Serine/threonine kinase which acts as an essential component of the MAP kinase signal transduction pathway. MAPK14 is one of the four p38 MAPKs which play an important role in the cascades of cellular responses evoked by extracellular stimuli such as proinflammatory cytokines or physical stress leading to direct activation of transcription factors. Accordingly, p38 MAPKs phosphorylate a broad range of proteins and it has been estimated that they may have approximately 200 to 300 substrates each. Some of the targets are downstream kinases which are activated through phosphorylation and further phosphorylate additional targets. RPS6KA5/MSK1 and RPS6KA4/MSK2 can directly phosphorylate and activate transcription factors such as CREB1, ATF1, the NF-kappa-B isoform RELA/NFKB3, STAT1 and STAT3, but can also phosphorylate histone H3 and the nucleosomal protein HMGN1. RPS6KA5/MSK1 and RPS6KA4/MSK2 play important roles in the rapid induction of immediate-early genes in response to stress or mitogenic stimuli, either by inducing chromatin remodeling or by recruiting the transcription machinery. On the other hand, two other kinase targets, MAPKAPK2/MK2 and MAPKAPK3/MK3, participate in the control of gene expression mostly at the post-transcriptional level, by phosphorylating ZFP36 (tristetraprolin) and ELAVL1, and by regulating EEF2K, which is important for the elongation of mRNA during translation. MKNK1/MNK1 and MKNK2/MNK2, two other kinases activated by p38 MAPKs, regulate protein synthesis by phosphorylating the initiation factor EIF4E2. MAPK14 interacts also with casein kinase II, leading to its activation through autophosphorylation and further phosphorylation of TP53/p53. In the cytoplasm, the p38 MAPK pathway is an important regulator of protein turnover. For example, CFLAR is an inhibitor of TNF-induced apoptosis whose proteasome-mediated degradation is regulated by p38 MAPK phosphorylation. In a similar way, MAPK14 phosphorylates the ubiquitin ligase SIAH2, regulating its activity towards EGLN3. MAPK14 may also inhibit the lysosomal degradation pathway of autophagy by interfering with the intracellular trafficking of the transmembrane protein ATG9. Another function of MAPK14 is to regulate the endocytosis of membrane receptors by different mechanisms that impinge on the small GTPase RAB5A. In addition, clathrin-mediated EGFR internalization induced by inflammatory cytokines and UV irradiation depends on MAPK14-mediated phosphorylation of EGFR itself as well as of RAB5A effectors. Ectodomain shedding of transmembrane proteins is regulated by p38 MAPKs as well. In response to inflammatory stimuli, p38 MAPKs phosphorylate the membrane-associated metalloprotease ADAM17. Such phosphorylation is required for ADAM17-mediated ectodomain shedding of TGF-alpha family ligands, which results in the activation of EGFR signaling and cell proliferation. Another p38 MAPK substrate is FGFR1. FGFR1 can be translocated from the extracellular space into the cytosol and nucleus of target cells, and regulates processes such as rRNA synthesis and cell growth. FGFR1 translocation requires p38 MAPK activation. In the nucleus, many transcription factors are phosphorylated and activated by p38 MAPKs in response to different stimuli. Classical examples include ATF1, ATF2, ATF6, ELK1, PTPRH, DDIT3, TP53/p53 and MEF2C and MEF2A. The p38 MAPKs are emerging as important modulators of gene expression by regulating chromatin modifiers and remodelers. The promoters of several genes involved in the inflammatory response, such as IL6, IL8 and IL12B, display a p38 MAPK-dependent enrichment of histone H3 phosphorylation on 'Ser-10' (H3S10ph) in LPS-stimulated myeloid cells. This phosphorylation enhances the accessibility of the cryptic NF-kappa-B-binding sites marking promoters for increased NF-kappa-B recruitment. Phosphorylates CDC25B and CDC25C which is required for binding to 14-3-3 proteins and leads to initiation of a G2 delay after ultraviolet radiation. Phosphorylates TIAR following DNA damage, releasing TIAR from GADD45A mRNA and preventing mRNA degradation. The p38 MAPKs may also have kinase-independent roles, which are thought to be due to the binding to targets in the absence of phosphorylation. Protein O-Glc-N-acylation catalyzed by the OGT is regulated by MAPK14, and, although OGT does not seem to be phosphorylated by MAPK14, their interaction increases upon MAPK14 activation induced by glucose deprivation. This interaction may regulate OGT activity by recruiting it to specific targets such as neurofilament H, stimulating its O-Glc-N-acylation. Required in mid-fetal development for the growth of embryo-derived blood vessels in the labyrinth layer of the placenta. Also plays an essential role in developmental and stress-induced erythropoiesis, through regulation of EPO gene expression.
Mitogen-activated protein (MAP) kinase-mediated signalling to the nucleus is an important event in the conversion of extracellular signals into a cellular response. However, the existence of multiple MAP kinases which phosphorylate similar phosphoacceptor motifs poses a problem in maintaining substrate specificity and hence the correct biological response. Both the extracellular signal-regulated kinase (ERK) and c-Jun NH2-terminal kinase (JNK) subfamilies of MAP kinases use a second specificity determinant and require docking to their transcription factor substrates to achieve maximal substrate activation. In this study, we demonstrate that among the different MAP kinases, the MADS-box transcription factors MEF2A and MEF2C are preferentially phosphorylated and activated by the p38 subfamily members p38alpha and p38beta2. The efficiency of phosphorylation in vitro and transcriptional activation in vivo of MEF2A and MEF2C by these p38 subtypes requires the presence of a kinase docking domain (D-domain). Furthermore, the D-domain from MEF2A is sufficient to confer p38 responsiveness on different transcription factors, and reciprocal effects are observed upon the introduction of alternative D-domains into MEF2A. These results therefore contribute to our understanding of signalling to MEF2 transcription factors and demonstrate that the requirement for substrate binding by MAP kinases is an important facet of three different subclasses of MAP kinases (ERK, JNK, and p38).
The epidermal growth factor receptor (EGFR) frequently associates with cancer and already serves as a target for therapy. We report that inflammatory cytokines and ultraviolet (UV) irradiation respectively induce transient or sustained phosphorylation of EGFR. Subsequently, EGFR internalizes via a Clathrin-mediated process. In cytokine-stimulated cells, EGFR recycles back to the cell surface, whereas in irradiated cells it arrests in Rab5-containing endosomes. Under both conditions, receptor internalization is instigated by the p38 stress-induced kinase. The underlying mechanism entails phosphorylation of EGFR at a short segment (amino acids 1002-1022) containing multiple serines and threonines, as well as phosphorylation of two Rab5 effectors, EEA1 and GDI. Like UV irradiation, a chemotherapeutic agent activates p38 and accelerates receptor internalization. We demonstrate that abrogating EGFR internalization reduces the efficacy of chemotherapy-induced cell death. Hence, by preventing EGFR-mediated survival signaling, the internalization route we uncovered enhances the cytotoxic effect of drugs like cis-platinum, which may underlie interactions between chemotherapy and EGFR-targeting drugs.
Autophagy, a lysosomal degradation pathway, is essential for homeostasis, development, neurological diseases, and cancer. Regulation of autophagy in human disease is not well understood. Atg9 is a transmembrane protein required for autophagy, and it has been proposed that trafficking of Atg9 may regulate autophagy. Mammalian Atg9 traffics between the TGN and endosomes in basal conditions, and newly formed autophagosomes in response to signals inducing autophagy. We identified p38IP as a new mAtg9 interactor and showed that this interaction is regulated by p38alpha MAPK. p38IP is required for starvation-induced mAtg9 trafficking and autophagosome formation. Manipulation of p38IP and p38alpha alters mAtg9 localization, suggesting p38alpha regulates, through p38IP, the starvation-induced mAtg9 trafficking to forming autophagosomes. Furthermore, we show that p38alpha is a negative regulator of both basal autophagy and starvation-induced autophagy, and suggest that this regulation may be through a direct competition with mAtg9 for binding to p38IP. Our results provide evidence for a link between the MAPK pathway and the control of autophagy through mAtg9 and p38IP.
We have identified a novel mitogen- and stress-activated protein kinase (MSK1) that contains two protein kinase domains in a single polypeptide. MSK1 is activated in vitro by MAPK2/ERK2 or SAPK2/p38. Endogenous MSK1 is activated in 293 cells by either growth factor/phorbol ester stimulation, or by exposure to UV radiation, and oxidative and chemical stress. The activation of MSK1 by growth factors/phorbol esters is prevented by PD 98059, which suppresses activation of the MAPK cascade, while the activation of MSK1 by stress stimuli is prevented by SB 203580, a specific inhibitor of SAPK2/p38. In HeLa, PC12 and SK-N-MC cells, PD 98059 and SB 203580 are both required to suppress the activation of MSK1 by TNF, NGF and FGF, respectively, because these agonists activate both the MAPK/ERK and SAPK2/p38 cascades. MSK1 is localized in the nucleus of unstimulated or stimulated cells, and phosphorylates CREB at Ser133 with a Km value far lower than PKA, MAPKAP-K1(p90Rsk) and MAPKAP-K2. The effects of SB 203580, PD 98059 and Ro 318220 on agonist-induced activation of CREB and ATF1 in four cell-lines mirror the effects of these inhibitors on MSK1 activation, and exclude a role for MAPKAP-K1 and MAPKAP-K2/3 in this process. These findings, together with other observations, suggest that MSK1 may mediate the growth-factor and stress-induced activation of CREB.
