demonstrate that phenylarsine oxide can inhibit tyrosine phosphatases while leaving tyrosine kinase function intact. We use this ...

  16. A p130Cas tyrosine phosphorylated substrate domain decoy disrupts v-Crk signaling

    Directory of Open Access Journals (Sweden)

    Hanafusa Hidesaburo

    2002-07-01

    Full Text Available Abstract Background The adaptor protein p130Cas (Cas has been shown to be involved in different cellular processes including cell adhesion, migration and transformation. This protein has a substrate domain with up to 15 tyrosines that are potential kinase substrates, able to serve as docking sites for proteins with SH2 or PTB domains. Cas interacts with focal adhesion plaques and is phosphorylated by the tyrosine kinases FAK and Src. A number of effector molecules have been shown to interact with Cas and play a role in its function, including c-crk and v-crk, two adaptor proteins involved in intracellular signaling. Cas function is dependent on tyrosine phosphorylation of its substrate domain, suggesting that tyrosine phosphorylation of Cas in part regulates its control of adhesion and migration. To determine whether the substrate domain alone when tyrosine phosphorylated could signal, we have constructed a chimeric Cas molecule that is phosphorylated independently of upstream signals. Results We found that a tyrosine phosphorylated Cas substrate domain acts as a dominant negative mutant by blocking Cas-mediated signaling events, including JNK activation by the oncogene v-crk in transient and stable lines and v-crk transformation. This block was the result of competition for binding partners as the chimera competed for binding to endogenous c-crk and exogenously expressed v-crk. Conclusion Our approach suggests a novel method to study adaptor proteins that require phosphorylation, and indicates that mere tyrosine phosphorylation of the substrate domain of Cas is not sufficient for its function.

  17. The Contribution of Serine 194 Phosphorylation to Steroidogenic Acute Regulatory Protein Function

    OpenAIRE

    Sasaki, Goro; Zubair, Mohamad; Ishii, Tomohiro; Mitsui, Toshikatsu; Hasegawa, Tomonobu; Auchus, Richard J.

    2014-01-01

    The steroidogenic acute regulatory protein (StAR) facilitates the delivery of cholesterol to the inner mitochondrial membrane, where the cholesterol side-chain cleavage enzyme catalyzes the initial step of steroid hormone biosynthesis. StAR was initially identified in adrenocortical cells as a phosphoprotein, the expression and phosphorylation of which were stimulated by corticotropin. A number of in vitro studies have implicated cAMP-dependent phosphorylation at serine 194 (S194, S195 in hum...

  18. Phosphorylated derivatives of anabasine: synthesis, constitution and complex forming properties

    International Nuclear Information System (INIS)

    Babaev, B.N.

    2004-01-01

    Full text: With the purpose of detection of new effective extra gents of metals from number of sulphur containing derivatives of acids of phosphorus with different functional groups, analysis of effect of the different factors on selectivity of allocation of metals, installation of optimum conditions of an extraction, the detections of effective extra gents of noble metals from industrial sewage waters, are synthesized phosphorylated derivatives of the anabasine - O-alkyl-O-(anaba-sinoisopropyl)- and O-alkyl-O-(anabasinobutyn-2-yl)phenyl phosphonates and O-(anabasinoisopropyl)- and O-(anabasinobutyn-2-yl)diphenyl phosphonates In an IR-spectrum about O-pentyl-O-[anabasinoisopropyl]phenylphosphonate There are absorption band of the following functional groups (ν, cm -1 ): (P-O-C 5 H 11 ) 990-1000, (P = 0) 1250, (P-C 6 H 5 )1450, (C-N in cycle) 1550. In a spectrum PMR O-(anabasinobutyn-2-yl)phenylphosphonate in the field of a weak field apart from signals of two phenylic radicals the signals of a b-displaced pyridine, reference for a molecule anabasine are observed: Hαa-8,46 p.m., H α '-8,41 p.m., H γ -7,60 p.m. And H β -7,15 p.m. A double triplet at 4,70 p.m. And triplet at 3,05 p.m. Belong to signals OCH 2 and N-CH 2 of groups, accordingly, separated by acetylene bond. The signals of piperidine cycle of anabasine have the following chemical shifts: H 2a -3,27 p.m., H 6e -2,78 p.m., H 6a -2,45 p.m., and remaining protons (6H, m, CH 2 ) are in resonance in the field of 1,1-1,9 p.m. The analysis of mass-spectrometer decay of the synthesized connections has shown, that the mass-spectrometer fragmentation M * about - O-alkyl-O- (anabasinoisopropyl) phenylphosphonates flows past in different directions and is characterized, as against about O-alkyl-O-(anabasinobutyn-2-yl)phenyl-phosphonates, large number of phosphor containing ions; the availability of the second phenylic radical in molecules anabasincontaining derivatives of a diphenyl phosphinic acid essentially

  19. Quantifying Kinase-Specific Phosphorylation Stoichiometry Using Stable Isotope Labeling In a Reverse In-Gel Kinase Assay

    Energy Technology Data Exchange (ETDEWEB)

    Li, Xiang; Cox, Jonathan T.; Huang, Weiliang; Kane, Maureen; Tang, Keqi; Bieberich, Charles J.

    2016-12-06

    Reversible protein phosphorylation regulates essentially all cellular activities. Aberrant protein phosphorylation is an etiological factor in a wide array of diseases, including cancer1, diabetes2, and Alzheimer’s3. Given the broad impact of protein phosphorylation on cellular biology and organismal health, understanding how protein phosphorylation is regulated and the consequences of gain and loss of phosphoryl moieties from proteins is of primary importance. Advances in instrumentation, particularly in mass spectrometry, coupled with high throughput approaches have recently yielded large datasets cataloging tens of thousands of protein phosphorylation sites in multiple organisms4-6. While these studies are seminal in term of data collection, our understanding of protein phosphorylation regulation remains largely one-dimensional.

  20. Hydrogen bond based smart polymer for highly selective and tunable capture of multiply phosphorylated peptides.

    Science.gov (United States)

    Qing, Guangyan; Lu, Qi; Li, Xiuling; Liu, Jing; Ye, Mingliang; Liang, Xinmiao; Sun, Taolei

    2017-09-06

    Multisite phosphorylation is an important and common mechanism for finely regulating protein functions and subsequent cellular responses. However, this study is largely restricted by the difficulty to capture low-abundance multiply phosphorylated peptides (MPPs) from complex biosamples owing to the limitation of enrichment materials and their interactions with phosphates. Here we show that smart polymer can serve as an ideal platform to resolve this challenge. Driven by specific but tunable hydrogen bonding interactions, the smart polymer displays differential complexation with MPPs, singly phosphorylated and non-modified peptides. Importantly, MPP binding can be modulated conveniently and precisely by solution conditions, resulting in highly controllable MPP adsorption on material surface. This facilitates excellent performance in MPP enrichment and separation from model proteins and real biosamples. High enrichment selectivity and coverage, extraordinary adsorption capacities and recovery towards MPPs, as well as high discovery rates of unique phosphorylation sites, suggest its great potential in phosphoproteomics studies.Capture of low-abundance multiply phosphorylated peptides (MPPs) is difficult due to limitation of enrichment materials and their interactions with phosphates. Here the authors show, a smart polymer driven by specific but tunable hydrogen bonding interactions can differentially complex with MPPs, singly phosphorylated and non-modified peptides.

  1. Construction and Deciphering of Human Phosphorylation-Mediated Signaling Transduction Networks.

    Science.gov (United States)

    Zhang, Menghuan; Li, Hong; He, Ying; Sun, Han; Xia, Li; Wang, Lishun; Sun, Bo; Ma, Liangxiao; Zhang, Guoqing; Li, Jing; Li, Yixue; Xie, Lu

    2015-07-02

    Protein phosphorylation is the most abundant reversible covalent modification. Human protein kinases participate in almost all biological pathways, and approximately half of the kinases are associated with disease. PhoSigNet was designed to store and display human phosphorylation-mediated signal transduction networks, with additional information related to cancer. It contains 11 976 experimentally validated directed edges and 216 871 phosphorylation sites. Moreover, 3491 differentially expressed proteins in human cancer from dbDEPC, 18 907 human cancer variation sites from CanProVar, and 388 hyperphosphorylation sites from PhosphoSitePlus were collected as annotation information. Compared with other phosphorylation-related databases, PhoSigNet not only takes the kinase-substrate regulatory relationship pairs into account, but also extends regulatory relationships up- and downstream (e.g., from ligand to receptor, from G protein to kinase, and from transcription factor to targets). Furthermore, PhoSigNet allows the user to investigate the impact of phosphorylation modifications on cancer. By using one set of in-house time series phosphoproteomics data, the reconstruction of a conditional and dynamic phosphorylation-mediated signaling network was exemplified. We expect PhoSigNet to be a useful database and analysis platform benefiting both proteomics and cancer studies.

  2. Rapid changes in protein phosphorylation associated with gravity perception in corn roots

    International Nuclear Information System (INIS)

    McFadden, J.J.; Poovaiah, B.W.

    1987-01-01

    A previous paper from this laboratory showed calcium- and calmodulin-dependent in vivo protein phosphorylation in corn root tips. The authors show that rapid changes in calcium-dependent protein phosphorylation are involved in light-dependent graviperception in corn root tips. Corn seedlings (Zea mays L, cv Merit) were grown in the dark for 3 d, then apical root segments were harvested in dim green light to measure in vivo protein phosphorylation. Segments were incubated with 0.5 mCi 32 P for 1 h, then immediately frozen in liquid N 2 or first treated with either 7 min light, or 7 min light plus 1 mM EGTA and 10 μM A23187. Labeled proteins were separated by 2D gel electrophoresis and detected by autoradiography. Light caused rapid and specific promotion of phosphorylation of 5 polypeptides. The increases in protein phosphorylation were reversed by treating with EGTA and A23187. The authors postulate that these changes in protein phosphorylation are an essential part of the light-dependent gravity response in Merit roots

  3. The ATM homologue MEC1 is required for phosphorylation of replication protein A in yeast

    International Nuclear Information System (INIS)

    Brush, G.S.; Morrow, D.M.; Hieter, P.; Kelly, T.J.

    1996-01-01

    Replication protein A (RPA) is a highly conserved single-stranded DNA-binding protein, required for cellular DNA replication, repair, and recombination. In human cells, RPA is phosphorylated during the S and G2 phases of the cell cycle and also in response to ionizing or ultraviolet radiation. Saccharomyces cerevisiae exhibits a similar pattern of cell cycle-regulated RPA phosphorylation, and our studies indicate that the radiation-induced reactions occur in yeast as well. We have examined yeast RPA phosphorylation during the normal cell cycle and in response to environmental insult, and have demonstrated that the checkpoint gene MEC1 is required for the reaction under all conditions tested. Through examination of several checkpoint mutants, we have placed RPA phosphorylation in a novel pathway of the DNA damage response. MEC1 is similar in sequence to human ATM, the gene mutated in patients with ataxia-telangiectasia (A-T). A-T cells are deficient in multiple checkpoint pathways and are hypersensitive to killing by ionizing radiation. Because A-T cells exhibit a delay in ionizing radiation-induced RPA phosphorylation, our results indicate a functional similarity between MEC1 and ATM, and suggest that RPA phosphorylation is involved in a conserved eukaryotic DNA damage-response pathway defective in A-T

  4. Selective Sensing of Tyrosine Phosphorylation in Peptides Using Terbium(III Complexes

    Directory of Open Access Journals (Sweden)

    Jun Sumaoka

    2016-01-01

    Full Text Available Phosphorylation of tyrosine residues in proteins, as well as their dephosphorylation, is closely related to various diseases. However, this phosphorylation is usually accompanied by more abundant phosphorylation of serine and threonine residues in the proteins and covers only 0.05% of the total phosphorylation. Accordingly, highly selective detection of phosphorylated tyrosine in proteins is an urgent subject. In this review, recent developments in this field are described. Monomeric and binuclear TbIII complexes, which emit notable luminescence only in the presence of phosphotyrosine (pTyr, have been developed. There, the benzene ring of pTyr functions as an antenna and transfers its photoexcitation energy to the TbIII ion as the emission center. Even in the coexistence of phosphoserine (pSer and phosphothreonine (pThr, pTyr can be efficintly detected with high selectivity. Simply by adding these TbIII complexes to the solutions, phosphorylation of tyrosine in peptides by protein tyrosine kinases and dephosphorylation by protein tyrosine phosphatases can be successfully visualized in a real-time fashion. Furthermore, the activities of various inhibitors on these enzymes are quantitatively evaluated, indicating a strong potential of the method for efficient screening of eminent inhibitors from a number of candidates.

