CBL-interacting protein kinase 6 negatively regulates immune response to Pseudomonas syringae in Arabidopsis.

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Sardar, Atish; Nandi, Ashis Kumar; Chattopadhyay, Debasis

2017-06-15

Cytosolic calcium ion (Ca2+) is an essential mediator of the plant innate immune response. Here, we report that a calcium-regulated protein kinase Calcineurin B-like protein (CBL)-interacting protein kinase 6 (CIPK6) functions as a negative regulator of immunity against the bacterial pathogen Pseudomonas syringae in Arabidopsis thaliana. Arabidopsis lines with compromised expression of CIPK6 exhibited enhanced disease resistance to the bacterial pathogen and to P. syringae harboring certain but not all avirulent effectors, while restoration of CIPK6 expression resulted in abolition of resistance. Plants overexpressing CIPK6 were more susceptible to P. syringae. Enhanced resistance in the absence of CIPK6 was accompanied by increased accumulation of salicylic acid and elevated expression of defense marker genes. Salicylic acid accumulation was essential for improved immunity in the absence of CIPK6. CIPK6 negatively regulated the oxidative burst associated with perception of pathogen-associated microbial patterns (PAMPs) and bacterial effectors. Accelerated and enhanced activation of the mitogen-activated protein kinase cascade in response to bacterial and fungal elicitors was observed in the absence of CIPK6. The results of this study suggested that CIPK6 negatively regulates effector-triggered and PAMP-triggered immunity in Arabidopsis. © The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Protein tyrosine kinase and mitogen-activated protein kinase signalling pathways contribute to differences in heterophil-mediated innate immune responsiveness between two lines of broilers

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Protein tyrosine phosphorylation mediates signal transduction of cellular processes, with protein tyrosine kinases (PTKs) regulating virtually all signaling events. The mitogen-activated protein kinase (MAPK) super-family consists of three conserved pathways that convert receptor activation into ce...

Outer Membrane Protein 25 of Brucella Activates Mitogen-Activated Protein Kinase Signal Pathway in Human Trophoblast Cells

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Jing Zhang

2017-12-01

Full Text Available Outer membrane protein 25 (OMP25, a virulence factor from Brucella, plays an important role in maintaining the structural stability of Brucella. Mitogen-activated protein kinase (MAPK signal pathway widely exists in eukaryotic cells. In this study, human trophoblast cell line HPT-8 and BALB/c mice were infected with Brucella abortus 2308 strain (S2308 and 2308ΔOmp25 mutant strain. The expression of cytokines and activation of MAPK signal pathway were detected. We found that the expressions of tumor necrosis factor-α, interleukin-1, and interleukin-10 (IL-10 were increased in HPT-8 cells infected with S2308 and 2308ΔOmp25 mutant. S2308 also activated p38 phosphorylation protein, extracellular-regulated protein kinases (ERK, and Jun-N-terminal kinase (JNK from MAPK signal pathway. 2308ΔOmp25 could not activate p38, ERK, and JNK branches. Immunohistochemistry experiments showed that S2308 was able to activate phosphorylation of p38 and ERK in BABL/c mice. However, 2308ΔOmp25 could weakly activate phosphorylation of p38 and ERK. These results suggest that Omp25 played an important role in the process of Brucella activation of the MAPK signal pathway.

In vitro, inhibition of mitogen-activated protein kinase pathways protects against bupivacaine- and ropivacaine-induced neurotoxicity

NARCIS (Netherlands)

Lirk, Philipp; Haller, Ingrid; Colvin, Hans Peter; Lang, Leopold; Tomaselli, Bettina; Klimaschewski, Lars; Gerner, Peter

2008-01-01

Animal models show us that specific activation of the p38 mitogen-activated protein kinase (MAPK) may be a pivotal step in lidocaine neurotoxicity, but this has not been investigated in the case of two very widely used local anesthetics, bupivacaine and ropivacaine. We investigated the hypotheses

Mining the active proteome of Arabidopsis thaliana

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Renier A. L. Van Der Hoorn

2011-11-01

Full Text Available Assigning functions to the >30.000 proteins encoded by the Arabidopsis genome is a challenging task of the Arabidopsis Functional Genomics Network. Although genome-wide technologies like proteomics and transcriptomics have generated a wealth of information that significantly accelerated gene annotation, protein activities are poorly predicted by transcript or protein levels as protein activities are post-translationally regulated. To directly display protein activities in Arabidopsis proteomes, we developed and applied Activity-based Protein Profiling (ABPP. ABPP is based on the use of small molecule probes that react with the catalytic residues of distinct protein classes in an activity-dependent manner. Labeled proteins are separated and detected from proteins gels and purified and identified by mass spectrometry. Using probes of six different chemotypes we have displayed of activities of 76 Arabidopsis proteins. These proteins represent over ten different protein classes that contain over 250 Arabidopsis proteins, including cysteine- serine- and metallo-proteases, lipases, acyltransferases, and the proteasome. We have developed methods for identification of in vivo labeled proteins using click-chemistry and for in vivo imaging with fluorescent probes. In vivo labeling has revealed novel protein activities and unexpected subcellular activities of the proteasome. Labeling of extracts displayed several differential activities e.g. of the proteasome during immune response and methylesterases during infection. These studies illustrate the power of ABPP to display the functional proteome and testify to a successful interdisciplinary collaboration involving chemical biology, organic chemistry and proteomics.

The potent, indirect adenosine monophosphate-activated protein kinase activator R419 attenuates mitogen-activated protein kinase signaling, inhibits nociceptor excitability, and reduces pain hypersensitivity in mice

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Galo L. Mejia

2016-07-01

Full Text Available Abstract. There is a great need for new therapeutics for the treatment of pain. A possible avenue to development of such therapeutics is to interfere with signaling pathways engaged in peripheral nociceptors that cause these neurons to become hyperexcitable. There is strong evidence that mitogen-activated protein kinases and phosphoinositide 3-kinase (PI3K/mechanistic target of rapamycin signaling pathways are key modulators of nociceptor excitability in vitro and in vivo. Activation of adenosine monophosphate-activated protein kinase (AMPK can inhibit signaling in both of these pathways, and AMPK activators have been shown to inhibit nociceptor excitability and pain hypersensitivity in rodents. R419 is one of, if not the most potent AMPK activator described to date. We tested whether R419 activates AMPK in dorsal root ganglion (DRG neurons and if this leads to decreased pain hypersensitivity in mice. We find that R419 activates AMPK in DRG neurons resulting in decreased mitogen-activated protein kinase signaling, decreased nascent protein synthesis, and enhanced P body formation. R419 attenuates nerve growth factor (NGF-induced changes in excitability in DRG neurons and blocks NGF-induced mechanical pain amplification in vivo. Moreover, locally applied R419 attenuates pain hypersensitivity in a model of postsurgical pain and blocks the development of hyperalgesic priming in response to both NGF and incision. We conclude that R419 is a promising lead candidate compound for the development of potent and specific AMPK activation to inhibit pain hypersensitivity as a result of injury.

Mitogen-activated protein kinase phosphatase-1 expression in macrophages is controlled by lymphocytes during macrophage activation.

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Luo, Chong; Yang, Xiqiang; Yao, Lan; Jiang, Liping; Liu, Wei; Li, Xin; Wang, Lijia

2012-01-01

The viewpoints on the control of innate immune cells by the adaptive immune system during sepsis remain controversial. Mitogen-activated protein kinase phosphatase-1 (MKP-1) is essential to the negative control of innate immunity and suppresses the activation of macrophages by inhibiting activated mitogen-activated protein kinase (MAPK). The purpose of the current study was to observe inflammatory response and macrophage activation in mice with severe combined immunodeficiency (SCID) with endotoxemia and to determine the role of MKP-1 in the control of macrophage activation by the adaptive immune system. Endotoxemia was induced in wild-type and SCID mice by an intraperitoneal injection of lipopolysaccharide (LPS), and all of the SCID mice died. SCID mice produced more inflammatory cytokines than BALB/c mice systemically and locally. TNF-α mRNA expression was higher and MKP-1 mRNA expression was lower in peritoneal macrophages (PMa) from SCID mice compared to PMa from wild-type mice after and even before LPS injection. Thioglycollate-stimulated PMa from wild-type mice were stimulated with LPS in vitro in the presence or absence of pan-T cells. The levels of TNF-α and IL-6 were higher in the supernatants from PMa cultured alone compared to PMa co-cultured with pan-T cells, and PMa MKP-1 mRNA and protein expression were higher when PMa were co-cultured with pan-T cells. Therefore, pan-T cells can up-regulate MKP-1 expression in macrophages and inhibit the secretion of inflammatory cytokines secretion by macrophages. In SCID mice, lymphocyte deficiency, especially T cell deficiency, causes insufficient MKP-1 expression in macrophages, which can be responsible for the severe inflammation and bad prognosis of septic SCID mice. MKP-1 plays an important role in the control of macrophage activation by the adaptive immune system.

The mitogenic activities of bean proteins determined by assay of the incorporation of sup(3)H - thymidine by human lymphocytes

International Nuclear Information System (INIS)

Derbyshire, E.; Carvalho, M.T.V.; Vitti, D.M.S.; Costa, C.P. da

1988-01-01

The proteins in a saline extract from cotyledons of the bean cultivar Goiano precoce included a protein with electrophoretic mobility equal to that of a commercial preparation of bean mitogen. The crude extract stimulated the incorporation of sup(3)H-tymidine by cultures of human lymphocytes at concentrations of extracted protein from 30 mu g - 300 mu g/culture, and the existence of an optimal concentration in the vicinity of 175 mu g/culture was indicated by the data. The range of active concentrations and the optimal concentration of the heterogeneous extract were 12-15 times greater than the corresponding values obtained when the commercial mitogen was employed. Microscopic examinations showed the presence of blast cells and mitotic figures only in cultures which included seed extract or commercial mitogen. (author)

Mitogen-activated protein kinases interacting kinases are autoinhibited by a reprogrammed activation segment.

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Jauch, Ralf; Cho, Min-Kyu; Jäkel, Stefan; Netter, Catharina; Schreiter, Kay; Aicher, Babette; Zweckstetter, Markus; Jäckle, Herbert; Wahl, Markus C

2006-09-06

Autoinhibition is a recurring mode of protein kinase regulation and can be based on diverse molecular mechanisms. Here, we show by crystal structure analysis, nuclear magnetic resonance (NMR)-based nucleotide affinity studies and rational mutagenesis that nonphosphorylated mitogen-activated protein (MAP) kinases interacting kinase (Mnk) 1 is autoinhibited by conversion of the activation segment into an autoinhibitory module. In a Mnk1 crystal structure, the activation segment is repositioned via a Mnk-specific sequence insertion at the N-terminal lobe with the following consequences: (i) the peptide substrate binding site is deconstructed, (ii) the interlobal cleft is narrowed, (iii) an essential Lys-Glu pair is disrupted and (iv) the magnesium-binding loop is locked into an ATP-competitive conformation. Consistently, deletion of the Mnk-specific insertion or removal of a conserved phenylalanine side chain, which induces a blockade of the ATP pocket, increase the ATP affinity of Mnk1. Structural rearrangements required for the activation of Mnks are apparent from the cocrystal structure of a Mnk2 D228G -staurosporine complex and can be modeled on the basis of crystal packing interactions. Our data suggest a novel regulatory mechanism specific for the Mnk subfamily.

Pre-ERCP infusion of semapimod, a mitogen-activated protein kinases inhibitor, lowers post-ERCP hyperamylasemia but not pancreatitis incidence

NARCIS (Netherlands)

van Westerloo, David J.; Rauws, Erik A.; Hommes, Daan; de Vos, Alex F.; van der Poll, Tom; Powers, Barbara L.; Fockens, Paul; Dijkgraaf, Marcel G. W.; Bruno, Marco J.

2008-01-01

BACKGROUND: Acute pancreatitis and hyperamylasemia are frequent complications of an ERCP. Semapimod is a synthetic guanylhydrazone that inhibits the mitogen-activated protein kinase (MAPK) pathway, macrophage activation, and the production of several inflammatory cytokines. OBJECTIVE: This study

Cardiac imaging in RASopathies/mitogen activated protein kinase syndromes

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Rita Gravino

2014-07-01

Full Text Available RASopathies include a spectrum of disorders due to dysregulation of RAS/mitogen activated protein kinase pathway that plays an essential role in the control of the cell cycle and differentiation. As a consequence, its dysregulation has profound developmental consequences, in particular cardiac malformations. RASopathies with cardiac features are: Noonan syndrome, multiple lentigines syndrome, cardio-faciocutaneous syndrome, Costello syndrome, neurofibromatosis- 1, Legius syndrome, neurofibromatosis- Noonan syndrome. The former syndromes are associated with a high rate of cardiac involvement (60-85% and 12 genes: PTPN11, SOS1, RAF1, KRAS, HRAS, BRAF, MEK1/MAP2K1, MEK2/MAP2K2, NRAS, SHOC2, CBL and SPRED1. Although the majority of these diseases are readily distinguishable in clinical terms, an integrated imaging study of the cardiac condition associated to RASopathies helps to better define risk assessment, surveillance, and management of these patients.

Recruitment of focal adhesion kinase and paxillin to β1 integrin promotes cancer cell migration via mitogen activated protein kinase activation

International Nuclear Information System (INIS)

Crowe, David L; Ohannessian, Arthur

2004-01-01

Integrin-extracellular matrix interactions activate signaling cascades such as mitogen activated protein kinases (MAPK). Integrin binding to extracellular matrix increases tyrosine phosphorylation of focal adhesion kinase (FAK). Inhibition of FAK activity by expression of its carboxyl terminus decreases cell motility, and cells from FAK deficient mice also show reduced migration. Paxillin is a focal adhesion protein which is also phosphorylated on tyrosine. FAK recruitment of paxillin to the cell membrane correlates with Shc phosphorylation and activation of MAPK. Decreased FAK expression inhibits papilloma formation in a mouse skin carcinogenesis model. We previously demonstrated that MAPK activation was required for growth factor induced in vitro migration and invasion by human squamous cell carcinoma (SCC) lines. Adapter protein recruitment to integrin subunits was examined by co-immunoprecipitation in SCC cells attached to type IV collagen or plastic. Stable clones overexpressing FAK or paxillin were created using the lipofection technique. Modified Boyden chambers were used for invasion assays. In the present study, we showed that FAK and paxillin but not Shc are recruited to the β1 integrin cytoplasmic domain following attachment of SCC cells to type IV collagen. Overexpression of either FAK or paxillin stimulated cancer cell migration on type IV collagen and invasion through reconstituted basement membrane which was dependent on MAPK activity. We concluded that recruitment of focal adhesion kinase and paxillin to β1 integrin promoted cancer cell migration via the mitogen activated protein kinase pathway

Recruitment of focal adhesion kinase and paxillin to β1 integrin promotes cancer cell migration via mitogen activated protein kinase activation

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Ohannessian Arthur

2004-05-01

Full Text Available Abstract Background Integrin-extracellular matrix interactions activate signaling cascades such as mitogen activated protein kinases (MAPK. Integrin binding to extracellular matrix increases tyrosine phosphorylation of focal adhesion kinase (FAK. Inhibition of FAK activity by expression of its carboxyl terminus decreases cell motility, and cells from FAK deficient mice also show reduced migration. Paxillin is a focal adhesion protein which is also phosphorylated on tyrosine. FAK recruitment of paxillin to the cell membrane correlates with Shc phosphorylation and activation of MAPK. Decreased FAK expression inhibits papilloma formation in a mouse skin carcinogenesis model. We previously demonstrated that MAPK activation was required for growth factor induced in vitro migration and invasion by human squamous cell carcinoma (SCC lines. Methods Adapter protein recruitment to integrin subunits was examined by co-immunoprecipitation in SCC cells attached to type IV collagen or plastic. Stable clones overexpressing FAK or paxillin were created using the lipofection technique. Modified Boyden chambers were used for invasion assays. Results In the present study, we showed that FAK and paxillin but not Shc are recruited to the β1 integrin cytoplasmic domain following attachment of SCC cells to type IV collagen. Overexpression of either FAK or paxillin stimulated cancer cell migration on type IV collagen and invasion through reconstituted basement membrane which was dependent on MAPK activity. Conclusions We concluded that recruitment of focal adhesion kinase and paxillin to β1 integrin promoted cancer cell migration via the mitogen activated protein kinase pathway.

Mitogen-Activated Protein Kinase Phosphatase-2 Deletion Impairs Synaptic Plasticity and Hippocampal-Dependent Memory.

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Abdul Rahman, Nor Zaihana; Greenwood, Sam M; Brett, Ros R; Tossell, Kyoko; Ungless, Mark A; Plevin, Robin; Bushell, Trevor J

2016-02-24

Mitogen-activated protein kinases (MAPKs) regulate brain function and their dysfunction is implicated in a number of brain disorders, including Alzheimer's disease. Thus, there is great interest in understanding the signaling systems that control MAPK function. One family of proteins that contribute to this process, the mitogen-activated protein kinase phosphatases (MKPs), directly inactivate MAPKs through dephosphorylation. Recent studies have identified novel functions of MKPs in development, the immune system, and cancer. However, a significant gap in our knowledge remains in relation to their role in brain functioning. Here, using transgenic mice where the Dusp4 gene encoding MKP-2 has been knocked out (MKP-2(-/-) mice), we show that long-term potentiation is impaired in MKP-2(-/-) mice compared with MKP-2(+/+) controls whereas neuronal excitability, evoked synaptic transmission, and paired-pulse facilitation remain unaltered. Furthermore, spontaneous EPSC (sEPSC) frequency was increased in acute slices and primary hippocampal cultures prepared from MKP-2(-/-) mice with no effect on EPSC amplitude observed. An increase in synapse number was evident in primary hippocampal cultures, which may account for the increase in sEPSC frequency. In addition, no change in ERK activity was detected in both brain tissue and primary hippocampal cultures, suggesting that the effects of MKP-2 deletion were MAPK independent. Consistent with these alterations in hippocampal function, MKP-2(-/-) mice show deficits in spatial reference and working memory when investigated using the Morris water maze. These data show that MKP-2 plays a role in regulating hippocampal function and that this effect may be independent of MAPK signaling. Copyright © 2016 Abdul Rahman et al.

Heat Shock Proteins and Mitogen-activated Protein Kinases in Steatotic Livers Undergoing Ischemia-Reperfusion: Some Answers

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Massip-Salcedo, Marta; Casillas-Ramirez, Araní; Franco-Gou, Rosah; Bartrons, Ramón; Ben Mosbah, Ismail; Serafin, Anna; Roselló-Catafau, Joan; Peralta, Carmen

2006-01-01

Ischemic preconditioning protects steatotic livers against ischemia-reperfusion (I/R) injury, but just how this is achieved is poorly understood. Here, I/R or preconditioning plus I/R was induced in steatotic and nonsteatotic livers followed by investigating the effect of pharmacological treatments that modulate heat shock proteins (HSPs) and mitogen-activated protein kinases (MAPKs). MAPKs, HSPs, protein kinase C, and transaminase levels were measured after reperfusion. We report that preconditioning increased HSP72 and heme-oxygenase-1 (HO-1) at 6 and 24 hours of reperfusion, respectively. Unlike nonsteatotic livers, steatotic livers benefited from HSP72 activators (geranylgeranylacetone) throughout reperfusion. This protection seemed attributable to HO-1 induction. In steatotic livers, preconditioning and geranylgeranylacetone treatment (which are responsible for HO-1 induction) increased protein kinase C activity. HO-1 activators (cobalt(III) protoporphyrin IX) protected both liver types. Preconditioning reduced p38 MAPK and c-Jun N-terminal kinase (JNK), resulting in HSP72 induction though HO-1 remained unmodified. Like HSP72, both p38 and JNK appeared not to be crucial in preconditioning, and inhibitors of p38 (SB203580) and JNK (SP600125) were less effective against hepatic injury than HO-1 activators. These results provide new data regarding the mechanisms of preconditioning and may pave the way to the development of new pharmacological strategies in liver surgery. PMID:16651615

Angiotensin II regulation of neuromodulation: downstream signaling mechanism from activation of mitogen-activated protein kinase.

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Lu, D; Yang, H; Raizada, M K

1996-12-01

Angiotensin II (Ang II) stimulates expression of tyrosine hydroxylase and norepinephrine transporter genes in brain neurons; however, the signal-transduction mechanism is not clearly defined. This study was conducted to determine the involvement of the mitogen-activated protein (MAP) kinase signaling pathway in Ang II stimulation of these genes. MAP kinase was localized in the perinuclear region of the neuronal soma. Ang II caused activation of MAP kinase and its subsequent translocation from the cytoplasmic to nuclear compartment, both effects being mediated by AT1 receptor subtype. Ang II also stimulated SRE- and AP1-binding activities and fos gene expression and its translocation in a MAP kinase-dependent process. These observations are the first demonstration of a downstream signaling pathway involving MAP kinase in Ang II-mediated neuromodulation in noradrenergic neurons.

LmxMPK4, an essential mitogen-activated protein kinase of Leishmania mexicana is phosphorylated and activated by the STE7-like protein kinase LmxMKK5

DEFF Research Database (Denmark)

John von Freyend, Simona; Rosenqvist, Heidi; Fink, Annette

2010-01-01

The essential mitogen-activated protein kinase (MAP kinase), LmxMPK4, of Leishmania mexicana is minimally active when purified following recombinant expression in Escherichia coli and was therefore unsuitable for drug screening until now. Using an E. coli protein co-expression system we identified...... LmxMKK5, a STE7-like protein kinase from L. mexicana, which phosphorylates and activates recombinant LmxMPK4 in vitro. LmxMKK5 is comprised of 525 amino acids and has a calculated molecular mass of 55.9kDa. The co-expressed, purified LmxMPK4 showed strong phosphotransferase activity in radiometric...... kinase assays and was confirmed by immunoblot and tandem mass spectrometry analyses to be phosphorylated on threonine 190 and tyrosine 192 of the typical TXY MAP kinase activation motif. The universal protein kinase inhibitor staurosporine reduced the phosphotransferase activity of co...

Systems biology analysis of mitogen activated protein kinase inhibitor resistance in malignant melanoma.

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Zecena, Helma; Tveit, Daniel; Wang, Zi; Farhat, Ahmed; Panchal, Parvita; Liu, Jing; Singh, Simar J; Sanghera, Amandeep; Bainiwal, Ajay; Teo, Shuan Y; Meyskens, Frank L; Liu-Smith, Feng; Filipp, Fabian V

2018-04-04

Kinase inhibition in the mitogen activated protein kinase (MAPK) pathway is a standard therapy for cancer patients with activating BRAF mutations. However, the anti-tumorigenic effect and clinical benefit are only transient, and tumors are prone to treatment resistance and relapse. To elucidate mechanistic insights into drug resistance, we have established an in vitro cellular model of MAPK inhibitor resistance in malignant melanoma. The cellular model evolved in response to clinical dosage of the BRAF inhibitor, vemurafenib, PLX4032. We conducted transcriptomic expression profiling using RNA-Seq and RT-qPCR arrays. Pathways of melanogenesis, MAPK signaling, cell cycle, and metabolism were significantly enriched among the set of differentially expressed genes of vemurafenib-resistant cells vs control. The underlying mechanism of treatment resistance and pathway rewiring was uncovered to be based on non-genomic adaptation and validated in two distinct melanoma models, SK-MEL-28 and A375. Both cell lines have activating BRAF mutations and display metastatic potential. Downregulation of dual specific phosphatases, tumor suppressors, and negative MAPK regulators reengages mitogenic signaling. Upregulation of growth factors, cytokines, and cognate receptors triggers signaling pathways circumventing BRAF blockage. Further, changes in amino acid and one-carbon metabolism support cellular proliferation despite MAPK inhibitor treatment. In addition, treatment-resistant cells upregulate pigmentation and melanogenesis, pathways which partially overlap with MAPK signaling. Upstream regulator analysis discovered significant perturbation in oncogenic forkhead box and hypoxia inducible factor family transcription factors. The established cellular models offer mechanistic insight into cellular changes and therapeutic targets under inhibitor resistance in malignant melanoma. At a systems biology level, the MAPK pathway undergoes major rewiring while acquiring inhibitor resistance

Functional Roles of p38 Mitogen-Activated Protein Kinase in Macrophage-Mediated Inflammatory Responses

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Yanyan Yang

2014-01-01

Full Text Available Inflammation is a natural host defensive process that is largely regulated by macrophages during the innate immune response. Mitogen-activated protein kinases (MAPKs are proline-directed serine and threonine protein kinases that regulate many physiological and pathophysiological cell responses. p38 MAPKs are key MAPKs involved in the production of inflammatory mediators, including tumor necrosis factor-α (TNF-α and cyclooxygenase-2 (COX-2. p38 MAPK signaling plays an essential role in regulating cellular processes, especially inflammation. In this paper, we summarize the characteristics of p38 signaling in macrophage-mediated inflammation. In addition, we discuss the potential of using inhibitors targeting p38 expression in macrophages to treat inflammatory diseases.

The Mitogen-Activated Protein Kinase p38 alpha Regulates Tubular Damage in Murine Anti-Glomerular Basement Membrane Nephritis

NARCIS (Netherlands)

Mueller, Ralf; Daniel, Christoph; Hugo, Christian; Amann, Kerstin; Mielenz, Dirk; Endlich, Karlhans; Braun, Tobias; van der Veen, Betty; Heeringa, Peter; Schett, Georg; Zwerina, Jochen

2013-01-01

p38 mitogen-activated protein kinase (MAPK) is thought to play a central role in acute and chronic inflammatory responses. Whether p38MAPK plays a pathogenic role in crescentic GN (GN) and which of its four isoforms is preferentially involved in kidney inflammation is not definitely known. We thus

Constitutive Activity of the Arabidopsis MAP Kinase 3 Confers Resistance to Pseudomonas syringae and Drives Robust Immune Responses

KAUST Repository

Lang, Julien

2017-08-02

Mitogen Activated Protein Kinases (MAPKs) are known to be important mediators of plant responses to biotic and abiotic stresses. In a recent report, we enlarged the understanding of the Arabidopsis thaliana MPK3 functions showing that the expression of a constitutively active (CA) form of the protein led to auto-immune phenotypes. CA-MPK3 plants are dwarf and display defense responses that are characterized by the accumulation of salicylic acid and phytoalexins as well as by the upregulation of several defense genes. Consistently with these data, we present here results demonstrating that, compared to wild type controls, CA-MPK3 plants are more resistant to the hemibiotrophic pathogen Pseudomonas syringae DC3000. Based on our previous work, we also discuss the mechanisms of robust plant immunity controlled by sustained MPK3 activity, focusing especially on the roles of disease resistance proteins.

The mitogen-activated protein kinase (MAPK pathway: role in immune evasion by trypanosomatids

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Mercedes Carolina Soares-Silva

2016-02-01

Full Text Available Leishmania spp and Trypanosoma cruzi are the causative agents of leishmaniasis and Chagas' disease, respectively, two neglected tropical diseases that affect about 25 million people worldwide. These parasites belong to the family Trypanosomatidae and are both obligate intracellular parasites that manipulate host signaling pathways to establish the infection, and also subvert the host innate immune system. Mitogen-activated protein kinases (MAPKs are serine and threonine protein kinases, highly conserved in eukaryotes, and are involved in signal transduction pathways that are related to modulation of physiological and pathophysiological cell responses. This mini-review highlights the current knowledge about the mechanisms that Leishmania spp and T. cruzi have evolved to target host MAPK signaling pathway, highjack immune response, and in this manner, promote parasite maintenance in the host.

Cyclic nucleotides and mitogen-activated protein kinases: regulation of simvastatin in platelet activation

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Hou Ssu-Yu

2010-06-01

Full Text Available Abstract Background 3-Hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA reductase inhibitors (statins have been widely used to reduce cardiovascular risk. These statins (i.e., simvastatin may exert other effects besides from their cholesterol-lowering actions, including inhibition of platelet activation. Platelet activation is relevant to a variety of coronary heart diseases. Although the inhibitory effect of simvastatin in platelet activation has been studied; the detailed signal transductions by which simvastatin inhibit platelet activation has not yet been completely resolved. Methods The aim of this study was to systematically examine the detailed mechanisms of simvastatin in preventing platelet activation. Platelet aggregation, flow cytometric analysis, immunoblotting, and electron spin resonance studies were used to assess the antiplatelet activity of simvastatin. Results Simvastatin (20-50 μM exhibited more-potent activity of inhibiting platelet aggregation stimulated by collagen than other agonists (i.e., thrombin. Simvastatin inhibited collagen-stimulated platelet activation accompanied by [Ca2+]i mobilization, thromboxane A2 (TxA2 formation, and phospholipase C (PLCγ2, protein kinase C (PKC, and mitogen-activated protein kinases (i.e., p38 MAPK, JNKs phosphorylation in washed platelets. Simvastatin obviously increased both cyclic AMP and cyclic GMP levels. Simvastatin markedly increased NO release, vasodilator-stimulated phosphoprotein (VASP phosphorylation, and endothelial nitric oxide synthase (eNOS expression. SQ22536, an inhibitor of adenylate cyclase, markedly reversed the simvastatin-mediated inhibitory effects on platelet aggregation, PLCγ2 and p38 MAPK phosphorylation, and simvastatin-mediated stimulatory effects on VASP and eNOS phosphorylation. Conclusion The most important findings of this study demonstrate for the first time that inhibitory effect of simvastatin in platelet activation may involve activation of the cyclic AMP

Ménage à trois: the complex relationships between mitogen-activated protein kinases, WRKY transcription factors, and VQ-motif-containing proteins.

Science.gov (United States)

Weyhe, Martin; Eschen-Lippold, Lennart; Pecher, Pascal; Scheel, Dierk; Lee, Justin

2014-01-01

Out of the 34 members of the VQ-motif-containing protein (VQP) family, 10 are phosphorylated by the mitogen-activated protein kinases (MAPKs), MPK3 and MPK6. Most of these MPK3/6-targeted VQPs (MVQs) interacted with specific sub-groups of WRKY transcription factors in a VQ-motif-dependent manner. In some cases, the MAPK appears to phosphorylate either the MVQ or the WRKY, while in other cases, both proteins have been reported to act as MAPK substrates. We propose a network of dynamic interactions between members from the MAPK, MVQ and WRKY families - either as binary or as tripartite interactions. The compositions of the WRKY-MVQ transcriptional protein complexes may change - for instance, through MPK3/6-mediated modulation of protein stability - and therefore control defense gene transcription.

Involvement of protein kinase B and mitogen-activated protein kinases in experimental normothermic liver ischaemia-reperfusion injury.

Science.gov (United States)

Cursio, R; Filippa, N; Miele, C; Van Obberghen, E; Gugenheim, J

2006-06-01

This study evaluated the role of protein kinase B (PKB), phosphatidylinositol 3-kinase (PI3-K), Bcl-2-associated death protein (BAD) and mitogen-activated protein kinases (MAPKs) in normothermic ischaemia-reperfusion (IR)-induced apoptosis in rat liver. Rats were divided into two groups that received either phosphate-buffered saline (control) or the caspase inhibitor Z-Asp-2,6-dichorobenzoyloxymethylketone (Z-Asp-cmk), injected intravenously 2 min before the induction of 120 min of normothermic liver ischaemia. Liver apoptosis was assessed by the terminal deoxyribonucleotidyltransferase-mediated dUTP nick end labelling (TUNEL) method. PI3-K, PKB, BAD and MAPK activities were measured in ischaemic and non-ischaemic lobes at various times after reperfusion. The number of TUNEL-positive cells was significantly decreased after pretreatment with Z-Asp-cmk. In controls, PI3-K and PKB activities and BAD phosphorylation were inhibited in ischaemic liver lobes. The MAPKs (extracellular signal-regulated kinases, c-Jun N-terminal kinase and p38) showed different patterns of activation during IR. PKB activity was not modified by pretreatment with Z-Asp-cmk. Induction of apoptosis during IR liver injury might be triggered by inactivation of the antiapoptotic PI3-K-PKB pathway and activation of the proapoptotic MAPKs. Copyright (c) 2006 British Journal of Surgery Society Ltd. Published by John Wiley & Sons, Ltd.

Constitutively active Arabidopsis MAP Kinase 3 triggers defense responses involving salicylic acid and SUMM2 resistance protein

KAUST Repository

Genot, Baptiste

2017-04-12

Mitogen-activated protein kinases (MAPKs) are important regulators of plant immunity. Most of the knowledge about the function of these pathways is derived from loss-of-function approaches. Using a gain-of-function approach, we investigated the responses controlled by a constitutively active (CA) MPK3 in Arabidopsis thaliana. CA-MPK3 plants are dwarfed and display a massive de-repression of defense genes associated with spontaneous cell death as well as accumulation of reactive oxygen species (ROS), phytoalexins and the stress-related hormones ethylene and salicylic acid (SA). Remarkably CA-MPK3/sid2 and CA-MPK3/ein2-50 lines which are impaired in SA synthesis and ethylene signaling, respectively, retain most of the CA-MPK3-associated phenotypes, indicating that constitutive activity of MPK3 can bypass SA and ethylene signaling to activate defense responses. A comparative analysis of the molecular phenotypes of CA-MPK3 and mpk4 autoimmunity suggested convergence between the MPK3 and MPK4-guarding modules. In support of this model, CA-MPK3 crosses with summ1 and summ2, two known suppressors of mpk4, resulted in a partial reversion of the CA-MPK3 phenotypes. Overall, our data unravel a novel mechanism by which the MAPK signaling network contributes to a robust defense response system.

Toward a Comprehensive Phylogenetic Reconstruction of the Evolutionary History of Mitogen-Activated Protein Kinases in the Plant Kingdom

OpenAIRE

Janitza, Philipp; Ullrich, Kristian Karsten; Quint, Marcel

2012-01-01

The mitogen-activated protein kinase (MAPK) pathway is a three-tier signaling cascade that transmits cellular information from the plasma membrane to the cytoplasm where it triggers downstream responses. The MAPKs represent the last step in this cascade and are activated when both tyrosine and threonine residues in a conserved TxY motif are phosphorylated by MAPK kinases, which in turn are themselves activated by phosphorylation by MAPK kinase kinases. To understand the molecular evolution of...

Cocoa Procyanidins Suppress Transformation by Inhibiting Mitogen-activated Protein Kinase Kinase*S⃞

Science.gov (United States)

Kang, Nam Joo; Lee, Ki Won; Lee, Dong Eun; Rogozin, Evgeny A.; Bode, Ann M.; Lee, Hyong Joo; Dong, Zigang

2008-01-01

Cocoa was shown to inhibit chemically induced carcinogenesis in animals and exert antioxidant activity in humans. However, the molecular mechanisms of the chemopreventive potential of cocoa and its active ingredient(s) remain unknown. Here we report that cocoa procyanidins inhibit neoplastic cell transformation by suppressing the kinase activity of mitogen-activated protein kinase kinase (MEK). A cocoa procyanidin fraction (CPF) and procyanidin B2 at 5 μg/ml and 40 μm, respectively, inhibited 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced neoplastic transformation of JB6 P+ mouse epidermal (JB6 P+) cells by 47 and 93%, respectively. The TPA-induced promoter activity and expression of cyclooxygenase-2, which is involved in tumor promotion and inflammation, were dose-dependently inhibited by CPF or procyanidin B2. The activation of activator protein-1 and nuclear factor-κB induced by TPA was also attenuated by CPF or procyanidin B2. The TPA-induced phosphorylation of MEK, extracellular signal-regulated kinase, and p90 ribosomal s6 kinase was suppressed by CPF or procyanidin B2. In vitro and ex vivo kinase assay data demonstrated that CPF or procyanidin B2 inhibited the kinase activity of MEK1 and directly bound with MEK1. CPF or procyanidin B2 suppressed JB6 P+ cell transformation induced by epidermal growth factor or H-Ras, both of which are known to be involved in MEK/ERK signal activation. In contrast, theobromine (up to 80 μm) had no effect on TPA-induced transformation, cyclooxygenase-2 expression, the transactivation of activator protein-1 or nuclear factor-κB, or MEK. Notably, procyanidin B2 exerted stronger inhibitory effects compared with PD098059 (a well known pharmacological inhibitor of MEK) on MEK1 activity and neoplastic cell transformation. PMID:18519570

Blockades of mitogen-activated protein kinase and calcineurin both change fibre-type markers in skeletal muscle culture

DEFF Research Database (Denmark)

Higginson, James; Wackerhage, Henning; Woods, Niall

2002-01-01

A and mitogen-activated protein kinase kinase (MEK1/2) blockade with U0126 upon myosin heavy chain (MHC) isoform mRNA levels and activities of metabolic enzymes after 1 day, 3 days and 7 days of treatment in primary cultures of spontaneously twitching rat skeletal muscle. U0126 treatment significantly decreased......Activation of either the calcineurin or the extracellular signal-regulated kinase (ERK1/2) pathway increases the percentage of slow fibres in vivo suggesting that both pathways can regulate fibre phenotypes in skeletal muscle. We investigated the effect of calcineurin blockade with cyclosporin...

Mitigation of direct neurotoxic effects of lidocaine and amitriptyline by inhibition of p38 mitogen-activated protein kinase in vitro and in vivo

NARCIS (Netherlands)

Lirk, Philipp; Haller, Ingrid; Myers, Robert R.; Klimaschewski, Lars; Kau, Yi-Chuan; Hung, Yu-Chun; Gerner, Peter

2006-01-01

Local anesthetic-induced direct neurotoxicity (paresthesia, failure to regain normal sensory and motor function) is a potentially devastating complication of regional anesthesia. Local anesthetics activate the p38 mitogen-activated protein kinase (MAPK) system, which is involved in apoptotic cell

Toward a comprehensive phylogenetic reconstruction of the evolutionary history of mitogen-activated protein kinases in the plant kingdom.

Science.gov (United States)

Janitza, Philipp; Ullrich, Kristian Karsten; Quint, Marcel

2012-01-01

The mitogen-activated protein kinase (MAPK) pathway is a three-tier signaling cascade that transmits cellular information from the plasma membrane to the cytoplasm where it triggers downstream responses. The MAPKs represent the last step in this cascade and are activated when both tyrosine and threonine residues in a conserved TxY motif are phosphorylated by MAPK kinases, which in turn are themselves activated by phosphorylation by MAPK kinase kinases. To understand the molecular evolution of MAPKs in the plant kingdom, we systematically conducted a Hidden-Markov-Model based screen to identify MAPKs in 13 completely sequenced plant genomes. In this analysis, we included green algae, bryophytes, lycophytes, and several mono- and eudicotyledonous species covering >800 million years of evolution. The phylogenetic relationships of the 204 identified MAPKs based on Bayesian inference facilitated the retraction of the sequence of emergence of the four major clades that are characterized by the presence of a TDY or TEY-A/TEY-B/TEY-C type kinase activation loop. We present evidence that after the split of TDY- and TEY-type MAPKs, initially the TEY-C clade emerged. This was followed by the TEY-B clade in early land plants until the TEY-A clade finally emerged in flowering plants. In addition to these well characterized clades, we identified another highly conserved clade of 45 MAPK-likes, members of which were previously described as Mak-homologous kinases. In agreement with their essential functions, molecular population genetic analysis of MAPK genes in Arabidopsis thaliana accessions reveal that purifying selection drove the evolution of the MAPK family, implying strong functional constraints on MAPK genes. Closely related MAPKs most likely subfunctionalized, a process in which differential transcriptional regulation of duplicates may be involved.

Towards a comprehensive phylogenetic reconstruction of the evolutionary history of mitogen-activated protein kinases in the plant kingdom

Directory of Open Access Journals (Sweden)

Philipp eJanitza

2012-12-01

Full Text Available The mitogen-activated protein kinase (MAPK pathway is a three-tier signaling cascade that transmits cellular information from the plasma membrane to the cytoplasm where it triggers downstream responses. The MAPKs represent the last step in this cascade and are activated when both tyrosine and threonine residues in a conserved TxY motif are phosphorylated by MAPK kinases, which in turn are themselves activated by phosphorylation by MAPK kinase kinases. To understand the molecular evolution of MAPKs in the plant kingdom, we systematically conducted a Hidden-Markov-Model based screen to identify MAPKs in 13 completely sequenced plant genomes. In this analysis, we included green algae, bryophytes, lycophytes, and several mono- and dicotyledonous species covering >800 million years of evolution. The phylogenetic relationships of the 204 identified MAPKs based on Bayesian inference facilitated the retraction of the sequence of emergence of the four major clades that are characterized by the presence of a TDY or TEY-A/TEY-B/TEY-C type kinase activation loop. We present evidence that after the split of TDY- and TEY-type MAPKs, initially the TEY-C clade emerged. This was followed by the TEY-B clade in early land plants until the TEY-A clade finally emerged in flowering plants. In addition to these well characterized clades, we identified another highly conserved clade of 45 MAPK-likes, members of which were previously described as MHKs. In agreement with their essential functions, molecular population genetic analysis of MAPK genes in Arabidopsis thaliana accessions reveal that purifying selection drove the evolution of the MAPK family, implying strong functional constraints on MAPK genes. Closely related MAPKs most likely subfunctionalized, a process in which differential transcriptional regulation of duplicates may be involved.

Context Specificity of Stress-activated Mitogen-activated Protein (MAP) Kinase Signaling: The Story as Told by Caenorhabditis elegans*

Science.gov (United States)

Andrusiak, Matthew G.; Jin, Yishi

2016-01-01

Stress-associated p38 and JNK mitogen-activated protein (MAP) kinase signaling cascades trigger specific cellular responses and are involved in multiple disease states. At the root of MAP kinase signaling complexity is the differential use of common components on a context-specific basis. The roundworm Caenorhabditis elegans was developed as a system to study genes required for development and nervous system function. The powerful genetics of C. elegans in combination with molecular and cellular dissections has led to a greater understanding of how p38 and JNK signaling affects many biological processes under normal and stress conditions. This review focuses on the studies revealing context specificity of different stress-activated MAPK components in C. elegans. PMID:26907690

Eotaxin induces degranulation and chemotaxis of eosinophils through the activation of ERK2 and p38 mitogen-activated protein kinases

DEFF Research Database (Denmark)

Kampen, G T; Stafford, S; Adachi, T

2000-01-01

Eotaxin and other CC chemokines acting via CC chemokine receptor-3 (CCR3) are believed to play an integral role in the development of eosinophilic inflammation in asthma and allergic inflammatory diseases. However, little is known about the intracellular events following agonist binding to CCR3...... and the relationship of these events to the functional response of the cell. The objectives of this study were to investigate CCR3-mediated activation of the mitogen-activated protein (MAP) kinases extracellular signal-regulated kinase-2 (ERK2), p38, and c-jun N-terminal kinase (JNK) in eosinophils and to assess...... the requirement for MAP kinases in eotaxin-induced eosinophil cationic protein (ECP) release and chemotaxis. MAP kinase activation was studied in eotaxin-stimulated eosinophils (more than 97% purity) by Western blotting and immune-complex kinase assays. ECP release was measured by radioimmunoassay. Chemotaxis...

Expression of MMPs is dependent on the activity of mitogen-activated protein kinase in chondrosarcoma.

Science.gov (United States)

Yao, Min; Wang, Xiaomei; Zhao, Yufeng; Wang, Xiaomeng; Gao, Feng

2017-02-01

Matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinases (TIMPs) serve an important role in chondrosarcoma. The present study investigated whether the expression of MMPs was dependent on the activity of mitogen-activated protein kinase (MAPK) in chondrosarcoma. Surgical pathological specimens were collected to detect MMP-1, MMP-13, TIMP-1, type II collagen and phosphorylated MAPK levels in normal cartilage, enchondroma and chondrosarcoma tissues. The expression of MMP‑1, MMP‑13, TIMP‑1 and type II collagen was investigated utilizing MAPK inhibitors in chondrosarcoma cells. It was noted that the expression levels of MMP‑1, MMP‑13 and TIMP‑1 were increased in chondrosarcoma with the activity of MAPK. After chondrosarcoma cells were pretreated with MAPK inhibitors, the levels of MMP‑1, MMP‑13 and TIMP‑1 were inhibited. Furthermore, MMP‑1 and MMP‑13 are essential in regulating the degradation of type II collagen and decomposing cartilage matrix major. The high expression levels of MMP‑1 and MMP‑13 in chondrosarcoma expedite the invasion by chondrosarcoma cells and their expression can be depressed by MAPK inhibitors.

Aspergillus nidulans Natural Product Biosynthesis Is Regulated by MpkB, a Putative Pheromone Response Mitogen-Activated Protein Kinase

International Nuclear Information System (INIS)

Atoui, A.; Bao, D.; Kaur, N.; Grayburn, W.S.; Calvo, A.M.

2008-01-01

The Aspergillus nidulans putative mitogen-activated protein kinase encoded by mpkB has a role in natural product biosynthesis. An mpkB mutant exhibited a decrease in sterigmatocystin gene expression and low mycotoxin levels. The mutation also affected the expression of genes involved in penicillin and terrequinone A synthesis. mpkB was necessary for normal expression of laeA, which has been found to regulate secondary metabolism gene clusters. (author)

Context Specificity of Stress-activated Mitogen-activated Protein (MAP) Kinase Signaling: The Story as Told by Caenorhabditis elegans.

Science.gov (United States)

Andrusiak, Matthew G; Jin, Yishi

2016-04-08

Stress-associated p38 and JNK mitogen-activated protein (MAP) kinase signaling cascades trigger specific cellular responses and are involved in multiple disease states. At the root of MAP kinase signaling complexity is the differential use of common components on a context-specific basis. The roundwormCaenorhabditis eleganswas developed as a system to study genes required for development and nervous system function. The powerful genetics ofC. elegansin combination with molecular and cellular dissections has led to a greater understanding of how p38 and JNK signaling affects many biological processes under normal and stress conditions. This review focuses on the studies revealing context specificity of different stress-activated MAPK components inC. elegans. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Genes encoding calmodulin-binding proteins in the Arabidopsis genome

Science.gov (United States)

Reddy, Vaka S.; Ali, Gul S.; Reddy, Anireddy S N.

2002-01-01

Analysis of the recently completed Arabidopsis genome sequence indicates that approximately 31% of the predicted genes could not be assigned to functional categories, as they do not show any sequence similarity with proteins of known function from other organisms. Calmodulin (CaM), a ubiquitous and multifunctional Ca(2+) sensor, interacts with a wide variety of cellular proteins and modulates their activity/function in regulating diverse cellular processes. However, the primary amino acid sequence of the CaM-binding domain in different CaM-binding proteins (CBPs) is not conserved. One way to identify most of the CBPs in the Arabidopsis genome is by protein-protein interaction-based screening of expression libraries with CaM. Here, using a mixture of radiolabeled CaM isoforms from Arabidopsis, we screened several expression libraries prepared from flower meristem, seedlings, or tissues treated with hormones, an elicitor, or a pathogen. Sequence analysis of 77 positive clones that interact with CaM in a Ca(2+)-dependent manner revealed 20 CBPs, including 14 previously unknown CBPs. In addition, by searching the Arabidopsis genome sequence with the newly identified and known plant or animal CBPs, we identified a total of 27 CBPs. Among these, 16 CBPs are represented by families with 2-20 members in each family. Gene expression analysis revealed that CBPs and CBP paralogs are expressed differentially. Our data suggest that Arabidopsis has a large number of CBPs including several plant-specific ones. Although CaM is highly conserved between plants and animals, only a few CBPs are common to both plants and animals. Analysis of Arabidopsis CBPs revealed the presence of a variety of interesting domains. Our analyses identified several hypothetical proteins in the Arabidopsis genome as CaM targets, suggesting their involvement in Ca(2+)-mediated signaling networks.

Noise exposure immediately activates cochlear mitogen-activated protein kinase signaling

Directory of Open Access Journals (Sweden)

Kumar N Alagramam

2014-01-01

Full Text Available Noise-induced hearing loss (NIHL is a major public health issue worldwide. Uncovering the early molecular events associated with NIHL would reveal mechanisms leading to the hearing loss. Our aim is to investigate the immediate molecular responses after different levels of noise exposure and identify the common and distinct pathways that mediate NIHL. Previous work showed mice exposed to 116 decibels sound pressure level (dB SPL broadband noise for 1 h had greater threshold shifts than the mice exposed to 110 dB SPL broadband noise, hence we used these two noise levels in this study. Groups of 4-8-week-old CBA/CaJ mice were exposed to no noise (control or to broadband noise for 1 h, followed by transcriptome analysis of total cochlear RNA isolated immediately after noise exposure. Previously identified and novel genes were found in all data sets. Following exposure to noise at 116 dB SPL, the earliest responses included up-regulation of 243 genes and down-regulation of 61 genes, while a similar exposure at 110 dB SPL up-regulated 155 genes and down-regulated 221 genes. Bioinformatics analysis indicated that mitogen-activated protein kinase (MAPK signaling was the major pathway in both levels of noise exposure. Nevertheless, both qualitative and quantitative differences were noticed in some MAPK signaling genes, after exposure to different noise levels. Cacna1b , Cacna1g , and Pla2g6 , related to calcium signaling were down-regulated after 110 dB SPL exposure, while the fold increase in the expression of Fos was relatively lower than what was observed after 116 dB SPL exposure. These subtle variations provide insight on the factors that may contribute to the differences in NIHL despite the activation of a common pathway.

Ebselen impairs cellular oxidative state and induces endoplasmic reticulum stress and activation of crucial mitogen-activated protein kinases in pancreatic tumour AR42J cells.

Science.gov (United States)

Santofimia-Castaño, Patricia; Izquierdo-Alvarez, Alicia; Plaza-Davila, María; Martinez-Ruiz, Antonio; Fernandez-Bermejo, Miguel; Mateos-Rodriguez, Jose M; Salido, Gines M; Gonzalez, Antonio

2018-01-01

Ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one) is an organoselenium radical scavenger compound, which has strong antioxidant and anti-inflammatory effects. However, evidence suggests that this compound could exert deleterious actions on cell physiology. In this study, we have analyzed the effect of ebselen on rat pancreatic AR42J cells. Cytosolic free-Ca 2+ concentration ([Ca 2+ ] c ), cellular oxidative status, setting of endoplasmic reticulum stress, and phosphorylation of major mitogen-activated protein kinases were analyzed. Our results show that ebselen evoked a concentration-dependent increase in [Ca 2+ ] c . The compound induced an increase in the generation of reactive oxygen species in the mitochondria. We also observed an increase in global cysteine oxidation in the presence of ebselen. In the presence of ebselen an impairment of cholecystokinin-evoked amylase release was noted. Moreover, involvement of the unfolded protein response markers, ER chaperone and signaling regulator GRP78/BiP, eukaryotic translation initiation factor 2α and X-box binding protein 1 was detected. Finally, increases in the phosphorylation of SAPK/JNK, p38 MAPK, and p44/42 MAPK in the presence of ebselen were also observed. Our results provide evidences for an impairment of cellular oxidative state and enzyme secretion, the induction of endoplasmic reticulum stress and the activation of crucial mitogen-activated protein kinases in the presence of ebselen. As a consequence ebselen exerts a potential toxic effect on AR42J cells. © 2017 Wiley Periodicals, Inc.

Evidence of a New Role for the High-Osmolarity Glycerol Mitogen-Activated Protein Kinase Pathway in Yeast: Regulating Adaptation to Citric Acid Stress†

OpenAIRE

Lawrence, Clare L.; Botting, Catherine H.; Antrobus, Robin; Coote, Peter J.

2004-01-01

Screening the Saccharomyces cerevisiae disruptome, profiling transcripts, and determining changes in protein expression have identified an important new role for the high-osmolarity glycerol (HOG) mitogen-activated protein kinase (MAPK) pathway in the regulation of adaptation to citric acid stress. Deletion of HOG1, SSK1, PBS2, PTC2, PTP2, and PTP3 resulted in sensitivity to citric acid. Furthermore, citric acid resulted in the dual phosphorylation, and thus activation, of Hog1p. Despite mino...

Functional characterization of Arabidopsis thaliana transthyretin-like protein.

Science.gov (United States)

Pessoa, João; Sárkány, Zsuzsa; Ferreira-da-Silva, Frederico; Martins, Sónia; Almeida, Maria R; Li, Jianming; Damas, Ana M

2010-02-18

Arabidopsis thaliana transthyretin-like (TTL) protein is a potential substrate in the brassinosteroid signalling cascade, having a role that moderates plant growth. Moreover, sequence homology revealed two sequence domains similar to 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline (OHCU) decarboxylase (N-terminal domain) and 5-hydroxyisourate (5-HIU) hydrolase (C-terminal domain). TTL is a member of the transthyretin-related protein family (TRP), which comprises a number of proteins with sequence homology to transthyretin (TTR) and the characteristic C-terminal sequence motif Tyr-Arg-Gly-Ser. TRPs are single domain proteins that form tetrameric structures with 5-HIU hydrolase activity. Experimental evidence is fundamental for knowing if TTL is a tetrameric protein, formed by the association of the 5-HIU hydrolase domains and, in this case, if the structural arrangement allows for OHCU decarboxylase activity. This work reports about the biochemical and functional characterization of TTL. The TTL gene was cloned and the protein expressed and purified for biochemical and functional characterization. The results show that TTL is composed of four subunits, with a moderately elongated shape. We also found evidence for 5-HIU hydrolase and OHCU decarboxylase activities in vitro, in the full-length protein. The Arabidopsis thaliana transthyretin-like (TTL) protein is a tetrameric bifunctional enzyme, since it has 5-HIU hydrolase and OHCU decarboxylase activities, which were simultaneously observed in vitro.

The Arabidopsis SOS2 protein kinase physically interacts with and is activated by the calcium-binding protein SOS3

OpenAIRE

Halfter, Ursula; Ishitani, Manabu; Zhu, Jian-Kang

2000-01-01

The Arabidopsis thaliana SOS2 and SOS3 genes are required for intracellular Na+ and K+ homeostasis and plant tolerance to high Na+ and low K+ environments. SOS3 is an EF hand type calcium-binding protein having sequence similarities with animal neuronal calcium sensors and the yeast calcineurin B. SOS2 is a serine/threonine protein kinase in the SNF1/AMPK family. We report here that SOS3 physically interacts with and activates SOS2 protein kinase. Genetically, sos2sos3 double mutant analysis ...

Variation in the Subcellular Localization and Protein Folding Activity among Arabidopsis thaliana Homologs of Protein Disulfide Isomerase

Directory of Open Access Journals (Sweden)

Christen Y. L. Yuen

2013-10-01

Full Text Available Protein disulfide isomerases (PDIs catalyze the formation, breakage, and rearrangement of disulfide bonds to properly fold nascent polypeptides within the endoplasmic reticulum (ER. Classical animal and yeast PDIs possess two catalytic thioredoxin-like domains (a, a′ and two non-catalytic domains (b, b′, in the order a-b-b′-a′. The model plant, Arabidopsis thaliana, encodes 12 PDI-like proteins, six of which possess the classical PDI domain arrangement (AtPDI1 through AtPDI6. Three additional AtPDIs (AtPDI9, AtPDI10, AtPDI11 possess two thioredoxin domains, but without intervening b-b′ domains. C-terminal green fluorescent protein (GFP fusions to each of the nine dual-thioredoxin PDI homologs localized predominantly to the ER lumen when transiently expressed in protoplasts. Additionally, expression of AtPDI9:GFP-KDEL and AtPDI10: GFP-KDDL was associated with the formation of ER bodies. AtPDI9, AtPDI10, and AtPDI11 mediated the oxidative folding of alkaline phosphatase when heterologously expressed in the Escherichia coli protein folding mutant, dsbA−. However, only three classical AtPDIs (AtPDI2, AtPDI5, AtPDI6 functionally complemented dsbA−. Interestingly, chemical inducers of the ER unfolded protein response were previously shown to upregulate most of the AtPDIs that complemented dsbA−. The results indicate that Arabidopsis PDIs differ in their localization and protein folding activities to fulfill distinct molecular functions in the ER.

Stress and vascular responses: atheroprotective effect of laminar fluid shear stress in endothelial cells: possible role of mitogen-activated protein kinases.

Science.gov (United States)

Yoshizumi, Masanori; Abe, Jun-Ichi; Tsuchiya, Koichiro; Berk, Bradford C; Tamaki, Toshiaki

2003-03-01

Atherosclerosis preferentially occurs in areas of turbulent blood flow and low fluid shear stress, whereas laminar blood flow and high shear stress are atheroprotective. Inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-alpha), stimulate expression of endothelial cell (EC) genes that may promote atherosclerosis. Recent findings suggest a steady laminar blood flow decreases EC apoptosis and inhibits TNF-mediated EC activation. EC apoptosis or activation is suggested to be involved in plaque erosion, which may lead to platelet aggregation. TNF-alpha regulates gene expression in ECs, in part, by stimulating mitogen-activated protein (MAP) kinases, which phosphorylate transcription factors. We hypothesized that steady laminar flow inhibits cytokine-mediated activation of MAP kinases in ECs. To test this hypothesis, we determined the effects of steady laminar flow (shear stress = 12 dynes/cm(2)) on TNF-alpha-stimulated activity of three MAP kinases in human umbilical vein ECs (HUVEC): extracellular signal-regulated kinase (ERK1/2), c-Jun N-terminal kinase (JNK), and p38. TNF-alpha activated ERK1/2, JNK, and p38 maximally at 15 min in HUVEC. Pre-exposing HUVEC for 10 min to flow inhibited TNF-alpha activation of JNK, but showed no significant effect on ERK1/2 or p38 activation. Incubation of HUVEC with PD98059, a specific ERK1/2 inhibitor, blocked the flow-mediated inhibition of TNF activation of JNK. Transfection studies with dominant-negative constructs of the protein kinase MEK5 suggested an important role for big mitogen-activated protein kinase 1 (BMK1) in flow-mediated regulation of EC activation by TNF-alpha. Understanding the mechanisms by which steady laminar flow regulates JNK activation by cytokines may provide insight into the atheroprotective mechanisms induced by laminar blood flow.

Phosphorylation of paxillin via the ERK mitogen-activated protein kinase cascade in EL4 thymoma cells.

Science.gov (United States)

Ku, H; Meier, K E

2000-04-14

Intracellular signals can regulate cell adhesion via several mechanisms in a process referred to as "inside-out" signaling. In phorbol ester-sensitive EL4 thymoma cells, phorbol-12-myristate 13-acetate (PMA) induces activation of extracellular signal-regulated kinase (ERK) mitogen-activated protein kinases and promotes cell adhesion. In this study, clonal EL4 cell lines with varying abilities to activate ERKs in response to PMA were used to examine signaling events occurring downstream of ERK activation. Paxillin, a multifunctional docking protein involved in cell adhesion, was phosphorylated on serine/threonine residues in response to PMA treatment. This response was correlated with the extent and time course of ERK activation. PMA-induced phosphorylation of paxillin was inhibited by compounds that block the ERK activation pathway in EL4 cells, primary murine thymocytes, and primary murine splenocytes. Paxillin was phosphorylated in vitro by purified active ERK2. Two-dimensional electrophoresis revealed that PMA treatment generated a complex pattern of phosphorylated paxillin species in intact cells, some of which were generated by ERK-mediated phosphorylation in vitro. An ERK pathway inhibitor interfered with PMA-induced adhesion of sensitive EL4 cells to substrate. These findings describe a novel inside-out signaling pathway by which the ERK cascade may regulate events involved in adhesion.

Functional characterization of Arabidopsis thaliana transthyretin-like protein

Directory of Open Access Journals (Sweden)

Almeida Maria R

2010-02-01

Full Text Available Abstract Background Arabidopsis thaliana transthyretin-like (TTL protein is a potential substrate in the brassinosteroid signalling cascade, having a role that moderates plant growth. Moreover, sequence homology revealed two sequence domains similar to 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline (OHCU decarboxylase (N-terminal domain and 5-hydroxyisourate (5-HIU hydrolase (C-terminal domain. TTL is a member of the transthyretin-related protein family (TRP, which comprises a number of proteins with sequence homology to transthyretin (TTR and the characteristic C-terminal sequence motif Tyr-Arg-Gly-Ser. TRPs are single domain proteins that form tetrameric structures with 5-HIU hydrolase activity. Experimental evidence is fundamental for knowing if TTL is a tetrameric protein, formed by the association of the 5-HIU hydrolase domains and, in this case, if the structural arrangement allows for OHCU decarboxylase activity. This work reports about the biochemical and functional characterization of TTL. Results The TTL gene was cloned and the protein expressed and purified for biochemical and functional characterization. The results show that TTL is composed of four subunits, with a moderately elongated shape. We also found evidence for 5-HIU hydrolase and OHCU decarboxylase activities in vitro, in the full-length protein. Conclusions The Arabidopsis thaliana transthyretin-like (TTL protein is a tetrameric bifunctional enzyme, since it has 5-HIU hydrolase and OHCU decarboxylase activities, which were simultaneously observed in vitro.

Human prostatic cancer cells, PC3, elaborate mitogenic activity which selectively stimulates human bone cells

International Nuclear Information System (INIS)

Perkel, V.S.; Mohan, S.; Herring, S.J.; Baylink, D.J.; Linkhart, T.A.

1990-01-01

Prostatic cancer typically produces osteoblastic metastases which are not attended by marrow fibrosis. In the present study we sought to test the hypothesis that prostatic cancer cells produce factor(s) which act selectively on human osteoblasts. Such a paracrine mechanism would explain the observed increase in osteoblasts, unaccompanied by an increase in marrow fibroblasts. To test this hypothesis we investigated the mitogenic activity released by the human prostatic tumor cell line, PC3. PC3 cells have been reported previously to produce mitogenic activity for cells that was relatively specific for rat osteoblasts compared to rat fibroblasts. However, the effects of this activity on human cells has not been examined previously. PC3-conditioned medium (CM) (5-50 micrograms CM protein/ml) stimulated human osteoblast proliferation by 200-950% yet did not stimulate human fibroblast proliferation ([3H]thymidine incorporation). PC3 CM also increased cell numbers in human osteoblast but not fibroblast cell cultures. To determine whether the osteoblast-specific mitogenic activity could be attributed to known bone growth factors, specific assays for these growth factors were performed. PC3 CM contained 10 pg insulin-like growth factor (IGF) I, less than 2 pg IGF II, 54 pg basic fibroblast growth factor, and 16 pg transforming growth factor beta/microgram CM protein. None of these growth factors alone or in combination could account for the observed osteoblast-specific PC3 cell-derived mitogenic activity. Furthermore, when 5 micrograms/ml PC3 CM was tested in combination with maximally effective concentrations of either basic fibroblast growth factor, IGF I, IGF II, or transforming growth factor beta, it produced an additive effect suggesting that PC3 CM stimulates osteoblast proliferation by a mechanism independent of these bone mitogens

The Na+/H+ exchanger, NHE1, differentially regulates mitogen-activated protein kinase subfamilies after osmotic shrinkage in Ehrlich Lettre Ascites cells

DEFF Research Database (Denmark)

Petersen, Stine Helene Falsig; Rasmussen, Maria; Darborg, Barbara Vasek

2007-01-01

Osmotic stress modulates mitogen activated protein kinase (MAPK) activities, leading to altered gene transcription and cell death/survival balance, however, the mechanisms involved are incompletely elucidated. Here, we show, using a combination of biochemical and molecular biology approaches...... by human (h) NHE1 expression in cells lacking endogenous NHE1 activity. The effect of NHE1 on ERK1/2 was pH(i)-independent and upstream of MEK1/2. Shrinkage-activation of JNK1/2 was attenuated by EIPA, augmented by hNHE1 expression, and abolished in the presence of HCO(3)(-). Basal JNK activity...

Identification of Adenyl Cyclase Activity in a Disease Resistance Protein in Arabidopsis thaliana

KAUST Repository

Hussein, Rana

2012-11-01

Cyclic nucleotide, cAMP, is an important signaling molecule in animals and plants. However, in plants the enzymes that synthesize this second messenger, adenyl cyclases (ACs), remain elusive. Given the physiological importance of cAMP in signaling, particularly in response to biotic and abiotic stresses, it is thus important to identify and characterize ACs in higher plants. Using computational approaches, a disease resistance protein from Arabidopsis thaliana, At3g04220 was found to have an AC catalytic center motif. In an attempt to prove that this candidate has adenyl cyclases activity in vitro, the coding sequence of the putative AC catalytic domain of this protein was cloned and expressed in E. coli and the recombinant protein was purified. The nucleotide cyclase activity of the recombinant protein was examined using cyclic nucleotide enzyme immunoassays. In parallel, the expression of At3g04220 was measured in leaves under three different stress conditions in order to determine under which conditions the disease resistance protein could function. Results show that the purified recombinant protein has Mn2+ dependent AC activity in vitro, and the expression analysis supports a role for At3g04220 and cAMP in plant defense.

Regulation of Schistosoma mansoni development and reproduction by the mitogen-activated protein kinase signaling pathway.

Science.gov (United States)

Andrade, Luiza Freire de; Mourão, Marina de Moraes; Geraldo, Juliana Assis; Coelho, Fernanda Sales; Silva, Larissa Lopes; Neves, Renata Heisler; Volpini, Angela; Machado-Silva, José Roberto; Araujo, Neusa; Nacif-Pimenta, Rafael; Caffrey, Conor R; Oliveira, Guilherme

2014-06-01

Protein kinases are proven targets for drug development with an increasing number of eukaryotic Protein Kinase (ePK) inhibitors now approved as drugs. Mitogen-activated protein kinase (MAPK) family members connect cell-surface receptors to regulatory targets within cells and influence a number of tissue-specific biological activities such as cell proliferation, differentiation and survival. However, the contributions of members of the MAPK pathway to schistosome development and survival are unclear. We employed RNA interference (RNAi) to elucidate the functional roles of five S. mansoni genes (SmCaMK2, SmJNK, SmERK1, SmERK2 and SmRas) involved in MAPK signaling pathway. Mice were injected with post-infective larvae (schistosomula) subsequent to RNAi and the development of adult worms observed. The data demonstrate that SmJNK participates in parasite maturation and survival of the parasites, whereas SmERK are involved in egg production as infected mice had significantly lower egg burdens with female worms presenting underdeveloped ovaries. Furthermore, it was shown that the c-fos transcription factor was overexpressed in parasites submitted to RNAi of SmERK1, SmJNK and SmCaMK2 indicating its putative involvement in gene regulation in this parasite's MAPK signaling cascade. We conclude that MAPKs proteins play important roles in the parasite in vivo survival, being essential for normal development and successful survival and reproduction of the schistosome parasite. Moreover SmERK and SmJNK are potential targets for drug development.

Modulation of Cyclins, p53 and Mitogen-Activated Protein Kinases Signaling in Breast Cancer Cell Lines by 4-(3,4,5-Trimethoxyphenoxybenzoic Acid

Directory of Open Access Journals (Sweden)

Kuan-Han Lee

2014-01-01

Full Text Available Despite the advances in cancer therapy and early detection, breast cancer remains a leading cause of cancer-related deaths among females worldwide. The aim of the current study was to investigate the antitumor activity of a novel compound, 4-(3,4,5-trimethoxyphenoxybenzoic acid (TMPBA and its mechanism of action, in breast cancer. Results indicated the relatively high sensitivity of human breast cancer cell-7 and MDA-468 cells towards TMPBA with IC50 values of 5.9 and 7.9 µM, respectively compared to hepatocarcinoma cell line Huh-7, hepatocarcinoma cell line HepG2, and cervical cancer cell line Hela cells. Mechanistically, TMPBA induced apoptotic cell death in MCF-7 cells as indicated by 4',6-diamidino-2-phenylindole (DAPI nuclear staining, cell cycle analysis and the activation of caspase-3. Western blot analysis revealed the ability of TMPBA to target pathways mediated by mitogen-activated protein (MAP kinases, 5' adenosine monophosphate-activated protein kinase (AMPK, and p53, of which the concerted action underlined its antitumor efficacy. In addition, TMPBA induced alteration of cyclin proteins’ expression and consequently modulated the cell cycle. Taken together, the current study underscores evidence that TMPBA induces apoptosis in breast cancer cells via the modulation of cyclins and p53 expression as well as the modulation of AMPK and mitogen-activated protein kinases (MAPK signaling. These findings support TMPBA’s clinical promise as a potential candidate for breast cancer therapy.