The cap-binding translation initiation factor eukaryotic initiation factor 4E (eIF4E) is phosphorylated in vivo at Ser209 in response to a variety of stimuli. In this paper, we show that the mitogen-activated protein kinase (MAPK) signal-integrating kinase Mnk2 phosphorylates eIF4E at this residue. Mnk2 binds to the scaffolding protein eIF4G, and overexpression of Mnk2 results in increased phosphorylation of endogenous eIF4E, showing that it can act as an eIF4E kinase in vivo. We have identified eight phosphorylation sites in Mnk2, of which at least three potential MAPK sites are likely to be essential for Mnk2 activity. In contrast to that of Mnk1, the activity of overexpressed Mnk2 is high under control conditions and could only be reduced substantially by a combination of PD98059 and SB203580, while the activity of endogenous Mnk2 in Swiss 3T3 cells was hardly affected upon treatment with these inhibitors. These compounds did not abolish phosphorylation of eIF4E, implying that Mnk2 may mediate phosphorylation of eIF4E in Swiss 3T3 cells. In vitro phosphorylation studies show that Mnk2 is a significantly better substrate than Mnk1 for extracellular signal-regulated kinase 2 (ERK2), p38MAPKalpha, and p38MAPKbeta. Therefore, the high levels of activity of Mnk2 under several conditions may be explained by efficient activation of Mnk2 by low levels of activity of the upstream kinases. Interestingly, we found that the association of both Mnk1 and Mnk2 with eIF4G increased upon inhibition of the MAPK pathways while activation of ERK resulted in decreased binding to eIF4G. This might reflect a mechanism to ensure rapid, but transient, phosphorylation of eIF4E upon stimulation of the MAPK pathways.
The RING finger ubiquitin ligase Siah2 controls the stability of various substrates involved in stress and hypoxia responses, including the PHD3, which controls the stability of HIF-1alpha. In the present study we determined the role of Siah2 phosphorylation in the regulation of its activity toward PHD3. We show that Siah2 is subject to phosphorylation by p38 MAPK, which increases Siah2-mediated degradation of PHD3. Consistent with these findings, MKK3/MKK6 double-deficient cells, which cannot activate p38 kinases, exhibit impaired Siah2-dependent degradation of PHD3. Phosphopeptide mapping identified T24 and S29 as the primary phospho-acceptor sites. Phospho-mutant forms of Siah2 (S29A or T24A/S29A) exhibit impaired degradation of PHD3, particularly after hypoxia. Conversely, a phospho-mimic form of Siah2 (T24E/S29D) exhibits stronger degradation of PHD3, compared with wild type Siah2. Whereas phospho-mutant Siah2 exhibits weaker association with PHD3, phospho-mimic Siah2 associates as well as wild type and is localized within the perinuclear region, suggesting that phosphorylation of Siah2 affects its subcellular localization and, consequently, the degree of its association with PHD3. In all, our findings reveal the phosphorylation of Siah2 by p38 and the implications of such phosphorylation for Siah2 activity toward PHD3.
Response to genotoxic stress can be considered as a multistage process involving initiation of cell-cycle arrest and maintenance of arrest during DNA repair. Although maintenance of G2/M checkpoints is known to involve Chk1, Chk2/Rad53 and upstream components, the mechanisms involved in its initiation are less well defined. Here we report that p38 kinase has a critical role in the initiation of a G2 delay after ultraviolet radiation. Inhibition of p38 blocks the rapid initiation of this checkpoint in both human and murine cells after ultraviolet radiation. In vitro, p38 binds and phosphorylates Cdc25B at serines 309 and 361, and Cdc25C at serine 216; phosphorylation of these residues is required for binding to 14-3-3 proteins. In vivo, inhibition of p38 prevents both phosphorylation of Cdc25B at serine 309 and 14-3-3 binding after ultraviolet radiation, and mutation of this site is sufficient to inhibit the checkpoint initiation. In contrast, in vivo Cdc25C binding to 14-3-3 is not affected by p38 inhibition after ultraviolet radiation. We propose that regulation of Cdc25B phosphorylation by p38 is a critical event for initiating the G2/M checkpoint after ultraviolet radiation.
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 to be an allosteric mechanism. Furthermore, we demonstrate that anisomycin- and tumor necrosis factor-alpha-induced phosphorylation of p53 at Ser-392, which is important for the transcriptional activity of this growth suppressor protein, requires p38 MAP kinase and CK2 activities.
J. Biol. Chem. 273, 1741-1748 (1998)[PubMed:9430721]
The cellular response to treatment with proinflammatory cytokines or exposure to environmental stress is mediated, in part, by the p38 group of mitogen-activated protein (MAP) kinases. We report the molecular cloning of a novel isoform of p38 MAP kinase, p38 beta 2. This p38 MAP kinase, like p38 alpha, is inhibited by the pyridinyl imidazole drug SB203580. The p38 MAP kinase kinase MKK6 is identified as a common activator of p38 alpha, p38 beta 2, and p38 gamma MAP kinase isoforms, while MKK3 activates only p38 alpha and p38 gamma MAP kinase isoforms. The MKK3 and MKK6 signal transduction pathways are therefore coupled to distinct, but overlapping, groups of p38 MAP kinases.
p38 MAPK family consists of four isoform proteins (alpha, beta, gamma, and delta) that are activated by the same stimuli, but the information about how these proteins act together to yield a biological response is missing. Here we show a feed-forward mechanism by which p38alpha may regulate Ras transformation and stress response through depleting its family member p38gamma protein via c-Jun-dependent ubiquitin-proteasome pathways. Analyses of MAPK kinase 6 (MKK6)-p38 fusion proteins showed that constitutively active p38alpha (MKK6-p38alpha) and p38gamma (MKK6-p38gamma) stimulates and inhibits c-Jun phosphorylation respectively, leading to a distinct AP-1 regulation. Depending on cell type and/or stimuli, p38alpha phosphorylation results in either Ras-transformation inhibition or a cell-death escalation that invariably couples with a decrease in p38gamma protein expression. p38gamma, on the other hand, increases Ras-dependent growth or inhibits stress induced cell-death independent of phosphorylation. In cells expressing both proteins, p38alpha phosphorylation decreases p38gamma protein expression, whereas its inhibition increases cellular p38gamma concentrations, indicating an active role of p38alpha phosphorylation in negatively regulating p38gamma protein expression. Mechanistic analyses show that p38alpha requires c-Jun activation to deplete p38gamma proteins by ubiquitin-proteasome pathways. These results suggest that p38alpha may, upon phosphorylation, act as a gatekeeper of the p38 MAPK family to yield a coordinative biological response through disrupting its antagonistic p38gamma family protein.
J. Biol. Chem. 273, 29661-29671 (1998)[PubMed:9792677]
A novel ribosomal S6 kinase (RSK) family member, RSK-B, was identified in a p38alphaMAPK-baited intracellular interaction screen. RSK-B presents two catalytic domains typical for the RSK family. The protein kinase C-like N-terminal and the calcium/calmodulin kinase-like C-terminal domains both contain conserved ATP-binding and activation consensus sequences. RSK-B is a p38alphaMAPK substrate, and activated by p38alphaMAPK and, more weakly, by ERK1. RSK-B phosphorylates the cAMP response element-binding protein (CREB) and c-Fos peptides. In intracellular assays, RSK-B drives cAMP response element- and AP1-dependent reporter expression. RSK-B locates to the cell nucleus and co-translocates p38alphaMAPK. In conclusion, RSK-B is a novel CREB kinase under dominant p38alphaMAPK control, also phosphorylating additional substrates.
The p38 family of mitogen-activated protein kinases is composed of several isoforms. Mxi2 is a splicing variant of p38alpha that harbors a unique carboxy-terminus. Here we show that this sole divergence results in remarkable differences between Mxi2 and p38alpha. Mxi2 is distinctively activated by stress stimuli and potently activated by mitogens. Mxi2 affinity for bona fide p38 substrates is remarkably diminished and Mxi2 activity is largely unaffected by the phosphatase CL100. Also, Mxi2 sensitivity to inhibition by SB203580 is greatly reduced. Interestingly, we show that the p38 C-terminus is involved in conferring sensitivity to this compound. Overall, our results point to the p38 carboxy-terminus as a key determinant of the biochemical properties of this family of kinases.
Inflammatory stimuli activate ectodomain shedding of TNF-alpha, L-selectin, and other transmembrane proteins. We show that p38 MAP kinase, which is activated in response to inflammatory or stress signals, directly activates TACE, a membrane-associated metalloprotease that is also known as ADAM17 and effects shedding in response to growth factors and Erk MAP kinase activation. p38alpha MAP kinase interacts with the cytoplasmic domain of TACE and phosphorylates it on Thr(735), which is required for TACE-mediated ectodomain shedding. Activation of TACE by p38 MAP kinase results in the release of TGF-alpha family ligands, which activate EGF receptor signaling, leading to enhanced cell proliferation. Conversely, depletion of p38alpha MAP kinase activity suppresses EGF receptor signaling and downstream Erk MAP kinase signaling, as well as autocrine EGF receptor-dependent proliferation. Autocrine EGF receptor activation through TACE-mediated ectodomain shedding intimately links inflammation and cancer progression and may play a role in stress and conditions that relate to p38 MAP kinase activation.
J. Immunol. 174, 7257-7267 (2005)[PubMed:15905572]
The targets of the p38 MAPK pathway that mediate neutrophil functional responses are largely unknown. To identify p38 MAPK targets, a proteomic approach was applied in which recombinant active p38 MAPK and [(32)P]ATP were added to lysates from unstimulated human neutrophils. Proteins were separated by two-dimensional gel electrophoresis, and phosphoproteins were visualized by autoradiography and identified by MALDI-TOF. Myeloid-related protein-14 (MRP-14) was identified as a candidate p38 MAPK substrate. MRP-14 phosphorylation by p38 MAPK was confirmed by an in vitro kinase reaction using purified MRP-14/MRP-8 complexes. The site of MRP-14 phosphorylation by p38 MAPK was identified by tandem mass spectrometry and site-directed mutagenesis to be Thr(113). MRP-14 phosphorylation by p38 MAPK in intact neutrophils was confirmed by [(32)P]orthophosphate loading, followed by fMLP stimulation in the presence and absence of a p38 MAPK inhibitor, SB203580. Confocal microscopy of Triton X-100 permeabilized neutrophils showed that a small amount of MRP-14 was associated with cortical F-actin in unstimulated cells. fMLP stimulation resulted in a p38 MAPK-dependent increase in MRP-14 staining at the base of lamellipodia. By immunoblot analysis, MRP-14 was present in plasma membrane/secretory vesicle fractions and gelatinase and specific granules, but not in azurophil granules. The amount of MRP-14 associated with plasma membrane/secretory vesicle and gelatinase granule fractions increased after fMLP stimulation in a p38 MAPK-dependent manner. Direct phosphorylation of the MRP-14/MRP-8 complex by p38 MAPK increased actin binding in vitro by 2-fold. These results indicate that MRP-14 is a potential mediator of p38 MAPK-dependent functional responses in human neutrophils.