  5. Lead induced changes in phosphorylation of PSII proteins in low light grown pea plants.

    Science.gov (United States)

    Wioleta, Wasilewska; Anna, Drożak; Ilona, Bacławska; Kamila, Kąkol; Elżbieta, Romanowska

    2015-02-01

    Light-intensity and redox-state induced thylakoid proteins phosphorylation involved in structural changes and in regulation of protein turnover. The presence of heavy metal ions triggers a wide range of cellular responses including changes in plant growth and photosynthesis. Plants have evolved a number of mechanisms to protect photosynthetic apparatus. We have characterized the effect of lead on PSII protein phosphorylation in pea (Pisum sativum L.) plants grown in low light conditions. Pb ions affected only slightly photochemical efficiency of PSII and had no effect on organization of thylakoid complexes. Lead activated strongly phosphorylation of PSII core D1 protein and dephosphorylation of this protein did not proceed in far red light. D1 protein was also not degraded in this conditions. However, phosphorylation of LHCII proteins was not affected by lead. These results indicate that Pb(2+) stimulate the phosphorylation of PSII core proteins and by disturbing the disassembly of supercomplexes play a role in PSII repair mechanism. LHCII phosphorylation could control the distribution of energy between the photosystems in low light conditions. This demonstrates that plants may respond to heavy metals by induction different pathways responsible for protein protection under stress conditions.

  6. Kinase-loaded magnetic beads for sequential in vitro phosphorylation of peptides and proteins.

    Science.gov (United States)

    Hromadkova, Lenka; Kupcik, Rudolf; Vajrychova, Marie; Prikryl, Petr; Charvatova, Andrea; Jankovicova, Barbora; Ripova, Daniela; Bilkova, Zuzana; Slovakova, Marcela

    2018-01-15

    Post-translational modifications, including phosphorylation, greatly impact the physiological function of proteins, especially those that are natively unfolded and implicated in many neurodegenerative diseases. However, structural and functional studies of such proteins require fully defined phosphorylation, including those that are not physiological. Thus, the kinases ERK2 and GSK-3β were immobilized to various superparamagnetic beads with carboxylic, aldehyde, Ni 2+ , or Co 3+ functional groups, with a view to efficiently phosphorylate peptides and proteins in vitro. Full phosphorylation of specific synthetic peptides confirmed that beads were successfully loaded with kinases. Remarkably, enzymes covalently immobilized on carboxylated SeraMag beads remained active upon reuse, with residual activity after 10 uses 99.5 ± 0.34% for GSK-3β and 36.2 ± 2.01% for ERK2. The beads were also used to sequentially phosphorylate recombinant tau, which in vivo is a biomarker of Alzheimer's disease. Thus, a system consisting of two fully active kinases immobilized to magnetic beads is demonstrated for the first time. In comparison to soluble enzymes, the beads are easier to handle, reusable, and thus low-cost. Importantly, these beads are also convenient to remove from reactions to minimize contamination of phosphorylated products or to exchange with other kinases.

  7. Phosphorylation and nuclear accumulation are distinct events contributing to the activation of p53

    International Nuclear Information System (INIS)

    O'Hagan, Heather M.; Ljungman, Mats

    2004-01-01

    It has been recently shown that ionizing radiation (IR) and the mRNA synthesis inhibitor 5,6-dichloro-1-b-D-ribofuranosylbenzimidazole (DRB) act in synergy to induce p53-mediated transactivation of reporter plasmids in human cells [Oncogene 19 (2000) 3829]. We have extended these studies and show that ionizing radiation and DRB also act in synergy to induce ATM-mediated phosphorylation of the ser15 site of p53 and enhance the expression of endogenous p21 protein. Examination of the localization of p53 revealed that while DRB did not induce phosphorylation of the ser15 site of p53 but efficiently accumulated p53 in the nucleus, ionizing radiation induced phosphorylation of the ser15 site of p53 without prolonged nuclear accumulation. Importantly, the combination of DRB and IR resulted in a strong accumulation of phosphorylated p53 in the nucleus that was more persistent then p53 accumulation after IR alone. Furthermore, the nuclear export inhibitor leptomycin B showed a similar synergy with IR as did DRB regarding ser15 phosphorylation of p53 and p21 induction. These results suggest that the synergistic activation of the p53 response by the combination treatment is due to the activation of two distinct pathways where DRB causes the prolonged nuclear accumulation of p53 while ionizing radiation activates p53 by ATM-mediated phosphorylation

  8. The selective phosphorylation of a guanine nucleotide-binding regulatory protein

    International Nuclear Information System (INIS)

    Carlson, K.E.

    1989-01-01

    Receptor-activated signal transduction pathways regulate the responsiveness of cells to external stimuli. These transduction pathways themselves are subject to regulation, most commonly by phosphorylation. Guanine nucleotide-binding regulatory proteins (G Proteins), as requisite signal transducing elements for many plasma membrane receptors, are considered likely targets for regulation by phosphorylation. Protein kinase C (PKC) has been shown to phosphorylate the α subunit of G i and other G proteins in solution. However, the occurrence of the phosphorylation of G 1 within intact cells in response to activation of PKC has not been rigorously demonstrated. In this thesis, the extent to which the α subunits of G i undergo phosphorylation within human platelets in response to activation of PKC was examined by means of radiolabeling and immunoprecipitation. Incubation of platelets with phorbol-12-myristate-13-acetate (PMA), a potent activator of PKC, promoted the phosphorylation of several proteins within saponin-permeabilized and intact platelets incubated with [γ 32 P]ATP and [ 32 P]H 3 PO 4 , respectively. None of the phosphoproteins, however, were precipitated by either of two antisera containing antibodies differing in specificities for epitopes within G iα -despite precipitation of a substantial fraction of the subunit itself. In contrast, other antisera, containing antibodies specific for the recently describe G zα , or antibodies for both G zα and G iα , precipitated a 40-kDa phosphoprotein

  9. Exposure to Tumescent Solution Significantly Increases Phosphorylation of Perilipin in Adipocytes.

    Science.gov (United States)

    Keskin, Ilknur; Sutcu, Mustafa; Eren, Hilal; Keskin, Mustafa

    2017-02-01

    Lidocaine and epinephrine could potentially decrease adipocyte viability, but these effects have not been substantiated. The phosphorylation status of perilipin in adipocytes may be predictive of cell viability. Perilipin coats lipid droplets and restricts access of lipases; phospho-perilipin lacks this protective function. The authors investigated the effects of tumescent solution containing lidocaine and epinephrine on the phosphorylation status of perilipin in adipocytes. In this in vitro study, lipoaspirates were collected before and after tumescence from 15 women who underwent abdominoplasty. Fat samples were fixed, sectioned, and stained for histologic and immunohistochemical analyses. Relative phosphorylation of perilipin was inferred from pixel intensities of immunostained adipocytes observed with confocal microscopy. For adipocytes collected before tumescent infiltration, 10.08% of total perilipin was phosphorylated. In contrast, 30.62% of total perilipin was phosphorylated for adipocytes collected from tumescent tissue (P < .01). The tumescent technique increases the relative phosphorylation of perilipin in adipocytes, making these cells more vulnerable to lipolysis. Tumescent solution applied for analgesia or hemostasis of the donor site should contain the lowest possible concentrations of lidocaine and epinephrine. LEVEL OF EVIDENCE 5. © 2016 The American Society for Aesthetic Plastic Surgery, Inc. Reprints and permission: journals.permissions@oup.com.

  10. Combined functional CT/FDG-PET: demonstrates reduced hepatic phosphorylation of glucose in advanced colorectal cancer

    International Nuclear Information System (INIS)

    Miles, K.A.; Keith, C.J.; Griffiths, M.R.; Fuentes, M.; Bunce, I.

    2002-01-01

    Full text: This study describes a technique to quantify hepatic glucose phosphorylation using combined data from functional CT and FDG-PET and assesses the differences in phosphorylation between patients with either early or advanced colorectal cancer. Functional CT and FDG-PET were utilised to obtain measurements of perfusion and glucose uptake respectively within the livers of a series of 35 patients with colorectal cancer. Patients with PET evidence of extrahepatic tumour were considered to have advanced disease. The net influx constant (Ki) for FDG was determined from the liver SUV. CT measurements of hepatic perfusion were incorporated into FDG kinetic analysis to determine hepatic glucose phosphorylation fraction. SUV and Ki were significantly lower in the 12 patients with advanced disease (p=0.015 and p=0.013 respectively) whereas portal and total hepatic perfusion were increased (p=0.013 and p=0.008 respectively). Combining the PET and CT data yielded phosphorylation fractions of 1.14% and 0.74% for early and advanced disease respectively (p=0.002). Hepatic glucose phosphorylation can be determined by combining functional CT measurements of perfusion with PET measurements of FDG and is significantly reduced in patients with more advanced malignancy. Reduced hepatic glucose phosphorylation may be an important mechanism in the development of cancer cachexia. Copyright (2002) The Australian and New Zealand Society of Nuclear Medicine Inc

  11. Combined functional CT/FDG-PET: demonstrates reduced hepatic phosphorylation of glucose in advanced colorectal cancer

    Energy Technology Data Exchange (ETDEWEB)

    Miles, K A [Southernex Imaging Group, QLD (Australia); Queensland University of Technology, QLD (Australia); Keith, C J [Southernex Imaging Group, QLD (Australia); Wesley Research Institute, QLD (Australia); Griffiths, M R [Queensland University of Technology, QLD (Australia); Fuentes, M [Southernex Imaging Group, QLD (Australia); Bunce, I [Wesley Research Institute, QLD (Australia)

    2002-07-01

    Full text: This study describes a technique to quantify hepatic glucose phosphorylation using combined data from functional CT and FDG-PET and assesses the differences in phosphorylation between patients with either early or advanced colorectal cancer. Functional CT and FDG-PET were utilised to obtain measurements of perfusion and glucose uptake respectively within the livers of a series of 35 patients with colorectal cancer. Patients with PET evidence of extrahepatic tumour were considered to have advanced disease. The net influx constant (Ki) for FDG was determined from the liver SUV. CT measurements of hepatic perfusion were incorporated into FDG kinetic analysis to determine hepatic glucose phosphorylation fraction. SUV and Ki were significantly lower in the 12 patients with advanced disease (p=0.015 and p=0.013 respectively) whereas portal and total hepatic perfusion were increased (p=0.013 and p=0.008 respectively). Combining the PET and CT data yielded phosphorylation fractions of 1.14% and 0.74% for early and advanced disease respectively (p=0.002). Hepatic glucose phosphorylation can be determined by combining functional CT measurements of perfusion with PET measurements of FDG and is significantly reduced in patients with more advanced malignancy. Reduced hepatic glucose phosphorylation may be an important mechanism in the development of cancer cachexia. Copyright (2002) The Australian and New Zealand Society of Nuclear Medicine Inc.