Induction of viral, 7-methyl-guanosine cap-independent translation and oncolysis by mitogen-activated protein kinase-interacting kinase-mediated effects on the serine/arginine-rich protein kinase.

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Brown, Michael C; Bryant, Jeffrey D; Dobrikova, Elena Y; Shveygert, Mayya; Bradrick, Shelton S; Chandramohan, Vidyalakshmi; Bigner, Darell D; Gromeier, Matthias

2014-11-01

Protein synthesis, the most energy-consuming process in cells, responds to changing physiologic priorities, e.g., upon mitogen- or stress-induced adaptations signaled through the mitogen-activated protein kinases (MAPKs). The prevailing status of protein synthesis machinery is a viral pathogenesis factor, particularly for plus-strand RNA viruses, where immediate translation of incoming viral RNAs shapes host-virus interactions. In this study, we unraveled signaling pathways centered on the ERK1/2 and p38α MAPK-interacting kinases MNK1/2 and their role in controlling 7-methyl-guanosine (m(7)G) "cap"-independent translation at enterovirus type 1 internal ribosomal entry sites (IRESs). Activation of Raf-MEK-ERK1/2 signals induced viral IRES-mediated translation in a manner dependent on MNK1/2. This effect was not due to MNK's known functions as eukaryotic initiation factor (eIF) 4G binding partner or eIF4E(S209) kinase. Rather, MNK catalytic activity enabled viral IRES-mediated translation/host cell cytotoxicity through negative regulation of the Ser/Arg (SR)-rich protein kinase (SRPK). Our investigations suggest that SRPK activity is a major determinant of type 1 IRES competency, host cell cytotoxicity, and viral proliferation in infected cells. We are targeting unfettered enterovirus IRES activity in cancer with PVSRIPO, the type 1 live-attenuated poliovirus (PV) (Sabin) vaccine containing a human rhinovirus type 2 (HRV2) IRES. A phase I clinical trial of PVSRIPO with intratumoral inoculation in patients with recurrent glioblastoma (GBM) is showing early promise. Viral translation proficiency in infected GBM cells is a core requirement for the antineoplastic efficacy of PVSRIPO. Therefore, it is critically important to understand the mechanisms controlling viral cap-independent translation in infected host cells. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Mitogen activated protein kinases selectively regulate palytoxin-stimulated gene expression in mouse keratinocytes

International Nuclear Information System (INIS)

Zeliadt, Nicholette A.; Warmka, Janel K.; Wattenberg, Elizabeth V.

2003-01-01

We have been investigating how the novel skin tumor promoter palytoxin transmits signals through mitogen activated protein kinases (MAPKs). Palytoxin activates three major MAPKs, extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38, in a keratinocyte cell line derived from initiated mouse skin (308). We previously showed that palytoxin requires ERK to increase matrix metalloproteinase-13 (MMP-13) gene expression, an enzyme implicated in carcinogenesis. Diverse stimuli require JNK and p38 to increase MMP-13 gene expression, however. We therefore used the JNK and p38 inhibitors SP 600125 and SB 202190, respectively, to investigate the role of these MAPKs in palytoxin-induced MMP-13 gene expression. Surprisingly, palytoxin does not require JNK and p38 to increase MMP-13 gene expression. Accordingly, ERK activation, independent of palytoxin and in the absence of JNK and p38 activation, is sufficient to induce MMP-13 gene expression in 308 keratinocytes. Dexamethasone, a synthetic glucocorticoid that inhibits activator protein-1 (AP-1), blocked palytoxin-stimulated MMP-13 gene expression. Therefore, the AP-1 site present in the promoter of the MMP-13 gene appears to be functional and to play a key role in palytoxin-stimulated gene expression. Previous studies showed that palytoxin simulates an ERK-dependent selective increase in the c-Fos content of AP-1 complexes that bind to the promoter of the MMP-13 gene. JNK and p38 can also modulate c-Fos. Palytoxin does not require JNK or p38 to increase c-Fos binding, however. Altogether, these studies indicate that ERK plays a distinctly essential role in transmitting palytoxin-stimulated signals to specific nuclear targets in keratinocytes derived from initiated mouse skin

Molecular Cloning and Characterization of a P38-Like Mitogen-Activated Protein Kinase from Echinococcus granulosus.

Science.gov (United States)

Lü, Guodong; Li, Jing; Zhang, Chuanshan; Li, Liang; Bi, Xiaojuan; Li, Chaowang; Fan, Jinliang; Lu, Xiaomei; Vuitton, Dominique A; Wen, Hao; Lin, Renyong

2016-12-01

Cystic echinococcosis (CE) treatment urgently requires a novel drug. The p38 mitogen-activated protein kinases (MAPKs) are a family of Ser/Thr protein kinases, but still have to be characterized in Echinococcus granulosus . We identified a 1,107 bp cDNA encoding a 368 amino acid MAPK protein (Egp38) in E. granulosus . Egp38 exhibits 2 distinguishing features of p38-like kinases: a highly conserved T-X-Y motif and an activation loop segment. Structural homology modeling indicated a conserved structure among Egp38, EmMPK2, and H. sapiens p38α, implying a common binding mechanism for the ligand domain and downstream signal transduction processing similar to that described for p38α. Egp38 and its phosphorylated form are expressed in the E. granulosus larval stages vesicle and protoscolices during intermediate host infection of an intermediate host. Treatment of in vitro cultivated protoscolices with the p38-MAPK inhibitor ML3403 effectively suppressed Egp38 activity and led to significant protoscolices death within 5 days. Treatment of in vitro-cultivated protoscolices with TGF-β1 effectively induced Egp38 phosphorylation. In summary, the MAPK, Egp38, was identified in E. granulosus , as an anti-CE drug target and participates in the interplay between the host and E. granulosus via human TGF-β1.

A membrane protein / signaling protein interaction network for Arabidopsis version AMPv2

Directory of Open Access Journals (Sweden)

Sylvie Lalonde

2010-09-01

Full Text Available Interactions between membrane proteins and the soluble fraction are essential for signal transduction and for regulating nutrient transport. To gain insights into the membrane-based interactome, 3,852 open reading frames (ORFs out of a target list of 8,383 representing membrane and signaling proteins from Arabidopsis thaliana were cloned into a Gateway compatible vector. The mating-based split-ubiquitin system was used to screen for potential protein-protein interactions (pPPIs among 490 Arabidopsis ORFs. A binary robotic screen between 142 receptor-like kinases, 72 transporters, 57 soluble protein kinases and phosphatases, 40 glycosyltransferases, 95 proteins of various functions and 89 proteins with unknown function detected 387 out of 90,370 possible PPIs. A secondary screen confirmed 343 (of 387 pPPIs between 179 proteins, yielding a scale-free network (r2=0.863. Eighty of 142 transmembrane receptor-like kinases (RLK tested positive, identifying three homomers, 63 heteromers and 80 pPPIs with other proteins. Thirty-one out of 142 RLK interactors (including RLKs had previously been found to be phosphorylated; thus interactors may be substrates for respective RLKs. None of the pPPIs described here had been reported in the major interactome databases, including potential interactors of G protein-coupled receptors, phospholipase C, and AMT ammonium transporters. Two RLKs found as putative interactors of AMT1;1 were independently confirmed using a split luciferase assay in Arabidopsis protoplasts. These RLKs may be involved in ammonium-dependent phosphorylation of the C-terminus and regulation of ammonium uptake activity. The robotic screening method established here will enable a systematic analysis of membrane protein interactions in fungi, plants and metazoa.

Mitogen-activated protein kinase phosphatase (MKP)-1 as a neuroprotective agent: promotion of the morphological development of midbrain dopaminergic neurons.

Science.gov (United States)

Collins, Louise M; O'Keeffe, Gerard W; Long-Smith, Caitriona M; Wyatt, Sean L; Sullivan, Aideen M; Toulouse, André; Nolan, Yvonne M

2013-06-01

A greater understanding of the mechanisms that promote the survival and growth of dopaminergic neurons is essential for the advancement of cell replacement therapies for Parkinson's disease (PD). Evidence supports a role for the mitogen-activated protein kinase p38 in the demise of dopaminergic neurons, while mitogen-activated protein kinase phosphatase-1 (MKP-1), which negatively regulates p38 activity, has not yet been investigated in this context. Here, we show that MKP-1 is expressed in dopaminergic neurons cultured from E14 rat ventral mesencephalon (VM). When dopaminergic neurons were transfected to overexpress MKP-1, they displayed a more complex morphology than their control counterparts in vitro. Specifically, MKP-1-transfection induced significant increases in neurite length and branching with a maximum increase observed in primary branches. We demonstrate that inhibition of dopaminergic neurite growth induced by treatment of rat VM neurons with the dopaminergic neurotoxin 6-hydroxydopamine (6-OHDA) in vitro is mediated by p38 and is concomitant with a significant and selective decrease in MKP-1 expression in these neurons. We further show that overexpression of MKP-1 in dopaminergic neurons contributes to neuroprotection against the effects of 6-OHDA. Collectively, we report that MKP-1 can promote the growth and elaboration of dopaminergic neuronal processes and can help protect them from the neurotoxic effects of 6-OHDA. Thus, we propose that strategies aimed at augmenting MKP-1 expression or activity may be beneficial in protecting dopaminergic neurons and may provide potential therapeutic approaches for PD.

Repeated pulses of serotonin required for long-term facilitation activate mitogen-activated protein kinase in sensory neurons of Aplysia

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Michael, Dan; Martin, Kelsey C.; Seger, Rony; Ning, Ming-Ming; Baston, Rene; Kandel, Eric R.

1998-01-01

Long-term facilitation of the connections between the sensory and motor neurons of the gill-withdrawal reflex in Aplysia requires five repeated pulses of serotonin (5-HT). The repeated pulses of 5-HT initiate a cascade of gene activation that leads ultimately to the growth of new synaptic connections. Several genes in this process have been identified, including the transcriptional regulators apCREB-1, apCREB-2, apC/EBP, and the cell adhesion molecule apCAM, which is thought to be involved in the formation of new synaptic connections. Here we report that the transcriptional regulators apCREB-2 and apC/EBP, as well as a peptide derived from the cytoplasmic domain of apCAM, are phosphorylated in vitro by Aplysia mitogen-activated protein kinase (apMAPK). We have cloned the cDNA encoding apMAPK and show that apMAPK activity is increased in sensory neurons treated with repeated pulses of 5-HT and by the cAMP pathway. These results suggest that apMAPK may participate with cAMP-dependent protein kinase during long-term facilitation in sensory cells by modifying some of the key elements involved in the consolidation of short- to long-lasting changes in synaptic strength. PMID:9465108

Structural and functional analysis of VQ motif-containing proteins in Arabidopsis as interacting proteins of WRKY transcription factors.

Science.gov (United States)

Cheng, Yuan; Zhou, Yuan; Yang, Yan; Chi, Ying-Jun; Zhou, Jie; Chen, Jian-Ye; Wang, Fei; Fan, Baofang; Shi, Kai; Zhou, Yan-Hong; Yu, Jing-Quan; Chen, Zhixiang

2012-06-01

WRKY transcription factors are encoded by a large gene superfamily with a broad range of roles in plants. Recently, several groups have reported that proteins containing a short VQ (FxxxVQxLTG) motif interact with WRKY proteins. We have recently discovered that two VQ proteins from Arabidopsis (Arabidopsis thaliana), SIGMA FACTOR-INTERACTING PROTEIN1 and SIGMA FACTOR-INTERACTING PROTEIN2, act as coactivators of WRKY33 in plant defense by specifically recognizing the C-terminal WRKY domain and stimulating the DNA-binding activity of WRKY33. In this study, we have analyzed the entire family of 34 structurally divergent VQ proteins from Arabidopsis. Yeast (Saccharomyces cerevisiae) two-hybrid assays showed that Arabidopsis VQ proteins interacted specifically with the C-terminal WRKY domains of group I and the sole WRKY domains of group IIc WRKY proteins. Using site-directed mutagenesis, we identified structural features of these two closely related groups of WRKY domains that are critical for interaction with VQ proteins. Quantitative reverse transcription polymerase chain reaction revealed that expression of a majority of Arabidopsis VQ genes was responsive to pathogen infection and salicylic acid treatment. Functional analysis using both knockout mutants and overexpression lines revealed strong phenotypes in growth, development, and susceptibility to pathogen infection. Altered phenotypes were substantially enhanced through cooverexpression of genes encoding interacting VQ and WRKY proteins. These findings indicate that VQ proteins play an important role in plant growth, development, and response to environmental conditions, most likely by acting as cofactors of group I and IIc WRKY transcription factors.

The atypical mitogen-activated protein kinase ERK3 is essential for establishment of epithelial architecture.

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Takahashi, Chika; Miyatake, Koichi; Kusakabe, Morioh; Nishida, Eisuke

2018-06-01

Epithelia contribute to physical barriers that protect internal tissues from the external environment and also support organ structure. Accordingly, establishment and maintenance of epithelial architecture are essential for both embryonic development and adult physiology. Here, using gene knockout and knockdown techniques along with gene profiling, we show that extracellular signal-regulated kinase 3 (ERK3), a poorly characterized atypical mitogen-activated protein kinase (MAPK), regulates the epithelial architecture in vertebrates. We found that in Xenopus embryonic epidermal epithelia, ERK3 knockdown impairs adherens and tight-junction protein distribution, as well as tight-junction barrier function, resulting in epidermal breakdown. Moreover, in human epithelial breast cancer cells, inhibition of ERK3 expression induced thickened epithelia with aberrant adherens and tight junctions. Results from microarray analyses suggested that transcription factor AP-2α (TFAP2A), a transcriptional regulator important for epithelial gene expression, is involved in ERK3-dependent changes in gene expression. Of note, TFAP2A knockdown phenocopied ERK3 knockdown in both Xenopus embryos and human cells, and ERK3 was required for full activation of TFAP2A-dependent transcription. Our findings reveal that ERK3 regulates epithelial architecture, possibly together with TFAP2A. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Ibuprofen abates cypermethrin-induced expression of pro-inflammatory mediators and mitogen-activated protein kinases and averts the nigrostriatal dopaminergic neurodegeneration.

Science.gov (United States)

Singh, Ashish; Tripathi, Pratibha; Prakash, Om; Singh, Mahendra Pratap

2016-12-01

Cypermethrin induces oxidative stress, microglial activation, inflammation and apoptosis leading to Parkinsonism in rats. While ibuprofen, a non-steroidal anti-inflammatory drug, relieves from inflammation, its efficacy against cypermethrin-induced Parkinsonism has not yet been investigated. The study aimed to explore the protective role of ibuprofen in cypermethrin-induced Parkinsonism, an environmentally relevant model of Parkinson's disease (PD), along with its underlying mechanism. Animals were treated with/without cypermethrin in the presence/absence of ibuprofen. Behavioural, immunohistochemical and biochemical parameters of Parkinsonism and expression of pro-inflammatory and pro-apoptotic proteins along with mitogen-activated protein kinases (MAPKs) were determined. Ibuprofen resisted cypermethrin-induced behavioural impairments, striatal dopamine depletion, oxidative stress in the nigrostriatal tissues and loss of the nigral dopamine producing cells and increase in microglial activation along with atypical expression of pro-inflammatory and apoptotic proteins that include cyclooxygenase-2, tumour necrosis factor-α, MAPKs (c-Jun N-terminal kinase, p38 and extracellular signal-regulated kinase), B cell lymphoma 2-associated protein X, tumour suppressor protein p53, cytochrome c and caspase-3 in the nigrostriatal tissue. The results obtained thus demonstrate that ibuprofen lessens inflammation and regulates MAPKs expression thereby averts cypermethrin-induced Parkinsonism.

Glucose, other secretagogues, and nerve growth factor stimulate mitogen-activated protein kinase in the insulin-secreting beta-cell line, INS-1

DEFF Research Database (Denmark)

Frödin, M; Sekine, N; Roche, E

1995-01-01

The signaling pathways whereby glucose and hormonal secretagogues regulate insulin-secretory function, gene transcription, and proliferation of pancreatic beta-cells are not well defined. We show that in the glucose-responsive beta-cell line INS-1, major secretagogue-stimulated signaling pathways...... converge to activate 44-kDa mitogen-activated protein (MAP) kinase. Thus, glucose-induced insulin secretion was found to be associated with a small stimulatory effect on 44-kDa MAP kinase, which was synergistically enhanced by increased levels of intracellular cAMP and by the hormonal secretagogues......-1. Phorbol ester, an activator of protein kinase C, stimulated 44-kDa MAP kinase by both Ca(2+)-dependent and -independent pathways. Nerve growth factor, independently of changes in cytosolic Ca2+, efficiently stimulated 44-kDa MAP kinase without causing insulin release, indicating that activation...

p38gamma and p38delta mitogen activated protein kinases (MAPKs, new stars in the MAPK galaxy

Directory of Open Access Journals (Sweden)

Alejandra eEscós

2016-04-01

Full Text Available The protein kinases p38γ and p38δ belong to the p38 mitogen-activated protein kinase (MAPK family. p38MAPK signalling controls many cellular processes and is one of the most conserved mechanisms in eukaryotes for the cellular response to environmental stress and inflammation. Although p38γ and p38δ are widely expressed, it is likely that they perform specific functions in different tissues. Their involvement in human pathologies such as inflammation-related diseases or cancer is starting to be uncovered. In this article we give a general overview and highlight recent advances made in defining the functions of p38γ and p38δ, focusing in innate immunity and inflammation. We consider the potential of the pharmacological targeting of MAPK pathways to treat autoimmune and inflammatory diseases and cancer

Activation of Extracellular Signal-Regulated Kinase but Not of p38 Mitogen-Activated Protein Kinase Pathways in Lymphocytes Requires Allosteric Activation of SOS

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Jun, Jesse E.; Yang, Ming; Chen, Hang; Chakraborty, Arup K.

2013-01-01

Thymocytes convert graded T cell receptor (TCR) signals into positive selection or deletion, and activation of extracellular signal-related kinase (ERK), p38, and Jun N-terminal protein kinase (JNK) mitogen-activated protein kinases (MAPKs) has been postulated to play a discriminatory role. Two families of Ras guanine nucleotide exchange factors (RasGEFs), SOS and RasGRP, activate Ras and the downstream RAF-MEK-ERK pathway. The pathways leading to lymphocyte p38 and JNK activation are less well defined. We previously described how RasGRP alone induces analog Ras-ERK activation while SOS and RasGRP cooperate to establish bimodal ERK activation. Here we employed computational modeling and biochemical experiments with model cell lines and thymocytes to show that TCR-induced ERK activation grows exponentially in thymocytes and that a W729E allosteric pocket mutant, SOS1, can only reconstitute analog ERK signaling. In agreement with RasGRP allosterically priming SOS, exponential ERK activation is severely decreased by pharmacological or genetic perturbation of the phospholipase Cγ (PLCγ)-diacylglycerol-RasGRP1 pathway. In contrast, p38 activation is not sharply thresholded and requires high-level TCR signal input. Rac and p38 activation depends on SOS1 expression but not allosteric activation. Based on computational predictions and experiments exploring whether SOS functions as a RacGEF or adaptor in Rac-p38 activation, we established that the presence of SOS1, but not its enzymatic activity, is critical for p38 activation. PMID:23589333

Characterization and enzymatic properties of protein kinase ACR4 from Arabidopsis thaliana.

Science.gov (United States)

Zhao, Yu; Liu, Xuehe; Xu, Ziyan; Yang, Hui; Li, Jixi

2017-07-22

Serine/threonine-protein kinase-like protein ARABIDOPSIS CRINKLY4 (ACR4), a transmembrane protein of Arabidopsis thaliana, plays important roles in cell division and differentiation. Although accumulating studies shed light on the function of ACR4, the structure and catalytic mechanism of ACR4 remain to be elucidated. Here, we report the purification and enzymatic properties of the intracellular kinase domain (residues 464-799) of ACR4 (ACR4 IKD ). Through Ni-affinity chromatography and gel filter chromatography methods, we successfully obtain high-purity ACR4 IKD protein from Escherichia coli. Dynamic light scattering and gel-filtration methods reveal that ACR4 IKD distributes with high homogeneity and exists as a monomer in solution. In addition, the ACR4 IKD protein has typical kinase activity with myelin basic protein (MBP) as the substrate. Our study may lay the foundation for structure determination of ACR4 IKD and further functional research, for example, screening significant substrates of ACR4 in Arabidopsis thaliana. Copyright © 2017 Elsevier Inc. All rights reserved.

Arabidopsis dynamin-related protein 1A polymers bind, but do not tubulate, liposomes

International Nuclear Information System (INIS)

Backues, Steven K.; Bednarek, Sebastian Y.

2010-01-01

The Arabidopsis dynamin-related protein 1A (AtDRP1A) is involved in endocytosis and cell plate maturation in Arabidopsis. Unlike dynamin, AtDRP1A does not have any recognized membrane binding or protein-protein interaction domains. We report that GTPase active AtDRP1A purified from Escherichia coli as a fusion to maltose binding protein forms homopolymers visible by negative staining electron microscopy. These polymers interact with protein-free liposomes whose lipid composition mimics that of the inner leaflet of the Arabidopsis plasma membrane, suggesting that lipid-binding may play a role in AtDRP1A function. However, AtDRP1A polymers do not appear to assemble and disassemble in a dynamic fashion and do not have the ability to tubulate liposomes in vitro, suggesting that additional factors or modifications are necessary for AtDRP1A's in vivo function.

p38 mitogen-activated protein kinase mediates IL-8 induction by the ribotoxin deoxynivalenol in human monocytes

International Nuclear Information System (INIS)

Islam, Zahidul; Gray, Jennifer S.; Pestka, James J.

2006-01-01

The effects of the ribotoxic trichothecene deoxynivalenol (DON) on mitogen-activated protein kinase (MAPK)-mediated IL-8 expression were investigated in cloned human monocytes and peripheral blood mononuclear cells (PBMC). DON (250 to 1000 ng/ml) induced both IL-8 mRNA and IL-8 heteronuclear RNA (hnRNA), an indicator of IL-8 transcription, in the human U937 monocytic cell line in a concentration-dependent manner. Expression of IL-8 hnRNA, mRNA and protein correlated with p38 phosphorylation and was completely abrogated by the p38 MAPK inhibitor SB203580. DON at 500 ng/ml similarly induced p38-dependent IL-8 protein and mRNA expression in PBMC cultures from healthy volunteers. Significantly increased IL-6 and IL-1β intracellular protein and mRNA expression was also observed in PBMC treated with DON (500 ng/ml) which were also partially p38-dependent. Flow cytometry of PBMC revealed that DON-induced p38 phosphorylation varied among individuals relative to both threshold toxin concentrations (25-100 ng/ml) and relative increases in percentages of phospho-p38 + cells. DON-induced p38 activation occurred exclusively in the CD14 + monocyte population. DON was devoid of agonist activity for human Toll-like receptors 2, 3, 4, 5, 7, 8 and 9. However, two other ribotoxins, emetine and anisomycin, induced p38 phosphorylation in PBMC similarly to DON. Taken together, these data suggest that (1) p38 activation was required for induction of IL-8 and proinflammatory gene expression in the monocyte and (2) DON induced p38 activation in human monocytes via the ribotoxic stress response

Identification of novel PAMP-triggered phosphorylation and dephosphorylation events in arabidopsis thaliana by quantitative phosphoproteomic analysis

KAUST Repository

Rayapuram, Naganand; Bonhomme, Ludovic; Bigeard, Jean; Haddadou, Kahina; Przybylski, Cé dric; Hirt, Heribert; Pflieger, Delphine

2014-01-01

Signaling cascades rely strongly on protein kinase-mediated substrate phosphorylation. Currently a major challenge in signal transduction research is to obtain high confidence substrate phosphorylation sites and assign them to specific kinases. In response to bacterial flagellin, a pathogen-associated molecular pattern (PAMP), we searched for rapidly phosphorylated proteins in Arabidopsis thaliana by combining multistage activation (MSA) and electron transfer dissociation (ETD) fragmentation modes, which generate complementary spectra and identify phosphopeptide sites with increased reliability. Of a total of 825 phosphopeptides, we identified 58 to be differentially phosphorylated. These peptides harbor kinase motifs of mitogen-activated protein kinases (MAPKs) and calcium-dependent protein kinases (CDPKs), as well as yet unknown protein kinases. Importantly, 12 of the phosphopeptides show reduced phosphorylation upon flagellin treatment. Since protein abundance levels did not change, these results indicate that flagellin induces not only various protein kinases but also protein phosphatases, even though a scenario of inhibited kinase activity may also be possible. © 2014 American Chemical Society.

Identification of novel PAMP-triggered phosphorylation and dephosphorylation events in arabidopsis thaliana by quantitative phosphoproteomic analysis

KAUST Repository

Rayapuram, Naganand

2014-04-04

Signaling cascades rely strongly on protein kinase-mediated substrate phosphorylation. Currently a major challenge in signal transduction research is to obtain high confidence substrate phosphorylation sites and assign them to specific kinases. In response to bacterial flagellin, a pathogen-associated molecular pattern (PAMP), we searched for rapidly phosphorylated proteins in Arabidopsis thaliana by combining multistage activation (MSA) and electron transfer dissociation (ETD) fragmentation modes, which generate complementary spectra and identify phosphopeptide sites with increased reliability. Of a total of 825 phosphopeptides, we identified 58 to be differentially phosphorylated. These peptides harbor kinase motifs of mitogen-activated protein kinases (MAPKs) and calcium-dependent protein kinases (CDPKs), as well as yet unknown protein kinases. Importantly, 12 of the phosphopeptides show reduced phosphorylation upon flagellin treatment. Since protein abundance levels did not change, these results indicate that flagellin induces not only various protein kinases but also protein phosphatases, even though a scenario of inhibited kinase activity may also be possible. © 2014 American Chemical Society.

Mitogen-activated protein kinase signaling in plants

DEFF Research Database (Denmark)

Rodriguez, Maria Cristina Suarez; Petersen, Morten; Mundy, John

2010-01-01

crossinhibition, feedback control, and scaffolding. Plant MAPK cascades regulate numerous processes, including stress and hormonal responses, innate immunity, and developmental programs. Genetic analyses have uncovered several predominant MAPK components shared by several of these processes including...... of substrate proteins, whose altered activities mediate a wide array of responses, including changes in gene expression. Cascades may share kinase components, but their signaling specificity is maintained by spaciotemporal constraints and dynamic protein-protein interactions and by mechanisms that include...

Adult-onset hyperthyroidism impairs spatial learning: possible involvement of mitogen-activated protein kinase signaling pathways.

Science.gov (United States)

Bitiktaş, Soner; Kandemir, Başak; Tan, Burak; Kavraal, Şehrazat; Liman, Narin; Dursun, Nurcan; Dönmez-Altuntaş, Hamiyet; Aksan-Kurnaz, Işil; Suer, Cem

2016-08-03

Given evidence that mitogen-activated protein kinase (MAPK) activation is part of the nongenomic actions of thyroid hormones, we investigated the possible consequences of hyperthyroidism for the cognitive functioning of adult rats. Young adult rats were treated with L-thyroxine or saline. Twenty rats in each group were exposed to Morris water maze testing, measuring their performance in a hidden-platform spatial task. In a separate set of rats not exposed to Morris water maze testing (untrained rats), the expression and phosphorylated levels of p38-MAPK and of its two downstream effectors, Elk-1 and cAMP response element-binding protein, were evaluated using quantitative reverse transcriptase-PCR and western blotting. Rats with hyperthyroidism showed delayed acquisition of learning compared with their wild-type counterparts, as shown by increased escape latencies and distance moved on the last two trials of daily training in the water maze. The hyperthyroid rats, however, showed no difference during probe trials. Western blot analyses of the hippocampus showed that hyperthyroidism increased phosphorylated p38-MAPK levels in untrained rats. Although our study is correlative in nature and does not exclude the contribution of other molecular targets, our findings suggest that the observed impairments in acquisition during actual learning in rats with hyperthyroidism may result from the increased phosphorylation of p38-MAPK.

The Role of Unfolded Protein Response and Mitogen-Activated Protein Kinase Signaling in Neurodegenerative Diseases with Special Focus on Prion Diseases

Directory of Open Access Journals (Sweden)

Lifeng Yang

2017-05-01

Full Text Available Prion diseases are neurodegenerative pathologies characterized by the accumulation of a protease-resistant form of the cellular prion protein named prion protein scrapie (PrPSc in the brain. PrPSc accumulation in the endoplasmic reticulum (ER result in a dysregulated calcium (Ca2+ homeostasis and subsequent initiation of unfolded protein response (UPR leading to neuronal dysfunction and apoptosis. The molecular mechanisms for the transition between adaptation to ER stress and ER stress-induced apoptosis are still unclear. Mitogen-activated protein kinases (MAPKs are serine/threonine protein kinases that rule the signaling of many extracellular stimuli from plasma membrane to the nucleus. However the identification of numerous points of cross talk between the UPR and MAPK signaling pathways may contribute to our understanding of the consequences of ER stress in prion diseases. Indeed the MAPK signaling network is known to regulate cell cycle progression and cell survival or death responses following a variety of stresses including misfolded protein response stress. In this article, we review the UPR signaling in prion diseases and discuss the triad of MAPK signaling pathways. We also describe the role played by MAPK signaling cascades in Alzheimer’s (AD and Parkinson’s disease (PD. We will also overview the mechanisms of cell death and the role of MAPK signaling in prion disease progression and highlight potential avenues for therapeutic intervention.

Mitogen-activated protein kinase (MAPK) dynamics determine cell fate in the yeast mating response.

Science.gov (United States)

Li, Yang; Roberts, Julie; AkhavanAghdam, Zohreh; Hao, Nan

2017-12-15

In the yeast Saccharomyces cerevisiae , the exposure to mating pheromone activates a prototypic mitogen-activated protein kinase (MAPK) cascade and triggers a dose-dependent differentiation response. Whereas a high pheromone dose induces growth arrest and formation of a shmoo-like morphology in yeast cells, lower pheromone doses elicit elongated cell growth. Previous population-level analysis has revealed that the MAPK Fus3 plays an important role in mediating this differentiation switch. To further investigate how Fus3 controls the fate decision process at the single-cell level, we developed a specific translocation-based reporter for monitoring Fus3 activity in individual live cells. Using this reporter, we observed strikingly different dynamic patterns of Fus3 activation in single cells differentiated into distinct fates. Cells committed to growth arrest and shmoo formation exhibited sustained Fus3 activation. In contrast, most cells undergoing elongated growth showed either a delayed gradual increase or pulsatile dynamics of Fus3 activity. Furthermore, we found that chemically perturbing Fus3 dynamics with a specific inhibitor could effectively redirect the mating differentiation, confirming the causative role of Fus3 dynamics in driving cell fate decisions. MAPKs mediate proliferation and differentiation signals in mammals and are therapeutic targets in many cancers. Our results highlight the importance of MAPK dynamics in regulating single-cell responses and open up the possibility that MAPK signaling dynamics could be a pharmacological target in therapeutic interventions. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Proteomic identification of S-nitrosylated proteins in Arabidopsis

DEFF Research Database (Denmark)

Lindermayr, C.; Saalbach, G.; Durner, J.

2005-01-01

Although nitric oxide (NO) has grown into a key signaling molecule in plants during the last few years, less is known about how NO regulates different events in plants. Analyses of NO-dependent processes in animal systems have demonstrated protein S-nitrosylation of cysteine (Cys) residues...... to be one of the dominant regulation mechanisms for many animal proteins. For plants, the principle of S-nitrosylation remained to be elucidated. We generated S-nitrosothiols by treating extracts from Arabidopsis (Arabidopsis thaliana) cell suspension cultures with the NO-donor S......-nitrosoglutathione. Furthermore, Arabidopsis plants were treated with gaseous NO to analyze whether S-nitrosylation can occur in the specific redox environment of a plant cell in vivo. S-Nitrosylated proteins were detected by a biotin switch method, converting S-nitrosylated Cys to biotinylated Cys. Biotin-labeled proteins were...

Mercuric ions inhibit mitogen-activated protein kinase dephosphorylation by inducing reactive oxygen species

International Nuclear Information System (INIS)

Haase, Hajo; Engelhardt, Gabriela; Hebel, Silke; Rink, Lothar

2011-01-01

Mercury intoxication profoundly affects the immune system, in particular, signal transduction of immune cells. However, the mechanism of the interaction of mercury with cellular signaling pathways, such as mitogen activated protein kinases (MAPK), remains elusive. Therefore, the objective of this study is to investigate three potential ways in which Hg 2+ ions could inhibit MAPK dephosphorylation in the human T-cell line Jurkat: (1) by direct binding to phosphatases; (2) by releasing cellular zinc (Zn 2+ ); and (3) by inducing reactive oxygen species (ROS). Hg 2+ causes production of ROS, measured by dihydrorhodamine 123, and triggers ROS-mediated Zn 2+ release, detected with FluoZin-3. Yet, phosphatase-inhibition is not mediated by binding of Zn 2+ or Hg 2+ . Rather, phosphatases are inactivated by at least two forms of thiol oxidation; initial inhibition is reversible with reducing agents such as Tris(2-carboxyethyl)phosphine. Prolonged inhibition leads to non-reversible phosphatase oxidation, presumably oxidizing the cysteine thiol to sulfinic- or sulfonic acid. Notably, phosphatases are a particularly sensitive target for Hg 2+ -induced oxidation, because phosphatase activity is inhibited at concentrations of Hg 2+ that have only minor impact on over all thiol oxidation. This phosphatase inhibition results in augmented, ROS-dependent MAPK phosphorylation. MAPK are important regulators of T-cell function, and MAPK-activation by inhibition of phosphatases seems to be one of the molecular mechanisms by which mercury affects the immune system.

Functionally Similar WRKY Proteins Regulate Vacuolar Acidification in Petunia and Hair Development in Arabidopsis.

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Verweij, Walter; Spelt, Cornelis E; Bliek, Mattijs; de Vries, Michel; Wit, Niek; Faraco, Marianna; Koes, Ronald; Quattrocchio, Francesca M

2016-03-01

The WD40 proteins ANTHOCYANIN11 (AN11) from petunia (Petunia hybrida) and TRANSPARENT TESTA GLABRA1 (TTG1) from Arabidopsis thaliana and associated basic helix-loop-helix (bHLH) and MYB transcription factors activate a variety of differentiation processes. In petunia petals, AN11 and the bHLH protein AN1 activate, together with the MYB protein AN2, anthocyanin biosynthesis and, together with the MYB protein PH4, distinct genes, such as PH1 and PH5, that acidify the vacuole. To understand how AN1 and AN11 activate anthocyanin biosynthetic and PH genes independently, we isolated PH3. We found that PH3 is a target gene of the AN11-AN1-PH4 complex and encodes a WRKY protein that can bind to AN11 and is required, in a feed-forward loop, together with AN11-AN1-PH4 for transcription of PH5. PH3 is highly similar to TTG2, which regulates hair development, tannin accumulation, and mucilage production in Arabidopsis. Like PH3, TTG2 can bind to petunia AN11 and the Arabidopsis homolog TTG1, complement ph3 in petunia, and reactivate the PH3 target gene PH5. Our findings show that the specificity of WD40-bHLH-MYB complexes is in part determined by interacting proteins, such as PH3 and TTG2, and reveal an unanticipated similarity in the regulatory circuitry that controls petunia vacuolar acidification and Arabidopsis hair development. © 2016 American Society of Plant Biologists. All rights reserved.

Involvement of p38 mitogen-activated protein kinase in acquired gemcitabine-resistant human urothelial carcinoma sublines

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Yu-Ting Kao

2014-07-01

Full Text Available Resistance to chemotherapeutic drugs is one of the major challenges in the treatment of cancer. A better understanding of how resistance arises and what molecular alterations correlate with resistance is the key to developing novel effective therapeutic strategies. To investigate the underlying mechanisms of gemcitabine (Gem resistance and provide possible therapeutic options, three Gem-resistant urothelial carcinoma sublines were established (NG0.6, NG0.8, and NG1.0. These cells were cross-resistant to arabinofuranosyl cytidine and cisplatin, but sensitive to 5-fluorouracil. The resistant cells expressed lower values of [hENT1 × dCK/RRM1 × RRM2] mRNA ratio. Two adenosine triphosphate-binding cassette proteins ABCD1 as well as multidrug resistance protein 1 were elevated. Moreover, cyclin D1, cyclin-dependent kinases 2 and 4 were upregulated, whereas extracellular signal-regulated kinase 1/2 and p38 mitogen-activated protein kinase (MAPK activity were repressed significantly. Administration of p38 MAPK inhibitor significantly reduced the Gem sensitivity in NTUB1 cells, whereas that of an extracellular signal-regulated kinase MAPK inhibitor did not. Furthermore, the Gem-resistant sublines also exhibited higher migration ability. Forced expression of p38 MAPK impaired the cell migration activity and augmented Gem sensitivity in NG1.0 cells. Taken together, these results demonstrate that complex mechanisms were merged in acquiring Gem resistance and provide information that can be important for developing therapeutic targets for treating Gem-resistant tumors.

Antifungal Effect of Arabidopsis SGT1 Proteins via Mitochondrial Reactive Oxygen Species.

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Park, Seong-Cheol; Cheong, Mi Sun; Kim, Eun-Ji; Kim, Jin Hyo; Chi, Yong Hun; Jang, Mi-Kyeong

2017-09-27

The highly conserved SGT1 (suppressor of the G2 alleles of skp1) proteins from Arabidopsis are known to contribute to plant resistance to pathogens. While SGT1 proteins respond to fungal pathogens, their antifungal activity is not reported and the mechanism for this inhibition is not well understood. Therefore, recombinant Arabidopsis SGT1 proteins were cloned, expressed, and purified to evaluate their antifungal activity, resulting in their potent inhibition of pathogen growth. Dye-labeled proteins are localized to the cytosol of Candida albicans cells without the disruption of the cell membrane. Moreover, we showed that entry of the proteins into C. albicans cells resulted in the accumulation of reactive oxygen species (ROS) and cell death via altered mitochondrial potential. Morphological changes of C. albicans cells in the presence of proteins were visualized by scanning electron microscopy. Our data suggest that AtSGT1 proteins play a critical role in plant resistance to pathogenic fungal infection and they can be classified to a new plant antifungal protein.

Excessive L-cysteine induces vacuole-like cell death by activating endoplasmic reticulum stress and mitogen-activated protein kinase signaling in intestinal porcine epithelial cells.

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Ji, Yun; Wu, Zhenlong; Dai, Zhaolai; Sun, Kaiji; Zhang, Qing; Wu, Guoyao

2016-01-01

High intake of dietary cysteine is extremely toxic to animals and the underlying mechanism remains largely unknown. This study was conducted to test the hypothesis that excessive L-cysteine induces cell death by activating endoplasmic reticulum (ER) stress and mitogen-activated protein kinase (MAPK) signaling in intestinal porcine epithelial cells. Jejunal enterocytes were cultured in the presence of 0-10 mmol/L L-cysteine. Cell viability, morphologic alterations, mRNA levels for genes involved in ER stress, protein abundances for glucose-regulated protein 78, C/EBP homologous protein (CHOP), alpha subunit of eukaryotic initiation factor-2 (eIF2α), extracellular signal-regulated kinase (ERK1/2), p38 MAPK, and c-Jun N-terminal protein kinase (JNK1/2) were determined. The results showed that L-cysteine (5-10 mmol/L) reduced cell viability (P L-cysteine were not affected by the autophagy inhibitor 3-methyladenine. The protein abundances for CHOP, phosphorylated (p)-eIF2α, p-JNK1/2, p-p38 MAPK, and the spliced form of XBP-1 mRNA were enhanced (P L-cysteine induces vacuole-like cell death via the activation of ER stress and MAPK signaling in small intestinal epithelial cells. These signaling pathways may be potential targets for developing effective strategies to prevent the toxicity of dietary cysteine.

Similar Pathogen Targets in Arabidopsis thaliana and Homo sapiens Protein Networks

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2012-09-21

Similar Pathogen Targets in Arabidopsis thaliana and Homo sapiens Protein Networks Paulo Shakarian1*, J. Kenneth Wickiser2 1 Paulo Shakarian...significantly attacked. Citation: Shakarian P, Wickiser JK (2012) Similar Pathogen Targets in Arabidopsis thaliana and Homo sapiens Protein Networks...to 00-00-2012 4. TITLE AND SUBTITLE Similar Pathogen Targets in Arabidopsis thaliana and Homo sapiens Protein Networks 5a. CONTRACT NUMBER 5b

p38 mitogen-activated protein kinase plays a key role in regulating MAPKAPK2 expression

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Sudo, Tatsuhiko; Kawai, Kayoko; Matsuzaki, Hiroshi; Osada, Hiroyuki

2005-01-01

One of three major families of the mitogen-activated kinases (MAPK), p38 as well as JNK, has been shown to transduce extracellular stress stimuli into cellular responses by phospho-relay cascades. Among p38 families, p38α is a widely characterized isoform and the biological phenomena are explained by its kinase activity regulating functions of its downstream substrates. However, its specific contributions to each phenomenon are yet not fully elucidated. For better understanding of the role of MAPKs, especially p38α, we utilized newly established mouse fibroblast cell lines originated from a p38α null mouse, namely, a parental cell line without p38α gene locus, knockout of p38α (KOP), Zeosin-resistant (ZKOP), revertant of p38α (RKOP), and Exip revertant (EKOP). EKOP is smaller in size but grows faster than the others. Although comparable amounts of ERK and JNK are expressed in each cell line, ERK is highly phosphorylated in EKOP even in normal culture conditions. Serum stimulation after serum starvation led to ERK phosphorylation in RKOP and ZKOP, but not in EKOP as much. On the contrary, relative phosphorylation level of JNK to total JNK in response to UV was low in RKOP. And its phosphorylation as well as total JNK is slightly lower in EKOP. RKOP is less sensitive to UV irradiation as judged by the survival rate. Stress response upon UV or sorbitol stimuli, leading to mitogen activate protein kinase activated kinase 2 (MAPKAPK2) phosphorylation, was only observed in RKOP. Further experiments reveal that MAPKAPK2 expression is largely suppressed in ZKOP and EKOP. Its expression was recovered by re-introduction of p38α. The loss of MAPKAPK2 expression accompanied by the defect of p38α is confirmed in an embryonic extract prepared from p38α null mice. These data demonstrate that p38 signal pathway is regulated not only by phosphorylation but also by modulation of the expression of its component. Together, we have established cell lines that can be used in

SMALL GRAIN 1, which encodes a mitogen-activated protein kinase kinase 4, influences grain size in rice.

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Duan, Penggen; Rao, Yuchun; Zeng, Dali; Yang, Yaolong; Xu, Ran; Zhang, Baolan; Dong, Guojun; Qian, Qian; Li, Yunhai

2014-02-01

Although grain size is one of the most important components of grain yield, little information is known about the mechanisms that determine final grain size in crops. Here we characterize rice small grain1 (smg1) mutants, which exhibit small and light grains, dense and erect panicles and comparatively slightly shorter plants. The short grain and panicle phenotypes of smg1 mutants are caused by a defect in cell proliferation. The smg1 mutations were identified, using a map-based cloning approach, in mitogen-activated protein kinase kinase 4 (OsMKK4). Relatively higher expression of OsMKK4/SMG1 was detected in younger organs than in older ones, consistent with its role in cell proliferation. Green fluorescent protein (GFP)-OsMKK4/SMG1 fusion proteins appear to be distributed ubiquitously in plant cells. Further results revealed that OsMKK4 influenced brassinosteroid (BR) responses and the expression of BR-related genes. Thus, our findings have identified OsMKK4 as a factor for grain size, and suggest a possible link between the MAPK pathways and BRs in grain growth. © 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd.

Large-scale protein-protein interaction analysis in Arabidopsis mesophyll protoplasts by split firefly luciferase complementation.

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Li, Jian-Feng; Bush, Jenifer; Xiong, Yan; Li, Lei; McCormack, Matthew

2011-01-01

Protein-protein interactions (PPIs) constitute the regulatory network that coordinates diverse cellular functions. There are growing needs in plant research for creating protein interaction maps behind complex cellular processes and at a systems biology level. However, only a few approaches have been successfully used for large-scale surveys of PPIs in plants, each having advantages and disadvantages. Here we present split firefly luciferase complementation (SFLC) as a highly sensitive and noninvasive technique for in planta PPI investigation. In this assay, the separate halves of a firefly luciferase can come into close proximity and transiently restore its catalytic activity only when their fusion partners, namely the two proteins of interest, interact with each other. This assay was conferred with quantitativeness and high throughput potential when the Arabidopsis mesophyll protoplast system and a microplate luminometer were employed for protein expression and luciferase measurement, respectively. Using the SFLC assay, we could monitor the dynamics of rapamycin-induced and ascomycin-disrupted interaction between Arabidopsis FRB and human FKBP proteins in a near real-time manner. As a proof of concept for large-scale PPI survey, we further applied the SFLC assay to testing 132 binary PPIs among 8 auxin response factors (ARFs) and 12 Aux/IAA proteins from Arabidopsis. Our results demonstrated that the SFLC assay is ideal for in vivo quantitative PPI analysis in plant cells and is particularly powerful for large-scale binary PPI screens.

Large-scale protein-protein interaction analysis in Arabidopsis mesophyll protoplasts by split firefly luciferase complementation.

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Jian-Feng Li

Full Text Available Protein-protein interactions (PPIs constitute the regulatory network that coordinates diverse cellular functions. There are growing needs in plant research for creating protein interaction maps behind complex cellular processes and at a systems biology level. However, only a few approaches have been successfully used for large-scale surveys of PPIs in plants, each having advantages and disadvantages. Here we present split firefly luciferase complementation (SFLC as a highly sensitive and noninvasive technique for in planta PPI investigation. In this assay, the separate halves of a firefly luciferase can come into close proximity and transiently restore its catalytic activity only when their fusion partners, namely the two proteins of interest, interact with each other. This assay was conferred with quantitativeness and high throughput potential when the Arabidopsis mesophyll protoplast system and a microplate luminometer were employed for protein expression and luciferase measurement, respectively. Using the SFLC assay, we could monitor the dynamics of rapamycin-induced and ascomycin-disrupted interaction between Arabidopsis FRB and human FKBP proteins in a near real-time manner. As a proof of concept for large-scale PPI survey, we further applied the SFLC assay to testing 132 binary PPIs among 8 auxin response factors (ARFs and 12 Aux/IAA proteins from Arabidopsis. Our results demonstrated that the SFLC assay is ideal for in vivo quantitative PPI analysis in plant cells and is particularly powerful for large-scale binary PPI screens.

Mitogen-Activated Protein Kinase Kinase 3 Regulates Seed Dormancy in Barley.

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Nakamura, Shingo; Pourkheirandish, Mohammad; Morishige, Hiromi; Kubo, Yuta; Nakamura, Masako; Ichimura, Kazuya; Seo, Shigemi; Kanamori, Hiroyuki; Wu, Jianzhong; Ando, Tsuyu; Hensel, Goetz; Sameri, Mohammad; Stein, Nils; Sato, Kazuhiro; Matsumoto, Takashi; Yano, Masahiro; Komatsuda, Takao

2016-03-21

Seed dormancy has fundamental importance in plant survival and crop production; however, the mechanisms regulating dormancy remain unclear [1-3]. Seed dormancy levels generally decrease during domestication to ensure that crops successfully germinate in the field. However, reduction of seed dormancy can cause devastating losses in cereals like wheat (Triticum aestivum L.) and barley (Hordeum vulgare L.) due to pre-harvest sprouting, the germination of mature seed (grain) on the mother plant when rain occurs before harvest. Understanding the mechanisms of dormancy can facilitate breeding of crop varieties with the appropriate levels of seed dormancy [4-8]. Barley is a model crop [9, 10] and has two major seed dormancy quantitative trait loci (QTLs), SD1 and SD2, on chromosome 5H [11-19]. We detected a QTL designated Qsd2-AK at SD2 as the single major determinant explaining the difference in seed dormancy between the dormant cultivar "Azumamugi" (Az) and the non-dormant cultivar "Kanto Nakate Gold" (KNG). Using map-based cloning, we identified the causal gene for Qsd2-AK as Mitogen-activated Protein Kinase Kinase 3 (MKK3). The dormant Az allele of MKK3 is recessive; the N260T substitution in this allele decreases MKK3 kinase activity and appears to be causal for Qsd2-AK. The N260T substitution occurred in the immediate ancestor allele of the dormant allele, and the established dormant allele became prevalent in barley cultivars grown in East Asia, where the rainy season and harvest season often overlap. Our findings show fine-tuning of seed dormancy during domestication and provide key information for improving pre-harvest sprouting tolerance in barley and wheat. Copyright © 2016 Elsevier Ltd. All rights reserved.

Structural and Functional Analysis of VQ Motif-Containing Proteins in Arabidopsis as Interacting Proteins of WRKY Transcription Factors1[W][OA

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Cheng, Yuan; Zhou, Yuan; Yang, Yan; Chi, Ying-Jun; Zhou, Jie; Chen, Jian-Ye; Wang, Fei; Fan, Baofang; Shi, Kai; Zhou, Yan-Hong; Yu, Jing-Quan; Chen, Zhixiang

2012-01-01

WRKY transcription factors are encoded by a large gene superfamily with a broad range of roles in plants. Recently, several groups have reported that proteins containing a short VQ (FxxxVQxLTG) motif interact with WRKY proteins. We have recently discovered that two VQ proteins from Arabidopsis (Arabidopsis thaliana), SIGMA FACTOR-INTERACTING PROTEIN1 and SIGMA FACTOR-INTERACTING PROTEIN2, act as coactivators of WRKY33 in plant defense by specifically recognizing the C-terminal WRKY domain and stimulating the DNA-binding activity of WRKY33. In this study, we have analyzed the entire family of 34 structurally divergent VQ proteins from Arabidopsis. Yeast (Saccharomyces cerevisiae) two-hybrid assays showed that Arabidopsis VQ proteins interacted specifically with the C-terminal WRKY domains of group I and the sole WRKY domains of group IIc WRKY proteins. Using site-directed mutagenesis, we identified structural features of these two closely related groups of WRKY domains that are critical for interaction with VQ proteins. Quantitative reverse transcription polymerase chain reaction revealed that expression of a majority of Arabidopsis VQ genes was responsive to pathogen infection and salicylic acid treatment. Functional analysis using both knockout mutants and overexpression lines revealed strong phenotypes in growth, development, and susceptibility to pathogen infection. Altered phenotypes were substantially enhanced through cooverexpression of genes encoding interacting VQ and WRKY proteins. These findings indicate that VQ proteins play an important role in plant growth, development, and response to environmental conditions, most likely by acting as cofactors of group I and IIc WRKY transcription factors. PMID:22535423

Specific and differential activation of mitogen-activated protein kinase cascades by unfamiliar taste in the insular cortex of the behaving rat.

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Berman, D E; Hazvi, S; Rosenblum, K; Seger, R; Dudai, Y

1998-12-01

Rats were given to drink an unfamiliar taste solution under conditions that result in long-term memory of that taste. The insular cortex, which contains the taste cortex, was then removed and assayed for activation of mitogen-activated protein kinase (MAPK) cascades by using antibodies to the activated forms of various MAPKs. Extracellular responsive kinase 1-2 (ERK1-2) in the cortical homogenate was significantly activated within taste solution, without alteration in the total level of the ERK1-2 proteins. The activity subsided to basal levels within ERK1-2 was not activated when the taste was made familiar. The effect of the unfamiliar taste was specific to the insular cortex. Jun N-terminal kinase 1-2 (JNK1-2) was activated by drinking the taste but with a delayed time course, whereas the activity of Akt kinase and p38MAPK remained unchanged. Elk-1, a member of the ternary complex factor and an ERK/JNK downstream substrate, was activated with a time course similar to that of ERK1-2. Microinjection of a reversible inhibitor of MAPK/ERK kinase into the insular cortex shortly before exposure to the novel taste in a conditioned taste aversion training paradigm attenuated long-term taste aversion memory without significantly affecting short-term memory or the sensory, motor, and motivational faculties required to express long-term taste aversion memory. It was concluded that ERK and JNK are specifically and differentially activated in the insular cortex after exposure to a novel taste, and that this activation is required for consolidation of long-term taste memory.

Induction of glutathione synthesis in human hepatocytes by acute and chronic arsenic exposure: Differential roles of mitogen-activated protein kinases

International Nuclear Information System (INIS)

Hou, Yongyong; Wang, Yi; Wang, Huihui; Xu, Yuanyuan

2014-01-01

Highlights: • Arsenic exposure increased intracellular levels of glutathione. • Mitogen-activated protein kinases were involved in glutathione homeostasis. • ERK contributed to glutathione synthesis during acute arsenic exposure. • Glutathione synthesis was regulated by p38 at least in part independent of NRF2 during chronic arsenic exposure. - Abstract: Glutathione (GSH) is a vital component of antioxidant defense which protects cells from toxic insults. Previously we found intracellular GSH was involved in cell resistance against arsenic-induced cytotoxicity. However, molecular mechanisms of GSH homeostasis during arsenic exposure are largely undefined. Here, we investigated roles of mitogen-activated protein kinases (MAPKs) in GSH synthesis pathway with two arsenic exposure strategies by using Chang human hepatocytes. In one strategy, acute arsenic exposure (20 μM, 24 h) was applied, as MAPK signaling is generally considered to be transient. In the other one, chronic arsenic exposure (500 nM, 20 weeks) was applied, which mimicked the general human exposure to arsenic. We found that acute arsenic exposure activated extracellular signal-regulated 1/2 kinases (ERK1/2) and c-Jun N-terminal kinase (JNK) in parallel with increased transcription and nuclear translocation of factor-erythroid 2-related factor 2 (NRF2) and enhanced expression of γ-glutamyl cysteine ligase catalytic subunit (GCLC), resulting in elevated intracellular GSH levels. Specific ERK inhibitor abolished arsenic-induced NRF2 nuclear translocation and GSH synthesis. During chronic arsenic exposure which induced a malignant cellular phenotype, continuous p38 activation and NRF2 nuclear translocation were observed with enhanced GSH synthesis. Specific p38 inhibitor attenuated arsenic-enhanced GSH synthesis without changing NRF2 nuclear translocation. Taken together, our results indicate MAPK pathways play an important role in cellular GSH homeostasis in response to arsenic. However, the

An Ime2-like mitogen-activated protein kinase is involved in cellulase expression in the filamentous fungus Trichoderma reesei.

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Chen, Fei; Chen, Xiu-Zhen; Su, Xiao-Yun; Qin, Li-Na; Huang, Zhen-Bang; Tao, Yong; Dong, Zhi-Yang

2015-10-01

Eukaryotic mitogen-activated protein kinases (MAPKs) play crucial roles in transducing environmental and developmental signals inside the cell and regulating gene expression, however, the roles of MAPKs remain largely unknown in Trichoderma reesei. T. reesei ime2 (TrIme2) encodes an Ime2-like MAPK in T. reesei. The deletion of the TrIme2 gene led to 90% increase in cellulase activity against filter paper during earlier period time of cellulase induction as well as the extracellular protein production. Compared to the parent strain, the transcriptional levels of the three major cellulase genes cbh1,cbh2, egl1 were increased by about 9 times, 4 times, 2 times, respectively, at 8 h after cellulase induction in the ΔTrIme2 mutant. In addition, the disruption of TrIme2 caused over 50% reduction of the transcript levels of cellulase transcriptional regulators cre1 and xyr1. TrIme2 functions in regulation of the expression of cellulase gene in T.reesei, and is a good candidate for genetically engineering of T. reesei for higher cellulase production.

Effects of Butyltins (BTs) on Mitogen-Activated-Protein Kinase Kinase Kinase (MAP3K) and Ras Activity in Human Natural Killer Cells

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Celada, Lindsay J.; Whalen, Margaret M.

2013-01-01

Butyltins (BTs) contaminate the environment and are found in human blood. BTs, tributyltin (TBT) and dibutyltin (DBT), diminish the cytotoxic function and levels of key proteins of human natural killer (NK) cells. NK cells are an initial immune defense against tumors, virally-infected cells and antibody-coated cells and thus critical to human health. The signaling pathways that regulate NK cell functions include mitogen-activated protein kinases (MAPKs). Studies have shown that exposure to BTs leads to the activation of specific MAPKs and MAPK kinases (MAP2Ks) in human NK cells. MAP2K kinases (MAP3Ks) are upstream activators of MAP2Ks, which then activate MAPKs. The current study examined if BT-induced activation of MAP3Ks was responsible for MAP2K and thus, MAPK activation. This study examines the effects of TBT and DBT on the total levels of two MAP3Ks, c-Raf and ASK1, as well as activating and inhibitory phosphorylation sites on these MAP3Ks. In addition, the immediate upstream activator of c-Raf, Ras, was examined for BT-induced alterations. Our results show significant activation of the MAP3K, c-Raf, in human NK cells within 10 minutes of TBT exposure and the MAP3K, ASK1, after one hour exposures to TBT. In addition, our results suggest that both TBT and DBT are impacting the regulation of c-Raf. PMID:24038145

A Role for Protein Phosphatase 2A in Regulating p38 Mitogen Activated Protein Kinase Activation and Tumor Necrosis Factor-Alpha Expression during Influenza Virus Infection

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Anna H. Y. Law

2013-04-01

Full Text Available Influenza viruses of avian origin continue to pose pandemic threats to human health. Some of the H5N1 and H9N2 virus subtypes induce markedly elevated cytokine levels when compared with the seasonal H1N1 virus. We previously showed that H5N1/97 hyperinduces tumor necrosis factor (TNF-alpha through p38 mitogen activated protein kinase (MAPK. However, the detailed mechanisms of p38MAPK activation and TNF-alpha hyperinduction following influenza virus infections are not known. Negative feedback regulations of cytokine expression play important roles in avoiding overwhelming production of proinflammatory cytokines. Here we hypothesize that protein phosphatases are involved in the regulation of cytokine expressions during influenza virus infection. We investigated the roles of protein phosphatases including MAPK phosphatase-1 (MKP-1 and protein phosphatase type 2A (PP2A in modulating p38MAPK activation and downstream TNF-alpha expressions in primary human monocyte-derived macrophages (PBMac infected with H9N2/G1 or H1N1 influenza virus. We demonstrate that H9N2/G1 virus activated p38MAPK and hyperinduced TNF-alpha production in PBMac when compared with H1N1 virus. H9N2/G1 induced PP2A activity in PBMac and, with the treatment of a PP2A inhibitor, p38MAPK phosphorylation and TNF-alpha production were further increased in the virus-infected macrophages. However, H9N2/G1 did not induce the expression of PP2A indicating that the activation of PP2A is not mediated by p38MAPK in virus-infected PBMac. On the other hand, PP2A may not be the targets of H9N2/G1 in the upstream of p38MAPK signaling pathways since H1N1 also induced PP2A activation in primary macrophages. Our results may provide new insights into the control of cytokine dysregulation.

TRAF6 promotes myogenic differentiation via the TAK1/p38 mitogen-activated protein kinase and Akt pathways.

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Fang Xiao

Full Text Available p38 mitogen-activated protein kinase (MAPK is an essential kinase involved in myogenic differentiation. Although many substrates of p38 MAPK have been identified, little is known about its upstream activators during myogenic differentiation. TRAF6 is known to function in cytokine signaling during inflammatory responses. However, not much is known about its role in myogenic differentiation and muscle regeneration. We showed here that TRAF6 and its intrinsic ubiquitin E3 ligase activity are required for myogenic differentiation. In mouse myoblasts, knockdown of TRAF6 compromised the p38 MAPK and Akt pathways, while deliberate activation of either pathway rescued the differentiation defect caused by TRAF6 knockdown. TAK1 acted as a key signal transducer downstream of TRAF6 in myogenic differentiation. In vivo, knockdown of TRAF6 in mouse muscles compromised the injury-induced muscle regeneration without impairing macrophage infiltration and myoblast proliferation. Collectively, we demonstrated that TRAF6 promotes myogenic differentiation and muscle regeneration via the TAK1/p38 MAPK and Akt pathways.

An inhibition of p38 mitogen activated protein kinase delays the platelet storage lesion.

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Andrey Skripchenko

Full Text Available BACKGROUND AND OBJECTIVES: Platelets during storage undergo diverse alterations collectively known as the platelet storage lesion, including metabolic, morphological, functional and structural changes. Some changes correlate with activation of p38 mitogen activated protein kinase (p38 MAPK. Another MAPK, extracellular signal-related kinase (ERK, is involved in PLT activation. The aim of this study was to compare the properties of platelets stored in plasma in the presence or absence of p38 and ERK MAPK inhibitors. MATERIALS AND METHODS: A single Trima apheresis platelet unit (n = 12 was aliquoted into five CLX storage bags. Two aliquots were continuously agitated with or without MAPK inhibitors. Two aliquots were subjected to 48 hours of interruption of agitation with or without MAPK inhibitors. One aliquot contained the same amount of solvent vehicle used to deliver the inhibitor. Platelets were stored at 20-24°C for 7 days and sampled on Days 1, 4, and 7 for 18 in vitro parameters. RESULTS: Inhibition of p38 MAPK by VX-702 leads to better maintenance of all platelet in vitro storage parameters including platelet mitochondrial function. Accelerated by interruption of agitation, the platelet storage lesion of units stored with VX-702 was diminished to that of platelets stored with continuous agitation. Inhibition of ERK MAPK did not ameliorate decrements in any in vitro platelet properties. CONCLUSION: Signaling through p38 MAPK, but not ERK, is associated with platelet deterioration during storage.

Identification of proteins interacting with Arabidopsis ACD11

DEFF Research Database (Denmark)

Petersen, Nikolaj H T; Joensen, Jan; McKinney, Lea V

2009-01-01

The Arabidopsis ACD11 gene encodes a sphingosine transfer protein and was identified by the accelerated cell death phenotype of the loss of function acd11 mutant, which exhibits heightened expression of genes involved in the disease resistance hypersensitive response (HR). We used ACD11 as bait...... in a yeast two-hybrid screen of an Arabidopsis cDNA library to identify ACD11 interacting proteins. One interactor identified is a protein of unknown function with an RNA recognition motif (RRM) designated BPA1 (binding partner of ACD11). Co-immunoprecipitation experiments confirmed the ACD11-BPA1...

The F-box protein Fbp1 functions in the invasive growth and cell wall integrity mitogen-activated protein kinase (MAPK) pathways in Fusarium oxysporum.

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Miguel-Rojas, Cristina; Hera, Concepcion

2016-01-01

F-box proteins determine substrate specificity of the ubiquitin-proteasome system. Previous work has demonstrated that the F-box protein Fbp1, a component of the SCF(Fbp1) E3 ligase complex, is essential for invasive growth and virulence of the fungal plant pathogen Fusarium oxysporum. Here, we show that, in addition to invasive growth, Fbp1 also contributes to vegetative hyphal fusion and fungal adhesion to tomato roots. All of these functions have been shown previously to require the mitogen-activated protein kinase (MAPK) Fmk1. We found that Fbp1 is required for full phosphorylation of Fmk1, indicating that Fbp1 regulates virulence and invasive growth via the Fmk1 pathway. Moreover, the Δfbp1 mutant is hypersensitive to sodium dodecylsulfate (SDS) and calcofluor white (CFW) and shows reduced phosphorylation levels of the cell wall integrity MAPK Mpk1 after SDS treatment. Collectively, these results suggest that Fbp1 contributes to both the invasive growth and cell wall integrity MAPK pathways of F. oxysporum. © 2015 BSPP AND JOHN WILEY & SONS LTD.

Beyond Histones: New Substrate Proteins of Lysine Deacetylases in Arabidopsis Nuclei

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Magdalena Füßl

2018-04-01

Full Text Available The reversible acetylation of lysine residues is catalyzed by the antagonistic action of lysine acetyltransferases and deacetylases, which can be considered as master regulators of their substrate proteins. Lysine deacetylases, historically referred to as histone deacetylases, have profound functions in regulating stress defenses and development in plants. Lysine acetylation of the N-terminal histone tails promotes gene transcription and decondensation of chromatin, rendering the DNA more accessible to the transcription machinery. In plants, the classical lysine deacetylases from the RPD3/HDA1-family have thus far mainly been studied in the context of their deacetylating activities on histones, and their versatility in molecular activities is still largely unexplored. Here we discuss the potential impact of lysine acetylation on the recently identified nuclear substrate proteins of lysine deacetylases from the Arabidopsis RPD3/HDA1-family. Among the deacetylase substrate proteins, many interesting candidates involved in nuclear protein import, transcriptional regulation, and chromatin remodeling have been identified. These candidate proteins represent key starting points for unraveling new molecular functions of the Arabidopsis lysine deacetylases. Site-directed engineering of lysine acetylation sites on these target proteins might even represent a new approach for optimizing plant growth under climate change conditions.

p75 Neurotrophin Receptor Signaling Activates Sterol Regulatory Element-binding Protein-2 in Hepatocyte Cells via p38 Mitogen-activated Protein Kinase and Caspase-3.

Science.gov (United States)

Pham, Dan Duc; Do, Hai Thi; Bruelle, Céline; Kukkonen, Jyrki P; Eriksson, Ove; Mogollón, Isabel; Korhonen, Laura T; Arumäe, Urmas; Lindholm, Dan

2016-05-13

Nerve growth factor (NGF) influences the survival and differentiation of a specific population of neurons during development, but its role in non-neuronal cells has been less studied. We observed here that NGF and its pro-form, pro-NGF, are elevated in fatty livers from leptin-deficient mice compared with controls, concomitant with an increase in low density lipoprotein receptors (LDLRs). Stimulation of mouse primary hepatocytes with NGF or pro-NGF increased LDLR expression through the p75 neurotrophin receptor (p75NTR). Studies using Huh7 human hepatocyte cells showed that the neurotrophins activate the sterol regulatory element-binding protein-2 (SREBP2) that regulates genes involved in lipid metabolism. The mechanisms for this were related to stimulation of p38 mitogen-activated protein kinase (p38 MAPK) and activation of caspase-3 and SREBP2 cleavage following NGF and pro-NGF stimulations. Cell fractionation experiments showed that caspase-3 activity was increased particularly in the membrane fraction that harbors SREBP2 and caspase-2. Experiments showed further that caspase-2 interacts with pro-caspase-3 and that p38 MAPK reduced this interaction and caused caspase-3 activation. Because of the increased caspase-3 activity, the cells did not undergo cell death following p75NTR stimulation, possibly due to concomitant activation of nuclear factor-κB (NF-κB) pathway by the neurotrophins. These results identify a novel signaling pathway triggered by ligand-activated p75NTR that via p38 MAPK and caspase-3 mediate the activation of SREBP2. This pathway may regulate LDLRs and lipid uptake particularly after injury or during tissue inflammation accompanied by an increased production of growth factors, including NGF and pro-NGF. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

P-proteins in Arabidopsis are heteromeric structures involved in rapid sieve tube sealing.