Members of the MEF2 family of transcription factors bind as homo- and heterodimers to the MEF2 site found in the promoter regions of numerous muscle-specific, growth- or stress-induced genes. We showed previously that the transactivation activity of MEF2C is stimulated by p38 mitogen-activated protein (MAP) kinase. In this study, we examined the potential role of the p38 MAP kinase pathway in regulating the other MEF2 family members. We found that MEF2A, but not MEF2B or MEF2D, is a substrate for p38. Among the four p38 group members, p38 is the most potent kinase for MEF2A. Threonines 312 and 319 within the transcription activation domain of MEF2A are the regulatory sites phosphorylated by p38. Phosphorylation of MEF2A in a MEF2A-MEF2D heterodimer enhances MEF2-dependent gene expression. These results demonstrate that the MAP kinase signaling pathway can discriminate between different MEF2 isoforms and can regulate MEF2-dependent genes through posttranslational activation of preexisting MEF2 protein.
Activity of the p38alpha MAP kinase is stimulated by various stresses and hematopoietic growth factors. A role for p38alpha in mouse development and physiology was investigated by targeted disruption of the p38alpha locus. Whereas some p38alpha(-/-) embryos die between embryonic days 11.5 and 12.5, those that develop past this stage have normal morphology but are anemic owing to failed definitive erythropoiesis, caused by diminished erythropoietin (Epo) gene expression. As p38alpha-deficient hematopoietic stem cells reconstitute lethally irradiated hosts, p38alpha function is not required downstream of Epo receptor. Inhibition of p38 activity also interferes with stabilization of Epo mRNA in human hepatoma cells undergoing hypoxic stress. The p38alpha MAP kinase plays a critical role linking developmental and stress-induced erythropoiesis through regulation of Epo expression.
Following genotoxic stress, cells activate a complex kinase-based signaling network to arrest the cell cycle and initiate DNA repair. p53-defective tumor cells rewire their checkpoint response and become dependent on the p38/MK2 pathway for survival after DNA damage, despite a functional ATR-Chk1 pathway. We used functional genetics to dissect the contributions of Chk1 and MK2 to checkpoint control. We show that nuclear Chk1 activity is essential to establish a G(2)/M checkpoint, while cytoplasmic MK2 activity is critical for prolonged checkpoint maintenance through a process of posttranscriptional mRNA stabilization. Following DNA damage, the p38/MK2 complex relocalizes from nucleus to cytoplasm where MK2 phosphorylates hnRNPA0, to stabilize Gadd45α mRNA, while p38 phosphorylates and releases the translational inhibitor TIAR. In addition, MK2 phosphorylates PARN, blocking Gadd45α mRNA degradation. Gadd45α functions within a positive feedback loop, sustaining the MK2-dependent cytoplasmic sequestration of Cdc25B/C to block mitotic entry in the presence of unrepaired DNA damage. Our findings demonstrate a critical role for the MK2 pathway in the posttranscriptional regulation of gene expression as part of the DNA damage response in cancer cells.
Activity of the p38alpha MAP kinase is stimulated by various stresses and hematopoietic growth factors. A role for p38alpha in mouse development and physiology was investigated by targeted disruption of the p38alpha locus. Whereas some p38alpha(-/-) embryos die between embryonic days 11.5 and 12.5, those that develop past this stage have normal morphology but are anemic owing to failed definitive erythropoiesis, caused by diminished erythropoietin (Epo) gene expression. As p38alpha-deficient hematopoietic stem cells reconstitute lethally irradiated hosts, p38alpha function is not required downstream of Epo receptor. Inhibition of p38 activity also interferes with stabilization of Epo mRNA in human hepatoma cells undergoing hypoxic stress. The p38alpha MAP kinase plays a critical role linking developmental and stress-induced erythropoiesis through regulation of Epo expression.
J. Biol. Chem. 273, 29661-29671 (1998)[PubMed:9792677]
A novel ribosomal S6 kinase (RSK) family member, RSK-B, was identified in a p38alphaMAPK-baited intracellular interaction screen. RSK-B presents two catalytic domains typical for the RSK family. The protein kinase C-like N-terminal and the calcium/calmodulin kinase-like C-terminal domains both contain conserved ATP-binding and activation consensus sequences. RSK-B is a p38alphaMAPK substrate, and activated by p38alphaMAPK and, more weakly, by ERK1. RSK-B phosphorylates the cAMP response element-binding protein (CREB) and c-Fos peptides. In intracellular assays, RSK-B drives cAMP response element- and AP1-dependent reporter expression. RSK-B locates to the cell nucleus and co-translocates p38alphaMAPK. In conclusion, RSK-B is a novel CREB kinase under dominant p38alphaMAPK control, also phosphorylating additional substrates.
J. Biol. Chem. 273, 1741-1748 (1998)[PubMed:9430721]
The cellular response to treatment with proinflammatory cytokines or exposure to environmental stress is mediated, in part, by the p38 group of mitogen-activated protein (MAP) kinases. We report the molecular cloning of a novel isoform of p38 MAP kinase, p38 beta 2. This p38 MAP kinase, like p38 alpha, is inhibited by the pyridinyl imidazole drug SB203580. The p38 MAP kinase kinase MKK6 is identified as a common activator of p38 alpha, p38 beta 2, and p38 gamma MAP kinase isoforms, while MKK3 activates only p38 alpha and p38 gamma MAP kinase isoforms. The MKK3 and MKK6 signal transduction pathways are therefore coupled to distinct, but overlapping, groups of p38 MAP kinases.
The p38 family of mitogen-activated protein kinases is composed of several isoforms. Mxi2 is a splicing variant of p38alpha that harbors a unique carboxy-terminus. Here we show that this sole divergence results in remarkable differences between Mxi2 and p38alpha. Mxi2 is distinctively activated by stress stimuli and potently activated by mitogens. Mxi2 affinity for bona fide p38 substrates is remarkably diminished and Mxi2 activity is largely unaffected by the phosphatase CL100. Also, Mxi2 sensitivity to inhibition by SB203580 is greatly reduced. Interestingly, we show that the p38 C-terminus is involved in conferring sensitivity to this compound. Overall, our results point to the p38 carboxy-terminus as a key determinant of the biochemical properties of this family of kinases.
Response to genotoxic stress can be considered as a multistage process involving initiation of cell-cycle arrest and maintenance of arrest during DNA repair. Although maintenance of G2/M checkpoints is known to involve Chk1, Chk2/Rad53 and upstream components, the mechanisms involved in its initiation are less well defined. Here we report that p38 kinase has a critical role in the initiation of a G2 delay after ultraviolet radiation. Inhibition of p38 blocks the rapid initiation of this checkpoint in both human and murine cells after ultraviolet radiation. In vitro, p38 binds and phosphorylates Cdc25B at serines 309 and 361, and Cdc25C at serine 216; phosphorylation of these residues is required for binding to 14-3-3 proteins. In vivo, inhibition of p38 prevents both phosphorylation of Cdc25B at serine 309 and 14-3-3 binding after ultraviolet radiation, and mutation of this site is sufficient to inhibit the checkpoint initiation. In contrast, in vivo Cdc25C binding to 14-3-3 is not affected by p38 inhibition after ultraviolet radiation. We propose that regulation of Cdc25B phosphorylation by p38 is a critical event for initiating the G2/M checkpoint after ultraviolet radiation.
The cap-binding translation initiation factor eukaryotic initiation factor 4E (eIF4E) is phosphorylated in vivo at Ser209 in response to a variety of stimuli. In this paper, we show that the mitogen-activated protein kinase (MAPK) signal-integrating kinase Mnk2 phosphorylates eIF4E at this residue. Mnk2 binds to the scaffolding protein eIF4G, and overexpression of Mnk2 results in increased phosphorylation of endogenous eIF4E, showing that it can act as an eIF4E kinase in vivo. We have identified eight phosphorylation sites in Mnk2, of which at least three potential MAPK sites are likely to be essential for Mnk2 activity. In contrast to that of Mnk1, the activity of overexpressed Mnk2 is high under control conditions and could only be reduced substantially by a combination of PD98059 and SB203580, while the activity of endogenous Mnk2 in Swiss 3T3 cells was hardly affected upon treatment with these inhibitors. These compounds did not abolish phosphorylation of eIF4E, implying that Mnk2 may mediate phosphorylation of eIF4E in Swiss 3T3 cells. In vitro phosphorylation studies show that Mnk2 is a significantly better substrate than Mnk1 for extracellular signal-regulated kinase 2 (ERK2), p38MAPKalpha, and p38MAPKbeta. Therefore, the high levels of activity of Mnk2 under several conditions may be explained by efficient activation of Mnk2 by low levels of activity of the upstream kinases. Interestingly, we found that the association of both Mnk1 and Mnk2 with eIF4G increased upon inhibition of the MAPK pathways while activation of ERK resulted in decreased binding to eIF4G. This might reflect a mechanism to ensure rapid, but transient, phosphorylation of eIF4E upon stimulation of the MAPK pathways.
