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MAPK Phosphatase (mapk + phosphatase)
Selected AbstractsInducible expression of a MAP kinase phosphatase-3-GFP chimera specifically blunts fibroblast growth and ras-dependent tumor formation in nude mice,JOURNAL OF CELLULAR PHYSIOLOGY, Issue 3 2004S. Marchetti The p42/p44 mitogen activated protein kinase (MAPK) pathway participates in a wide range of cellular programs including proliferation, migration, differentiation, and survival. Specific pharmacological inhibitors, like PD98059 and U0126, are often used to inhibit p42/p44 MAPK signaling. However, these inhibitors are not appropriate to study the function of these kinases in whole organisms. We thus developed an inducible system designed to inhibit p42/p44 MAPK activity through the expression of a phosphatase specific for these two kinases, the MAPK phosphatase 3 (MKP-3). A fibroblast cell line was established in which MKP-3 expression is controlled by tetracycline. Tetracycline-induced MKP-3 resulted in partial de-phosphorylation of p42/p44 MAPKs in serum-stimulated cells. However, we could improve MKP-3 stability and thereby the rate of MAPK de-phosphorylation, when the C-terminal end of MKP-3 was fused to the green fluorescent protein (GFP). Importantly, the fusion of GFP to MKP-3 did not alter the specificity of the phosphatase towards its MAPK substrates. We further show that conditional expression of MKP-3-GFP in this fibroblast cell line results in the inhibition of: (a) the phosphorylation of the p42/p44 MAPK substrates Elk1 and HIF-1,, (b) vascular endothelial growth factor (VEGF), cyclin D1, and c-fos gene transcription in response to MAPK pathway activation, and (c) cell proliferation. Finally, the MKP-3-GFP inducible cell line was transformed by Ha-ras and injected into nude mice. Treatment of mice with the tetracycline analog doxycycline resulted in a large delay in tumor emergence and growth as compared to the untreated control group, indicating that MKP-3-GFP activity is maintained in vivo. Altogether, these results show that inducible expression of MKP-3-GFP constitutes a valuable tool to study the role of p42/p44 MAPKs in various cellular responses in both cultured cell and animal models, a tool that may also be used to block unwanted cell growth in pathological conditions. © 2004 Wiley-Liss, Inc. [source] MAPK phosphatase MKP2 mediates disease responses in Arabidopsis and functionally interacts with MPK3 and MPK6THE PLANT JOURNAL, Issue 6 2010Victoria Lumbreras Summary Mitogen-activated protein kinase (MAPK) cascades have important functions in plant stress responses and development and are key players in reactive oxygen species (ROS) signalling and in innate immunity. In Arabidopsis, the transmission of ROS and pathogen signalling by MAPKs involves the coordinated activation of MPK6 and MPK3; however, the specificity of their negative regulation by phosphatases is not fully known. Here, we present genetic analyses showing that MAPK phosphatase 2 (MKP2) regulates oxidative stress and pathogen defence responses and functionally interacts with MPK3 and MPK6. We show that plants lacking a functional MKP2 gene exhibit delayed wilting symptoms in response to Ralstonia solanacearum and, by contrast, acceleration of disease progression during Botrytis cinerea infection, suggesting that this phosphatase plays differential functions in biotrophic versus necrotrophic pathogen-induced responses. MKP2 function appears to be linked to MPK3 and MPK6 regulation, as indicated by BiFC experiments showing that MKP2 associates with MPK3 and MPK6 in vivo and that in response to fungal elicitors MKP2 exerts differential affinity versus both kinases. We also found that MKP2 interacts with MPK6 in HR-like responses triggered by fungal elicitors, suggesting that MPK3 and MPK6 are subject to differential regulation by MKP2 in this process. We propose that MKP2 is a key regulator of MPK3 and MPK6 networks controlling both abiotic and specific pathogen responses in plants. [source] Arabidopsis mitogen-activated protein kinase MPK12 interacts with the MAPK phosphatase IBR5 and regulates auxin signalingTHE PLANT JOURNAL, Issue 6 2009Jin Suk Lee Summary Mitogen-activated protein kinase (MAPK) phosphatases are important negative regulators in the MAPK signaling pathways responsible for many essential processes in plants, including development, stress management and hormonal responses. A mutation in INDOLE-3-BUTYRIC ACID-RESPONSE5 (IBR5), which is predicted to encode a dual-specificity MAPK phosphatase, was previously reported to confer reduced sensitivity to auxin and ABA in Arabidopsis roots. To further characterize IBR5, and