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Jekat, Stephan B; Ernst, Antonia M; von Bohl, Andreas; Zielonka, Sascia; Twyman, Richard M; Noll, Gundula A; Prüfer, Dirk

2013-01-01

Structural phloem proteins (P-proteins) are characteristic components of the sieve elements in all dicotyledonous and many monocotyledonous angiosperms. Tobacco P-proteins were recently confirmed to be encoded by the widespread sieve element occlusion (SEO) gene family, and tobacco SEO proteins were shown to be directly involved in sieve tube sealing thus preventing the loss of photosynthate. Analysis of the two Arabidopsis SEO proteins (AtSEOa and AtSEOb) indicated that the corresponding P-protein subunits do not act in a redundant manner. However, there are still pending questions regarding the interaction properties and specific functions of AtSEOa and AtSEOb as well as the general function of structural P-proteins in Arabidopsis. In this study, we characterized the Arabidopsis P-proteins in more detail. We used in planta bimolecular fluorescence complementation assays to confirm the predicted heteromeric interactions between AtSEOa and AtSEOb. Arabidopsis mutants depleted for one or both AtSEO proteins lacked the typical P-protein structures normally found in sieve elements, underlining the identity of AtSEO proteins as P-proteins and furthermore providing the means to determine the role of Arabidopsis P-proteins in sieve tube sealing. We therefore developed an assay based on phloem exudation. Mutants with reduced AtSEO expression levels lost twice as much photosynthate following injury as comparable wild-type plants, confirming that Arabidopsis P-proteins are indeed involved in sieve tube sealing.

P-proteins in Arabidopsis are heteromeric structures involved in rapid sieve tube sealing

Directory of Open Access Journals (Sweden)

Stephan B Jekat

2013-07-01

Full Text Available Structural phloem proteins (P-proteins are characteristic components of the sieve elements in all dicotyledonous and many monocotyledonous angiosperms. Tobacco P-proteins were recently evidenced to be encoded by the widespread SEO gene family, and tobacco SEO proteins were shown to be directly involved in sieve tube sealing thus preventing the loss of photosynthate. Analysis of the two Arabidopsis SEO proteins (AtSEOa and AtSEOb indicated that the corresponding P-protein subunits do not act in a redundant manner. However, there are still pending questions regarding the interaction properties and specific functions of AtSEOa and AtSEOb as well as the general function of structural P-proteins in Arabidopsis. In this study, we characterized the Arabidopsis P-proteins in more detail. We used in planta bimolecular fluorescence complementation assays to confirm the predicted heteromeric interactions between AtSEOa and AtSEOb. Arabidopsis mutants depleted for one or both AtSEO proteins lacked the typical P-protein structures normally found in sieve elements, underlining the identity of AtSEO proteins as P-proteins and furthermore providing the means to determine the role of Arabidopsis P-proteins in sieve tube sealing. We therefore developed an assay based on phloem exudation. Mutants with reduced AtSEO expression levels lost twice as much photosynthate following injury as comparable wild-type plants, confirming that Arabidopsis P-proteins are indeed involved in sieve tube sealing. 

Uncaria rhynchophylla inhibits the production of nitric oxide and interleukin-1β through blocking nuclear factor κB, Akt, and mitogen-activated protein kinase activation in macrophages.

Science.gov (United States)

Kim, Ji-Hee; Bae, Chang Hwan; Park, Sun Young; Lee, Sang Joon; Kim, YoungHee

2010-10-01

The stems with hook of Uncaria rhynchophylla have been used in traditional medicine as an antipyretic, antihypertensive, and anticonvulsant in China and Korea. In this study, we investigated the mechanism responsible for anti-inflammatory effects of U. rhynchophylla in RAW 264.7 macrophages. The aqueous extract of U. rhynchophylla inhibited lipopolysaccharide (LPS)-induced nitric oxide (NO) and interleukin (IL)-1β secretion as well as inducible NO synthase (iNOS) expression, without affecting cell viability. Furthermore, U. rhynchophylla suppressed LPS-induced nuclear factor κB (NF-κB) activation, phosphorylation, and degradation of inhibitory protein IκB (IκB)-α, phosphorylation of Akt, extracellular signal-regulated kinase 1/2, p38 kinase, and c-Jun N-terminal kinase. These results suggest that U. rhynchophylla has the inhibitory effects on LPS-induced NO and IL-1β production in macrophages through blockade in the phosphorylation of Akt and mitogen-activated protein kinases, following IκB-α degradation and NF-κB activation.

Nitric oxide mediates the indole acetic acid induction activation of a mitogen-activated protein kinase cascade involved in adventitious root development.

Science.gov (United States)

Pagnussat, Gabriela Carolina; Lanteri, María Luciana; Lombardo, María Cristina; Lamattina, Lorenzo

2004-05-01

Recently, it was demonstrated that nitric oxide (NO) and cGMP are involved in the auxin response during the adventitious rooting process in cucumber (Cucumis sativus; Pagnussat et al., 2002, 2003). However, not much is known about the complex molecular network operating during the cell proliferation and morphogenesis triggered by auxins and NO in that process. Anatomical studies showed that formation of adventitious root primordia was clearly detected in indole acetic acid (IAA)- and NO-treated cucumber explants, while neither cell proliferation nor differentiation into root primordia could be observed in control explants 3 d after primary root was removed. In order to go further with signal transduction mechanisms that operate during IAA- and NO-induced adventitious root formation, experiments were designed to test the involvement of a mitogen-activated protein kinase (MAPK) cascade in that process. Cucumber explants were treated with the NO-donor sodium nitroprusside (SNP) or with SNP plus the specific NO-scavenger cPTIO. Protein extracts from those explants were assayed for protein kinase (PK) activity by using myelin basic protein (MBP) as substrate in both in vitro and in-gel assays. The activation of a PK of approximately 48 kD could be detected 1 d after NO treatment with a maximal activation after 3 d of treatment. In control explants, a PK activity was detected only after 4 d of treatment. The MBP-kinase activity was also detected in extracts from IAA-treated explants, while no signal was observed in IAA + cPTIO treatments. The PK activity could be inhibited by the cell-permeable MAPK kinase inhibitor PD098059, suggesting that the NO-dependent MBP-kinase activity is a MAPK. Furthermore, when PD098059 was administered to explants treated with SNP or IAA, it produced a delay in root emergence and a dose-dependent reduction in root number. Altogether, our results suggest that a MAPK signaling cascade is activated during the adventitious rooting process

Effects of phorbol ester on mitogen-activated protein kinase kinase activity in wild-type and phorbol ester-resistant EL4 thymoma cells.

Science.gov (United States)

Gause, K C; Homma, M K; Licciardi, K A; Seger, R; Ahn, N G; Peterson, M J; Krebs, E G; Meier, K E

1993-08-05

Phorbol ester-sensitive and -resistant EL4 thymoma cell lines differ in their ability to activate mitogen-activated protein kinase (MAPK) in response to phorbol ester. Treatment of wild-type EL4 cells with phorbol ester results in the rapid activations of MAPK and pp90rsk kinase, a substrate for MAPK, while neither kinase is activated in response to phorbol ester in variant EL4 cells. This study examines the activation of MAPK kinase (MAPKK), an activator of MAPK, in wild-type and variant EL4 cells. Phosphorylation of a 40-kDa substrate, identified as MAPK, was observed following in vitro phosphorylation reactions using cytosolic extracts or Mono Q column fractions prepared from phorbol ester-treated wild-type EL4 cells. MAPKK activity coeluted with a portion of the inactive MAPK upon Mono Q anion-exchange chromatography, permitting detection of the MAPKK activity in fractions containing both kinases. This MAPKK activity was present in phorbol ester-treated wild-type cells, but not in phorbol ester-treated variant cells or in untreated wild-type or variant cells. The MAPKK from wild-type cells was able to activate MAPK prepared from either wild-type or variant cells. MAPKK activity could be stimulated in both wildtype and variant EL4 cells in response to treatment of cells with okadaic acid. These results indicate that the failure of variant EL4 cells to activate MAP kinase in response to phorbol ester is due to a failure to activate MAPKK. Therefore, the step that confers phorbol ester resistance to variant EL4 cells lies between the activation of protein kinase C and the activation of MAPKK.

Constitutive Activation of the Fission Yeast Pheromone-Responsive Pathway Induces Ectopic Meiosis and Reveals Ste11 as a Mitogen-Activated Protein Kinase Target

DEFF Research Database (Denmark)

Kjærulff, Søren; Lautrup-Larsen, I.; Truelsen, S.

2005-01-01

In the fission yeast Schizosaccharomyces pombe, meiosis normally takes place in diploid zygotes resulting from conjugation of haploid cells. In the present study, we report that the expression of a constitutively activated version of the pheromone-responsive mitogen-activated protein kinase kinase...... found that haploid meiosis was dramatically reduced when Ste11 was mutated to mimic phosphorylation by Pat1. The mutation of two putative MAPK sites in Ste11 also dramatically reduced the level of haploid meiosis, suggesting that Ste11 is a direct target of Spk1. Supporting this, we show that Spk1 can...... interact physically with Ste11 and also phosphorylate the transcription factor in vitro. Finally, we demonstrate that ste11 is required for pheromone-induced G1 arrest. Interestingly, when we mutated Ste11 in the sites for Pat1 and Spk1 phosphorylation simultaneously, the cells could still arrest in G1...

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo.

Science.gov (United States)

Komar, Dorota N; Mouriz, Alfonso; Jarillo, José A; Piñeiro, Manuel

2016-01-14

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and silencing of genes mediate the response of plants to environmental signals and developmental cues. Therefore, insights into the mechanisms that control plant gene expression are essential to gain a deep understanding of how biological processes are regulated in plants. The chromatin immunoprecipitation (ChIP) technique described here is a procedure to identify the DNA-binding sites of proteins in genes or genomic regions of the model species Arabidopsis thaliana. The interactions with DNA of proteins of interest such as transcription factors, chromatin proteins or posttranslationally modified versions of histones can be efficiently analyzed with the ChIP protocol. This method is based on the fixation of protein-DNA interactions in vivo, random fragmentation of chromatin, immunoprecipitation of protein-DNA complexes with specific antibodies, and quantification of the DNA associated with the protein of interest by PCR techniques. The use of this methodology in Arabidopsis has contributed significantly to unveil transcriptional regulatory mechanisms that control a variety of plant biological processes. This approach allowed the identification of the binding sites of the Arabidopsis chromatin protein EBS to regulatory regions of the master gene of flowering FT. The impact of this protein in the accumulation of particular histone marks in the genomic region of FT was also revealed through ChIP analysis.

The cis decoy against the estrogen response element suppresses breast cancer cells via target disrupting c-fos not mitogen-activated protein kinase activity.

Science.gov (United States)

Wang, Li Hua; Yang, Xiao Yi; Zhang, Xiaohu; Mihalic, Kelly; Xiao, Weihua; Farrar, William L

2003-05-01

Breast cancer, the most common malignancy in women, has been demonstrated to be associated with the steroid hormone estrogen and its receptor (ER), a ligand-activated transcription factor. Therefore, we developed a phosphorothiolate cis-element decoy against the estrogen response element (ERE decoy) to target disruption of ER DNA binding and transcriptional activity. Here, we showed that the ERE decoy potently ablated the 17beta-estrogen-inducible cell proliferation and induced apoptosis of human breast carcinoma cells by functionally affecting expression of c-fos gene and AP-1 luciferase gene reporter activity. Specificity of the decoy was demonstrated by its ability to directly block ER binding to a cis-element probe and transactivation. Moreover, the decoy failed to inhibit ER-mediated mitogen-activated protein kinase signaling pathways and cell growth of ER-negative breast cancer cells. Taken together, these data suggest that estrogen-mediated cell growth of breast cancer cells can be preferentially restricted via targeted disruption of ER at the level of DNA binding by a novel and specific decoy strategy applied to steroid nuclear receptors.

Protein Kinase Mitogen-activated Protein Kinase Kinase Kinase Kinase 4 (MAP4K4) Promotes Obesity-induced Hyperinsulinemia.

Science.gov (United States)

Roth Flach, Rachel J; Danai, Laura V; DiStefano, Marina T; Kelly, Mark; Menendez, Lorena Garcia; Jurczyk, Agata; Sharma, Rohit B; Jung, Dae Young; Kim, Jong Hun; Kim, Jason K; Bortell, Rita; Alonso, Laura C; Czech, Michael P

2016-07-29

Previous studies revealed a paradox whereby mitogen-activated protein kinase kinase kinase kinase 4 (Map4k4) acted as a negative regulator of insulin sensitivity in chronically obese mice, yet systemic deletion of Map4k4 did not improve glucose tolerance. Here, we report markedly reduced glucose-responsive plasma insulin and C-peptide levels in whole body Map4k4-depleted mice (M4K4 iKO) as well as an impaired first phase of insulin secretion from islets derived from M4K4 iKO mice ex vivo After long-term high fat diet (HFD), M4K4 iKO mice pancreata also displayed reduced β cell mass, fewer proliferating β cells and reduced islet-specific gene mRNA expression compared with controls, although insulin content was normal. Interestingly, the reduced plasma insulin in M4K4 iKO mice exposed to chronic (16 weeks) HFD was not observed in response to acute HFD challenge or short term treatment with the insulin receptor antagonist S961. Furthermore, the improved insulin sensitivity in obese M4K4 iKO mice was abrogated by high exogenous insulin over the course of a euglycemic clamp study, indicating that hypoinsulinemia promotes insulin sensitivity in chronically obese M4K4 iKO mice. These results demonstrate that protein kinase Map4k4 drives obesity-induced hyperinsulinemia and insulin resistance in part by promoting insulin secretion from β cells in mice. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Mesothelioma Cells Escape Heat Stress by Upregulating Hsp40/Hsp70 Expression via Mitogen-Activated Protein Kinases

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Michael Roth

2009-01-01

Full Text Available Therapy with hyperthermal chemotherapy in pleural diffuse malignant mesothelioma had limited benefits for patients. Here we investigated the effect of heat stress on heat shock proteins (HSP, which rescue tumour cells from apoptosis. In human mesothelioma and mesothelial cells heat stress (39–42°C induced the phosphorylation of two mitogen activated kinases (MAPK Erk1/2 and p38, and increased Hsp40, and Hsp70 expression. Mesothelioma cells expressed more Hsp40 and were less sensitive to heat stress compared to mesothelial cells. Inhibition of Erk1/2 MAPK by PD98059 or by Erk1 siRNA down-regulated heat stress-induced Hsp40 and Hsp70 expression and reduced mesothelioma cell survival. Inhibition of p38MAPK by SB203580 or siRNA reduced Hsp40, but not Hsp70, expression and also increased mesothelioma cell death. Thus hyperthermia combined with suppression of p38 MAPK or Hsp40 may represent a novel approach to improve mesothelioma therapy.

Brominated flame retardants, tetrabromobisphenol A and hexabromocyclododecane, activate mitogen-activated protein kinases (MAPKs) in human natural killer cells.

Science.gov (United States)

Cato, Anita; Celada, Lindsay; Kibakaya, Esther Caroline; Simmons, Nadia; Whalen, Margaret M

2014-12-01

Natural killer (NK) cells provide a vital surveillance against virally infected cells, tumor cells, and antibody-coated cells through the release of cytolytic mediators and gamma interferon (IFN-γ). Hexabromocyclododecane (HBCD) is a brominated flame retardant used primarily in expanded (EPS) and extruded (XPS) polystyrene foams for thermal insulation in the building and construction industry. Tetrabromobisphenol A (TBBPA) is used both as a reactive and an additive flame retardant in a variety of materials. HBCD and TBBPA contaminate the environment and are found in human blood samples. In previous studies, we have shown that other environmental contaminants, such as the dibutyltin (DBT) and tributyltin (TBT), decrease NK lytic function by activating mitogen-activated protein kinases (MAPKs) in the NK cells. HBCD and TBBPA also interfere with NK cell(s) lytic function. The current study evaluates whether HBCD and/or TBBPA have the capacity to activate MAPKs and MAPK kinases (MAP2Ks). The effects of concentrations of HBCD and TBBPA that inhibited lytic function on the phosphorylation state and total levels of the MAPKs (p44/42, p38, and JNK) and the phosphorylation and total levels of the MAP2Ks (MEK1/2 and MKK3/6) were examined. Results indicate that exposure of human NK cells to 10-0.5 μM HBCD or TBBPA activate MAPKs and MAP2Ks. This HBCD and TBBPA-induced activation of MAPKs may leave them unavailable for activation by virally infected or tumor target cells and thus contributes to the observed decreases in lytic function seen in NK cells exposed to HBCD and TBBPA.

Loss of mitogen-activated protein kinase kinase kinase 4 (MAP3K4 reveals a requirement for MAPK signalling in mouse sex determination.

Directory of Open Access Journals (Sweden)

Debora Bogani

2009-09-01

Full Text Available Sex determination in mammals is controlled by the presence or absence of the Y-linked gene SRY. In the developing male (XY gonad, sex-determining region of the Y (SRY protein acts to up-regulate expression of the related gene, SOX9, a transcriptional regulator that in turn initiates a downstream pathway of testis development, whilst also suppressing ovary development. Despite the requirement for a number of transcription factors and secreted signalling molecules in sex determination, intracellular signalling components functioning in this process have not been defined. Here we report a role for the phylogenetically ancient mitogen-activated protein kinase (MAPK signalling pathway in mouse sex determination. Using a forward genetic screen, we identified the recessive boygirl (byg mutation. On the C57BL/6J background, embryos homozygous for byg exhibit consistent XY gonadal sex reversal. The byg mutation is an A to T transversion causing a premature stop codon in the gene encoding MAP3K4 (also known as MEKK4, a mitogen-activated protein kinase kinase kinase. Analysis of XY byg/byg gonads at 11.5 d post coitum reveals a growth deficit and a failure to support mesonephric cell migration, both early cellular processes normally associated with testis development. Expression analysis of mutant XY gonads at the same stage also reveals a dramatic reduction in Sox9 and, crucially, Sry at the transcript and protein levels. Moreover, we describe experiments showing the presence of activated MKK4, a direct target of MAP3K4, and activated p38 in the coelomic region of the XY gonad at 11.5 d post coitum, establishing a link between MAPK signalling in proliferating gonadal somatic cells and regulation of Sry expression. Finally, we provide evidence that haploinsufficiency for Map3k4 accounts for T-associated sex reversal (Tas. These data demonstrate that MAP3K4-dependent signalling events are required for normal expression of Sry during testis development, and

Loss of Mitogen-Activated Protein Kinase Kinase Kinase 4 (MAP3K4) Reveals a Requirement for MAPK Signalling in Mouse Sex Determination

Science.gov (United States)

Bogani, Debora; Siggers, Pam; Brixey, Rachel; Warr, Nick; Beddow, Sarah; Edwards, Jessica; Williams, Debbie; Wilhelm, Dagmar; Koopman, Peter; Flavell, Richard A.; Chi, Hongbo; Ostrer, Harry; Wells, Sara; Cheeseman, Michael; Greenfield, Andy

2009-01-01

Sex determination in mammals is controlled by the presence or absence of the Y-linked gene SRY. In the developing male (XY) gonad, sex-determining region of the Y (SRY) protein acts to up-regulate expression of the related gene, SOX9, a transcriptional regulator that in turn initiates a downstream pathway of testis development, whilst also suppressing ovary development. Despite the requirement for a number of transcription factors and secreted signalling molecules in sex determination, intracellular signalling components functioning in this process have not been defined. Here we report a role for the phylogenetically ancient mitogen-activated protein kinase (MAPK) signalling pathway in mouse sex determination. Using a forward genetic screen, we identified the recessive boygirl (byg) mutation. On the C57BL/6J background, embryos homozygous for byg exhibit consistent XY gonadal sex reversal. The byg mutation is an A to T transversion causing a premature stop codon in the gene encoding MAP3K4 (also known as MEKK4), a mitogen-activated protein kinase kinase kinase. Analysis of XY byg/byg gonads at 11.5 d post coitum reveals a growth deficit and a failure to support mesonephric cell migration, both early cellular processes normally associated with testis development. Expression analysis of mutant XY gonads at the same stage also reveals a dramatic reduction in Sox9 and, crucially, Sry at the transcript and protein levels. Moreover, we describe experiments showing the presence of activated MKK4, a direct target of MAP3K4, and activated p38 in the coelomic region of the XY gonad at 11.5 d post coitum, establishing a link between MAPK signalling in proliferating gonadal somatic cells and regulation of Sry expression. Finally, we provide evidence that haploinsufficiency for Map3k4 accounts for T-associated sex reversal (Tas). These data demonstrate that MAP3K4-dependent signalling events are required for normal expression of Sry during testis development, and create a novel

The p38 mitogen-activated protein kinase signaling pathway is involved in regulating low-density lipoprotein receptor-related protein 1-mediated β-amyloid protein internalization in mouse brain.

Science.gov (United States)

Ma, Kai-Ge; Lv, Jia; Hu, Xiao-Dan; Shi, Li-Li; Chang, Ke-Wei; Chen, Xin-Lin; Qian, Yi-Hua; Yang, Wei-Na; Qu, Qiu-Min

2016-07-01

Alzheimer's disease (AD) is one of the most common neurodegenerative diseases. Recently, increasing evidence suggests that intracellular β-amyloid protein (Aβ) alone plays a pivotal role in the progression of AD. Therefore, understanding the signaling pathway and proteins that control Aβ internalization may provide new insight for regulating Aβ levels. In the present study, the regulation of Aβ internalization by p38 mitogen-activated protein kinases (MAPK) through low-density lipoprotein receptor-related protein 1 (LRP1) was analyzed in vivo. The data derived from this investigation revealed that Aβ1-42 were internalized by neurons and astrocytes in mouse brain, and were largely deposited in mitochondria and lysosomes, with some also being found in the endoplasmic reticulum. Aβ1-42-LRP1 complex was formed during Aβ1-42 internalization, and the p38 MAPK signaling pathway was activated by Aβ1-42 via LRP1. Aβ1-42 and LRP1 were co- localized in the cells of parietal cortex and hippocampus. Furthermore, the level of LRP1-mRNA and LRP1 protein involved in Aβ1-42 internalization in mouse brain. The results of this investigation demonstrated that Aβ1-42 induced an LRP1-dependent pathway that related to the activation of p38 MAPK resulting in internalization of Aβ1-42. These results provide evidence supporting a key role for the p38 MAPK signaling pathway which is involved in the regulation of Aβ1-42 internalization in the parietal cortex and hippocampus of mouse through LRP1 in vivo. Copyright © 2016 Elsevier Ltd. All rights reserved.

Nodularin induces tumor necrosis factor-alpha and mitogen-activated protein kinases (MAPK) and leads to induction of endoplasmic reticulum stress

Energy Technology Data Exchange (ETDEWEB)

Meili, Nicole; Christen, Verena [University of Applied Sciences and Arts Northwestern Switzerland (FHNW), Gründenstrasse 40, CH-4132 Muttenz (Switzerland); Fent, Karl, E-mail: karl.fent@fhnw.ch [University of Applied Sciences and Arts Northwestern Switzerland (FHNW), Gründenstrasse 40, CH-4132 Muttenz (Switzerland); Swiss Federal Institute of Technology Zürich (ETH Zürich), Department of Environmental Systems Science, CH-8092 Zürich (Switzerland)

2016-06-01

Nodularin is produced by the cyanobacterium Nodularia spumigena. It is of concern due to hepatotoxicity in humans and animals. Here we investigated unexplored molecular mechanisms by transcription analysis in human liver cells, focusing on induction of pro-inflammatory cytokines, the tumor necrosis factor α (TNF-α), endoplasmic reticulum (ER) stress and components of the activator protein-1 complex in human hepatoma cells (Huh7) exposed to non-cytotoxic (0.1 and 1 μM) and toxic concentrations (5 μM) for 24, 48, and 72 h. Transcripts of TNF-α and ER stress marker genes were strongly induced at 1 and 5 μM at all time-points. TNF-α led to induction of mitogen-activated protein kinases (MAPK), as demonstrated by induction of CJUN and CFOS, which form the AP-1 complex. Human primary liver cells reacted more sensitive than Huh7 cells. They showed higher cytotoxicity and induction of TNF-α and ER stress at 2.5 nM, while HepG2 cells were insensitive up to 10 μM due to low expression of organic anion transporting polypeptides. Furthermore, nodularin led to induction of TNF-α protein, and CCAAT/enhancer-binding protein-homologous (CHOP) protein. Our data indicate that nodularin induces inflammation and ER stress and leads to activation of MAPK in liver cells. All of these activated pathways, which were analysed here for the first time in detail, may contribute to the hepatotoxic, and tumorigenic action of nodularin. - Highlights: • Toxicity of nodularin and its mechanisms of action are poorly understood. • We investigated mechanisms of nodularin toxicity in human liver cell lines and human hepatocytes. • We identified several pathways involved in nodularin toxicity. • Nodularin induces TNF-α, MAPK pathway and ER stress • These activated pathways may contribute to the hepatotoxic and tumorigenic action of nodularin.

Nodularin induces tumor necrosis factor-alpha and mitogen-activated protein kinases (MAPK) and leads to induction of endoplasmic reticulum stress

International Nuclear Information System (INIS)

Meili, Nicole; Christen, Verena; Fent, Karl

2016-01-01

Nodularin is produced by the cyanobacterium Nodularia spumigena. It is of concern due to hepatotoxicity in humans and animals. Here we investigated unexplored molecular mechanisms by transcription analysis in human liver cells, focusing on induction of pro-inflammatory cytokines, the tumor necrosis factor α (TNF-α), endoplasmic reticulum (ER) stress and components of the activator protein-1 complex in human hepatoma cells (Huh7) exposed to non-cytotoxic (0.1 and 1 μM) and toxic concentrations (5 μM) for 24, 48, and 72 h. Transcripts of TNF-α and ER stress marker genes were strongly induced at 1 and 5 μM at all time-points. TNF-α led to induction of mitogen-activated protein kinases (MAPK), as demonstrated by induction of CJUN and CFOS, which form the AP-1 complex. Human primary liver cells reacted more sensitive than Huh7 cells. They showed higher cytotoxicity and induction of TNF-α and ER stress at 2.5 nM, while HepG2 cells were insensitive up to 10 μM due to low expression of organic anion transporting polypeptides. Furthermore, nodularin led to induction of TNF-α protein, and CCAAT/enhancer-binding protein-homologous (CHOP) protein. Our data indicate that nodularin induces inflammation and ER stress and leads to activation of MAPK in liver cells. All of these activated pathways, which were analysed here for the first time in detail, may contribute to the hepatotoxic, and tumorigenic action of nodularin. - Highlights: • Toxicity of nodularin and its mechanisms of action are poorly understood. • We investigated mechanisms of nodularin toxicity in human liver cell lines and human hepatocytes. • We identified several pathways involved in nodularin toxicity. • Nodularin induces TNF-α, MAPK pathway and ER stress • These activated pathways may contribute to the hepatotoxic and tumorigenic action of nodularin.

Proteomic and phosphoproteomic analyses of chromatin-associated proteins from Arabidopsis thaliana

KAUST Repository

Bigeard, Jean

2014-07-10

The nucleus is the organelle where basically all DNA-related processes take place in eukaryotes, such as replication, transcription, and splicing as well as epigenetic regulation. The identification and description of the nuclear proteins is one of the requisites toward a comprehensive understanding of the biological functions accomplished in the nucleus. Many of the regulatory mechanisms of protein functions rely on their PTMs among which phosphorylation is probably one of the most important properties affecting enzymatic activity, interaction with other molecules, localization, or stability. So far, the nuclear and subnuclear proteome and phosphoproteome of the model plant Arabidopsis thaliana have been the subject of very few studies. In this work, we developed a purification protocol of Arabidopsis chromatin-associated proteins and performed proteomic and phosphoproteomic analyses identifying a total of 879 proteins of which 198 were phosphoproteins that were mainly involved in chromatin remodeling, transcriptional regulation, and RNA processing. From 230 precisely localized phosphorylation sites (phosphosites), 52 correspond to hitherto unidentified sites. This protocol and data thereby obtained should be a valuable resource for many domains of plant research.

Proteomic and phosphoproteomic analyses of chromatin-associated proteins from Arabidopsis thaliana

KAUST Repository

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

2014-01-01

The nucleus is the organelle where basically all DNA-related processes take place in eukaryotes, such as replication, transcription, and splicing as well as epigenetic regulation. The identification and description of the nuclear proteins is one of the requisites toward a comprehensive understanding of the biological functions accomplished in the nucleus. Many of the regulatory mechanisms of protein functions rely on their PTMs among which phosphorylation is probably one of the most important properties affecting enzymatic activity, interaction with other molecules, localization, or stability. So far, the nuclear and subnuclear proteome and phosphoproteome of the model plant Arabidopsis thaliana have been the subject of very few studies. In this work, we developed a purification protocol of Arabidopsis chromatin-associated proteins and performed proteomic and phosphoproteomic analyses identifying a total of 879 proteins of which 198 were phosphoproteins that were mainly involved in chromatin remodeling, transcriptional regulation, and RNA processing. From 230 precisely localized phosphorylation sites (phosphosites), 52 correspond to hitherto unidentified sites. This protocol and data thereby obtained should be a valuable resource for many domains of plant research.

Arabidopsis protein phosphatase DBP1 nucleates a protein network with a role in regulating plant defense.

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José Luis Carrasco

Full Text Available Arabidopsis thaliana DBP1 belongs to the plant-specific family of DNA-binding protein phosphatases. Although recently identified as a novel host factor mediating susceptibility to potyvirus, little is known about DBP1 targets and partners and the molecular mechanisms underlying its function. Analyzing changes in the phosphoproteome of a loss-of-function dbp1 mutant enabled the identification of 14-3-3λ isoform (GRF6, a previously reported DBP1 interactor, and MAP kinase (MAPK MPK11 as components of a small protein network nucleated by DBP1, in which GRF6 stability is modulated by MPK11 through phosphorylation, while DBP1 in turn negatively regulates MPK11 activity. Interestingly, grf6 and mpk11 loss-of-function mutants showed altered response to infection by the potyvirus Plum pox virus (PPV, and the described molecular mechanism controlling GRF6 stability was recapitulated upon PPV infection. These results not only contribute to a better knowledge of the biology of DBP factors, but also of MAPK signalling in plants, with the identification of GRF6 as a likely MPK11 substrate and of DBP1 as a protein phosphatase regulating MPK11 activity, and unveils the implication of this protein module in the response to PPV infection in Arabidopsis.

Functionally Similar WRKY Proteins Regulate Vacuolar Acidification in Petunia and Hair Development in Arabidopsis

NARCIS (Netherlands)

Verweij, W.; Spelt, C.E.; Bliek, M.; de Vries, M.; Wit, N.; Faraco, M.; Koes, R.; Quattrocchio, F.

2016-01-01

The WD40 proteins ANTHOCYANIN11 (AN11) from petunia (Petunia hybrida) and TRANSPARENT TESTA GLABRA1 (TTG1) fromArabidopsis thalianaand associated basic helix-loop-helix (bHLH) and MYB transcription factors activate a variety of differentiation processes. In petunia petals, AN11 and the bHLH protein

Genome-wide identification and expression analysis of the mitogen-activated protein kinase gene family in cassava

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Yan Yan

2016-08-01

Full Text Available Mitogen-activated protein kinases (MAPKs play central roles in plant developmental processes, hormone signaling transduction, and responses to abiotic stress. However, no data are currently available about the MAPK family in cassava, an important tropical crop. Herein, 21 MeMAPK genes were identified from cassava. Phylogenetic analysis indicated that MeMAPKs could be classified into four subfamilies. Gene structure analysis demonstrated that the number of introns in MeMAPK genes ranged from 1 to 10, suggesting large variation among cassava MAPK genes. Conserved motif analysis indicated that all MeMAPKs had typical protein kinase domains. Transcriptomic analysis suggested that MeMAPK genes showed differential expression patterns in distinct tissues and in response to drought stress between wild subspecies and cultivated varieties. Interaction networks and co-expression analyses revealed that crucial pathways controlled by MeMAPK networks may be involved in the differential response to drought stress in different accessions of cassava. Expression of nine selected MAPK genes showed that these genes could comprehensively respond to osmotic, salt, cold, oxidative stressors, and abscisic acid (ABA signaling. These findings yield new insights into the transcriptional control of MAPK gene expression, provide an improved understanding of abiotic stress responses and signaling transduction in cassava, and lead to potential applications in the genetic improvement of cassava cultivars.

Investigating the Role of the Arabidopsis Homologue of the Human G3BP in RNA Metabolism, Cellular Stress Responses and Innate Immunity

KAUST Repository

Abulfaraj, Aala A.

2018-04-01

Mitogen-activated protein kinases (MAPKs) belong to the most conserved signaling pathways and are found in all eukaryotes, including humans where they play important roles in various diseases and cancer. Stimulation of this signal transduction pathway by microbe-associated molecular patterns (MAMP) results in a multitude of events to regulate innate immune responses in Arabidopsis thaliana stimulating large-scale changes in gene expression. Starting from a phosphoproteomic screen in Arabidopsis thaliana wild type and mpk3, mpk4 and mpk6 mutants following microbe-associated molecular pattern (MAMP) treatment, several novel chromatin-associated proteins were identified that are differentially phosphorylated by stress-induced protein kinases. Arabidopsis Ras GTPase-activating protein SH3-domain-binding protein (AtG3BP-1) is a downstream putative substrate of the MAMP-stimulated MAPK pathway that is phosphorylated by MPK3, 4 and 6 in in vitro kinase assays. AtG3BP1 belongs to a highly conserved family of RNA-binding proteins in eukaryotes that link kinase receptormediated signaling to RNA metabolism. Here, we report the characterization of the Arabidopsis homolog of human G3BP1 in plant innate immunity. AtG3BP1 negatively regulates plant immunity and defense immune responses. Atg3bp1 mutant lines show constitutive stomata closure, expression of a number of key defense marker genes, and accumulate salicylic acid but not jasmonic acid. Furthermore, Atg3bp1 plants exhibit enhanced resistance to the biotrophic pathogen Pseudomonas syringae pv. tomato. Pathogen resistance was mediated by stomatal and apoplastic immunity in Atg3bp1. More generally, our data reinforce that AtG3BP1 is a key mediator of plant defense responses and transient expression of AtG3BP1 delivered striking disease resistance in the absence of yield penalty, highlighting a potential application of this gene in crop protection.

Expression of the Grb2-related protein of the lymphoid system in B cell subsets enhances B cell antigen receptor signaling through mitogen-activated protein kinase pathways.

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Yankee, Thomas M; Solow, Sasha A; Draves, Kevin D; Clark, Edward A

2003-01-01

Adapter proteins play a critical role in regulating signals triggered by Ag receptor cross-linking. These small molecules link receptor proximal events with downstream signaling pathways. In this study, we explore the expression and function of the Grb2-related protein of the lymphoid system (GrpL)/Grb2-related adaptor downstream of Shc adapter protein in human B cells. GrpL is expressed in naive B cells and is down-regulated following B cell Ag receptor ligation. By contrast, germinal center and memory B cells express little or no GrpL. Using human B cell lines, we detected constitutive interactions between GrpL and B cell linker protein, Src homology (SH)2 domain-containing leukocyte protein of 76 kDa, hemopoietic progenitor kinase 1, and c-Cbl. The N-terminal SH3 domain of GrpL binds c-Cbl while the C-terminal SH3 domain binds B cell linker protein and SH2 domain-containing leukocyte protein of 76 kDa. Exogenous expression of GrpL in a GrpL-negative B cell line leads to enhanced Ag receptor-induced extracellular signal-related kinase and p38 mitogen-activated protein kinase phosphorylation. Thus, GrpL expression in human B cell subsets appears to regulate Ag receptor-mediated signaling events.

Parathyroid mitogenic activity in plasma from patients with familial multiple endocrine neoplasia type 1

International Nuclear Information System (INIS)

Brandi, M.L.; Aurbach, G.D.; Fitzpatrick, L.A.; Quarto, R.; Spiegel, A.M.; Bliziotes, M.M.; Norton, J.A.; Doppman, J.L.; Marx, S.J.

1986-01-01

Hyperplasia of the parathyroid glands is a central feature of familial multiple endocrine neoplasia type 1. We used cultured bovine parathyroid cells to test for mitogenic activity in plasma from patients with this disorder. Normal plasma stimulated [ 3 H]thymidine incorporation, on the average, to the same extent as it was stimulated in a plasma-free control culture. This contrasted with the results of the tests with plasma from patients with familial multiple endocrine neoplasia type 1, in which parathyroid mitogenic activity increased 2400 percent over the control value (P less than 0.001). Plasma from these patients also stimulated the proliferation of bovine parathyroid cells in culture, whereas plasma from normal subjects inhibited it. Parathyroid mitogenic activity in plasma from the patients with familial multiple endocrine neoplasia type 1 was greater than that in plasma from patients with various other disorders, including sporadic primary hyperparathyroidism (with adenoma, hyperplasia, or cancer of the parathyroid), sporadic primary hypergastrinemia, sporadic pituitary tumor, familial hypocalciuric hypercalcemia, and multiple endocrine neoplasia type 2 (P less than 0.05). Parathyroid mitogenic activity in the plasma of patients with familial multiple endocrine neoplasia type 1 persisted for up to four years after total parathyroidectomy. The plasma also had far more mitogenic activity in cultures of parathyroid cells than did optimal concentrations of known growth factors or of any parathyroid secretagogue. This mitogenic activity had an apparent molecular weight of 50,000 to 55,000. We conclude that primary hyperparathyroidism in familial multiple endocrine neoplasia type 1 may have a humoral cause

Inhibition of gap-junctional intercellular communication and activation of mitogen-activated protein kinases by cyanobacterial extracts--indications of novel tumor-promoting cyanotoxins?

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Bláha, Ludĕk; Babica, Pavel; Hilscherová, Klára; Upham, Brad L

2010-01-01

Toxicity and liver tumor promotion of cyanotoxins microcystins have been extensively studied. However, recent studies document that other metabolites present in the complex cyanobacterial water blooms may also have adverse health effects. In this study we used rat liver epithelial stem-like cells (WB-F344) to examine the effects of cyanobacterial extracts on two established markers of tumor promotion, inhibition of gap-junctional intercellular communication (GJIC) and activation of mitogen-activated protein kinases (MAPKs) - ERK1/2. Extracts of cyanobacteria (laboratory cultures of Microcystis aeruginosa and Aphanizomenon flos-aquae and water blooms dominated by these species) inhibited GJIC and activated MAPKs in a dose-dependent manner (effective concentrations ranging 0.5-5mgd.w./mL). Effects were independent of the microcystin content and the strongest responses were elicited by the extracts of Aphanizomenon sp. Neither pure microcystin-LR nor cylindrospermopsin inhibited GJIC or activated MAPKs. Modulations of GJIC and MAPKs appeared to be specific to cyanobacterial extracts since extracts from green alga Chlamydomonas reinhardtii, heterotrophic bacterium Klebsiella terrigena, and isolated bacterial lipopolysaccharides had no comparable effects. Our study provides the first evidence on the existence of unknown cyanobacterial toxic metabolites that affect in vitro biomarkers of tumor promotion, i.e. inhibition of GJIC and activation of MAPKs.

Modulation of mitogen-activated protein kinase-activated protein kinase 3 by hepatitis C virus core protein

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Ngo, HT; Pham, Long; Kim, JW

2013-01-01

Hepatitis C virus (HCV) is highly dependent on cellular proteins for its own propagation. In order to identify the cellular factors involved in HCV propagation, we performed protein microarray assays using the HCV core protein as a probe. Of ~9,000 host proteins immobilized in a microarray...... inducers. Binding of HCV core to MAPKAPK3 was confirmed by in vitro pulldown assay and further verified by coimmunoprecipitation assay. HCV core protein interacted with MAPKAPK3 through amino acid residues 41 to 75 of core and the N-terminal half of kinase domain of MAPKAPK3. In addition, both RNA...... increased HCV IRES-mediated translation and MAPKAPK3-dependent HCV IRES activity was further increased by core protein. These data suggest that HCV core may modulate MAPKAPK3 to facilitate its own propagation....

A chimeric protein of aluminum-activated malate transporter generated from wheat and Arabidopsis shows enhanced response to trivalent cations.

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Sasaki, Takayuki; Tsuchiya, Yoshiyuki; Ariyoshi, Michiyo; Ryan, Peter R; Yamamoto, Yoko

2016-07-01

TaALMT1 from wheat (Triticum aestivum) and AtALMT1 from Arabidopsis thaliana encode aluminum (Al)-activated malate transporters, which confer acid-soil tolerance by releasing malate from roots. Chimeric proteins from TaALMT1 and AtALMT1 (Ta::At, At::Ta) were previously analyzed in Xenopus laevis oocytes. Those studies showed that Al could activate malate efflux from the Ta::At chimera but not from At::Ta. Here, functions of TaALMT1, AtALMT1 and the chimeric protein Ta::At were compared in cultured tobacco BY-2 cells. We focused on the sensitivity and specificity of their activation by trivalent cations. The activation of malate efflux by Al was at least two-fold greater in the chimera than the native proteins. All proteins were also activated by lanthanides (erbium, ytterbium, gadolinium, and lanthanum), but the chimera again released more malate than TaALMT1 or AtALMT1. In Xenopus oocytes, Al, ytterbium, and erbium activated inward currents from the native TaALMT1 and the chimeric protein, but gadolinium only activated currents from the chimera. Lanthanum inhibited currents from both proteins. These results demonstrated that function of the chimera protein was altered compared to the native proteins and was more responsive to a range of trivalent cations when expressed in plant cells. Copyright © 2016 Elsevier B.V. All rights reserved.

The yeast mitogen-activated protein kinase Slt2 is involved in the cellular response to genotoxic stress

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Soriano-Carot María

2012-02-01

Full Text Available Abstract Background The maintenance of genomic integrity is essential for cell viability. Complex signalling pathways (DNA integrity checkpoints mediate the response to genotoxic stresses. Identifying new functions involved in the cellular response to DNA-damage is crucial. The Saccharomyces cerevisiae SLT2 gene encodes a member of the mitogen-activated protein kinase (MAPK cascade whose main function is the maintenance of the cell wall integrity. However, different observations suggest that SLT2 may also have a role related to DNA metabolism. Results This work consisted in a comprehensive study to connect the Slt2 protein to genome integrity maintenance in response to genotoxic stresses. The slt2 mutant strain was hypersensitive to a variety of genotoxic treatments, including incubation with hydroxyurea (HU, methylmetanosulfonate (MMS, phleomycin or UV irradiation. Furthermore, Slt2 was activated by all these treatments, which suggests that Slt2 plays a central role in the cellular response to genotoxic stresses. Activation of Slt2 was not dependent on the DNA integrity checkpoint. For MMS and UV, Slt2 activation required progression through the cell cycle. In contrast, HU also activated Slt2 in nocodazol-arrested cells, which suggests that Slt2 may respond to dNTP pools alterations. However, neither the protein level of the distinct ribonucleotide reductase subunits nor the dNTP pools were affected in a slt2 mutant strain. An analysis of the checkpoint function revealed that Slt2 was not required for either cell cycle arrest or the activation of the Rad53 checkpoint kinase in response to DNA damage. However, slt2 mutant cells showed an elongated bud and partially impaired Swe1 degradation after replicative stress, indicating that Slt2 could contribute, in parallel with Rad53, to bud morphogenesis control after genotoxic stresses. Conclusions Slt2 is activated by several genotoxic treatments and is required to properly cope with DNA damage. Slt

p38 Mitogen Activated Protein Kinase (MAPK): A New Therapeutic Target for Reducing the Risk of Adverse Pregnancy Outcomes

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Menon, Ramkumar; Papaconstantinou, John

2016-01-01

Introduction Spontaneous preterm birth (PTB) and preterm premature rupture of the membranes (pPROM) remain as a major clinical and therapeutic problem for intervention and management. Current strategies, based on our knowledge of pathways of preterm labor, have only been effective, in part, due to major gaps in our existing knowledge of risks and risk specific pathways. Areas covered Recent literature has identified physiologic aging of fetal tissues as a potential mechanistic feature of normal parturition. This process is affected by telomere dependent and p38 mitogen activated protein kinase (MAPK) induced senescence activation. Pregnancy associated risk factors can cause pathologic activation of this pathway that can cause oxidative stress induced p38 MAPK activation leading to senescence and premature aging of fetal tissues. Premature aging is associated with sterile inflammation capable of triggering preterm labor or preterm premature rupture of membranes. Preterm activation of p38MAPK can be considered as a key contributor to adverse pregnancies. Expert Opinion This review considers p38MAPK activation as a potential target for therapeutic interventions to prevent adverse pregnancy outcomes mediated by stress factors. In this review, we propose multiple strategies to prevent p38MAPK activation and its functional effects. PMID:27459026

Fisetin Ameliorated Photodamage by Suppressing the Mitogen-Activated Protein Kinase/Matrix Metalloproteinase Pathway and Nuclear Factor-κB Pathways.

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Chiang, Hsiu-Mei; Chan, Shih-Yun; Chu, Yin; Wen, Kuo-Ching

2015-05-13

Ultraviolet (UV) irradiation is one of the most important extrinsic factors contributing to skin photodamage. After UV irradiation, a series of signal transductions in the skin will be activated, leading to inflammatory response and photoaged skin. In this study, fisetin, a flavonol that exists in fruits and vegetables, was investigated for its photoprotective effects. The results revealed that 5-25 μM fisetin inhibits cyclooxygenase-2 (COX-2) and matrix metalloproteinase (MMP)-1, MMP-3, MMP-9 expression induced by ultraviolet B (UVB) irradiation in human skin fibroblasts. In addition, fisetin suppressed UVB-induced collagen degradation. With regard to its effect on upper-stream signal transduction, we found that fisetin reduced the expression of ultraviolet (UV)-induced ERK, JNK, and p38 phosphorylation in the mitogen-activated protein kinase (MAP kinase) pathway. Furthermore, fisetin reduced inhibitor κB (IκB) degradation and increased the amount of p65, which is a major subunit of nuclear factor-κB (NF-κB), in cytoplasm. It also suppressed NF-κB translocated to the nucleus and inhibited cAMP response element-binding protein (CREB) Ser-133 phosphorylation level in the phosphoinositide 3-kinase/protein kinase B/CREB (PI3K/AKT/CREB) pathway. Finally, fisetin inhibited UV-induced intracellular reactive oxygen species (ROS), prostaglandin E2 (PGE2), and nitric oxide (NO) generation. The mentioned effects and mechanisms suggest that fisetin can be used in the development of photoprotective agents.

Identification and molecular properties of SUMO-binding proteins in arabidopsis

KAUST Repository

Park, Hyeongcheol; Choi, Wonkyun; Park, Heejin; Cheong, Misun; Koo, Yoonduck; Shin, Gilok; Chung, Woosik; Kim, Woeyeon; Kim, Mingab; Bressan, Ray Anthony; Bohnert, Hans Jü rgen; Lee, Sangyeol; Yun, Daejin

2011-01-01

in Arabidopsis and to probe for biological functions of SUMO proteins, we constructed 6xHis-3xFLAG fused AtSUMO1 (HFAtSUMO1) controlled by the CaMV35S promoter for transformation into Arabidopsis Col-0. After heat treatment, an increased sumoylation pattern

Predicting protein-protein interactions in Arabidopsis thaliana through integration of orthology, gene ontology and co-expression

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Vandepoele Klaas

2009-06-01

Full Text Available Abstract Background Large-scale identification of the interrelationships between different components of the cell, such as the interactions between proteins, has recently gained great interest. However, unraveling large-scale protein-protein interaction maps is laborious and expensive. Moreover, assessing the reliability of the interactions can be cumbersome. Results In this study, we have developed a computational method that exploits the existing knowledge on protein-protein interactions in diverse species through orthologous relations on the one hand, and functional association data on the other hand to predict and filter protein-protein interactions in Arabidopsis thaliana. A highly reliable set of protein-protein interactions is predicted through this integrative approach making use of existing protein-protein interaction data from yeast, human, C. elegans and D. melanogaster. Localization, biological process, and co-expression data are used as powerful indicators for protein-protein interactions. The functional repertoire of the identified interactome reveals interactions between proteins functioning in well-conserved as well as plant-specific biological processes. We observe that although common mechanisms (e.g. actin polymerization and components (e.g. ARPs, actin-related proteins exist between different lineages, they are active in specific processes such as growth, cancer metastasis and trichome development in yeast, human and Arabidopsis, respectively. Conclusion We conclude that the integration of orthology with functional association data is adequate to predict protein-protein interactions. Through this approach, a high number of novel protein-protein interactions with diverse biological roles is discovered. Overall, we have predicted a reliable set of protein-protein interactions suitable for further computational as well as experimental analyses.

Requirement for Pectin Methyl Esterase and Preference for Fragmented over Native Pectins for Wall-associated Kinase-activated, EDS1/PAD4-dependent Stress Response in Arabidopsis*

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Kohorn, Bruce D.; Kohorn, Susan L.; Saba, Nicholas J.; Martinez, Victoriano Meco

2014-01-01

The wall-associated kinases (WAKs) have a cytoplasmic protein kinase domain that spans the plasma membrane and binds pectin in the extracellular matrix of plants. WAKs are required for cell expansion during Arabidopsis seedling development but are also an integral part of the response to pathogens and stress that present oligogalacturonides (OGs), which subsequently bind to WAKs and activate a MPK6 (mitogen-activated protein kinase)-dependent pathway. It was unclear how WAKs distinguish native pectin polymers and OGs to activate one or the other of these two pathways. A dominant allele of WAK2 constitutively activates the stress response, and we show here that the effect is dependent upon EDS1 and PAD4, transcriptional activators involved in the pathogen response. Moreover, the WAK2 dominant allele is suppressed by a null allele of a pectin methyl esterase (PME3) whose activity normally leads to cross-linking of pectins in the cell wall. Although OGs activate a transcriptional response in wild type, the response is enhanced in a pme3/pme3 null, consistent with a competition by OG and native polymers for activation of WAKs. This provides a plausible mechanism for WAKs to distinguish an expansion from a stress pathway. PMID:24855660

Requirement for pectin methyl esterase and preference for fragmented over native pectins for wall-associated kinase-activated, EDS1/PAD4-dependent stress response in Arabidopsis.

Science.gov (United States)

Kohorn, Bruce D; Kohorn, Susan L; Saba, Nicholas J; Martinez, Victoriano Meco

2014-07-04

The wall-associated kinases (WAKs) have a cytoplasmic protein kinase domain that spans the plasma membrane and binds pectin in the extracellular matrix of plants. WAKs are required for cell expansion during Arabidopsis seedling development but are also an integral part of the response to pathogens and stress that present oligogalacturonides (OGs), which subsequently bind to WAKs and activate a MPK6 (mitogen-activated protein kinase)-dependent pathway. It was unclear how WAKs distinguish native pectin polymers and OGs to activate one or the other of these two pathways. A dominant allele of WAK2 constitutively activates the stress response, and we show here that the effect is dependent upon EDS1 and PAD4, transcriptional activators involved in the pathogen response. Moreover, the WAK2 dominant allele is suppressed by a null allele of a pectin methyl esterase (PME3) whose activity normally leads to cross-linking of pectins in the cell wall. Although OGs activate a transcriptional response in wild type, the response is enhanced in a pme3/pme3 null, consistent with a competition by OG and native polymers for activation of WAKs. This provides a plausible mechanism for WAKs to distinguish an expansion from a stress pathway. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Genome-wide identification of mitogen-activated protein kinase gene family in Gossypium raimondii and the function of their corresponding orthologs in tetraploid cultivated cotton.

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Zhang, Xueying; Wang, Liman; Xu, Xiaoyang; Cai, Caiping; Guo, Wangzhen

2014-12-10

Mitogen-activated protein kinase (MAPK) cascades play a crucial role in plant growth and development as well as biotic and abiotic stress responses. Knowledge about the MAPK gene family in cotton is limited, and systematic investigation of MAPK family proteins has not been reported. By performing a bioinformatics homology search, we identified 28 putative MAPK genes in the Gossypium raimondii genome. These MAPK members were anchored onto 11 chromosomes in G. raimondii, with uneven distribution. Phylogenetic analysis showed that the MAPK candidates could be classified into the four known A, B, C and D groups, with more MAPKs containing the TEY phosphorylation site (18 members) than the TDY motif (10 members). Furthermore, 21 cDNA sequences of MAPKs with complete open reading frames (ORFs) were identified in G. hirsutum via PCR-based approaches, including 13 novel MAPKs and eight with homologs reported previously in tetraploid cotton. The expression patterns of 23 MAPK genes reveal their important roles in diverse functions in cotton, in both various developmental stages of vegetative and reproductive growth and in the stress response. Using a reverse genetics approach based on tobacco rattle virus-induced gene silencing (TRV-VIGS), we further verified that MPK9, MPK13 and MPK25 confer resistance to defoliating isolates of Verticillium dahliae in cotton. Silencing of MPK9, MPK13 and MPK25 can significantly enhance cotton susceptibility to this pathogen. This study presents a comprehensive identification of 28 mitogen-activated protein kinase genes in G. raimondii. Their phylogenetic relationships, transcript expression patterns and responses to various stressors were verified. This study provides the first systematic analysis of MAPKs in cotton, improving our understanding of defense responses in general and laying the foundation for future crop improvement using MAPKs.

Immunomodulatory Activity of Ganoderma atrum Polysaccharide on Purified T Lymphocytes through Ca2+/CaN and Mitogen-Activated Protein Kinase Pathway Based on RNA Sequencing.

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Xiang, Quan-Dan; Yu, Qiang; Wang, Hui; Zhao, Ming-Ming; Liu, Shi-Yu; Nie, Shao-Ping; Xie, Ming-Yong

2017-07-05

Our previous study has demonstrated that Ganoderma atrum polysaccharide (PSG-1) has immunomodulatory activity on spleen lymphocytes. However, how PSG-1 exerts its effect on purified lymphocytes is still obscure. Thus, this study aimed to investigate the immunomodulatory activity of PSG-1 on purified T lymphocytes and further elucidate the underlying mechanism based on RNA sequencing (RNA-seq). Our results showed that PSG-1 promoted T lymphocytes proliferation and increased the production of IL-2, IFN-γ, and IL-12. Meanwhile, RNA-seq analysis found 394 differentially expressed genes. KEGG pathway analysis identified 20 significant canonical pathways and seven biological functions. Furthermore, PSG-1 elevated intracellular Ca 2+ concentration and calcineurin (CaN) activity and raised the p-ERK, p-JNK, and p-p38 expression levels. T lymphocytes proliferation and the production of IL-2, IFN-γ, and IL-12 were decreased by the inhibitors of calcium channel and mitogen-activated protein kinases (MAPKs). These results indicated that PSG-1 possesses immunomodulatory activity on purified T lymphocytes, in which Ca 2+ /CaN and MAPK pathways play essential roles.

Phylogeny-dominant classification of J-proteins in Arabidopsis thaliana and Brassica oleracea.

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Zhang, Bin; Qiu, Han-Lin; Qu, Dong-Hai; Ruan, Ying; Chen, Dong-Hong

2018-04-05

Hsp40s or DnaJ/J-proteins are evolutionarily conserved in all organisms as co-chaperones of molecular chaperone HSP70s that mainly participate in maintaining cellular protein homeostasis, such as protein folding, assembly, stabilization, and translocation under normal conditions as well as refolding and degradation under environmental stresses. It has been reported that Arabidopsis J-proteins are classified into four classes (types A-D) according to domain organization, but their phylogenetic relationships are unknown. Here, we identified 129 J-proteins in the world-wide popular vegetable Brassica oleracea, a close relative of the model plant Arabidopsis, and also revised the information of Arabidopsis J-proteins based on the latest online bioresources. According to phylogenetic analysis with domain organization and gene structure as references, the J-proteins from Arabidopsis and B. oleracea were classified into 15 main clades (I-XV) separated by a number of undefined small branches with remote relationship. Based on the number of members, they respectively belong to multigene clades, oligo-gene clades, and mono-gene clades. The J-protein genes from different clades may function together or separately to constitute a complicated regulatory network. This study provides a constructive viewpoint for J-protein classification and an informative platform for further functional dissection and resistant genes discovery related to genetic improvement of crop plants.

Moringa oleifera fruit induce apoptosis via reactive oxygen species-dependent activation of mitogen-activated protein kinases in human melanoma A2058 cells.

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Guon, Tae Eun; Chung, Ha Sook

2017-08-01

The present study was performed to determine the effect of Moringa oleifera fruit extract on the apoptosis of human melanoma A2058 cells. A2058 cells were treated for 72 h with Moringa oleifera fruit extract at 50-100 µg/ml, and cell viability with apoptotic changes was examined. The involvement of reactive oxygen species (ROS) and mitogen-activated protein kinases (MAPKs) was examined. It was revealed that Moringa oleifera fruit extract significantly inhibited the cell viability and promoted apoptosis of A2058 cells in a concentration-dependent manner. Moringa oleifera fruit extract-treated A2058 cells exhibited increased activities of cleaved caspase-9 and caspase-3. It also caused an enhancement of MAPK phosphorylation and ROS production. The pro-apoptotic activity of Moringa oleifera fruit extract was significantly reversed by pretreatment with the c-Jun N-terminal kinase (JNK) inhibitor SP600125, extracellular-signal-regulated kinase (ERK) inhibitor PD98058 or ROS inhibitor N-acetyl-L-cysteine. Taken together, Moringa oleifera fruit extract is effective in inducing mitochondrial apoptosis of A2058 cells, which is mediated through induction of ROS formation, and JNK and ERK activation. Moringa oleifera fruit extract may thus have therapeutic benefits for human melanoma A2058 cells.

Biochemical function of typical and variant Arabidopsis thaliana U-box E3 ubiquitin-protein ligases.

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Wiborg, Jakob; O'Shea, Charlotte; Skriver, Karen

2008-08-01

The variance of the U-box domain in 64 Arabidopsis thaliana (thale cress) E3s (ubiquitin-protein ligases) was used to examine the interactions between E3s and E2s (ubiquitin-conjugating enzymes). E2s and E3s are components of the ubiquitin protein degradation pathway. Seven U-box proteins were analysed for their ability to ubiquitinate proteins in vitro in co-operation with different E2s. All U-box domains exhibited ubiquitination activity and interacted productively with UBC4/5-type E2s. Three and four of the U-box domains mediated ubiquitin addition in the presence of UBC13 and UBC7 E2s respectively, but no productive interaction was observed with the UBC15 E2 tested. The activity of AtPUB54 [Arabidopsis thaliana (thale cress) plant U-box 54 protein] was dependent on Trp(266) in the E2-binding cleft, and the E2 selectivity was changed by substitution of this position. The function of the distant U-box protein, AtPUB49, representing a large family of eukaryotic proteins containing a U-box linked to a cyclophilin-like peptidyl-prolyl cis-trans isomerase domain, was characterized biochemically. AtPUB49 functioned both as a prolyl isomerase and a chaperone by catalysing cis-trans isomerization of peptidyl-prolyl bonds and dissolving protein aggregates. In conclusion, both typical and atypical Arabidopsis U-box proteins were active E3s. The overlap in the E3/E2 selectivity suggests that in vivo specificity is not determined only by the E3-E2 interactions, but also by other parameters, e.g. co-existence or interactions with additional domains. The biochemical functions of AtPUB49 suggest that the protein can be involved in folding or degradation of protein substrates. Similar functions can also be retained within a protein complex with separate chaperone and U-box proteins.

Enhanced expression of WD repeat-containing protein 35 (WDR35 stimulated by domoic acid in rat hippocampus: involvement of reactive oxygen species generation and p38 mitogen-activated protein kinase activation

Directory of Open Access Journals (Sweden)

Tsunekawa Koji

2013-01-01

Full Text Available Abstract Background Domoic acid (DA is an excitatory amino acid analogue of kainic acid (KA that acts via activation of glutamate receptors to elicit a rapid and potent excitotoxic response, resulting in neuronal cell death. Recently, DA was shown to elicit reactive oxygen species (ROS production and induce apoptosis accompanied by activation of p38 mitogen-activated protein kinase (MAPK in vitro. We have reported that WDR35, a WD-repeat protein, may mediate apoptosis in several animal models. In the present study, we administered DA to rats intraperitoneally, then used liquid chromatography/ion trap tandem mass spectrometry (LC-MS/MS to identify and quantify DA in the brains of the rats and performed histological examinations of the hippocampus. We further investigated the potential involvement of glutamate receptors, ROS, p38 MAPK, and WDR35 in DA-induced toxicity in vivo. Results Our results showed that intraperitoneally administered DA was present in the brain and induced neurodegenerative changes including apoptosis in the CA1 region of the hippocampus. DA also increased the expression of WDR35 mRNA and protein in a dose- and time-dependent manner in the hippocampus. In experiments using glutamate receptor antagonists, the AMPA/KA receptor antagonist NBQX significantly attenuated the DA-induced increase in WDR35 protein expression, but the NMDA receptor antagonist MK-801 did not. In addition, the radical scavenger edaravone significantly attenuated the DA-induced increase in WDR35 protein expression. Furthermore, NBQX and edaravone significantly attenuated the DA-induced increase in p38 MAPK phosphorylation. Conclusion In summary, our results indicated that DA activated AMPA/KA receptors and induced ROS production and p38 MAPK phosphorylation, resulting in an increase in the expression of WDR35 in vivo.

LSM Proteins Provide Accurate Splicing and Decay of Selected Transcripts to Ensure Normal Arabidopsis Development[W

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Perea-Resa, Carlos; Hernández-Verdeja, Tamara; López-Cobollo, Rosa; Castellano, María del Mar; Salinas, Julio

2012-01-01

In yeast and animals, SM-like (LSM) proteins typically exist as heptameric complexes and are involved in different aspects of RNA metabolism. Eight LSM proteins, LSM1 to 8, are highly conserved and form two distinct heteroheptameric complexes, LSM1-7 and LSM2-8,that function in mRNA decay and splicing, respectively. A search of the Arabidopsis thaliana genome identifies 11 genes encoding proteins related to the eight conserved LSMs, the genes encoding the putative LSM1, LSM3, and LSM6 proteins being duplicated. Here, we report the molecular and functional characterization of the Arabidopsis LSM gene family. Our results show that the 11 LSM genes are active and encode proteins that are also organized in two different heptameric complexes. The LSM1-7 complex is cytoplasmic and is involved in P-body formation and mRNA decay by promoting decapping. The LSM2-8 complex is nuclear and is required for precursor mRNA splicing through U6 small nuclear RNA stabilization. More importantly, our results also reveal that these complexes are essential for the correct turnover and splicing of selected development-related mRNAs and for the normal development of Arabidopsis. We propose that LSMs play a critical role in Arabidopsis development by ensuring the appropriate development-related gene expression through the regulation of mRNA splicing and decay. PMID:23221597

Arabidopsis Yak1 protein (AtYak1) is a dual specificity protein kinase

KAUST Repository

Kim, Dongjin; Ntui, Valentine Otang; Zhang, Nianshu; Xiong, Liming

2015-01-01

Yak1 is a member of dual-specificity Tyr phosphorylation-regulated kinases (DYRKs) that are evolutionarily conserved. The downstream targets of Yak1 and their functions are largely unknown. Here, a homologous protein AtYAK1 was identified in Arabidopsis thaliana and the phosphoprotein profiles of the wild type and an atyak1 mutant were compared on two-dimensional gel following Pro-Q Diamond phosphoprotein gel staining. Annexin1, Annexin2 and RBD were phosphorylated at serine/ threonine residues by the AtYak1 kinase. Annexin1, Annexin2 and Annexin4 were also phosphorylated at tyrosine residues. Our study demonstrated that AtYak1 is a dual specificity protein kinase in Arabidopsis that may regulate the phosphorylation status of the annexin family proteins.

Arabidopsis Yak1 protein (AtYak1) is a dual specificity protein kinase

KAUST Repository

Kim, Dongjin

2015-10-09

Yak1 is a member of dual-specificity Tyr phosphorylation-regulated kinases (DYRKs) that are evolutionarily conserved. The downstream targets of Yak1 and their functions are largely unknown. Here, a homologous protein AtYAK1 was identified in Arabidopsis thaliana and the phosphoprotein profiles of the wild type and an atyak1 mutant were compared on two-dimensional gel following Pro-Q Diamond phosphoprotein gel staining. Annexin1, Annexin2 and RBD were phosphorylated at serine/ threonine residues by the AtYak1 kinase. Annexin1, Annexin2 and Annexin4 were also phosphorylated at tyrosine residues. Our study demonstrated that AtYak1 is a dual specificity protein kinase in Arabidopsis that may regulate the phosphorylation status of the annexin family proteins.

Monitoring protein phosphorylation by acrylamide pendant Phos-Tag™ in various plants

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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.

The p38 mitogen activated protein kinase regulates β-amyloid protein internalization through the α7 nicotinic acetylcholine receptor in mouse brain.

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Ma, Kai-Ge; Lv, Jia; Yang, Wei-Na; Chang, Ke-Wei; Hu, Xiao-Dan; Shi, Li-Li; Zhai, Wan-Ying; Zong, Hang-Fan; Qian, Yi-Hua

2018-03-01

Alzheimer's disease (AD) is one of the most devastating neurodegenerative disorders. Intracellular β-amyloid protein (Aβ) is an early event in AD. It induces the formation of amyloid plaques and neuron damage. The α7 nicotinic acetylcholine receptor (α7nAChR) has been suggested to play an important role in Aβ caused cognition. It has high affinity with Aβ and could mediate Aβ internalization in vitro. However, whether in mouse brain the p38 MAPK signaling pathway is involved in the regulation of the α7nAChR mediated Aβ internalization and their role in mitochondria remains little known. Therefore, in this study, we revealed that Aβ is internalized by cholinergic and GABAergic neurons. The internalized Aβ were found deposits in lysosomes/endosomes and mitochondria. Aβ could form Aβ-α7nAChR complex with α7nAChR, activates the p38 mitogen activated protein kinase (MAPK). And the increasing of α7nAChR could in return mediate Aβ internalization in the cortex and hippocampus. In addition, by using the α7nAChR agonist PNU282987, the p38 phosphorylation level decreases, rescues the biochemical changes which are tightly associated with Aβ-induced apoptosis, such as Bcl2/Bax level, cytochrome c (Cyt c) release. Collectively, the p38 MAPK signaling pathway could regulate the α7nAChR-mediated internalization of Aβ. The activation of α7nAChR or the inhibition of p38 MAPK signaling pathway may be a beneficial therapy to AD. Copyright © 2017 Elsevier Inc. All rights reserved.

Detection of phosphorylated mitogen-activated protein kinase in the developing spinal cord of the mouse embryo

International Nuclear Information System (INIS)

Teraishi, Toshiya; Miura, Kenji

2011-01-01

Highlights: → We detected physiologically phosphorylated MAPKs in developing spinal cord. → We detected physiologically phosphorylated MAPKs by an improved method. → p-ERK1/2 and p-JNK1/2 were detected in the marginal layer and the dorsal horn. → p-ERK1/2 and p-JNK1/2 might play critical roles in the developing spinal cord. → Constructing phosphoprotein atlases will be possible if expanding this work. -- Abstract: Global understanding of the proteome is a major research topic. The comprehensive visualization of the distribution of proteins in vivo or the construction of in situ protein atlases may be a valuable strategy for proteomic researchers. Information about the distribution of various proteins under physiological and pathological conditions should be extremely valuable for the basic and clinical sciences. The mitogen-activated protein kinase (MAPK) cascade plays an essential role in intracellular signaling in organisms. This cascade also regulates biological processes involving development, differentiation, and proliferation. Phosphorylation and dephosphorylation are integral reactions in regulating the activity of MAPKs. Changes in the phosphorylation state of MAPKs are rapid and reversible; therefore, the localizations of physiologically phosphorylated MAPKs in vivo are difficult to accurately detect. Furthermore, phosphorylated MAPKs are likely to change phosphorylated states through commonly used experimental manipulations. In the present study, as a step toward the construction of in situ phosphoprotein atlases, we attempted to detect physiologically phosphorylated MAPKs in vivo in developing spinal cords of mice. We previously reported an improved immunohistochemical method for detecting unstable phosphorylated MAPKs. The distribution patterns of phosphorylated MAPKs in the spinal cords of embryonic mice from embryonic day 13 (E13) to E17 were observed with an improved immunohistochemical method. Phosphorylated extracellular signal

The functional interplay between protein kinase CK2 and CCA1 transcriptional activity is essential for clock temperature compensation in Arabidopsis.