  12. Phosphorylation-induced changes in the energetic frustration in human Tank binding kinase 1.

    Science.gov (United States)

    Husain, Shahrukh; Kumar, Vijay; Hassan, Md Imtaiyaz

    2018-07-14

    Tank binding kinase 1 (TBK-1) plays an important role in immunity, inflammation, autophagy, cell growth and proliferation. Nevertheless, a key molecular and structural detail of TBK-1 phosphorylation and activation has been largely unknown. Here we investigated the energy landscape of phosphorylated (active) and unphosphorylated (inactive) forms of human TBK-1 to characterize the interplay between phosphorylation and local frustration. By employing the algorithm equipped with energy function and implemented in Frustratometer web-server (http://www.frustratometer.tk), we quantify the role of frustration in the activation of TBK-1. Accordingly, the conformational changes were observed in phosphoregulated active and inactive TBK-1. Substantial changes in frustration, flexibility and interatomic motions were observed among different forms of TBK-1. Structurally rigid kinase domain constitutes a minimally frustrated hub in the core of the catalytic domain, and highly frustrated clusters mainly at the C-lobe might enable the conformational transitions during activation. Also, a large network of highly frustrated interactions is found in the SDD domain of TBK-1 involved in protein-protein interactions and dimerization. The contact maps of the activation loop and α-C helix of kinase domain showed significant changes upon phosphorylation. Cross correlation analysis indicate that both intra and inter subunit correlated motions increases with phosphorylation of TBK-1. Phosphorylation thus introduces subtle changes in long-range contacts that might lead to significant conformational change of TBK-1. Copyright © 2018 Elsevier Ltd. All rights reserved.

  13. LOK is a major ERM kinase in resting lymphocytes and regulates cytoskeletal rearrangement through ERM phosphorylation.

    Science.gov (United States)

    Belkina, Natalya V; Liu, Yin; Hao, Jian-Jiang; Karasuyama, Hajime; Shaw, Stephen

    2009-03-24

    ERM (ezrin-radixin-moesin) proteins mediate linkage of actin cytoskeleton to plasma membrane in many cells. ERM activity is regulated in part by phosphorylation at a C-terminal threonine, but the identity of ERM kinases is unknown in lymphocytes and incompletely defined in other mammalian cells. Our studies show that lymphocyte-oriented kinase (LOK) is an ERM kinase in vitro and in vivo. Mass spectrometric analysis indicates LOK is abundant at the lymphocyte plasma membrane and immunofluorescence studies show LOK enrichment at the plasma membrane near ERM. In vitro peptide specificity analyses characterize LOK as a basophilic kinase whose optimal substrate sequence resembles the ERM site, including unusual preference for tyrosine at P-2. LOK's activity on moesin peptide and protein was comparable to reported ERM kinases ROCK and PKC but unlike them LOK displayed preferential specificity for moesin compared to traditional basophilic kinase substrates. Two genetic approaches demonstrate a role for LOK in ERM phosphorylation: cell transfection with LOK kinase domain augments ERM phosphorylation and lymphocytes from LOK knockout mice have >50% reduction in ERM phosphorylation. The findings on localization and specificity argue that LOK is a direct ERM kinase. The knockout mice have normal hematopoietic cell development but notably lymphocyte migration and polarization in response to chemokine are enhanced. These functional alterations fit the current understanding of the role of ERM phosphorylation in regulating cortical reorganization. Thus, these studies identify a new ERM kinase of importance in lymphocytes and confirm the role of ERM phosphorylation in regulating cell shape and motility.

  14. Inhibition of oxidative phosphorylation in ascites tumor mitochondria and cells by intramitochondrial Ca2+.

    Science.gov (United States)

    Villalobo, A; Lehninger, A L

    1980-03-25

    Accumulation of Ca2+ (+ phosphate) by respiring mitochondria from Ehrlich ascites or AS30-D hepatoma tumor cells inhibits subsequent phosphorylating respiration in response to ADP. The respiratory chain is still functional since a proton-conducting uncoupler produces a normal stimulation of electron transport. The inhibition of phosphorylating respiration is caused by intramitochondrial Ca2+ (+ phosphate). ATP + Mg2+ together, but not singly, prevents the inhibitory action of Ca2+. Neither AMP, GTP, GDP, nor any other nucleoside 5'-triphosphate or 5'-diphosphate could replace ATP in this effect. Phosphorylating respiration on NAD(NADP)-linked substrates was much more susceptible to the inhibitory effect of intramitochondrial Ca2+ than succinate-linked respiration. Significant inhibition of oxidative phosphorylation is given by the endogenous Ca2+ present in freshly isolated tumor mitochondria. The phosphorylating respiration of permeabilized Ehrlich ascites tumor cells is also inhibited by Ca2+ accumulated by the mitochondria in situ. Possible causes of the Ca2+-induced inhibition of oxidative phosphorylation are considered.

  15. Hemin inhibits internalization of transferrin by reticulocytes and promotes phosphorylation of the membrane transferrin receptor

    International Nuclear Information System (INIS)

    Cox, T.M.; O'Donnell, M.W.; Aisen, P.; London, I.M.

    1985-01-01

    Addition of hemin to reticulocytes inhibits incorporation of iron from transferrin. Heme also regulates protein synthesis in immature erythroid cells through its effects on phosphorylation of the initiation factor eIF-2. The authors have examined its effects on endocytosis of iron-transferrin and phosphorylation of the transferrin receptor. Hemin reduced iron transport but increased cell-associated transferrin. During uptake of 125 I-labeled transferrin in the steady state, the use of a washing technique to dissociate bound transferrin on the cell membrane showed that radioligand accumulated on the surface of hemin-treated cells. Receptor phosphorylation was investigated by immunoprecipitation of reticulocyte extracts after metabolic labeling with [ 32 P]P/sub i/. In the absence of ligand, phosphorylated receptor was chiefly localized on cell stroma. Exposure to transferrin increased cytosolic phosphorylated receptor from 15-30% to approximately 50% of the total, an effect overcome by hemin treatment. The findings suggest a possible relationship of phosphorylation to endocytosis of the transferrin receptor in reticulocytes

  16. ATM phosphorylation in HepG2 cells following continuous low dose-rate irradiation

    International Nuclear Information System (INIS)

    Mei Quelin; Du Duanming; Chen Zaizhong; Liu Pengcheng; Yang Jianyong; Li Yanhao

    2008-01-01

    Objective: To investigate the change of ATM phosphorylation in HepG2 cells following a continuous low dose-rate irradiation. Methods: Cells were persistently exposed to low dose-rate (8.28 cGy/h) irradiation. Indirect immunofluorescence and Western blot were used to detect the expression of ATM phosphorylated proteins. Colony forming assay was used to observe the effect of a low dose-rate irradiation on HepG2 cell survival. Results: After 30 min of low dose-rate irradiation, the phosphorylation of ATM occurred. After 6 h persistent irradiation, the expression of ATM phosphorylated protein reached the peak value, then gradually decreased. After ATM phosphorylation was inhibited with Wortmannin, the surviving fraction of HepG2 cells was lower than that of the irradiation alone group at each time point (P<0.05). Conclusions: Continuous low dose-rate irradiation attenuated ATM phosphorylation, suggesting that continuous low dose-rate irradiation has a potential effect for increasing the radiosensitivity of HepG2 cells. (authors)

  17. Phosphopeptide derivatization signatures to identify serine and threonine phosphorylated peptides by mass spectrometry.

    Science.gov (United States)

    Molloy, M P; Andrews, P C

    2001-11-15

    The development of rapid, global methods for monitoring states of protein phosphorylation would provide greater insight for understanding many fundamental biological processes. Current best practices use mass spectrometry (MS) to profile digests of purified proteins for evidence of phosphorylation. However, this approach is beset by inherent difficulties in both identifying phosphopeptides from within a complex mixture containing many other unmodified peptides and ionizing phosphopeptides in positive-ion MS. We have modified an approach that uses barium hydroxide to rapidly eliminate the phosphoryl group of serine and threonine modified amino acids, creating dehydroamino acids that are susceptible to nucleophilic derivatization. By derivatizing a protein digest with a mixture of two different alkanethiols, phosphopeptide-specific derivatives were readily distinguished by MS due to their characteristic ion-pair signature. The resulting tagged ion pairs accommodate simple and rapid screening for phosphopeptides in a protein digest, obviating the use of isotopically labeled samples for qualitative phosphopeptide detection. MALDI-MS is used in a first pass manner to detect derivatized phosphopeptides, while the remaining sample is available for tandem MS to reveal the site of derivatization and, thus, phosphorylation. We demonstrated the technique by identifying phosphopeptides from beta-casein and ovalbumin. The approach was further used to examine in vitro phosphorylation of recombinant human HSP22 by protein kinase C, revealing phosphorylation of Thr-63.

  18. Inhibition of nucleoside diphosphate kinase activity by in vitro phosphorylation by protein kinase CK2. Differential phosphorylation of NDP kinases in HeLa cells in culture

    DEFF Research Database (Denmark)

    Biondi, R M; Engel, M; Sauane, M

    1996-01-01

    that in vitro protein kinase CK2 catalyzed phosphorylation of human NDPK A inhibits its enzymatic activity by inhibiting the first step of its ping-pong mechanism of catalysis: its autophosphorylation. Upon in vivo 32P labeling of HeLa cells, we observed that both human NDPKs, A and B, were autophosphorylated...

  19. Is phosphorylated tau unique to chronic traumatic encephalopathy? Phosphorylated tau in epileptic brain and chronic traumatic encephalopathy.

    Science.gov (United States)

    Puvenna, Vikram; Engeler, Madeline; Banjara, Manoj; Brennan, Chanda; Schreiber, Peter; Dadas, Aaron; Bahrami, Ashkon; Solanki, Jesal; Bandyopadhyay, Anasua; Morris, Jacqueline K; Bernick, Charles; Ghosh, Chaitali; Rapp, Edward; Bazarian, Jeffrey J; Janigro, Damir

    2016-01-01

    Repetitive traumatic brain injury (rTBI) is one of the major risk factors for the abnormal deposition of phosphorylated tau (PT) in the brain and chronic traumatic encephalopathy (CTE). CTE and temporal lobe epilepsy (TLE) affect the limbic system, but no comparative studies on PT distribution in TLE and CTE are available. It is also unclear whether PT pathology results from repeated head hits (rTBI). These gaps prevent a thorough understanding of the pathogenesis and clinical significance of PT, limiting our ability to develop preventative and therapeutic interventions. We quantified PT in TLE and CTE to unveil whether a history of rTBI is a prerequisite for PT accumulation in the brain. Six postmortem CTE (mean 73.3 years) and age matched control samples were compared to 19 surgically resected TLE brain specimens (4 months-58 years; mean 27.6 years). No history of TBI was present in TLE or control; all CTE patients had a history of rTBI. TLE and CTE brain displayed increased levels of PT as revealed by immunohistochemistry. No age-dependent changes were noted, as PT was present as early as 4 months after birth. In TLE and CTE, cortical neurons, perivascular regions around penetrating pial vessels and meninges were immunopositive for PT; white matter tracts also displayed robust expression of extracellular PT organized in bundles parallel to venules. Microscopically, there were extensive tau-immunoreactive neuronal, astrocytic and degenerating neurites throughout the brain. In CTE perivascular tangles were most prominent. Overall, significant differences in staining intensities were found between CTE and control (P<0.01) but not between CTE and TLE (P=0.08). pS199 tau analysis showed that CTE had the most high molecular weight tangle-associated tau, whereas epileptic brain contained low molecular weight tau. Tau deposition may not be specific to rTBI since TLE recapitulated most of the pathological features of CTE. Copyright © 2015 Elsevier B.V. All rights

  20. Smooth muscle myosin light chain kinase efficiently phosphorylates serine 15 of cardiac myosin regulatory light chain

    Energy Technology Data Exchange (ETDEWEB)

    Josephson, Matthew P.; Sikkink, Laura A. [Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN 55905 (United States); Penheiter, Alan R. [Molecular Medicine Program, Mayo Clinic, Rochester, MN 55905 (United States); Burghardt, Thomas P., E-mail: burghardt@mayo.edu [Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN 55905 (United States); Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, MN 55905 (United States); Ajtai, Katalin [Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN 55905 (United States)