The RING finger ubiquitin ligase Siah2 controls the stability of various substrates involved in stress and hypoxia responses, including the PHD3, which controls the stability of HIF-1alpha. In the present study we determined the role of Siah2 phosphorylation in the regulation of its activity toward PHD3. We show that Siah2 is subject to phosphorylation by p38 MAPK, which increases Siah2-mediated degradation of PHD3. Consistent with these findings, MKK3/MKK6 double-deficient cells, which cannot activate p38 kinases, exhibit impaired Siah2-dependent degradation of PHD3. Phosphopeptide mapping identified T24 and S29 as the primary phospho-acceptor sites. Phospho-mutant forms of Siah2 (S29A or T24A/S29A) exhibit impaired degradation of PHD3, particularly after hypoxia. Conversely, a phospho-mimic form of Siah2 (T24E/S29D) exhibits stronger degradation of PHD3, compared with wild type Siah2. Whereas phospho-mutant Siah2 exhibits weaker association with PHD3, phospho-mimic Siah2 associates as well as wild type and is localized within the perinuclear region, suggesting that phosphorylation of Siah2 affects its subcellular localization and, consequently, the degree of its association with PHD3. In all, our findings reveal the phosphorylation of Siah2 by p38 and the implications of such phosphorylation for Siah2 activity toward PHD3.
Members of the MEF2 family of transcription factors bind as homo- and heterodimers to the MEF2 site found in the promoter regions of numerous muscle-specific, growth- or stress-induced genes. We showed previously that the transactivation activity of MEF2C is stimulated by p38 mitogen-activated protein (MAP) kinase. In this study, we examined the potential role of the p38 MAP kinase pathway in regulating the other MEF2 family members. We found that MEF2A, but not MEF2B or MEF2D, is a substrate for p38. Among the four p38 group members, p38 is the most potent kinase for MEF2A. Threonines 312 and 319 within the transcription activation domain of MEF2A are the regulatory sites phosphorylated by p38. Phosphorylation of MEF2A in a MEF2A-MEF2D heterodimer enhances MEF2-dependent gene expression. These results demonstrate that the MAP kinase signaling pathway can discriminate between different MEF2 isoforms and can regulate MEF2-dependent genes through posttranslational activation of preexisting MEF2 protein.
Following genotoxic stress, cells activate a complex kinase-based signaling network to arrest the cell cycle and initiate DNA repair. p53-defective tumor cells rewire their checkpoint response and become dependent on the p38/MK2 pathway for survival after DNA damage, despite a functional ATR-Chk1 pathway. We used functional genetics to dissect the contributions of Chk1 and MK2 to checkpoint control. We show that nuclear Chk1 activity is essential to establish a G(2)/M checkpoint, while cytoplasmic MK2 activity is critical for prolonged checkpoint maintenance through a process of posttranscriptional mRNA stabilization. Following DNA damage, the p38/MK2 complex relocalizes from nucleus to cytoplasm where MK2 phosphorylates hnRNPA0, to stabilize Gadd45α mRNA, while p38 phosphorylates and releases the translational inhibitor TIAR. In addition, MK2 phosphorylates PARN, blocking Gadd45α mRNA degradation. Gadd45α functions within a positive feedback loop, sustaining the MK2-dependent cytoplasmic sequestration of Cdc25B/C to block mitotic entry in the presence of unrepaired DNA damage. Our findings demonstrate a critical role for the MK2 pathway in the posttranscriptional regulation of gene expression as part of the DNA damage response in cancer cells.
The epidermal growth factor receptor (EGFR) frequently associates with cancer and already serves as a target for therapy. We report that inflammatory cytokines and ultraviolet (UV) irradiation respectively induce transient or sustained phosphorylation of EGFR. Subsequently, EGFR internalizes via a Clathrin-mediated process. In cytokine-stimulated cells, EGFR recycles back to the cell surface, whereas in irradiated cells it arrests in Rab5-containing endosomes. Under both conditions, receptor internalization is instigated by the p38 stress-induced kinase. The underlying mechanism entails phosphorylation of EGFR at a short segment (amino acids 1002-1022) containing multiple serines and threonines, as well as phosphorylation of two Rab5 effectors, EEA1 and GDI. Like UV irradiation, a chemotherapeutic agent activates p38 and accelerates receptor internalization. We demonstrate that abrogating EGFR internalization reduces the efficacy of chemotherapy-induced cell death. Hence, by preventing EGFR-mediated survival signaling, the internalization route we uncovered enhances the cytotoxic effect of drugs like cis-platinum, which may underlie interactions between chemotherapy and EGFR-targeting drugs.
p38 MAPK family consists of four isoform proteins (alpha, beta, gamma, and delta) that are activated by the same stimuli, but the information about how these proteins act together to yield a biological response is missing. Here we show a feed-forward mechanism by which p38alpha may regulate Ras transformation and stress response through depleting its family member p38gamma protein via c-Jun-dependent ubiquitin-proteasome pathways. Analyses of MAPK kinase 6 (MKK6)-p38 fusion proteins showed that constitutively active p38alpha (MKK6-p38alpha) and p38gamma (MKK6-p38gamma) stimulates and inhibits c-Jun phosphorylation respectively, leading to a distinct AP-1 regulation. Depending on cell type and/or stimuli, p38alpha phosphorylation results in either Ras-transformation inhibition or a cell-death escalation that invariably couples with a decrease in p38gamma protein expression. p38gamma, on the other hand, increases Ras-dependent growth or inhibits stress induced cell-death independent of phosphorylation. In cells expressing both proteins, p38alpha phosphorylation decreases p38gamma protein expression, whereas its inhibition increases cellular p38gamma concentrations, indicating an active role of p38alpha phosphorylation in negatively regulating p38gamma protein expression. Mechanistic analyses show that p38alpha requires c-Jun activation to deplete p38gamma proteins by ubiquitin-proteasome pathways. These results suggest that p38alpha may, upon phosphorylation, act as a gatekeeper of the p38 MAPK family to yield a coordinative biological response through disrupting its antagonistic p38gamma family protein.
Autophagy, a lysosomal degradation pathway, is essential for homeostasis, development, neurological diseases, and cancer. Regulation of autophagy in human disease is not well understood. Atg9 is a transmembrane protein required for autophagy, and it has been proposed that trafficking of Atg9 may regulate autophagy. Mammalian Atg9 traffics between the TGN and endosomes in basal conditions, and newly formed autophagosomes in response to signals inducing autophagy. We identified p38IP as a new mAtg9 interactor and showed that this interaction is regulated by p38alpha MAPK. p38IP is required for starvation-induced mAtg9 trafficking and autophagosome formation. Manipulation of p38IP and p38alpha alters mAtg9 localization, suggesting p38alpha regulates, through p38IP, the starvation-induced mAtg9 trafficking to forming autophagosomes. Furthermore, we show that p38alpha is a negative regulator of both basal autophagy and starvation-induced autophagy, and suggest that this regulation may be through a direct competition with mAtg9 for binding to p38IP. Our results provide evidence for a link between the MAPK pathway and the control of autophagy through mAtg9 and p38IP.
J. Immunol. 174, 7257-7267 (2005)[PubMed:15905572]
The targets of the p38 MAPK pathway that mediate neutrophil functional responses are largely unknown. To identify p38 MAPK targets, a proteomic approach was applied in which recombinant active p38 MAPK and [(32)P]ATP were added to lysates from unstimulated human neutrophils. Proteins were separated by two-dimensional gel electrophoresis, and phosphoproteins were visualized by autoradiography and identified by MALDI-TOF. Myeloid-related protein-14 (MRP-14) was identified as a candidate p38 MAPK substrate. MRP-14 phosphorylation by p38 MAPK was confirmed by an in vitro kinase reaction using purified MRP-14/MRP-8 complexes. The site of MRP-14 phosphorylation by p38 MAPK was identified by tandem mass spectrometry and site-directed mutagenesis to be Thr(113). MRP-14 phosphorylation by p38 MAPK in intact neutrophils was confirmed by [(32)P]orthophosphate loading, followed by fMLP stimulation in the presence and absence of a p38 MAPK inhibitor, SB203580. Confocal microscopy of Triton X-100 permeabilized neutrophils showed that a small amount of MRP-14 was associated with cortical F-actin in unstimulated cells. fMLP stimulation resulted in a p38 MAPK-dependent increase in MRP-14 staining at the base of lamellipodia. By immunoblot analysis, MRP-14 was present in plasma membrane/secretory vesicle fractions and gelatinase and specific granules, but not in azurophil granules. The amount of MRP-14 associated with plasma membrane/secretory vesicle and gelatinase granule fractions increased after fMLP stimulation in a p38 MAPK-dependent manner. Direct phosphorylation of the MRP-14/MRP-8 complex by p38 MAPK increased actin binding in vitro by 2-fold. These results indicate that MRP-14 is a potential mediator of p38 MAPK-dependent functional responses in human neutrophils.
We have identified a novel mitogen- and stress-activated protein kinase (MSK1) that contains two protein kinase domains in a single polypeptide. MSK1 is activated in vitro by MAPK2/ERK2 or SAPK2/p38. Endogenous MSK1 is activated in 293 cells by either growth factor/phorbol ester stimulation, or by exposure to UV radiation, and oxidative and chemical stress. The activation of MSK1 by growth factors/phorbol esters is prevented by PD 98059, which suppresses activation of the MAPK cascade, while the activation of MSK1 by stress stimuli is prevented by SB 203580, a specific inhibitor of SAPK2/p38. In HeLa, PC12 and SK-N-MC cells, PD 98059 and SB 203580 are both required to suppress the activation of MSK1 by TNF, NGF and FGF, respectively, because these agonists activate both the MAPK/ERK and SAPK2/p38 cascades. MSK1 is localized in the nucleus of unstimulated or stimulated cells, and phosphorylates CREB at Ser133 with a Km value far lower than PKA, MAPKAP-K1(p90Rsk) and MAPKAP-K2. The effects of SB 203580, PD 98059 and Ro 318220 on agonist-induced activation of CREB and ATF1 in four cell-lines mirror the effects of these inhibitors on MSK1 activation, and exclude a role for MAPKAP-K1 and MAPKAP-K2/3 in this process. These findings, together with other observations, suggest that MSK1 may mediate the growth-factor and stress-induced activation of CREB.