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Sergi Portolés

2010-11-01

Full Text Available Circadian rhythms are daily biological oscillations driven by an endogenous mechanism known as circadian clock. The protein kinase CK2 is one of the few clock components that is evolutionary conserved among different taxonomic groups. CK2 regulates the stability and nuclear localization of essential clock proteins in mammals, fungi, and insects. Two CK2 regulatory subunits, CKB3 and CKB4, have been also linked with the Arabidopsis thaliana circadian system. However, the biological relevance and the precise mechanisms of CK2 function within the plant clockwork are not known. By using ChIP and Double-ChIP experiments together with in vivo luminescence assays at different temperatures, we were able to identify a temperature-dependent function for CK2 modulating circadian period length. Our study uncovers a previously unpredicted mechanism for CK2 antagonizing the key clock regulator CIRCADIAN CLOCK-ASSOCIATED 1 (CCA1. CK2 activity does not alter protein accumulation or subcellular localization but interferes with CCA1 binding affinity to the promoters of the oscillator genes. High temperatures enhance the CCA1 binding activity, which is precisely counterbalanced by the CK2 opposing function. Altering this balance by over-expression, mutation, or pharmacological inhibition affects the temperature compensation profile, providing a mechanism by which plants regulate circadian period at changing temperatures. Therefore, our study establishes a new model demonstrating that two opposing and temperature-dependent activities (CCA1-CK2 are essential for clock temperature compensation in Arabidopsis.

Involvement of mitogen-activated protein kinases and NFκB in LPS-induced CD40 expression on human monocytic cells

International Nuclear Information System (INIS)

Wu Weidong; Alexis, Neil E.; Chen Xian; Bromberg, Philip A.; Peden, David B.

2008-01-01

CD40 is a costimulatory molecule linking innate and adaptive immune responses to bacterial stimuli, as well as a critical regulator of functions of other costimulatory molecules. The mechanisms regulating lipopolysaccharide (LPS)-induced CD40 expression have not been adequately characterized in human monocytic cells. In this study we used a human monocytic cell line, THP-1, to investigate the possible mechanisms of CD40 expression following LPS exposure. Exposure to LPS resulted in a dose- and time-dependent increase in CD40 expression. Further studies using immunoblotting and pharmacological inhibitors revealed that mitogen-activated protein kinases (MAPKs) and NFκB were activated by LPS exposure and involved in LPS-induced CD40 expression. Activation of MAPKs was not responsible for LPS-induced NFκB activation. TLR4 was expressed on THP-1 cells and pretreatment of cells with a Toll-like receptor 4 (TLR4) neutralizing antibody (HTA125) significantly blunted LPS-induced MAPK and NFκB activation and ensuing CD40 expression. Additional studies with murine macrophages expressing wild type and mutated TLR4 showed that TLR4 was implicated in LPS-induced ERK and NFκB activation, and CD40 expression. Moreover, blockage of MAPK and NFκB activation inhibited LPS-induced TLR4 expression. In summary, LPS-induced CD40 expression in monocytic cells involves MAPKs and NFκB

Maize and Arabidopsis ARGOS Proteins Interact with Ethylene Receptor Signaling Complex, Supporting a Regulatory Role for ARGOS in Ethylene Signal Transduction[OPEN

Science.gov (United States)

Shi, Jinrui; Wang, Hongyu; Habben, Jeffrey E.

2016-01-01

The phytohormone ethylene regulates plant growth and development as well as plant response to environmental cues. ARGOS genes reduce plant sensitivity to ethylene when overexpressed in transgenic Arabidopsis (Arabidopsis thaliana) and maize (Zea mays). A previous genetic study suggested that the endoplasmic reticulum and Golgi-localized maize ARGOS1 targets the ethylene signal transduction components at or upstream of CONSTITUTIVE TRIPLE RESPONSE1, but the mechanism of ARGOS modulating ethylene signaling is unknown. Here, we demonstrate in Arabidopsis that ZmARGOS1, as well as the Arabidopsis ARGOS homolog ORGAN SIZE RELATED1, physically interacts with Arabidopsis REVERSION-TO-ETHYLENE SENSITIVITY1 (RTE1), an ethylene receptor interacting protein that regulates the activity of ETHYLENE RESPONSE1. The protein-protein interaction was also detected with the yeast split-ubiquitin two-hybrid system. Using the same yeast assay, we found that maize RTE1 homolog REVERSION-TO-ETHYLENE SENSITIVITY1 LIKE4 (ZmRTL4) and ZmRTL2 also interact with maize and Arabidopsis ARGOS proteins. Like AtRTE1 in Arabidopsis, ZmRTL4 and ZmRTL2 reduce ethylene responses when overexpressed in maize, indicating a similar mechanism for ARGOS regulating ethylene signaling in maize. A polypeptide fragment derived from ZmARGOS8, consisting of a Pro-rich motif flanked by two transmembrane helices that are conserved among members of the ARGOS family, can interact with AtRTE1 and maize RTL proteins in Arabidopsis. The conserved domain is necessary and sufficient to reduce ethylene sensitivity in Arabidopsis and maize. Overall, these results suggest a physical association between ARGOS and the ethylene receptor signaling complex via AtRTE1 and maize RTL proteins, supporting a role for ARGOS in regulating ethylene perception and the early steps of signal transduction in Arabidopsis and maize. PMID:27268962

Mitogen-activated protein kinase phosphatase-1 modulates regional effects of injurious mechanical ventilation in rodent lungs.

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Park, Moo Suk; He, Qianbin; Edwards, Michael G; Sergew, Amen; Riches, David W H; Albert, Richard K; Douglas, Ivor S

2012-07-01

Mechanical ventilation induces heterogeneous lung injury by mitogen-activated protein kinase (MAPK) and nuclear factor-κB. Mechanisms regulating regional injury and protective effects of prone positioning are unclear. To determine the key regulators of the lung regional protective effects of prone positioning in rodent lungs exposed to injurious ventilation. Adult rats were ventilated with high (18 ml/kg, positive end-expiratory pressure [PEEP] 0) or low Vt (6 ml/kg; PEEP 3 cm H(2)O; 3 h) in supine or prone position. Dorsal-caudal lung mRNA was analyzed by microarray and MAPK phosphatases (MKP)-1 quantitative polymerase chain reaction. MKP-1(-/-) or wild-type mice were ventilated with very high (24 ml/kg; PEEP 0) or low Vt (6-7 ml/kg; PEEP 3 cm H(2)O). The MKP-1 regulator PG490-88 (MRx-108; 0.75 mg/kg) or phosphate-buffered saline was administered preventilation. Injury was assessed by lung mechanics, bronchioalveolar lavage cell counts, protein content, and lung injury scoring. Immunoblotting for MKP-1, and IκBα and cytokine ELISAs were performed on lung lysates. Prone positioning was protective against injurious ventilation in rats. Expression profiling demonstrated MKP-1 20-fold higher in rats ventilated prone rather than supine and regional reduction in p38 and c-jun N-terminal kinase activation. MKP-1(-/-) mice experienced amplified injury. PG490-88 improved static lung compliance and injury scores, reduced bronchioalveolar lavage cell counts and cytokine levels, and induced MKP-1 and IκBα. Injurious ventilation induces MAPK in an MKP-1-dependent fashion. Prone positioning is protective and induces MKP-1. PG490-88 induced MKP-1 and was protective against high Vt in a nuclear factor-κB-dependent manner. MKP-1 is a potential target for modulating regional effects of injurious ventilation.

cDNA Library Screening Identifies Protein Interactors Potentially Involved in Non-telomeric Roles of Arabidopsis Telomerase

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Ladislav eDokládal

2015-11-01

Full Text Available Telomerase-reverse transcriptase (TERT plays an essential catalytic role in maintaining telomeres. However, in animal systems telomerase plays additional non-telomeric functional roles. We previously screened an Arabidopsis cDNA library for proteins that interact with the C-terminal extension (CTE TERT domain and identified a nuclear-localized protein that contains a RNA recognition motif (RRM. This RRM-protein forms homodimers in both plants and yeast. Mutation of the gene encoding the RRM-protein had no detectable effect on plant growth and development, nor did it affect telomerase activity or telomere length in vivo, suggesting a non-telomeric role for TERT/RRM-protein complexes. The gene encoding the RRM-protein is highly expressed in leaf and reproductive tissues. We further screened an Arabidopsis cDNA library for proteins that interact with the RRM-protein and identified five interactors. These proteins are involved in numerous non-telomere-associated cellular activities. In plants, the RRM-protein, both alone and in a complex with its interactors, localizes to nuclear speckles. Transcriptional analyses in wild-type and rrm mutant plants, as well as transcriptional co-analyses, suggest that TERT, the RRM-protein, and the RRM-protein interactors may play important roles in non-telomeric cellular functions.

Receptor trafficking via the perinuclear recycling compartment accompanied by cell division is necessary for permanent neurotensin cell sensitization and leads to chronic mitogen-activated protein kinase activation.

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Toy-Miou-Leong, Mireille; Cortes, Catherine Llorens; Beaudet, Alain; Rostène, William; Forgez, Patricia

2004-03-26

Most G protein-coupled receptors are internalized after interaction with their respective ligand, a process that subsequently contributes to cell desensitization, receptor endocytosis, trafficking, and finally cell resensitization. Although cellular mechanisms leading to cell desensitization have been widely studied, those responsible for cell resensitization are still poorly understood. We examined here the traffic of the high affinity neurotensin receptor (NT1 receptor) following prolonged exposure to high agonist concentration. Fluorescence and confocal microscopy of Chinese hamster ovary, human neuroblastoma (CHP 212), and murine neuroblastoma (N1E-115) cells expressing green fluorescent protein-tagged NT1 receptor revealed that under prolonged treatment with saturating concentrations of neurotensin (NT) agonist, NT1 receptor and NT transiently accumulated in the perinuclear recycling compartment (PNRC). During this cellular event, cell surface receptors remained markedly depleted as detected by both confocal microscopy and (125)I-NT binding assays. In dividing cells, we observed that following prolonged NT agonist stimulation, NT1 receptors were removed from the PNRC, accumulated in dispersed vesicles inside the cytoplasm, and subsequently reappeared at the cell surface. This NT binding recovery allowed for constant cell sensitization and led to a chronic activation of mitogen-activated protein kinases p42 and p44. Under these conditions, the constant activation of NT1 receptor generates an oncogenic regulation. These observations support the potent role for neuropeptides, such as NT, in cancer progression.

Involvement of YODA and mitogen activated protein kinase 6 in Arabidopsis post-embryogenic root development through auxin up-regulation and cell division plane orientation

Czech Academy of Sciences Publication Activity Database

Smékalová, V.; Luptovčiak, I.; Komis, G.; Šamajová, O.; Ovečka, M.; Doskočilová, A.; Takáč, T.; Vadovič, P.; Novák, Ondřej; Pechan, T.; Ziemann, A.; Košútová, P.; Šamaj, J.

2014-01-01

Roč. 203, č. 4 (2014), s. 1175-1193 ISSN 0028-646X R&D Projects: GA MŠk(CZ) LO1204 Institutional support: RVO:61389030 Keywords : Arabidopsis * cell division plane * MAP65-1 Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 7.672, year: 2014

Functional analysis of the Hikeshi-like protein and its interaction with HSP70 in Arabidopsis

Energy Technology Data Exchange (ETDEWEB)

Koizumi, Shinya; Ohama, Naohiko; Mizoi, Junya [Laboratory of Plant Molecular Physiology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657 (Japan); Shinozaki, Kazuo [RIKEN Plant Science Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama, Kanagawa 230-0045 (Japan); Yamaguchi-Shinozaki, Kazuko, E-mail: akys@mail.ecc.u-tokyo.ac.jp [Laboratory of Plant Molecular Physiology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657 (Japan)

2014-07-18

Highlights: • HKL, a Hikeshi homologous gene is identified in Arabidopsis. • HKL interacts with two HSP70 isoforms and regulates the subcellular localization of HSC70-1. • The two HSP70 translocate into nucleus in response to heat stress. • Overexpression of HKL confers thermotolerance in transgenic plants. - Abstract: Heat shock proteins (HSPs) refold damaged proteins and are an essential component of the heat shock response. Previously, the 70 kDa heat shock protein (HSP70) has been reported to translocate into the nucleus in a heat-dependent manner in many organisms. In humans, the heat-induced translocation of HSP70 requires the nuclear carrier protein Hikeshi. In the Arabidopsis genome, only one gene encodes a protein with high homology to Hikeshi, and we named this homolog Hikeshi-like (HKL) protein. In this study, we show that two Arabidopsis HSP70 isoforms accumulate in the nucleus in response to heat shock and that HKL interacts with these HSP70s. Our histochemical analysis revealed that HKL is predominantly expressed in meristematic tissues, suggesting the potential importance of HKL during cell division in Arabidopsis. In addition, we show that HKL regulates HSP70 localization, and HKL overexpression conferred thermotolerance to transgenic Arabidopsis plants. Our results suggest that HKL plays a positive role in the thermotolerance of Arabidopsis plants and cooperatively interacts with HSP70.

An Arabidopsis kinase cascade influences auxin-responsive cell expansion.

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Enders, Tara A; Frick, Elizabeth M; Strader, Lucia C

2017-10-01

Mitogen-activated protein kinase (MPK) cascades are conserved mechanisms of signal transduction across eukaryotes. Despite the importance of MPK proteins in signaling events, specific roles for many Arabidopsis MPK proteins remain unknown. Multiple studies have suggested roles for MPK signaling in a variety of auxin-related processes. To identify MPK proteins with roles in auxin response, we screened mpk insertional alleles and identified mpk1-1 as a mutant that displays hypersensitivity in auxin-responsive cell expansion assays. Further, mutants defective in the upstream MAP kinase kinase MKK3 also display hypersensitivity in auxin-responsive cell expansion assays, suggesting that this MPK cascade affects auxin-influenced cell expansion. We found that MPK1 interacts with and phosphorylates ROP BINDING PROTEIN KINASE 1 (RBK1), a protein kinase that interacts with members of the Rho-like GTPases from Plants (ROP) small GTPase family. Similar to mpk1-1 and mkk3-1 mutants, rbk1 insertional mutants display auxin hypersensitivity, consistent with a possible role for RBK1 downstream of MPK1 in influencing auxin-responsive cell expansion. We found that RBK1 directly phosphorylates ROP4 and ROP6, supporting the possibility that RBK1 effects on auxin-responsive cell expansion are mediated through phosphorylation-dependent modulation of ROP activity. Our data suggest a MKK3 • MPK1 • RBK1 phosphorylation cascade that may provide a dynamic module for altering cell expansion. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

AMP N1-Oxide, a Unique Compound of Royal Jelly, Induces Neurite Outgrowth from PC12 Vells via Signaling by Protein Kinase A Independent of that by Mitogen-Activated Protein Kinase

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Noriko Hattori

2010-01-01

Full Text Available Earlier we identified adenosine monophosphate (AMP N1-oxide as a unique compound of royal jelly (RJ that induces neurite outgrowth (neuritegenesis from cultured rat pheochromocytoma PC12 cells via the adenosine A2A receptor. Now, we found that AMP N1-oxide stimulated the phosphorylation of not only mitogen-activated protein kinase (MAPK but also that of cAMP/calcium-response element-binding protein (CREB in a dose-dependent manner. Inhibition of MAPK activation by a MEK inhibitor, PD98059, did not influence the AMP N1-oxide-induced neuritegenesis, whereas that of protein kinase A (PKA by a selective inhibitor, KT5720, significantly reduced neurite outgrowth. AMP N1-oxide also had the activity of suppressing the growth of PC12 cells, which correlated well with the neurite outgrowth-promoting activity. KT5720 restored the growth of AMP N1-oxide-treated PC12 cells. It is well known that nerve growth factor suppresses proliferation of PC12 cells before causing stimulation of neuronal differentiation. Thus, AMP N1-oxide elicited neuronal differentiation of PC12 cells, as evidenced by generation of neurites, and inhibited cell growth through adenosine A2A receptor-mediated PKA signaling, which may be responsible for characteristic actions of RJ.

The Salmonella effector protein SpvC, a phosphothreonine lyase is functional in plant cells

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Christina eNeumann

2014-10-01

Full Text Available Salmonella is one of the most prominent causes of food poisoning and growing evidence indicates that contaminated fruits and vegetables are an increasing concern for human health. Successful infection demands the suppression of the host immune system, which is often achieved via injection of bacterial effector proteins into host cells. In this report we present the function of Salmonella effector protein in plant cell, supporting the new concept of trans-kingdom competence of this bacterium. We screened a range of Salmonella Typhimurium effector proteins for interference with plant immunity. Among these, the phosphothreonine lyase SpvC attenuated the induction of immunity-related genes when present in plant cells. Using in vitro and in vivo systems we show that this effector protein interacts with and dephosphorylates activated Arabidopsis Mitogen-activated Protein Kinase 6 (MPK6, thereby inhibiting defense signaling. Moreover, the requirement of Salmonella SpvC was shown by the decreased proliferation of the ΔspvC mutant in Arabidopsis plants. These results suggest that some Salmonella effector proteins could have a conserved function during proliferation in different hosts. The fact that Salmonella and other Enterobacteriaceae use plants as hosts strongly suggests that plants represent a much larger reservoir for animal pathogens than so far estimated.

The Salmonella effector protein SpvC, a phosphothreonine lyase is functional in plant cells

KAUST Repository

Neumann, Christina

2014-10-17

Salmonella is one of the most prominent causes of food poisoning and growing evidence indicates that contaminated fruits and vegetables are an increasing concern for human health. Successful infection demands the suppression of the host immune system, which is often achieved via injection of bacterial effector proteins into host cells. In this report we present the function of Salmonella effector protein in plant cell, supporting the new concept of trans-kingdom competence of this bacterium. We screened a range of Salmonella Typhimurium effector proteins for interference with plant immunity. Among these, the phosphothreonine lyase SpvC attenuated the induction of immunity-related genes when present in plant cells. Using in vitro and in vivo systems we show that this effector protein interacts with and dephosphorylates activated Arabidopsis Mitogen-activated Protein Kinase 6 (MPK6), thereby inhibiting defense signaling. Moreover, the requirement of Salmonella SpvC was shown by the decreased proliferation of the ΔspvC mutant in Arabidopsis plants. These results suggest that some Salmonella effector proteins could have a conserved function during proliferation in different hosts. The fact that Salmonella and other Enterobacteriaceae use plants as hosts strongly suggests that plants represent a much larger reservoir for animal pathogens than so far estimated.

Novel Ribonuclease Activity Differs between Fibrillarins from Arabidopsis thaliana

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Ulises Rodriguez-Corona

2017-10-01

Full Text Available Fibrillarin is one of the most important nucleolar proteins that have been shown as essential for life. Fibrillarin localizes primarily at the periphery between fibrillar center and dense fibrillar component as well as in Cajal bodies. In most plants there are at least two different genes for fibrillarin. In Arabidopsis thaliana both genes show high level of expression in transcriptionally active cells. Here, we focus on two important differences between A. thaliana fibrillarins. First and most relevant is the enzymatic activity by AtFib2. The AtFib2 shows a novel ribonuclease activity that is not seen with AtFib1. Second is a difference in the ability to interact with phosphoinositides and phosphatidic acid between both proteins. We also show that the novel ribonuclease activity as well as the phospholipid binding region of fibrillarin is confine to the GAR domain. The ribonuclease activity of fibrillarin reveals in this study represents a new role for this protein in rRNA processing.

An early nodulin-like protein accumulates in the sieve element plasma membrane of Arabidopsis

DEFF Research Database (Denmark)

Khan, Junaid A.; Wang, Qi; Sjölund, Richard D.

2007-01-01

Membrane proteins within the sieve element-companion cell complex have essential roles in the physiological functioning of the phloem. The monoclonal antibody line RS6, selected from hybridomas raised against sieve elements isolated from California shield leaf (Streptanthus tortuosus; Brassicaceae...... was revealed by reverse transcription-PCR of Arabidopsis leaf RNA using degenerate primers to be an early nodulin (ENOD)-like protein that is encoded by the expressed gene At3g20570. Arabidopsis ENOD-like proteins are encoded by a multigene family composed of several types of structurally related phytocyanins...... from the precursor protein, resulting in a mature peptide of approximately 15 kD that is attached to the sieve element plasma membrane via a carboxy-terminal glycosylphosphatidylinositol membrane anchor. Many of the Arabidopsis ENOD-like proteins accumulate in gametophytic tissues, whereas in both...

The C-terminal domain of the Arabidopsis AtMBD7 protein confers strong chromatin binding activity

International Nuclear Information System (INIS)

Zemach, Assaf; Paul, Laju K.; Stambolsky, Perry; Efroni, Idan; Rotter, Varda; Grafi, Gideon

2009-01-01

The Arabidopsis MBD7 (AtMBD7) - a naturally occurring poly MBD protein - was previously found to be functional in binding methylated-CpG dinucleotides in vitro and localized to highly methylated chromocenters in vivo. Furthermore, AtMBD7 has significantly lower mobility within the nucleus conferred by cooperative activity of its three MBD motifs. Here we show that besides the MBD motifs, AtMBD7 possesses a strong chromatin binding domain located at its C-terminus designated sticky-C (StkC). Mutational analysis showed that a glutamic acid residue near the C-terminus is essential though not sufficient for the StkC function. Further analysis demonstrated that this motif can render nuclear proteins highly immobile both in plant and animal cells, without affecting their native subnuclear localization. Thus, the C-terminal, StkC motif plays an important role in fastening AtMBD7 to its chromosomal, CpG-methylated sites. It may be possible to utilize this motif for fastening nuclear proteins to their chromosomal sites both in plant and animal cells for research and gene therapy applications.

Lactococcus lactis, an alternative system for functional expression of peripheral and intrinsic Arabidopsis membrane proteins.

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Annie Frelet-Barrand

Full Text Available BACKGROUND: Despite their functional and biotechnological importance, the study of membrane proteins remains difficult due to their hydrophobicity and their low natural abundance in cells. Furthermore, into established heterologous systems, these proteins are frequently only produced at very low levels, toxic and mis- or unfolded. Lactococcus lactis, a gram-positive lactic bacterium, has been traditionally used in food fermentations. This expression system is also widely used in biotechnology for large-scale production of heterologous proteins. Various expression vectors, based either on constitutive or inducible promoters, are available for this system. While previously used to produce bacterial and eukaryotic membrane proteins, the ability of this system to produce plant membrane proteins was until now not tested. METHODOLOGY/PRINCIPAL FINDINGS: The aim of this work was to test the expression, in Lactococcus lactis, of either peripheral or intrinsic Arabidopsis membrane proteins that could not be produced, or in too low amount, using more classical heterologous expression systems. In an effort to easily transfer genes from Gateway-based Arabidopsis cDNA libraries to the L. lactis expression vector pNZ8148, we first established a cloning strategy compatible with Gateway entry vectors. Interestingly, the six tested Arabidopsis membrane proteins could be produced, in Lactococcus lactis, at levels compatible with further biochemical analyses. We then successfully developed solubilization and purification processes for three of these proteins. Finally, we questioned the functionality of a peripheral and an intrinsic membrane protein, and demonstrated that both proteins were active when produced in this system. CONCLUSIONS/SIGNIFICANCE: Altogether, these data suggest that Lactococcus lactis might be an attractive system for the efficient and functional production of difficult plant membrane proteins.

p38 mitogen-activated protein kinase is involved in arginase-II-mediated eNOS-uncoupling in obesity.

Science.gov (United States)

Yu, Yi; Rajapakse, Angana G; Montani, Jean-Pierre; Yang, Zhihong; Ming, Xiu-Fen

2014-07-18

Endothelial nitric oxide synthase (eNOS)-uncoupling links obesity-associated insulin resistance and type-II diabetes to the increased incidence of cardiovascular disease. Studies have indicated that increased arginase is involved in eNOS-uncoupling through competing with the substrate L-arginine. Given that arginase-II (Arg-II) exerts some of its biological functions through crosstalk with signal transduction pathways, and that p38 mitogen-activated protein kinase (p38mapk) is involved in eNOS-uncoupling, we investigated here whether p38mapk is involved in Arg-II-mediated eNOS-uncoupling in a high fat diet (HFD)-induced obesity mouse model. Obesity was induced in wild type (WT) and Arg-II-deficient (Arg-II(-/-)) mice on C57BL/6 J background by high-fat diet (HFD, 55% fat) for 14 weeks starting from age of 7 weeks. The entire aortas were isolated and subjected to 1) immunoblotting analysis of the protein level of eNOS, Arg-II and p38mapk activation; 2) arginase activity assay; 3) endothelium-dependent and independent vasomotor responses; 4) en face staining of superoxide anion and NO production with Dihydroethidium and 4,5-Diaminofluorescein Diacetate, respectively, to assess eNOS-uncoupling. To evaluate the role of p38mapk, isolated aortas were treated with p38mapk inhibitor SB203580 (10 μmol/L, 1 h) prior to the analysis. In addition, the role of p38mapk in Arg-II-induced eNOS-uncoupling was investigated in cultured human endothelial cells overexpressing Arg-II in the absence or presence of shRNA against p38mapk. HFD enhanced Arg-II expression/activity and p38mapk activity, which was associated with eNOS-uncoupling as revealed by decreased NO and enhanced L-NAME-inhibitable superoxide in aortas of WT obese mice. In accordance, WT obese mice revealed decreased endothelium-dependent relaxations to acetylcholine despite of higher eNOS protein level, whereas Arg-II(-/-) obese mice were protected from HFD-induced eNOS-uncoupling and endothelial dysfunction, which

Regulation of p53, nuclear factor κB and cyclooxygenase-2 expression by bromelain through targeting mitogen-activated protein kinase pathway in mouse skin

International Nuclear Information System (INIS)

Kalra, Neetu; Bhui, Kulpreet; Roy, Preeti; Srivastava, Smita; George, Jasmine; Prasad, Sahdeo; Shukla, Yogeshwer

2008-01-01

Bromelain is a pharmacologically active compound, present in stems and immature fruits of pineapples (Ananas cosmosus), which has been shown to have anti-edematous, anti-inflammatory, anti-thrombotic and anti-metastatic properties. In the present study, antitumorigenic activity of bromelain was recorded in 7,12-dimethylbenz(a)anthracene (DMBA)-initiated and 12-O-tetradecanoylphorbol-13-acetate (TPA)-promoted 2-stage mouse skin model. Results showed that bromelain application delayed the onset of tumorigenesis and reduced the cumulative number of tumors, tumor volume and the average number of tumors/mouse. To establish a cause and effect relationship, we targeted the proteins involved in the cell death pathway. Bromelain treatment resulted in upregulation of p53 and Bax and subsequent activation of caspase 3 and caspase 9 with concomitant decrease in antiapoptotic protein Bcl-2 in mouse skin. Since persistent induction of cyclooxygenase-2 (Cox-2) is frequently implicated in tumorigenesis and is regulated by nuclear factor-kappa B (NF-κB), we also investigated the effect of bromelain on Cox-2 and NF-κB expression. Results showed that bromelain application significantly inhibited Cox-2 and inactivated NF-κB by blocking phosphorylation and subsequent degradation of IκBα. In addition, bromelain treatment attenuated DMBA-TPA-induced phosphorylation of extracellular signal-regulated protein kinase (ERK1/2), mitogen-activated protein kinase (MAPK) and Akt. Taken together, we conclude that bromelain induces apoptosis-related proteins along with inhibition of NF-κB-driven Cox-2 expression by blocking the MAPK and Akt/protein kinase B signaling in DMBA-TPA-induced mouse skin tumors, which may account for its anti-tumorigenic effects

Amitriptyline induces early growth response-1 gene expression via ERK and JNK mitogen-activated protein kinase pathways in rat C6 glial cells.

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Chung, Eun Young; Shin, Soon Young; Lee, Young Han

2007-07-05

Astrocytes play important roles in guiding the construction of the nervous system, controlling extracellular ions and neurotransmitters, and regulating CNS synaptogenesis. Egr-1 is a transcription factor involved in neuronal differentiation and astrocyte cell proliferation. In this study, we investigated whether the tricyclic antidepressant (TCA) amitriptyline induces Egr-1 expression in astrocytes using rat C6 glioma cells as a model. We found that amitriptyline increased the expression of Egr-1 in a dose- and time-dependent manner. The amitriptyline-induced Egr-1 expression was mediated through serum response elements (SREs) in the Egr-1 promoter. SREs were activated by the Ets-domain transcription factor Elk-1 through the ERK and JNK mitogen-activated protein (MAP) kinase pathways. The inhibition of the ERK and JNK MAP kinase signals attenuated amitriptyline-induced transactivation of Gal4-Elk-1 and Egr-1 promoter activity. Our findings suggest that the induction of Egr-1 expression in astrocytes may be required to attain the therapeutic effects of antidepressant drugs.

Electrical stimulation with periodic alternating intervals stimulates neuronal cells to produce neurotrophins and cytokines through activation of mitogen-activated protein kinase pathways.

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Yamamoto, Kenta; Yamamoto, Toshiro; Honjo, Kenichi; Ichioka, Hiroaki; Oseko, Fumishige; Kishida, Tsunao; Mazda, Osam; Kanamura, Narisato

2015-12-01

Peripheral neuropathy is a representative complication of dental surgery. Electrical therapy, based on electrical stimulation with periodic alternating intervals (ES-PAI), may promote nerve regeneration after peripheral nerve injury in a non-invasive manner, potentially providing an effective therapy for neuropathy. This study aimed to analyze the molecular mechanisms underlying the nerve recovery stimulated by ES-PAI. In brief, ES-PAI was applied to a neuronal cell line, Neuro2A, at various intensities using the pulse generator apparatus, FREUDE. Cell viability, neurotrophin mRNA expression, and cytokine production were examined using a tetrazolium-based assay, real-time RT-PCR, and ELISA, respectively. Mitogen-activated protein kinase (MAPK) signaling was assessed using flow cytometry. It was found that ES-PAI increased the viability of cells and elevated expression of nerve growth factor (NGF) and neurotrophin-3 (NT-3); ESPAI also augmented vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) expression, which was restored by addition of p38 inhibitors. Phosphorylation of p38 and extracellular signal-regulated kinase 1/2 (ERK-1/2) was augmented by ES-PAI. Hence, ES-PAI may ameliorate peripheral neuropathy by promoting neuronal cell proliferation and production of neurogenic factors by activating p38 and ERK-1/2 pathways. © 2015 Eur J Oral Sci.

The RNA-binding protein repertoire of Arabidopsis thaliana

KAUST Repository

Marondedze, Claudius

2016-07-11

RNA-binding proteins (RBPs) have essential roles in determining the fate of RNA from synthesis to decay and have been studied on a protein-by-protein basis, or computationally based on a number of well-characterised RNA-binding domains. Recently, high-throughput methods enabled the capture of mammalian RNA-binding proteomes. To gain insight into the role of Arabidopsis thaliana RBPs at the systems level, we have employed interactome capture techniques using cells from different ecotypes grown in cultures and leaves. In vivo UV-crosslinking of RNA to RBPs, oligo(dT) capture and mass spectrometry yielded 1,145 different proteins including 550 RBPs that either belong to the functional category ‘RNA-binding’, have known RNA-binding domains or have orthologs identified in mammals, C. elegans, or S. cerevisiae in addition to 595 novel candidate RBPs. We noted specific subsets of RBPs in cultured cells and leaves and a comparison of Arabidopsis, mammalian, C. elegans, and S. cerevisiae RBPs reveals a common set of proteins with a role in intermediate metabolism, as well as distinct differences suggesting that RBPs are also species and tissue specific. This study provides a foundation for studies that will advance our understanding of the biological significance of RBPs in plant developmental and stimulus specific responses.

Scaffold Protein Ahk1, Which Associates with Hkr1, Sho1, Ste11, and Pbs2, Inhibits Cross Talk Signaling from the Hkr1 Osmosensor to the Kss1 Mitogen-Activated Protein Kinase

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Nishimura, Akiko; Yamamoto, Katsuyoshi; Oyama, Masaaki; Kozuka-Hata, Hiroko

2016-01-01

In the budding yeast Saccharomyces cerevisiae, osmostress activates the Hog1 mitogen-activated protein kinase (MAPK), which regulates diverse osmoadaptive responses. Hkr1 is a large, highly glycosylated, single-path transmembrane protein that is a putative osmosensor in one of the Hog1 upstream pathways termed the HKR1 subbranch. The extracellular region of Hkr1 contains both a positive and a negative regulatory domain. However, the function of the cytoplasmic domain of Hkr1 (Hkr1-cyto) is unknown. Here, using a mass spectrometric method, we identified a protein, termed Ahk1 (Associated with Hkr1), that binds to Hkr1-cyto. Deletion of the AHK1 gene (in the absence of other Hog1 upstream branches) only partially inhibited osmostress-induced Hog1 activation. In contrast, Hog1 could not be activated by constitutively active mutants of the Hog1 pathway signaling molecules Opy2 or Ste50 in ahk1Δ cells, whereas robust Hog1 activation occurred in AHK1+ cells. In addition to Hkr1-cyto binding, Ahk1 also bound to other signaling molecules in the HKR1 subbranch, including Sho1, Ste11, and Pbs2. Although osmotic stimulation of Hkr1 does not activate the Kss1 MAPK, deletion of AHK1 allowed Hkr1 to activate Kss1 by cross talk. Thus, Ahk1 is a scaffold protein in the HKR1 subbranch and prevents incorrect signal flow from Hkr1 to Kss1. PMID:26787842

Buddleja officinalis suppresses high glucose-induced vascular smooth muscle cell proliferation: role of mitogen-activated protein kinases, nuclear factor-kappaB and matrix metalloproteinases.

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Lee, Yun Jung; Kim, Jin Sook; Kang, Dae Gill; Lee, Ho Sub

2010-02-01

Diabetes mellitus is a well-established risk factor for vascular diseases caused by atherosclerosis. In the development of diabetic atherogenesis, vascular smooth muscle cell proliferation is recognized as a key event. Thus, we aimed to investigate whether an ethanol extract of Buddleja officinalis (EBO) suppresses high glucose-induced proliferation in primary cultured human aortic smooth muscle cells (HASMC). [(3)H]-thymidine incorporation revealed that incubation of HASMC with a high concentration of glucose (25 mmol/L) increased cell proliferation. The expression levels of cell cycle protein were also increased by treatment with high glucose concentration. Pretreatment of HASMC with EBO significantly attenuated the increase of high glucose-induced cell proliferation as well as p38 mitogen-activated protein kinases (MAPK) and JNK phosphorylation. EBO suppressed high glucose-induced matrix metalloproteinase (MMP)-9 activity in a dose-dependent manner. In addition, EBO suppressed nuclear factor-kappaB (NF-kappaB) nuclear translocation and transcriptional activity in high glucose conditions. Taken together, the present data suggest that EBO could suppress high glucose-induced atherosclerotic processes through inhibition of p38, JNK, NF-kappaB and MMP signal pathways in HASMC.

Purification and characterization of a mucin specific mycelial lectin from Aspergillus gorakhpurensis: application for mitogenic and antimicrobial activity.

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Ram Sarup Singh

Full Text Available Lectins are carbohydrate binding proteins or glycoproteins that bind reversibly to specific carbohydrates present on the apposing cells, which are responsible for their ability to agglutinate red blood cells, lymphocytes, fibroblasts, etc. Interest in lectins has been intensified due to their carbohydrate specificity as they can be valuable reagents for the investigation of cell surface sugars, purification and characterization of glycoproteins. The present study reports the purification, characterization and evaluation of mitogenic and antimicrobial potential of a mycelial lectin from Aspergillus gorakhpurensis.Affinity chromatography on mucin-sepharose column was carried out for purification of Aspergillus gorakhpurensis lectin. The lectin was characterized for physico-chemical parameters. Mitogenic potential of the lectin was evaluated against splenocytes of Swiss albino mice by MTT assay. Antimicrobial activity of the purified lectin has also been evaluated by disc diffusion assay.Single-step affinity purification resulted in 18.6-fold purification of the mycelial lectin. The molecular mass of the lectin was found to be 70 kDa and it was composed of two subunits of 34.8 kDa as determined by gel filtration chromatography, SDS-PAGE and MALDI-TOF analysis. pH optima of the lectin was found to be 6.5-9.5, while optimum temperature for lectin activity was 20-30 °C. Lectin was stable within a pH range of 7.0-10.5 and showed fair thermostability. EDTA did not affect lectin activity whereas it was found susceptible to the denaturants tested. MTT assay revealed strong mitogenic potential of A. gorakhpurensis lectin at a concentration upto 150 µg/mL. Antimicrobial activity assay showed its potent antibacterial activity against Bacillus cereus, Staphylococcous aureus and Escherichia coli and marginal antifungal activity against Saccharomyces cerevisiae.This is the first report on the mitogenic and antimicrobial potential of Aspergillus gorakhpurensis

Purification and characterization of a mucin specific mycelial lectin from Aspergillus gorakhpurensis: application for mitogenic and antimicrobial activity.

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Singh, Ram Sarup; Kaur, Hemant Preet; Singh, Jatinder

2014-01-01

Lectins are carbohydrate binding proteins or glycoproteins that bind reversibly to specific carbohydrates present on the apposing cells, which are responsible for their ability to agglutinate red blood cells, lymphocytes, fibroblasts, etc. Interest in lectins has been intensified due to their carbohydrate specificity as they can be valuable reagents for the investigation of cell surface sugars, purification and characterization of glycoproteins. The present study reports the purification, characterization and evaluation of mitogenic and antimicrobial potential of a mycelial lectin from Aspergillus gorakhpurensis. Affinity chromatography on mucin-sepharose column was carried out for purification of Aspergillus gorakhpurensis lectin. The lectin was characterized for physico-chemical parameters. Mitogenic potential of the lectin was evaluated against splenocytes of Swiss albino mice by MTT assay. Antimicrobial activity of the purified lectin has also been evaluated by disc diffusion assay. Single-step affinity purification resulted in 18.6-fold purification of the mycelial lectin. The molecular mass of the lectin was found to be 70 kDa and it was composed of two subunits of 34.8 kDa as determined by gel filtration chromatography, SDS-PAGE and MALDI-TOF analysis. pH optima of the lectin was found to be 6.5-9.5, while optimum temperature for lectin activity was 20-30 °C. Lectin was stable within a pH range of 7.0-10.5 and showed fair thermostability. EDTA did not affect lectin activity whereas it was found susceptible to the denaturants tested. MTT assay revealed strong mitogenic potential of A. gorakhpurensis lectin at a concentration upto 150 µg/mL. Antimicrobial activity assay showed its potent antibacterial activity against Bacillus cereus, Staphylococcous aureus and Escherichia coli and marginal antifungal activity against Saccharomyces cerevisiae. This is the first report on the mitogenic and antimicrobial potential of Aspergillus gorakhpurensis lectin. The

The Development of Protein Microarrays and Their Applications in DNA-Protein and Protein-Protein Interaction Analyses of Arabidopsis Transcription Factors

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Gong, Wei; He, Kun; Covington, Mike; Dinesh-Kumar, S. P.; Snyder, Michael; Harmer, Stacey L.; Zhu, Yu-Xian; Deng, Xing Wang

2009-01-01

We used our collection of Arabidopsis transcription factor (TF) ORFeome clones to construct protein microarrays containing as many as 802 TF proteins. These protein microarrays were used for both protein-DNA and protein-protein interaction analyses. For protein-DNA interaction studies, we examined AP2/ERF family TFs and their cognate cis-elements. By careful comparison of the DNA-binding specificity of 13 TFs on the protein microarray with previous non-microarray data, we showed that protein microarrays provide an efficient and high throughput tool for genome-wide analysis of TF-DNA interactions. This microarray protein-DNA interaction analysis allowed us to derive a comprehensive view of DNA-binding profiles of AP2/ERF family proteins in Arabidopsis. It also revealed four TFs that bound the EE (evening element) and had the expected phased gene expression under clock-regulation, thus providing a basis for further functional analysis of their roles in clock regulation of gene expression. We also developed procedures for detecting protein interactions using this TF protein microarray and discovered four novel partners that interact with HY5, which can be validated by yeast two-hybrid assays. Thus, plant TF protein microarrays offer an attractive high-throughput alternative to traditional techniques for TF functional characterization on a global scale. PMID:19802365

Membrane-localized extra-large G proteins and Gbg of the heterotrimeric G proteins form functional complexes engaged in plant immunity in Arabidopsis.

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Maruta, Natsumi; Trusov, Yuri; Brenya, Eric; Parekh, Urvi; Botella, José Ramón

2015-03-01

In animals, heterotrimeric G proteins, comprising Ga, Gb, and Gg subunits, are molecular switches whose function tightly depends on Ga and Gbg interaction. Intriguingly, in Arabidopsis (Arabidopsis thaliana), multiple defense responses involve Gbg, but not Ga. We report here that the Gbg dimer directly partners with extra-large G proteins (XLGs) to mediate plant immunity. Arabidopsis mutants deficient in XLGs, Gb, and Gg are similarly compromised in several pathogen defense responses, including disease development and production of reactive oxygen species. Genetic analysis of double, triple, and quadruple mutants confirmed that XLGs and Gbg functionally interact in the same defense signaling pathways. In addition, mutations in XLG2 suppressed the seedling lethal and cell death phenotypes of BRASSINOSTEROID INSENSITIVE1-associated receptor kinase1-interacting receptor-like kinase1 mutants in an identical way as reported for Arabidopsis Gb-deficient mutants. Yeast (Saccharomyces cerevisiae) three-hybrid and bimolecular fluorescent complementation assays revealed that XLG2 physically interacts with all three possible Gbg dimers at the plasma membrane. Phylogenetic analysis indicated a close relationship between XLGs and plant Ga subunits, placing the divergence point at the dawn of land plant evolution. Based on these findings, we conclude that XLGs form functional complexes with Gbg dimers, although the mechanism of action of these complexes, including activation/deactivation, must be radically different form the one used by the canonical Ga subunit and are not likely to share the same receptors. Accordingly, XLGs expand the repertoire of heterotrimeric G proteins in plants and reveal a higher level of diversity in heterotrimeric G protein signaling.

Alternative splicing affects the targeting sequence of peroxisome proteins in Arabidopsis.

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An, Chuanjing; Gao, Yuefang; Li, Jinyu; Liu, Xiaomin; Gao, Fuli; Gao, Hongbo

2017-07-01

A systematic analysis of the Arabidopsis genome in combination with localization experiments indicates that alternative splicing affects the peroxisomal targeting sequence of at least 71 genes in Arabidopsis. Peroxisomes are ubiquitous eukaryotic cellular organelles that play a key role in diverse metabolic functions. All peroxisome proteins are encoded by nuclear genes and target to peroxisomes mainly through two types of targeting signals: peroxisomal targeting signal type 1 (PTS1) and PTS2. Alternative splicing (AS) is a process occurring in all eukaryotes by which a single pre-mRNA can generate multiple mRNA variants, often encoding proteins with functional differences. However, the effects of AS on the PTS1 or PTS2 and the targeting of the protein were rarely studied, especially in plants. Here, we systematically analyzed the genome of Arabidopsis, and found that the C-terminal targeting sequence PTS1 of 66 genes and the N-terminal targeting sequence PTS2 of 5 genes are affected by AS. Experimental determination of the targeting of selected protein isoforms further demonstrated that AS at both the 5' and 3' region of a gene can affect the inclusion of PTS2 and PTS1, respectively. This work underscores the importance of AS on the global regulation of peroxisome protein targeting.

Mitogen-activated protein kinase 1 from disk abalone (Haliotis discus discus): Roles in early development and immunity-related transcriptional responses.

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Perera, N C N; Godahewa, G I; Lee, Jehee

2016-12-01

Mitogen-activated protein kinase (MAPK) is involved in the regulation of cellular events by mediating signal transduction pathways. MAPK1 is a member of the extracellular-signal regulated kinases (ERKs), playing roles in cell proliferation, differentiation, and development. This is mainly in response to growth factors, mitogens, and many environmental stresses. In the current study, we have characterized the structural features of a homolog of MAPK1 from disk abalone (AbMAPK1). Further, we have unraveled its expressional kinetics against different experimental pathogenic infections or related chemical stimulants. AbMAPK1 harbors a 5' untranslated region (UTR) of 23 bps, a coding sequence of 1104 bps, and a 3' UTR of 448 bp. The putative peptide comprises a predicted molecular mass of 42.2 kDa, with a theoretical pI of 6.28. Based on the in silico analysis, AbMAPK1 possesses two N-glycosylation sites, one S_TK catalytic domain, and a conserved His-Arg-Asp domain (HRD). In addition, a conservative glycine rich ATP-phosphate-binding loop and a threonine-x-tyrosine motif (TEY) important for the autophosphorylation were also identified in the protein. Homology assessment of AbMAPK1 showed several conserved regions, and ark clam (Aplysia californica) showed the highest sequence identity (87.9%). The phylogenetic analysis supported close evolutionary kinship with molluscan orthologs. Constitutive expression of AbMAPK1 was observed in six different tissues of disk abalone, with the highest expression in the digestive tract, followed by the gills and hemocytes. Highest AbMAPK1 mRNA expression level was detected at the trochophore developmental stage, suggesting its role in abalone cell differentiation and proliferation. Significant modulation of AbMAPK1 expression under pathogenic stress suggested its putative involvement in the immune defense mechanism. Copyright © 2016 Elsevier Ltd. All rights reserved.

Syndecan-1/CD147 association is essential for cyclophilin B-induced activation of p44/42 mitogen-activated protein kinases and promotion of cell adhesion and chemotaxis.

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Pakula, Rachel; Melchior, Aurélie; Denys, Agnès; Vanpouille, Christophe; Mazurier, Joël; Allain, Fabrice

2007-05-01

Many of the biological functions attributed to cell surface proteoglycans are dependent on the interaction with extracellular mediators through their heparan sulphate (HS) moieties and the participation of their core proteins in signaling events. A class of recently identified inflammatory mediators is secreted cyclophilins, which are mostly known as cyclosporin A-binding proteins. We previously demonstrated that cyclophilin B (CyPB) triggers chemotaxis and integrin-mediated adhesion of T lymphocytes mainly of the CD4+/CD45RO+ phenotype. These activities are related to interactions with two types of binding sites, CD147 and cell surface HS. Here, we demonstrate that CyPB-mediated adhesion of CD4+/CD45RO+ T cells is related to p44/42 mitogen-activated protein kinase (MAPK) activation by a mechanism involving CD147 and HS proteoglycans (HSPG). Although HSPG core proteins are represented by syndecan-1, -2, -4, CD44v3 and betaglycan in CD4+/CD45RO+ T cells, we found that only syndecan-1 is physically associated with CD147. The intensity of the heterocomplex increased in response to CyPB, suggesting a transient enhancement and/or stabilization in the association of CD147 to syndecan-1. Pretreatment with anti-syndecan-1 antibodies or knockdown of syndecan-1 expression by RNA interference dramatically reduced CyPB-induced p44/p42 MAPK activation and consequent migration and adhesion, supporting the model in which syndecan-1 serves as a binding subunit to form the fully active receptor of CyPB. Altogether, our findings provide a novel example of a soluble mediator in which a member of the syndecan family plays a critical role in efficient interaction with signaling receptors and initiation of cellular responses.

Heat- and radiation effects on the hemaglutinating- and mitogenic activity of phytohemaglutinins

International Nuclear Information System (INIS)

Mancini Filho, J.; Vizeu, D.M.; Lajolo, F.M.

1975-01-01

The effect of ionizing radiation on hemaglutinating and mitogenic activity of phytohemaglutinins (PHA) in solution is studied. 10 Krad (electron beam) are neede for the destruction of 50% of the aglutinating capacity. The mitogenic effect is more resistent to irradiation (70 Krad for 50% inactivation) may be because both effects are due to different molecules. Changes were also followed by electrophoresis in polyacrylamida. The resistence to irradiation showed to be exponential function of the concentration of PHA in solution. (author) [pt

The pearl millet mitogen-activated protein kinase PgMPK4 is involved in responses to downy mildew infection and in jasmonic- and salicylic acid-mediated defense.

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Melvin, Prasad; Prabhu, S Ashok; Veena, Mariswamy; Shailasree, Sekhar; Petersen, Morten; Mundy, John; Shetty, Shekar H; Kini, K Ramachandra

2015-02-01

Plant mitogen-activated protein kinases (MPKs) transduce signals required for the induction of immunity triggered by host recognition of pathogen-associated molecular patterns. We isolated a full-length cDNA of a group B MPK (PgMPK4) from pearl millet. Autophosphorylation assay of recombinant PgMPK4 produced in Escherichia coli confirmed it as a kinase. Differential accumulation of PgMPK4 mRNA and kinase activity was observed between pearl millet cultivars 852B and IP18292 in response to inoculation with the downy mildew oomycete pathogen Sclerospora graminicola. This increased accumulation of PgMPK4 mRNA, kinase activity as well as nuclear-localization of PgMPK protein(s) was only detected in the S. graminicola resistant cultivar IP18292 with a ~tenfold peak at 9 h post inoculation. In the susceptible cultivar 852B, PgMPK4 mRNA and immuno-detectable nuclear PgMPK could be induced by application of the chemical elicitor β-amino butyric acid, the non-pathogenic bacteria Pseudomonas fluorescens, or by the phytohormones jasmonic acid (JA) or salicylic acid (SA). Furthermore, kinase inhibitor treatments indicated that PgMPK4 is involved in the JA- and SA-mediated expression of three defense genes, lipoxygenase, catalase 3 and polygalacturonase-inhibitor protein. These findings indicate that PgMPK/s contribute to pearl millet defense against the downy mildew pathogen by activating the expression of defense proteins.

p38 mitogen-activated protein kinase up-regulates LPS-induced NF-κB activation in the development of lung injury and RAW 264.7 macrophages

International Nuclear Information System (INIS)

Kim, Hee J.; Lee, Hui S.; Chong, Young H.; Kang, Jihee Lee

2006-01-01

Clarification of the key regulatory steps that lead to nuclear factor-kappa B (NF-κB) under cellular and pathological conditions is very important. The action of p38 mitogen-activated protein kinase (MAPK) on the upstream of NF-κB activation remains controversial. To examine this issue using an in vivo lung injury model, SB203580, a p38 MAPK inhibitor was given intraorally 1 h prior to lipopolysaccharide (LPS) treatment (intratracheally). The mice were sacrificed 4 h after LPS treatment. SB203580 substantially suppressed LPS-induced rises in p38 MAPK phosphorylation, neutrophil recruitment, total protein content in bronchoalveolar lavage fluid, and apoptosis of bronchoalveolar cells. Furthermore, SB203580 blocked LPS-induced NF-κB activation in lung tissue through down-regulation of serine phosphorylation, degradation of IκB-α, and consequent translocation of the p65 subunit of NF-κB to the nucleus. It is likely that, in cultured RAW 264.7 macrophages, SB203580 also blocked LPS-induced NF-κB activation in a dose-dependent manner. SB203580 inhibited LPS-induced serine phosphorylation, degradation of IκB-α, and tyrosine phosphorylation of p65 NF-κB. These data indicate that p38 MAPK acts upstream of LPS-induced NF-κB activation by modulating the phosphorylation of IκB-α and p65 NF-κB during acute lung injury. Because LPS-stimulated macrophages may contribute to inflammatory lung injury, the inhibition of the p38 MAPK-mediated intracellular signaling pathway leading to NF-κB activation represents a target for the attenuation of lung inflammation and parenchymal damage

Mitogen-activated protein kinase signaling in the heart: angels versus demons in a heart-breaking tale.

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Rose, Beth A; Force, Thomas; Wang, Yibin

2010-10-01

Among the myriad of intracellular signaling networks that govern the cardiac development and pathogenesis, mitogen-activated protein kinases (MAPKs) are prominent players that have been the focus of extensive investigations in the past decades. The four best characterized MAPK subfamilies, ERK1/2, JNK, p38, and ERK5, are the targets of pharmacological and genetic manipulations to uncover their roles in cardiac development, function, and diseases. However, information reported in the literature from these efforts has not yet resulted in a clear view about the roles of specific MAPK pathways in heart. Rather, controversies from contradictive results have led to a perception that MAPKs are ambiguous characters in heart with both protective and detrimental effects. The primary object of this review is to provide a comprehensive overview of the current progress, in an effort to highlight the areas where consensus is established verses the ones where controversy remains. MAPKs in cardiac development, cardiac hypertrophy, ischemia/reperfusion injury, and pathological remodeling are the main focuses of this review as these represent the most critical issues for evaluating MAPKs as viable targets of therapeutic development. The studies presented in this review will help to reveal the major challenges in the field and the limitations of current approaches and point to a critical need in future studies to gain better understanding of the fundamental mechanisms of MAPK function and regulation in the heart.

Integrative Identification of Arabidopsis Mitochondrial Proteome and Its Function Exploitation through Protein Interaction Network

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Cui, Jian; Liu, Jinghua; Li, Yuhua; Shi, Tieliu

2011-01-01

Mitochondria are major players on the production of energy, and host several key reactions involved in basic metabolism and biosynthesis of essential molecules. Currently, the majority of nucleus-encoded mitochondrial proteins are unknown even for model plant Arabidopsis. We reported a computational framework for predicting Arabidopsis mitochondrial proteins based on a probabilistic model, called Naive Bayesian Network, which integrates disparate genomic data generated from eight bioinformatics tools, multiple orthologous mappings, protein domain properties and co-expression patterns using 1,027 microarray profiles. Through this approach, we predicted 2,311 candidate mitochondrial proteins with 84.67% accuracy and 2.53% FPR performances. Together with those experimental confirmed proteins, 2,585 mitochondria proteins (named CoreMitoP) were identified, we explored those proteins with unknown functions based on protein-protein interaction network (PIN) and annotated novel functions for 26.65% CoreMitoP proteins. Moreover, we found newly predicted mitochondrial proteins embedded in particular subnetworks of the PIN, mainly functioning in response to diverse environmental stresses, like salt, draught, cold, and wound etc. Candidate mitochondrial proteins involved in those physiological acitivites provide useful targets for further investigation. Assigned functions also provide comprehensive information for Arabidopsis mitochondrial proteome. PMID:21297957

A large complement of the predicted Arabidopsis ARM repeat proteins are members of the U-box E3 ubiquitin ligase family.

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Mudgil, Yashwanti; Shiu, Shin-Han; Stone, Sophia L; Salt, Jennifer N; Goring, Daphne R

2004-01-01

The Arabidopsis genome was searched to identify predicted proteins containing armadillo (ARM) repeats, a motif known to mediate protein-protein interactions in a number of different animal proteins. Using domain database predictions and models generated in this study, 108 Arabidopsis proteins were identified that contained a minimum of two ARM repeats with the majority of proteins containing four to eight ARM repeats. Clustering analysis showed that the 108 predicted Arabidopsis ARM repeat proteins could be divided into multiple groups with wide differences in their domain compositions and organizations. Interestingly, 41 of the 108 Arabidopsis ARM repeat proteins contained a U-box, a motif present in a family of E3 ligases, and these proteins represented the largest class of Arabidopsis ARM repeat proteins. In 14 of these U-box/ARM repeat proteins, there was also a novel conserved domain identified in the N-terminal region. Based on the phylogenetic tree, representative U-box/ARM repeat proteins were selected for further study. RNA-blot analyses revealed that these U-box/ARM proteins are expressed in a variety of tissues in Arabidopsis. In addition, the selected U-box/ARM proteins were found to be functional E3 ubiquitin ligases. Thus, these U-box/ARM proteins represent a new family of E3 ligases in Arabidopsis.

Trafficking of endoplasmic reticulum-retained recombinant proteins is unpredictable in Arabidopsis thaliana

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Thomas eDe Meyer

2014-09-01

Full Text Available A wide variety of recombinant proteins has been produced in the dicot model plant, Arabidopsis thaliana. Many of these proteins are targeted for secretion by means of an N terminal endoplasmic reticulum (ER signal peptide. In addition, they can also be designed for ER retention by adding a C terminal H/KDEL-tag. Despite extensive knowledge of the protein trafficking pathways, the final protein destination, especially of such H/KDEL-tagged recombinant proteins, is unpredictable. In this respect, glycoproteins are ideal study objects. Microscopy experiments reveal their deposition pattern and characterization of their N-glycans aids in elucidating the trafficking. Here, we combine microscopy and N glycosylation data generated in Arabidopsis leaves and seeds, and highlight the lack of a decent understanding of heterologous protein trafficking.

Cross activity of orthologous WRKY transcription factors in wheat and Arabidopsis

NARCIS (Netherlands)

Poietti, S.; Bertini, L.; Ent, S. van der; Leon Reyes, H.A.; Pieterse, C.M.J.; Tucci, M.; Caporale, C.; Caruso, C.

2011-01-01

WRKY proteins are transcription factors involved in many plant processes including plant responses to pathogens. Here, the cross activity of TaWRKY78 from the monocot wheat and AtWRKY20 from the dicot Arabidopsis on the cognate promoters of the orthologous PR4-type genes wPR4e and AtHEL of wheat and

GmGBP1, a homolog of human ski interacting protein in soybean, regulates flowering and stress tolerance in Arabidopsis

Directory of Open Access Journals (Sweden)

Zhang Yanwei

2013-02-01

Full Text Available Abstract Background SKIP is a transcription cofactor in many eukaryotes. It can regulate plant stress tolerance in rice and Arabidopsis. But the homolog of SKIP protein in soybean has been not reported up to now. Results In this study, the expression patterns of soybean GAMYB binding protein gene (GmGBP1 encoding a homolog of SKIP protein were analyzed in soybean under abiotic stresses and different day lengths. The expression of GmGBP1 was induced by polyethyleneglycol 6000, NaCl, gibberellin, abscisic acid and heat stress. GmGBP1 had transcriptional activity in C-terminal. GmGBP1 could interact with R2R3 domain of GmGAMYB1 in SKIP domain to take part in gibberellin flowering pathway. In long-day (16 h-light condition, transgenic Arabidopsis with the ectopic overexpression of GmGBP1 exhibited earlier flowering and less number of rosette leaves; Suppression of AtSKIP in Arabidopsis resulted in growth arrest, flowering delay and down-regulation of many flowering-related genes (CONSTANS, FLOWERING LOCUS T, LEAFY; Arabidopsis myb33 mutant plants with ectopic overexpression of GmGBP1 showed the same flowering phenotype with wild type. In short-day (8 h-light condition, transgenic Arabidopsis plants with GmGBP1 flowered later and showed a higher level of FLOWERING LOCUS C compared with wild type. When treated with abiotic stresses, transgenic Arabidopsis with the ectopic overexpression of GmGBP1 enhanced the tolerances to heat and drought stresses but reduced the tolerance to high salinity, and affected the expressions of several stress-related genes. Conclusions In Arabidopsis, GmGBP1 might positively regulate the flowering time by affecting CONSTANS, FLOWERING LOCUS T, LEAFY and GAMYB directly or indirectly in photoperiodic and gibberellin pathways in LDs, but GmGBP1 might represse flowering by affecting FLOWERING LOCUS C and SHORT VEGETATIVE PHASE in autonomous pathway in SDs. GmGBP1 might regulate the activity of ROS-eliminating to improve the

Phosphorylation of mitogen-activated protein kinase (MAPK) is required for cytokinesis and progression of cell cycle in tobacco BY-2 cells.

Science.gov (United States)

Ma, Zhaowu; Yu, Guanghui

2010-02-15

The role of mitogen-activated protein kinase (MAPK) in plant cytokinesis remains largely uncharacterized. To elucidate its role, tobacco Bright Yellow-2 (BY-2) cells have been synchronized using a two-step procedure, and the different phases of the cell cycle identified by Histone 4 gene expression and the mitotic index. MAPK expression was analyzed by semi-quantitative (SQ) RT-PCR and protein gel blot analysis for phosphorylated MAPK during cell cycle progression. The SQ RT-PCR analysis indicated that MAPK expression is lower in mitosis than in interphase (G1, G2 and S). However, the amount of phosphorylated MAPK remained stable throughout the cell cycle, indicating that MAPK activity is predominantly regulated at the post-translational level and that phosphorylation of MAPK plays an important role in mitosis. Application of the specific MAPK phosphorylation inhibitor U0126 revealed that while U0126 treatment decreases the phosphorylation of MAPK and the progression from telophase to early cytokinesis is significantly inhibited. The formation of the phragmoplast is also negatively affected at this stage. These results demonstrate that MAPK phosphorylation is involved in the formation of the cell plate within the phragmoplast during cytokinesis and that MAPK predominantly functions during the cytokinesis stage of the cell cycle in tobacco BY-2 cells. Copyright 2009 Elsevier GmbH. All rights reserved.

Reduced response of splenocytes after mitogen-stimulation in the prion protein (PrP) gene-deficient mouse: PrPLP/Doppel production and cerebral degeneration

International Nuclear Information System (INIS)

Kim, Chi-Kyeong; Hirose, Yuko; Sakudo, Akikazu; Takeyama, Natsumi; Kang, Chung-Boo; Taniuchi, Yojiro; Matsumoto, Yoshitsugu; Itohara, Shigeyoshi; Sakaguchi, Suehiro; Onodera, Takashi

2007-01-01

Splenocytes of wild-type (Prnp +/+ ) and prion protein gene-deficient (Prnp -/- ) mice were treated with various activation stimuli such as T cell mitogen concanavalin A (ConA), phorbol 12-myristate 13-acetate (PMA) + ionomycin (Io), or B cell mitogen lipopolysaccharide (LPS). Cellular prion protein (PrP C ) expression was enhanced following ConA stimulation, but not PMA + Io or LPS in Prnp +/+ splenocytes. Rikn Prnp -/- splenocytes elicited lower cell proliferations than Prnp +/+ or Zrch I Prnp -/- splenocytes after LPS stimulation and showed sporadic nerve cells in the cerebral cortex and deeper structure. Around the degenerated nerve cells, mild vacuolation in the neuropil was observed. This neural alteration correlated well to the suppressed response of B cells in the spleen. The finding that discrete lesions within the central nervous systems induced marked modulation of immune function probably indicates the existence of a delicately balanced neural-endocrine network by PrP C and PrPLP/Doppel

The Arabidopsis mitogen-activated protein kinase 6 is associated with γ-tubulin on microtubules, phosphorylates EB1c and maintains spindle orientation under nitrosative stress

Czech Academy of Sciences Publication Activity Database

Kohoutová, Lucie; Kourová, Hana; Nagy, S. K.; Volc, Jindřich; Halada, Petr; Mészáros, T.; Meskiene, I.; Bögre, L.; Binarová, Pavla

2015-01-01

Roč. 207, č. 4 (2015), s. 1061-1074 ISSN 0028-646X R&D Projects: GA MŠk 7AMB13AT013; GA ČR GAP501/12/2333 Institutional support: RVO:61388971 Keywords : Arabidopsis * cell division * EB1c Subject RIV: EE - Microbiology, Virology Impact factor: 7.210, year: 2015

Bacteroides fragilis Enterotoxin Induces Formation of Autophagosomes in Endothelial Cells but Interferes with Fusion with Lysosomes for Complete Autophagic Flux through a Mitogen-Activated Protein Kinase-, AP-1-, and C/EBP Homologous Protein-Dependent Pathway.

Science.gov (United States)

Ko, Su Hyuk; Jeon, Jong Ik; Myung, Hyun Soo; Kim, Young-Jeon; Kim, Jung Mogg

2017-10-01

Bacteroides fragilis enterotoxin (BFT), a virulence factor of enterotoxigenic B. fragilis (ETBF), plays an essential role in mucosal inflammation. Although autophagy contributes to the pathogenesis of diverse infectious diseases, little is known about autophagy in ETBF infection. This study was conducted to investigate the role of BFT in the autophagic process in endothelial cells (ECs). Stimulation of human umbilical vein ECs (HUVECs) with BFT increased light chain 3 protein II (LC3-II) conversion from LC3-I and protein expression of p62, Atg5, and Atg12. In addition, BFT-exposed ECs showed increased indices of autophagosomal fusion with lysosomes such as LC3-lysosome-associated protein 2 (LAMP2) colocalization and the percentage of red vesicles monitored by the expression of dual-tagged LC3B. BFT also upregulated expression of C/EBP homologous protein (CHOP), and inhibition of CHOP significantly increased indices of autophagosomal fusion with lysosomes. BFT activated an AP-1 transcription factor, in which suppression of AP-1 activity significantly downregulated CHOP and augmented autophagosomal fusion with lysosomes. Furthermore, suppression of Jun N-terminal protein kinase (JNK) mitogen-activated protein kinase (MAPK) significantly inhibited the AP-1 and CHOP signals, leading to an increase in autophagosomal fusion with lysosomes in BFT-stimulated ECs. These results suggest that BFT induced accumulation of autophagosomes in ECs, but activation of a signaling pathway involving JNK, AP-1, and CHOP may interfere with complete autophagy. Copyright © 2017 American Society for Microbiology.

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

Science.gov (United States)

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

2016-01-01

Tumor-associated macrophages (TAM) play pivotal roles in cancer initiation and progression. Monocytes, the precursors of TAMs, normally undergo spontaneous apoptosis within 2 days, but can subsist in the inflammatory tumor microenvironment for continuous survival and generation of sufficient TAMs. The mechanisms underlying tumor-driving monocyte survival remain obscure. Here we report that cancer cell-derived exosomes were crucial mediators for monocyte survival in the inflammatory niche. Analysis of the survival-promoting molecules in monocytes revealed that cancer cell-derived exosomes activated Ras and extracellular signal-regulated kinases in the mitogen-activated protein kinase (MAPK) pathway, resulting in the prevention of caspase cleavage. Phosphorylated receptor tyrosine kinases (RTKs), such as phosphorylated epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER-2), were abundantly expressed in cancer cell-derived exosomes. Knock-out of EGFR or/and HER-2, or alternatively, inhibitors against their phosphorylation significantly disturbed the exosome-mediated activation of the MAPK pathway, inhibition of caspase cleavage, and increase in survival rate in monocytes. Moreover, the deprived survival-stimulating activity of exosomes due to null expression of EGFR and HER-2 could be restored by activation of another RTK, insulin receptor. Overall, our study uncovered a mechanism of tumor-associated monocyte survival and demonstrated that cancer cell-derived exosomes can stimulate the MAPK pathway in monocytes through transport of functional RTKs, leading to inactivation of apoptosis-related caspases. This work provides insights into the long sought question on monocyte survival prior to formation of plentiful TAMs in the tumor microenvironment. PMID:26895960

Dynamic Proteomics Emphasizes the Importance of Selective mRNA Translation and Protein Turnover during Arabidopsis Seed Germination*

Science.gov (United States)

Galland, Marc; Huguet, Romain; Arc, Erwann; Cueff, Gwendal; Job, Dominique; Rajjou, Loïc

2014-01-01

During seed germination, the transition from a quiescent metabolic state in a dry mature seed to a proliferative metabolic state in a vigorous seedling is crucial for plant propagation as well as for optimizing crop yield. This work provides a detailed description of the dynamics of protein synthesis during the time course of germination, demonstrating that mRNA translation is both sequential and selective during this process. The complete inhibition of the germination process in the presence of the translation inhibitor cycloheximide established that mRNA translation is critical for Arabidopsis seed germination. However, the dynamics of protein turnover and the selectivity of protein synthesis (mRNA translation) during Arabidopsis seed germination have not been addressed yet. Based on our detailed knowledge of the Arabidopsis seed proteome, we have deepened our understanding of seed mRNA translation during germination by combining two-dimensional gel-based proteomics with dynamic radiolabeled proteomics using a radiolabeled amino acid precursor, namely [35S]-methionine, in order to highlight de novo protein synthesis, stability, and turnover. Our data confirm that during early imbibition, the Arabidopsis translatome keeps reflecting an embryonic maturation program until a certain developmental checkpoint. Furthermore, by dividing the seed germination time lapse into discrete time windows, we highlight precise and specific patterns of protein synthesis. These data refine and deepen our knowledge of the three classical phases of seed germination based on seed water uptake during imbibition and reveal that selective mRNA translation is a key feature of seed germination. Beyond the quantitative control of translational activity, both the selectivity of mRNA translation and protein turnover appear as specific regulatory systems, critical for timing the molecular events leading to successful germination and seedling establishment. PMID:24198433

Ethanol extract of the seed of Zizyphus jujuba var. spinosa potentiates hippocampal synaptic transmission through mitogen-activated protein kinase, adenylyl cyclase, and protein kinase A pathways.