    2011-12-16

    Highlights: Black-Right-Pointing-Pointer Cardiac myosin regulatory light chain (MYL2) is phosphorylated at S15. Black-Right-Pointing-Pointer Smooth muscle myosin light chain kinase (smMLCK) is a ubiquitous kinase. Black-Right-Pointing-Pointer It is a widely believed that MYL2 is a poor substrate for smMLCK. Black-Right-Pointing-Pointer In fact, smMLCK efficiently and rapidly phosphorylates S15 in MYL2. Black-Right-Pointing-Pointer Phosphorylation kinetics measured by novel fluorescence method without radioactivity. -- Abstract: Specific phosphorylation of the human ventricular cardiac myosin regulatory light chain (MYL2) modifies the protein at S15. This modification affects MYL2 secondary structure and modulates the Ca{sup 2+} sensitivity of contraction in cardiac tissue. Smooth muscle myosin light chain kinase (smMLCK) is a ubiquitous kinase prevalent in uterus and present in other contracting tissues including cardiac muscle. The recombinant 130 kDa (short) smMLCK phosphorylated S15 in MYL2 in vitro. Specific modification of S15 was verified using the direct detection of the phospho group on S15 with mass spectrometry. SmMLCK also specifically phosphorylated myosin regulatory light chain S15 in porcine ventricular myosin and chicken gizzard smooth muscle myosin (S20 in smooth muscle) but failed to phosphorylate the myosin regulatory light chain in rabbit skeletal myosin. Phosphorylation kinetics, measured using a novel fluorescence method eliminating the use of radioactive isotopes, indicates similar Michaelis-Menten V{sub max} and K{sub M} for regulatory light chain S15 phosphorylation rates in MYL2, porcine ventricular myosin, and chicken gizzard myosin. These data demonstrate that smMLCK is a specific and efficient kinase for the in vitro phosphorylation of MYL2, cardiac, and smooth muscle myosin. Whether smMLCK plays a role in cardiac muscle regulation or response to a disease causing stimulus is unclear but it should be considered a potentially significant

  1. Insulin rapidly stimulates phosphorylation of a 46-kDa membrane protein on tyrosine residues as well as phosphorylation of several soluble proteins in intact fat cells

    International Nuclear Information System (INIS)

    Haering, H.U.; White, M.F.; Machicao, F.; Ermel, B.; Schleicher, E.; Obermaier, B.

    1987-01-01

    It is speculated that the transmission of an insulin signal across the plasma membrane of cells occurs through activation of the tyrosine-specific receptor kinase, autophosphorylation of the receptor, and subsequent phosphorylation of unidentified substrates in the cell. In an attempt to identify possible substrates, the authors labeled intact rat fat cells with [ 32 P]orthophosphate and used an antiphosphotyrosine antibody to identify proteins that become phosphorylated on tyrosine residues in an insulin-stimulated way. In the membrane fraction of the fat cells, they found, in addition to the 95-kDa β-subunit of the receptor, a 46-kDa phosphoprotein that is phosphorylated exclusively on tyrosine residues. This protein is not immunoprecipitated by antibodies against different regions of the insulin receptor and its HPLC tryptic peptide map is different from the tryptic peptide map of the insulin receptor, suggesting that it is not derived from the receptor β-subunit. Insulin stimulates the tyrosine phosphorylation of the 46-kDa protein within 150 sec in the intact cell 3- to 4-fold in a dose-dependent way at insulin concentrations between 0.5 nM and 100 nM. Insulin (0.5 nM, 100 nM) stimulated within 2 min the 32 P incorporation into a 116-kDa band, a 62 kDa band, and three bands between 45 kDa and 50 kDa 2- to 10-fold. They suggest that the 46-kDa membrane protein and possibly also the soluble proteins are endogenous substrates of the receptor tyrosine kinase in fat cells and that their phosphorylation is an early step in insulin signal transmission

  2. Towards the systematic discovery of signal transduction networks using phosphorylation dynamics data

    Directory of Open Access Journals (Sweden)

    Yachie Nozomu

    2010-05-01

    Full Text Available Abstract Background Phosphorylation is a ubiquitous and fundamental regulatory mechanism that controls signal transduction in living cells. The number of identified phosphoproteins and their phosphosites is rapidly increasing as a result of recent mass spectrometry-based approaches. Results We analyzed time-course phosphoproteome data obtained previously by liquid chromatography mass spectrometry with the stable isotope labeling using amino acids in cell culture (SILAC method. This provides the relative phosphorylation activities of digested peptides at each of five time points after stimulating HeLa cells with epidermal growth factor (EGF. We initially calculated the correlations between the phosphorylation dynamics patterns of every pair of peptides and connected the strongly correlated pairs to construct a network. We found that peptides extracted from the same intracellular fraction (nucleus vs. cytoplasm tended to be close together within this phosphorylation dynamics-based network. The network was then analyzed using graph theory and compared with five known signal-transduction pathways. The dynamics-based network was correlated with known signaling pathways in the NetPath and Phospho.ELM databases, and especially with the EGF receptor (EGFR signaling pathway. Although the phosphorylation patterns of many proteins were drastically changed by the EGF stimulation, our results suggest that only EGFR signaling transduction was both strongly activated and precisely controlled. Conclusions The construction of a phosphorylation dynamics-based network provides a useful overview of condition-specific intracellular signal transduction using quantitative time-course phosphoproteome data under specific experimental conditions. Detailed prediction of signal transduction based on phosphoproteome dynamics remains challenging. However, since the phosphorylation profiles of kinase-substrate pairs on the specific pathway were localized in the dynamics

  3. Phos-tag-based analysis of myosin regulatory light chain phosphorylation in human uterine myocytes.

    Directory of Open Access Journals (Sweden)

    Hector N Aguilar

    Full Text Available The 'phosphate-binding tag' (phos-tag reagent enables separation of phospho-proteins during SDS-PAGE by impeding migration proportional to their phosphorylation stoichiometry. Western blotting can then be used to detect and quantify the bands corresponding to the phospho-states of a target protein. We present a method for quantification of data regarding phospho-states derived from phos-tag SDS-PAGE. The method incorporates corrections for lane-to-lane loading variability and for the effects of drug vehicles thus enabling the comparison of multiple treatments by using the untreated cellular set-point as a reference. This method is exemplified by quantifying the phosphorylation of myosin regulatory light chain (RLC in cultured human uterine myocytes.We have evaluated and validated the concept that, when using an antibody (Ab against the total-protein, the sum of all phosphorylation states in a single lane represents a 'closed system' since all possible phospho-states and phosphoisotypes are detected. Using this approach, we demonstrate that oxytocin (OT and calpeptin (Calp induce RLC kinase (MLCK- and rho-kinase (ROK-dependent enhancements in phosphorylation of RLC at T18 and S19. Treatment of myocytes with a phorbol ester (PMA induced phosphorylation of S1-RLC, which caused a mobility shift in the phos-tag matrices distinct from phosphorylation at S19.We have presented a method for analysis of phospho-state data that facilitates quantitative comparison to a reference control without the use of a traditional 'loading' or 'reference' standard. This analysis is useful for assessing effects of putative agonists and antagonists where all phospho-states are represented in control and experimental samples. We also demonstrated that phosphorylation of RLC at S1 is inducible in intact uterine myocytes, though the signal in the resting samples was not sufficiently abundant to allow quantification by the approach used here.

  4. Ca(2+)-calmodulin-dependent phosphorylation of islet secretory granule proteins

    International Nuclear Information System (INIS)

    Watkins, D.T.

    1991-01-01

    The effect of Ca2+ and calmodulin on phosphorylation of islet secretory granule proteins was studied. Secretory granules were incubated in a phosphorylation reaction mixture containing [32P]ATP and test reagents. The 32P-labeled proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the 32P content was visualized by autoradiography, and the relative intensities of specific bands were quantitated. When the reaction mixture contained EGTA and no added Ca2+, 32P was incorporated into two proteins with molecular weights of 45,000 and 13,000. When 10(-4) M Ca2+ was added without EGTA, two additional proteins (58,000 and 48,000 Mr) were phosphorylated, and the 13,000-Mr protein was absent. The addition of 2.4 microM calmodulin markedly enhanced the phosphorylation of the 58,000- and 48,000-Mr proteins and resulted in the phosphorylation of a major protein whose molecular weight (64,000 Mr) is identical to that of one of the calmodulin binding proteins located on the granule surface. Calmodulin had no effect on phosphorylation in the absence of Ca2+ but was effective in the presence of calcium between 10 nM and 50 microM. Trifluoperazine and calmidazolium, calmodulin antagonists, produced a dose-dependent inhibition of the calmodulin effect. 12-O-tetradecanoylphorbol 13-acetate, a phorbol ester that activates protein kinase C, produced no increase in phosphorylation, and 1-(5-isoquinoline sulfonyl)-2-methyl piperazine dihydrochloride, an inhibitor of protein kinase C, had no effect. These results indicate that Ca(2+)-calmodulin-dependent protein kinases and endogenous substrates are present in islet secretory granules

  5. Accurate determination of the oxidative phosphorylation affinity for ADP in isolated mitochondria.

    Directory of Open Access Journals (Sweden)

    Gilles Gouspillou

    Full Text Available BACKGROUND: Mitochondrial dysfunctions appear strongly implicated in a wide range of pathologies. Therefore, there is a growing need in the determination of the normal and pathological integrated response of oxidative phosphorylation to cellular ATP demand. The present study intends to address this issue by providing a method to investigate mitochondrial oxidative phosphorylation affinity for ADP in isolated mitochondria. METHODOLOGY/PRINCIPAL FINDINGS: The proposed method is based on the simultaneous monitoring of substrate oxidation (determined polarographically and phosphorylation (determined using the glucose-hexokinase glucose-6-phosphate dehydrogenase-NADP(+ enzymatic system rates, coupled to the determination of actual ADP and ATP concentrations by bioluminescent assay. This enzymatic system allows the study of oxidative phosphorylation during true steady states in a wide range of ADP concentrations. We demonstrate how the application of this method allows an accurate determination of mitochondrial affinity for ADP from both oxidation (K(mVox and phosphorylation (K(mVp rates. We also demonstrate that determination of K(mVox leads to an important overestimation of the mitochondrial affinity for ADP, indicating that mitochondrial affinity for ADP should be determined using phosphorylation rate. Finally, we show how this method allows the direct and precise determination of the mitochondrial coupling efficiency. Data obtained from rat skeletal muscle and liver mitochondria illustrate the discriminating capabilities of this method. CONCLUSIONS/SIGNIFICANCE: Because the proposed method allows the accurate determination of mitochondrial oxidative phosphorylation affinity for ADP in isolated mitochondria, it also opens the route to a better understanding of functional consequences of mitochondrial adaptations/dysfunctions arising in various physiological/pathophysiological conditions.

  6. Cyclin dependent kinase 5 regulates endocytosis in nerve terminals via dynamin I phosphorylation

    International Nuclear Information System (INIS)

    Tan, T.C.; Hansra, G.; Calova, V.; Cousin, M.; Robinson, P.J.