Mitogen-activated protein (MAP) kinase-mediated signalling to the nucleus is an important event in the conversion of extracellular signals into a cellular response. However, the existence of multiple MAP kinases which phosphorylate similar phosphoacceptor motifs poses a problem in maintaining substrate specificity and hence the correct biological response. Both the extracellular signal-regulated kinase (ERK) and c-Jun NH2-terminal kinase (JNK) subfamilies of MAP kinases use a second specificity determinant and require docking to their transcription factor substrates to achieve maximal substrate activation. In this study, we demonstrate that among the different MAP kinases, the MADS-box transcription factors MEF2A and MEF2C are preferentially phosphorylated and activated by the p38 subfamily members p38alpha and p38beta2. The efficiency of phosphorylation in vitro and transcriptional activation in vivo of MEF2A and MEF2C by these p38 subtypes requires the presence of a kinase docking domain (D-domain). Furthermore, the D-domain from MEF2A is sufficient to confer p38 responsiveness on different transcription factors, and reciprocal effects are observed upon the introduction of alternative D-domains into MEF2A. These results therefore contribute to our understanding of signalling to MEF2 transcription factors and demonstrate that the requirement for substrate binding by MAP kinases is an important facet of three different subclasses of MAP kinases (ERK, JNK, and p38).
Inflammatory stimuli activate ectodomain shedding of TNF-alpha, L-selectin, and other transmembrane proteins. We show that p38 MAP kinase, which is activated in response to inflammatory or stress signals, directly activates TACE, a membrane-associated metalloprotease that is also known as ADAM17 and effects shedding in response to growth factors and Erk MAP kinase activation. p38alpha MAP kinase interacts with the cytoplasmic domain of TACE and phosphorylates it on Thr(735), which is required for TACE-mediated ectodomain shedding. Activation of TACE by p38 MAP kinase results in the release of TGF-alpha family ligands, which activate EGF receptor signaling, leading to enhanced cell proliferation. Conversely, depletion of p38alpha MAP kinase activity suppresses EGF receptor signaling and downstream Erk MAP kinase signaling, as well as autocrine EGF receptor-dependent proliferation. Autocrine EGF receptor activation through TACE-mediated ectodomain shedding intimately links inflammation and cancer progression and may play a role in stress and conditions that relate to p38 MAP kinase activation.
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 to be an allosteric mechanism. Furthermore, we demonstrate that anisomycin- and tumor necrosis factor-alpha-induced phosphorylation of p53 at Ser-392, which is important for the transcriptional activity of this growth suppressor protein, requires p38 MAP kinase and CK2 activities.
The RING finger ubiquitin ligase Siah2 controls the stability of various substrates involved in stress and hypoxia responses, including the PHD3, which controls the stability of HIF-1alpha. In the present study we determined the role of Siah2 phosphorylation in the regulation of its activity toward PHD3. We show that Siah2 is subject to phosphorylation by p38 MAPK, which increases Siah2-mediated degradation of PHD3. Consistent with these findings, MKK3/MKK6 double-deficient cells, which cannot activate p38 kinases, exhibit impaired Siah2-dependent degradation of PHD3. Phosphopeptide mapping identified T24 and S29 as the primary phospho-acceptor sites. Phospho-mutant forms of Siah2 (S29A or T24A/S29A) exhibit impaired degradation of PHD3, particularly after hypoxia. Conversely, a phospho-mimic form of Siah2 (T24E/S29D) exhibits stronger degradation of PHD3, compared with wild type Siah2. Whereas phospho-mutant Siah2 exhibits weaker association with PHD3, phospho-mimic Siah2 associates as well as wild type and is localized within the perinuclear region, suggesting that phosphorylation of Siah2 affects its subcellular localization and, consequently, the degree of its association with PHD3. In all, our findings reveal the phosphorylation of Siah2 by p38 and the implications of such phosphorylation for Siah2 activity toward PHD3.
The epidermal growth factor receptor (EGFR) frequently associates with cancer and already serves as a target for therapy. We report that inflammatory cytokines and ultraviolet (UV) irradiation respectively induce transient or sustained phosphorylation of EGFR. Subsequently, EGFR internalizes via a Clathrin-mediated process. In cytokine-stimulated cells, EGFR recycles back to the cell surface, whereas in irradiated cells it arrests in Rab5-containing endosomes. Under both conditions, receptor internalization is instigated by the p38 stress-induced kinase. The underlying mechanism entails phosphorylation of EGFR at a short segment (amino acids 1002-1022) containing multiple serines and threonines, as well as phosphorylation of two Rab5 effectors, EEA1 and GDI. Like UV irradiation, a chemotherapeutic agent activates p38 and accelerates receptor internalization. We demonstrate that abrogating EGFR internalization reduces the efficacy of chemotherapy-induced cell death. Hence, by preventing EGFR-mediated survival signaling, the internalization route we uncovered enhances the cytotoxic effect of drugs like cis-platinum, which may underlie interactions between chemotherapy and EGFR-targeting drugs.
Response to genotoxic stress can be considered as a multistage process involving initiation of cell-cycle arrest and maintenance of arrest during DNA repair. Although maintenance of G2/M checkpoints is known to involve Chk1, Chk2/Rad53 and upstream components, the mechanisms involved in its initiation are less well defined. Here we report that p38 kinase has a critical role in the initiation of a G2 delay after ultraviolet radiation. Inhibition of p38 blocks the rapid initiation of this checkpoint in both human and murine cells after ultraviolet radiation. In vitro, p38 binds and phosphorylates Cdc25B at serines 309 and 361, and Cdc25C at serine 216; phosphorylation of these residues is required for binding to 14-3-3 proteins. In vivo, inhibition of p38 prevents both phosphorylation of Cdc25B at serine 309 and 14-3-3 binding after ultraviolet radiation, and mutation of this site is sufficient to inhibit the checkpoint initiation. In contrast, in vivo Cdc25C binding to 14-3-3 is not affected by p38 inhibition after ultraviolet radiation. We propose that regulation of Cdc25B phosphorylation by p38 is a critical event for initiating the G2/M checkpoint after ultraviolet radiation.
p38 MAPK family consists of four isoform proteins (alpha, beta, gamma, and delta) that are activated by the same stimuli, but the information about how these proteins act together to yield a biological response is missing. Here we show a feed-forward mechanism by which p38alpha may regulate Ras transformation and stress response through depleting its family member p38gamma protein via c-Jun-dependent ubiquitin-proteasome pathways. Analyses of MAPK kinase 6 (MKK6)-p38 fusion proteins showed that constitutively active p38alpha (MKK6-p38alpha) and p38gamma (MKK6-p38gamma) stimulates and inhibits c-Jun phosphorylation respectively, leading to a distinct AP-1 regulation. Depending on cell type and/or stimuli, p38alpha phosphorylation results in either Ras-transformation inhibition or a cell-death escalation that invariably couples with a decrease in p38gamma protein expression. p38gamma, on the other hand, increases Ras-dependent growth or inhibits stress induced cell-death independent of phosphorylation. In cells expressing both proteins, p38alpha phosphorylation decreases p38gamma protein expression, whereas its inhibition increases cellular p38gamma concentrations, indicating an active role of p38alpha phosphorylation in negatively regulating p38gamma protein expression. Mechanistic analyses show that p38alpha requires c-Jun activation to deplete p38gamma proteins by ubiquitin-proteasome pathways. These results suggest that p38alpha may, upon phosphorylation, act as a gatekeeper of the p38 MAPK family to yield a coordinative biological response through disrupting its antagonistic p38gamma family protein.
Inflammatory stimuli activate ectodomain shedding of TNF-alpha, L-selectin, and other transmembrane proteins. We show that p38 MAP kinase, which is activated in response to inflammatory or stress signals, directly activates TACE, a membrane-associated metalloprotease that is also known as ADAM17 and effects shedding in response to growth factors and Erk MAP kinase activation. p38alpha MAP kinase interacts with the cytoplasmic domain of TACE and phosphorylates it on Thr(735), which is required for TACE-mediated ectodomain shedding. Activation of TACE by p38 MAP kinase results in the release of TGF-alpha family ligands, which activate EGF receptor signaling, leading to enhanced cell proliferation. Conversely, depletion of p38alpha MAP kinase activity suppresses EGF receptor signaling and downstream Erk MAP kinase signaling, as well as autocrine EGF receptor-dependent proliferation. Autocrine EGF receptor activation through TACE-mediated ectodomain shedding intimately links inflammation and cancer progression and may play a role in stress and conditions that relate to p38 MAP kinase activation.
J. Biol. Chem. 273, 1741-1748 (1998)[PubMed:9430721]
The cellular response to treatment with proinflammatory cytokines or exposure to environmental stress is mediated, in part, by the p38 group of mitogen-activated protein (MAP) kinases. We report the molecular cloning of a novel isoform of p38 MAP kinase, p38 beta 2. This p38 MAP kinase, like p38 alpha, is inhibited by the pyridinyl imidazole drug SB203580. The p38 MAP kinase kinase MKK6 is identified as a common activator of p38 alpha, p38 beta 2, and p38 gamma MAP kinase isoforms, while MKK3 activates only p38 alpha and p38 gamma MAP kinase isoforms. The MKK3 and MKK6 signal transduction pathways are therefore coupled to distinct, but overlapping, groups of p38 MAP kinases.
The p38 family of mitogen-activated protein kinases is composed of several isoforms. Mxi2 is a splicing variant of p38alpha that harbors a unique carboxy-terminus. Here we show that this sole divergence results in remarkable differences between Mxi2 and p38alpha. Mxi2 is distinctively activated by stress stimuli and potently activated by mitogens. Mxi2 affinity for bona fide p38 substrates is remarkably diminished and Mxi2 activity is largely unaffected by the phosphatase CL100. Also, Mxi2 sensitivity to inhibition by SB203580 is greatly reduced. Interestingly, we show that the p38 C-terminus is involved in conferring sensitivity to this compound. Overall, our results point to the p38 carboxy-terminus as a key determinant of the biochemical properties of this family of kinases.