Science.gov (United States)

Jo, So Yeon; Jung, In Ho; Yi, Jee Hyun; Choi, Tae Joon; Lee, Seungheon; Jung, Ji Wook; Yun, Jeanho; Lee, Young Choon; Ryu, Jong Hoon; Kim, Dong Hyun

2017-03-22

As the seed of Zizyphus jujuba var. spinosa (Bunge) Hu ex H.F. Chow (Rhamnaceae) has been used to sleep disturbances in traditional Chinese and Korean medicine, many previous studies have focused on its sedative effect. Recently, we reported the neuroprotective effect of the effect of Z. jujuba var. spinosa. However, its effects on synaptic function have not yet been studied. In this project, we examined the action of ethanol extract of the seed of Z. jujuba var. spinosa (DHP1401) on synaptic transmission in the hippocampus. To investigate the effects of DHP1401, field recordings were conducted using hippocampal slices (400µm). Object recognition test was introduced to examine whether DHP1401 affect normal recognition memory. DHP1401 (50μg/ml) induced a significant increase in synaptic activity in Shaffer collateral pathway in a concentration-dependent manner. This increase of synaptic responses was blocked by NBQX, a broad spectrum α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor antagonist, but not IEM-1460, a Ca 2+ -permeable AMPAR blocker. Moreover, U0126, a mitogen-activated protein kinase inhibitor, SQ22536, an adenylyl cyclase inhibitor, and PKI, a protein kinase A inhibitor, blocked DHP1401-induced increase in synaptic transmission. Finally, DHP1401 facilitated object recognition memory. These results suggest that DHP1401 increase synaptic transmission through increase of synaptic AMPAR transmission via MAPK, AC and PAK. Copyright © 2017 Elsevier Ireland Ltd. All rights reserved.

Aqueous fraction from Cuscuta japonica seed suppresses melanin synthesis through inhibition of the p38 mitogen-activated protein kinase signaling pathway in B16F10 cells.

Science.gov (United States)

Jang, Ji Yeon; Kim, Ha Neui; Kim, Yu Ri; Choi, Yung Hyun; Kim, Byung Woo; Shin, Hwa Kyoung; Choi, Byung Tae

2012-05-07

Semen cuscutae has been used traditionally to treat pimples and alleviate freckles and melasma in Korea. The present study aimed to investigate the inhibitory effect of Cuscuta japonica Choisy seeds on alpha-melanocyte-stimulating hormone (α-MSH)-induced melanogenesis. The aqueous fraction from Semen cuscutae (AFSC) was used to determine anti-melanogenic effects by examination of cellular melanin contents, tyrosinase activity assay, cAMP assay and Western blot analysis for melanin synthesis-related signaling proteins in B16F10 mouse melanoma cells. AFSC markedly inhibited α-MSH-induced melanin synthesis and tyrosinase activity, and also decreased α-MSH-induced expression of microphthalmia-associated transcription factor (MITF) and tyrosinase-related proteins (TRPs). Moreover, AFSC significantly decreased the level of phosphorylated p38 mitogen-activated protein kinase (MAPK) signaling through the down-regulation of α-MSH-induced cAMP. Furthermore, we confirmed that the specific inhibitor of p38 MAPK (SB203580)-mediated suppressed melanin synthesis and tyrosinase activity was further attenuated by AFSC. AFSC also further decreased SB203580-mediated suppression of MITF and TRP expression. These results indicate that AFSC inhibits p38 MAPK phosphorylation with suppressed cAMP levels and subsequently down-regulate MITF and TRP expression, which results in a marked reduction of melanin synthesis and tyrosinase activity in α-MSH-stimulated B16F10 cells. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

Regulation of brain capillary endothelial cells by P2Y receptors coupled to Ca2+, phospholipase C and mitogen-activated protein kinase.

Science.gov (United States)

Albert, J L; Boyle, J P; Roberts, J A; Challiss, R A; Gubby, S E; Boarder, M R

1997-11-01

1. The blood-brain barrier is formed by capillary endothelial cells and is regulated by cell-surface receptors, such as the G protein-coupled P2Y receptors for nucleotides. Here we investigated some of the characteristics of control of brain endothelial cells by these receptors, characterizing the phospholipase C and Ca2+ response and investigating the possible involvement of mitogen-activated protein kinases (MAPK). 2. Using an unpassaged primary culture of rat brain capillary endothelial cells we showed that ATP, UTP and 2-methylthio ATP (2MeSATP) give similar and substantial increases in cytosolic Ca2+, with a rapid rise to peak followed by a slower decline towards basal or to a sustained plateau. Removal of extracellular Ca2+ had little effect on the peak Ca2+-response, but resulted in a more rapid decline to basal. There was no response to alpha,beta-MethylATP (alpha,beta MeATP) in these unpassaged cells, but a response to this P2X agonist was seen after a single passage. 3. ATP (log EC50 -5.1+/-0.2) also caused an increase in the total [3H]-inositol (poly)phosphates ([3H]-InsPx) in the presence of lithium with a rank order of agonist potency of ATP=UTP=UDP>ADP, with 2MeSATP and alpha,beta MeATP giving no detectable response. 4. Stimulating the cells with ATP or UTP gave a rapid rise in the level of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3), with a peak at 10 s followed by a decline to a sustained plateau phase. 2MeSATP gave no detectable increase in the level of Ins(1,4,5)P3. 5. None of the nucleotides tested affected basal cyclic AMP, while ATP and ATPgammaS, but not 2MeSATP, stimulated cyclic AMP levels in the presence of 5 microM forskolin. 6. Both UTP and ATP stimulated tyrosine phosphorylation of p42 and p44 mitogen-activated protein kinase (MAPK), while 2MeSATP gave a smaller increase in this index of MAPK activation. By use of a peptide kinase assay, UTP gave a substantial increase in MAPK activity with a concentration-dependency consistent with

A Large Complement of the Predicted Arabidopsis ARM Repeat Proteins Are Members of the U-Box E3 Ubiquitin Ligase Family1[w

Science.gov (United States)

Mudgil, Yashwanti; Shiu, Shin-Han; Stone, Sophia L.; Salt, Jennifer N.; Goring, Daphne R.

2004-01-01

The Arabidopsis genome was searched to identify predicted proteins containing armadillo (ARM) repeats, a motif known to mediate protein-protein interactions in a number of different animal proteins. Using domain database predictions and models generated in this study, 108 Arabidopsis proteins were identified that contained a minimum of two ARM repeats with the majority of proteins containing four to eight ARM repeats. Clustering analysis showed that the 108 predicted Arabidopsis ARM repeat proteins could be divided into multiple groups with wide differences in their domain compositions and organizations. Interestingly, 41 of the 108 Arabidopsis ARM repeat proteins contained a U-box, a motif present in a family of E3 ligases, and these proteins represented the largest class of Arabidopsis ARM repeat proteins. In 14 of these U-box/ARM repeat proteins, there was also a novel conserved domain identified in the N-terminal region. Based on the phylogenetic tree, representative U-box/ARM repeat proteins were selected for further study. RNA-blot analyses revealed that these U-box/ARM proteins are expressed in a variety of tissues in Arabidopsis. In addition, the selected U-box/ARM proteins were found to be functional E3 ubiquitin ligases. Thus, these U-box/ARM proteins represent a new family of E3 ligases in Arabidopsis. PMID:14657406

Identification and molecular properties of SUMO-binding proteins in arabidopsis

KAUST Repository

Park, Hyeongcheol

2011-05-20

Reversible conjugation of the small ubiquitin modifier (SUMO) peptide to proteins (SUMOylation) plays important roles in cellular processes in animals and yeasts. However, little is known about plant SUMO targets. To identify SUMO substrates in Arabidopsis and to probe for biological functions of SUMO proteins, we constructed 6xHis-3xFLAG fused AtSUMO1 (HFAtSUMO1) controlled by the CaMV35S promoter for transformation into Arabidopsis Col-0. After heat treatment, an increased sumoylation pattern was detected in the transgenic plants. SUMO1-modified proteins were selected after two-dimensional gel electrophoresis (2-DE) image analysis and identified using matrix-assisted laser-desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). We identified 27 proteins involved in a variety of processes such as nucleic acid metabolism, signaling, metabolism, and including proteins of unknown functions. Binding and sumoylation patterns were confirmed independently. Surprisingly, MCM3 (At5G46280), a DNA replication licensing factor, only interacted with and became sumoylated by AtSUMO1, but not by SUMO1ΔGG or AtSUMO3. The results suggest specific interactions between sumoylation targets and particular sumoylation enzymes. ©2011 KSMCB.

SnRK1 activates autophagy via the TOR signaling pathway in Arabidopsis thaliana.

Science.gov (United States)

Soto-Burgos, Junmarie; Bassham, Diane C

2017-01-01

Autophagy is a degradation process in which cells break down and recycle their cytoplasmic contents when subjected to environmental stress or during cellular remodeling. The Arabidopsis thaliana SnRK1 complex is a protein kinase that senses changes in energy levels and triggers downstream responses to enable survival. Its mammalian ortholog, AMPK, and yeast ortholog, Snf-1, activate autophagy in response to low energy conditions. We therefore hypothesized that SnRK1 may play a role in the regulation of autophagy in response to nutrient or energy deficiency in Arabidopsis. To test this hypothesis, we determined the effect of overexpression or knockout of the SnRK1 catalytic subunit KIN10 on autophagy activation by abiotic stresses, including nutrient deficiency, salt, osmotic, oxidative, and ER stress. While wild-type plants had low basal autophagy activity in control conditions, KIN10 overexpression lines had increased autophagy under these conditions, indicating activation of autophagy by SnRK1. A kin10 mutant had a basal level of autophagy under control conditions similar to wild-type plants, but activation of autophagy by most abiotic stresses was blocked, indicating that SnRK1 is required for autophagy induction by a wide variety of stress conditions. In mammals, TOR is a negative regulator of autophagy, and AMPK acts to activate autophagy both upstream of TOR, by inhibiting its activity, and in a parallel pathway. Inhibition of Arabidopsis TOR leads to activation of autophagy; inhibition of SnRK1 did not block this activation. Furthermore, an increase in SnRK1 activity was unable to induce autophagy when TOR was also activated. These results demonstrate that SnRK1 acts upstream of TOR in the activation of autophagy in Arabidopsis.

Mitogenic activity of new lectins from seeds of wild Artocarpus species from Vietnam.

Science.gov (United States)

Blasco, E; Ngoc, L D; Aucouturier, P; Preud'Homme, J L; Barra, A

1996-05-01

Proliferative response of human peripheral blood mononuclear cells (PBMC) stimulated by new lectins purified from seeds of differents Artocarpus species from Vietnam (A. asperulus, A. heterophyllus, A. masticata, A. melinoxylus, A. parva and A. petelotii) was studied and compared to those of the lectin jacalin purified from jackfruit (A. heterophyllus) seeds collected in the island La Réunion. All lectins stimulated human PBMC to proliferate, with a variable efficiency of the mitogenic activity. Phenotypic analysis of cells recovered after 7 day-cultures showed that these lectins mostly stimulated CD4+ T lymphocytes. These results suggest that these lectins from different Artocarpus species are similar in terms of their mitogenic activity although their structural features are not identical.

Expression of human ARGONAUTE 2 inhibits endogenous microRNA activity in Arabidopsis

Directory of Open Access Journals (Sweden)

Ira eDeveson

2013-04-01

Full Text Available Plant and animal microRNA (miRNA pathways share many analogous components, the ARGONAUTE (AGO proteins being foremost among them. We sought to ascertain the degree of functional conservation shared by Homo sapiens ARGONAUTE 2 (HsAGO2 and Arabidopsis thaliana ARGONAUTE 1 (AtAGO1, which are the predominant AGO family members involved with miRNA activity in their respective species. Transgenic Arabidopsis plants expressing HsAGO2 were indistinguishable from counterparts over-expressing AtAGO1, each group exhibiting the morphological and molecular hallmarks of miRNA-pathway loss-of-function alleles. However, unlike AtAGO1, HsAGO2 was unable to rescue the ago1-27 allele. We conclude that, despite the evolutionary gulf between them, HsAGO2 is likely capable of interacting with some component/s of the Arabidopsis miRNA pathway, thereby perturbing its operation, although differences have arisen such that HsAGO2 alone is insufficient to confer efficient silencing of miRNA targets in planta.

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

Science.gov (United States)

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

2016-04-15

Tumor-associated macrophages (TAM) play pivotal roles in cancer initiation and progression. Monocytes, the precursors of TAMs, normally undergo spontaneous apoptosis within 2 days, but can subsist in the inflammatory tumor microenvironment for continuous survival and generation of sufficient TAMs. The mechanisms underlying tumor-driving monocyte survival remain obscure. Here we report that cancer cell-derived exosomes were crucial mediators for monocyte survival in the inflammatory niche. Analysis of the survival-promoting molecules in monocytes revealed that cancer cell-derived exosomes activated Ras and extracellular signal-regulated kinases in the mitogen-activated protein kinase (MAPK) pathway, resulting in the prevention of caspase cleavage. Phosphorylated receptor tyrosine kinases (RTKs), such as phosphorylated epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER-2), were abundantly expressed in cancer cell-derived exosomes. Knock-out of EGFR or/and HER-2, or alternatively, inhibitors against their phosphorylation significantly disturbed the exosome-mediated activation of the MAPK pathway, inhibition of caspase cleavage, and increase in survival rate in monocytes. Moreover, the deprived survival-stimulating activity of exosomes due to null expression of EGFR and HER-2 could be restored by activation of another RTK, insulin receptor. Overall, our study uncovered a mechanism of tumor-associated monocyte survival and demonstrated that cancer cell-derived exosomes can stimulate the MAPK pathway in monocytes through transport of functional RTKs, leading to inactivation of apoptosis-related caspases. This work provides insights into the long sought question on monocyte survival prior to formation of plentiful TAMs in the tumor microenvironment. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

FYVE zinc-finger proteins in the plant model Arabidopsis thaliana

DEFF Research Database (Denmark)

Jensen, R B; La Cour, T; Albrethsen, J

2001-01-01

Classic FYVE zinc-finger domains recognize the phosphoinositide signal PtdIns3P and share the basic (R/K)(1)(R/K)HHCR(6) (single-letter amino acid codes) consensus sequence. This domain is present in predicted PtdIns3P 5-kinases and lipases from Arabidopsis thaliana. Other Arabidopsis proteins......) of the basic motif. Dot-blot and liposome-binding assays were used in vitro to examine the phospholipid-binding ability of isolated PRAF domains. Whereas the PH domain preferentially bound PtdIns(4,5)P(2), the variant FYVE domain showed a weaker charge-dependent binding of phosphoinositides. In contrast....... A biochemical function for PRAF was indicated by its ability to catalyse guanine nucleotide exchange on some of the small GTPases of the Rab family, permitting a discussion of the biological roles of plant FYVE proteins and their regulation by phosphoinositides....

Transcriptional regulation of receptor-like protein genes by environmental stresses and hormones and their overexpression activities in Arabidopsis thaliana

NARCIS (Netherlands)

Wu, Jinbin; Liu, Zhijun; Zhang, Zhao; Lv, Yanting; Yang, Nan; Zhang, Guohua; Wu, Menyao; Lv, Shuo; Pan, Lixia; Joosten, Matthieu H.A.J.; Wang, Guodong

2016-01-01

Receptor-like proteins (RLPs) have been implicated in multiple biological processes, including plant development and immunity to microbial infection. Fifty-seven AtRLP genes have been identified in Arabidopsis, whereas only a few have been functionally characterized. This is due to the lack of

Phosphorylation and activation of p42 and p44 mitogen-activated protein kinase are required for the P2 purinoceptor stimulation of endothelial prostacyclin production.

Science.gov (United States)

Patel, V; Brown, C; Goodwin, A; Wilkie, N; Boarder, M R

1996-11-15

Extracellular ATP and ADP, released from platelets and other sites stimulate the endothelial production of prostacyclin (PGI2) by acting on G-protein-coupled P2Y2 and P2Y2 purinoceptors, contributing to the maintenance of a non-thrombogenic surface. The mechanism, widely described as being dependent on elevated cytosolic [Ca2+], also requires protein tyrosine phosphorylation. Here we show that activation of both these P2 receptor types leads to the tyrosine phosphorylation and activation of both the p42 and p44 forms of mitogen-activated protein kinase (MAPK). 2-Methylthio-ATP and UTP, selectively activating P2Y1 and P2Y2 purinoceptors respectively, and ATP, a non-selective agonist at these two receptors, stimulate the tyrosine phosphorylation of both p42mapk and p44mapk, as revealed by Western blots with an antiserum specific for the tyrosine-phosphorylated forms of the enzymes. By using separation on Resource Q columns, peptide kinase activity associated with the phosphorylated MAPK enzymes distributes into two peaks, one mainly p42mapk and one mainly p44mapk, both of which are stimulated by ATP with respect to kinase activity and phospho-MAPK immunoreactivity. Stimulation of P2Y1 or P2Y2 purinoceptors leads to a severalfold increase in PGI2 efflux; this was blocked in a dose-dependent manner by the selective MAPK kinase inhibitor PD98059. This drug also blocked the agonist-stimulated increase in phospho-MAPK immunoreactivity for both p42mapk and p44mapk but left the phospholipase C response to P2 agonists essentially unchanged. Olomoucine has been reported to inhibit p44mapk activity. Here we show that in the same concentration range olomoucine inhibits activity in both peaks from the Resource Q column and also the agonist stimulation of 6-keto-PGF1, but has no effect on agonist-stimulated phospho-MAPK immunoreactivity. These results provide direct evidence for the involvement of p42 and p44 MAPK in the PGI2 response of intact endothelial cells: we have shown

Growth enhancement and gene expression of Arabidopsis thaliana irradiated with active oxygen species

Science.gov (United States)

Watanabe, Satoshi; Ono, Reoto; Hayashi, Nobuya; Shiratani, Masaharu; Tashiro, Kosuke; Kuhara, Satoru; Inoue, Asami; Yasuda, Kaori; Hagiwara, Hiroko

2016-07-01

The characteristics of plant growth enhancement effect and the mechanism of the enhancement induced by plasma irradiation are investigated using various active species in plasma. Active oxygen species in oxygen plasma are effective for growth enhancement of plants. DNA microarray analysis of Arabidopsis thaliana indicates that the genes coding proteins that counter oxidative stresses by eliminating active oxygen species are expressed at significantly high levels. The size of plant cells increases owing to oxygen plasma irradiation. The increases in gene expression levels and cell size suggest that the increase in the expression level of the expansin protein is essential for plant growth enhancement phenomena.

Pentachlorophenol-Induced Cytotoxic, Mitogenic, and Endocrine-Disrupting Activities in Channel Catfish, Ictalurus punctatus

Directory of Open Access Journals (Sweden)

Paul B. Tchounwou

2004-09-01

Full Text Available Pentachlorophenol (PCP is an organochlorine compound that has been widely used as a biocide in several industrial, agricultural, and domestic applications. Although it has been shown to induce systemic toxicity and carcinogenesis in several experimental studies, the literature is scarce regarding its toxic mechanisms of action at the cellular and molecular levels. Recent investigations in our laboratory have shown that PCP induces cytotoxicity and transcriptionally activates stress genes in human liver carcinoma (HepG2 cells [1]. In this research, we hypothesize that environmental exposure to PCP may trigger cytotoxic, mitogenic, and endocrine-disrupting activities in aquatic organisms including fish. To test this hypothesis, we carried out in vitro cultures of male channel catfish hepatocytes, and performed the fluorescein diacetate assay (FDA to assess for cell viability, and the Western Blot analysis to assess for vitellogenin expression following exposure to PCP. Data obtained from FDA experiments indicated a strong dose-response relationship with respect to PCP cytotoxicity. Upon 48 hrs of exposure, the chemical dose required to cause 50% reduction in cell viability (LD50 was computed to be 1,987.0 + 9.6 μg PCP/mL. The NOAEL and LOAEL were 62.5 + 10.3 μg PCP/mL and 125.0+15.2 μg PCP/mL, respectively. At lower levels of exposure, PCP was found to be mitogenic, showing a strong dose- and time-dependent response with regard to cell proliferation. Western Blot analysis demonstrated the potential of PCP to cause endocrine-disrupting activity, as evidenced by the up regulation of the 125-kDa vitellogenin protein the hepatocytes of male channel catfish.

The Histone Deacetylase Inhibitors MS-275 and SAHA Suppress the p38 Mitogen-Activated Protein Kinase Signaling Pathway and Chemotaxis in Rheumatoid Arthritic Synovial Fibroblastic E11 Cells

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Hai-Shu Lin

2013-11-01

Full Text Available MS-275 (entinostat and SAHA (vorinostat, two histone deacetylase (HDAC inhibitors currently in oncological trials, have displayed potent anti-rheumatic activities in rodent models of rheumatoid arthritis (RA. To further elucidate their anti-inflammatory mechanisms, the impact of MS-275 and SAHA on the p38 mitogen-activated protein kinase (MAPK signaling pathway and chemotaxis was assessed in human rheumatoid arthritic synovial fibroblastic E11 cells. MS-275 and SAHA significantly suppressed the expression of p38α  MAPK, but induced the expression of MAPK phosphatase-1 (MKP-1, an endogenous suppressor of p38α  in E11 cells. At the same time, the association between p38α and MKP-1 was up-regulated and consequently, the activation (phosphorylation of p38α  was inhibited. Moreover, MS-275 and SAHA suppressed granulocyte chemotactic protein-2 (GCP-2, monocyte chemotactic protein-2 (MCP-2 and macrophage migration inhibitory factor (MIF in E11 cells in a concentration-dependent manner. Subsequently, E11-driven migration of THP-1 and U937 monocytes was inhibited. In summary, suppression of the p38 MAPK signaling pathway and chemotaxis appear to be important anti-rheumatic mechanisms of action of these HDAC inhibitors.

Carprofen induction of p75NTR-dependent apoptosis via the p38 mitogen-activated protein kinase pathway in prostate cancer cells.

Science.gov (United States)

Khwaja, Fatima S; Quann, Emily J; Pattabiraman, Nagarajan; Wynne, Shehla; Djakiew, Daniel

2008-11-01

The p75 neurotrophin receptor (p75(NTR)) functions as a tumor suppressor in prostate epithelial cells, where its expression declines with progression to malignant cancer. Previously, we showed that treatment with R-flurbiprofen or ibuprofen induced p75(NTR) expression in several prostate cancer cell lines leading to p75(NTR)-mediated decreased survival. Using the 2-phenyl propionic acid moiety of these profens as a pharmacophore, we screened an in silico database of 30 million compounds and identified carprofen as having an order of magnitude greater activity for induction of p75(NTR) levels and inhibition of cell survival. Prostate (PC-3 and DU-145) and bladder (T24) cancer cells were more sensitive to carprofen induction of p75(NTR)-associated loss of survival than breast (MCF-7) and fibroblast (3T3) cells. Transfection of prostate cell lines with a dominant-negative form of p75(NTR) before carprofen treatment partially rescued cell survival, showing a cause-and-effect relationship between carprofen induction of p75(NTR) levels and inhibition of survival. Carprofen induced apoptotic nuclear fragmentation in prostate but not in MCF-7 and 3T3 cells. Furthermore, small interfering RNA knockdown of the p38 mitogen-activated protein kinase (MAPK) protein prevented induction of p75(NTR) by carprofen in both prostate cell lines. Carprofen treatment induced phosphorylation of p38 MAPK as early as within 1 min. Expression of a dominant-negative form of MK2, the kinase downstream of p38 MAPK frequently associated with signaling cascades leading to apoptosis, prevented carprofen induction of the p75(NTR) protein. Collectively, we identify carprofen as a highly potent profen capable of inducing p75(NTR)-dependent apoptosis via the p38 MAPK pathway in prostate cancer cells.

The Air Liquid-interface, a Skin Microenvironment, Promotes Growth of Melanoma Cells, but not Their Apoptosis and Invasion, through Activation of Mitogen-activated Protein Kinase

International Nuclear Information System (INIS)

Hong Yee, Chong; Aoki, Shigehisa; Uchihashi, Kazuyoshi; Matsunobu, Aki; Yamasaki, Fumio; Misago, Noriyuki; Piao, Meihua; Tetsuji, Uemura; Yonemitsu, Nobuhisa; Sugihara, Hajime; Toda, Shuji

2010-01-01

The air-liquid interface (ALI) is a common microenvironment of the skin, but it is unknown whether the ALI affects melanoma cell behaviors. Using a collagen gel invasion assay, immunohistochemistry, and Western blots, here we show that melanoma cell proliferation in cultures with an ALI is higher than melanoma cell proliferation in submerged cultures. Bromodeoxyuridine (BrdU) uptake, an indicator of cell proliferation, of melanoma cells at the ALI was about 3 times that of submerged cells, while ALI and submerged melanoma cells had similar levels of single-stranded DNA (a marker of apoptosis). The ALI enhanced the expression of Raf-1, MEK-1 and pERK-1/2 components of the mitogen-activated protein kinase (MAPK) cascade, in cells more than the submerged condition did. The increases in BrdU uptake and pERK-1/2 expression promoted by ALI was abolished by the MEK inhibitor, PD-98059. ALI-treated and submerged melanoma cells did not infiltrate into the collagen gel, and they showed no significant difference in the expression of the invasion- and motility-related molecules, matrix metalloproteinase-1 and -9, laminin 5, and filamin A. Our data indicate that the ALI, a skin microenvironment, accelerates the growth, but not the apoptosis or invasion, of melanoma cells through MAPK activation

Arabidopsis mRNA polyadenylation machinery: comprehensive analysis of protein-protein interactions and gene expression profiling

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Mo Min

2008-05-01

Full Text Available Abstract Background The polyadenylation of mRNA is one of the critical processing steps during expression of almost all eukaryotic genes. It is tightly integrated with transcription, particularly its termination, as well as other RNA processing events, i.e. capping and splicing. The poly(A tail protects the mRNA from unregulated degradation, and it is required for nuclear export and translation initiation. In recent years, it has been demonstrated that the polyadenylation process is also involved in the regulation of gene expression. The polyadenylation process requires two components, the cis-elements on the mRNA and a group of protein factors that recognize the cis-elements and produce the poly(A tail. Here we report a comprehensive pairwise protein-protein interaction mapping and gene expression profiling of the mRNA polyadenylation protein machinery in Arabidopsis. Results By protein sequence homology search using human and yeast polyadenylation factors, we identified 28 proteins that may be components of Arabidopsis polyadenylation machinery. To elucidate the protein network and their functions, we first tested their protein-protein interaction profiles. Out of 320 pair-wise protein-protein interaction assays done using the yeast two-hybrid system, 56 (~17% showed positive interactions. 15 of these interactions were further tested, and all were confirmed by co-immunoprecipitation and/or in vitro co-purification. These interactions organize into three distinct hubs involving the Arabidopsis polyadenylation factors. These hubs are centered around AtCPSF100, AtCLPS, and AtFIPS. The first two are similar to complexes seen in mammals, while the third one stands out as unique to plants. When comparing the gene expression profiles extracted from publicly available microarray datasets, some of the polyadenylation related genes showed tissue-specific expression, suggestive of potential different polyadenylation complex configurations. Conclusion An

Auxin efflux by PIN-FORMED proteins is activated by two different protein kinases, D6 PROTEIN KINASE and PINOID

KAUST Repository

Zourelidou, Melina; Absmanner, Birgit; Weller, Benjamin; Barbosa, Inê s CR; Willige, Bjö rn C; Fastner, Astrid; Streit, Verena; Port, Sarah A; Colcombet, Jean; de la Fuente van Bentem, Sergio; Hirt, Heribert; Kuster, Bernhard; Schulze, Waltraud X; Hammes, Ulrich Z; Schwechheimer, Claus

2014-01-01

The development and morphology of vascular plants is critically determined by synthesis and proper distribution of the phytohormone auxin. The directed cell-to-cell distribution of auxin is achieved through a system of auxin influx and efflux transporters. PIN-FORMED (PIN) proteins are proposed auxin efflux transporters, and auxin fluxes can seemingly be predicted based on the-in many cells-asymmetric plasma membrane distribution of PINs. Here, we show in a heterologous Xenopus oocyte system as well as in Arabidopsis thaliana inflorescence stems that PIN-mediated auxin transport is directly activated by D6 PROTEIN KINASE (D6PK) and PINOID (PID)/WAG kinases of the Arabidopsis AGCVIII kinase family. At the same time, we reveal that D6PKs and PID have differential phosphosite preferences. Our study suggests that PIN activation by protein kinases is a crucial component of auxin transport control that must be taken into account to understand auxin distribution within the plant.

Auxin efflux by PIN-FORMED proteins is activated by two different protein kinases, D6 PROTEIN KINASE and PINOID

KAUST Repository

Zourelidou, Melina

2014-06-19

The development and morphology of vascular plants is critically determined by synthesis and proper distribution of the phytohormone auxin. The directed cell-to-cell distribution of auxin is achieved through a system of auxin influx and efflux transporters. PIN-FORMED (PIN) proteins are proposed auxin efflux transporters, and auxin fluxes can seemingly be predicted based on the-in many cells-asymmetric plasma membrane distribution of PINs. Here, we show in a heterologous Xenopus oocyte system as well as in Arabidopsis thaliana inflorescence stems that PIN-mediated auxin transport is directly activated by D6 PROTEIN KINASE (D6PK) and PINOID (PID)/WAG kinases of the Arabidopsis AGCVIII kinase family. At the same time, we reveal that D6PKs and PID have differential phosphosite preferences. Our study suggests that PIN activation by protein kinases is a crucial component of auxin transport control that must be taken into account to understand auxin distribution within the plant.

Novel mitogen-activated protein kinase MpkC of Aspergillus fumigatus is required for utilization of polyalcohol sugars.

Science.gov (United States)

Reyes, Guadalupe; Romans, Angela; Nguyen, C Kim; May, Gregory S

2006-11-01

The genome of Aspergillus fumigatus has four genes that encode mitogen-activated protein kinases (MAPKs), sakA/hogA, mpkA, mpkB, and mpkC. The functions of the MpkB and MpkC MAPKs are unknown for A. fumigatus and the closely related and genetically amenable species Aspergillus nidulans. mpkC deletion mutants of A. fumigatus were made and their phenotypes characterized. The mpkC deletion mutants were viable and had normal conidial germination and hyphal growth on minimal or complete media. This is in contrast to deletion mutants with deletions in the closely related MAPK gene sakA/hogA that we previously reported had a nitrogen source-dependent germination phenotype. Similarly, the growth of the mpkC deletion mutants was wild type on high-osmolarity medium. Consistent with these two MAP kinase genes regulating different cellular responses, we determined that the mpkC deletion mutants were unable to grow on minimal medium with sorbitol or mannitol as the sole carbon source. This result implicates MpkC signaling in carbon source utilization. Changes in mRNA levels for sakA and mpkC were measured in response to hypertonic stress, oxidative stress, and a shift from glucose to sorbitol to determine if there was overlap in the SakA and MpkC signaling pathways. These studies demonstrated that SakA- and MpkC-dependent patterns of change in mRNA levels are distinct and have minimal overlap in response to these environmental stresses.

Influence of protein depletion and repletion on lymphocyte activation in adult mice

International Nuclear Information System (INIS)

Elliott, T.S.; Olson, L.M.; Visek, W.J.

1986-01-01

Male 8 month old Balb/c mice were fed 0.5% (Depletion, n = 126) or 6% (Control, n = 16) protein diets for 5 wks. All control and 16 depleted mice were killed (time 0), and the remaining 110 mice were randomly assigned to 1 of 3 diets (3, 6, or 12% protein) and fed for 4, 8, or 32 additional days. Diets were formulated following AIN-76A guidelines with skim milk powder used as the source of protein. Serum albumin and 3 H-thymidine incorporation into splenocytes stimulated by the T-cell mitogens concanavalin A (Con A), or phytohemagglutinin (PHA), and the B-cell mitogen lipopolysaccharide (LPS) were determined. At time 0, mice fed 0.5% protein had lost 32% of their initial body weight and weighed significantly less than controls (19.5 +/- 0.1 g vs 29.9 +/- 0.7 g, p < 0.0001). Protein level did not affect food intake or spleen weight/body weight. Depletion significantly decreased serum albumin concentrations (4.6 +/- 0.1 g/dl vs 6.4 +/- 0.6 g/dl) and responses to PHA and LPS. During repletion mice fed 3% protein weighted significantly less than mice fed 6 or 12% ptn (p < 0.001) despite a significantly higher food intake. The activation of splenocytes by all mitogens increased with time of repletion but was independent of dietary protein concentration

Moss Pathogenesis-Related-10 protein enhances resistance to Pythium irregulare in Physcomitrella patens and Arabidopsis thaliana

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Alexandra eCastro

2016-04-01

Full Text Available Plants respond to pathogen infection by activating signaling pathways leading to the accumulation of proteins with diverse roles in defense. Here, we addressed the functional role of PpPR-10, a pathogenesis-related (PR-10 gene, of the moss Physcomitrella patens, in response to biotic stress. PpPR-10 belongs to a multigene family and encodes a protein twice the usual size of PR-10 proteins due to the presence of two Bet v1 domains. Moss PR-10 genes are differentially regulated during development and inoculation with the fungal pathogen Botrytis cinerea. Specifically, PpPR-10 transcript levels increase significantly by treatments with elicitors of Pectobacterium carotovorum subsp. carotovorum, spores of B. cinerea, and the defense hormone salicylic acid. To characterize the role of PpPR-10 in plant defense against pathogens, we conducted overexpression analysis in P. patens and in Arabidopsis thaliana. We demonstrate that constitutive expression of PpPR-10 in moss tissues increased resistance against the oomycete Pythium irregulare. PpPR-10 overexpressing moss plants developed less symptoms and decreased mycelium growth than wild type plants. In addition, PpPR-10 overexpressing plants constitutively produced cell wall depositions in protonemal tissue. Ectopic expression of PpPR-10 in Arabidopsis resulted in increased resistance against P. irregulare as well, evidenced by smaller lesions and less cellular damage compared to wild type plants. These results indicate that PpPR-10 is functionally active in the defense against the pathogen P. irregulare, in both P. patens and Arabidopsis, two evolutionary distant plants. Thus, P. patens can serve as an interesting source of genes to improve resistance against pathogen infection in flowering plants.

A nuclear-encoded chloroplast protein harboring a single CRM domain plays an important role in the Arabidopsis growth and stress response.

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Lee, Kwanuk; Lee, Hwa Jung; Kim, Dong Hyun; Jeon, Young; Pai, Hyun-Sook; Kang, Hunseung

2014-04-16

Although several chloroplast RNA splicing and ribosome maturation (CRM) domain-containing proteins have been characterized for intron splicing and rRNA processing during chloroplast gene expression, the functional role of a majority of CRM domain proteins in plant growth and development as well as chloroplast RNA metabolism remains largely unknown. Here, we characterized the developmental and stress response roles of a nuclear-encoded chloroplast protein harboring a single CRM domain (At4g39040), designated CFM4, in Arabidopsis thaliana. Analysis of CFM4-GFP fusion proteins revealed that CFM4 is localized to chloroplasts. The loss-of-function T-DNA insertion mutants for CFM4 (cfm4) displayed retarded growth and delayed senescence, suggesting that CFM4 plays a role in growth and development of plants under normal growth conditions. In addition, cfm4 mutants showed retarded seed germination and seedling growth under stress conditions. No alteration in the splicing patterns of intron-containing chloroplast genes was observed in the mutant plants, but the processing of 16S and 4.5S rRNAs was abnormal in the mutant plants. Importantly, CFM4 was determined to possess RNA chaperone activity. These results suggest that the chloroplast-targeted CFM4, one of two Arabidopsis genes encoding a single CRM domain-containing protein, harbors RNA chaperone activity and plays a role in the Arabidopsis growth and stress response by affecting rRNA processing in chloroplasts.

MAP kinase cascades in Arabidopsis innate immunity

DEFF Research Database (Denmark)

Rasmussen, Magnus Wohlfahrt; Roux, Milena Edna; Petersen, Morten

2012-01-01

Plant mitogen-activated protein kinase (MAPK) cascades generally transduce extracellular stimuli into cellular responses. These stimuli include the perception of pathogen-associated molecular patterns (PAMPs) by host transmembrane pattern recognition receptors which trigger MAPK-dependent innate ...

A role for seed storage proteins in Arabidopsis seed longevity

NARCIS (Netherlands)

Nguyen, Thu-Phuong|info:eu-repo/dai/nl/328228818; Cueff, Gwendal; Hegedus, Dwayne D; Rajjou, Loïc; Bentsink, Leónie|info:eu-repo/dai/nl/241338735

2015-01-01

Proteomics approaches have been a useful tool for determining the biological roles and functions of individual proteins and identifying the molecular mechanisms that govern seed germination, vigour and viability in response to ageing. In this work the dry seed proteome of four Arabidopsis thaliana

The Arabidopsis Malectin-Like/LRR-RLK IOS1 Is Critical for BAK1-Dependent and BAK1-Independent Pattern-Triggered Immunity

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Kadota, Yasuhiro; Huang, Pin-Yao; Chien, Hsiao-Chiao; Chu, Po-Wei; Zimmerli, Laurent

2016-01-01

Plasma membrane-localized pattern recognition receptors (PRRs) such as FLAGELLIN SENSING2 (FLS2), EF-TU RECEPTOR (EFR), and CHITIN ELICITOR RECEPTOR KINASE1 (CERK1) recognize microbe-associated molecular patterns (MAMPs) to activate pattern-triggered immunity (PTI). A reverse genetics approach on genes responsive to the priming agent β-aminobutyric acid (BABA) revealed IMPAIRED OOMYCETE SUSCEPTIBILITY1 (IOS1) as a critical PTI player. Arabidopsis thaliana ios1 mutants were hypersusceptible to Pseudomonas syringae bacteria. Accordingly, ios1 mutants showed defective PTI responses, notably delayed upregulation of the PTI marker gene FLG22-INDUCED RECEPTOR-LIKE KINASE1, reduced callose deposition, and mitogen-activated protein kinase activation upon MAMP treatment. Moreover, Arabidopsis lines overexpressing IOS1 were more resistant to bacteria and showed a primed PTI response. In vitro pull-down, bimolecular fluorescence complementation, coimmunoprecipitation, and mass spectrometry analyses supported the existence of complexes between the membrane-localized IOS1 and BRASSINOSTEROID INSENSITIVE1-ASSOCIATED KINASE1 (BAK1)-dependent PRRs FLS2 and EFR, as well as with the BAK1-independent PRR CERK1. IOS1 also associated with BAK1 in a ligand-independent manner and positively regulated FLS2-BAK1 complex formation upon MAMP treatment. In addition, IOS1 was critical for chitin-mediated PTI. Finally, ios1 mutants were defective in BABA-induced resistance and priming. This work reveals IOS1 as a novel regulatory protein of FLS2-, EFR-, and CERK1-mediated signaling pathways that primes PTI activation. PMID:27317676

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.

Comparative molecular modeling study of Arabidopsis NADPH-dependent thioredoxin reductase and its hybrid protein.

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Yuno Lee

Full Text Available 2-Cys peroxiredoxins (Prxs play important roles in the protection of chloroplast proteins from oxidative damage. Arabidopsis NADPH-dependent thioredoxin reductase isotype C (AtNTRC was identified as efficient electron donor for chloroplastic 2-Cys Prx-A. There are three isotypes (A, B, and C of thioredoxin reductase (TrxR in Arabidopsis. AtNTRA contains only TrxR domain, but AtNTRC consists of N-terminal TrxR and C-terminal thioredoxin (Trx domains. AtNTRC has various oligomer structures, and Trx domain is important for chaperone activity. Our previous experimental study has reported that the hybrid protein (AtNTRA-(Trx-D, which was a fusion of AtNTRA and Trx domain from AtNTRC, has formed variety of structures and shown strong chaperone activity. But, electron transfer mechanism was not detected at all. To find out the reason of this problem with structural basis, we performed two different molecular dynamics (MD simulations on AtNTRC and AtNTRA-(Trx-D proteins with same cofactors such as NADPH and flavin adenine dinucleotide (FAD for 50 ns. Structural difference has found from superimposition of two structures that were taken relatively close to average structure. The main reason that AtNTRA-(Trx-D cannot transfer the electron from TrxR domain to Trx domain is due to the difference of key catalytic residues in active site. The long distance between TrxR C153 and disulfide bond of Trx C387-C390 has been observed in AtNTRA-(Trx-D because of following reasons: i unstable and unfavorable interaction of the linker region, ii shifted Trx domain, and iii different or weak interface interaction of Trx domains. This study is one of the good examples for understanding the relationship between structure formation and reaction activity in hybrid protein. In addition, this study would be helpful for further study on the mechanism of electron transfer reaction in NADPH-dependent thioredoxin reductase proteins.

MAPK phosphatase AP2C3 induces ectopic proliferation of epidermal cells leading to stomata development in Arabidopsis.

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Julija Umbrasaite

2010-12-01

Full Text Available In plant post-embryonic epidermis mitogen-activated protein kinase (MAPK signaling promotes differentiation of pavement cells and inhibits initiation of stomata. Stomata are cells specialized to modulate gas exchange and water loss. Arabidopsis MAPKs MPK3 and MPK6 are at the core of the signaling cascade; however, it is not well understood how the activity of these pleiotropic MAPKs is constrained spatially so that pavement cell differentiation is promoted only outside the stomata lineage. Here we identified a PP2C-type phosphatase termed AP2C3 (Arabidopsis protein phosphatase 2C that is expressed distinctively during stomata development as well as interacts and inactivates MPK3, MPK4 and MPK6. AP2C3 co-localizes with MAPKs within the nucleus and this localization depends on its N-terminal extension. We show that other closely related phosphatases AP2C2 and AP2C4 are also MAPK phosphatases acting on MPK6, but have a distinct expression pattern from AP2C3. In accordance with this, only AP2C3 ectopic expression is able to stimulate cell proliferation leading to excess stomata development. This function of AP2C3 relies on the domains required for MAPK docking and intracellular localization. Concomitantly, the constitutive and inducible AP2C3 expression deregulates E2F-RB pathway, promotes the abundance and activity of CDKA, as well as changes of CDKB1;1 forms. We suggest that AP2C3 downregulates the MAPK signaling activity to help maintain the balance between differentiation of stomata and pavement cells.

Stress-induced activation of protein kinase CK2 by direct interaction with p38 mitogen-activated protein kinase

DEFF Research Database (Denmark)

Sayed, M; Kim, S O; Salh, B S

2000-01-01

Protein kinase CK2 has been implicated in the regulation of a wide range of proteins that are important in cell proliferation and differentiation. Here we demonstrate that the stress signaling agents anisomycin, arsenite, and tumor necrosis factor-alpha stimulate the specific enzyme activity of CK2...... in the human cervical carcinoma HeLa cells by up to 8-fold, and this could be blocked by the p38 MAP kinase inhibitor SB203580. We show that p38alpha MAP kinase, in a phosphorylation-dependent manner, can directly interact with the alpha and beta subunits of CK2 to activate the holoenzyme through what appears...

Proteomics reveal energy metabolism and mitogen-activated protein kinase signal transduction perturbation in human Borna disease virus Hu-H1-infected oligodendroglial cells.

Science.gov (United States)

Liu, X; Yang, Y; Zhao, M; Bode, L; Zhang, L; Pan, J; Lv, L; Zhan, Y; Liu, S; Zhang, L; Wang, X; Huang, R; Zhou, J; Xie, P

2014-05-30

Borna disease virus (BDV) is a neurotropic, non-cytolytic RNA virus which replicates in the cell nucleus targeting mainly hippocampal neurons, but also astroglial and oligodendroglial cells in the brain. BDV is associated with a large spectrum of neuropsychiatric pathologies in animals. Its relationship to human neuropsychiatric illness still remains controversial. We could recently demonstrate that human BDV strain Hu-H1 promoted apoptosis and inhibited cell proliferation in a human oligodendroglial cell line (OL cells) whereas laboratory BDV strain V acted contrariwise. Here, differential protein expression between BDV Hu-H1-infected OL cells and non-infected OL cells was assessed through a proteomics approach, using two-dimensional electrophoresis followed by matrix-assisted laser desorption ionization-time of flight tandem mass spectrometry. A total of 63 differential host proteins were identified in BDV Hu-H1-infected OL cells compared to non-infected OL cells. We found that most changes referred to alterations related to the pentose phosphate pathway, glyoxylate and dicarboxylate metabolism, the tricarboxylic acid (TCA) cycle, and glycolysis /gluconeogenesis. By manual querying, two differential proteins were found to be associated with mitogen-activated protein kinase (MAPK) signal transduction. Five key signaling proteins of this pathway (i.e., p-Raf, p-MEK, p-ERK1/2, p-RSK, and p-MSK) were selected for Western blotting validation. p-ERK1/2 and p-RSK were found to be significantly up-regulated, and p-MSK was found to be significantly down-regulated in BDV Hu-H1-infected OL cells compared to non-infected OL cell. Although BDV Hu-H1 constitutively activated the ERK-RSK pathway, host cell proliferation and nuclear translocation of activated pERK in BDV Hu-H1-infected OL cells were impaired. These findings indicate that BDV Hu-H1 infection of human oligodendroglial cells significantly perturbs host energy metabolism, activates the downstream ERK-RSK complex of

Monosodium iodoacetate-induced joint pain is associated with increased phosphorylation of mitogen activated protein kinases in the rat spinal cord

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Jarvis Michael F

2011-05-01

Full Text Available Abstract Background Intra-articular injection of monosodium iodoacetate (MIA in the knee joint of rats disrupts chondrocyte metabolism resulting in cartilage degeneration and subsequent nociceptive behavior that has been described as a model of osteoarthritis (OA pain. Central sensitization through activation of mitogen activated protein kinases (MAPKs is recognized as a pathogenic mechanism in chronic pain. In the present studies, induction of central sensitization as indicated by spinal dorsal horn MAPK activation, specifically ERK and p38 phosphorylation, was assessed in the MIA-OA model. Results Behaviorally, MIA-injected rats displayed reduced hind limb grip force 1, 2, and 3 weeks post-MIA treatment. In the same animals, activation of phospho ERK1/2 was gradually increased, reaching a significant level at post injection week 3. Conversely, phosphorylation of p38 MAPK was enhanced maximally at post injection week 1 and decreased, but remained elevated, thereafter. Double labeling from 3-wk MIA rats demonstrated spinal pERK1/2 expression in neurons, but not glia. In contrast, p-p38 was expressed by microglia and a subpopulation of neurons, but not astrocytes. Additionally, there was increased ipsilateral expression of microglia, but not astrocytes, in 3-wk MIA-OA rats. Consistent with increased MAPK immunoreactivity in the contralateral dorsal horn, mechanical allodynia to the contralateral hind-limb was observed 3-wk following MIA. Finally, intrathecal injection of the MEK1 inhibitor PD98059 blocked both reduced hind-limb grip force and pERK1/2 induction in MIA-OA rats. Conclusion Results of these studies support the role of MAPK activation in the progression and maintenance of central sensitization in the MIA-OA experimental pain model.

Sorbitol dehydrogenase is a cytosolic protein required for sorbitol metabolism in Arabidopsis thaliana.

Science.gov (United States)

Aguayo, María Francisca; Ampuero, Diego; Mandujano, Patricio; Parada, Roberto; Muñoz, Rodrigo; Gallart, Marta; Altabella, Teresa; Cabrera, Ricardo; Stange, Claudia; Handford, Michael

2013-05-01

Sorbitol is converted to fructose in Rosaceae species by SORBITOL DEHYDROGENASE (SDH, EC 1.1.1.14), especially in sink organs. SDH has also been found in non-Rosaceae species and here we show that the protein encoded by At5g51970 in Arabidopsis thaliana (L.) Heynh. possesses the molecular characteristics of an SDH. Using a green fluorescent protein-tagged version and anti-SDH antisera, we determined that SDH is cytosolically localized, consistent with bioinformatic predictions. We also show that SDH is widely expressed, and that SDH protein accumulates in both source and sink organs. In the presence of NAD+, recombinant SDH exhibited greatest oxidative activity with sorbitol, ribitol and xylitol as substrates; other sugar alcohols were oxidized to a lesser extent. Under standard growth conditions, three independent sdh- mutants developed as wild-type. Nevertheless, all three exhibited reduced dry weight and primary root length compared to wild-type when grown in the presence of sorbitol. Additionally, under short-day conditions, the mutants were more resistant to dehydration stress, as shown by a reduced loss of leaf water content when watering was withheld, and a greater survival rate on re-watering. This evidence suggests that limitations in the metabolism of sugar alcohols alter the growth of Arabidopsis and its response to drought. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Site-directed mutagenesis of the Arabidopsis heterotrimeric G protein β subunit suggests divergent mechanisms of effector activation between plant and animal G proteins.

Science.gov (United States)

Chakravorty, David; Trusov, Yuri; Botella, José Ramón

2012-03-01

Heterotrimeric G proteins are integral components of signal transduction in humans and other mammals and have been therefore extensively studied. However, while they are known to mediate many processes, much less is currently known about the effector pathways and molecular mechanisms used by these proteins to regulate effectors in plants. We designed a complementation strategy to study G protein signaling in Arabidopsis thaliana, particularly the mechanism of action of AGB1, the sole identified β subunit. We used biochemical and effector regulation data from human G protein studies to identify four potentially important residues for site-directed mutagenesis (T65, M111, D250 and W361 of AGB1). Each residue was individually mutated and the resulting mutated protein introduced in the agb1-2 mutant background under the control of the native AGB1 promoter. Interestingly, even though these mutations have been shown to have profound effects on effector signaling in humans, all the mutated subunits were able to restore thirteen of the fifteen Gβ-deficient phenotypes characterized in this study. Only one mutated protein, T65A was unable to complement the hypersensitivity to mannitol during germination observed in agb1 mutants; while only D250A failed to restore lateral root numbers in the agb1 mutant to wild-type levels. Our results suggest that the mechanisms used in mammalian G protein signaling are not well conserved in plant G protein signaling, and that either the effectors used by plant G proteins, or the mechanisms used to activate them, are at least partially divergent from the well-studied mammalian G proteins.

The investigation of Mitogen-Activated Protein kinase Phosphatase-1 as a potential pharmacological target in non-small cell lung carcinomas, assisted by non-invasive molecular imaging

International Nuclear Information System (INIS)

Tai, Cheng-Jeng; Lee, Horng-Mo; Deng, Win-Ping; Wu, Alexander TH; Chiou, Jeng-Feng; Jan, Hsun-Jin; Wei, Hon-Jian; Hsu, Chung-Huei; Lin, Che-Tong; Chiu, Wen-Ta; Wu, Cheng-Wen

2010-01-01

Invasiveness and metastasis are the most common characteristics of non small cell lung cancer (NSCLC) and causes of tumour-related morbidity and mortality. Mitogen-activated protein kinases (MAPKs) signalling pathways have been shown to play critical roles in tumorigenesis. However, the precise pathological role(s) of mitogen-activated protein kinase phosphatase-1 (MKP-1) in different cancers has been controversial such that the up-regulation of MKP-1 in different cancers does not always correlate to a better prognosis. In this study, we showed that the induction of MKP-1 lead to a significant retardation of proliferation and metastasis in NSCLC cells. We also established that rosiglitazone (a PPARγ agonist) elevated MKP-1 expression level in NSCLC cells and inhibited tumour metastasis. Both wildtype and dominant negative forms of MKP-1 were constitutively expressed in NSCLC cell line H441GL. The migration and invasion abilities of these cells were examined in vitro. MKP-1 modulating agents such as rosiglitazone and triptolide were used to demonstrate MKP-1's role in tumorigenesis. Bioluminescent imaging was utilized to study tumorigenesis of MKP-1 over-expressing H441GL cells and anti-metastatic effect of rosiglitazone. Over-expression of MKP-1 reduced NSCLC cell proliferation rate as well as cell invasive and migratory abilities, evident by the reduced expression levels of MMP-2 and CXCR4. Mice inoculated with MKP-1 over-expressing H441 cells did not develop NSCLC while their control wildtype H441 inoculated littermates developed NSCLC and bone metastasis. Pharmacologically, rosiglitazone, a peroxisome proliferator activated receptor-γ (PPARγ) agonist appeared to induce MKP-1 expression while reduce MMP-2 and CXCR4 expression. H441GL-inoculated mice receiving daily oral rosiglitazone treatment demonstrated a significant inhibition of bone metastasis when compared to mice receiving sham treatment. We found that rosiglitazone treatment impeded the ability

Lysophosphatidylcholine Promotes Phagosome Maturation and Regulates Inflammatory Mediator Production Through the Protein Kinase A–Phosphatidylinositol 3 Kinase–p38 Mitogen-Activated Protein Kinase Signaling Pathway During Mycobacterium tuberculosis Infection in Mouse Macrophages

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Hyo-Ji Lee

2018-04-01

Full Text Available Tuberculosis is caused by the infectious agent Mycobacterium tuberculosis (Mtb. Mtb has various survival strategies, including blockade of phagosome maturation and inhibition of antigen presentation. Lysophosphatidylcholine (LPC is a major phospholipid component of oxidized low-density lipoprotein and is involved in various cellular responses, such as activation of second messengers and bactericidal activity in neutrophils. In this study, macrophages were infected with a low infectious dose of Mtb and treated with LPC to investigate the bactericidal activity of LPC against Mtb. In macrophages infected with Mtb strain, H37Ra or H37Rv, LPC suppressed bacterial growth; however, this effect was suppressed in bone marrow-derived macrophages (BMDMs isolated from G2A (a G protein-coupled receptor involved in some LPC actions knockout mice. LPC also promoted phagosome maturation via phosphatidylinositol 3 kinase (PI3K–p38 mitogen-activated protein kinase (MAPK-mediated reactive oxygen species production and intracellular Ca2+ release during Mtb infection. In addition, LPC induced increased levels of intracellular cyclic adenosine monophosphate (cAMP and phosphorylated glycogen synthase kinase 3 beta (GSK3β in Mtb-infected macrophages. Protein kinase A (PKA-induced phosphorylation of GSK3β suppressed activation of NF-κB in LPC-treated macrophages during Mtb infection, leading to decreased secretion of pro-inflammatory cytokines and increased secretion of anti-inflammatory cytokines. These results suggest that LPC can effectively control Mtb growth by promoting phagosome maturation via cAMP-induced activation of the PKA–PI3K–p38 MAPK pathway. Moreover, LPC can regulate excessive production of pro-inflammatory cytokines associated with bacterial infection of macrophages.

Arabidopsis SEIPIN Proteins Modulate Triacylglycerol Accumulation and Influence Lipid Droplet Proliferation[OPEN

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2015-01-01

The lipodystrophy protein SEIPIN is important for lipid droplet (LD) biogenesis in human and yeast cells. In contrast with the single SEIPIN genes in humans and yeast, there are three SEIPIN homologs in Arabidopsis thaliana, designated SEIPIN1, SEIPIN2, and SEIPIN3. Essentially nothing is known about the functions of SEIPIN homologs in plants. Here, a yeast (Saccharomyces cerevisiae) SEIPIN deletion mutant strain and a plant (Nicotiana benthamiana) transient expression system were used to test the ability of Arabidopsis SEIPINs to influence LD morphology. In both species, expression of SEIPIN1 promoted accumulation of large-sized lipid droplets, while expression of SEIPIN2 and especially SEIPIN3 promoted small LDs. Arabidopsis SEIPINs increased triacylglycerol levels and altered composition. In tobacco, endoplasmic reticulum (ER)-localized SEIPINs reorganized the normal, reticulated ER structure into discrete ER domains that colocalized with LDs. N-terminal deletions and swapping experiments of SEIPIN1 and 3 revealed that this region of SEIPIN determines LD size. Ectopic overexpression of SEIPIN1 in Arabidopsis resulted in increased numbers of large LDs in leaves, as well as in seeds, and increased seed oil content by up to 10% over wild-type seeds. By contrast, RNAi suppression of SEIPIN1 resulted in smaller seeds and, as a consequence, a reduction in the amount of oil per seed compared with the wild type. Overall, our results indicate that Arabidopsis SEIPINs are part of a conserved LD biogenesis machinery in eukaryotes and that in plants these proteins may have evolved specialized roles in the storage of neutral lipids by differentially modulating the number and sizes of lipid droplets. PMID:26362606

Arabidopsis RIBA Proteins: Two out of Three Isoforms Have Lost Their Bifunctional Activity in Riboflavin Biosynthesis

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Hiltunen, Hanna-Maija; Illarionov, Boris; Hedtke, Boris; Fischer, Markus; Grimm, Bernhard

2012-01-01

Riboflavin serves as a precursor for flavocoenzymes (FMN and FAD) and is essential for all living organisms. The two committed enzymatic steps of riboflavin biosynthesis are performed in plants by bifunctional RIBA enzymes comprised of GTP cyclohydrolase II (GCHII) and 3,4-dihydroxy-2-butanone-4-phosphate synthase (DHBPS). Angiosperms share a small RIBA gene family consisting of three members. A reduction of AtRIBA1 expression in the Arabidopsis rfd1mutant and in RIBA1 antisense lines is not complemented by the simultaneously expressed isoforms AtRIBA2 and AtRIBA3. The intensity of the bleaching leaf phenotype of RIBA1 deficient plants correlates with the inactivation of AtRIBA1 expression, while no significant effects on the mRNA abundance of AtRIBA2 and AtRIBA3 were observed. We examined reasons why both isoforms fail to sufficiently compensate for a lack of RIBA1 expression. All three RIBA isoforms are shown to be translocated into chloroplasts as GFP fusion proteins. Interestingly, both AtRIBA2 and AtRIBA3 have amino acid exchanges in conserved peptides domains that have been found to be essential for the two enzymatic functions. In vitro activity assays of GCHII and DHBPS with all of the three purified recombinant AtRIBA proteins and complementation of E. coli ribA and ribB mutants lacking DHBPS and GCHII expression, respectively, confirmed the loss of bifunctionality for AtRIBA2 and AtRIBA3. Phylogenetic analyses imply that the monofunctional, bipartite RIBA3 proteins, which have lost DHBPS activity, evolved early in tracheophyte evolution. PMID:23203051

KFC, a Ste20-like kinase with mitogenic potential and capability to activate the SAPK/JNK pathway.

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Yustein, J T; Li, D; Robinson, D; Kung, H J

2000-02-03

The Sterile-20 (Ste20) family of serine-threonine kinases has been implicated in the activation of the stress-activated protein kinase pathways. However, the physiological role has remained ambiguous for most of the investigated mammalian Ste20's. Here we report the cloning of a novel Ste20-like kinase, from chicken embryo fibroblast (CEF) cells, which we have named KFC, for Kinase From Chicken. The 898 amino acid full-length KFC protein contains an amino-terminal kinase domain, an adjacent downstream serine-rich region, and a C-terminal tail containing a coiled-coil domain. Here we show that the coiled-coil domain of KFC negatively regulates the intrinsic kinase activity. We have also identified a splice variant of KFC in which there is a 207 nucleotide in-frame deletion. This deletion of 69 amino acids encompasses the serine-rich region. These two isoforms, called KFCL, for full-length, and KFCS for spliced (or short) form, not only differ in structure, but also in biological properties. Stable CEF cells overexpressing KFCL, but not KFCS, have a significant increase in growth rate when compared to parental cells. This mitogenic effect is the first such reported for this family of kinases. Finally, we found that KFC, when activated by truncation of the regulatory C-terminus, has a specific activation of the stress-activated protein kinase (SAPK/JNK) pathway.

p38 mitogen-activated protein kinase (p38MAPK) upregulates catalase levels in response to low dose H2O2 treatment through enhancement of mRNA stability.

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Sen, Prosenjit; Chakraborty, Prabir Kumar; Raha, Sanghamitra

2005-08-15

V79 fibroblasts were repetitively stressed through multiple exposures to a low dose (30 microM) H2O2 in culture for 4 weeks. Catalase activity, protein levels and mRNA levels increased markedly (5-6-fold) during this time and these augmentations were inhibited by the simultaneous presence of SB203580, an inhibitor of p38 mitogen-activated protein kinase (p38MAPK). p38MAPK became dually phosphorylated and ATF-2, a p38MAPK substrate also became increasingly phosphorylated over the repetitive stress period. Short interfering RNA that induced effective silencing of p38MAPK, was used to silence p38MAPK in V79 fibroblasts. Silencing of p38MAPK drastically hindered the elevation in catalase (protein and mRNA) levels observed after a single low dose (50 microM) of H2O. The rise in catalase mRNA levels induced by low concentration (single and multiple dose) H2O2 treatment was established to be unconnected with transcriptional upregulation but was brought forth primarily by an enhancement in catalase mRNA stability through the action of p38MAPK. Therefore, our data strongly indicate that activation of p38MAPK is a key controlling step in the upregulation of catalase levels by low dose H2O2 treatment.

Novel Mitogen-Activated Protein Kinase MpkC of Aspergillus fumigatus Is Required for Utilization of Polyalcohol Sugars▿

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Reyes, Guadalupe; Romans, Angela; Nguyen, C. Kim; May, Gregory S.

2006-01-01

The genome of Aspergillus fumigatus has four genes that encode mitogen-activated protein kinases (MAPKs), sakA/hogA, mpkA, mpkB, and mpkC. The functions of the MpkB and MpkC MAPKs are unknown for A. fumigatus and the closely related and genetically amenable species Aspergillus nidulans. mpkC deletion mutants of A. fumigatus were made and their phenotypes characterized. The mpkC deletion mutants were viable and had normal conidial germination and hyphal growth on minimal or complete media. This is in contrast to deletion mutants with deletions in the closely related MAPK gene sakA/hogA that we previously reported had a nitrogen source-dependent germination phenotype. Similarly, the growth of the mpkC deletion mutants was wild type on high-osmolarity medium. Consistent with these two MAP kinase genes regulating different cellular responses, we determined that the mpkC deletion mutants were unable to grow on minimal medium with sorbitol or mannitol as the sole carbon source. This result implicates MpkC signaling in carbon source utilization. Changes in mRNA levels for sakA and mpkC were measured in response to hypertonic stress, oxidative stress, and a shift from glucose to sorbitol to determine if there was overlap in the SakA and MpkC signaling pathways. These studies demonstrated that SakA- and MpkC-dependent patterns of change in mRNA levels are distinct and have minimal overlap in response to these environmental stresses. PMID:16998074

Crosstalk between Smad and Mitogen-Activated Protein Kinases for the Regulation of Apoptosis in Cyclosporine A- Induced Renal Tubular Injury

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Hideyuki Iwayama

2011-10-01

Full Text Available Background/Aims: It remains elusive whether there is a crosstalk between Smad and mitogen-activated protein kinases (MAPKs and whether it regulates cyclosporine A (CyA-induced apoptosis in renal proximal tubular cells (RPTCs. Methods: The effect of CyA on nuclear translocation of Smad2/3 and MAPKs (measured by Western blotting or immunofluorescence and apoptosis (determined by Hoechst 33258 staining was examined in HK-2 cells. Results: CyA induced apoptosis at 24 h and nuclear translocation of phosphorylated (p-Smad2/3 at 3 h, which was continued till 24 h. CyA enhanced the expression of p-ERK at 1 h, which was continued till 24 h, and of p-p38MAPK at 1–6 h, which returned to control level at 12 h. CyA did not affect JNK. An inhibitor of ERK, PD98059, prevented CyA-induced nuclear translocation of Smad2/3 and apoptosis. An inhibitor of p38MAPK, SB202190, deteriorated CyA-induced nuclear translocation of p-Smad2/3. Epidermal growth factor (EGF activated ERK and p38MAPK but not JNK. EGF-induced activation of MAPKs ameliorated CyA-induced nuclear translocation of p-Smad2/3 and apoptosis. Inhibition of p38MAPK but not of ERK abolished the protective effect of EGF on CyA-induced nuclear translocation of p-Smad2/3 and apoptosis. Conclusion: Crosstalk between R-Smad and p38MAPK/ERK, but not JNK differentially regulates apoptosis in CyA-induced RPTC injury.

Arabidopsis Lectin Receptor Kinases LecRK-IX.1 and LecRK-IX.2 Are Functional Analogs in Regulating Phytophthora Resistance and Plant Cell Death.

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Wang, Yan; Cordewener, Jan H G; America, Antoine H P; Shan, Weixing; Bouwmeester, Klaas; Govers, Francine

2015-09-01

L-type lectin receptor kinases (LecRK) are potential immune receptors. Here, we characterized two closely-related Arabidopsis LecRK, LecRK-IX.1 and LecRK-IX.2, of which T-DNA insertion mutants showed compromised resistance to Phytophthora brassicae and Phytophthora capsici, with double mutants showing additive susceptibility. Overexpression of LecRK-IX.1 or LecRK-IX.2 in Arabidopsis and transient expression in Nicotiana benthamiana increased Phytophthora resistance but also induced cell death. Phytophthora resistance required both the lectin domain and kinase activity, but for cell death, the lectin domain was not needed. Silencing of the two closely related mitogen-activated protein kinase genes NbSIPK and NbNTF4 in N. benthamiana completely abolished LecRK-IX.1-induced cell death but not Phytophthora resistance. Liquid chromatography-mass spectrometry analysis of protein complexes coimmunoprecipitated in planta with LecRK-IX.1 or LecRK-IX.2 as bait, resulted in the identification of the N. benthamiana ABC transporter NbPDR1 as a potential interactor of both LecRK. The closest homolog of NbPDR1 in Arabidopsis is ABCG40, and coimmunoprecipitation experiments showed that ABCG40 associates with LecRK-IX.1 and LecRK-IX.2 in planta. Similar to the LecRK mutants, ABCG40 mutants showed compromised Phytophthora resistance. This study shows that LecRK-IX.1 and LecRK-IX.2 are Phytophthora resistance components that function independent of each other and independent of the cell-death phenotype. They both interact with the same ABC transporter, suggesting that they exploit similar signal transduction pathways.

Comparative differential gene expression analysis of nucleus-encoded proteins for Rafflesia cantleyi against Arabidopsis thaliana

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Ng, Siuk-Mun; Lee, Xin-Wei; Wan, Kiew-Lian; Firdaus-Raih, Mohd

2015-09-01

Regulation of functional nucleus-encoded proteins targeting the plastidial functions was comparatively studied for a plant parasite, Rafflesia cantleyi versus a photosynthetic plant, Arabidopsis thaliana. This study involved two species of different feeding modes and different developmental stages. A total of 30 nucleus-encoded proteins were found to be differentially-regulated during two stages in the parasite; whereas 17 nucleus-encoded proteins were differentially-expressed during two developmental stages in Arabidopsis thaliana. One notable finding observed for the two plants was the identification of genes involved in the regulation of photosynthesis-related processes where these processes, as expected, seem to be present only in the autotroph.

17beta-estradiol promotes the odonto/osteogenic differentiation of stem cells from apical papilla via mitogen-activated protein kinase pathway.