    2002-01-01

    Full text: Synaptic vesicle endocytosis (SVE) in nerve terminals is essential for normal synaptic transmission and for memory retrieval. Dynamin I is a 96kDa nerve terminal phosphoprotein necessary for synaptic vesicle endocytosis in the nerve terminal. Dynamin I is dephosphorylated and rephosphorylated in a cyclical fashion with nerve terminal depolarisation and repolarisation. A number of kinases phosphorylate dynamin I in vitro including PKC, MAP kinase and cdc2. PKC phosphorylates dynamin in the proline rich domain on Ser 795 and is also thought to be the in vivo kinase for dynamin I. Another candidate is the neuron specific kinase cdk5, crucial for CNS development. The aim of this study is to identify the kinase which phosphorylates dynamin I in intact nerve terminals. Here we show that cyclin-dependent kinase 5 (cdk5) phosphorylates dynamin I in the proline-rich tail on Ser-774 or Ser-778. The phosphorylation of these sites but not Ser-795 also occurred in intact nerve terminals suggesting that cdk5 is the physiologically relevant enzyme for dynamin I. Synaptosomes prepared from rat brains (after cervical dislocations) and labelled with 32 Pi, were incubated with 100 M roscovitine (a selective inhibitor of cdks), 10 M Ro 31-8220 (a selective PKC inhibitor) and 100 M PD 98059 (a MEK kinase inhibitor). Dynamin rephosphorylation during repolarisation was reduced in synaptosomes treated with roscovitine and Ro 38-8220 but not in synaptosomes treated with PD 98059. Fluorimetric experiments on intact synaptosomes utilising FM-210 (a fluorescent dye) indicate that endocytosis was reduced in synaptosomes treated with 100 M roscovitine. Our results suggest that dynamin phosphorylation in intact nerve terminals may not be regulated by PKC or MAP kinase and that dynamin phosphorylation by cdk5 may regulate endocytosis. Copyright (2002) Australian Neuroscience Society

  7. Tyrosine phosphorylation of LRP6 by Src and Fer inhibits Wnt/β-catenin signalling

    Science.gov (United States)

    Chen, Qing; Su, Yi; Wesslowski, Janine; Hagemann, Anja I; Ramialison, Mirana; Wittbrodt, Joachim; Scholpp, Steffen; Davidson, Gary

    2014-01-01

    Low-density lipoprotein receptor-related proteins 5 and 6 (LRP5/6) function as transmembrane receptors to transduce Wnt signals. A key mechanism for signalling is Wnt-induced serine/threonine phosphorylation at conserved PPPSPxS motifs in the LRP6 cytoplasmic domain, which promotes pathway activation. Conserved tyrosine residues are positioned close to all PPPSPxS motifs, which suggests they have a functional significance. Using a cell culture-based cDNA expression screen, we identified the non-receptor tyrosine kinases Src and Fer as novel LRP6 modifiers. Both Src and Fer associate with LRP6 and phosphorylate LRP6 directly. In contrast to the known PPPSPxS Ser/Thr kinases, tyrosine phosphorylation by Src and Fer negatively regulates LRP6-Wnt signalling. Epistatically, they function upstream of β-catenin to inhibit signalling and in agreement with a negative role in regulating LRP6, MEF cells lacking these kinases show enhanced Wnt signalling. Wnt3a treatment of cells enhances tyrosine phosphorylation of endogenous LRP6 and, mechanistically, Src reduces cell surface LRP6 levels and disrupts LRP6 signalosome formation. Interestingly, CK1γ inhibits Fer-induced LRP6 phosphorylation, suggesting a mechanism whereby CK1γ acts to de-represses inhibitory LRP6 tyrosine phosphorylation. We propose that LRP6 tyrosine phosphorylation by Src and Fer serves a negative regulatory function to prevent over-activation of Wnt signalling at the level of the Wnt receptor, LRP6. Subject Categories Membrane & Intracellular Transport; Post-translational Modifications, Proteolysis & Proteomics PMID:25391905

  8. Cell stress promotes the association of phosphorylated HspB1 with F-actin.

    Directory of Open Access Journals (Sweden)

    Joseph P Clarke

    Full Text Available Previous studies have suggested that the small heat shock protein, HspB1, has a direct influence on the dynamics of cytoskeletal elements, in particular, filamentous actin (F-actin polymerization. In this study we have assessed the influence of HspB1 phosphorylation on its interaction(s with F-actin. We first determined the distribution of endogenous non-phosphorylated HspB1, phosphorylated HspB1 and F-actin in neuroendocrine PC12 cells by immunocytochemistry and confocal microscopy. We then investigated a potential direct interaction between HspB1 with F-actin by precipitating F-actin directly with biotinylated phalloidin followed by Western analyses; the reverse immunoprecipitation of HspB1 was also carried out. The phosphorylation influence of HspB1 in this interaction was investigated by using pharmacologic inhibition of p38 MAPK. In control cells, HspB1 interacts with F-actin as a predominantly non-phosphorylated protein, but subsequent to stress there is a redistribution of HspB1 to the cytoskeletal fraction and a significantly increased association of pHspB1 with F-actin. Our data demonstrate HspB1 is found in a complex with F-actin both in phosphorylated and non-phosphorylated forms, with an increased association of pHspB1 with F-actin after heat stress. Overall, our study combines both cellular and biochemical approaches to show cellular localization and direct demonstration of an interaction between endogenous HspB1 and F-actin using methodolgy that specifically isolates F-actin.

  9. Kinome analysis of receptor-induced phosphorylation in human natural killer cells.

    Directory of Open Access Journals (Sweden)

    Sebastian König

    Full Text Available BACKGROUND: Natural killer (NK cells contribute to the defense against infected and transformed cells through the engagement of multiple germline-encoded activation receptors. Stimulation of the Fc receptor CD16 alone is sufficient for NK cell activation, whereas other receptors, such as 2B4 (CD244 and DNAM-1 (CD226, act synergistically. After receptor engagement, protein kinases play a major role in signaling networks controlling NK cell effector functions. However, it has not been characterized systematically which of all kinases encoded by the human genome (kinome are involved in NK cell activation. RESULTS: A kinase-selective phosphoproteome approach enabled the determination of 188 kinases expressed in human NK cells. Crosslinking of CD16 as well as 2B4 and DNAM-1 revealed a total of 313 distinct kinase phosphorylation sites on 109 different kinases. Phosphorylation sites on 21 kinases were similarly regulated after engagement of either CD16 or co-engagement of 2B4 and DNAM-1. Among those, increased phosphorylation of FYN, KCC2G (CAMK2, FES, and AAK1, as well as the reduced phosphorylation of MARK2, were reproducibly observed both after engagement of CD16 and co-engagement of 2B4 and DNAM-1. Notably, only one phosphorylation on PAK4 was differentally regulated. CONCLUSIONS: The present study has identified a significant portion of the NK cell kinome and defined novel phosphorylation sites in primary lymphocytes. Regulated phosphorylations observed in the early phase of NK cell activation imply these kinases are involved in NK cell signaling. Taken together, this study suggests a largely shared signaling pathway downstream of distinct activation receptors and constitutes a valuable resource for further elucidating the regulation of NK cell effector responses.

  10. Band 3 Erythrocyte Membrane Protein Acts as Redox Stress Sensor Leading to Its Phosphorylation by p72 Syk

    Directory of Open Access Journals (Sweden)

    Antonella Pantaleo

    2016-01-01

    Full Text Available In erythrocytes, the regulation of the redox sensitive Tyr phosphorylation of band 3 and its functions are still partially defined. A role of band 3 oxidation in regulating its own phosphorylation has been previously suggested. The current study provides evidences to support this hypothesis: (i in intact erythrocytes, at 2 mM concentration of GSH, band 3 oxidation, and phosphorylation, Syk translocation to the membrane and Syk phosphorylation responded to the same micromolar concentrations of oxidants showing identical temporal variations; (ii the Cys residues located in the band 3 cytoplasmic domain are 20-fold more reactive than GSH; (iii disulfide linked band 3 cytoplasmic domain docks Syk kinase; (iv protein Tyr phosphatases are poorly inhibited at oxidant concentrations leading to massive band 3 oxidation and phosphorylation. We also observed that hemichromes binding to band 3 determined its irreversible oxidation and phosphorylation, progressive hemolysis, and serine hyperphosphorylation of different cytoskeleton proteins. Syk inhibitor suppressed the phosphorylation of band 3 also preventing serine phosphorylation changes and hemolysis. Our data suggest that band 3 acts as redox sensor regulating its own phosphorylation and that hemichromes leading to the protracted phosphorylation of band 3 may trigger a cascade of events finally leading to hemolysis.

  11. Akt regulates the subcellular localization of the Rab27a-binding protein JFC1 by phosphorylation.

    Science.gov (United States)

    Johnson, Jennifer L; Pacquelet, Sandrine; Lane, William S; Eam, Boreth; Catz, Sergio D

    2005-08-01

    Here, we show that the Rab27a-binding protein JFC1/Slp1 (synaptotagmin-like protein) is regulated by Akt-mediated phosphorylation. Using the phosphatase and tensin homolog-null LNCaP cells and the phosphatidylinositol 3-kinase inhibitor LY294002, we show that the phosphorylation of endogenous JFC1 is dependent on the phosphatidylinositol 3-kinase/Akt pathway. JFC1 was phosphorylated in cells expressing a constitutively active Akt, confirming that it is an Akt substrate in vivo. Direct phosphorylation of JFC1 by Akt was confirmed in vitro. Using microcapillary high-performance liquid chromatography tandem mass spectrometry, we identified five Akt-phosphorylation sites in JFC1. By mutagenesis analysis and subsequent immunoprecipitation (IP), we established that Akt phosphorylates JFC1 at serine 241. JFC1 and Rab27a colocalize in the proximity of the plasma membrane in LNCaP cells. The interaction was confirmed by IP analysis and was abolished by the point mutation W83S in JFC1. Phosphorylation did not alter the ability of JFC1 to bind to Rab27a. Instead, phosphorylation by Akt dramatically decreased when JFC1 was bound to Rab27a. Finally, we show that as a consequence of in vivo phosphorylation, JFC1 dissociates from the membrane, promoting JFC1 redistribution to the cytosol. Our results suggest that Akt regulates JFC1/Slp1 function by phosphorylation and may have implications on Rab27a-containing vesicle secretion.

  12. Novel Tyrosine Phosphorylation Sites in Rat Skeletal Muscle Revealed by Phosphopeptide Enrichment and HPLC-ESI-MS/MS

    Science.gov (United States)

    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 (tyrosine phosphorylation sites have been identified in mammalian skeletal muscle to date. Here, we used immunoprecipitation of phosphotyrosine peptides prior to HPLC-ESI-MS/MS analysis to improve the discovery of tyrosine phosphorylation in relatively small skeletal muscle biopsies from rats. This resulted in the identification of 87 distinctly localized tyrosine phosphorylation sites in 46 muscle proteins. Among them, 31 appear to be novel. The tyrosine phosphorylated proteins included major enzymes in the glycolytic pathway and glycogen metabolism, sarcomeric proteins, and proteins involved in Ca2+ homeostasis and phosphocreatine resynthesis. Among proteins regulated by insulin, we found tyrosine phosphorylation sites in glycogen synthase, and two of its inhibitors, GSK-3α and DYRK1A. Moreover, tyrosine phosphorylation sites were identified in several MAP kinases and a protein tyrosine phosphatase, SHPTP2. These results provide the largest catalogue of mammalian skeletal muscle tyrosine phosphorylation sites to date and provide novel targets for the investigation of human skeletal muscle phosphoproteins in various disease states. PMID:22609512

  13. In vitro phosphorylation of insulin receptor substrate 1 by protein kinase C-zeta: functional analysis and identification of novel phosphorylation sites.