We have identified a novel mitogen- and stress-activated protein kinase (MSK1) that contains two protein kinase domains in a single polypeptide. MSK1 is activated in vitro by MAPK2/ERK2 or SAPK2/p38. Endogenous MSK1 is activated in 293 cells by either growth factor/phorbol ester stimulation, or by exposure to UV radiation, and oxidative and chemical stress. The activation of MSK1 by growth factors/phorbol esters is prevented by PD 98059, which suppresses activation of the MAPK cascade, while the activation of MSK1 by stress stimuli is prevented by SB 203580, a specific inhibitor of SAPK2/p38. In HeLa, PC12 and SK-N-MC cells, PD 98059 and SB 203580 are both required to suppress the activation of MSK1 by TNF, NGF and FGF, respectively, because these agonists activate both the MAPK/ERK and SAPK2/p38 cascades. MSK1 is localized in the nucleus of unstimulated or stimulated cells, and phosphorylates CREB at Ser133 with a Km value far lower than PKA, MAPKAP-K1(p90Rsk) and MAPKAP-K2. The effects of SB 203580, PD 98059 and Ro 318220 on agonist-induced activation of CREB and ATF1 in four cell-lines mirror the effects of these inhibitors on MSK1 activation, and exclude a role for MAPKAP-K1 and MAPKAP-K2/3 in this process. These findings, together with other observations, suggest that MSK1 may mediate the growth-factor and stress-induced activation of CREB.
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 to be an allosteric mechanism. Furthermore, we demonstrate that anisomycin- and tumor necrosis factor-alpha-induced phosphorylation of p53 at Ser-392, which is important for the transcriptional activity of this growth suppressor protein, requires p38 MAP kinase and CK2 activities.
J. Immunol. 174, 7257-7267 (2005)[PubMed:15905572]
The targets of the p38 MAPK pathway that mediate neutrophil functional responses are largely unknown. To identify p38 MAPK targets, a proteomic approach was applied in which recombinant active p38 MAPK and [(32)P]ATP were added to lysates from unstimulated human neutrophils. Proteins were separated by two-dimensional gel electrophoresis, and phosphoproteins were visualized by autoradiography and identified by MALDI-TOF. Myeloid-related protein-14 (MRP-14) was identified as a candidate p38 MAPK substrate. MRP-14 phosphorylation by p38 MAPK was confirmed by an in vitro kinase reaction using purified MRP-14/MRP-8 complexes. The site of MRP-14 phosphorylation by p38 MAPK was identified by tandem mass spectrometry and site-directed mutagenesis to be Thr(113). MRP-14 phosphorylation by p38 MAPK in intact neutrophils was confirmed by [(32)P]orthophosphate loading, followed by fMLP stimulation in the presence and absence of a p38 MAPK inhibitor, SB203580. Confocal microscopy of Triton X-100 permeabilized neutrophils showed that a small amount of MRP-14 was associated with cortical F-actin in unstimulated cells. fMLP stimulation resulted in a p38 MAPK-dependent increase in MRP-14 staining at the base of lamellipodia. By immunoblot analysis, MRP-14 was present in plasma membrane/secretory vesicle fractions and gelatinase and specific granules, but not in azurophil granules. The amount of MRP-14 associated with plasma membrane/secretory vesicle and gelatinase granule fractions increased after fMLP stimulation in a p38 MAPK-dependent manner. Direct phosphorylation of the MRP-14/MRP-8 complex by p38 MAPK increased actin binding in vitro by 2-fold. These results indicate that MRP-14 is a potential mediator of p38 MAPK-dependent functional responses in human neutrophils.
Following genotoxic stress, cells activate a complex kinase-based signaling network to arrest the cell cycle and initiate DNA repair. p53-defective tumor cells rewire their checkpoint response and become dependent on the p38/MK2 pathway for survival after DNA damage, despite a functional ATR-Chk1 pathway. We used functional genetics to dissect the contributions of Chk1 and MK2 to checkpoint control. We show that nuclear Chk1 activity is essential to establish a G(2)/M checkpoint, while cytoplasmic MK2 activity is critical for prolonged checkpoint maintenance through a process of posttranscriptional mRNA stabilization. Following DNA damage, the p38/MK2 complex relocalizes from nucleus to cytoplasm where MK2 phosphorylates hnRNPA0, to stabilize Gadd45α mRNA, while p38 phosphorylates and releases the translational inhibitor TIAR. In addition, MK2 phosphorylates PARN, blocking Gadd45α mRNA degradation. Gadd45α functions within a positive feedback loop, sustaining the MK2-dependent cytoplasmic sequestration of Cdc25B/C to block mitotic entry in the presence of unrepaired DNA damage. Our findings demonstrate a critical role for the MK2 pathway in the posttranscriptional regulation of gene expression as part of the DNA damage response in cancer cells.
The cap-binding translation initiation factor eukaryotic initiation factor 4E (eIF4E) is phosphorylated in vivo at Ser209 in response to a variety of stimuli. In this paper, we show that the mitogen-activated protein kinase (MAPK) signal-integrating kinase Mnk2 phosphorylates eIF4E at this residue. Mnk2 binds to the scaffolding protein eIF4G, and overexpression of Mnk2 results in increased phosphorylation of endogenous eIF4E, showing that it can act as an eIF4E kinase in vivo. We have identified eight phosphorylation sites in Mnk2, of which at least three potential MAPK sites are likely to be essential for Mnk2 activity. In contrast to that of Mnk1, the activity of overexpressed Mnk2 is high under control conditions and could only be reduced substantially by a combination of PD98059 and SB203580, while the activity of endogenous Mnk2 in Swiss 3T3 cells was hardly affected upon treatment with these inhibitors. These compounds did not abolish phosphorylation of eIF4E, implying that Mnk2 may mediate phosphorylation of eIF4E in Swiss 3T3 cells. In vitro phosphorylation studies show that Mnk2 is a significantly better substrate than Mnk1 for extracellular signal-regulated kinase 2 (ERK2), p38MAPKalpha, and p38MAPKbeta. Therefore, the high levels of activity of Mnk2 under several conditions may be explained by efficient activation of Mnk2 by low levels of activity of the upstream kinases. Interestingly, we found that the association of both Mnk1 and Mnk2 with eIF4G increased upon inhibition of the MAPK pathways while activation of ERK resulted in decreased binding to eIF4G. This might reflect a mechanism to ensure rapid, but transient, phosphorylation of eIF4E upon stimulation of the MAPK pathways.
Autophagy, a lysosomal degradation pathway, is essential for homeostasis, development, neurological diseases, and cancer. Regulation of autophagy in human disease is not well understood. Atg9 is a transmembrane protein required for autophagy, and it has been proposed that trafficking of Atg9 may regulate autophagy. Mammalian Atg9 traffics between the TGN and endosomes in basal conditions, and newly formed autophagosomes in response to signals inducing autophagy. We identified p38IP as a new mAtg9 interactor and showed that this interaction is regulated by p38alpha MAPK. p38IP is required for starvation-induced mAtg9 trafficking and autophagosome formation. Manipulation of p38IP and p38alpha alters mAtg9 localization, suggesting p38alpha regulates, through p38IP, the starvation-induced mAtg9 trafficking to forming autophagosomes. Furthermore, we show that p38alpha is a negative regulator of both basal autophagy and starvation-induced autophagy, and suggest that this regulation may be through a direct competition with mAtg9 for binding to p38IP. Our results provide evidence for a link between the MAPK pathway and the control of autophagy through mAtg9 and p38IP.
Activity of the p38alpha MAP kinase is stimulated by various stresses and hematopoietic growth factors. A role for p38alpha in mouse development and physiology was investigated by targeted disruption of the p38alpha locus. Whereas some p38alpha(-/-) embryos die between embryonic days 11.5 and 12.5, those that develop past this stage have normal morphology but are anemic owing to failed definitive erythropoiesis, caused by diminished erythropoietin (Epo) gene expression. As p38alpha-deficient hematopoietic stem cells reconstitute lethally irradiated hosts, p38alpha function is not required downstream of Epo receptor. Inhibition of p38 activity also interferes with stabilization of Epo mRNA in human hepatoma cells undergoing hypoxic stress. The p38alpha MAP kinase plays a critical role linking developmental and stress-induced erythropoiesis through regulation of Epo expression.
Members of the MEF2 family of transcription factors bind as homo- and heterodimers to the MEF2 site found in the promoter regions of numerous muscle-specific, growth- or stress-induced genes. We showed previously that the transactivation activity of MEF2C is stimulated by p38 mitogen-activated protein (MAP) kinase. In this study, we examined the potential role of the p38 MAP kinase pathway in regulating the other MEF2 family members. We found that MEF2A, but not MEF2B or MEF2D, is a substrate for p38. Among the four p38 group members, p38 is the most potent kinase for MEF2A. Threonines 312 and 319 within the transcription activation domain of MEF2A are the regulatory sites phosphorylated by p38. Phosphorylation of MEF2A in a MEF2A-MEF2D heterodimer enhances MEF2-dependent gene expression. These results demonstrate that the MAP kinase signaling pathway can discriminate between different MEF2 isoforms and can regulate MEF2-dependent genes through posttranslational activation of preexisting MEF2 protein.
Mitogen-activated protein (MAP) kinase-mediated signalling to the nucleus is an important event in the conversion of extracellular signals into a cellular response. However, the existence of multiple MAP kinases which phosphorylate similar phosphoacceptor motifs poses a problem in maintaining substrate specificity and hence the correct biological response. Both the extracellular signal-regulated kinase (ERK) and c-Jun NH2-terminal kinase (JNK) subfamilies of MAP kinases use a second specificity determinant and require docking to their transcription factor substrates to achieve maximal substrate activation. In this study, we demonstrate that among the different MAP kinases, the MADS-box transcription factors MEF2A and MEF2C are preferentially phosphorylated and activated by the p38 subfamily members p38alpha and p38beta2. The efficiency of phosphorylation in vitro and transcriptional activation in vivo of MEF2A and MEF2C by these p38 subtypes requires the presence of a kinase docking domain (D-domain). Furthermore, the D-domain from MEF2A is sufficient to confer p38 responsiveness on different transcription factors, and reciprocal effects are observed upon the introduction of alternative D-domains into MEF2A. These results therefore contribute to our understanding of signalling to MEF2 transcription factors and demonstrate that the requirement for substrate binding by MAP kinases is an important facet of three different subclasses of MAP kinases (ERK, JNK, and p38).