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Li, Yao; Yan, Ming; Wang, Zilu; Zheng, Yangyu; Li, Junjun; Ma, Shu; Liu, Genxia; Yu, Jinhua

2014-11-17

Estrogen plays an important role in the osteogenic differentiation of mesenchymal stem cells, while stem cells from apical papilla (SCAP) can contribute to the formation of dentin/bone-like tissues. To date, the effects of estrogen on the differentiation of SCAP remain unclear. SCAP was isolated and treated with 10⁻⁷ M 17beta-estradiol (E2). The odonto/osteogenic potency and the involvement of mitogen-activated protein kinase (MAPK) signaling pathway were subsequently investigated by using methyl-thiazolyl-tetrazolium (MTT) assay, and other methods. MTT and flow cytometry results demonstrated that E2 treatment had no effect on the proliferation of SCAP in vitro, while alkaline phosphatase (ALP) assay and alizarin red staining showed that E2 can significantly promote ALP activity and mineralization ability in SCAP. Real-time reverse transcription polymerase chain reaction (RT-PCR) and western blot assay revealed that the odonto/osteogenic markers (ALP, DMP1/DMP1, DSPP/DSP, RUNX2/RUNX2, OSX/OSX and OCN/OCN) were significantly upregulated in E2-treated SCAP. In addition, the expression of phosphor-p38 and phosphor-JNK in these stem cells was enhanced by E2 treatment, as was the expression of the nuclear downstream transcription factors including phosphor-Sp1, phosphor-Elk-1, phosphor-c-Jun and phosphor-c-Fos, indicating the activation of MAPK signaling pathway during the odonto/osteogenic differentiation of E2-treated SCAP. Conversely, the differentiation of E2-treated SCAP was inhibited in the presence of MAPK specific inhibitors. The ondonto/osteogenic differentiation of SCAP is enhanced by 10⁻⁷ M 17beta-estradiol via the activation of MAPK signaling pathway.

A double-mutant collection targeting MAP kinase related genes in Arabidopsis for studying genetic interactions.

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Su, Shih-Heng; Krysan, Patrick J

2016-12-01

Mitogen-activated protein kinase cascades are conserved in all eukaryotes. In Arabidopsis thaliana there are approximately 80 genes encoding MAP kinase kinase kinases (MAP3K), 10 genes encoding MAP kinase kinases (MAP2K), and 20 genes encoding MAP kinases (MAPK). Reverse genetic analysis has failed to reveal abnormal phenotypes for a majority of these genes. One strategy for uncovering gene function when single-mutant lines do not produce an informative phenotype is to perform a systematic genetic interaction screen whereby double-mutants are created from a large library of single-mutant lines. Here we describe a new collection of 275 double-mutant lines derived from a library of single-mutants targeting genes related to MAP kinase signaling. To facilitate this study, we developed a high-throughput double-mutant generating pipeline using a system for growing Arabidopsis seedlings in 96-well plates. A quantitative root growth assay was used to screen for evidence of genetic interactions in this double-mutant collection. Our screen revealed four genetic interactions, all of which caused synthetic enhancement of the root growth defects observed in a MAP kinase 4 (MPK4) single-mutant line. Seeds for this double-mutant collection are publicly available through the Arabidopsis Biological Resource Center. Scientists interested in diverse biological processes can now screen this double-mutant collection under a wide range of growth conditions in order to search for additional genetic interactions that may provide new insights into MAP kinase signaling. © 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.

The FANTASTIC FOUR proteins influence shoot meristem size in Arabidopsis thaliana

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Brand Luise H

2010-12-01

Full Text Available Abstract Background Throughout their lives plants produce new organs from groups of pluripotent cells called meristems, located at the tips of the shoot and the root. The size of the shoot meristem is tightly controlled by a feedback loop, which involves the homeodomain transcription factor WUSCHEL (WUS and the CLAVATA (CLV proteins. This regulatory circuit is further fine-tuned by morphogenic signals such as hormones and sugars. Results Here we show that a family of four plant-specific proteins, encoded by the FANTASTIC FOUR (FAF genes, has the potential to regulate shoot meristem size in Arabidopsis thaliana. FAF2 and FAF4 are expressed in the centre of the shoot meristem, overlapping with the site of WUS expression. Consistent with a regulatory interaction between the FAF gene family and WUS, our experiments indicate that the FAFs can repress WUS, which ultimately leads to an arrest of meristem activity in FAF overexpressing lines. The finding that meristematic expression of FAF2 and FAF4 is under negative control by CLV3 further supports the hypothesis that the FAFs are modulators of the genetic circuit that regulates the meristem. Conclusion This study reports the initial characterization of the Arabidopsis thaliana FAF gene family. Our data indicate that the FAF genes form a plant specific gene family, the members of which have the potential to regulate the size of the shoot meristem by modulating the CLV3-WUS feedback loop.

The FANTASTIC FOUR proteins influence shoot meristem size in Arabidopsis thaliana.

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Wahl, Vanessa; Brand, Luise H; Guo, Ya-Long; Schmid, Markus

2010-12-22

Throughout their lives plants produce new organs from groups of pluripotent cells called meristems, located at the tips of the shoot and the root. The size of the shoot meristem is tightly controlled by a feedback loop, which involves the homeodomain transcription factor WUSCHEL (WUS) and the CLAVATA (CLV) proteins. This regulatory circuit is further fine-tuned by morphogenic signals such as hormones and sugars. Here we show that a family of four plant-specific proteins, encoded by the FANTASTIC FOUR (FAF) genes, has the potential to regulate shoot meristem size in Arabidopsis thaliana. FAF2 and FAF4 are expressed in the centre of the shoot meristem, overlapping with the site of WUS expression. Consistent with a regulatory interaction between the FAF gene family and WUS, our experiments indicate that the FAFs can repress WUS, which ultimately leads to an arrest of meristem activity in FAF overexpressing lines. The finding that meristematic expression of FAF2 and FAF4 is under negative control by CLV3 further supports the hypothesis that the FAFs are modulators of the genetic circuit that regulates the meristem. This study reports the initial characterization of the Arabidopsis thaliana FAF gene family. Our data indicate that the FAF genes form a plant specific gene family, the members of which have the potential to regulate the size of the shoot meristem by modulating the CLV3-WUS feedback loop.

Arabidopsis mitochondrial protein slow embryo development1 is essential for embryo development

International Nuclear Information System (INIS)

Ju, Yan; Liu, Chunying; Lu, Wenwen; Zhang, Quan; Sodmergen

2016-01-01

The plant seeds formation are crucial parts in reproductive process in seed plants as well as food source for humans. Proper embryo development ensure viable seed formation. Here, we showed an Arabidopsis T-DNA insertion mutant slow embryo development1 (sed1) which exhibited retarded embryogenesis, led to aborted seeds. Embryo without SED1 developed slower compared to normal one and could be recognized at early globular stage by its white appearance. In later development stage, storage accumulated poorly with less protein and lipid body production. In vitro culture did not rescue albino embryo. SED1 encoded a protein targeted to mitochondria. Transmission electron microscopic analysis revealed that mitochondria developed abnormally, and more strikingly plastid failed to construct grana in time in sed1/sed1 embryo. These data indicated that SED1 is indispensable for embryogenesis in Arabidopsis, and the mitochondria may be involved in the regulation of many aspects of seed development. -- Highlights: •Arabidopsis SED1 is essential for embryo development. •The sed1 embryo accumulates less storage and has abnormal ultrastructure. •SED1 localizes to the mitochondrion.

Arabidopsis mitochondrial protein slow embryo development1 is essential for embryo development

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Ju, Yan; Liu, Chunying; Lu, Wenwen; Zhang, Quan; Sodmergen, E-mail: sodmergn@pku.edu.cn

2016-05-27

The plant seeds formation are crucial parts in reproductive process in seed plants as well as food source for humans. Proper embryo development ensure viable seed formation. Here, we showed an Arabidopsis T-DNA insertion mutant slow embryo development1 (sed1) which exhibited retarded embryogenesis, led to aborted seeds. Embryo without SED1 developed slower compared to normal one and could be recognized at early globular stage by its white appearance. In later development stage, storage accumulated poorly with less protein and lipid body production. In vitro culture did not rescue albino embryo. SED1 encoded a protein targeted to mitochondria. Transmission electron microscopic analysis revealed that mitochondria developed abnormally, and more strikingly plastid failed to construct grana in time in sed1/sed1 embryo. These data indicated that SED1 is indispensable for embryogenesis in Arabidopsis, and the mitochondria may be involved in the regulation of many aspects of seed development. -- Highlights: •Arabidopsis SED1 is essential for embryo development. •The sed1 embryo accumulates less storage and has abnormal ultrastructure. •SED1 localizes to the mitochondrion.

Abscisic Acid (ABA) Regulation of Arabidopsis SR Protein Gene Expression

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Cruz, Tiago M. D.; Carvalho, Raquel F.; Richardson, Dale N.; Duque, Paula

2014-01-01

Serine/arginine-rich (SR) proteins are major modulators of alternative splicing, a key generator of proteomic diversity and flexible means of regulating gene expression likely to be crucial in plant environmental responses. Indeed, mounting evidence implicates splicing factors in signal transduction of the abscisic acid (ABA) phytohormone, which plays pivotal roles in the response to various abiotic stresses. Using real-time RT-qPCR, we analyzed total steady-state transcript levels of the 18 SR and two SR-like genes from Arabidopsis thaliana in seedlings treated with ABA and in genetic backgrounds with altered expression of the ABA-biosynthesis ABA2 and the ABA-signaling ABI1 and ABI4 genes. We also searched for ABA-responsive cis elements in the upstream regions of the 20 genes. We found that members of the plant-specific SC35-Like (SCL) Arabidopsis SR protein subfamily are distinctively responsive to exogenous ABA, while the expression of seven SR and SR-related genes is affected by alterations in key components of the ABA pathway. Finally, despite pervasiveness of established ABA-responsive promoter elements in Arabidopsis SR and SR-like genes, their expression is likely governed by additional, yet unidentified cis-acting elements. Overall, this study pinpoints SR34, SR34b, SCL30a, SCL28, SCL33, RS40, SR45 and SR45a as promising candidates for involvement in ABA-mediated stress responses. PMID:25268622

Lack of direct mitogenic activity of dichloroacetate and trichloroacetate in cultured rat hepatocytes

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Walgren, Jennie L.; Kurtz, David T.; McMillan, JoEllyn M.

2005-01-01

Dichloroacetate (DCA) and trichloroacetate (TCA) are hepatocarcinogenic metabolites of the common groundwater contaminant, 1,1,2-trichloroethylene. DCA and TCA have been shown to induce hepatocyte proliferation in vivo, but it is not known if this response is the result of direct mitogenic activity or whether cell replication occurs indirectly in response to tissue injury or inflammation. In this study we used primary cultures of rat hepatocytes, a species susceptible to DCA- but not TCA-induced hepatocarcinogenesis, to determine whether DCA and TCA are direct hepatocyte mitogens. Rat hepatocytes, cultured in growth factor-free medium, were treated with 0.01-1.0 mM DCA or TCA for 10-40 h; cell replication was then assessed by measuring incorporation of 3 H-thymidine into DNA and by cell counts. DCA or TCA treatment did not alter 3 H-thymidine incorporation in the cultured hepatocytes. Although an increase in cell number was not observed, DCA treatment significantly abrogated the normal background cell loss, suggesting an ability to inhibit apoptotic cell death in primary hepatocyte cultures. Furthermore, treatment with DCA synergistically enhanced the mitogenic response to epidermal growth factor. The data indicate that DCA and TCA are not direct mitogens in hepatocyte cultures, which is of interest in view of their ability to stimulate hepatocyte replication in vivo. Nevertheless, the synergistic enhancement of epidermal growth factor-induced hepatocyte replication by DCA is of particular interest and warrants further study

3D structure prediction of histone acetyltransferase (HAC proteins of the p300/CBP family and their interactome in Arabidopsis thaliana

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Amar Cemanovic

2014-09-01

Full Text Available Histone acetylation is an important posttranslational modification correlated with gene activation. In Arabidopsis thaliana the histone acetyltransferase (HAC proteins of the CBP family are homologous to animal p300/CREB (cAMP-responsive element-binding proteins, which are important histone acetyltransferases participating in many physiological processes, including proliferation, differentiation, and apoptosis. In this study the 3-D structure of all HAC protein subunits in Arabidopsis thaliana: HAC1, HAC2, HAC4, HAC5 and HAC12 is predicted by homology modeling and confirmed by Ramachandran plot analysis. The amino acid sequences HAC family members are highly similar to the sequences of the homologous human p300/CREB protein. Conservation of p300/CBP domains among the HAC proteins was examined further by sequence alignment and pattern search. The domains of p300/CBP required for the HAC function, such as PHD, TAZ and ZZ domains, are conserved in all HAC proteins. Interactome analysis revealed that HAC1, HAC5 and HAC12 proteins interact with S-adenosylmethionine-dependent methyltransferase domaincontaining protein that shows methyltransferase activity, suggesting an additional function of the HAC proteins. Additionally, HAC5 has a strong interaction value for the putative c-myb-like transcription factor MYB3R-4, which suggests that it also may have a function in regulation of DNA replication.

Induced overexpression of protein kinase D1 stimulates mitogenic signaling in human pancreatic carcinoma PANC-1 cells.

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Kisfalvi, Krisztina; Hurd, Cliff; Guha, Sushovan; Rozengurt, Enrique

2010-05-01

Neurotensin (NT) stimulates protein kinase D1 (PKD1), extracellular signal regulated kinase (ERK), c-Jun N-terminal Kinase (JNK), and DNA synthesis in the human pancreatic adenocarcinoma cell line PANC-1. To determine the effect of PKD1 overexpression on these biological responses, we generated inducible stable PANC-1 clones that express wild-type (WT) or kinase-dead (K618N) forms of PKD1 in response to the ecdysone analog ponasterone-A (PonA). NT potently stimulated c-Jun Ser(63) phosphorylation in both wild type and clonal derivatives of PANC-1 cells. PonA-induced expression of WT, but not K618N PKD1, rapidly blocked NT-mediated c-Jun Ser(63) phosphorylation either at the level of or upstream of MKK4, a dual-specificity kinase that leads to JNK activation. This is the first demonstration that PKD1 suppresses NT-induced JNK/cJun activation in PANC-1 cells. In contrast, PKD1 overexpression markedly increased the duration of NT-induced ERK activation in these cells. The reciprocal influence of PKD1 signaling on pro-mitogenicERK and pro-apopotic JNK/c-Jun pathways prompted us to examine whether PKD1 overexpression promotes DNA synthesis and proliferation of PANC-1 cells. Our results show that PKD1 overexpression increased DNA synthesis and cell numbers of PANC-1 cells cultured in regular dishes or in polyhydroxyethylmethacrylate [Poly-(HEMA)]-coated dishes to eliminate cell adhesion (anchorage-independent growth). Furthermore, PKD1 overexpression markedly enhanced DNA synthesis induced by NT (1-10 nM). These results indicate that PKD1 mediates mitogenic signaling in PANC-1 and suggests that this enzyme could be a novel target for the development of therapeutic drugs that restrict the proliferation of these cells.

Differential expression of insulin like growth factor I and other fibroblast mitogens in porcine colostrum and milk

International Nuclear Information System (INIS)

Tan, T.J.; Simmen, R.C.M.; Simmen, F.A.

1987-01-01

Sow mammary secretions contain at least 3 distinct growth factor activities, distinguished by their size and relative abundance in colostrum or later milk. Gel filtration of colostrum in Sephadex G-200 columns, followed by acid-ethanol extraction and radioimmunoassay (RIA) for insulin like growth factor I (IGF-I) revealed high levels of this factor in the 150K and 50K MW regions, characteristic of IGF-I: binding protein complexes. Acid treatment of these fractions yielded free IGF-I peptide (7.5K). Parallel mitogen assays with a fibroblast cell line (AKR-2B) demonstrated a predominant peak of high MW activity (sow colostral growth factor-I, SCGF-I) eluting near the column void volume (MW > 150K). Treatment of SCGF-I with 1M acetic acid resulted in a size reduction of the mitogenic activity (MW < 10K), suggesting association of SCGF-I with a binding protein. The SCGF-I peptide was noncompetitive in IGF-I RIA, was distinct in MW from free IGF-I, and was not mitogenic for chick embryo fibroblasts. Sow milk contains less IGF-I and SCGF-I but does display a predominant peak of small MW (∼ 3K) AKR-2B activity. The changes in expression of these growth factors during lactation may reflect differing roles in lactogenesis and/or neonatal growth and development

ASH1L Suppresses Matrix Metalloproteinase through Mitogen-activated Protein Kinase Signaling Pathway in Pulpitis.

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Bei, Yin; Tianqian, Hui; Fanyuan, Yu; Haiyun, Luo; Xueyang, Liao; Jing, Yang; Chenglin, Wang; Ling, Ye

2017-02-01

Pulpitis is an inflammation of dental pulp produced by a response to external stimuli. The response entails substantial cellular and molecular activities. Both genetic and epigenetic regulators contribute to the occurrence of pulpitis. However, the epigenetic mechanisms are still poorly understood. In this research, we studied the role of the absent, small, or homeotic-like (ASH1L) gene in the process of pulpitis. Human dental pulp cells (HDPCs) were stimulated with proinflammatory cytokine tumor necrosis factor alpha (TNF-α). Gene expression profiling was performed to assess the occurrence of epigenetic regulators. Pulp tissue from rat experimental pulpitis was subjected to immunofluorescence to detect the occurrence of ASH1L and trimethylation of lysine 4 histone 3 (H3K4me3). The presence of ASH1L in HDPCs that had been generated by TNF-α stimulation was analyzed by Western blot procedures and cellular immunofluorescence. Once detected, ASH1L was silenced through the use of specific small interfering RNA. The effects of ASH1L on the occurrence and operation of matrix metalloproteinases (MMPs) were then tested by analysis of quantitative polymerase chain reactions, Western blotting, and zymography. Chromatin immunoprecipitation was performed to detect whether ASH1L and H3K4me3 were present in the promoter regions of MMPs. We then used Western blot procedures to examine the nuclear factor kappa B and the mitogen-activated protein kinase (MAPK) responses to the silencing of ASH1L. We also examined the specific pathway involved in ASH1L regulation of the MMPs. After stimulating HDPCs with TNF-α, ASH1L emerged as 1 of the most strongly induced epigenetic mediators. We found that TNF-α treatment induced the expression of ASH1L through the nuclear factor kappa B and MAPK signal pathways. ASH1L was found in both the nucleus and the cytoplasm. TNF-α treatment was particularly active in inducing the accumulation of ASH1L in cellular cytoplasm. As is also consistent

A rapid chemical method for lysing Arabidopsis cells for protein analysis

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Takano Tetsuo

2011-07-01

Full Text Available Abstract Background Protein extraction is a frequent procedure in biological research. For preparation of plant cell extracts, plant materials usually have to be ground and homogenized to physically break the robust cell wall, but this step is laborious and time-consuming when a large number of samples are handled at once. Results We developed a chemical method for lysing Arabidopsis cells without grinding. In this method, plants are boiled for just 10 minutes in a solution containing a Ca2+ chelator and detergent. Cell extracts prepared by this method were suitable for SDS-PAGE and immunoblot analysis. This method was also applicable to genomic DNA extraction for PCR analysis. Our method was applied to many other plant species, and worked well for some of them. Conclusions Our method is rapid and economical, and allows many samples to be prepared simultaneously for protein analysis. Our method is useful not only for Arabidopsis research but also research on certain other species.

Direct transcriptional activation of BT genes by NLP transcription factors is a key component of the nitrate response in Arabidopsis.

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Sato, Takeo; Maekawa, Shugo; Konishi, Mineko; Yoshioka, Nozomi; Sasaki, Yuki; Maeda, Haruna; Ishida, Tetsuya; Kato, Yuki; Yamaguchi, Junji; Yanagisawa, Shuichi

2017-01-29

Nitrate modulates growth and development, functioning as a nutrient signal in plants. Although many changes in physiological processes in response to nitrate have been well characterized as nitrate responses, the molecular mechanisms underlying the nitrate response are not yet fully understood. Here, we show that NLP transcription factors, which are key regulators of the nitrate response, directly activate the nitrate-inducible expression of BT1 and BT2 encoding putative scaffold proteins with a plant-specific domain structure in Arabidopsis. Interestingly, the 35S promoter-driven expression of BT2 partially rescued growth inhibition caused by reductions in NLP activity in Arabidopsis. Furthermore, simultaneous disruption of BT1 and BT2 affected nitrate-dependent lateral root development. These results suggest that direct activation of BT1 and BT2 by NLP transcriptional activators is a key component of the molecular mechanism underlying the nitrate response in Arabidopsis. Copyright © 2016 Elsevier Inc. All rights reserved.

Differential binding of calmodulin-related proteins to their targets revealed through high-density Arabidopsis protein microarrays

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Popescu, Sorina C.; Popescu, George V.; Bachan, Shawn; Zhang, Zimei; Seay, Montrell; Gerstein, Mark; Snyder, Michael; Dinesh-Kumar, S. P.

2007-01-01

Calmodulins (CaMs) are the most ubiquitous calcium sensors in eukaryotes. A number of CaM-binding proteins have been identified through classical methods, and many proteins have been predicted to bind CaMs based on their structural homology with known targets. However, multicellular organisms typically contain many CaM-like (CML) proteins, and a global identification of their targets and specificity of interaction is lacking. In an effort to develop a platform for large-scale analysis of proteins in plants we have developed a protein microarray and used it to study the global analysis of CaM/CML interactions. An Arabidopsis thaliana expression collection containing 1,133 ORFs was generated and used to produce proteins with an optimized medium-throughput plant-based expression system. Protein microarrays were prepared and screened with several CaMs/CMLs. A large number of previously known and novel CaM/CML targets were identified, including transcription factors, receptor and intracellular protein kinases, F-box proteins, RNA-binding proteins, and proteins of unknown function. Multiple CaM/CML proteins bound many binding partners, but the majority of targets were specific to one or a few CaMs/CMLs indicating that different CaM family members function through different targets. Based on our analyses, the emergent CaM/CML interactome is more extensive than previously predicted. Our results suggest that calcium functions through distinct CaM/CML proteins to regulate a wide range of targets and cellular activities. PMID:17360592

Nicotine stimulates urokinase-type plasminogen activator receptor expression and cell invasiveness through mitogen-activated protein kinase and reactive oxygen species signaling in ECV304 endothelial cells

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Khoi, Pham Ngoc; Park, Jung Sun; Kim, Nam Ho; Jung, Young Do, E-mail: ydjung@chonnam.ac.kr

2012-03-01

Urokinase-type plasminogen activator receptor (uPAR) expression is elevated during inflammation, tissue remodeling and in many human cancers. This study investigated the effect of nicotine, a major alkaloid in tobacco, on uPAR expression and cell invasiveness in ECV304 endothelial cells. Nicotine stimulated uPAR expression in a dose-dependent manner and activated extracellular signal-regulated kinases-1/2 (Erk-1/2), c-Jun amino-terminal kinase (JNK) and p38 mitogen activated protein kinase (MAPK). Specific inhibitors of MEK-1 (PD98059) and JNK (SP600125) inhibited the nicotine-induced uPAR expression, while the p38 MAPK inhibitor SB203580 did not. Expression vectors encoding dominant negative MEK-1 (pMCL-K97M) and JNK (TAM67) also prevented nicotine-induced uPAR promoter activity. The intracellular hydrogen peroxide (H{sub 2}O{sub 2}) content was increased by nicotine treatment. The antioxidant N-acetylcysteine prevented nicotine-activated production of reactive oxygen species (ROS) and uPAR expression. Furthermore, exogenous H{sub 2}O{sub 2} increased uPAR mRNA expression. Deleted and site-directed mutagenesis demonstrated the involvement of the binding sites of transcription factor nuclear factor-kappaB (NF-κB) and activator protein (AP)-1 in the nicotine-induced uPAR expression. Studies with expression vectors encoding mutated NF-κB signaling molecules and AP-1 decoy confirmed that NF-κB and AP-1 were essential for the nicotine-stimulated uPAR expression. MAPK (Erk-1/2 and JNK) and ROS functioned as upstream signaling molecules in the activation of AP-1 and NF-κB, respectively. In addition, ECV304 endothelial cells treated with nicotine displayed markedly enhanced invasiveness, which was partially abrogated by uPAR neutralizing antibodies. The data indicate that nicotine induces uPAR expression via the MAPK/AP-1 and ROS/NF-κB signaling pathways and, in turn, stimulates invasiveness in human ECV304 endothelial cells. -- Highlights: ► Endothelial cells

Nicotine stimulates urokinase-type plasminogen activator receptor expression and cell invasiveness through mitogen-activated protein kinase and reactive oxygen species signaling in ECV304 endothelial cells

International Nuclear Information System (INIS)

Khoi, Pham Ngoc; Park, Jung Sun; Kim, Nam Ho; Jung, Young Do

2012-01-01

Urokinase-type plasminogen activator receptor (uPAR) expression is elevated during inflammation, tissue remodeling and in many human cancers. This study investigated the effect of nicotine, a major alkaloid in tobacco, on uPAR expression and cell invasiveness in ECV304 endothelial cells. Nicotine stimulated uPAR expression in a dose-dependent manner and activated extracellular signal-regulated kinases-1/2 (Erk-1/2), c-Jun amino-terminal kinase (JNK) and p38 mitogen activated protein kinase (MAPK). Specific inhibitors of MEK-1 (PD98059) and JNK (SP600125) inhibited the nicotine-induced uPAR expression, while the p38 MAPK inhibitor SB203580 did not. Expression vectors encoding dominant negative MEK-1 (pMCL-K97M) and JNK (TAM67) also prevented nicotine-induced uPAR promoter activity. The intracellular hydrogen peroxide (H 2 O 2 ) content was increased by nicotine treatment. The antioxidant N-acetylcysteine prevented nicotine-activated production of reactive oxygen species (ROS) and uPAR expression. Furthermore, exogenous H 2 O 2 increased uPAR mRNA expression. Deleted and site-directed mutagenesis demonstrated the involvement of the binding sites of transcription factor nuclear factor-kappaB (NF-κB) and activator protein (AP)-1 in the nicotine-induced uPAR expression. Studies with expression vectors encoding mutated NF-κB signaling molecules and AP-1 decoy confirmed that NF-κB and AP-1 were essential for the nicotine-stimulated uPAR expression. MAPK (Erk-1/2 and JNK) and ROS functioned as upstream signaling molecules in the activation of AP-1 and NF-κB, respectively. In addition, ECV304 endothelial cells treated with nicotine displayed markedly enhanced invasiveness, which was partially abrogated by uPAR neutralizing antibodies. The data indicate that nicotine induces uPAR expression via the MAPK/AP-1 and ROS/NF-κB signaling pathways and, in turn, stimulates invasiveness in human ECV304 endothelial cells. -- Highlights: ► Endothelial cells treated with nicotine

Calcium-Dependent Protein Kinases from Arabidopsis show substrate specificity differences in an analysis of 103 substrates

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Amy eCurran

2011-08-01

Full Text Available The identification of substrates represents a critical challenge for understanding any protein kinase-based signal transduction pathway. In Arabidopsis, there are more than 1000 different protein kinases, 34 of which belong to a family of Ca2+-dependent protein kinases (CPKs. While CPKs are implicated in regulating diverse aspects of plant biology, from ion transport to transcription, relatively little is known about isoform-specific differences in substrate specificity, or the number of phosphorylation targets. Here, in vitro kinase assays were used to compare phosphorylation targets of four CPKs from Arabidopsis (CPK1, 10, 16 and 34. Significant differences in substrate specificity for each kinase were revealed by assays using 103 different substrates. For example CPK16 phosphorylated Serine 109 in a peptide from the stress-regulated protein, Di19-2 with KM ~70 µM, but this site was not phosphorylated significantly by CPKs 1, 10, or 34. In contrast, CPKs 1, 10, and 34 phosphorylated 93 other peptide substrates not recognized by CPK16. Examples of substrate specificity differences among all four CPKs were verified by kinetic analyses. To test the correlation between in vivo phosphorylation events and in vitro kinase activities, assays were performed with 274 synthetic peptides that contained phosphorylation sites previously mapped in proteins isolated from plants (in vivo-mapped sites. Of these, 74 (27% were found to be phosphorylated by at least one of the four CPKs tested. This 27% success rate validates a robust strategy for linking the activities of specific kinases, such as CPKs, to the thousands of in planta phosphorylation sites that are being uncovered by emerging technologies.

Ascorbic acid deficiency activates cell death and disease resistance responses in Arabidopsis.

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Pavet, Valeria; Olmos, Enrique; Kiddle, Guy; Mowla, Shaheen; Kumar, Sanjay; Antoniw, John; Alvarez, María E; Foyer, Christine H

2005-11-01

Programmed cell death, developmental senescence, and responses to pathogens are linked through complex genetic controls that are influenced by redox regulation. Here we show that the Arabidopsis (Arabidopsis thaliana) low vitamin C mutants, vtc1 and vtc2, which have between 10% and 25% of wild-type ascorbic acid, exhibit microlesions, express pathogenesis-related (PR) proteins, and have enhanced basal resistance against infections caused by Pseudomonas syringae. The mutants have a delayed senescence phenotype with smaller leaf cells than the wild type at maturity. The vtc leaves have more glutathione than the wild type, with higher ratios of reduced glutathione to glutathione disulfide. Expression of green fluorescence protein (GFP) fused to the nonexpressor of PR protein 1 (GFP-NPR1) was used to detect the presence of NPR1 in the nuclei of transformed plants. Fluorescence was observed in the nuclei of 6- to 8-week-old GFP-NPR1 vtc1 plants, but not in the nuclei of transformed GFP-NPR1 wild-type plants at any developmental stage. The absence of senescence-associated gene 12 (SAG12) mRNA at the time when constitutive cell death and basal resistance were detected confirms that elaboration of innate immune responses in vtc plants does not result from activation of early senescence. Moreover, H2O2-sensitive genes are not induced at the time of systemic acquired resistance execution. These results demonstrate that ascorbic acid abundance modifies the threshold for activation of plant innate defense responses via redox mechanisms that are independent of the natural senescence program.

Reference: 510 [Arabidopsis Phenome Database[Archive

Lifescience Database Archive (English)

Full Text Available in support of PSII activity, whereas the interaction of PsbO2 with PSII regulates the turnover... its degradation. The Arabidopsis PsbO2 protein regulates dephosphorylation and turnover of the photosystem

Expression Profiling of Mitogen-Activated Protein Kinase Genes Reveals Their Evolutionary and Functional Diversity in Different Rubber Tree (Hevea brasiliensis Cultivars

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Xiang Jin

2017-10-01

Full Text Available Rubber tree (Hevea brasiliensis is the only commercially cultivated plant for producing natural rubber, one of the most essential industrial raw materials. Knowledge of the evolutionary and functional characteristics of kinases in H. brasiliensis is limited because of the long growth period and lack of well annotated genome information. Here, we reported mitogen-activated protein kinases in H. brasiliensis (HbMPKs by manually checking and correcting the rubber tree genome. Of the 20 identified HbMPKs, four members were validated by proteomic data. Protein motif and phylogenetic analyses classified these members into four known groups comprising Thr-Glu-Tyr (TEY and Thr-Asp-Tyr (TDY domains, respectively. Evolutionary and syntenic analyses suggested four duplication events: HbMPK3/HbMPK6, HbMPK8/HbMPK9/HbMPK15, HbMPK10/HbMPK12 and HbMPK11/HbMPK16/HbMPK19. Expression profiling of the identified HbMPKs in roots, stems, leaves and latex obtained from three cultivars with different latex yield ability revealed tissue- and variety-expression specificity of HbMPK paralogues. Gene expression patterns under osmotic, oxidative, salt and cold stresses, combined with cis-element distribution analyses, indicated different regulation patterns of HbMPK paralogues. Further, Ka/Ks and Tajima analyses suggested an accelerated evolutionary rate in paralogues HbMPK10/12. These results revealed HbMPKs have diverse functions in natural rubber biosynthesis, and highlighted the potential possibility of using MPKs to improve stress tolerance in future rubber tree breeding.

Simplified Enrichment of Plasma Membrane Proteins from Arabidopsis thaliana Seedlings Using Differential Centrifugation and Brij-58 Treatment.

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Collins, Carina A; Leslie, Michelle E; Peck, Scott C; Heese, Antje

2017-01-01

The plasma membrane (PM) forms a barrier between a plant cell and its environment. Proteins at this subcellular location play diverse and complex roles, including perception of extracellular signals to coordinate cellular changes. Analyses of PM proteins, however, are often limited by the relatively low abundance of these proteins in the total cellular protein pool. Techniques traditionally used for enrichment of PM proteins are time consuming, tedious, and require extensive optimization. Here, we provide a simple and reproducible enrichment procedure for PM proteins from Arabidopsis thaliana seedlings starting from total microsomal membranes isolated by differential centrifugation. To enrich for PM proteins, total microsomes are treated with the nonionic detergent Brij-58 to decrease the abundance of contaminating organellar proteins. This protocol combined with the genetic resources available in Arabidopsis provides a powerful tool that will enhance our understanding of proteins at the PM.

Salidroside pretreatment attenuates apoptosis and autophagy during hepatic ischemia–reperfusion injury by inhibiting the mitogen-activated protein kinase pathway in mice

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Feng J

2017-07-01

Full Text Available Jiao Feng,1,* Qinghui Zhang,2,* Wenhui Mo,3,* Liwei Wu,1 Sainan Li,1 Jingjing Li,1 Tong Liu,1 Shizan Xu,4 Xiaoming Fan,5 Chuanyong Guo1 1Department of Gastroenterology, Shanghai Tenth People’s Hospital, Tongji University School of Medicine, Shanghai, 2Department of Clinical Laboratory, Kunshan First People’s Hospital Affiliated to Jiangsu University, Kunshan, JiangSu, 3Department of Gastroenterology, Minhang Hospital, Fudan University, Shanghai, 4Department of Gastroenterology, Shanghai Tenth People’s Hospital, School of Clinical Medicine of Nanjing Medical University, Shanghai, 5Department of Gastroenterology, Jinshan Hospital of Fudan University, Jinshan, Shanghai, China *These authors contributed equally to this work Abstract: Ischemia–reperfusion injury (IRI contributes to liver damage in many clinical situations, such as liver resection and liver transplantation. In the present study, we investigated the effects of the antioxidant, anti-inflammatory, and anticancer agent salidroside (Sal on hepatic IRI in mice. The mice were randomly divided into six groups: normal control, Sham, Sal (20 mg/kg, IRI, IRI + Sal (10 mg/kg, and IRI + Sal (20 mg/kg. We measured liver enzymes, proinflammatory cytokines, TNF-α and interleukin-6, and apoptosis- and autophagy-related marker proteins at 2, 8, and 24 hours after reperfusion. Components of mitogen-activated protein kinase (MAPK signaling, including P-38, jun N-terminal kinase (JNK, and extracellular signal-regulated kinase (ERK, were also measured using an MAPK activator anisomycin to deduce their roles in hepatic IRI. Our results show that Sal safely protects hepatocytes from IRI by reducing levels of liver enzymes in the serum. These findings were confirmed by histopathology. We concluded that Sal protects hepatocytes from IRI partly by inhibiting the activation of MAPK signaling, including the phosphorylation of P38, JNK, and ERK. This ameliorates inflammatory reactions, apoptosis, and

Arabidopsis calmodulin-like protein CML36 is a calcium (Ca2+) sensor that interacts with the plasma membrane Ca2+-ATPase isoform ACA8 and stimulates its activity.

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Astegno, Alessandra; Bonza, Maria Cristina; Vallone, Rosario; La Verde, Valentina; D'Onofrio, Mariapina; Luoni, Laura; Molesini, Barbara; Dominici, Paola

2017-09-08

Calmodulin-like (CML) proteins are major EF-hand-containing, calcium (Ca 2+ )-binding proteins with crucial roles in plant development and in coordinating plant stress tolerance. Given their abundance in plants, the properties of Ca 2+ sensors and identification of novel target proteins of CMLs deserve special attention. To this end, we recombinantly produced and biochemically characterized CML36 from Arabidopsis thaliana We analyzed Ca 2+ and Mg 2+ binding to the individual EF-hands, observed metal-induced conformational changes, and identified a physiologically relevant target. CML36 possesses two high-affinity Ca 2+ /Mg 2+ mixed binding sites and two low-affinity Ca 2+ -specific sites. Binding of Ca 2+ induced an increase in the α-helical content and a conformational change that lead to the exposure of hydrophobic regions responsible for target protein recognition. Cation binding, either Ca 2+ or Mg 2+ , stabilized the secondary and tertiary structures of CML36, guiding a large structural transition from a molten globule apo-state to a compact holoconformation. Importantly, through in vitro binding and activity assays, we showed that CML36 interacts directly with the regulative N terminus of the Arabidopsis plasma membrane Ca 2+ -ATPase isoform 8 (ACA8) and that this interaction stimulates ACA8 activity. Gene expression analysis revealed that CML36 and ACA8 are co-expressed mainly in inflorescences. Collectively, our results support a role for CML36 as a Ca 2+ sensor that binds to and modulates ACA8, uncovering a possible involvement of the CML protein family in the modulation of plant-autoinhibited Ca 2+ pumps. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Protection of Human Podocytes from Shiga Toxin 2-Induced Phosphorylation of Mitogen-Activated Protein Kinases and Apoptosis by Human Serum Amyloid P Component

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Dettmar, Anne K.; Binder, Elisabeth; Greiner, Friederike R.; Liebau, Max C.; Kurschat, Christine E.; Jungraithmayr, Therese C.; Saleem, Moin A.; Schmitt, Claus-Peter; Feifel, Elisabeth; Orth-Höller, Dorothea; Kemper, Markus J.; Pepys, Mark; Würzner, Reinhard

2014-01-01

Hemolytic uremic syndrome (HUS) is mainly induced by Shiga toxin 2 (Stx2)-producing Escherichia coli. Proteinuria can occur in the early phase of the disease, and its persistence determines the renal prognosis. Stx2 may injure podocytes and induce proteinuria. Human serum amyloid P component (SAP), a member of the pentraxin family, has been shown to protect against Stx2-induced lethality in mice in vivo, presumably by specific binding to the toxin. We therefore tested the hypothesis that SAP can protect against Stx2-induced injury of human podocytes. To elucidate the mechanisms underlying podocyte injury in HUS-associated proteinuria, we assessed Stx2-induced activation of mitogen-activated protein kinases (MAPKs) and apoptosis in immortalized human podocytes and evaluated the impact of SAP on Stx2-induced damage. Human podocytes express Stx2-binding globotriaosylceramide 3. Stx2 applied to cultured podocytes was internalized and then activated p38α MAPK and c-Jun N-terminal kinase (JNK), important signaling steps in cell differentiation and apoptosis. Stx2 also activated caspase 3, resulting in an increased level of apoptosis. Coincubation of podocytes with SAP and Stx2 mitigated the effects of Stx2 and induced upregulation of antiapoptotic Bcl2. These data suggest that podocytes are a target of Stx2 and that SAP protects podocytes against Stx2-induced injury. SAP may therefore be a useful therapeutic option. PMID:24566618

TMV-Cg Coat Protein stabilizes DELLA proteins and in turn negatively modulates salicylic acid-mediated defense pathway during Arabidopsis thaliana viral infection.

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Rodriguez, Maria Cecilia; Conti, Gabriela; Zavallo, Diego; Manacorda, Carlos Augusto; Asurmendi, Sebastian

2014-08-03

Plant viral infections disturb defense regulatory networks during tissue invasion. Emerging evidence demonstrates that a significant proportion of these alterations are mediated by hormone imbalances. Although the DELLA proteins have been reported to be central players in hormone cross-talk, their role in the modulation of hormone signaling during virus infections remains unknown. This work revealed that TMV-Cg coat protein (CgCP) suppresses the salicylic acid (SA) signaling pathway without altering defense hormone SA or jasmonic acid (JA) levels in Arabidopsis thaliana. Furthermore, it was observed that the expression of CgCP reduces plant growth and delays the timing of floral transition. Quantitative RT-qPCR analysis of DELLA target genes showed that CgCP alters relative expression of several target genes, indicating that the DELLA proteins mediate transcriptional changes produced by CgCP expression. Analyses by fluorescence confocal microscopy showed that CgCP stabilizes DELLA proteins accumulation in the presence of gibberellic acid (GA) and that the DELLA proteins are also stabilized during TMV-Cg virus infections. Moreover, DELLA proteins negatively modulated defense transcript profiles during TMV-Cg infection. As a result, TMV-Cg accumulation was significantly reduced in the quadruple-DELLA mutant Arabidopsis plants compared to wild type plants. Taken together, these results demonstrate that CgCP negatively regulates the salicylic acid-mediated defense pathway by stabilizing the DELLA proteins during Arabidopsis thaliana viral infection, suggesting that CgCP alters the stability of DELLAs as a mechanism of negative modulation of antiviral defense responses.

Extract of Polygala tenuifolia Alleviates Stress-Exacerbated Atopy-Like Skin Dermatitis through the Modulation of Protein Kinase A and p38 Mitogen-Activated Protein Kinase Signaling Pathway

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Bongjun Sur

2017-01-01

Full Text Available Atopic dermatitis (AD and stress create a vicious cycle: stress exacerbates atopic symptoms, and atopic disease elicits stress and anxiety. Targeting multiple pathways including stress and allergic inflammation is, therefore, important for treating AD. In this study, we investigated the remedial value of Polygala tenuifolia Willd. (PTW for treating immobilization (IMO stress-exacerbated atopy-like skin dermatitis and its underlying mechanism. Trimellitic anhydride (TMA was applied to dorsal skin for sensitization and subsequently both ears for eliciting T-cell-dependent contact hypersensitivity in mice, which underwent 2 h-IMO stress and PTW administration for the latter 6 and 9 days in the ear exposure period of TMA, respectively. To elicit in vitro degranulation of human mast cell line-1 (HMC-1, 10 µM substance P (SP and 200 nM corticotrophin-releasing factor (CRF were sequentially added with 48 h-interval. PTW extract (500 µg/mL was added 30 min before CRF treatment. IMO stress exacerbated TMA-induced scratching behavior by 252%, and increased their blood corticosterone levels by two-fold. Treatment with 250 mg/kg PTW significantly restored IMO stress-exacerbated scratching behavior and other indicators such as skin inflammation and water content, lymph node weights, and serum histamine and immunoglobulin E (lgE levels. Furthermore, it also reversed TMA-stimulated expression of tumor necrosis factor (TNF-α and interleukin (IL-4 mRNAs in ear tissues. PTW significantly inhibited SP/CRF-stimulated degranulation of HMC-1 cells, subsequent tryptase secretion, and protein kinase A (PKA activity. PTW also selectively inhibited p38 mitogen-activated protein kinase (MAPK phosphorylation in SP/CRF-treated HMC-1 cells. PTW significantly inhibited HMC-1 cell degranulation and alleviated IMO stress-exacerbated atopic dermatitis symptoms by modulating the PKA/p38 MAPK signaling pathway.

Growth rate correlates negatively with protein turnover in Arabidopsis accessions.

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Ishihara, Hirofumi; Moraes, Thiago Alexandre; Pyl, Eva-Theresa; Schulze, Waltraud X; Obata, Toshihiro; Scheffel, André; Fernie, Alisdair R; Sulpice, Ronan; Stitt, Mark

2017-08-01

Previous studies with Arabidopsis accessions revealed that biomass correlates negatively to dusk starch content and total protein, and positively to the maximum activities of enzymes in photosynthesis. We hypothesized that large accessions have lower ribosome abundance and lower rates of protein synthesis, and that this is compensated by lower rates of protein degradation. This would increase growth efficiency and allow more investment in photosynthetic machinery. We analysed ribosome abundance and polysome loading in 19 accessions, modelled the rates of protein synthesis and compared them with the observed rate of growth. Large accessions contained less ribosomes than small accessions, due mainly to cytosolic ribosome abundance falling at night in large accessions. The modelled rates of protein synthesis resembled those required for growth in large accessions, but were up to 30% in excess in small accessions. We then employed 13 CO 2 pulse-chase labelling to measure the rates of protein synthesis and degradation in 13 accessions. Small accessions had a slightly higher rate of protein synthesis and much higher rates of protein degradation than large accessions. Protein turnover was negligible in large accessions but equivalent to up to 30% of synthesised protein day -1 in small accessions. We discuss to what extent the decrease in growth in small accessions can be quantitatively explained by known costs of protein turnover and what factors may lead to the altered diurnal dynamics and increase of ribosome abundance in small accessions, and propose that there is a trade-off between protein turnover and maximisation of growth rate. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

Three Fusarium oxysporum mitogen-activated protein kinases (MAPKs) have distinct and complementary roles in stress adaptation and cross-kingdom pathogenicity.

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Segorbe, David; Di Pietro, Antonio; Pérez-Nadales, Elena; Turrà, David

2017-09-01

Mitogen-activated protein kinase (MAPK) cascades mediate cellular responses to environmental signals. Previous studies in the fungal pathogen Fusarium oxysporum have revealed a crucial role of Fmk1, the MAPK orthologous to Saccharomyces cerevisiae Fus3/Kss1, in vegetative hyphal fusion and plant infection. Here, we genetically dissected the individual and combined contributions of the three MAPKs Fmk1, Mpk1 and Hog1 in the regulation of development, stress response and virulence of F. oxysporum on plant and animal hosts. Mutants lacking Fmk1 or Mpk1 were affected in reactive oxygen species (ROS) homeostasis and impaired in hyphal fusion and aggregation. Loss of Mpk1 also led to increased sensitivity to cell wall and heat stress, which was exacerbated by simultaneous inactivation of Fmk1, suggesting that both MAPKs contribute to cellular adaptation to high temperature, a prerequisite for mammalian pathogens. Deletion of Hog1 caused increased sensitivity to hyperosmotic stress and resulted in partial rescue of the restricted colony growth phenotype of the mpk1Δ mutant. Infection assays on tomato plants and the invertebrate animal host Galleria mellonella revealed distinct and additive contributions of the different MAPKs to virulence. Our results indicate that positive and negative cross-talk between the three MAPK pathways regulates stress adaptation, development and virulence in the cross-kingdom pathogen F. oxysporum. © 2016 BSPP AND JOHN WILEY & SONS LTD.

Multilayered Regulation of Ethylene Induction Plays a Positive Role in Arabidopsis Resistance against Pseudomonas syringae1[OPEN

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Guan, Rongxia; Su, Jianbin; Meng, Xiangzong; Li, Sen; Liu, Yidong; Xu, Juan; Zhang, Shuqun

2015-01-01

Ethylene, a key phytohormone involved in plant-pathogen interaction, plays a positive role in plant resistance against fungal pathogens. However, its function in plant bacterial resistance remains unclear. Here, we report a detailed analysis of ethylene induction in Arabidopsis (Arabidopsis thaliana) in response to Pseudomonas syringae pv tomato DC3000 (Pst). Ethylene biosynthesis is highly induced in both pathogen/microbe-associated molecular pattern (PAMP)-triggered immunity and effector-triggered immunity (ETI), and the induction is potentiated by salicylic acid (SA) pretreatment. In addition, Pst actively suppresses PAMP-triggered ethylene induction in a type III secretion system-dependent manner. SA potentiation of ethylene induction is dependent mostly on MITOGEN-ACTIVATED PROTEIN KINASE6 (MPK6) and MPK3 and their downstream ACS2 and ACS6, two type I isoforms of 1-aminocyclopropane-1-carboxylic acid synthases (ACSs). ACS7, a type III ACS whose expression is enhanced by SA pretreatment, is also involved. Pst expressing the avrRpt2 effector gene (Pst-avrRpt2), which is capable of triggering ETI, induces a higher level of ethylene production, and the elevated portion is dependent on SALICYLIC ACID INDUCTION DEFICIENT2 and NONEXPRESSER OF PATHOGENESIS-RELATED GENE1, two key players in SA biosynthesis and signaling. High-order ACS mutants with reduced ethylene induction are more susceptible to both Pst and Pst-avrRpt2, demonstrating a positive role of ethylene in plant bacterial resistance mediated by both PAMP-triggered immunity and ETI. PMID:26265775

Expression of pH-sensitive green fluorescent protein in Arabidopsis thaliana

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Moseyko, N.; Feldman, L. J.

2001-01-01

This is the first report on using green fluorescent protein (GFP) as a pH reporter in plants. Proton fluxes and pH regulation play important roles in plant cellular activity and therefore, it would be extremely helpful to have a plant gene reporter system for rapid, non-invasive visualization of intracellular pH changes. In order to develop such a system, we constructed three vectors for transient and stable transformation of plant cells with a pH-sensitive derivative of green fluorescent protein. Using these vectors, transgenic Arabidopsis thaliana and tobacco plants were produced. Here the application of pH-sensitive GFP technology in plants is described and, for the first time, the visualization of pH gradients between different developmental compartments in intact whole-root tissues of A. thaliana is reported. The utility of pH-sensitive GFP in revealing rapid, environmentally induced changes in cytoplasmic pH in roots is also demonstrated.

Biochemical function of typical and variant Arabidopsis thaliana U-box E3 ubiquitin-protein ligases

DEFF Research Database (Denmark)

Wiborg, Jakob; O'Shea, Charlotte; Skriver, Karen

2008-01-01

of the distant U-box protein, AtPUB49, representing a large family of eukaryotic proteins containing a U-box linked to a cyclophilin-like peptidyl-prolyl cis-trans isomerase domain, was characterized biochemically. AtPUB49 functioned both as a prolyl isomerase and a chaperone by catalysing cis......The variance of the U-box domain in 64 Arabidopsis thaliana (thale cress) E3s (ubiquitin-protein ligases) was used to examine the interactions between E3s and E2s (ubiquitin-conjugating enzymes). E2s and E3s are components of the ubiquitin protein degradation pathway. Seven U-box proteins were...... analysed for their ability to ubiquitinate proteins in vitro in co-operation with different E2s. All U-box domains exhibited ubiquitination activity and interacted productively with UBC4/5-type E2s. Three and four of the U-box domains mediated ubiquitin addition in the presence of UBC13 and UBC7 E2s...

RNA-Binding Proteins Revisited – The Emerging Arabidopsis mRNA Interactome

KAUST Repository

Köster, Tino

2017-04-13

RNA–protein interaction is an important checkpoint to tune gene expression at the RNA level. Global identification of proteins binding in vivo to mRNA has been possible through interactome capture – where proteins are fixed to target RNAs by UV crosslinking and purified through affinity capture of polyadenylated RNA. In Arabidopsis over 500 RNA-binding proteins (RBPs) enriched in UV-crosslinked samples have been identified. As in mammals and yeast, the mRNA interactomes came with a few surprises. For example, a plethora of the proteins caught on RNA had not previously been linked to RNA-mediated processes, for example proteins of intermediary metabolism. Thus, the studies provide unprecedented insights into the composition of the mRNA interactome, highlighting the complexity of RNA-mediated processes.

RNA-Binding Proteins Revisited – The Emerging Arabidopsis mRNA Interactome

KAUST Repository

Kö ster, Tino; Marondedze, Claudius; Meyer, Katja; Staiger, Dorothee

2017-01-01

RNA–protein interaction is an important checkpoint to tune gene expression at the RNA level. Global identification of proteins binding in vivo to mRNA has been possible through interactome capture – where proteins are fixed to target RNAs by UV crosslinking and purified through affinity capture of polyadenylated RNA. In Arabidopsis over 500 RNA-binding proteins (RBPs) enriched in UV-crosslinked samples have been identified. As in mammals and yeast, the mRNA interactomes came with a few surprises. For example, a plethora of the proteins caught on RNA had not previously been linked to RNA-mediated processes, for example proteins of intermediary metabolism. Thus, the studies provide unprecedented insights into the composition of the mRNA interactome, highlighting the complexity of RNA-mediated processes.

NDR proteins: Lessons learned from Arabidopsis and animal cells prompt a testable hypothesis

OpenAIRE

Mudgil, Yashwanti; Jones, Alan M

2010-01-01

N-myc downregulated (NDR) genes were discovered more than fifteen years ago. Indirect evidence support a role in tumor progression and cellular differentiation, but their biochemical function is still unknown. Our detailed analyses on Arabidopsis NDR proteins (deisgnated NDR-like, NDL) show their involvement in altering auxin transport, local auxin gradients and expression level of auxin transport proteins. Animal NDL proteins may be involved in membrane recycling of E-cadherin and effector f...

Multilayered Regulation of Ethylene Induction Plays a Positive Role in Arabidopsis Resistance against Pseudomonas syringae.

Science.gov (United States)

Guan, Rongxia; Su, Jianbin; Meng, Xiangzong; Li, Sen; Liu, Yidong; Xu, Juan; Zhang, Shuqun

2015-09-01

Ethylene, a key phytohormone involved in plant-pathogen interaction, plays a positive role in plant resistance against fungal pathogens. However, its function in plant bacterial resistance remains unclear. Here, we report a detailed analysis of ethylene induction in Arabidopsis (Arabidopsis thaliana) in response to Pseudomonas syringae pv tomato DC3000 (Pst). Ethylene biosynthesis is highly induced in both pathogen/microbe-associated molecular pattern (PAMP)-triggered immunity and effector-triggered immunity (ETI), and the induction is potentiated by salicylic acid (SA) pretreatment. In addition, Pst actively suppresses PAMP-triggered ethylene induction in a type III secretion system-dependent manner. SA potentiation of ethylene induction is dependent mostly on MITOGEN-ACTIVATED PROTEIN KINASE6 (MPK6) and MPK3 and their downstream ACS2 and ACS6, two type I isoforms of 1-aminocyclopropane-1-carboxylic acid synthases (ACSs). ACS7, a type III ACS whose expression is enhanced by SA pretreatment, is also involved. Pst expressing the avrRpt2 effector gene (Pst-avrRpt2), which is capable of triggering ETI, induces a higher level of ethylene production, and the elevated portion is dependent on SALICYLIC ACID INDUCTION DEFICIENT2 and NONEXPRESSER OF PATHOGENESIS-RELATED GENE1, two key players in SA biosynthesis and signaling. High-order ACS mutants with reduced ethylene induction are more susceptible to both Pst and Pst-avrRpt2, demonstrating a positive role of ethylene in plant bacterial resistance mediated by both PAMP-triggered immunity and ETI. © 2015 American Society of Plant Biologists. All Rights Reserved.

Regulatory Proteolysis in Arabidopsis-Pathogen Interactions.

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Pogány, Miklós; Dankó, Tamás; Kámán-Tóth, Evelin; Schwarczinger, Ildikó; Bozsó, Zoltán

2015-09-24

Approximately two and a half percent of protein coding genes in Arabidopsis encode enzymes with known or putative proteolytic activity. Proteases possess not only common housekeeping functions by recycling nonfunctional proteins. By irreversibly cleaving other proteins, they regulate crucial developmental processes and control responses to environmental changes. Regulatory proteolysis is also indispensable in interactions between plants and their microbial pathogens. Proteolytic cleavage is simultaneously used both by plant cells, to recognize and inactivate invading pathogens, and by microbes, to overcome the immune system of the plant and successfully colonize host cells. In this review, we present available results on the group of proteases in the model plant Arabidopsis thaliana whose functions in microbial pathogenesis were confirmed. Pathogen-derived proteolytic factors are also discussed when they are involved in the cleavage of host metabolites. Considering the wealth of review papers available in the field of the ubiquitin-26S proteasome system results on the ubiquitin cascade are not presented. Arabidopsis and its pathogens are conferred with abundant sets of proteases. This review compiles a list of those that are apparently involved in an interaction between the plant and its pathogens, also presenting their molecular partners when available.

Molecular and Functional Characterization of a Polygalacturonase-Inhibiting Protein from Cynanchum komarovii That Confers Fungal Resistance in Arabidopsis.

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Nana Liu

Full Text Available Compliance with ethical standards: This study did not involve human participants and animals, and the plant of interest is not an endangered species. Polygalacturonase-inhibiting proteins (PGIPs are leucine-rich repeat proteins that plants produce against polygalacturonase, a key virulence agent in pathogens. In this paper, we cloned and purified CkPGIP1, a gene product from Cynanchum komarovii that effectively inhibits polygalacturonases from Botrytis cinerea and Rhizoctonia solani. We found the expression of CkPGIP1 to be induced in response to salicylic acid, wounding, and infection with B. cinerea and R. solani. In addition, transgenic overexpression in Arabidopsis enhanced resistance against B. cinerea. Furthermore, CkPGIP1 obtained from transgenic Arabidopsis inhibited the activity of B. cinerea and R. solani polygalacturonases by 62.7-66.4% and 56.5-60.2%, respectively. Docking studies indicated that the protein interacts strongly with the B1-sheet at the N-terminus of the B. cinerea polygalacturonase, and with the C-terminus of the polygalacturonase from R. solani. This study highlights the significance of CkPGIP1 in plant disease resistance, and its possible application to manage fungal pathogens.

Na+/H+ exchange activity in the plasma membrane of Arabidopsis.

Science.gov (United States)

Qiu, Quan-Sheng; Barkla, Bronwyn J; Vera-Estrella, Rosario; Zhu, Jian-Kang; Schumaker, Karen S

2003-06-01

In plants, Na+/H+ exchangers in the plasma membrane are critical for growth in high levels of salt, removing toxic Na+ from the cytoplasm by transport out of the cell. The molecular identity of a plasma membrane Na+/H+ exchanger in Arabidopsis (SOS1) has recently been determined. In this study, immunological analysis provided evidence that SOS1 localizes to the plasma membrane of leaves and roots. To characterize the transport activity of this protein, purified plasma membrane vesicles were isolated from leaves of Arabidopsis. Na+/H+ exchange activity, monitored as the ability of Na to dissipate an established pH gradient, was absent in plants grown without salt. However, exchange activity was induced when plants were grown in 250 mm NaCl and increased with prolonged salt exposure up to 8 d. H+-coupled exchange was specific for Na, because chloride salts of other monovalent cations did not dissipate the pH gradient. Na+/H+ exchange activity was dependent on Na (substrate) concentration, and kinetic analysis indicated that the affinity (apparent Km) of the transporter for Na+ is 22.8 mm. Data from two experimental approaches supports electroneutral exchange (one Na+ exchanged for one proton): (a) no change in membrane potential was measured during the exchange reaction, and (b) Na+/H+ exchange was unaffected by the presence or absence of a membrane potential. Results from this research provide a framework for future studies into the regulation of the plant plasma membrane Na+/H+ exchanger and its relative contribution to the maintenance of cellular Na+ homeostasis during plant growth in salt.

Arabidopsis Intracellular NHX-Type Sodium-Proton Antiporters are Required for Seed Storage Protein Processing.

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Ashnest, Joanne R; Huynh, Dung L; Dragwidge, Jonathan M; Ford, Brett A; Gendall, Anthony R

2015-11-01

The Arabidopsis intracellular sodium-proton exchanger (NHX) proteins AtNHX5 and AtNHX6 have a well-documented role in plant development, and have been used to improve salt tolerance in a variety of species. Despite evidence that intracellular NHX proteins are important in vacuolar trafficking, the mechanism of this role is poorly understood. Here we show that NHX5 and NHX6 are necessary for processing of the predominant seed storage proteins, and also influence the processing and activity of a vacuolar processing enzyme. Furthermore, we show by yeast two-hybrid and bimolecular fluorescence complementation (BiFC) technology that the C-terminal tail of NHX6 interacts with a component of Retromer, another component of the cell sorting machinery, and that this tail is critical for NHX6 activity. These findings demonstrate that NHX5 and NHX6 are important in processing and activity of vacuolar cargo, and suggest a mechanism by which NHX intracellular (IC)-II antiporters may be involved in subcellular trafficking. © The Author 2015. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Arabidopsis GCP3-interacting protein 1/MOZART 1 is an integral component of the γ-tubulin-containing microtubule nucleating complex.

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Nakamura, Masayoshi; Yagi, Noriyoshi; Kato, Takehide; Fujita, Satoshi; Kawashima, Noriyuki; Ehrhardt, David W; Hashimoto, Takashi

2012-07-01

Microtubules in eukaryotic cells are nucleated from ring-shaped complexes that contain γ-tubulin and a family of homologous γ-tubulin complex proteins (GCPs), but the subunit composition of the complexes can vary among fungi, animals and plants. Arabidopsis GCP3-interacting protein 1 (GIP1), a small protein with no homology to the GCP family, interacts with GCP3 in vitro, and is a plant homolog of vertebrate mitotic-spindle organizing protein associated with a ring of γ-tubulin 1 (MOZART1), a recently identified component of the γ-tubulin complex in human cell lines. In this study, we characterized two closely related Arabidopsis GIP1s: GIP1a and GIP1b. Single mutants of gip1a and gip1b were indistinguishable from wild-type plants, but their double mutant was embryonic lethal, and showed impaired development of male gametophytes. Functional fusions of GIP1a with green fluorescent protein (GFP) were used to purify GIP1a-containing complexes from Arabidopsis plants, which contained all the subunits (except NEDD1) previously identified in the Arabidopsis γ-tubulin complexes. GIP1a and GIP1b interacted specifically with Arabidopsis GCP3 in yeast. GFP-GIP1a labeled mitotic microtubule arrays in a pattern largely consistent with, but partly distinct from, the localization of the γ-tubulin complex containing GCP2 or GCP3 in planta. In interphase cortical arrays, the labeled complexes were preferentially recruited to existing microtubules, from which new microtubules were efficiently nucleated. However, in contrast to complexes labeled with tagged GCP2 or GCP3, their recruitment to cortical areas with no microtubules was rarely observed. These results indicate that GIP1/MOZART1 is an integral component of a subset of the Arabidopsis γ-tubulin complexes. © 2012 The Authors. The Plant Journal © 2012 Blackwell Publishing Ltd.

The genetic basis of constitutive and herbivore-induced ESP-independent nitrile formation in Arabidopsis.

Science.gov (United States)

Burow, Meike; Losansky, Anja; Müller, René; Plock, Antje; Kliebenstein, Daniel J; Wittstock, Ute

2009-01-01

Glucosinolates are a group of thioglucosides that are components of an activated chemical defense found in the Brassicales. Plant tissue damage results in hydrolysis of glucosinolates by endogenous thioglucosidases known as myrosinases. Spontaneous rearrangement of the aglucone yields reactive isothiocyanates that are toxic to many organisms. In the presence of specifier proteins, alternative products, namely epithionitriles, simple nitriles, and thiocyanates with different biological activities, are formed at the expense of isothiocyanates. Recently, simple nitriles were recognized to serve distinct functions in plant-insect interactions. Here, we show that simple nitrile formation in Arabidopsis (Arabidopsis thaliana) ecotype Columbia-0 rosette leaves increases in response to herbivory and that this increase is independent of the known epithiospecifier protein (ESP). We combined phylogenetic analysis, a screen of Arabidopsis mutants, recombinant protein characterization, and expression quantitative trait locus mapping to identify a gene encoding a nitrile-specifier protein (NSP) responsible for constitutive and herbivore-induced simple nitrile formation in Columbia-0 rosette leaves. AtNSP1 is one of five Arabidopsis ESP homologues that promote simple nitrile, but not epithionitrile or thiocyanate, formation. Four of these homologues possess one or two lectin-like jacalin domains, which share a common ancestry with the jacalin domains of the putative Arabidopsis myrosinase-binding proteins MBP1 and MBP2. A sixth ESP homologue lacked specifier activity and likely represents the ancestor of the gene family with a different biochemical function. By illuminating the genetic and biochemical bases of simple nitrile formation, our study provides new insights into the evolution of metabolic diversity in a complex plant defense system.

Genome-wide cloning and sequence analysis of leucine-rich repeat receptor-like protein kinase genes in Arabidopsis thaliana

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Yuan Tong

2010-01-01

Full Text Available Abstract Background Transmembrane receptor kinases play critical roles in both animal and plant signaling pathways regulating growth, development, differentiation, cell death, and pathogenic defense responses. In Arabidopsis thaliana, there are at least 223 Leucine-rich repeat receptor-like kinases (LRR-RLKs, representing one of the largest protein families. Although functional roles for a handful of LRR-RLKs have been revealed, the functions of the majority of members in this protein family have not been elucidated. Results As a resource for the in-depth analysis of this important protein family, the complementary DNA sequences (cDNAs of 194 LRR-RLKs were cloned into the GatewayR donor vector pDONR/ZeoR and analyzed by DNA sequencing. Among them, 157 clones showed sequences identical to the predictions in the Arabidopsis sequence resource, TAIR8. The other 37 cDNAs showed gene structures distinct from the predictions of TAIR8, which was mainly caused by alternative splicing of pre-mRNA. Most of the genes have been further cloned into GatewayR destination vectors with GFP or FLAG epitope tags and have been transformed into Arabidopsis for in planta functional analysis. All clones from this study have been submitted to the Arabidopsis Biological Resource Center (ABRC at Ohio State University for full accessibility by the Arabidopsis research community. Conclusions Most of the Arabidopsis LRR-RLK genes have been isolated and the sequence analysis showed a number of alternatively spliced variants. The generated resources, including cDNA entry clones, expression constructs and transgenic plants, will facilitate further functional analysis of the members of this important gene family.

NDR proteins: lessons learned from Arabidopsis and animal cells prompt a testable hypothesis.

Science.gov (United States)

Mudgil, Yashwanti; Jones, Alan M

2010-08-01

N-myc Down Regulated (NDR) genes were discovered more than fifteen years ago. Indirect evidence support a role in tumor progression and cellular differentiation, but their biochemical function is still unknown. Our detailed analyses on Arabidopsis NDL proteins show their involvement in altering auxin transport, local auxin gradients and expression level of auxin transport proteins. Animal NDL proteins may be involved in membrane recycling of E-cadherin and effector for the small GTPase. In light of these findings, we hypothesize that NDL proteins regulate vesicular trafficking of auxin transport facilitator PIN proteins by biochemically alterating the local lipid environment of PIN proteins.

Evolution of a signalling system that incorporates both redundancy and diversity: Arabidopsis SUMOylation

Science.gov (United States)

Chosed, Renee; Mukherjee, Sohini; Lois, Luisa Maria; Orth, Kim

2006-01-01

The reversible post-translational modifier, SUMO (small ubiquitin-related modifier), modulates the activity of a diverse set of target proteins, resulting in important consequences to the cellular machinery. Conjugation machinery charges the processed SUMO so that it can be linked via an isopeptide bond to a target protein. The removal of SUMO moieties from conjugated proteins by isopeptidases regenerates pools of processed SUMOs and unmodified target proteins. The evolutionarily conserved SUMO-conjugating proteins, E1 and E2, recognize a diverse set of Arabidopsis SUMO proteins using them to modify protein substrates. In contrast, the deSUMOylating enzymes differentially recognize the Arabidopsis SUMO proteins, resulting in specificity of the deconjugating machinery. The specificity of the Arabidopsis deSUMOylating enzymes is further diversified by the addition of regulatory domains. Therefore the SUMO proteins, in this signalling system, have evolved to contain information that allows not only redundancy with the conjugation system but also diversity with the deconjugating enzymes. PMID:16740136

Involvement of a novel p38 mitogen-activated protein kinase in larval metamorphosis of the polychaete Hydroides elegans (Haswell)

KAUST Repository

Wang, Hao

2010-04-19

Hydroides elegans is a common marine fouling organism in most tropical and subtropical waters. The life cycle of H. elegans includes a planktonic larval stage in which swimming larvae normally take 5 days to attain competency to settle. Larval metamorphosis marks the beginning of its benthic life; however, the endogenous molecular mechanisms that regulate metamorphosis remain largely unknown. In this study, a PCR-based suppressive subtractive hybridization (SSH) library was constructed to screen the genes expressed in competent larvae but not in precompetent larvae. Among the transcripts isolated from the library, 21 significantly matched sequences in the GenBank. Many of these isolated transcripts have putative roles in the reactive oxygen species (ROS) signal transduction pathway or in response to ROS stress. A putative novel p38 mitogen-activated protein kinase (MAPK), which was also isolated with SSH screen, was then cloned and characterized. The MAPK inhibitors assay showed that both p38 MAPK inhibitors SB202190 and SB203580 effectively inhibited the biofilm-induced metamorphosis of H. elegans. A cell stressors assay showed that H2O2 effectively induced larval metamorphosis of H. elegans, but the inductivity of H2O2 was also inhibited by both SB inhibitors. The catalase assay showed that the catalase could effetely inhibit H. elegans larvae from responding to inductive biofilm. These results showed that the p38 MAPK-dependent pathway plays critical role in controlling larval metamorphosis of the marine polychaete H. elegans, and the reactive oxygen radicals produced by biofilm could be the cue inducing larval metamorphosis. © 2010 Wiley-Liss, Inc.