    Science.gov (United States)

    Sommerfeld, Mark R; Metzger, Sabine; Stosik, Magdalene; Tennagels, Norbert; Eckel, Jürgen

    2004-05-18

    Protein kinase C-zeta (PKC-zeta) participates both in downstream insulin signaling and in the negative feedback control of insulin action. Here we used an in vitro approach to identify PKC-zeta phosphorylation sites within insulin receptor substrate 1 (IRS-1) and to characterize the functional implications. A recombinant IRS-1 fragment (rIRS-1(449)(-)(664)) containing major tyrosine motifs for interaction with phosphatidylinositol (PI) 3-kinase strongly associated to the p85alpha subunit of PI 3-kinase after Tyr phosphorylation by the insulin receptor. Phosphorylation of rIRS-1(449)(-)(664) by PKC-zeta induced a prominent inhibition of this process with a mixture of classical PKC isoforms being less effective. Both PKC-zeta and the classical isoforms phosphorylated rIRS-1(449)(-)(664) on Ser(612). However, modification of this residue did not reduce the affinity of p85alpha binding to pTyr-containing peptides (amino acids 605-615 of rat IRS-1), as determined by surface plasmon resonance. rIRS-1(449)(-)(664) was then phosphorylated by PKC-zeta using [(32)P]ATP and subjected to tryptic phosphopeptide mapping based on two-dimensional HPLC coupled to mass spectrometry. Ser(498) and Ser(570) were identified as novel phosphoserine sites targeted by PKC-zeta. Both sites were additionally confirmed by phosphopeptide mapping of the corresponding Ser --> Ala mutants of rIRS-1(449)(-)(664). Ser(570) was specifically targeted by PKC-zeta, as shown by immunoblotting with a phosphospecific antiserum against Ser(570) of IRS-1. Binding of p85alpha to the S570A mutant was less susceptible to inhibition by PKC-zeta, when compared to the S612A mutant. In conclusion, our in vitro data demonstrate a strong inhibitory action of PKC-zeta at the level of IRS-1/PI 3-kinase interaction involving multiple serine phosphorylation sites. Whereas Ser(612) appears not to participate in the negative control of insulin signaling, Ser(570) may at least partly contribute to this process.

  14. Proteomic Analysis of Calcium- and Phosphorylation-dependentCalmodulin Complexes in Mammalian Cells

    Energy Technology Data Exchange (ETDEWEB)

    Jang, Deok-Jin; Wang, Daojing

    2006-05-26

    Protein conformational changes due to cofactor binding (e.g. metal ions, heme) and/or posttranslational modifications (e.g. phosphorylation) modulate dynamic protein complexes. Calmodulin (CaM) plays an essential role in regulating calcium (Ca{sup 2+}) signaling and homeostasis. No systematic approach on the identification of phosphorylation-dependent Ca{sup 2+}/CaM binding proteins has been published. Herein, we report a proteome-wide study of phosphorylation-dependent CaM binding proteins from mammalian cells. This method, termed 'Dynamic Phosphoprotein Complex Trapping', 'DPPC Trapping' for short, utilizes a combination of in vivo and in vitro assays. The basic strategy is to drastically shift the equilibrium towards endogenous phosphorylation of Ser, Thr, and Tyr at the global scale by inhibiting corresponding phosphatases in vivo. The phosphorylation-dependent calmodulin-binding proteins are then trapped in vitro in a Ca{sup 2+}-dependent manner by CaM-Sepharose chromatography. Finally, the isolated calmodulin-binding proteins are separated by SDS-PAGE and identified by LC/MS/MS. In parallel, the phosphorylation-dependent binding is visualized by silver staining and/or Western blotting. Using this method, we selectively identified over 120 CaM-associated proteins including many previously uncharacterized. We verified ubiquitin-protein ligase EDD1, inositol 1, 4, 5-triphosphate receptor type 1 (IP{sub 3}R1), and ATP-dependent RNA helicase DEAD box protein 3 (DDX3), as phosphorylation-dependent CaM binding proteins. To demonstrate the utilities of our method in understanding biological pathways, we showed that pSer/Thr of IP{sub 3}R1 in vivo by staurosporine-sensitive kinase(s), but not by PKA/PKG/PKC, significantly reduced the affinity of its Ca{sup 2+}-dependent CaM binding. However, pSer/Thr of IP{sub 3}R1 did not substantially affect its Ca{sup 2+}-independent CaM binding. We further showed that phosphatase PP1, but not PP2A or PP2B

  15. Dynamic Phosphorylation of VP30 Is Essential for Ebola Virus Life Cycle.

    Science.gov (United States)

    Biedenkopf, Nadine; Lier, Clemens; Becker, Stephan

    2016-05-15

    Ebola virus is the causative agent of a severe fever with high fatality rates in humans and nonhuman primates. The regulation of Ebola virus transcription and replication currently is not well understood. An important factor regulating viral transcription is VP30, an Ebola virus-specific transcription factor associated with the viral nucleocapsid. Previous studies revealed that the phosphorylation status of VP30 impacts viral transcription. Together with NP, L, and the polymerase cofactor VP35, nonphosphorylated VP30 supports viral transcription. Upon VP30 phosphorylation, viral transcription ceases. Phosphorylation weakens the interaction between VP30 and the polymerase cofactor VP35 and/or the viral RNA. VP30 thereby is excluded from the viral transcription complex, simultaneously leading to increased viral replication which is supported by NP, L, and VP35 alone. Here, we use an infectious virus-like particle assay and recombinant viruses to show that the dynamic phosphorylation of VP30 is critical for the cotransport of VP30 with nucleocapsids to the sites of viral RNA synthesis, where VP30 is required to initiate primary viral transcription. We further demonstrate that a single serine residue at amino acid position 29 was sufficient to render VP30 active in primary transcription and to generate a recombinant virus with characteristics comparable to those of wild-type virus. In contrast, the rescue of a recombinant virus with a single serine at position 30 in VP30 was unsuccessful. Our results indicate critical roles for phosphorylated and dephosphorylated VP30 during the viral life cycle. The current Ebola virus outbreak in West Africa has caused more than 28,000 cases and 11,000 fatalities. Very little is known regarding the molecular mechanisms of how the Ebola virus transcribes and replicates its genome. Previous investigations showed that the transcriptional support activity of VP30 is activated upon VP30 dephosphorylation. The current study reveals that

  16. Structure of smAKAP and its regulation by PKA-mediated phosphorylation

    Science.gov (United States)

    Burgers, Pepijn P.; Bruystens, Jessica; Burnley, Rebecca J.; Nikolaev, Viacheslav O.; Keshwani, Malik; Wu, Jian; Janssen, Bert J. C.; Taylor, Susan S.; Heck, Albert J. R.; Scholten, Arjen

    2016-01-01

    The A-kinase anchoring protein (AKAP) smAKAP has three extraordinary features; it is very small, it is anchored directly to membranes by acyl motifs, and it interacts almost exclusively with the type I regulatory subunits (RI) of cAMP-dependent kinase (PKA). Here, we determined the crystal structure of smAKAP’s A-kinase binding domain (smAKAP-AKB) in complex with the dimerization/docking (D/D) domain of RIα which reveals an extended hydrophobic interface with unique interaction pockets that drive smAKAP’s high specificity for RI subunits. We also identify a conserved PKA phosphorylation site at Ser66 in the AKB domain which we predict would cause steric clashes and disrupt binding. This correlates with in vivo colocalization and fluorescence polarization studies, where Ser66 AKB phosphorylation ablates RI binding. Hydrogen/deuterium exchange studies confirm that the AKB helix is accessible and dynamic. Furthermore, full-length smAKAP as well as the unbound AKB is predicted to contain a break at the phosphorylation site, and circular dichroism measurements confirm that the AKB domain loses its helicity following phosphorylation. As the active site of PKA’s catalytic subunit does not accommodate α-helices, we predict that the inherent flexibility of the AKB domain enables its phosphorylation by PKA. This represents a novel mechanism, whereby activation of anchored PKA can terminate its binding to smAKAP affecting the regulation of localized cAMP signaling events. PMID:27028580

  17. Growth hormone-promoted tyrosyl phosphorylation of SHC proteins and SHC association with Grb2

    DEFF Research Database (Denmark)

    VanderKuur, J; Allevato, G; Billestrup, Nils

    1995-01-01

    . To gain insight into pathways coupling GH receptor (GHR) to MAP kinase activation and signaling molecules that might interact with GHR and its associated tyrosine kinase JAK2, we examined whether SHC and Grb2 proteins serve as signaling molecules for GH. Human GH was shown to promote the rapid tyrosyl...... phosphorylation of 66-, 52-, and 46-kDa SHC proteins in 3T3-F442A fibroblasts. GH also promoted binding of GHR and JAK2 to the SH2 domain of 46/52-kDa SHC protein fused to glutathione S-transferase (GST). Constitutively phosphorylated JAK2, from COS-7 cells transiently transfected with murine JAK2 cDNA, bound......-638 and GHR1-638(Y333,338F), GH stimulated phosphorylation of all 3 SHC proteins whereas GH stimulated phosphorylation of only the 66- and 52-kDa SHC proteins in cells expressing GHR1-454. GH had no effect on SHC phosphorylation in cells expressing GHR1-294 or GHR delta P, the latter lacking amino acids 297...

  18. Protein phosphorylation in pancreatic islets induced by 3-phosphoglycerate and 2-phosphoglycerate

    International Nuclear Information System (INIS)

    Pek, S.B.; Usami, Masaru; Bilir, N.; Fischer-Bovenkerk, C.; Ueda, Tetsufumi

    1990-01-01

    The authors have shown previously that 3-phosphoglycerate, which is a glycolytic metabolite of glucose, induces protein phosphorylation in bovine and rat brain and in rat heart, kidney, liver, lung, and whole pancreas. Since glycolytic metabolism of glucose is of paramount importance in insulin release, they considered the possibility that 3-phosphoglycerate may act as a coupling factor, and they searched for evidence for the existence of 3-phosphoglycerate-dependent protein phosphorylation systems in freshly isolated normal rat pancreatic islets. Membrane and cytosol fractions were incubated with [γ- 32 P]ATP and appropriate test substances and were subjected to NaDodSO 4 /PAGE and autoradiography. As little as 0.005 mM 3-phosphoglycerate or 2-phosphoglycerate stimulated the phosphorylation of 65-kDa cytosol protein by as early as 0.25 min. The phosphate bond of the 65-kDa phosphoprotein was sufficiently stable to withstand dialysis; the radioactivity could not be chased out by subsequent exposure to ATP, ADP, 3-phosphoglycerate, or 2,3-bisphosphoglycerate. Moreover, cAMP, cGMP, phorbol 12-myristate 13-acetate, or calcium failed to stimulate the phosphorylation of the 65-kDa protein. Phosphoglycerate-dependent protein phosphorylation in islets may have relevance to stimulation of insulin secretion

  19. Phosphorylation of protein synthesis initiation factor 2 (elF-2) in the yeast Saccharomyces cerevisiae

    International Nuclear Information System (INIS)

    Romero, D.P.

    1986-01-01

    Initiation Factor 2 (elF-2) in the yeast Saccharomyces cerevisiae is comprised of 3 subunits. The control of protein synthesis in mammalian cells have been shown to involve the phosphorylation of the small (alpha) subunit by a specific protein kinase. Phosphorylation results in an inhibition of protein synthesis. In order to determine whether or not an analogous system is operative in yeast, the phosphorylation state of the alpha subunit of elF-2 in Saccharomyces was determined during various growth and nongrowth conditions. Cells were radiolabelled with 32 P and 35 S, and the whole cell lysates were analyzed by two dimensional gel electrophoresis. These experiments revealed that the smallest subunit (alpha, M/sub r/ = 31,000) is a phosphoprotein in vivo under a variety of growth and nongrowth conditions. This is in direct contrast to the pattern exhibited in mammalian cells. The fact that the small subunit of elF-2 in yeast is phosphorylated under a variety of physiological conditions indicates that such a covalent modification is important for some aspects of elF-2 function. In order to investigate this problem further, a protein kinase that specifically labels the alpha subunit of elF-2 in vitro was isolated. The kinase is not autophosphorylating, utilizes ATP as a phosphate donor, phosphorylates an exogenous protein, casein, modifies serine residues in elF-2, is cyclic nucleotide-independent, and is strongly inhibited by heparin

  20. Limits to sustainable muscle performance: interaction between glycolysis and oxidative phosphorylation.

    Science.gov (United States)

    Conley, K E; Kemper, W F; Crowther, G J

    2001-09-01

    This paper proposes a mechanism responsible for setting the sustainable level of muscle performance. Our contentions are that the sustainable work rate is determined (i) at the muscle level, (ii) by the ability to maintain ATP supply and (iii) by the products of glycolysis that may inhibit the signal for oxidative phosphorylation. We argue below that no single factor 'limits' sustainable performance, but rather that the flux through and the interaction between glycolysis and oxidative phosphorylation set the level of sustainable ATP supply. This argument is based on magnetic resonance spectroscopy measurements of the sources and sinks for energy in vivo in human muscle and rattlesnake tailshaker muscle during sustained contractions. These measurements show that glycolysis provides between 20% (human muscle) and 40% (tailshaker muscle) of the ATP supply during sustained contractions in these muscles. We cite evidence showing that this high glycolytic flux does not reflect an O(2) limitation or mitochondria operating at their capacity. Instead, this flux reflects a pathway independent of oxidative phosphorylation for ATP supply during aerobic exercise. The consequence of this high glycolytic flux is accumulation of H(+), which we argue inhibits the rise in the signal activating oxidative phosphorylation, thereby restricting oxidative ATP supply to below the oxidative capacity. Thus, both glycolysis and oxidative phosphorylation play important roles in setting the highest steady-state ATP synthesis flux and thereby determine the sustainable level of work by exercising muscle.