to understand how it might help integrate MAPK cascades with hormone signaling, we searched for IBR5-interacting MAPKs. Yeast two-hybrid assays, in vitro binding assays and in vivo protein co-immunoprecipitation studies demonstrated that MPK12 and IBR5 are physically coupled. The C-terminus of MPK12 appears to be essential for its interaction with IBR5, and in vitro dephosphorylation and immunocomplex kinase assays indicated that activated MPK12 is efficiently dephosphorylated and inactivated by IBR5. MPK12 and IBR5 mRNAs are both widely expressed across Arabidopsis tissues, and at the subcellular level each protein is predominantly localized in the nucleus. In transgenic plants with reduced expression of the MPK12 gene, root growth is hypersensitive to exogenous auxins, but shows normal ABA sensitivity. MPK12 suppression in an ibr5 background partially complements the ibr5 auxin-insensitivity phenotype. Our results demonstrate that IBR5 is a bona fide MAPK phosphatase, and suggest that MPK12 is both a physiological substrate of IBR5 and a novel negative regulator of auxin signaling in Arabidopsis. [source] Aberrant hypertrophy in Smad3-deficient murine chondrocytes is rescued by restoring transforming growth factor ,,activated kinase 1/activating transcription factor 2 signaling: A potential clinical implication for osteoarthritisARTHRITIS & RHEUMATISM, Issue 8 2010Tian-Fang Li Objective To investigate the biologic significance of Smad3 in the progression of osteoarthritis (OA), the crosstalk between Smad3 and activating transcription factor 2 (ATF-2) in the transforming growth factor , (TGF,) signaling pathway, and the effects of ATF-2 overexpression and p38 activation in chondrocyte differentiation. Methods Joint disease in Smad3-knockout (Smad3,/,) mice was examined by microfocal computed tomography and histologic analysis. Numerous in vitro methods including immunostaining, real-time polymerase chain reaction, Western blotting, an ATF-2 DNA-binding assay, and a p38 kinase activity assay were used to study the various signaling responses and protein interactions underlying the altered chondrocyte phenotype in Smad3,/, mice. Results In Smad3,/, mice, an end-stage OA phenotype gradually developed. TGF,-activated kinase 1 (TAK1)/ATF-2 signaling was disrupted in Smad3,/, mouse chondrocytes at the level of p38 MAP kinase (MAPK) activation, resulting in reduced ATF-2 phosphorylation and transcriptional activity. Reintroduction of Smad3 into Smad3,/, cells restored the normal p38 response to TGF,. Phosphorylated p38 formed a complex with Smad3 by binding to a portion of Smad3 containing both the MAD homology 1 and linker domains. Additionally, Smad3 inhibited the dephosphorylation of p38 by MAPK phosphatase 1 (MKP-1). Both ATF-2 overexpression and p38 activation repressed type X collagen expression in wild-type and Smad3,/, chondrocytes. P38 was detected in articular cartilage and perichondrium; articular and sternal chondrocytes expressed p38 isoforms ,, ,, and ,, but not ,. Conclusion Smad3 is involved in both the onset and progression of OA. Loss of Smad3 abrogates TAK1/ATF-2 signaling, most likely by disrupting the Smad3,phosphorylated p38 complex, thereby promoting p38 dephosphorylation and inactivation by MKP-1. ATF-2 and p38 activation inhibit chondrocyte hypertrophy. Modulation of p38 isoform activity may provide a new therapeutic approach for OA. [source] Aurothiomalate inhibits cyclooxygenase 2, matrix metalloproteinase 3, and interleukin-6 expression in chondrocytes by increasing MAPK phosphatase 1 expression and decreasing p38 phosphorylation: MAPK phosphatase 1 as a novel target for antirheumatic drugsARTHRITIS & RHEUMATISM, Issue 6 2010Riina Nieminen Objective Aurothiomalate is a disease-modifying antirheumatic drug that suppresses inflammation and retards cartilage degradation and bone erosion in arthritis. The molecular mechanisms of action of aurothiomalate are not known in detail. MAPK pathways are major signaling pathways in inflammation that regulate the production of many inflammatory and destructive factors in arthritis. The purpose of the present study was to investigate the effects of aurothiomalate on the activity of p38 MAPK and on the expression of MAPK phosphatase 1 (MKP-1), cyclooxygenase 2 (COX-2), matrix metalloproteinase 3 (MMP-3), and interleukin-6 (IL-6) in immortalized murine H4 chondrocytes and in intact human and murine cartilage. Methods Protein expression was examined by Western blotting or by enzyme-linked immunosorbent assay, and messenger RNA (mRNA) expression was examined by real-time reverse transcription,polymerase chain reaction analysis. The mediator role of MKP-1 was