Colletotrichum higginsianum Mitogen-Activated Protein Kinase ChMK1: Role in Growth, Cell Wall Integrity, Colony Melanization and Pathogenicity

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Wei Wei

2016-08-01

Full Text Available Colletotrichum higginsianum is an economically important pathogen that causes anthracnose disease in a wide range of cruciferous crops. To facilitate the efficient control of anthracnose disease, it will be important to understand the mechanism by which the cruciferous crops and C. higginsianum interact. A key step in understanding this interaction is characterizing the mitogen-activated protein kinases (MAPK signaling pathway of C. higginsianum. MAPK plays important roles in diverse physiological processes of multiple pathogens. In this study, a Fus3/Kss1-related MAPK gene, ChMK1, from C. higginsianum was analyzed. The results showed that the Fus3/Kss1-related MAPK ChMK1 plays a significant role in cell wall integrity. Targeted deletion of ChMK1 resulted in a hypersensitivity to cell wall inhibitors, reduced conidiation and albinistic colonies. Further, the deletion mutant was also unable to form melanized appressorium, a specialized infection structure that is necessary for successful infection. Therefore, the deletion mutant loses pathogenicity on A. thaliana leaves, demonstrating that ChMK1 plays an essential role in the early infection step. In addition, the ChMK1 deletion mutant showed an attenuated growth rate that is different from that of its homologue in C. lagenarium, indicating the diverse roles that Fus3/Kss1-related MAPKs plays in phytopathogenic fungi. Furthermore, the expression level of three melanin synthesis associated genes were clearly decreased in the albinistic ChMK1 mutant compared to that of the wild type strain, suggesting that ChMK1 is also required for colony melanization in C. higginsianum.

Looking deep inside: detection of low-abundance proteins in leaf extracts of Arabidopsis and phloem exudates of pumpkin.

Science.gov (United States)

Fröhlich, Andreas; Gaupels, Frank; Sarioglu, Hakan; Holzmeister, Christian; Spannagl, Manuel; Durner, Jörg; Lindermayr, Christian

2012-07-01

The field of proteomics suffers from the immense complexity of even small proteomes and the enormous dynamic range of protein concentrations within a given sample. Most protein samples contain a few major proteins, which hamper in-depth proteomic analysis. In the human field, combinatorial hexapeptide ligand libraries (CPLL; such as ProteoMiner) have been used for reduction of the dynamic range of protein concentrations; however, this technique is not established in plant research. In this work, we present the application of CPLL to Arabidopsis (Arabidopsis thaliana) leaf proteins. One- and two-dimensional gel electrophoresis showed a decrease in high-abundance proteins and an enrichment of less abundant proteins in CPLL-treated samples. After optimization of the CPLL protocol, mass spectrometric analyses of leaf extracts led to the identification of 1,192 proteins in control samples and an additional 512 proteins after the application of CPLL. Upon leaf infection with virulent Pseudomonas syringae DC3000, CPLL beads were also used for investigating the bacterial infectome. In total, 312 bacterial proteins could be identified in infected Arabidopsis leaves. Furthermore, phloem exudates of pumpkin (Cucurbita maxima) were analyzed. CPLL prefractionation caused depletion of the major phloem proteins 1 and 2 and improved phloem proteomics, because 67 of 320 identified proteins were detectable only after CPLL treatment. In sum, our results demonstrate that CPLL beads are a time- and cost-effective tool for reducing major proteins, which often interfere with downstream analyses. The concomitant enrichment of less abundant proteins may facilitate a deeper insight into the plant proteome.

EphA2 modulates radiosensitive of hepatocellular carcinoma cells via p38/mitogen-activated protein kinase-mediated signal pathways

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Qiao Jin

2015-10-01

Full Text Available This experiment was conducted to investigate the role of EPH receptor A2 (EphA2 in the modulation of radiosensitivity of hepatic cellular cancer (HCC cells and to determine whether p38/mitogen-activated protein kinase (p38MAPK signaling mediated EphA2 function in this respect. The protein expressions of EphA2 and phosphorylated p38MAPK were tested in HCC and normal hepatic tissues. In HCC 97H cells, EphA2 was overexpressed and knocked out by transfection with EphA2 expression vector and EphA2-ShRNA, respectively, prior to cell exposure to low-dose irradiation. Significantly upregulated EphA2 and phosphorylated p38MAPK were observed in HCC tissues, compared with those in normal hepatic tissues. Low-dose irradiation (1 Gy only caused minor damage to HCC 97H cells, as assessed by alterations in cell viability, apoptosis rate, and cell healing capacity (p = 0.072, p = 0.078, and p = 0.069 respectively. However, EphA2 knock-out in HCC 97H cells induced significant reduction in cell viability and cell healing capacity after these cells were subjected to low-dose irradiation. Apoptosis rate underwent dramatic increase (p < 0.01. By contrast, EphA2 overexpression in HCC 97H cells reversed these effects and enhanced cell colony formation rate, thus displaying remarkable attenuation of radiosensitivity of HCC 97H cells. Further, SB203580, a specific inhibitor of p38MAPK, was added to HCC 97H cells over-expressing EphA2. The effect of EphA2 overexpression on the radiosensitivity of HCC 97H cells was abrogated. Thus, the present study indicates that EphA2 have the ability to negatively regulate the radiosensitivity of HCC 97H cells, which mainly depends on 38MAPK-mediated signal pathways.

Mitogen-activated protein kinases mediate Mycobacterium ...

Indian Academy of Sciences (India)

2012-01-19

Jan 19, 2012 ... effector molecules and also in the control of intracellular bacterial replication ..... H37Ra in THP-1 cells. The fall and rise in the activation of .... use this distinct role of p38 MAPK to balance the expression of CD44 during ...

Effects of protein kinase C activators on phorbol ester-sensitive and -resistant EL4 thymoma cells.

Science.gov (United States)

Sansbury, H M; Wisehart-Johnson, A E; Qi, C; Fulwood, S; Meier, K E

1997-09-01

Phorbol ester-sensitive EL4 murine thymoma cells respond to phorbol 12-myristate 13-acetate with activation of ERK mitogen-activated protein kinases, synthesis of interleukin-2, and death, whereas phorbol ester-resistant variants of this cell line do not exhibit these responses. Additional aspects of the resistant phenotype were examined, using a newly-established resistant cell line. Phorbol ester induced morphological changes, ERK activation, calcium-dependent activation of the c-Jun N-terminal kinase (JNK), interleukin-2 synthesis, and growth inhibition in sensitive but not resistant cells. A series of protein kinase C activators caused membrane translocation of protein kinase C's (PKCs) alpha, eta, and theta in both cell lines. While PKC eta was expressed at higher levels in sensitive than in resistant cells, overexpression of PKC eta did not restore phorbol ester-induced ERK activation to resistant cells. In sensitive cells, PKC activators had similar effects on cell viability and ERK activation, but differed in their abilities to induce JNK activation and interleukin-2 synthesis. PD 098059, an inhibitor of the mitogen activated protein (MAP)/ERK kinase kinase MEK, partially inhibited ERK activation and completely blocked phorbol ester-induced cell death in sensitive cells. Thus MEK and/or ERK activation, but not JNK activation or interleukin-2 synthesis, appears to be required for phorbol ester-induced toxicity. Alterations in phorbol ester response pathways, rather than altered expression of PKC isoforms, appear to confer phorbol ester resistance to EL4 cells.

Induced Mitogenic Activity in AML-12 Mouse Hepatocytes Exposed to Low-dose Ultra-Wideband Electromagnetic Radiation

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P. B. Tchounwou

2005-04-01

Full Text Available Ultra–wideband (UWB technology has increased with the use of various civilian and military applications. In the present study, we hypothesized that low-dose UWB electromagnetic radiation (UWBR could elicit a mitogenic effect in AML-12 mouse hepatocytes, in vitro. To test this hypothesis, we exposed AML-12 mouse hepatocytes, to UWBR in a specially constructed gigahertz transverse electromagnetic mode (GTEM cell. Cells were exposed to UWBR for 2 h at a temperature of 23°C, a pulse width of 10 ns, a repetition rate of 1 kHz, and field strength of 5-20 kV/m. UWB pulses were triggered by an external pulse generator for UWBR exposure but were not triggered for the sham exposure. We performed an MTT Assay to assess cell viability for UWBR-treated and sham-exposed hepatocytes. Data from viability studies indicated a time-related increase in hepatocytes at time intervals from 8-24 h post exposure. UWBR exerted a statistically significant (p < 0.05 dose-dependent response in cell viability in both serum-treated and serum free medium (SFM -treated hepatocytes. Western blot analysis of hepatocyte lysates demonstrated that cyclin A protein was induced in hepatocytes, suggesting that increased MTT activity after UWBR exposure was due to cell proliferation. This study indicates that UWBR has a mitogenic effect on AML-12 mouse hepatocytes and implicates a possible role for UWBR in hepatocarcinoma.

Short communication: Camel milk ameliorates inflammatory responses and oxidative stress and downregulates mitogen-activated protein kinase signaling pathways in lipopolysaccharide-induced acute respiratory distress syndrome in rats.

Science.gov (United States)

Zhu, Wei-Wei; Kong, Gui-Qing; Ma, Ming-Ming; Li, Yan; Huang, Xiao; Wang, Li-Peng; Peng, Zhen-Yi; Zhang, Xiao-Hua; Liu, Xiang-Yong; Wang, Xiao-Zhi

2016-01-01

Acute respiratory distress syndrome (ARDS) is a complex syndrome disorder with high mortality rate. Camel milk (CM) contains antiinflammatory and antioxidant properties and protects against numerous diseases. This study aimed to demonstrate the function of CM in lipopolysaccharide (LPS)-induced ARDS in rats. Camel milk reduced the lung wet:dry weight ratio and significantly reduced LPS-induced increases in neutrophil infiltration, interstitial and intra-alveolar edema, thickness of the alveolar wall, and lung injury scores of lung tissues. It also had antiinflammatory and antioxidant effects on LPS-induced ARDS. After LPS stimulation, the levels of proinflammatory cytokines (tumor necrosis factor-α, IL-10, and IL-1β) in serum and oxidative stress markers (malondialdehyde, myeloperoxidase, and total antioxidant capacity) in lung tissue were notably attenuated by CM. Camel milk also downregulated mitogen-activated protein kinase signaling pathways. Given these results, CM is a potential complementary food for ARDS treatment. Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Pathogen-secreted proteases activate a novel plant immune pathway.

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Cheng, Zhenyu; Li, Jian-Feng; Niu, Yajie; Zhang, Xue-Cheng; Woody, Owen Z; Xiong, Yan; Djonović, Slavica; Millet, Yves; Bush, Jenifer; McConkey, Brendan J; Sheen, Jen; Ausubel, Frederick M

2015-05-14

Mitogen-activated protein kinase (MAPK) cascades play central roles in innate immune signalling networks in plants and animals. In plants, however, the molecular mechanisms of how signal perception is transduced to MAPK activation remain elusive. Here we report that pathogen-secreted proteases activate a previously unknown signalling pathway in Arabidopsis thaliana involving the Gα, Gβ, and Gγ subunits of heterotrimeric G-protein complexes, which function upstream of an MAPK cascade. In this pathway, receptor for activated C kinase 1 (RACK1) functions as a novel scaffold that binds to the Gβ subunit as well as to all three tiers of the MAPK cascade, thereby linking upstream G-protein signalling to downstream activation of an MAPK cascade. The protease-G-protein-RACK1-MAPK cascade modules identified in these studies are distinct from previously described plant immune signalling pathways such as that elicited by bacterial flagellin, in which G proteins function downstream of or in parallel to an MAPK cascade without the involvement of the RACK1 scaffolding protein. The discovery of the new protease-mediated immune signalling pathway described here was facilitated by the use of the broad host range, opportunistic bacterial pathogen Pseudomonas aeruginosa. The ability of P. aeruginosa to infect both plants and animals makes it an excellent model to identify novel immunoregulatory strategies that account for its niche adaptation to diverse host tissues and immune systems.

A Member of the p38 Mitogen-Activated Protein Kinase Family Is Responsible for Transcriptional Induction of Dopa decarboxylase in the Epidermis of Drosophila melanogaster during the Innate Immune Response▿ †

Science.gov (United States)

Davis, Monica M.; Primrose, David A.; Hodgetts, Ross B.

2008-01-01

Drosophila innate immunity is controlled primarily by the activation of IMD (immune deficiency) or Toll signaling leading to the production of antimicrobial peptides (AMPs). IMD signaling also activates the JUN N-terminal kinase (JNK) cascade, which is responsible for immune induction of non-antimicrobial peptide immune gene transcription though the transcription factor AP-1. Transcription of the Dopa decarboxylase (Ddc) gene is induced in response to gram-negative and gram-positive septic injury, but not aseptic wounding. Transcription is induced throughout the epidermis and not specifically at the site of infection. Ddc transcripts are detectible within 2 h and remain high for several hours following infection with either gram-negative or gram-positive bacteria. Using Ddc-green fluorescent protein (GFP) reporter gene constructs, we show that a conserved consensus AP-1 binding site upstream of the Ddc transcription start site is required for induction. However, neither the Toll, IMD, nor JNK pathway is involved. Rather, Ddc transcription depends on a previously uncharacterized member of the p38 mitogen-activated protein kinase family, p38c. We propose that the involvement of DDC in a new pathway involved in Drosophila immunity increases the levels of dopamine, which is metabolized to produce reactive quinones that exert an antimicrobial effect on invading bacteria. PMID:18519585

Regulator of differentiation 1 (ROD1) binds to the amphipathic C-terminal peptide of thrombospondin-4 and is involved in its mitogenic activity.

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Sadvakassova, Gulzhakhan; Dobocan, Monica C; Difalco, Marcos R; Congote, Luis F

2009-09-01

The matrix protein thrombospondin-4 has an acidic amphipathic C-terminal peptide (C21) which stimulates erythroid cell proliferation. Here we show that C21 stimulates red cell formation in anemic mice in vivo. In vitro experiments indicated that the peptide-mediated increase of erythroid colony formation in cultures of human CD34+ hematopoietic progenitor cells was possible only under continuous presence of erythropoietin. In the absence of this cytokine, C21 stimulated exclusively myeloid colony formation. Therefore, the peptide is not a specific erythroid differentiation factor. In fact, it is mitogenic in non-erythroid cells, such as skin fibroblasts and kidney epithelial cells. In erythroleukemic TF-1 cells, it actually decreased the production of the erythroid differentiation marker glycophorin A. C21-affinity chromatography revealed regulator of differentiation 1 (ROD1) as a major C21-binding protein. ROD1 is the hematopoietic cell paralog of polypyrimidine tract binding proteins (PTBs), RNA splice regulators which regulate differentiation by repressing tissue-specific exons. ROD1 binding to C21 was strongly inhibited by synthetic RNAs in the order poly A > poly U > poly G = poly C and was weakly inhibited by a synthetic phosphorylated peptide mimicking the C-terminal domain of RNA polymerase II. Cellular overexpression or knockdown experiments of ROD1 suggest a role for this protein in the mitogenic activity of C21. Since the nuclear proteins ROD1 and PTBs regulate differentiation at a posttranscriptional level and there is a fast nuclear uptake of C21, we put forward the idea that the peptide is internalized, goes to the nucleus and maintains cells in a proliferative state by supporting ROD1-mediated inhibition of differentiation.

A Lipid Transfer Protein Increases the Glutathione Content and Enhances Arabidopsis Resistance to a Trichothecene Mycotoxin.

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John E McLaughlin

Full Text Available Fusarium head blight (FHB or scab is one of the most important plant diseases worldwide, affecting wheat, barley and other small grains. Trichothecene mycotoxins such as deoxynivalenol (DON accumulate in the grain, presenting a food safety risk and health hazard to humans and animals. Despite considerable breeding efforts, highly resistant wheat or barley cultivars are not available. We screened an activation tagged Arabidopsis thaliana population for resistance to trichothecin (Tcin, a type B trichothecene in the same class as DON. Here we show that one of the resistant lines identified, trichothecene resistant 1 (trr1 contains a T-DNA insertion upstream of two nonspecific lipid transfer protein (nsLTP genes, AtLTP4.4 and AtLTP4.5. Expression of both nsLTP genes was induced in trr1 over 10-fold relative to wild type. Overexpression of AtLTP4.4 provided greater resistance to Tcin than AtLTP4.5 in Arabidopsis thaliana and in Saccharomyces cerevisiae relative to wild type or vector transformed lines, suggesting a conserved protection mechanism. Tcin treatment increased reactive oxygen species (ROS production in Arabidopsis and ROS stain was associated with the chloroplast, the cell wall and the apoplast. ROS levels were attenuated in Arabidopsis and in yeast overexpressing AtLTP4.4 relative to the controls. Exogenous addition of glutathione and other antioxidants enhanced resistance of Arabidopsis to Tcin while the addition of buthionine sulfoximine, an inhibitor of glutathione synthesis, increased sensitivity, suggesting that resistance was mediated by glutathione. Total glutathione content was significantly higher in Arabidopsis and in yeast overexpressing AtLTP4.4 relative to the controls, highlighting the importance of AtLTP4.4 in maintaining the redox state. These results demonstrate that trichothecenes cause ROS accumulation and overexpression of AtLTP4.4 protects against trichothecene-induced oxidative stress by increasing the glutathione

Arabidopsis annexin1 mediates the radical-activated plasma membrane Ca²+- and K+-permeable conductance in root cells.

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Laohavisit, Anuphon; Shang, Zhonglin; Rubio, Lourdes; Cuin, Tracey A; Véry, Anne-Aliénor; Wang, Aihua; Mortimer, Jennifer C; Macpherson, Neil; Coxon, Katy M; Battey, Nicholas H; Brownlee, Colin; Park, Ohkmae K; Sentenac, Hervé; Shabala, Sergey; Webb, Alex A R; Davies, Julia M

2012-04-01

Plant cell growth and stress signaling require Ca²⁺ influx through plasma membrane transport proteins that are regulated by reactive oxygen species. In root cell growth, adaptation to salinity stress, and stomatal closure, such proteins operate downstream of the plasma membrane NADPH oxidases that produce extracellular superoxide anion, a reactive oxygen species that is readily converted to extracellular hydrogen peroxide and hydroxyl radicals, OH•. In root cells, extracellular OH• activates a plasma membrane Ca²⁺-permeable conductance that permits Ca²⁺ influx. In Arabidopsis thaliana, distribution of this conductance resembles that of annexin1 (ANN1). Annexins are membrane binding proteins that can form Ca²⁺-permeable conductances in vitro. Here, the Arabidopsis loss-of-function mutant for annexin1 (Atann1) was found to lack the root hair and epidermal OH•-activated Ca²⁺- and K⁺-permeable conductance. This manifests in both impaired root cell growth and ability to elevate root cell cytosolic free Ca²⁺ in response to OH•. An OH•-activated Ca²⁺ conductance is reconstituted by recombinant ANN1 in planar lipid bilayers. ANN1 therefore presents as a novel Ca²⁺-permeable transporter providing a molecular link between reactive oxygen species and cytosolic Ca²⁺ in plants.

Unravelling Protein-Protein Interaction Networks Linked to Aliphatic and Indole Glucosinolate Biosynthetic Pathways in Arabidopsis

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Sebastian J. Nintemann

2017-11-01

Full Text Available Within the cell, biosynthetic pathways are embedded in protein-protein interaction networks. In Arabidopsis, the biosynthetic pathways of aliphatic and indole glucosinolate defense compounds are well-characterized. However, little is known about the spatial orchestration of these enzymes and their interplay with the cellular environment. To address these aspects, we applied two complementary, untargeted approaches—split-ubiquitin yeast 2-hybrid and co-immunoprecipitation screens—to identify proteins interacting with CYP83A1 and CYP83B1, two homologous enzymes specific for aliphatic and indole glucosinolate biosynthesis, respectively. Our analyses reveal distinct functional networks with substantial interconnection among the identified interactors for both pathway-specific markers, and add to our knowledge about how biochemical pathways are connected to cellular processes. Specifically, a group of protein interactors involved in cell death and the hypersensitive response provides a potential link between the glucosinolate defense compounds and defense against biotrophic pathogens, mediated by protein-protein interactions.

Genome-wide identification and comparative analysis of squamosa-promoter binding proteins (sbp) transcription factor family in gossypium raimondii and arabidopsis thaliana

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Ali, M.A.; Alia, K.B.; Atif, R.M.; Rasulj, I.; Nadeem, H.U.; Shahid, A.; Azeem, F

2017-01-01

SQUAMOSA-Promoter Binding Proteins (SBP) are class of transcription factors that play vital role in regulation of plant tissue growth and development. The genes encoding these proteins have not yet been identified in diploid cotton. Thus here, a comprehensive genome wide analysis of SBP genes/proteins was carried out to identify the genes encoding SBP proteins in Gossypium raimondii and Arabidopsis thaliana. We identified 17 SBP genes from Arabidopsis thaliana genome and 30 SBP genes from Gossypium raimondii. Chromosome localization studies revealed the uneven distribution of SBP encoding genes both in the genomes of A. thaliana and G. raimondii. In cotton, five SBP genes were located on chromosome no. 2, while no gene was found on chromosome 9. In A. thaliana, maximum seven SBP genes were identified on chromosome 9, while chromosome 4 did not have any SBP gene. Thus, the SBP gene family might have expanded as a result of segmental as well as tandem duplications in these species. The comparative phylogenetic analysis of Arabidopsis and cotton SBPs revealed the presence of eight groups. The gene structure analysis of SBP encoding genes revealed the presence of one to eleven inrons in both Arabidopsis and G. raimondii. The proteins sharing the same phyletic group mostly demonstrated the similar intron-exon occurrence pattern; and share the common conserved domains. The SBP DNA-binding domain shared 24 absolutely conserved residues in Arabidopsis. The present study can serve as a base for the functional characterization of SBP gene family in Gossypium raimondii. (author)

Systemic acquired resistance in soybean is regulated by two proteins, Orthologous to Arabidopsis NPR1

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Sandhu Devinder

2009-08-01

Full Text Available Abstract Background Systemic acquired resistance (SAR is induced in non-inoculated leaves following infection with certain pathogenic strains. SAR is effective against many pathogens. Salicylic acid (SA is a signaling molecule of the SAR pathway. The development of SAR is associated with the induction of pathogenesis related (PR genes. Arabidopsis non-expressor of PR1 (NPR1 is a regulatory gene of the SA signal pathway 123. SAR in soybean was first reported following infection with Colletotrichum trancatum that causes anthracnose disease. We investigated if SAR in soybean is regulated by a pathway, similar to the one characterized in Arabidopsis. Results Pathogenesis-related gene GmPR1 is induced following treatment of soybean plants with the SAR inducer, 2,6-dichloroisonicotinic acid (INA or infection with the oomycete pathogen, Phytophthora sojae. In P. sojae-infected plants, SAR was induced against the bacterial pathogen, Pseudomonas syringae pv. glycinea. Soybean GmNPR1-1 and GmNPR1-2 genes showed high identities to Arabidopsis NPR1. They showed similar expression patterns among the organs, studied in this investigation. GmNPR1-1 and GmNPR1-2 are the only soybean homologues of NPR1and are located in homoeologous regions. In GmNPR1-1 and GmNPR1-2 transformed Arabidopsis npr1-1 mutant plants, SAR markers: (i PR-1 was induced following INA treatment and (ii BGL2 following infection with Pseudomonas syringae pv. tomato (Pst, and SAR was induced following Pst infection. Of the five cysteine residues, Cys82, Cys150, Cys155, Cys160, and Cys216 involved in oligomer-monomer transition in NPR1, Cys216 in GmNPR1-1 and GmNPR1-2 proteins was substituted to Ser and Leu, respectively. Conclusion Complementation analyses in Arabidopsis npr1-1 mutants revealed that homoeologous GmNPR1-1 and GmNPR1-2 genes are orthologous to Arabidopsis NPR1. Therefore, SAR pathway in soybean is most likely regulated by GmNPR1 genes. Substitution of Cys216 residue, essential

Systemic acquired resistance in soybean is regulated by two proteins, Orthologous to Arabidopsis NPR1.

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Sandhu, Devinder; Tasma, I Made; Frasch, Ryan; Bhattacharyya, Madan K

2009-08-05

Systemic acquired resistance (SAR) is induced in non-inoculated leaves following infection with certain pathogenic strains. SAR is effective against many pathogens. Salicylic acid (SA) is a signaling molecule of the SAR pathway. The development of SAR is associated with the induction of pathogenesis related (PR) genes. Arabidopsis non-expressor of PR1 (NPR1) is a regulatory gene of the SA signal pathway 123. SAR in soybean was first reported following infection with Colletotrichum trancatum that causes anthracnose disease. We investigated if SAR in soybean is regulated by a pathway, similar to the one characterized in Arabidopsis. Pathogenesis-related gene GmPR1 is induced following treatment of soybean plants with the SAR inducer, 2,6-dichloroisonicotinic acid (INA) or infection with the oomycete pathogen, Phytophthora sojae. In P. sojae-infected plants, SAR was induced against the bacterial pathogen, Pseudomonas syringae pv. glycinea. Soybean GmNPR1-1 and GmNPR1-2 genes showed high identities to Arabidopsis NPR1. They showed similar expression patterns among the organs, studied in this investigation. GmNPR1-1 and GmNPR1-2 are the only soybean homologues of NPR1and are located in homoeologous regions. In GmNPR1-1 and GmNPR1-2 transformed Arabidopsis npr1-1 mutant plants, SAR markers: (i) PR-1 was induced following INA treatment and (ii) BGL2 following infection with Pseudomonas syringae pv. tomato (Pst), and SAR was induced following Pst infection. Of the five cysteine residues, Cys82, Cys150, Cys155, Cys160, and Cys216 involved in oligomer-monomer transition in NPR1, Cys216 in GmNPR1-1 and GmNPR1-2 proteins was substituted to Ser and Leu, respectively. Complementation analyses in Arabidopsis npr1-1 mutants revealed that homoeologous GmNPR1-1 and GmNPR1-2 genes are orthologous to Arabidopsis NPR1. Therefore, SAR pathway in soybean is most likely regulated by GmNPR1 genes. Substitution of Cys216 residue, essential for oligomer-monomer transition of Arabidopsis NPR1

Wheat Transcription Factor TaAREB3 Participates in Drought and Freezing Tolerances in Arabidopsis.

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Wang, Jingyi; Li, Qian; Mao, Xinguo; Li, Ang; Jing, Ruilian

2016-01-01

AREB (ABA response element binding) proteins in plants play direct regulatory roles in response to multiple stresses, but their functions in wheat (Triticum aestivum L.) are not clear. In the present study, TaAREB3, a new member of the AREB transcription factor family, was isolated from wheat. Sequence analysis showed that the TaAREB3 protein is composed of three parts, a conserved N-terminal, a variable M region, and a conserved C-terminal with a bZIP domain. It belongs to the group A subfamily of bZIP transcription factors. TaAREB3 was constitutively expressed in stems, leaves, florets, anthers, pistils, seeds, and most highly, in roots. TaAREB3 gene expression was induced with abscisic acid (ABA) and low temperature stress, and its protein was localized in the nucleus when transiently expressed in tobacco epidermal cells and stably expressed in transgenic Arabidopsis. TaAREB3 protein has transcriptional activation activity, and can bind to the ABRE cis-element in vitro. Overexpression of TaAREB3 in Arabidopsis not only enhanced ABA sensitivity, but also strengthened drought and freezing tolerances. TaAREB3 also activated RD29A, RD29B, COR15A, and COR47 by binding to their promoter regions in transgenic Arabidopsis. These results demonstrated that TaAREB3 plays an important role in drought and freezing tolerances in Arabidopsis.

Na+/H+ Exchange Activity in the Plasma Membrane of Arabidopsis1

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Qiu, Quan-Sheng; Barkla, Bronwyn J.; Vera-Estrella, Rosario; Zhu, Jian-Kang; Schumaker, Karen S.

2003-01-01

In plants, Na+/H+ exchangers in the plasma membrane are critical for growth in high levels of salt, removing toxic Na+ from the cytoplasm by transport out of the cell. The molecular identity of a plasma membrane Na+/H+ exchanger in Arabidopsis (SOS1) has recently been determined. In this study, immunological analysis provided evidence that SOS1 localizes to the plasma membrane of leaves and roots. To characterize the transport activity of this protein, purified plasma membrane vesicles were isolated from leaves of Arabidopsis. Na+/H+ exchange activity, monitored as the ability of Na to dissipate an established pH gradient, was absent in plants grown without salt. However, exchange activity was induced when plants were grown in 250 mm NaCl and increased with prolonged salt exposure up to 8 d. H+-coupled exchange was specific for Na, because chloride salts of other monovalent cations did not dissipate the pH gradient. Na+/H+ exchange activity was dependent on Na (substrate) concentration, and kinetic analysis indicated that the affinity (apparent Km) of the transporter for Na+ is 22.8 mm. Data from two experimental approaches supports electroneutral exchange (one Na+ exchanged for one proton): (a) no change in membrane potential was measured during the exchange reaction, and (b) Na+/H+ exchange was unaffected by the presence or absence of a membrane potential. Results from this research provide a framework for future studies into the regulation of the plant plasma membrane Na+/H+ exchanger and its relative contribution to the maintenance of cellular Na+ homeostasis during plant growth in salt. PMID:12805632

The Zinc-Finger Thylakoid-Membrane Protein FIP Is Involved With Abiotic Stress Response in Arabidopsis thaliana

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Karina L. Lopes

2018-04-01

Full Text Available Many plant genes have their expression modulated by stress conditions. Here, we used Arabidopsis FtsH5 protease, which expression is regulated by light stress, as bait in a yeast two-hybrid screen to search for new proteins involved in the stress response. As a result, we found FIP (FtsH5 Interacting Protein, which possesses an amino proximal cleavable transit peptide, a hydrophobic membrane-anchoring region, and a carboxyl proximal C4-type zinc-finger domain. In vivo experiments using FIP fused to green fluorescent protein (GFP showed a plastid localization. This finding was corroborated by chloroplast import assays that showed FIP inserted in the thylakoid membrane. FIP expression was down-regulated in plants exposed to high light intensity, oxidative, salt, and osmotic stresses, whereas mutant plants expressing low levels of FIP were more tolerant to these abiotic stresses. Our data shows a new thylakoid-membrane protein involved with abiotic stress response in Arabidopsis thaliana.

Proteomic identification of early salicylate- and flg22-responsive redox-sensitive proteins in Arabidopsis

KAUST Repository

Liu, Peng; Zhang, Huoming; Yu, Boying; Xiong, Liming; Xia, Yiji

2015-01-01

in Arabidopsis cells during the early response to salicylate or flg22, two defense pathway elicitors that are known to disturb cellular redox homeostasis. Among the salicylate- and/or flg22-responsive redox-sensitive proteins are those involved in transcriptional

A mannose-specific tetrameric lectin with mitogenic and antibacterial activities from the ovary of a teleost, the cobia (Rachycentron canadum).

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Ngai, Patrick H K; Ng, T B

2007-02-01

A tetrameric lectin, with hemagglutinating activity toward rabbit erythrocytes and with specificity toward D-mannosamine and D(+)-mannose, was isolated from the ovaries of a teleost, the cobia Rachycentron canadum. The isolation protocol comprised ion exchange chromatography on CM-cellulose and Q-Sepharose, ion exchange chromatography by fast protein liquid chromatography (FPLC) on Mono Q, and finally gel filtration by FPLC on Superose 12. The lectin was adsorbed on all ion exchangers used. It exhibited a molecular mass of 180 kDa in gel filtration on Superose 12 and a single 45-kDa band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating that it is a tetrameric protein. The hemagglutinating activity of the lectin was stable up to 40 degrees C and between pH 4 and pH 10. All hemagglutinating activity disappeared at 60 degrees C and at pH 1 and pH 13. The hemagglutinating activity was doubled in the presence of 0.1 microM FeCl3. The lectin exerted antibacterial activity against Escherichia coli with 50% inhibition at 250 microg. There was no antifungal activity toward Coprinus comatus, Fusarium oxysporum, Mycosphaerella arachidicola, and Rhizoctonia solani at a dose of 300 microg. The lectin exhibited maximal mitogenic response from mouse splenocytes at a concentration of 14 microM.

Genome-wide analysis of protein disorder in Arabidopsis thaliana: implications for plant environmental adaptation.

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Pietrosemoli, Natalia; García-Martín, Juan A; Solano, Roberto; Pazos, Florencio

2013-01-01

Intrinsically disordered proteins/regions (IDPs/IDRs) are currently recognized as a widespread phenomenon having key cellular functions. Still, many aspects of the function of these proteins need to be unveiled. IDPs conformational flexibility allows them to recognize and interact with multiple partners, and confers them larger interaction surfaces that may increase interaction speed. For this reason, molecular interactions mediated by IDPs/IDRs are particularly abundant in certain types of protein interactions, such as those of signaling and cell cycle control. We present the first large-scale study of IDPs in Arabidopsis thaliana, the most widely used model organism in plant biology, in order to get insight into the biological roles of these proteins in plants. The work includes a comparative analysis with the human proteome to highlight the differential use of disorder in both species. Results show that while human proteins are in general more disordered, certain functional classes, mainly related to environmental response, are significantly more enriched in disorder in Arabidopsis. We propose that because plants cannot escape from environmental conditions as animals do, they use disorder as a simple and fast mechanism, independent of transcriptional control, for introducing versatility in the interaction networks underlying these biological processes so that they can quickly adapt and respond to challenging environmental conditions.

Polycomb Group Proteins RING1A and RING1B Regulate the Vegetative Phase Transition in Arabidopsis

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Jian Li

2017-05-01

Full Text Available Polycomb group (PcG protein-mediated gene silencing is a major regulatory mechanism in higher eukaryotes that affects gene expression at the transcriptional level. Here, we report that two conserved homologous PcG proteins, RING1A and RING1B (RING1A/B, are required for global H2A monoubiquitination (H2Aub in Arabidopsis. The mutation of RING1A/B increased the expression of members of the SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL gene family and caused an early vegetative phase transition. The early vegetative phase transition observed in ring1a ring1b double mutant plants was dependent on an SPL family gene, and the H2Aub status of the chromatin at SPL locus was dependent on RING1A/B. Moreover, mutation in RING1A/B affected the miRNA156a-mediated vegetative phase transition, and RING1A/B and the AGO7-miR390-TAS3 pathway were found to additively regulate this transition in Arabidopsis. Together, our results demonstrate that RING1A/B regulates the vegetative phase transition in Arabidopsis through the repression of SPL family genes.

The Arabidopsis CROWDED NUCLEI genes regulate seed germination by modulating degradation of ABI5 protein.

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Zhao, Wenming; Guan, Chunmei; Feng, Jian; Liang, Yan; Zhan, Ni; Zuo, Jianru; Ren, Bo

2016-07-01

In Arabidopsis, the phytohormone abscisic acid (ABA) plays a vital role in inhibiting seed germination and in post-germination seedling establishment. In the ABA signaling pathway, ABI5, a basic Leu zipper transcription factor, has important functions in the regulation of seed germination. ABI5 protein localizes in nuclear bodies, along with AFP, COP1, and SIZ1, and was degraded through the 26S proteasome pathway. However, the mechanisms of ABI5 nuclear body formation and ABI5 protein degradation remain obscure. In this study, we found that the Arabidopsis CROWDED NUCLEI (CRWN) proteins, predicted nuclear matrix proteins essential for maintenance of nuclear morphology, also participate in ABA-controlled seed germination by regulating the degradation of ABI5 protein. During seed germination, the crwn mutants are hypersensitive to ABA and have higher levels of ABI5 protein compared to wild type. Genetic analysis suggested that CRWNs act upstream of ABI5. The observation that CRWN3 colocalizes with ABI5 in nuclear bodies indicates that CRWNs might participate in ABI5 protein degradation in nuclear bodies. Moreover, we revealed that the extreme C-terminal of CRWN3 protein is necessary for its function in the response to ABA in germination. Our results suggested important roles of CRWNs in ABI5 nuclear body organization and ABI5 protein degradation during seed germination. © 2015 Institute of Botany, Chinese Academy of Sciences.

Role of Akt/PKB and PFKFB isoenzymes in the control of glycolysis, cell proliferation and protein synthesis in mitogen-stimulated thymocytes.

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Houddane, Amina; Bultot, Laurent; Novellasdemunt, Laura; Johanns, Manuel; Gueuning, Marie-Agnès; Vertommen, Didier; Coulie, Pierre G; Bartrons, Ramon; Hue, Louis; Rider, Mark H

2017-06-01

Proliferating cells depend on glycolysis mainly to supply precursors for macromolecular synthesis. Fructose 2,6-bisphosphate (Fru-2,6-P 2 ) is the most potent positive allosteric effector of 6-phosphofructo-1-kinase (PFK-1), and hence of glycolysis. Mitogen stimulation of rat thymocytes with concanavalin A (ConA) led to time-dependent increases in lactate accumulation (6-fold), Fru-2,6-P 2 content (4-fold), 6-phosphofructo-2-kinase (PFK-2)/fructose-2,6-bisphosphatase isoenzyme 3 and 4 (PFKFB3 and PFKFB4) protein levels (~2-fold and ~15-fold, respectively) and rates of cell proliferation (~40-fold) and protein synthesis (10-fold) after 68h of incubation compared with resting cells. After 54h of ConA stimulation, PFKFB3 mRNA levels were 45-fold higher than those of PFKFB4 mRNA. Although PFKFB3 could be phosphorylated at Ser461 by protein kinase B (PKB) in vitro leading to PFK-2 activation, PFKFB3 Ser461 phosphorylation was barely detectable in resting cells and only increased slightly in ConA-stimulated cells. On the other hand, PFKFB3 and PFKFB4 mRNA levels were decreased (90% and 70%, respectively) by exposure of ConA-stimulated cells to low doses of PKB inhibitor (MK-2206), suggesting control of expression of the two PFKFB isoenzymes by PKB. Incubation of thymocytes with ConA resulted in increased expression and phosphorylation of the translation factors eukaryotic initiation factor-4E-binding protein-1 (4E-BP1) and ribosomal protein S6 (rpS6). Treatment of ConA-stimulated thymocytes with PFK-2 inhibitor (3PO) or MK-2206 led to significant decreases in Fru-2,6-P 2 content, medium lactate accumulation and rates of cell proliferation and protein synthesis. These data were confirmed by using siRNA knockdown of PFKFB3, PFKFB4 and PKB α/β in the more easily transfectable Jurkat E6-1 cell line. The findings suggest that increased PFKFB3 and PFKFB4 expression, but not increased PFKFB3 Ser461 phosphorylation, plays a role in increasing glycolysis in mitogen

ABA-Induced Stomatal Closure Involves ALMT4, a Phosphorylation-Dependent Vacuolar Anion Channel of Arabidopsis[OPEN

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Baetz, Ulrike; Huck, Nicola V.; Zhang, Jingbo

2017-01-01

Stomatal pores are formed between a pair of guard cells and allow plant uptake of CO2 and water evaporation. Their aperture depends on changes in osmolyte concentration of guard cell vacuoles, specifically of K+ and Mal2−. Efflux of Mal2− from the vacuole is required for stomatal closure; however, it is not clear how the anion is released. Here, we report the identification of ALMT4 (ALUMINUM ACTIVATED MALATE TRANSPORTER4) as an Arabidopsis thaliana ion channel that can mediate Mal2− release from the vacuole and is required for stomatal closure in response to abscisic acid (ABA). Knockout mutants showed impaired stomatal closure in response to the drought stress hormone ABA and increased whole-plant wilting in response to drought and ABA. Electrophysiological data show that ALMT4 can mediate Mal2− efflux and that the channel activity is dependent on a phosphorylatable C-terminal serine. Dephosphomimetic mutants of ALMT4 S382 showed increased channel activity and Mal2− efflux. Reconstituting the active channel in almt4 mutants impaired growth and stomatal opening. Phosphomimetic mutants were electrically inactive and phenocopied the almt4 mutants. Surprisingly, S382 can be phosphorylated by mitogen-activated protein kinases in vitro. In brief, ALMT4 likely mediates Mal2− efflux during ABA-induced stomatal closure and its activity depends on phosphorylation. PMID:28874508

Similar pathogen targets in Arabidopsis thaliana and homo sapiens protein networks.

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Paulo Shakarian

Full Text Available We study the behavior of pathogens on host protein networks for humans and Arabidopsis - noting striking similarities. Specifically, we preform [Formula: see text]-shell decomposition analysis on these networks - which groups the proteins into various "shells" based on network structure. We observe that shells with a higher average degree are more highly targeted (with a power-law relationship and that highly targeted nodes lie in shells closer to the inner-core of the network. Additionally, we also note that the inner core of the network is significantly under-targeted. We show that these core proteins may have a role in intra-cellular communication and hypothesize that they are less attacked to ensure survival of the host. This may explain why certain high-degree proteins are not significantly attacked.

Discovery and Characterization of Non-ATP Site Inhibitors of the Mitogen Activated Protein (MAP) Kinases

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Comess, Kenneth M.; Sun, Chaohong; Abad-Zapatero, Cele; Goedken, Eric R.; Gum, Rebecca J.; Borhani, David W.; Argiriadi, Maria; Groebe, Duncan R.; Jia, Yong; Clampit, Jill E.; Haasch, Deanna L.; Smith, Harriet T.; Wang, Sanyi; Song, Danying; Coen, Michael L.; Cloutier, Timothy E.; Tang, Hua; Cheng, Xueheng; Quinn, Christopher; Liu, Bo; Xin, Zhili; Liu, Gang; Fry, Elizabeth H.; Stoll, Vincent; Ng, Teresa I.; Banach, David; Marcotte, Doug; Burns, David J.; Calderwood, David J.; Hajduk, Philip J. (Abbott)

2012-03-02

Inhibition of protein kinases has validated therapeutic utility for cancer, with at least seven kinase inhibitor drugs on the market. Protein kinase inhibition also has significant potential for a variety of other diseases, including diabetes, pain, cognition, and chronic inflammatory and immunologic diseases. However, as the vast majority of current approaches to kinase inhibition target the highly conserved ATP-binding site, the use of kinase inhibitors in treating nononcology diseases may require great selectivity for the target kinase. As protein kinases are signal transducers that are involved in binding to a variety of other proteins, targeting alternative, less conserved sites on the protein may provide an avenue for greater selectivity. Here we report an affinity-based, high-throughput screening technique that allows nonbiased interrogation of small molecule libraries for binding to all exposed sites on a protein surface. This approach was used to screen both the c-Jun N-terminal protein kinase Jnk-1 (involved in insulin signaling) and p38{alpha} (involved in the formation of TNF{alpha} and other cytokines). In addition to canonical ATP-site ligands, compounds were identified that bind to novel allosteric sites. The nature, biological relevance, and mode of binding of these ligands were extensively characterized using two-dimensional {sup 1}H/{sup 13}C NMR spectroscopy, protein X-ray crystallography, surface plasmon resonance, and direct enzymatic activity and activation cascade assays. Jnk-1 and p38{alpha} both belong to the MAP kinase family, and the allosteric ligands for both targets bind similarly on a ledge of the protein surface exposed by the MAP insertion present in the CMGC family of protein kinases and distant from the active site. Medicinal chemistry studies resulted in an improved Jnk-1 ligand able to increase adiponectin secretion in human adipocytes and increase insulin-induced protein kinase PKB phosphorylation in human hepatocytes, in

Arabidopsis N-MYC DOWNREGULATED-LIKE1, a positive regulator of auxin transport in a G protein-mediated pathway.

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Mudgil, Yashwanti; Uhrig, Joachm F; Zhou, Jiping; Temple, Brenda; Jiang, Kun; Jones, Alan M

2009-11-01

Root architecture results from coordinated cell division and expansion in spatially distinct cells of the root and is established and maintained by gradients of auxin and nutrients such as sugars. Auxin is transported acropetally through the root within the central stele and then, upon reaching the root apex, auxin is transported basipetally through the outer cortical and epidermal cells. The two Gbetagamma dimers of the Arabidopsis thaliana heterotrimeric G protein complex are differentially localized to the central and cortical tissues of the Arabidopsis roots. A null mutation in either the single beta (AGB1) or the two gamma (AGG1 and AGG2) subunits confers phenotypes that disrupt the proper architecture of Arabidopsis roots and are consistent with altered auxin transport. Here, we describe an evolutionarily conserved interaction between AGB1/AGG dimers and a protein designated N-MYC DOWNREGULATED-LIKE1 (NDL1). The Arabidopsis genome encodes two homologs of NDL1 (NDL2 and NDL3), which also interact with AGB1/AGG1 and AGB1/AGG2 dimers. We show that NDL proteins act in a signaling pathway that modulates root auxin transport and auxin gradients in part by affecting the levels of at least two auxin transport facilitators. Reduction of NDL family gene expression and overexpression of NDL1 alter root architecture, auxin transport, and auxin maxima. AGB1, auxin, and sugars are required for NDL1 protein stability in regions of the root where auxin gradients are established; thus, the signaling mechanism contains feedback loops.

Harpin-induced expression and transgenic overexpression of the phloem protein gene AtPP2-A1 in Arabidopsis repress phloem feeding of the green peach aphid Myzus persicae

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Sun Weiwei

2011-01-01

Full Text Available Abstract Background Treatment of plants with HrpNEa, a protein of harpin group produced by Gram-negative plant pathogenic bacteria, induces plant resistance to insect herbivores, including the green peach aphid Myzus persicae, a generalist phloem-feeding insect. Under attacks by phloem-feeding insects, plants defend themselves using the phloem-based defense mechanism, which is supposed to involve the phloem protein 2 (PP2, one of the most abundant proteins in the phloem sap. The purpose of this study was to obtain genetic evidence for the function of the Arabidopsis thaliana (Arabidopsis PP2-encoding gene AtPP2-A1 in resistance to M. persicae when the plant was treated with HrpNEa and after the plant was transformed with AtPP2-A1. Results The electrical penetration graph technique was used to visualize the phloem-feeding activities of apterous agamic M. persicae females on leaves of Arabidopsis plants treated with HrpNEa and an inactive protein control, respectively. A repression of phloem feeding was induced by HrpNEa in wild-type (WT Arabidopsis but not in atpp2-a1/E/142, the plant mutant that had a defect in the AtPP2-A1 gene, the most HrpNEa-responsive of 30 AtPP2 genes. In WT rather than atpp2-a1/E/142, the deterrent effect of HrpNEa treatment on the phloem-feeding activity accompanied an enhancement of AtPP2-A1 expression. In PP2OETAt (AtPP2-A1-overexpression transgenic Arabidopsis thaliana plants, abundant amounts of the AtPP2-A1 gene transcript were detected in different organs, including leaves, stems, calyces, and petals. All these organs had a deterrent effect on the phloem-feeding activity compared with the same organs of the transgenic control plant. When a large-scale aphid population was monitored for 24 hours, there was a significant decrease in the number of aphids that colonized leaves of HrpNEa-treated WT and PP2OETAt plants, respectively, compared with control plants. Conclusions The repression in phloem-feeding activities of

Harpin-induced expression and transgenic overexpression of the phloem protein gene AtPP2-A1 in Arabidopsis repress phloem feeding of the green peach aphid Myzus persicae.

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Zhang, Chunling; Shi, Haojie; Chen, Lei; Wang, Xiaomeng; Lü, Beibei; Zhang, Shuping; Liang, Yuan; Liu, Ruoxue; Qian, Jun; Sun, Weiwei; You, Zhenzhen; Dong, Hansong

2011-01-13

Treatment of plants with HrpNEa, a protein of harpin group produced by Gram-negative plant pathogenic bacteria, induces plant resistance to insect herbivores, including the green peach aphid Myzus persicae, a generalist phloem-feeding insect. Under attacks by phloem-feeding insects, plants defend themselves using the phloem-based defense mechanism, which is supposed to involve the phloem protein 2 (PP2), one of the most abundant proteins in the phloem sap. The purpose of this study was to obtain genetic evidence for the function of the Arabidopsis thaliana (Arabidopsis) PP2-encoding gene AtPP2-A1 in resistance to M. persicae when the plant was treated with HrpNEa and after the plant was transformed with AtPP2-A1. The electrical penetration graph technique was used to visualize the phloem-feeding activities of apterous agamic M. persicae females on leaves of Arabidopsis plants treated with HrpNEa and an inactive protein control, respectively. A repression of phloem feeding was induced by HrpNEa in wild-type (WT) Arabidopsis but not in atpp2-a1/E/142, the plant mutant that had a defect in the AtPP2-A1 gene, the most HrpNEa-responsive of 30 AtPP2 genes. In WT rather than atpp2-a1/E/142, the deterrent effect of HrpNEa treatment on the phloem-feeding activity accompanied an enhancement of AtPP2-A1 expression. In PP2OETAt (AtPP2-A1-overexpression transgenic Arabidopsis thaliana) plants, abundant amounts of the AtPP2-A1 gene transcript were detected in different organs, including leaves, stems, calyces, and petals. All these organs had a deterrent effect on the phloem-feeding activity compared with the same organs of the transgenic control plant. When a large-scale aphid population was monitored for 24 hours, there was a significant decrease in the number of aphids that colonized leaves of HrpNEa-treated WT and PP2OETAt plants, respectively, compared with control plants. The repression in phloem-feeding activities of M. persicae as a result of AtPP2-A1 overexpression, and

Crystal structure of the Epithiospecifier Protein, ESP from Arabidopsis thaliana provides insights into its product specificity.

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Zhang, Weiwei; Wang, Wenhe; Liu, Zihe; Xie, Yongchao; Wang, Hao; Mu, Yajuan; Huang, Yao; Feng, Yue

2016-09-16

Specifier proteins are important components of the glucosinolate-myrosinase system, which mediate plant defense against herbivory and pathogen attacks. Upon tissue disruption, glucosinolates are hydrolyzed to instable aglucones by myrosinases, and then aglucones will rearrange to form defensive isothiocyanates. Specifier proteins can redirect this reaction to form other products, such as simple nitriles, epithionitriles and organic thiocyanates instead of isothiocyanates based on the side chain structure of glucosinolate and the type of the specifier proteins. Nevertheless, the molecular mechanism underlying the different product spectrums of various specifier proteins was not fully understood. Here in this study, we solved the crystal structure of the Epithiospecifier Protein, ESP from Arabidopsis thaliana (AtESP) at 2.3 Å resolution. Structural comparisons with the previously solved structure of thiocyanate forming protein, TFP from Thlaspi arvense (TaTFP) reveal that AtESP shows a dimerization pattern different from TaTFP. Moreover, AtESP harbors a slightly larger active site pocket than TaTFP and several residues around the active site are different between the two proteins, which might account for the different product spectrums of the two proteins. Together, our structural study provides important insights into the molecular mechanisms of specifier proteins and shed light on the basis of their different product spectrums. Copyright © 2016 Elsevier Inc. All rights reserved.

mCSF1, a nucleus-encoded CRM protein required for the processing of many mitochondrial introns, is involved in the biogenesis of respiratory complexes I and IV in Arabidopsis.

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Zmudjak, Michal; Colas des Francs-Small, Catherine; Keren, Ido; Shaya, Felix; Belausov, Eduard; Small, Ian; Ostersetzer-Biran, Oren

2013-07-01

The coding regions of many mitochondrial genes in plants are interrupted by intervening sequences that are classified as group II introns. Their splicing is essential for the expression of the genes they interrupt and hence for respiratory function, and is facilitated by various protein cofactors. Despite the importance of these cofactors, only a few of them have been characterized. CRS1-YhbY domain (CRM) is a recently recognized RNA-binding domain that is present in several characterized splicing factors in plant chloroplasts. The Arabidopsis genome encodes 16 CRM proteins, but these are largely uncharacterized. Here, we analyzed the intracellular location of one of these hypothetical proteins in Arabidopsis, mitochondrial CAF-like splicing factor 1 (mCSF1; At4 g31010), and analyzed the growth phenotypes and organellar activities associated with mcsf1 mutants in plants. Our data indicated that mCSF1 resides within mitochondria and its functions are essential during embryogenesis. Mutant plants with reduced mCSF1 displayed inhibited germination and retarded growth phenotypes that were tightly associated with reduced complex I and IV activities. Analogously to the functions of plastid-localized CRM proteins, analysis of the RNA profiles in wildtype and mcsf1 plants showed that mCSF1 acts in the splicing of many of the group II intron RNAs in Arabidopsis mitochondria. © 2013 The Authors. New Phytologist © 2013 New Phytologist Trust.

Differential processing of Arabidopsis ubiquitin-like Atg8 autophagy proteins by Atg4 cysteine proteases

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Woo, Jongchan; Park, Eunsook; Dinesh-Kumar, S. P.

2014-01-01

Autophagy is a highly conserved biological process during which double membrane bound autophagosomes carry intracellular cargo material to the vacuole or lysosome for degradation and/or recycling. Autophagosome biogenesis requires Autophagy 4 (Atg4) cysteine protease-mediated processing of ubiquitin-like Atg8 proteins. Unlike single Atg4 and Atg8 genes in yeast, the Arabidopsis genome contains two Atg4 (AtAtg4a and AtAtg4b) and nine Atg8 (AtAtg8a–AtAtg8i) genes. However, we know very little about specificity of different AtAtg4s for processing of different AtAtg8s. Here, we describe a unique bioluminescence resonance energy transfer-based AtAtg8 synthetic substrate to assess AtAtg4 activity in vitro and in vivo. In addition, we developed a unique native gel assay of superhRLUC catalytic activity assay to monitor cleavage of AtAtg8s in vitro. Our results indicate that AtAtg4a is the predominant protease and that it processes AtAtg8a, AtAtg8c, AtAtg8d, and AtAtg8i better than AtAtg4b in vitro. In addition, kinetic analyses indicate that although both AtAtg4s have similar substrate affinity, AtAtg4a is more active than AtAtg4b in vitro. Activity of AtAtg4s is reversibly inhibited in vitro by reactive oxygen species such as H2O2. Our in vivo bioluminescence resonance energy transfer analyses in Arabidopsis transgenic plants indicate that the AtAtg8 synthetic substrate is efficiently processed and this is AtAtg4 dependent. These results indicate that the synthetic AtAtg8 substrate is used efficiently in the biogenesis of autophagosomes in vivo. Transgenic Arabidopsis plants expressing the AtAtg8 synthetic substrate will be a valuable tool to dissect autophagy processes and the role of autophagy during different biological processes in plants. PMID:24379391

The Genetic Basis of Constitutive and Herbivore-Induced ESP-Independent Nitrile Formation in Arabidopsis1[W][OA

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Burow, Meike; Losansky, Anja; Müller, René; Plock, Antje; Kliebenstein, Daniel J.; Wittstock, Ute

2009-01-01

Glucosinolates are a group of thioglucosides that are components of an activated chemical defense found in the Brassicales. Plant tissue damage results in hydrolysis of glucosinolates by endogenous thioglucosidases known as myrosinases. Spontaneous rearrangement of the aglucone yields reactive isothiocyanates that are toxic to many organisms. In the presence of specifier proteins, alternative products, namely epithionitriles, simple nitriles, and thiocyanates with different biological activities, are formed at the expense of isothiocyanates. Recently, simple nitriles were recognized to serve distinct functions in plant-insect interactions. Here, we show that simple nitrile formation in Arabidopsis (Arabidopsis thaliana) ecotype Columbia-0 rosette leaves increases in response to herbivory and that this increase is independent of the known epithiospecifier protein (ESP). We combined phylogenetic analysis, a screen of Arabidopsis mutants, recombinant protein characterization, and expression quantitative trait locus mapping to identify a gene encoding a nitrile-specifier protein (NSP) responsible for constitutive and herbivore-induced simple nitrile formation in Columbia-0 rosette leaves. AtNSP1 is one of five Arabidopsis ESP homologues that promote simple nitrile, but not epithionitrile or thiocyanate, formation. Four of these homologues possess one or two lectin-like jacalin domains, which share a common ancestry with the jacalin domains of the putative Arabidopsis myrosinase-binding proteins MBP1 and MBP2. A sixth ESP homologue lacked specifier activity and likely represents the ancestor of the gene family with a different biochemical function. By illuminating the genetic and biochemical bases of simple nitrile formation, our study provides new insights into the evolution of metabolic diversity in a complex plant defense system. PMID:18987211

Targeting p38 Mitogen-Activated Protein Kinase Signaling Restores Subventricular Zone Neural Stem Cells and Corrects Neuromotor Deficits in Atm Knockout Mouse

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Kim, Jeesun

2012-01-01

Ataxia-telangiectasia (A-T) is a progressive degenerative disorder that results in major neurological disability. In A-T patients, necropsy has revealed atrophy of cerebellar cortical layers along with Purkinje and granular cell loss. We have previously identified an oxidative stress-mediated increase in phospho-p38 mitogen-activated protein kinase (MAPK) and the resultant downregulation of Bmi-1 and upregulation of p21 as key components of the mechanism causing defective proliferation of neural stem cells (NSCs) isolated from the subventricular zone (SVZ) of Atm−/− mice. However, the in vivo aspect of alteration in SVZ tissue and the functional significance of p38MAPK activation in NSCs for neuropathogenesis of ATM deficiency remain unknown. Here we show that the NSC population was abnormally decreased in the SVZ of 3-month-old Atm−/− mice; this decrease was accompanied by p38MAPK activation. However, after a 2-month treatment with the p38MAPK inhibitor SB203580, starting at 1 month old, Atm−/− mice showed restoration of normal levels of Bmi-1 and p21 with the rescue of NSC population in the SVZ. In addition, treated Atm−/− mice exhibited more Purkinje cells in the cerebellum. Most importantly, motor coordination of Atm−/− mice was significantly improved in the treatment group. Our results show for the first time in vivo evidence of depleted NSCs in the SVZ of Atm−/− mice and also demonstrate that pharmacologic inhibition of p38MAPK signaling has the potential to treat neurological defects of A-T. This study provides a promising approach targeting the oxidative stress-dependent p38 signaling pathway not only for A-T but also for other neurodegenerative disorders. PMID:23197859

Looking Deep Inside: Detection of Low-Abundance Proteins in Leaf Extracts of Arabidopsis and Phloem Exudates of Pumpkin1[W

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Fröhlich, Andreas; Gaupels, Frank; Sarioglu, Hakan; Holzmeister, Christian; Spannagl, Manuel; Durner, Jörg; Lindermayr, Christian

2012-01-01

The field of proteomics suffers from the immense complexity of even small proteomes and the enormous dynamic range of protein concentrations within a given sample. Most protein samples contain a few major proteins, which hamper in-depth proteomic analysis. In the human field, combinatorial hexapeptide ligand libraries (CPLL; such as ProteoMiner) have been used for reduction of the dynamic range of protein concentrations; however, this technique is not established in plant research. In this work, we present the application of CPLL to Arabidopsis (Arabidopsis thaliana) leaf proteins. One- and two-dimensional gel electrophoresis showed a decrease in high-abundance proteins and an enrichment of less abundant proteins in CPLL-treated samples. After optimization of the CPLL protocol, mass spectrometric analyses of leaf extracts led to the identification of 1,192 proteins in control samples and an additional 512 proteins after the application of CPLL. Upon leaf infection with virulent Pseudomonas syringae DC3000, CPLL beads were also used for investigating the bacterial infectome. In total, 312 bacterial proteins could be identified in infected Arabidopsis leaves. Furthermore, phloem exudates of pumpkin (Cucurbita maxima) were analyzed. CPLL prefractionation caused depletion of the major phloem proteins 1 and 2 and improved phloem proteomics, because 67 of 320 identified proteins were detectable only after CPLL treatment. In sum, our results demonstrate that CPLL beads are a time- and cost-effective tool for reducing major proteins, which often interfere with downstream analyses. The concomitant enrichment of less abundant proteins may facilitate a deeper insight into the plant proteome. PMID:22555880

Dominant negative RPW8.2 fusion proteins reveal the importance of haustorium-oriented protein trafficking for resistance against powdery mildew in Arabidopsis.

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Zhang, Qiong; Berkey, Robert; Pan, Zhiyong; Wang, Wenming; Zhang, Yi; Ma, Xianfeng; King, Harlan; Xiao, Shunyuan

2015-01-01

Powdery mildew fungi form feeding structures called haustoria inside epidermal cells of host plants to extract photosynthates for their epiphytic growth and reproduction. The haustorium is encased by an interfacial membrane termed the extrahaustorial membrane (EHM). The atypical resistance protein RPW8.2 from Arabidopsis is specifically targeted to the EHM where RPW8.2 activates haustorium-targeted (thus broad-spectrum) resistance against powdery mildew fungi. EHM-specific localization of RPW8.2 suggests the existence of an EHM-oriented protein/membrane trafficking pathway during EHM biogenesis. However, the importance of this specific trafficking pathway for host defense has not been evaluated via a genetic approach without affecting other trafficking pathways. Here, we report that expression of EHM-oriented, nonfunctional RPW8.2 chimeric proteins exerts dominant negative effect over functional RPW8.2 and potentially over other EHM-localized defense proteins, thereby compromising both RPW8.2-mediated and basal resistance to powdery mildew. Thus, our results highlight the importance of the EHM-oriented protein/membrane trafficking pathway for host resistance against haustorium-forming pathogens such as powdery mildew fungi.

Inhibition of mitogen-activated protein kinase kinase, DNA methyltransferase, and transforming growth factor-β promotes differentiation of human induced pluripotent stem cells into enterocytes.

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Kodama, Nao; Iwao, Takahiro; Kabeya, Tomoki; Horikawa, Takashi; Niwa, Takuro; Kondo, Yuki; Nakamura, Katsunori; Matsunaga, Tamihide

2016-06-01

We previously reported that small-molecule compounds were effective in generating pharmacokinetically functional enterocytes from human induced pluripotent stem (iPS) cells. In this study, to determine whether the compounds promote the differentiation of human iPS cells into enterocytes, we investigated the effects of a combination of mitogen-activated protein kinase kinase (MEK), DNA methyltransferase (DNMT), and transforming growth factor (TGF)-β inhibitors on intestinal differentiation. Human iPS cells cultured on feeder cells were differentiated into endodermal cells by activin A. These endodermal-like cells were then differentiated into intestinal stem cells by fibroblast growth factor 2. Finally, the cells were differentiated into enterocyte cells by epidermal growth factor and small-molecule compounds. After differentiation, mRNA expression levels and drug-metabolizing enzyme activities were measured. The mRNA expression levels of the enterocyte marker sucrase-isomaltase and the major drug-metabolizing enzyme cytochrome P450 (CYP) 3A4 were increased by a combination of MEK, DNMT, and TGF-β inhibitors. The mRNA expression of CYP3A4 was markedly induced by 1α,25-dihydroxyvitamin D3. Metabolic activities of CYP1A1/2, CYP2B6, CYP2C9, CYP2C19, CYP3A4/5, UDP-glucuronosyltransferase, and sulfotransferase were also observed in the differentiated cells. In conclusion, MEK, DNMT, and TGF-β inhibitors can be used to promote the differentiation of human iPS cells into pharmacokinetically functional enterocytes. Copyright © 2016 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.

Xingshentongqiao Decoction Mediates Proliferation, Apoptosis, Orexin-A Receptor and Orexin-B Receptor Messenger Ribonucleic Acid Expression and Represses Mitogen-activated Protein Kinase Signaling

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Yuanli Dong

2015-01-01

Full Text Available Background: Hypocretin (HCRT signaling plays an important role in the pathogenesis of narcolepsy and can be significantly influenced by Chinese herbal therapy. Our previous study showed that xingshentongqiao decoction (XSTQ is clinically effective for the treatment of narcolepsy. To determine whether XSTQ improves narcolepsy by modulating HCRT signaling, we investigated its effects on SH-SY5Y cell proliferation, apoptosis, and HCRT receptor 1/2 (orexin receptor 1 [OX1R] and orexin receptor 2 [OX2R] expression. The signaling pathways involved in these processes were also assessed. Methods: The effects of XSTQ on proliferation and apoptosis in SH-SY5Y cells were assessed using cell counting kit-8 and annexin V-fluorescein isothiocyanate assays. OX1R and OX2R expression was assessed by quantitative real-time polymerase chain reaction analysis. Western blotting for mitogen-activated protein kinase (MAPK pathway activation was performed to further assess the signaling mechanism of XSTQ. Results: XSTQ reduced the proliferation and induced apoptosis of SH-SY5Y cells. This effect was accompanied by the upregulation of OX1R and OX2R expression and the reduced phosphorylation of extracellular signal-regulated kinase (Erk 1/2, p38 MAPK and c-Jun N-terminal kinase (JNK. Conclusions: XSTQ inhibits proliferation and induces apoptosis in SH-SY5Y cells. XSTQ also promotes OX1R and OX2R expression. These effects are associated with the repression of the Erk1/2, p38 MAPK, and JNK signaling pathways. These results define a molecular mechanism for XSTQ in regulating HCRT and MAPK activation, which may explain its ability to treat narcolepsy.

Different myrosinase and idioblast distribution in Arabidopsis and Brassica napus

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Andreasson, Erik; Jørgensen, Lise Bolt; Höglund, Anna-Stina

2001-01-01

Arabidopsis, Brassica napus, Myrosinase, Myrosinase Binding Protein, Glucosinolates, Myrosin Cell, Immunocytochemistry......Arabidopsis, Brassica napus, Myrosinase, Myrosinase Binding Protein, Glucosinolates, Myrosin Cell, Immunocytochemistry...

Protein intrinsic disorder in Arabidopsis NAC transcription factors

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O'Shea, Charlotte; Jensen, Mikael Kryger; Stender, Emil G.P.

2015-01-01

of differences in binding mechanisms. Although substitution of both hydrophobic and acidic residues of the ANAC046 MoRF region abolished binding, substitution of other residues, even with α-helix-breaking proline, was less disruptive. Together, the biophysical analyses suggest that RCD1-ANAC046 complex formation......Protein ID (intrinsic disorder) plays a significant, yet relatively unexplored role in transcription factors (TFs). In the present paper, analysis of the transcription regulatory domains (TRDs) of six phylogenetically representative, plant-specific NAC [no apical meristem, ATAF (Arabidopsis...

Overexpression of a SNARE protein AtBS14b alters BR response in Arabidopsis.

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Zhu, Zhong Xin; Ye, Hong Bo; Xuan, Yuan Hu; Yao, Da Nian

2014-12-01

N-ethyl-maleimide sensitive factor adaptor protein receptor (SNAREs) domain-containing proteins were known as key players in vesicle-associated membrane fusion. Genetic screening has revealed the function of SNAREs in different aspects of plant biology, but the role of many SNAREs are still unknown. In this study, we have characterized the role of Arabidopsis Qc-SNARE protein AtBS14b in brassinosteroids (BRs) signaling pathway. AtBS14b overexpression (AtBS14b ox) plants exhibited short hypocotyl and petioles lengths as well as insensitivity to exogenously supplied BR, while AtBS14b mutants did not show any visible BR-dependent morphological differences. BR biosynthesis enzyme BR6OX2 expression was slightly lower in AtBS14b ox than in wild type plants. Further BR-mediated repression of BR6OX2, CPD and DWF4 was inhibited in AtBS14b ox plants. AtBS14b-mCherry fusion protein localized in vesicular compartments surrounding plasma membrane in N. benthamiana leaves. In addition, isolation of AtBS14b-interacting BR signaling protein, which localized in plasma membrane, showed that AtBS14b directly interacted with membrane steroid binding protein 1 (MSBP1), but did not interact with BAK1 or BRI1. These data suggested that Qc-SNARE protein AtBS14b is the first SNARE protein identified that interacts with MSBP1, and the overexpression of AtBS14b modulates BR response in Arabidopsis.