  1. Light-regulated phosphorylation of maize phosphoenolpyruvate carboxykinase plays a vital role in its activity.

    Science.gov (United States)

    Chao, Qing; Liu, Xiao-Yu; Mei, Ying-Chang; Gao, Zhi-Fang; Chen, Yi-Bo; Qian, Chun-Rong; Hao, Yu-Bo; Wang, Bai-Chen

    2014-05-01

    Phosphoenolpyruvate carboxykinase (PEPCK)-the major decarboxylase in PEPCK-type C4 plants-is also present in appreciable amounts in the bundle sheath cells of NADP-malic enzyme-type C4 plants, such as maize (Zea mays), where it plays an apparent crucial role during photosynthesis (Wingler et al., in Plant Physiol 120(2):539-546, 1999; Furumoto et al., in Plant Mol Biol 41(3):301-311, 1999). Herein, we describe the use of mass spectrometry to demonstrate phosphorylation of maize PEPCK residues Ser55, Thr58, Thr59, and Thr120. Western blotting indicated that the extent of Ser55 phosphorylation dramatically increases in the leaves of maize seedlings when the seedlings are transferred from darkness to light, and decreases in the leaves of seedlings transferred from light to darkness. The effect of light on phosphorylation of this residue is opposite that of the effect of light on PEPCK activity, with the decarboxylase activity of PEPCK being less in illuminated leaves than in leaves left in the dark. This inverse relationship between PEPCK activity and the extent of phosphorylation suggests that the suppressive effect of light on PEPCK decarboxylation activity might be mediated by reversible phosphorylation of Ser55.

  2. Trihydrophobin 1 Phosphorylation by c-Src Regulates MAPK/ERK Signaling and Cell Migration

    Science.gov (United States)

    Wu, Weibin; Sun, Zhichao; Wu, Jingwen; Peng, Xiaomin; Gan, Huacheng; Zhang, Chunyi; Ji, Lingling; Xie, Jianhui; Zhu, Haiyan; Ren, Shifang

    2012-01-01

    c-Src activates Ras-MAPK/ERK signaling pathway and regulates cell migration, while trihydrophobin 1 (TH1) inhibits MAPK/ERK activation and cell migration through interaction with A-Raf and PAK1 and inhibiting their kinase activities. Here we show that c-Src interacts with TH1 by GST-pull down assay, coimmunoprecipitation and confocal microscopy assay. The interaction leads to phosphorylation of TH1 at Tyr-6 in vivo and in vitro. Phosphorylation of TH1 decreases its association with A-Raf and PAK1. Further study reveals that Tyr-6 phosphorylation of TH1 reduces its inhibition on MAPK/ERK signaling, enhances c-Src mediated cell migration. Moreover, induced tyrosine phosphorylation of TH1 has been found by EGF and estrogen treatments. Taken together, our findings demonstrate a novel mechanism for the comprehensive regulation of Ras/Raf/MEK/ERK signaling and cell migration involving tyrosine phosphorylation of TH1 by c-Src. PMID:22238675

  3. Age-related changes in the synthesis and phosphorylation of proteins

    International Nuclear Information System (INIS)

    Butler, J.A.; Heydari, A.; Richardson, A.

    1986-01-01

    It is well documented that the protein synthetic activity of liver tissue decreases significantly with age. However, very little information is available on the effect of age on the synthesis or phosphorylation of individual proteins. Hepatocytes were isolated from 5- to 30-month-old male Fischer F344 rats, and proteins were labeled with either [ 3 H]-valine or [ 32 P]-phosphate. Two-dimensional polyacrylamide gel electrophoresis was used to monitor the synthesis and phosphorylation of a wide variety of proteins. A dramatic increase or decrease in the synthesis of approximately 2 to 3% of the proteins was observed. Most of the proteins whose synthesis increased with age were found to be plasma proteins, e.g., acute phase proteins, synthesized by the liver. In general, the synthesis of most proteins decreased 20 to 40% with age. The phosphorylation of most proteins (over 200) did not appear to change with age. However the phosphorylation of two acidic proteins (molecular weights of 148 Kd and 130 Kd and pIs of 5.4 and 5.36, respectively) decreased with age while the phosphorylation of a basic protein (molecular weight of 57 Kd and pI of 8.09) increased with age

  4. A unified molecular mechanism for the regulation of acetyl-CoA carboxylase by phosphorylation.

    Science.gov (United States)

    Wei, Jia; Zhang, Yixiao; Yu, Tai-Yuan; Sadre-Bazzaz, Kianoush; Rudolph, Michael J; Amodeo, Gabriele A; Symington, Lorraine S; Walz, Thomas; Tong, Liang

    2016-01-01

    Acetyl-CoA carboxylases (ACCs) are crucial metabolic enzymes and attractive targets for drug discovery. Eukaryotic acetyl-CoA carboxylases are 250 kDa single-chain, multi-domain enzymes and function as dimers and higher oligomers. Their catalytic activity is tightly regulated by phosphorylation and other means. Here we show that yeast ACC is directly phosphorylated by the protein kinase SNF1 at residue Ser1157, which potently inhibits the enzyme. Crystal structure of three ACC central domains (AC3-AC5) shows that the phosphorylated Ser1157 is recognized by Arg1173, Arg1260, Tyr1113 and Ser1159. The R1173A/R1260A double mutant is insensitive to SNF1, confirming that this binding site is crucial for regulation. Electron microscopic studies reveal dramatic conformational changes in the holoenzyme upon phosphorylation, likely owing to the dissociation of the biotin carboxylase domain dimer. The observations support a unified molecular mechanism for the regulation of ACC by phosphorylation as well as by the natural product soraphen A, a potent inhibitor of eukaryotic ACC. These molecular insights enhance our understanding of acetyl-CoA carboxylase regulation and provide a basis for drug discovery.

  5. Novel Serine 176 Phosphorylation of YBX1 Activates NF-κB in Colon Cancer.

    Science.gov (United States)

    Martin, Matthew; Hua, Laiqing; Wang, Benlian; Wei, Han; Prabhu, Lakshmi; Hartley, Antja-Voy; Jiang, Guanglong; Liu, Yunlong; Lu, Tao

    2017-02-24

    Y box protein 1 (YBX1) is a well known oncoprotein that has tumor-promoting functions. YBX1 is widely considered to be an attractive therapeutic target in cancer. To develop novel therapeutics to target YBX1, it is of great importance to understand how YBX1 is finely regulated in cancer. Previously, we have shown that YBX1 could function as a tumor promoter through phosphorylation of its Ser-165 residue, leading to the activation of the NF-κB signaling pathway (1). In this study, using mass spectrometry analysis, we discovered a distinct phosphorylation site, Ser-176, on YBX1. Overexpression of the YBX1-S176A (serine-to-alanine) mutant in either HEK293 cells or colon cancer HT29 cells showed dramatically reduced NF-κB-activating ability compared with that of WT-YBX1, confirming that Ser-176 phosphorylation is critical for the activation of NF-κB by YBX1. Importantly, the mutant of Ser-176 and the previously reported Ser-165 sites regulate distinct groups of NF-κB target genes, suggesting the unique and irreplaceable function of each of these two phosphorylated serine residues. Our important findings could provide a novel cancer therapy strategy by blocking either Ser-176 or Ser-165 phosphorylation or both of YBX1 in colon cancer. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  6. Phorbol ester induced phosphorylation of the estrogen receptor in intact MCF-7 human breast cancer cells

    International Nuclear Information System (INIS)

    Knabbe, C.; Lippman, M.E.; Greene, G.L.; Dickson, R.B.

    1986-01-01

    Recent studies with a variety of cellular receptors have shown that phorbol ester induced phosphorylation modulates ligand binding and function. In this study the authors present direct evidence that the estrogen receptor in MCF-7 human breast cancer cells is a phosphoprotein whose phosphorylation state can be enhanced specifically by phorbol-12-myristate-13-acetate (PMA). Cells were cultured to 6h in the presence of [ 32 P]-orthophosphate. Whole cell extracts were immunoprecipitated with a monoclonal antibody (D58) against the estrogen receptor and subjected to SDS-polyacrylamide electrophoresis. Autoradiography showed a specific band in the region of 60-62 kDa which was significantly increased in preparations from PMA treated cells. Phospho-amino acid analysis demonstrated specific phosphorylation of serine and threonine residues. Cholera toxin or forskolin did not change the phosphorylation state of this protein. In a parallel binding analysis PMA led to a rapid decrease of estrogen binding sites. The estrogen induction of both progesterone receptors and growth in semisolid medium was blocked by PMA, whereas the estrogen induction of the 8kDa protein corresponding to the ps2 gene product and of the 52 kDa protein was not affected. In conclusion, phorbol esters can induce phosphorylation of the estrogen receptor. This process may be associated with the inactivation of certain receptor functions

  7. The Serine/threonine kinase Stk33 exhibits autophosphorylation and phosphorylates the intermediate filament protein Vimentin

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    Herrmann Harald

    2008-09-01

    Full Text Available Abstract Background Colocalization of Stk33 with vimentin by double immunofluorescence in certain cells indicated that vimentin might be a target for phosphorylation by the novel kinase Stk33. We therefore tested in vitro the ability of Stk33 to phosphorylate recombinant full length vimentin and amino-terminal truncated versions thereof. In order to prove that Stk33 and vimentin are also in vivo associated proteins co-immunoprecipitation experiments were carried out. For testing the enzymatic activity of immunoprecipitated Stk33 we incubated precipitated Stk33 with recombinant vimentin proteins. To investigate whether Stk33 binds directly to vimentin, an in vitro co-sedimentation assay was performed. Results The results of the kinase assays demonstrate that Stk33 is able to specifically phosphorylate the non-α-helical amino-terminal domain of vimentin in vitro. Furthermore, co-immunoprecipitation experiments employing cultured cell extracts indicate that Stk33 and vimentin are associated in vivo. Immunoprecipitated Stk33 has enzymatic activity as shown by successful phosphorylation of recombinant vimentin proteins. The results of the co-sedimentation assay suggest that vimentin binds directly to Stk33 and that no additional protein mediates the association. Conclusion We hypothesize that Stk33 is involved in the in vivo dynamics of the intermediate filament cytoskeleton by phosphorylating vimentin.