investigated by using small interfering RNA (siRNA) methods to down-regulated MKP-1 expression in chondrocytes in culture and by comparing the responses in intact cartilage from MKP-1,deficient and wild-type mice. The effects of aurothiomalate were also confirmed in human rheumatoid cartilage by using tissue samples obtained at the time of total knee replacement surgery. Results Aurothiomalate inhibited IL-1,,induced COX-2 expression and prostaglandin E2 production by destabilizing COX-2 mRNA, as did the p38 MAPK inhibitor SB203580. Interestingly, aurothiomalate also increased the expression of MKP-1 and reduced the IL-1,,induced phosphorylation of p38 MAPK. Knockdown of MKP-1 by siRNA significantly impaired the ability of aurothiomalate to inhibit the phosphorylation of p38 MAPK and the expression of COX-2, MMP-3, and IL-6. Likewise, aurothiomalate reduced COX-2, MMP-3, and IL-6 expression in articular cartilage from patients with rheumatoid arthritis, as well as in articular cartilage from wild-type mice but not from MKP-1,/, mice. Conclusion Our findings indicate a novel mechanism for the antiinflammatory and antierosive actions of aurothiomalate, through increased expression of MKP-1, which leads to reduced activation of p38 MAPK and suppressed expression of COX-2, MMP-3, and IL-6. The results suggest that manipulation of MKP-1 levels is a promising new mechanism to be directed in the search and development of novel antiinflammatory and antierosive compounds that have the good efficacy of gold compounds but not their toxicity. [source] The role of NADPH oxidase and MAP kinase phosphatase in UV-B-dependent gene expression in ArabidopsisPLANT CELL & ENVIRONMENT, Issue 9 2006IRINA KALBINA ABSTRACT Plant responses to supplementary UV-B irradiation have been reported to include formation of reactive oxygen species (ROS), hydrogen peroxide, in particular, and regulation by mitogen-activated protein kinase (MAPK) cascades which in turn are fine-tuned by MAPK phosphatases (MKPs). Here we present direct genetic evidence for the involvement of plasma membrane NADPH oxidase, a source of superoxide and hydrogen peroxide in the apoplasts, in UV-B signalling in Arabidopsis thaliana, by analysis of gene expression of the UV-B molecular markers in NADPH oxidase (atrbohD, F and DF) and MAP kinase phosphatase 1 (MKP1) knockout mutants (mkp1). Whereas the NADPH oxidase mutants were affected in UV-B-dependent CHS, PYROA and MEB5.2 gene expression, the mkp1 mutant was affected in the general expression pattern of the pathogenesis-related (PR) and PDF1.2 genes. The results indicate involvement of MKP1 in repressive action on gene expression of more general stress response pathways, similar to those activated by pathogen attack, while NADPH oxidase is involved in quantitative (rather than absolute) regulation of more UV-B-specific genes. The expressions of the molecular markers in the knockout mutant mkp1 and in its complemented lines (lines 6 and 10) were similar, as opposed to the responses of the corresponding wild-type Wassilewskija-4 (Ws-4). Lines 6 and 10 showed much higher MKP1 mRNA than Ws-4 but did not complement the mutant. This suggests a complex dependency of the MAPK phosporylation level of the PR and PDF1.2 genes. Both NADPH oxidase mutants and the mkp1 mutant phenotypically responded to UV-B by growth retardation. [source] Overproduction, purification and structure determination of human dual-specificity phosphatase 14ACTA CRYSTALLOGRAPHICA SECTION D, Issue 10 2009George T. Lountos Dual-specificity phosphatases (DUSPs) are enzymes that participate in the regulation of biological processes such as cell growth, differentiation, transcription and metabolism. A number of DUSPs are able to dephosphorylate phosphorylated serine, threonine and tyrosine residues on mitogen-activated protein kinases (MAPKs) and thus are also classified as MAPK phosphatases (MKPs). As an increasing number of DUSPs are being identified and characterized, there is a growing need to understand their biological activities at the molecular level. There is also significant interest in identifying DUSPs that could be potential targets for drugs that modulate MAPK-dependent signaling and immune responses, which have been implicated in a variety of maladies including cancer, infectious diseases and inflammatory disorders. Here, the overproduction, purification and crystal structure at 1.88,Å resolution of human dual-specificity phosphatase 14, DUSP14 (MKP6), are reported. This structural information should accelerate the study of DUSP14 at the molecular level and may also accelerate the discovery and development of novel therapeutic agents. [source] | ||||||||||