Abscisic acid-regulated protein degradation causes osmotic stress-induced accumulation of branched-chain amino acids in Arabidopsis thaliana.

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Huang, Tengfang; Jander, Georg

2017-10-01

Whereas proline accumulates through de novo biosynthesis in plants subjected to osmotic stress, leucine, isoleucine, and valine accumulation in drought-stressed Arabidopsis thaliana is caused by abscisic acid-regulated protein degradation. In response to several kinds of abiotic stress, plants greatly increase their accumulation of free amino acids. Although stress-induced proline increases have been studied the most extensively, the fold-increase of other amino acids, in particular branched-chain amino acids (BCAAs; leucine, isoleucine, and valine), is often higher than that of proline. In Arabidopsis thaliana (Arabidopsis), BCAAs accumulate in response to drought, salt, mannitol, polyethylene glycol, herbicide treatment, and nitrogen starvation. Plants that are deficient in abscisic acid signaling accumulate lower amounts of BCAAs, but not proline and most other amino acids. Previous bioinformatic studies had suggested that amino acid synthesis, rather than protein degradation, is responsible for the observed BCAA increase in osmotically stressed Arabidopsis. However, whereas treatment with the protease inhibitor MG132 decreased drought-induced BCAA accumulation, inhibition of BCAA biosynthesis with the acetolactate synthase inhibitors chlorsulfuron and imazapyr did not. Additionally, overexpression of BRANCHED-CHAIN AMINO ACID TRANSFERASE2 (BCAT2), which is upregulated in response to osmotic stress and functions in BCAA degradation, decreased drought-induced BCAA accumulation. Together, these results demonstrate that BCAA accumulation in osmotically stressed Arabidopsis is primarily the result of protein degradation. After relief of the osmotic stress, BCAA homeostasis is restored over time by amino acid degradation involving BCAT2. Thus, drought-induced BCAA accumulation is different from that of proline, which is accumulated due to de novo synthesis in an abscisic acid-independent manner and remains elevated for a more prolonged period of time after removal of

RNA-guided transcriptional activation via CRISPR/dCas9 mimics overexpression phenotypes in Arabidopsis.

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Jong-Jin Park

Full Text Available Clustered regularly interspaced short palindromic repeats (CRISPR and the CRISPR associated protein 9 (Cas9 system allows effective gene modification through RNA-guided DNA targeting. The Cas9 has undergone a series of functional alterations from the original active endonuclease to partially or completely deactivated Cas9. The catalytically deactivated Cas9 (dCas9 offers a platform to regulate transcriptional expression with the addition of activator or repressor domains. We redesigned a CRISPR/Cas9 activation system by adding the p65 transactivating subunit of NF-kappa B and a heat-shock factor 1 (HSF activation domain to dCas9 bound with the VP64 (tetramer of VP16 activation domain for application in plants. The redesigned CRISPR/Cas9 activation system was tested in Arabidopsis to increase endogenous transcriptional levels of production of anthocyanin pigment 1 (PAP1 and Arabidopsis thaliana vacuolar H+-pyrophosphatase (AVP1. The expression of PAP1 was increased two- to three-fold and the activated plants exhibited purple leaves similar to that of PAP1 overexpressors. The AVP1 gene expression was increased two- to five-fold in transgenic plants. In comparison to the wild type, AVP1 activated plants had increased leaf numbers, larger single-leaf areas and improved tolerance to drought stress. The AVP1 activated plants showed similar phenotypes to AVP1 overexpressors. Therefore, the redesigned CRISPR/Cas9 activation system containing modified p65-HSF provides a simple approach for producing activated plants by upregulating endogenous transcriptional levels.

PROTEIN TARGETING TO STARCH is required for localising GRANULE-BOUND STARCH SYNTHASE to starch granules and for normal amylose synthesis in Arabidopsis.

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David Seung

2015-02-01

Full Text Available The domestication of starch crops underpinned the development of human civilisation, yet we still do not fully understand how plants make starch. Starch is composed of glucose polymers that are branched (amylopectin or linear (amylose. The amount of amylose strongly influences the physico-chemical behaviour of starchy foods during cooking and of starch mixtures in non-food manufacturing processes. The GRANULE-BOUND STARCH SYNTHASE (GBSS is the glucosyltransferase specifically responsible for elongating amylose polymers and was the only protein known to be required for its biosynthesis. Here, we demonstrate that PROTEIN TARGETING TO STARCH (PTST is also specifically required for amylose synthesis in Arabidopsis. PTST is a plastidial protein possessing an N-terminal coiled coil domain and a C-terminal carbohydrate binding module (CBM. We discovered that Arabidopsis ptst mutants synthesise amylose-free starch and are phenotypically similar to mutants lacking GBSS. Analysis of granule-bound proteins showed a dramatic reduction of GBSS protein in ptst mutant starch granules. Pull-down assays with recombinant proteins in vitro, as well as immunoprecipitation assays in planta, revealed that GBSS physically interacts with PTST via a coiled coil. Furthermore, we show that the CBM domain of PTST, which mediates its interaction with starch granules, is also required for correct GBSS localisation. Fluorescently tagged Arabidopsis GBSS, expressed either in tobacco or Arabidopsis leaves, required the presence of Arabidopsis PTST to localise to starch granules. Mutation of the CBM of PTST caused GBSS to remain in the plastid stroma. PTST fulfils a previously unknown function in targeting GBSS to starch. This sheds new light on the importance of targeting biosynthetic enzymes to sub-cellular sites where their action is required. Importantly, PTST represents a promising new gene target for the biotechnological modification of starch composition, as it is

Mitogen activated protein kinase phosphatase-1 prevents the development of tactile sensitivity in a rodent model of neuropathic pain

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Ndong Christian

2012-04-01

Full Text Available Abstract Background Neuropathic pain due to nerve injury is one of the most difficult types of pain to treat. Following peripheral nerve injury, neuronal and glial plastic changes contribute to central sensitization and perpetuation of mechanical hypersensitivity in rodents. The mitogen activated protein kinase (MAPK family is pivotal in this spinal cord plasticity. MAPK phosphatases (MKPs limit inflammatory processes by dephosphorylating MAPKs. For example, MKP-1 preferentially dephosphorylates p-p38. Since spinal p-p38 is pivotal for the development of chronic hypersensitivity in rodent models of pain, and p-p38 inhibitors have shown clinical potential in acute and chronic pain patients, we hypothesize that induction of spinal MKP-1 will prevent the development of peripheral nerve-injury-induced hypersensitivity and p-p38 overexpression. Results We cloned rat spinal cord MKP-1 and optimize MKP-1 cDNA in vitro using transfections to BV-2 cells. We observed that in vitro overexpression of MKP-1 blocked lipopolysaccharide-induced phosphorylation of p38 (and other MAPKs as well as release of pro-algesic effectors (i.e., cytokines, chemokines, nitric oxide. Using this cDNA MKP-1 and a non-viral, in vivo nanoparticle transfection approach, we found that spinal cord overexpression of MKP-1 prevented development of peripheral nerve-injury-induced tactile hypersensitivity and reduced pro-inflammatory cytokines and chemokines and the phosphorylated form of p38. Conclusions Our results indicate that MKP-1, the natural regulator of p-p38, mediates resolution of the spinal cord pro-inflammatory milieu induced by peripheral nerve injury, resulting in prevention of chronic mechanical hypersensitivity. We propose that MKP-1 is a potential therapeutic target for pain treatment or prevention.

Three mitogen-activated protein kinases required for cell wall integrity contribute greatly to biocontrol potential of a fungal entomopathogen.

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Ying Chen

Full Text Available Bck1, Mkk1 and Slt2 are three mitogen-activated protein (MAP kinases constituting cell wall integrity (CWI pathway that may control multi-stress responses via crosstalk with high-osmolarity glycerol (HOG pathway in budding yeast. In this study, Bck1, Mkk1 and Slt2 orthologues in Beauveria bassiana were confirmed as the three-module cascade essential for CWI because cell wall impairment occurred in the hyphae and conidia of Δbck1, Δmkk1 and Δslt2 examined in multiple experiments. Strikingly, all the deletion mutants became more sensitive to hyperosmotic NaCl and sorbitol with the Western blot of Hog1 phosphorylation being weakened in Δbck1 and absent in Δmkk1 and Δslt2. Apart from crossing responses to cell wall perturbation and high osmolarity, three deletion mutants exhibited faster growth and conidiation on nutrition-rich medium, much less virulence to Galleria mellonella larvae, and higher sensitivity to nutritional, fungicidal, thermal and UV-B irradiative stresses, accompanied with less accumulation of intracellular mannitol and trehalose. Moreover, Δmkk1 and Δslt2 were equally more sensitive to all the stresses of different types except wet-heat stress than wild type and more or less different from Δbck1 in sensitivity to most of the stresses despite their null responses to two oxidants. All the changes in three deletion mutants were restored by each targeted gene complementation. Taken together, the CWI-required Bck1, Mkk1 and Slt2 are all positive, but differential, regulators of multi-stress tolerance and virulence perhaps due to interplay with the HOG pathway essential for osmoregulation, thereby contributing greatly to the biocontrol potential of the fungal entomopathogen.

Quantitative phosphoproteomic analysis reveals shared and specific targets of Arabidopsis MPK3, MPK4 and MPK6

KAUST Repository

Rayapuram, Naganand; Bigeard, Jean; Alhoraibi, Hanna Mohsen Abdulrab; Bonhomme, Ludovic; Hesse, Anne-Marie; Vinh, Joelle; Hirt, Heribert; Pflieger, Delphine

2017-01-01

In Arabidopsis, mitogen-activated protein kinases MPK3, MPK4 and MPK6 constitute essential relays for a variety of functions including cell division, development and innate immunity. While some substrates of MPK3, MPK4 and MPK6 have been identified, the picture is still far from complete. To identify substrates of these MAPKs likely involved in cell division, growth and development we compared the phosphoproteomes of wild-type and mpk3, mpk4 and mpk6. To study the function of these MAPKs in innate immunity, we analyzed their phosphoproteomes following microbe-associated molecular pattern (MAMP) treatment. Partially overlapping substrates were retrieved for all three MAPKs, showing target specificity to one, two or all three MAPKs in different biological processes. More precisely, our results illustrate the fact that the entity to be defined as a specific or a shared substrate for MAPKs is not a phosphoprotein but a particular (S/T)P phosphorylation site in a given protein. As a whole, 152 peptides were identified to be differentially phosphorylated in response to MAMP treatment and/or when compared between genotypes and 70 of them could be classified as putative MAPK targets. Biochemical analysis of a number of putative MAPK substrates by phosphorylation and interaction assays confirmed the global phosphoproteome approach. Our study also expands the set of MAPK substrates to involve other protein kinases, including calcium-dependent (CDPK) and sugar non-fermenting (SnRK) protein kinases.

Quantitative phosphoproteomic analysis reveals shared and specific targets of Arabidopsis MPK3, MPK4 and MPK6

KAUST Repository

Rayapuram, Naganand

2017-11-23

In Arabidopsis, mitogen-activated protein kinases MPK3, MPK4 and MPK6 constitute essential relays for a variety of functions including cell division, development and innate immunity. While some substrates of MPK3, MPK4 and MPK6 have been identified, the picture is still far from complete. To identify substrates of these MAPKs likely involved in cell division, growth and development we compared the phosphoproteomes of wild-type and mpk3, mpk4 and mpk6. To study the function of these MAPKs in innate immunity, we analyzed their phosphoproteomes following microbe-associated molecular pattern (MAMP) treatment. Partially overlapping substrates were retrieved for all three MAPKs, showing target specificity to one, two or all three MAPKs in different biological processes. More precisely, our results illustrate the fact that the entity to be defined as a specific or a shared substrate for MAPKs is not a phosphoprotein but a particular (S/T)P phosphorylation site in a given protein. As a whole, 152 peptides were identified to be differentially phosphorylated in response to MAMP treatment and/or when compared between genotypes and 70 of them could be classified as putative MAPK targets. Biochemical analysis of a number of putative MAPK substrates by phosphorylation and interaction assays confirmed the global phosphoproteome approach. Our study also expands the set of MAPK substrates to involve other protein kinases, including calcium-dependent (CDPK) and sugar non-fermenting (SnRK) protein kinases.

The Arabidopsis RNA-Binding Protein AtRGGA Regulates Tolerance to Salt and Drought Stress

KAUST Repository

Ambrosone, Alfredo; Batelli, Giorgia; Nurcato, Roberta; Aurilia, Vincenzo; Punzo, Paola; Bangarusamy, Dhinoth Kumar; Ruberti, Ida; Sassi, Massimiliano; Leone, Antonietta; Costa, Antonello; Grillo, Stefania

2015-01-01

Salt and drought stress severely reduce plant growth and crop productivity worldwide. The identification of genes underlying stress response and tolerance is the subject of intense research in plant biology. Through microarray analyses, we previously identified in potato (Solanum tuberosum) StRGGA, coding for an Arginine Glycine Glycine (RGG) box-containing RNA-binding protein, whose expression was specifically induced in potato cell cultures gradually exposed to osmotic stress. Here, we show that the Arabidopsis (Arabidopsis thaliana) ortholog, AtRGGA, is a functional RNA-binding protein required for a proper response to osmotic stress. AtRGGA gene expression was up-regulated in seedlings after long-term exposure to abscisic acid (ABA) and polyethylene glycol, while treatments with NaCl resulted in AtRGGA down-regulation. AtRGGA promoter analysis showed activity in several tissues, including stomata, the organs controlling transpiration. Fusion of AtRGGA with yellow fluorescent protein indicated that AtRGGA is localized in the cytoplasm and the cytoplasmic perinuclear region. In addition, the rgga knockout mutant was hypersensitive to ABA in root growth and survival tests and to salt stress during germination and at the vegetative stage. AtRGGA-overexpressing plants showed higher tolerance to ABA and salt stress on plates and in soil, accumulating lower levels of proline when exposed to drought stress. Finally, a global analysis of gene expression revealed extensive alterations in the transcriptome under salt stress, including several genes such as ASCORBATE PEROXIDASE2, GLUTATHIONE S-TRANSFERASE TAU9, and several SMALL AUXIN UPREGULATED RNA-like genes showing opposite expression behavior in transgenic and knockout plants. Taken together, our results reveal an important role of AtRGGA in the mechanisms of plant response and adaptation to stress.

The Arabidopsis RNA-Binding Protein AtRGGA Regulates Tolerance to Salt and Drought Stress

KAUST Repository

Ambrosone, Alfredo

2015-03-17

Salt and drought stress severely reduce plant growth and crop productivity worldwide. The identification of genes underlying stress response and tolerance is the subject of intense research in plant biology. Through microarray analyses, we previously identified in potato (Solanum tuberosum) StRGGA, coding for an Arginine Glycine Glycine (RGG) box-containing RNA-binding protein, whose expression was specifically induced in potato cell cultures gradually exposed to osmotic stress. Here, we show that the Arabidopsis (Arabidopsis thaliana) ortholog, AtRGGA, is a functional RNA-binding protein required for a proper response to osmotic stress. AtRGGA gene expression was up-regulated in seedlings after long-term exposure to abscisic acid (ABA) and polyethylene glycol, while treatments with NaCl resulted in AtRGGA down-regulation. AtRGGA promoter analysis showed activity in several tissues, including stomata, the organs controlling transpiration. Fusion of AtRGGA with yellow fluorescent protein indicated that AtRGGA is localized in the cytoplasm and the cytoplasmic perinuclear region. In addition, the rgga knockout mutant was hypersensitive to ABA in root growth and survival tests and to salt stress during germination and at the vegetative stage. AtRGGA-overexpressing plants showed higher tolerance to ABA and salt stress on plates and in soil, accumulating lower levels of proline when exposed to drought stress. Finally, a global analysis of gene expression revealed extensive alterations in the transcriptome under salt stress, including several genes such as ASCORBATE PEROXIDASE2, GLUTATHIONE S-TRANSFERASE TAU9, and several SMALL AUXIN UPREGULATED RNA-like genes showing opposite expression behavior in transgenic and knockout plants. Taken together, our results reveal an important role of AtRGGA in the mechanisms of plant response and adaptation to stress.

Reference: 783 [Arabidopsis Phenome Database[Archive

Lifescience Database Archive (English)

Full Text Available xpression of the Arabidopsis 10-kilodalton acyl-coenzyme A-binding protein ACBP6 en...phospholipid metabolism in Arabidopsis, including the possibility of ACBP6 in the cytosolic trafficking of phosphatidylcholine. Overe

Nitrile-specifier Proteins Involved in Glucosinolate Hydrolysis in Arabidopsis thaliana*S⃞

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Kissen, Ralph; Bones, Atle M.

2009-01-01

Glucosinolates are plant secondary metabolites present in Brassicaceae plants such as the model plant Arabidopsis thaliana. Intact glucosinolates are believed to be biologically inactive, whereas degradation products after hydrolysis have multiple roles in growth regulation and defense. The degradation of glucosinolates is catalyzed by thioglucosidases called myrosinases and leads by default to the formation of isothiocyanates. The interaction of a protein called epithiospecifier protein (ESP) with myrosinase diverts the reaction toward the production of epithionitriles or nitriles depending on the glucosinolate structure. Here we report the identification of a new group of nitrile-specifier proteins (AtNSPs) in A. thaliana able to generate nitriles in conjunction with myrosinase and a more detailed characterization of one member (AtNSP2). Recombinant AtNSP2 expressed in Escherichia coli was used to test its impact on the outcome of glucosinolate hydrolysis using a gas chromatography-mass spectrometry approach. AtNSP proteins share 30–45% sequence homology with A. thaliana ESP. Although AtESP and AtNSP proteins can switch myrosinase-catalyzed degradation of 2-propenylglucosinolate from isothiocyanate to nitrile, only AtESP generates the corresponding epithionitrile. Using the aromatic benzylglucosinolate, recombinant AtNSP2 is also able to direct product formation to the nitrile. Analysis of glucosinolate hydrolysis profiles of transgenic A. thaliana plants overexpressing AtNSP2 confirms its nitrile-specifier activity in planta. In silico expression analysis reveals distinctive expression patterns of AtNSPs, which supports a biological role for these proteins. In conclusion, we show that AtNSPs belonging to a new family of A. thaliana proteins structurally related to AtESP divert product formation from myrosinase-catalyzed glucosinolate hydrolysis and, thereby, likely affect the biological consequences of glucosinolate degradation. We discuss similarities and

Promiscuous and specific phospholipid binding by domains in ZAC, a membrane-associated Arabidopsis protein with an ARF GAP zinc finger and a C2 domain

DEFF Research Database (Denmark)

Jensen, R B; Lykke-Andersen, K; Frandsen, G I

2000-01-01

domain are separated by a region without homology to other known proteins. Zac promoter/beta-glucuronidase reporter assays revealed highest expression levels in flowering tissue, rosettes and roots. ZAC protein was immuno-detected mainly in association with membranes and fractionated with Golgi...... and plasma membrane marker proteins. ZAC membrane association was confirmed in assays by a fusion between ZAC and the green fluorescence protein and prompted an analysis of the in vitro phospholipid-binding ability of ZAC. Phospholipid dot-blot and liposome-binding assays indicated that fusion proteins...... zinc finger motif, but proteins containing only the zinc finger domain (residues 1-105) did not bind PI-3-P. Recombinant ZAC possessed GTPase-activating activity on Arabidopsis ARF proteins. These data identify a novel PI-3-P-binding protein region and thereby provide evidence...

2,2',4,4'-Tetrachlorobiphenyl upregulates cyclooxygenase-2 in HL-60 cells via p38 mitogen-activated protein kinase and NF-κB

International Nuclear Information System (INIS)

Bezdecny, Steven A.; Karmaus, Peer; Roth, Robert A.; Ganey, Patricia E.

2007-01-01

Polychlorinated biphenyls (PCBs) are ubiquitous, persistent environmental contaminants that affect a number of cellular systems, including neutrophils. Among the effects caused by the noncoplanar PCB 2,2',4,4'-tetrachlorobiphenyl (2244-TCB) in granulocytic HL-60 cells are increases in superoxide anion production, activation of phospholipase A 2 with subsequent release of arachidonic acid (AA) and upregulation of the inflammatory gene cyclooxygenase-2 (COX-2). The objective of this study was to determine the signal transduction pathways involved in the upregulation of COX-2 by 2244-TCB. Treatment of HL-60 cells with 2244-TCB led to increased expression of COX-2 mRNA. This increase was prevented by the transcriptional inhibitor actinomycin D in cells pretreated with 2244-TCB for 10 min. The increase in COX-2 mRNA was associated with release of 3 H-AA, phosphorylation of p38 and extracellular signal-regulated kinase (ERK) mitogen-activated protein (MAP) kinases, increased levels of nuclear NF-κB and increased superoxide anion production. Bromoenol lactone, an inhibitor of the calcium-independent phospholipase A 2 , reduced 3 H-AA release but had no effect on COX-2 mRNA, protein or activity. Pretreatment with SB-202190 or SB-203580, inhibitors of the p38 MAP kinase pathway, prevented the 2244-TCB-mediated induction of COX-2 and phosphorylation of p38 and ERK MAP kinases. These inhibitors did not alter 3 H-AA release. Treatment with PD 98059 or U 0126, inhibitors of the MAP/ERK (MEK) pathway, prevented the 2244-TCB-mediated activation of ERK but had no effect on COX-2 induction or p38 phosphorylation. 2244-TCB treatment did not affect c-Jun N-terminal kinase (JNK) phosphorylation. 2244-TCB exposure increased the amount of nuclear NF-κB. This increase was prevented by pretreatment with p38 MAP kinase inhibitors, but not by pretreatment with MEK inhibitors. Pretreatment with inhibitors of NF-κB prevented the 2244-TCB-mediated induction of COX-2 mRNA. 2244-TCB

Sodium appetite elicited by low-sodium diet is dependent on p44/42 mitogen-activated protein kinase (extracellular signal-regulated kinase 1/2) activation in the brain.

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Monteiro, L R N; Marangon, P B; Elias, L L K; Reis, L C; Antunes-Rodrigues, J; Mecawi, A S

2017-09-01

Sodium appetite is regulated by several signalling molecules, among which angiotensin II (Ang II) serves as a key driver of robust salt intake by binding to Ang II type 1 receptors (AT1R) in several regions in the brain. The activation of these receptors recruits the mitogen-activated protein kinase (MAPK) pathway, which has previously been linked to Ang II-induced increases in sodium appetite. Thus, we addressed the involvement of MAPK signalling in the induction of sodium appetite after 4 days of low-sodium diet consumption. An increase in extracellular signal-regulated kinase (ERK) phosphorylation in the laminae terminalis and mediobasal hypothalamus was observed after low-sodium diet consumption. This response was reduced by i.c.v. microinjection of an AT1R antagonist into the laminae terminalis but not the hypothalamus. This result indicates that low-sodium diet consumption activates the MAPK pathway via Ang II/AT1R signalling on the laminae terminalis. On the other hand, activation of the MAPK pathway in the mediobasal hypothalamus after low-sodium diet consumption appears to involve another extracellular mediator. We also evaluated whether a low-sodium diet could increase the sensitivity for Ang II in the brain and activate the MAPK pathway. However, i.c.v. injection of Ang II increased ERK phosphorylation on the laminae terminalis and mediobasal hypothalamus; this increase achieved a response magnitude similar to those observed in both the normal and low-sodium diet groups. These data indicate that low-sodium diet consumption for 4 days is insufficient to change the ERK phosphorylation response to Ang II in the brain. To investigate whether the MAPK pathway is involved in sodium appetite after low-sodium diet consumption, we performed i.c.v. microinjections of a MAPK pathway inhibitor (PD98059). PD98059 inhibited both saline and water intake after low-sodium diet consumption. Thus, the MAPK pathway is involved in promoting the sodium appetite after low

Bioactive glass induced osteogenic differentiation of human adipose stem cells is dependent on cell attachment mechanism and mitogen-activated protein kinases

Directory of Open Access Journals (Sweden)

M Ojansivu

2018-02-01

Full Text Available Bioactive glasses (BaGs are widely utilised in bone tissue engineering (TE but the molecular response of cells to BaGs is poorly understood. To elucidate the mechanisms of cell attachment to BaGs and BaG-induced early osteogenic differentiation, we cultured human adipose stem cells (hASCs on discs of two silica-based BaGs S53P4 (23.0 Na2O - 20.0 CaO - 4.0 P2O5 - 53.0 SiO2 (wt-% and 1-06 (5.9 Na2O - 12.0 K2O - 5.3 MgO - 22.6 CaO - 4.0 P2O5 - 0.2 B2O3 - 50.0 SiO2 in the absence of osteogenic supplements. Both BaGs induced early osteogenic differentiation by increasing alkaline phosphatase activity (ALP and the expression of osteogenic marker genes RUNX2a and OSTERIX. Based on ALP activity, the slower reacting 1-06 glass was a stronger osteoinducer. Regarding the cell attachment, cells cultured on BaGs had enhanced integrinβ1 and vinculin production, and mature focal adhesions were smaller but more dispersed than on cell culture plastic (polystyrene. Focal adhesion kinase (FAK, extracellular signal-regulated kinase (ERK1/2 and c-Jun N-terminal kinase (JNK-induced c-Jun phosphorylations were upregulated by glass contact. Moreover, the BaG-stimulated osteoinduction was significantly reduced by FAK and mitogen-activated protein kinase (MAPK inhibitors, indicating an important role for FAK and MAPKs in the BaG-induced early osteogenic commitment of hASCs. Upon indirect insert culture, the ions released from the BaG discs could not reproduce the observed cellular changes, which highlighted the role of direct cell-BaG interactions in the osteopotential of BaGs. These findings gave valuable insight into the mechanism of BaG-induced osteogenic differentiation and therefore provided knowledge to aid the future design of new functional biomaterials to meet the increasing demand for clinical bone TE treatments.

Calcium Hydroxide-induced Proliferation, Migration, Osteogenic Differentiation, and Mineralization via the Mitogen-activated Protein Kinase Pathway in Human Dental Pulp Stem Cells.

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Chen, Luoping; Zheng, Lisha; Jiang, Jingyi; Gui, Jinpeng; Zhang, Lingyu; Huang, Yan; Chen, Xiaofang; Ji, Jing; Fan, Yubo

2016-09-01

Calcium hydroxide has been extensively used as the gold standard for direct pulp capping in clinical dentistry. It induces proliferation, migration, and mineralization in dental pulp stem cells (DPSCs), but the underlying mechanisms are still unclear. The aim of this study was to investigate the role of the mitogen-activated protein (MAP) kinase pathway in calcium hydroxide-induced proliferation, migration, osteogenic differentiation, and mineralization in human DPSCs. Human DPSCs between passages 3 and 6 were used. DPSCs were preincubated with inhibitors of MAP kinases and cultured with calcium hydroxide. The phosphorylated MAP kinases were detected by Western blot analysis. Cell viability was analyzed via the methylthiazol tetrazolium assay. Cell migration was estimated using the wound healing assay. Alkaline phosphatase (ALP) expression was analyzed using the ALP staining assay. Mineralization was studied by alizarin red staining analysis. Calcium hydroxide significantly promoted the phosphorylation of the c-Jun N-terminal kinase (JNK), p38, and extracellular signal-regulated kinase. The inhibition of JNK and p38 signaling abolished calcium hydroxide-induced proliferation of DPSCs. The inhibition of JNK, p38, and extracellular signal-regulated kinase signaling suppressed the migration, ALP expression, and mineralization of DPSCs. Our study showed that the MAP kinase pathway was involved in calcium hydroxide-induced proliferation, migration, osteogenic differentiation, and mineralization in human DPSCs. Copyright © 2016 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

Novel nuclear-encoded proteins interacting with a plastid sigma factor, Sig1, in Arabidopsis thaliana.

Science.gov (United States)

Morikawa, Kazuya; Shiina, Takashi; Murakami, Shinya; Toyoshima, Yoshinori

2002-03-13

Sigma factor binding proteins are involved in modifying the promoter preferences of the RNA polymerase in bacteria. We found the nuclear encoded protein (SibI) that is transported into chloroplasts and interacts specifically with the region 4 of Sig1 in Arabidopsis. SibI and its homologue, T3K9.5 are novel proteins, which are not homologous to any protein of known function. The expression of sibI was tissue specific, light dependent, and developmentally timed. We suggest the transcriptional regulation by sigma factor binding proteins to function in the plastids of higher plant.

Carotenoid crystal formation in Arabidopsis and carrot roots caused by increased phytoene synthase protein levels.

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Dirk Maass

Full Text Available BACKGROUND: As the first pathway-specific enzyme in carotenoid biosynthesis, phytoene synthase (PSY is a prime regulatory target. This includes a number of biotechnological approaches that have successfully increased the carotenoid content in agronomically relevant non-green plant tissues through tissue-specific PSY overexpression. We investigated the differential effects of constitutive AtPSY overexpression in green and non-green cells of transgenic Arabidopsis lines. This revealed striking similarities to the situation found in orange carrot roots with respect to carotenoid amounts and sequestration mechanism. METHODOLOGY/PRINCIPAL FINDINGS: In Arabidopsis seedlings, carotenoid content remained unaffected by increased AtPSY levels although the protein was almost quantitatively imported into plastids, as shown by western blot analyses. In contrast, non-photosynthetic calli and roots overexpressing AtPSY accumulated carotenoids 10 and 100-fold above the corresponding wild-type tissues and contained 1800 and 500 microg carotenoids per g dry weight, respectively. This increase coincided with a change of the pattern of accumulated carotenoids, as xanthophylls decreased relative to beta-carotene and carotene intermediates accumulated. As shown by polarization microscopy, carotenoids were found deposited in crystals, similar to crystalline-type chromoplasts of non-green tissues present in several other taxa. In fact, orange-colored carrots showed a similar situation with increased PSY protein as well as carotenoid levels and accumulation patterns whereas wild white-rooted carrots were similar to Arabidopsis wild type roots in this respect. Initiation of carotenoid crystal formation by increased PSY protein amounts was further confirmed by overexpressing crtB, a bacterial PSY gene, in white carrots, resulting in increased carotenoid amounts deposited in crystals. CONCLUSIONS: The sequestration of carotenoids into crystals can be driven by the

Phosphorylation of Icariin Can Alleviate the Oxidative Stress Caused by the Duck Hepatitis Virus A through Mitogen-Activated Protein Kinases Signaling Pathways

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Wen Xiong

2017-09-01

Full Text Available The duck virus hepatitis (DVH caused by the duck hepatitis virus A (DHAV has produced extensive economic losses to the duck industry. The currently licensed commercial vaccine has shown some defects and does not completely prevent the DVH. Accordingly, a new alternative treatment for this disease is urgently needed. Previous studies have shown that icariin (ICA and its phosphorylated derivative (pICA possessed good anti-DHAV effects through direct and indirect antiviral pathways, such as antioxidative stress. But the antioxidant activity showed some differences between ICA and pICA. The aim of this study is to prove that ICA and pICA attenuate oxidative stress caused by DHAV in vitro and in vivo, and to investigate their mechanism of action to explain their differences in antioxidant activities. In vivo, the dynamic deaths, oxidative evaluation indexes and hepatic pathological change scores were detected. When was added the hinokitiol which showed the pro-oxidative effect as an intervention method, pICA still possessed more treatment effect than ICA. The strong correlation between mortality and oxidative stress proves that ICA and pICA alleviate oxidative stress caused by DHAV. This was also demonstrated by the addition of hydrogen peroxide (H2O2 as an intervention method in vitro. pICA can be more effective than ICA to improve duck embryonic hepatocytes (DEHs viability and reduce the virulence of DHAV. The strong correlation between TCID50 and oxidative stress demonstrates that ICA and pICA can achieve anti-DHAV effects by inhibiting oxidative stress. In addition, the superoxide dismutase (SOD and glutathione peroxidase (GSH-Px of ICA and pICA showed significant difference. pICA could significantly inhibit the phosphorylation of p38, extra cellular signal regulated Kinase (ERK 1/2 and c-Jun N-terminal kinase (JNK, which were related to mitogen-activated protein kinases (MAPKs signaling pathways. Ultimately, compared to ICA, pICA exhibited more

Arabidopsis KHZ1 and KHZ2, two novel non-tandem CCCH zinc-finger and K-homolog domain proteins, have redundant roles in the regulation of flowering and senescence.

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Yan, Zongyun; Jia, Jianheng; Yan, Xiaoyuan; Shi, Huiying; Han, Yuzhen

2017-12-01

The two novel CCCH zinc-finger and K-homolog (KH) proteins, KHZ1 and KHZ2, play important roles in regulating flowering and senescence redundantly in Arabidopsis. The CCCH zinc-finger proteins and K-homolog (KH) proteins play important roles in plant development and stress responses. However, the biological functions of many CCCH zinc-finger proteins and KH proteins remain uncharacterized. In Arabidopsis, KHZ1 and KHZ2 are characterized as two novel CCCH zinc-finger and KH domain proteins which belong to subfamily VII in CCCH family. We obtained khz1, khz2 mutants and khz1 khz2 double mutants, as well as overexpression (OE) lines of KHZ1 and KHZ2. Compared with the wild type (WT), the khz2 mutants displayed no defects in growth and development, and the khz1 mutants were slightly late flowering, whereas the khz1 khz2 double mutants showed a pronounced late flowering phenotype. In contrast, artificially overexpressing KHZ1 and KHZ2 led to the early flowering. Consistent with the late flowering phenotype, the expression of flowering repressor gene FLC was up-regulated, while the expression of flowering integrator and floral meristem identity (FMI) genes were down-regulated significantly in khz1 khz2. In addition, we also observed that the OE plants of KHZ1 and KHZ2 showed early leaf senescence significantly, whereas the khz1 khz2 double mutants showed delayed senescence of leaf and the whole plant. Both KHZ1 and KHZ2 were ubiquitously expressed throughout the tissues of Arabidopsis. KHZ1 and KHZ2 were localized to the nucleus, and possessed both transactivation activities and RNA-binding abilities. Taken together, we conclude that KHZ1 and KHZ2 have redundant roles in the regulation of flowering and senescence in Arabidopsis.

A Single Amino Acid Substitution in an ORANGE Protein Promotes Carotenoid Overaccumulation in Arabidopsis1[OPEN

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Yuan, Hui; Owsiany, Katherine; Sheeja, T.E.; Zhou, Xiangjun; Rodriguez, Caroline; Li, Yongxi; Welsch, Ralf; Chayut, Noam; Yang, Yong; Thannhauser, Theodore W.; Parthasarathy, Mandayam V.; Xu, Qiang; Deng, Xiuxin; Fei, Zhangjun; Schaffer, Ari; Katzir, Nurit; Burger, Joseph; Tadmor, Yaakov; Li, Li

2015-01-01

Carotenoids are crucial for plant growth and human health. The finding of ORANGE (OR) protein as a pivotal regulator of carotenogenesis offers a unique opportunity to comprehensively understand the regulatory mechanisms of carotenoid accumulation and develop crops with enhanced nutritional quality. Here, we demonstrated that alteration of a single amino acid in a wild-type OR greatly enhanced its ability to promote carotenoid accumulation. Whereas overexpression of OR from Arabidopsis (Arabidopsis thaliana; AtOR) or from the agronomically important crop sorghum (Sorghum bicolor; SbOR) increased carotenoid levels up to 2-fold, expression of AtORHis (R90H) or SbORHis (R104H) variants dramatically enhanced carotenoid accumulation by up to 7-fold in the Arabidopsis calli. Moreover, we found that AtORAla (R90A) functioned similarly to AtORHis to promote carotenoid overproduction. Neither AtOR nor AtORHis greatly affected carotenogenic gene expression. AtORHis exhibited similar interactions with phytoene synthase (PSY) as AtOR in posttranscriptionally regulating PSY protein abundance. AtORHis triggered biogenesis of membranous chromoplasts in the Arabidopsis calli, which shared structures similar to chromoplasts found in the curd of the orange cauliflower (Brassica oleracea) mutant. By contrast, AtOR did not cause plastid-type changes in comparison with the controls, but produced plastids containing larger and electron-dense plastoglobuli. The unique ability of AtORHis in mediating chromoplast biogenesis is responsible for its induced carotenoid overproduction. Our study demonstrates ORHis/Ala as powerful tools for carotenoid enrichment in plants, and provides insights into the mechanisms underlying ORHis-regulated carotenoid accumulation. PMID:26224804

PSM/SH2-B distributes selected mitogenic receptor signals to distinct components in the PI3-kinase and MAP kinase signaling pathways.

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Deng, Youping; Xu, Hu; Riedel, Heimo

2007-02-15

The Pro-rich, PH, and SH2 domain containing mitogenic signaling adapter PSM/SH2-B has been implicated as a cellular partner of various mitogenic receptor tyrosine kinases and related signaling mechanisms. Here, we report in a direct comparison of three peptide hormones, that PSM participates in the assembly of distinct mitogenic signaling complexes in response to insulin or IGF-I when compared to PDGF in cultured normal fibroblasts. The complex formed in response to insulin or IGF-I involves the respective peptide hormone receptor and presumably the established components leading to MAP kinase activation. However, our data suggest an alternative link from the PDGF receptor via PSM directly to MEK1/2 and consequently also to p44/42 activation, possibly through a scaffold protein. At least two PSM domains participate, the SH2 domain anticipated to link PSM to the respective receptor and the Pro-rich region in an association with an unidentified downstream component resulting in direct MEK1/2 and p44/42 regulation. The PDGF receptor signaling complex formed in response to PDGF involves PI 3-kinase in addition to the same components and interactions as described for insulin or IGF-I. PSM associates with PI 3-kinase via p85 and in addition the PSM PH domain participates in the regulation of PI 3-kinase activity, presumably through membrane interaction. In contrast, the PSM Pro-rich region appears to participate only in the MAP kinase signal. Both pathways contribute to the mitogenic response as shown by cell proliferation, survival, and focus formation. PSM regulates p38 MAP kinase activity in a pathway unrelated to the mitogenic response.

Variation of Enzyme Activities and Metabolite Levels in 24 Arabidopsis Accessions Growing in Carbon-Limited Conditions1[W

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Cross, Joanna M.; von Korff, Maria; Altmann, Thomas; Bartzetko, Linda; Sulpice, Ronan; Gibon, Yves; Palacios, Natalia; Stitt, Mark

2006-01-01

Our understanding of the interaction of carbon (C) metabolism with nitrogen (N) metabolism and growth is based mainly on studies of responses to environmental treatments, and studies of mutants and transformants. Here, we investigate which metabolic parameters vary and which parameters change in a coordinated manner in 24 genetically diverse Arabidopsis (Arabidopsis thaliana) accessions, grown in C-limited conditions. The accessions were grown in short days, moderate light, and high nitrate, and analyzed for rosette biomass, levels of structural components (protein, chlorophyll), total phenols and major metabolic intermediates (sugars, starch, nitrate, amino acids), and the activities of seven representative enzymes from central C and N metabolism. The largest variation was found for plant weight, reducing sugars, starch at the end of the night, and several enzyme activities. High levels of one sugar correlated with high levels of other sugars and starch, and a trend to increased amino acids, slightly lower nitrate, and higher protein. The activities of enzymes at the interface of C and N metabolism correlated with each other, but were unrelated to carbohydrates, amino acid levels, and total protein. Rosette weight was unrelated or showed a weak negative trend to sugar and amino acid contents at the end of the day in most of the accessions, and was negatively correlated with starch at the end of the night. Rosette weight was positively correlated with several enzyme activities. We propose that growth is not related to the absolute levels of starch, sugars, and amino acids; instead, it is related to flux, which is indicated by the enzymatic capacity to use these central resources. PMID:17085515

Arabidopsis and Maize RidA Proteins Preempt Reactive Enamine/Imine Damage to Branched-Chain Amino Acid Biosynthesis in Plastids[C][W][OPEN

Science.gov (United States)

Niehaus, Thomas D.; Nguyen, Thuy N.D.; Gidda, Satinder K.; ElBadawi-Sidhu, Mona; Lambrecht, Jennifer A.; McCarty, Donald R.; Downs, Diana M.; Cooper, Arthur J.L.; Fiehn, Oliver; Mullen, Robert T.; Hanson, Andrew D.

2014-01-01

RidA (for Reactive Intermediate Deaminase A) proteins are ubiquitous, yet their function in eukaryotes is unclear. It is known that deleting Salmonella enterica ridA causes Ser sensitivity and that S. enterica RidA and its homologs from other organisms hydrolyze the enamine/imine intermediates that Thr dehydratase forms from Ser or Thr. In S. enterica, the Ser-derived enamine/imine inactivates a branched-chain aminotransferase; RidA prevents this damage. Arabidopsis thaliana and maize (Zea mays) have a RidA homolog that is predicted to be plastidial. Expression of either homolog complemented the Ser sensitivity of the S. enterica ridA mutant. The purified proteins hydrolyzed the enamines/imines formed by Thr dehydratase from Ser or Thr and protected the Arabidopsis plastidial branched-chain aminotransferase BCAT3 from inactivation by the Ser-derived enamine/imine. In vitro chloroplast import assays and in vivo localization of green fluorescent protein fusions showed that Arabidopsis RidA and Thr dehydratase are chloroplast targeted. Disrupting Arabidopsis RidA reduced root growth and raised the root and shoot levels of the branched-chain amino acid biosynthesis intermediate 2-oxobutanoate; Ser treatment exacerbated these effects in roots. Supplying Ile reversed the root growth defect. These results indicate that plastidial RidA proteins can preempt damage to BCAT3 and Ile biosynthesis by hydrolyzing the Ser-derived enamine/imine product of Thr dehydratase. PMID:25070638

Sequence and 3D structure based analysis of TNT degrading proteins in Arabidopsis thaliana.

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Bhattacherjee, Amrita; Mandal, Rahul Shubhra; Das, Santasabuj; Kundu, Sudip

2014-03-01

TNT, accidentally released at several manufacturing sites, contaminates ground water and soil. It has a toxic effect to algae and invertebrate, and chronic exposure to TNT also causes harmful effects to human. On the other hand, many plants including Arabidopsis thaliana have the ability to metabolize TNT either completely or at least to a reduced less toxic form. In A. thaliana, the enzyme UDP glucosyltransferase (UDPGT) can further conjugate the reduced forms 2-HADNT and 4-HADNT (2-hydroxylamino-4, 6- dinitrotoluene and 4-hydroxylamino-2, 6- dinitrotoluene) of TNT. Based on the experimental analysis, existing literature and phylogenetic analysis, it is evident that among 107 UDPGT proteins only six are involved in the TNT degrading process. A total of 13 UDPGT proteins including five of these TNT degrading proteins fall within the same group of phylogeny. Thus, these 13 UDPGT proteins have been classified into two groups, TNT-degrading and TNT-non-degrading proteins. To understand the differences in TNT-degrading capacities; using homology modeling we first predicted two structures, taking one representative sequence from both the groups. Next, we performed molecular docking of the modeled structure and TNT reduced form 2-hydroxylamino-4, 6- dinitrotoluene (2-HADNT). We observed that while the Trp residue located within the active site region of the TNT- degrading protein showed π-Cation interaction; such type of interaction was absent in TNT-non-degrading protein, as the respective Trp residue lay outside of the pocket in this case. We observed the conservation of this π-Cation interaction during MD simulation of TNT-degrading protein. Thus, the position and the orientation of the active site residue Trp could explain the presence and absence of TNT-degrading capacity of the UDPGT proteins.

Localization of the CAPRICE-ENHANCER OF TRY AND CPC1 chimera protein in Arabidopsis root epidermis.

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Tominaga-Wada, Rumi; Kurata, Tetsuya; Wada, Takuji

2017-09-01

The CAPRICE (CPC) encodes an R3-type MYB transcription factor, which promotes root-hair differentiation. Previously, we showed that the CPC protein moves from the non-hair cell to the neighboring cell and induces root-hair differentiation in Arabidopsis. In addition, we proposed two cell-to-cell movement signal sequences, S1 and S2, in CPC. However, an S1:2xGFP:S2 chimera protein did not move between root epidermal cells. Here, we show that the S1 and S2 sequences do not confer cell-to-cell movement or nuclear localization ability to a GFP protein. The ENHANCER OF TRY AND CPC1 (ETC1) gene encodes the CPC homolog R3 MYB; this protein does not possess cell-to-cell movement ability or the S1 sequence. To elucidate whether the S1 sequence can induce cell-to-cell movement ability in ETC1, CPCp:S1:ETC1:2xGFP was constructed and introduced into Arabidopsis. Our results indicate that the addition of the S1 sequence was not sufficient for ETC1 to acquire cell-to-cell movement ability.

LIL3, a Light-Harvesting Complex Protein, Links Terpenoid and Tetrapyrrole Biosynthesis in Arabidopsis thaliana.

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Hey, Daniel; Rothbart, Maxi; Herbst, Josephine; Wang, Peng; Müller, Jakob; Wittmann, Daniel; Gruhl, Kirsten; Grimm, Bernhard

2017-06-01

The LIL3 protein of Arabidopsis ( Arabidopsis thaliana ) belongs to the light-harvesting complex (LHC) protein family, which also includes the light-harvesting chlorophyll-binding proteins of photosystems I and II, the early-light-inducible proteins, PsbS involved in nonphotochemical quenching, and the one-helix proteins and their cyanobacterial homologs designated high-light-inducible proteins. Each member of this family is characterized by one or two LHC transmembrane domains (referred to as the LHC motif) to which potential functions such as chlorophyll binding, protein interaction, and integration of interacting partners into the plastid membranes have been attributed. Initially, LIL3 was shown to interact with geranylgeranyl reductase (CHLP), an enzyme of terpene biosynthesis that supplies the hydrocarbon chain for chlorophyll and tocopherol. Here, we show another function of LIL3 for the stability of protochlorophyllide oxidoreductase (POR). Multiple protein-protein interaction analyses suggest the direct physical interaction of LIL3 with POR but not with chlorophyll synthase. Consistently, LIL3-deficient plants exhibit substantial loss of POR as well as CHLP, which is not due to defective transcription of the POR and CHLP genes but to the posttranslational modification of their protein products. Interestingly, in vitro biochemical analyses provide novel evidence that LIL3 shows high binding affinity to protochlorophyllide, the substrate of POR. Taken together, this study suggests a critical role for LIL3 in the organization of later steps in chlorophyll biosynthesis. We suggest that LIL3 associates with POR and CHLP and thus contributes to the supply of the two metabolites, chlorophyllide and phytyl pyrophosphate, required for the final step in chlorophyll a synthesis. © 2017 American Society of Plant Biologists. All Rights Reserved.

Dual localized AtHscB involved in iron sulfur protein biogenesis in Arabidopsis.

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Xiang Ming Xu

2009-10-01

Full Text Available Iron-sulfur clusters are ubiquitous structures which act as prosthetic groups for numerous proteins involved in several fundamental biological processes including respiration and photosynthesis. Although simple in structure both the assembly and insertion of clusters into apoproteins requires complex biochemical pathways involving a diverse set of proteins. In yeast, the J-type chaperone Jac1 plays a key role in the biogenesis of iron sulfur clusters in mitochondria.In this study we demonstrate that AtHscB from Arabidopsis can rescue the Jac1 yeast knockout mutant suggesting a role for AtHscB in iron sulfur protein biogenesis in plants. In contrast to mitochondrial Jac1, AtHscB localizes to both mitochondria and the cytosol. AtHscB interacts with AtIscU1, an Isu-like scaffold protein involved in iron-sulfur cluster biogenesis, and through this interaction AtIscU1 is most probably retained in the cytosol. The chaperone AtHscA can functionally complement the yeast Ssq1knockout mutant and its ATPase activity is enhanced by AtHscB and AtIscU1. Interestingly, AtHscA is also localized in both mitochondria and the cytosol. Furthermore, AtHscB is highly expressed in anthers and trichomes and an AtHscB T-DNA insertion mutant shows reduced seed set, a waxless phenotype and inappropriate trichome development as well as dramatically reduced activities of the iron-sulfur enzymes aconitase and succinate dehydrogenase.Our data suggest that AtHscB together with AtHscA and AtIscU1 plays an important role in the biogenesis of iron-sulfur proteins in both mitochondria and the cytosol.

Ectodomain of plasmodesmata-localized protein 5 in Arabidopsis: expression, purification, crystallization and crystallographic analysis.

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Wang, Xiaocui; Zhu, Peiyan; Qu, Shanshan; Zhao, Jie; Singh, Prashant K; Wang, Wei

2017-09-01

Plasmodesmata-localized protein 5 (PDLP5) is a cysteine-rich receptor-like protein which is localized on the plasmodesmata of Arabidopsis thaliana. Overexpression of PDLP5 can reduce the permeability of the plasmodesmata and further affect the cell-to-cell movement of viruses and macromolecules in plants. The ectodomain of PDLP5 contains two DUF26 domains; however, the function of these domains is still unknown. Here, the ectodomain of PDLP5 from Arabidopsis was cloned and overexpressed using an insect expression system and was then purified and crystallized. X-ray diffraction data were collected to 1.90 Å resolution and were indexed in space group P1, with unit-cell parameters a = 41.9, b = 48.1, c = 62.2 Å, α = 97.3, β = 103.1, γ = 99.7°. Analysis of the crystal content indicated that there are two molecules in the asymmetric unit, with a Matthews coefficient of 2.51 Å 3  Da -1 and a solvent content of 50.97%.

Asthmatic airway smooth muscle CXCL10 production: mitogen-activated protein kinase JNK involvement

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Alrashdan, Yazan A.; Alkhouri, Hatem; Chen, Emily; Lalor, Daniel J.; Poniris, Maree; Henness, Sheridan; Brightling, Christopher E.; Burgess, Janette K.; Armour, Carol L.; Ammit, Alaina J.

2012-01-01

CXCL10 (IP10) is involved in mast cell migration to airway smooth muscle (ASM) bundles in asthma. We aimed to investigate the role of cytokine-induced MAPK activation in CXCL10 production by ASM cells from people with and without asthma. Confluent growth-arrested ASM cells were treated with inhibitors of the MAPKs ERK, p38, and JNK and transcription factor NF-κB, or vehicle, and stimulated with IL-1β, TNF-α, or IFN-γ, alone or combined (cytomix). CXCL10 mRNA and protein, JNK, NF-κB p65 phosphorylation, and Iκ-Bα protein degradation were assessed using real-time PCR, ELISA, and immunoblotting, respectively. Cytomix, IL-1β, and TNF-α induced CXCL10 mRNA expression more rapidly in asthmatic than nonasthmatic ASM cells. IL-1β and/or TNF-α combined with IFN-γ synergistically increased asthmatic ASM cell CXCL10 release. Inhibitor effects were similar in asthmatic and nonasthmatic cells, but cytomix-induced release was least affected, with only JNK and NF-κB inhibitors halving it. Notably, JNK phosphorylation was markedly less in asthmatic compared with nonasthmatic cells. However, in both, the JNK inhibitor SP600125 reduced JNK phosphorylation and CXCL10 mRNA levels but did not affect CXCL10 mRNA stability or Iκ-Bα degradation. Together, the JNK and NF-κB inhibitors completely inhibited their CXCL10 release. We concluded that, in asthmatic compared with nonasthmatic ASM cells, JNK activation was reduced and CXCL10 gene expression was more rapid following cytomix stimulation. However, in both, JNK activation did not regulate early events leading to NF-κB activation. Thus JNK and NF-κB provide independent therapeutic targets for limiting CXCL10 production and mast cell migration to the ASM in asthma. PMID:22387292

Resveratrol inhibits PDGF receptor mitogenic signaling in mesangial cells: role of PTP1B

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Venkatesan, Balachandar; Ghosh-Choudhury, Nandini; Das, Falguni; Mahimainathan, Lenin; Kamat, Amrita; Kasinath, Balakuntalam S.; Abboud, Hanna E.; Choudhury, Goutam Ghosh

2008-01-01

Mesangioproliferative glomerulonephritis is associated with overactive PDGF receptor signal transduction. We show that the phytoalexin resveratrol dose dependently inhibits PDGF-induced DNA synthesis in mesangial cells with an IC50 of 10 μM without inducing apoptosis. Remarkably, the increased SIRT1 deacetylase activity induced by resveratrol was not necessary for this inhibitory effect. Resveratrol significantly blocked PDGF-stimulated c-Src and Akt kinase activation, resulting in reduced cyclin D1 expression and attenuated pRb phosphorylation and cyclin-dependent kinase-2 (CDK2) activity. Furthermore, resveratrol inhibited PDGFR phosphorylation at the PI 3 kinase and Grb-2 binding sites tyrosine-751 and tyrosine-716, respectively. This deficiency in PDGFR phosphorylation resulted in significant inhibition of PI 3 kinase and Erk1/2 MAPK activity. Interestingly, resveratrol increased the activity of protein tyrosine phosphatase PTP1B, which dephosphorylates PDGF-stimulated phosphorylation at tyrosine-751 and tyrosine-716 on PDGFR with concomitant reduction in Akt and Erk1/2 kinase activity. PTP1B significantly inhibited PDGF-induced DNA synthesis without inducing apoptosis. These results for the first time provide evidence that the stilbene resveratrol targets PTP1B to inhibit PDGFR mitogenic signaling.—Venkatesan, B., Ghosh-Choudhury, N., Das, F., Mahimainathan, L., Kamat, A., Kasinath, B. S., Abboud, H. E., Choudhury, G. G. Resveratrol inhibits PDGF receptor mitogenic signaling in mesangial cells: role of PTP1B. PMID:18567737

The structure of the karrikin-insensitive protein (KAI2) in Arabidopsis thaliana.

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Bythell-Douglas, Rohan; Waters, Mark T; Scaffidi, Adrian; Flematti, Gavin R; Smith, Steven M; Bond, Charles S

2013-01-01

KARRIKIN INSENSITIVE 2 (KAI2) is an α/β hydrolase involved in seed germination and seedling development. It is essential for plant responses to karrikins, a class of butenolide compounds derived from burnt plant material that are structurally similar to strigolactone plant hormones. The mechanistic basis for the function of KAI2 in plant development remains unclear. We have determined the crystal structure of Arabidopsis thaliana KAI2 in space groups P2(1) 2(1) 2(1) (a =63.57 Å, b =66.26 Å, c =78.25 Å) and P2(1) (a =50.20 Å, b =56.04 Å, c =52.43 Å, β =116.12°) to 1.55 and 2.11 Å respectively. The catalytic residues are positioned within a large hydrophobic pocket similar to that of DAD2, a protein required for strigolactone response in Petunia hybrida. KAI2 possesses a second solvent-accessible pocket, adjacent to the active site cavity, which offers the possibility of allosteric regulation. The structure of KAI2 is consistent with its designation as a serine hydrolase, as well as previous data implicating the protein in karrikin and strigolactone signalling.

The structure of the karrikin-insensitive protein (KAI2 in Arabidopsis thaliana.

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Rohan Bythell-Douglas

Full Text Available KARRIKIN INSENSITIVE 2 (KAI2 is an α/β hydrolase involved in seed germination and seedling development. It is essential for plant responses to karrikins, a class of butenolide compounds derived from burnt plant material that are structurally similar to strigolactone plant hormones. The mechanistic basis for the function of KAI2 in plant development remains unclear. We have determined the crystal structure of Arabidopsis thaliana KAI2 in space groups P2(1 2(1 2(1 (a =63.57 Å, b =66.26 Å, c =78.25 Å and P2(1 (a =50.20 Å, b =56.04 Å, c =52.43 Å, β =116.12° to 1.55 and 2.11 Å respectively. The catalytic residues are positioned within a large hydrophobic pocket similar to that of DAD2, a protein required for strigolactone response in Petunia hybrida. KAI2 possesses a second solvent-accessible pocket, adjacent to the active site cavity, which offers the possibility of allosteric regulation. The structure of KAI2 is consistent with its designation as a serine hydrolase, as well as previous data implicating the protein in karrikin and strigolactone signalling.

Proteomic and transcriptomic analysis of Arabidopsis seeds: molecular evidence for successive processing of seed proteins and its implication in the stress response to sulfur nutrition.

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Higashi, Yasuhiro; Hirai, Masami Yokota; Fujiwara, Toru; Naito, Satoshi; Noji, Masaaki; Saito, Kazuki

2006-11-01

Seed storage proteins are synthesized as sources of carbon, nitrogen and sulfur for the next generation of plants. Their composition changes according to nutritional conditions. Here, we report the precise molecular identification of seed proteins by proteomic analysis of wild-type Arabidopsis thaliana and methionine-over-accumulating mutant mto1-1 plants. The identities of 50 protein spots were determined in the protein extract of mature Arabidopsis seeds by two-dimensional (2D) gel electrophoresis and subsequent mass spectrometric analysis. Of these protein spots, 42 were identified as derived from 12S globulins or 2S albumins. These results indicate that approximately 84% of protein species in Arabidopsis seeds are derived from a few genes coding for 12S globulins and 2S albumins. Extensive mass spectrometric analysis of the 42 spots revealed that successive C-terminal degradation occurred on the 12S globulins. The feasibility of this C-terminal processing was rationalized by molecular modeling of the three-dimensional structure of 12S globulins. The C-terminal degradation at glutamic acid residues of the 12S globulin subunits was repressed under sulfur-deficient conditions. Transcriptome analysis was combined with proteomic analysis to elucidate the mechanism of changes in seed protein composition in response to sulfur deficiency. The results suggest that seed storage proteins in Arabidopsis undergo multi-layer regulation, with emphasis on post-translational modifications that enable the plant to respond to sulfur deficiency.

Arabidopsis IQM4, a Novel Calmodulin-Binding Protein, Is Involved With Seed Dormancy and Germination in Arabidopsis

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Yu Ping Zhou

2018-06-01

Full Text Available Seed dormancy and germination are regulated by complex mechanisms controlled by diverse hormones and environmental cues. Abscisic acid (ABA promotes seed dormancy and inhibits seed germination and post-germination growth. Calmodulin (CaM signals are involved with the inhibition of ABA during seed germination and seedling growth. In this study, we showed that Arabidopsis thaliana IQM4 could bind with calmodulin 5 (CaM5 both in vitro and in vivo, and that the interaction was the Ca2+-independent type. The IQM4 protein was localized in the chloroplast and the IQM4 gene was expressed in most tissues, especially the embryo and germinated seedlings. The T-DNA insertion mutants of IQM4 exhibited the reduced primary seed dormancy and lower ABA levels compared with wild type seeds. Moreover, IQM4 plays key roles in modulating the responses to ABA, salt, and osmotic stress during seed germination and post-germination growth. T-DNA insertion mutants exhibited ABA-insensitive and salt-hypersensitive phenotypes during seed germination and post-germination growth, whereas IQM4-overexpressing lines had ABA- and osmotic-hypersensitive, and salt-insensitive phenotypes. Gene expression analyses showed that mutation of IQM4 inhibited the expression of ABA biosynthetic genes NCED6 and NCED9, and seed maturation regulators LEC1, LEC2, ABI3, and ABI5 during the silique development, as well as promoted the expression of WRKY40 and inhibited that of ABI5 in ABA-regulated seed germination. These observations suggest that IQM4 is a novel Ca2+-independent CaM-binding protein, which is positively involved with seed dormancy and germination in Arabidopsis.

Duration of streptozotocin-induced diabetes differentially affects p38-mitogen-activated protein kinase (MAPK phosphorylation in renal and vascular dysfunction

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Gupta Akanksha

2005-03-01

Full Text Available Abstract Background In the present study we tested the hypothesis that progression of streptozotocin (STZ-induced diabetes (14-days to 28-days would produce renal and vascular dysfunction that correlate with altered p38- mitogen-activated protein kinase (p38-MAPK phosphorylation in kidneys and thoracic aorta. Methods Male Sprague Dawley rats (350–400 g were randomized into three groups: sham (N = 6, 14-days diabetic (N = 6 and 28-days diabetic rats (N = 6. Diabetes was induced using a single tail vein injection of STZ (60 mg/kg, I.V. on the first day. Rats were monitored for 28 days and food, water intake and plasma glucose levels were noted. At both 14-days and 28-days post diabetes blood samples were collected and kidney cortex, medulla and aorta were harvested from each rat. Results The diabetic rats lost body weight at both 14-days (-10% and 28-days (-13% more significantly as compared to sham (+10% group. Glucose levels were significantly elevated in the diabetic rats at both 14-days and 28-days post-STZ administration. Renal dysfunction as evidenced by renal hypertrophy, increased plasma creatinine concentration and reduced renal blood flow was observed in 14-days and 28-days diabetes. Vascular dysfunction as evidenced by decreased carotid blood flow was observed in 14-days and 28-days diabetes. We observed an up-regulation of inducible nitric oxide synthase (iNOS, prepro endothelin-1 (preproET-1 and phosphorylated p38-MAPK in thoracic aorta and kidney cortex but not in kidney medulla in 28-days diabetes group. Conclusion The study provides evidence that diabetes produces vascular and renal dysfunction with a profound effect on signaling mechanisms at later stage of diabetes.

Structure and biochemical function of a prototypical Arabidopsis U-box domain

DEFF Research Database (Denmark)

Andersen, Pernille; Kragelund, Birthe B; Olsen, Addie N

2004-01-01

U-box proteins, as well as other proteins involved in regulated protein degradation, are apparently over-represented in Arabidopsis compared with other model eukaryotes. The Arabidopsis protein AtPUB14 contains a typical U-box domain followed by an Armadillo repeat region, a domain organization t...

MaHog1, a Hog1-type mitogen-activated protein kinase gene, contributes to stress tolerance and virulence of the entomopathogenic fungus Metarhizium acridum.

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Jin, Kai; Ming, Yue; Xia, Yu Xian

2012-12-01

Fungal biocontrol agents have great potential in integrated pest management. However, poor efficacy and sensitivity to various adverse factors have hampered their wide application. In eukaryotic cells, Hog1 kinase plays a critical role in stress responses. In this study, MaHog1 (GenBank accession no. EFY85878), encoding a member of the Hog1/Sty1/p38 mitogen-activated protein kinase family in Metarhizium (Me.) acridum, was identified. Targeted gene disruption was used to analyse the role of MaHog1 in virulence and tolerance of adverse factors. Mutants with MaHog1 depletion showed increased sensitivity to high osmotic stress, high temperature and oxidative stress, and exhibited remarkable resistance to cell wall-disturbing agents. These results suggest that Hog1 kinase has a conserved function in regulating multistress responses among fungi, and that MaHog1 might influence cell wall biogenesis in Me. acridum. Bioassays conducted with topical inoculation and intrahaemocoel injection revealed that MaHog1 is required for both penetration and postpenetration development of Me. acridum. MaHog1 disruption resulted in a significant reduction in virulence, likely due to the combination of a decrease in conidial germination, a reduction in appressorium formation and a decline in growth rate in insect haemolymph, which might be caused by impairing fungal tolerance of various stresses during infection.

NTL8 Regulates Trichome Formation in Arabidopsis by Directly Activating R3 MYB Genes TRY and TCL1.

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Tian, Hainan; Wang, Xianling; Guo, Hongyan; Cheng, Yuxin; Hou, Chunjiang; Chen, Jin-Gui; Wang, Shucai

2017-08-01

The NAM, ATAF1/2, and CUC (NAC) are plant-specific transcription factors that regulate multiple aspects of plant growth and development and plant response to environmental stimuli. We report here the identification of NTM1-LIKE8 (NTL8), a membrane-associated NAC transcription factor, as a novel regulator of trichome formation in Arabidopsis ( Arabidopsis thaliana ). From an activation-tagged Arabidopsis population, we identified a dominant, gain-of-function mutant with glabrous inflorescence stem. By using plasmid rescue and RT-PCR analyses, we found that NTL8 was tagged; thus, the mutant was named ntl8-1 Dominant ( ntl8-1D ). Recapitulation experiment further confirmed that the phenotype observed in the ntl8-1D mutant was caused by elevated expression of NTL8 Quantitative RT-PCR results showed that the expression level of the single-repeat R3 MYB genes TRIPTYCHON ( TRY ) and TRICHOMELESS1 ( TCL1 ) was elevated in the ntl8-1D mutant. Genetic analyses demonstrated that NTL8 acts upstream of TRY and TCL1 in the regulation of trichome formation. When recruited to the promoter region of the reporter gene Gal4:GUS by a fused GAL4 DNA-binding domain, NTL8 activated the expression of the reporter gene. Chromatin immunoprecipitation results indicated that TRY and TCL1 are direct targets of NTL8. However, NTL8 did not interact with SQUAMOSA PROMOTER BINDING PROTEIN LIKE9, another transcription factor that regulates the expression of TRY and TCL1 , in yeast and plant cells. Taken together, our results suggest that NTL8 negatively regulates trichome formation in Arabidopsis by directly activating the expression of TRY and TCL1 . © 2017 American Society of Plant Biologists. All Rights Reserved.

Cloning, recombinant production, crystallization and preliminary X-ray diffraction analysis of SDF2-like protein from Arabidopsis thaliana

International Nuclear Information System (INIS)

Radzimanowski, Jens; Ravaud, Stephanie; Schott, Andrea; Strahl, Sabine; Sinning, Irmgard

2009-01-01

Overexpression, purification, crystallization and preliminary X-ray diffraction of the stromal-cell-derived factor 2-like protein of Arabidopsis thaliana are reported. The crystals belonged to the space group P6 1 and diffracted to 1.95 Å resolution. The stromal-cell-derived factor 2-like protein of Arabidopsis thaliana (AtSDL) has been shown to be highly up-regulated in response to unfolded protein response (UPR) inducing reagents, suggesting that it plays a crucial role in the plant UPR pathway. AtSDL has been cloned, overexpressed, purified and crystallized using the vapour-diffusion method. Two crystal forms have been obtained under very similar conditions. The needle-shaped crystals did not diffract X-rays, while the other form diffracted to 1.95 Å resolution using a synchrotron-radiation source and belonged to the hexagonal space group P6 1 , with unit-cell parameters a = b = 96.1, c = 69.3 Å

A plant small polypeptide is a novel component of DNA-binding protein phosphatase 1-mediated resistance to plum pox virus in Arabidopsis.

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Castelló, María José; Carrasco, Jose Luis; Navarrete-Gómez, Marisa; Daniel, Jacques; Granot, David; Vera, Pablo

2011-12-01

DNA-binding protein phosphatases (DBPs) have been identified as a novel class of plant-specific regulatory factors playing a role in plant-virus interactions. NtDBP1 from tobacco (Nicotiana tabacum) was shown to participate in transcriptional regulation of gene expression in response to virus infection in compatible interactions, and AtDBP1, its closest relative in the model plant Arabidopsis (Arabidopsis thaliana), has recently been found to mediate susceptibility to potyvirus, one of the most speciose taxa of plant viruses. Here, we report on the identification of a novel family of highly conserved small polypeptides that interact with DBP1 proteins both in tobacco and Arabidopsis, which we have designated DBP-interacting protein 2 (DIP2). The interaction of AtDIP2 with AtDBP1 was demonstrated in vivo by bimolecular fluorescence complementation, and AtDIP2 was shown to functionally interfere with AtDBP1 in yeast. Furthermore, reducing AtDIP2 gene expression leads to increased susceptibility to the potyvirus Plum pox virus and to a lesser extent also to Turnip mosaic virus, whereas overexpression results in enhanced resistance. Therefore, we describe a novel family of conserved small polypeptides in plants and identify AtDIP2 as a novel host factor contributing to resistance to potyvirus in Arabidopsis.

Activation of transcriptional activities of AP-1 and SRE by a new zinc-finger protein ZNF641