J. Biol. Chem. 273, 29661-29671 (1998)[PubMed:9792677]
A novel ribosomal S6 kinase (RSK) family member, RSK-B, was identified in a p38alphaMAPK-baited intracellular interaction screen. RSK-B presents two catalytic domains typical for the RSK family. The protein kinase C-like N-terminal and the calcium/calmodulin kinase-like C-terminal domains both contain conserved ATP-binding and activation consensus sequences. RSK-B is a p38alphaMAPK substrate, and activated by p38alphaMAPK and, more weakly, by ERK1. RSK-B phosphorylates the cAMP response element-binding protein (CREB) and c-Fos peptides. In intracellular assays, RSK-B drives cAMP response element- and AP1-dependent reporter expression. RSK-B locates to the cell nucleus and co-translocates p38alphaMAPK. In conclusion, RSK-B is a novel CREB kinase under dominant p38alphaMAPK control, also phosphorylating additional substrates.
Catalysis of the reaction: protein + ATP = protein phosphate + ADP. This reaction is the phosphorylation of proteins. Mitogen-activated protein kinase; a family of protein kinases that perform a crucial step in relaying signals from the plasma membrane to the nucleus. They are activated by a wide range of proliferation- or differentiation-inducing signals; activation is strong with agonists such as polypeptide growth factors and tumor-promoting phorbol esters, but weak (in most cell backgrounds) by stress stimuli.
Production of interleukin-1 and tumour necrosis factor from stimulated human monocytes is inhibited by a new series of pyridinyl-imidazole compounds. Using radiolabelled and radio-photoaffinity-labelled chemical probes, the target of these compounds was identified as a pair of closely related mitogen-activated protein kinase homologues, termed CSBPs. Binding of the pyridinyl-imidazole compounds inhibited CSBP kinase activity and could be directly correlated with their ability to inhibit cytokine production, suggesting that the CSBPs are critical for cytokine production.
Mitogen-activated protein (MAP) kinase-mediated signalling to the nucleus is an important event in the conversion of extracellular signals into a cellular response. However, the existence of multiple MAP kinases which phosphorylate similar phosphoacceptor motifs poses a problem in maintaining substrate specificity and hence the correct biological response. Both the extracellular signal-regulated kinase (ERK) and c-Jun NH2-terminal kinase (JNK) subfamilies of MAP kinases use a second specificity determinant and require docking to their transcription factor substrates to achieve maximal substrate activation. In this study, we demonstrate that among the different MAP kinases, the MADS-box transcription factors MEF2A and MEF2C are preferentially phosphorylated and activated by the p38 subfamily members p38alpha and p38beta2. The efficiency of phosphorylation in vitro and transcriptional activation in vivo of MEF2A and MEF2C by these p38 subtypes requires the presence of a kinase docking domain (D-domain). Furthermore, the D-domain from MEF2A is sufficient to confer p38 responsiveness on different transcription factors, and reciprocal effects are observed upon the introduction of alternative D-domains into MEF2A. These results therefore contribute to our understanding of signalling to MEF2 transcription factors and demonstrate that the requirement for substrate binding by MAP kinases is an important facet of three different subclasses of MAP kinases (ERK, JNK, and p38).
Following genotoxic stress, cells activate a complex kinase-based signaling network to arrest the cell cycle and initiate DNA repair. p53-defective tumor cells rewire their checkpoint response and become dependent on the p38/MK2 pathway for survival after DNA damage, despite a functional ATR-Chk1 pathway. We used functional genetics to dissect the contributions of Chk1 and MK2 to checkpoint control. We show that nuclear Chk1 activity is essential to establish a G(2)/M checkpoint, while cytoplasmic MK2 activity is critical for prolonged checkpoint maintenance through a process of posttranscriptional mRNA stabilization. Following DNA damage, the p38/MK2 complex relocalizes from nucleus to cytoplasm where MK2 phosphorylates hnRNPA0, to stabilize Gadd45α mRNA, while p38 phosphorylates and releases the translational inhibitor TIAR. In addition, MK2 phosphorylates PARN, blocking Gadd45α mRNA degradation. Gadd45α functions within a positive feedback loop, sustaining the MK2-dependent cytoplasmic sequestration of Cdc25B/C to block mitotic entry in the presence of unrepaired DNA damage. Our findings demonstrate a critical role for the MK2 pathway in the posttranscriptional regulation of gene expression as part of the DNA damage response in cancer cells.
Catalysis of the concomitant phosphorylation of threonine (T) and tyrosine (Y) residues in a Thr-Glu-Tyr (TEY) thiolester sequence in a MAP kinase (MAPK) substrate.
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 was assessed using Boyden microchambers. Eotaxin (10(-11) to 10(-7) mol/L) induced concentration-dependent phosphorylation of ERK2 and p38. Phosphorylation was detectable after 30 seconds, peaked at about 1 minute, and returned to baseline after 2 to 5 minutes. Phosphorylation of JNK above baseline could not be detected. The kinase activity of ERK2 and p38 paralleled phosphorylation. PD980 59, an inhibitor of the ERK2-activating enzyme MEK (MAP ERK kinase), blocked phosphorylation of ERK2 in a concentration-dependent manner. The functional relevance of ERK2 and p38 was studied using PD98 059 and the p38 inhibitor SB202 190. PD98 059 and SB202 190 both caused inhibition of eotaxin-induced ECP release and chemotaxis. We conclude that eotaxin induces a rapid concentration-dependent activation of ERK2 and p38 in eosinophils and that the activation of these MAP kinases is required for eotaxin-stimulated degranulation and directed locomotion. (Blood. 2000;95:1911-1917)
Interacting selectively and non-covalently with NFAT (nuclear factor of activated T cells) proteins, a family of transcription factors. NFAT proteins have crucial roles in the development and function of the immune system.
Interacting selectively and non-covalently with any protein or protein complex (a complex of two or more proteins that may include other nonprotein molecules).
Evidence
1:
Inferred from Physical InteractionIntAct
J. Biol. Chem. 273, 29661-29671 (1998)[PubMed:9792677]
A novel ribosomal S6 kinase (RSK) family member, RSK-B, was identified in a p38alphaMAPK-baited intracellular interaction screen. RSK-B presents two catalytic domains typical for the RSK family. The protein kinase C-like N-terminal and the calcium/calmodulin kinase-like C-terminal domains both contain conserved ATP-binding and activation consensus sequences. RSK-B is a p38alphaMAPK substrate, and activated by p38alphaMAPK and, more weakly, by ERK1. RSK-B phosphorylates the cAMP response element-binding protein (CREB) and c-Fos peptides. In intracellular assays, RSK-B drives cAMP response element- and AP1-dependent reporter expression. RSK-B locates to the cell nucleus and co-translocates p38alphaMAPK. In conclusion, RSK-B is a novel CREB kinase under dominant p38alphaMAPK control, also phosphorylating additional substrates.
Evidence
2:
Inferred from Physical InteractionIntAct
The phosphatidylinositol 3-kinase-mammalian target of rapamycin (PI3K-mTOR) pathway plays pivotal roles in cell survival, growth, and proliferation downstream of growth factors. Its perturbations are associated with cancer progression, type 2 diabetes, and neurological disorders. To better understand the mechanisms of action and regulation of this pathway, we initiated a large scale yeast two-hybrid screen for 33 components of the PI3K-mTOR pathway. Identification of 67 new interactions was followed by validation by co-affinity purification and exhaustive literature curation of existing information. We provide a nearly complete, functionally annotated interactome of 802 interactions for the PI3K-mTOR pathway. Our screen revealed a predominant place for glycogen synthase kinase-3 (GSK3) A and B and the AMP-activated protein kinase. In particular, we identified the deformed epidermal autoregulatory factor-1 (DEAF1) transcription factor as an interactor and in vitro substrate of GSK3A and GSK3B. Moreover, GSK3 inhibitors increased DEAF1 transcriptional activity on the 5-HT1A serotonin receptor promoter. We propose that DEAF1 may represent a therapeutic target of lithium and other GSK3 inhibitors used in bipolar disease and depression.
Evidence
3:
Inferred from Physical InteractionIntAct
Systematic mapping of protein-protein interactions, or 'interactome' mapping, was initiated in model organisms, starting with defined biological processes and then expanding to the scale of the proteome. Although far from complete, such maps have revealed global topological and dynamic features of interactome networks that relate to known biological properties, suggesting that a human interactome map will provide insight into development and disease mechanisms at a systems level. Here we describe an initial version of a proteome-scale map of human binary protein-protein interactions. Using a stringent, high-throughput yeast two-hybrid system, we tested pairwise interactions among the products of approximately 8,100 currently available Gateway-cloned open reading frames and detected approximately 2,800 interactions. This data set, called CCSB-HI1, has a verification rate of approximately 78% as revealed by an independent co-affinity purification assay, and correlates significantly with other biological attributes. The CCSB-HI1 data set increases by approximately 70% the set of available binary interactions within the tested space and reveals more than 300 new connections to over 100 disease-associated proteins. This work represents an important step towards a systematic and comprehensive human interactome project.