  8. Dopamine signaling negatively regulates striatal phosphorylation of Cdk5 at tyrosine 15 in mice.

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    Yukio eYamamura

    2013-02-01

    Full Text Available Striatal functions depend on the activity balance between the dopamine and glutamate neurotransmissions. Glutamate inputs activate cyclin-dependent kinase 5 (Cdk5, which inhibits postsynaptic dopamine signaling by phosphorylating DARPP-32 (dopamine- and cAMP-regulated phosphoprotein, 32 kDa at Thr75 in the striatum. c-Abelson tyrosine kinase (c-Abl is known to phosphorylate Cdk5 at Tyr15 (Tyr15-Cdk5 and thereby facilitates the Cdk5 activity. We here report that Cdk5 with Tyr15 phosphorylation (Cdk5-pTyr15 is enriched in the mouse striatum, where dopaminergic stimulation inhibited phosphorylation of Tyr15-Cdk5 by acting through the D2 class dopamine receptors. Moreover, in the 1-methyl-4-phenyl-1,2,4,6-tetrahydropyridine mouse model, dopamine deficiency caused increased phosphorylation of both Tyr15-Cdk5 and Thr75-DARPP-32 in the striatum, which could be attenuated by administration of L-3,4-dihydroxyphenylalanine and imatinib (STI-571, a selective c-Abl inhibitor. Our results suggest a functional link of Cdk5-pTyr15 with postsynaptic dopamine and glutamate signals through the c-Abl kinase activity in the striatum.

  9. Identification of phosphorylated butyrylcholinesterase in human plasma using immunoaffinity purification and mass spectrometry

    Energy Technology Data Exchange (ETDEWEB)

    Aryal, Uma K.; Lin, Chiann Tso; Kim, Jong Seo; Heibeck, Tyler H.; Wang, Jun; Qian, Weijun; Lin, Yuehe

    2012-04-20

    Paraoxon (diethyl 4-nitrophenyl phosphate) is an active metabolite of the common insecticide parathion and is acutely toxic due to the inhibition of cholinesterase (ChE) activity in the nervous systems. The Inhibition of butyrylcholinesterase (BChE) activity by paraoxon is due to the formation of phosphorylated BChE adduct, and the detection of the phosphorylated BChE adduct in human plasma can serve as an exposure biomarker of organophosphate pesticides and nerve agents. In this study, we performed immunoaffinity purification and liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis for identifying phosphorylated BChE in human plasma treated by paraoxon. BChE was captured by biotinylated anti-BChE polyclonal antibodies conjugated to streptavidin magnetic beads. Western blot analysis showed that the antibody was effective to recognize both native and modified BChE with high specificity. The exact phosphorylation site of BChE was confirmed on Serine 198 by MS/MS with a 108 Da modification mass and accurately measured parent ion masses. The phosphorylated BChE peptide was also successfully detected in the immunoaffinity purified sample from paraoxon treated human plasma. Thus, immunoaffinity purification combined with mass spectrometry represents a viable approach for the detection of paraoxon-modified BChE and other forms of modified BChE as exposure biomarkers of organophosphates and nerve agents.

  10. Calcium ion binding properties and the effect of phosphorylation on the intrinsically disordered Starmaker protein.

    Science.gov (United States)

    Wojtas, Magdalena; Hołubowicz, Rafał; Poznar, Monika; Maciejewska, Marta; Ożyhar, Andrzej; Dobryszycki, Piotr

    2015-10-27

    Starmaker (Stm) is an intrinsically disordered protein (IDP) involved in otolith biomineralization in Danio rerio. Stm controls calcium carbonate crystal formation in vivo and in vitro. Phosphorylation of Stm affects its biomineralization properties. This study examined the effects of calcium ions and phosphorylation on the structure of Stm. We have shown that CK2 kinase phosphorylates 25 or 26 residues in Stm. Furthermore, we have demonstrated that Stm's affinity for calcium binding is dependent on its phosphorylation state. Phosphorylated Stm (StmP) has an estimated 30 ± 1 calcium binding sites per protein molecule with a dissociation constant (KD) of 61 ± 4 μM, while the unphosphorylated protein has 28 ± 3 sites and a KD of 210 ± 22 μM. Calcium ion binding induces a compaction of the Stm molecule, causing a significant decrease in its hydrodynamic radius and the formation of a secondary structure. The screening effect of Na(+) ions on calcium binding was also observed. Analysis of the hydrodynamic properties of Stm and StmP showed that Stm and StmP molecules adopt the structure of native coil-like proteins.

  11. Phosphorylation of G Protein-Coupled Receptors: From the Barcode Hypothesis to the Flute Model.

    Science.gov (United States)

    Yang, Zhao; Yang, Fan; Zhang, Daolai; Liu, Zhixin; Lin, Amy; Liu, Chuan; Xiao, Peng; Yu, Xiao; Sun, Jin-Peng

    2017-09-01

    Seven transmembrane G protein-coupled receptors (GPCRs) are often phosphorylated at the C terminus and on intracellular loops in response to various extracellular stimuli. Phosphorylation of GPCRs by GPCR kinases and certain other kinases can promote the recruitment of arrestin molecules. The arrestins critically regulate GPCR functions not only by mediating receptor desensitization and internalization, but also by redirecting signaling to G protein-independent pathways via interactions with numerous downstream effector molecules. Accumulating evidence over the past decade has given rise to the phospho-barcode hypothesis, which states that ligand-specific phosphorylation patterns of a receptor direct its distinct functional outcomes. Our recent work using unnatural amino acid incorporation and fluorine-19 nuclear magnetic resonance ( 19 F-NMR) spectroscopy led to the flute model, which provides preliminary insight into the receptor phospho-coding mechanism, by which receptor phosphorylation patterns are recognized by an array of phosphate-binding pockets on arrestin and are translated into distinct conformations. These selective conformations are recognized by various effector molecules downstream of arrestin. The phospho-barcoding mechanism enables arrestin to recognize a wide range of phosphorylation patterns of GPCRs, contributing to their diverse functions. Copyright © 2017 by The Author(s).

  12. Rictor and integrin-linked kinase interact and regulate Akt phosphorylation and cancer cell survival.

    Science.gov (United States)

    McDonald, Paul C; Oloumi, Arusha; Mills, Julia; Dobreva, Iveta; Maidan, Mykola; Gray, Virginia; Wederell, Elizabeth D; Bally, Marcel B; Foster, Leonard J; Dedhar, Shoukat

    2008-03-15

    An unbiased proteomic screen to identify integrin-linked kinase (ILK) interactors revealed rictor as an ILK-binding protein. This finding was interesting because rictor, originally identified as a regulator of cytoskeletal dynamics, is also a component of mammalian target of rapamycin complex 2 (mTORC2), a complex implicated in Akt phosphorylation. These functions overlap with known ILK functions. Coimmunoprecipitation analyses confirmed this interaction, and ILK and rictor colocalized in membrane ruffles and leading edges of cancer cells. Yeast two-hybrid assays showed a direct interaction between the NH(2)- and COOH-terminal domains of rictor and the ILK kinase domain. Depletion of ILK and rictor in breast and prostate cancer cell lines resulted in inhibition of Akt Ser(473) phosphorylation and induction of apoptosis, whereas, in several cell lines, depletion of mTOR increased Akt phosphorylation. Akt and Ser(473)P-Akt were detected in ILK immunoprecipitates and small interfering RNA-mediated depletion of rictor, but not mTOR, inhibited the amount of Ser(473)P-Akt in the ILK complex. Expression of the NH(2)-terminal (1-398 amino acids) rictor domain also resulted in the inhibition of ILK-associated Akt Ser(473) phosphorylation. These data show that rictor regulates the ability of ILK to promote Akt phosphorylation and cancer cell survival.

  13. Structural Modulation of Phosducin by Phosphorylation and 14-3-3 Protein Binding

    Science.gov (United States)

    Rezabkova, Lenka; Kacirova, Miroslava; Sulc, Miroslav; Herman, Petr; Vecer, Jaroslav; Stepanek, Miroslav; Obsilova, Veronika; Obsil, Tomas

    2012-01-01

    Phosducin (Pdc), a highly conserved phosphoprotein, plays an important role in the regulation of G protein signaling, transcriptional control, and modulation of blood pressure. Pdc is negatively regulated by phosphorylation followed by binding to the 14-3-3 protein, whose role is still unclear. To gain insight into the role of 14-3-3 in the regulation of Pdc function, we studied structural changes of Pdc induced by phosphorylation and 14-3-3 protein binding using time-resolved fluorescence spectroscopy. Our data show that the phosphorylation of the N-terminal domain of Pdc at Ser-54 and Ser-73 affects the structure of the whole Pdc molecule. Complex formation with 14-3-3 reduces the flexibility of both the N- and C-terminal domains of phosphorylated Pdc, as determined by time-resolved tryptophan and dansyl fluorescence. Therefore, our data suggest that phosphorylated Pdc undergoes a conformational change when binding to 14-3-3. These changes involve the Gtβγ binding surface within the N-terminal domain of Pdc, and thus could explain the inhibitory effect of 14-3-3 on Pdc function. PMID:23199924

  14. Microgravity alters protein phosphorylation changes during initiation of sea urchin sperm motility

    Science.gov (United States)

    Tash, J. S.; Bracho, G. E.

    1999-01-01

    European Space Agency (ESA) studies demonstrated that bull sperm swim with higher velocity in microgravity (microG) than at 1 G. Coupling between protein phosphorylation and sperm motility during activation in microG and at 1 G was examined in the ESA Biorack on two space shuttle missions. Immotile sperm were activated to swim (86-90% motility) at launch +20 h by dilution into artificial seawater (ASW). Parallel ground controls were performed 2 h after the flight experiment. Activation after 0, 30, and 60 s was terminated with electrophoresis sample buffer and samples analyzed for phosphoamino acids by Western blotting. Phosphorylation of a 130-kDa phosphothreonine-containing protein (FP130) occurred three to four times faster in microG than at 1 G. A 32-kDa phosphoserine-containing protein was significantly stimulated at 30 s but returned to 1 G control levels at 60 s. The rate of FP130 phosphorylation in microG was attenuated by D2O, suggesting that changes in water properties participate in altering signal transduction. Changes in FP130 phosphorylation triggered by the egg peptide speract were delayed in microG. These results demonstrate that previously observed effects of microG on sperm motility are coupled to changes in phosphorylation of specific flagellar proteins and that early events of sperm activation and fertilization are altered in microG.

  15. A dual phosphorylation switch controls 14-3-3-dependent cell surface expression of TASK-1

    Science.gov (United States)

    Kilisch, Markus; Lytovchenko, Olga; Arakel, Eric C.; Bertinetti, Daniela; Schwappach, Blanche

    2016-01-01

    ABSTRACT The transport of the K+ channels TASK-1 and TASK-3 (also known as KCNK3 and KCNK9, respectively) to the cell surface is controlled by the binding of 14-3-3 proteins to a trafficking control region at the extreme C-terminus of the channels. The current model proposes that phosphorylation-dependent binding of 14-3-3 sterically masks a COPI-binding motif. However, the direct effects of phosphorylation on COPI binding and on the binding parameters of 14-3-3 isoforms are still unknown. We find that phosphorylation of the trafficking control region prevents COPI binding even in the absence of 14-3-3, and we present a quantitative analysis of the binding of all human 14-3-3 isoforms to the trafficking control regions of TASK-1 and TASK-3. Surprisingly, the affinities of 14-3-3 proteins for TASK-1 are two orders of magnitude lower than for TASK-3. Furthermore, we find that phosphorylation of a second serine residue in the C-terminus of TASK-1 inhibits 14-3-3 binding. Thus, phosphorylation of the trafficking control region can stimulate or inhibit transport of TASK-1 to the cell surface depending on the target serine residue. Our findings indicate that control of TASK-1 trafficking by COPI, kinases, phosphatases and 14-3-3 proteins is highly dynamic. PMID:26743085

  16. Melatonin synthesis in the human ciliary body triggered by TRPV4 activation: Involvement of AANAT phosphorylation.

    Science.gov (United States)

    Alkozi, Hanan Awad; Perez de Lara, María J; Pintor, Jesús

    2017-09-01

    Melatonin is a substance synthesized in the pineal gland as well as in other organs. This substance is involved in many ocular functions, giving its synthesis in numerous eye structures. Melatonin is synthesized from serotonin through two enzymes, the first limiting step into the synthesis of melatonin being aralkylamine N-acetyltransferase (AANAT). In this current study, A