Evidence
4:
Inferred from Physical InteractionBHF-UCL
Changes in the environment of a cell precipitate extracellular signals and sequential cascades of protein modification and elicit nuclear transcriptional responses. However, the functional links between intracellular signaling-dependent gene regulation and epigenetic regulation by chromatin-modifying proteins within the nucleus are largely unknown. Here, we describe novel epigenetic regulation by MAPK cascades that modulate formation of an ATP-dependent chromatin remodeling complex, WINAC (WSTF Including Nucleosome Assembly Complex), an SWI/SNF-type complex containing Williams syndrome transcription factor (WSTF). WSTF, a specific component of two chromatin remodeling complexes (SWI/SNF-type WINAC and ISWI-type WICH), was phosphorylated by the stimulation of MAPK cascades in vitro and in vivo. Ser-158 residue in the WAC (WSTF/Acf1/cbpq46) domain, located close to the N terminus of WSTF, was identified as a major phosphorylation target. Using biochemical analysis of a WSTF mutant (WSTF-S158A) stably expressing cell line, the phosphorylation of this residue (Ser-158) was found to be essential for maintaining the association between WSTF and core BAF complex components, thereby maintaining the ATPase activity of WINAC. WINAC-dependent transcriptional regulation of vitamin D receptor was consequently impaired by this WSTF mutation, but the recovery from DNA damage mediated by WICH was not impaired. Our results suggest that WSTF serves as a nuclear sensor of the extracellular signals to fine-tune the chromatin remodeling activity of WINAC. WINAC mediates a previously unknown MAPK-dependent step in epigenetic regulation, and this MAPK-dependent switching mechanism between the two functionally distinct WSTF-containing complexes might underlie the diverse functions of WSTF in various nuclear events.
Evidence
5:
Inferred from Physical InteractionIntAct
Mitogen-activated protein kinase (MAPK) pathways form the backbone of signal transduction in the mammalian cell. Here we applied a systematic experimental and computational approach to map 2,269 interactions between human MAPK-related proteins and other cellular machinery and to assemble these data into functional modules. Multiple lines of evidence including conservation with yeast supported a core network of 641 interactions. Using small interfering RNA knockdowns, we observed that approximately one-third of MAPK-interacting proteins modulated MAPK-mediated signaling. We uncovered the Na-H exchanger NHE1 as a potential MAPK scaffold, found links between HSP90 chaperones and MAPK pathways and identified MUC12 as the human analog to the yeast signaling mucin Msb2. This study makes available a large resource of MAPK interactions and clone libraries, and it illustrates a methodology for probing signaling networks based on functional refinement of experimentally derived protein-interaction maps.
Evidence
6:
Inferred from Physical InteractionIntAct
Proteome-scale protein interaction maps are available for many organisms, ranging from bacteria, yeast, worms and flies to humans. These maps provide substantial new insights into systems biology, disease research and drug discovery. However, only a small fraction of the total number of human protein-protein interactions has been identified. In this study, we map the interactions of an unbiased selection of 5026 human liver expression proteins by yeast two-hybrid technology and establish a human liver protein interaction network (HLPN) composed of 3484 interactions among 2582 proteins. The data set has a validation rate of over 72% as determined by three independent biochemical or cellular assays. The network includes metabolic enzymes and liver-specific, liver-phenotype and liver-disease proteins that are individually critical for the maintenance of liver functions. The liver enriched proteins had significantly different topological properties and increased our understanding of the functional relationships among proteins in a liver-specific manner. Our data represent the first comprehensive description of a HLPN, which could be a valuable tool for understanding the functioning of the protein interaction network of the human liver.
Evidence
7:
Inferred from Physical InteractionIntAct
The p38 signaling pathway is activated in response to cell stress and induces production of proinflammatory cytokines. P38alpha is phosphorylated and activated in response to cell stress by MKK3 and MKK6 and in turn phosphorylates a number of substrates, including MAPKAP kinase 2 (MK2). We have determined the crystal structure of the unphosphorylated p38alpha-MK2 heterodimer. The C-terminal regulatory domain of MK2 binds in the docking groove of p38alpha, and the ATP-binding sites of both kinases are at the heterodimer interface. The conformation suggests an extra mechanism in addition to the regulation of the p38alpha and MK2 phosphorylation states that prevents phosphorylation of substrates in the absence of cell stress. Addition of constitutively active MKK6-DD results in rapid phosphorylation of the p38alpha-MK2 heterodimer.
Evidence
8:
Inferred from Physical InteractionIntAct
During vertebrate gastrulation, an epithelial to mesenchymal transition (EMT) is necessary for migration of mesoderm from the primitive streak. We demonstrate that p38 MAP kinase and a p38-interacting protein (p38IP) are critically required for downregulation of E-cadherin during gastrulation. In an ENU-mutagenesis screen we identified the droopy eye (drey) mutation, which affects splicing of p38IP. p38IP(drey) mutant embryos display incompletely penetrant defects in neural tube closure, eye development, and gastrulation. A stronger allele (p38IP(RRK)) exhibits gastrulation defects in which mesoderm migration is defective due to deficiency in E-cadherin protein downregulation in the primitive streak. We show that p38IP binds directly to p38 and is required for p38 activation in vivo. Moreover, both p38 and p38IP are required for E-cadherin downregulation during gastrulation. Finally, p38 regulates E-cadherin protein expression downstream from NCK-interacting kinase (NIK) and independently of the regulation of transcription by Fibroblast Growth Factor (Fgf) signaling and Snail.
Evidence
9:
Inferred from Physical InteractionIntAct
The retinoblastoma protein (Rb) inhibits both cell division and apoptosis, but the mechanism by which Rb alternatively regulates these divergent outcomes remains poorly understood. Cyclin-dependent kinases (Cdks) promote cell division by phosphorylating and reversibly inactivating Rb by a hierarchical series of phosphorylation events and sequential conformational changes. The stress-regulated mitogen-activated protein kinase p38 also phosphorylates Rb, but it does so in a cell cycle-independent manner that is associated with apoptosis rather than with cell division. Here, we show that p38 phosphorylates Rb by a novel mechanism that is distinct from that of Cdks. p38 bypasses the cell cycle-associated hierarchical phosphorylation and directly phosphorylates Rb on Ser567, which is not phosphorylated during the normal cell cycle. Phosphorylation by p38, but not Cdks, triggers an interaction between Rb and the human homolog of murine double minute 2 (Hdm2), leading to degradation of Rb, release of E2F1 and cell death. These findings provide a mechanistic explanation as to how Rb regulates cell division and apoptosis through different kinases, and reveal how Hdm2 may functionally link the tumor suppressors Rb and p53.
Following genotoxic stress, cells activate a complex kinase-based signaling network to arrest the cell cycle and initiate DNA repair. p53-defective tumor cells rewire their checkpoint response and become dependent on the p38/MK2 pathway for survival after DNA damage, despite a functional ATR-Chk1 pathway. We used functional genetics to dissect the contributions of Chk1 and MK2 to checkpoint control. We show that nuclear Chk1 activity is essential to establish a G(2)/M checkpoint, while cytoplasmic MK2 activity is critical for prolonged checkpoint maintenance through a process of posttranscriptional mRNA stabilization. Following DNA damage, the p38/MK2 complex relocalizes from nucleus to cytoplasm where MK2 phosphorylates hnRNPA0, to stabilize Gadd45α mRNA, while p38 phosphorylates and releases the translational inhibitor TIAR. In addition, MK2 phosphorylates PARN, blocking Gadd45α mRNA degradation. Gadd45α functions within a positive feedback loop, sustaining the MK2-dependent cytoplasmic sequestration of Cdc25B/C to block mitotic entry in the presence of unrepaired DNA damage. Our findings demonstrate a critical role for the MK2 pathway in the posttranscriptional regulation of gene expression as part of the DNA damage response in cancer cells.
A programmed cell death process which begins when a cell receives an internal (e.g. DNA damage) or external signal (e.g. an extracellular death ligand), and proceeds through a series of biochemical events (signaling pathways) which typically lead to rounding-up of the cell, retraction of pseudopodes, reduction of cellular volume (pyknosis), chromatin condensation, nuclear fragmentation (karyorrhexis), plasma membrane blebbing and fragmentation of the cell into apoptotic bodies. The process ends when the cell has died. The process is divided into a signaling pathway phase, and an execution phase, which is triggered by the former.
A series of molecular signals initiated by activation of a receptor on the surface of a cell. The pathway begins with binding of an extracellular ligand to a cell surface receptor, or for receptors that signal in the absence of a ligand, by ligand-withdrawal or the activity of a constitutively active receptor. The pathway ends with regulation of a downstream cellular process, e.g. transcription.
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 was assessed using Boyden microchambers. Eotaxin (10(-11) to 10(-7) mol/L) induced concentration-dependent phosphorylation of ERK2 and p38. Phosphorylation was detectable after 30 seconds, peaked at about 1 minute, and returned to baseline after 2 to 5 minutes. Phosphorylation of JNK above baseline could not be detected. The kinase activity of ERK2 and p38 paralleled phosphorylation. PD980 59, an inhibitor of the ERK2-activating enzyme MEK (MAP ERK kinase), blocked phosphorylation of ERK2 in a concentration-dependent manner. The functional relevance of ERK2 and p38 was studied using PD98 059 and the p38 inhibitor SB202 190. PD98 059 and SB202 190 both caused inhibition of eotaxin-induced ECP release and chemotaxis. We conclude that eotaxin induces a rapid concentration-dependent activation of ERK2 and p38 in eosinophils and that the activation of these MAP kinases is required for eotaxin-stimulated degranulation and directed locomotion. (Blood. 2000;95:1911-1917)
Activation of alpha1B-adrenergic receptors ((alpha1B)AR) by phenylephrine (PE) induces scattering of HepG2 cells stably transfected with the (alpha1B)AR (TFG2 cells). Scattering was also observed after stimulation of TFG2 cells with phorbol myristate acetate (PMA) but not with hepatocyte growth factor/scatter factor, epidermal growth factor, or insulin. PMA but not phenylephrine rapidly activated PKCalpha in TFG2 cells, and the highly selective PKC inhibitor bisindolylmaleimide (GFX) completely abolished PMA-induced but not PE-induced scattering. PE rapidly activated p44/42 mitogen-activated protein kinase (MAPK), p38 MAPK, c-Jun N-terminal kinase (JNK), and AP1 (c-fos/c-jun). Selective blockade of p42/44 MAPK activity by PD98059 or by transfection of a MEK1 dominant negative adenovirus significantly inhibited the PE-induced scattering of TFG2 cells. Selective inhibition of p38 MAPK by SB203850 or SB202190 also blocked PE-induced scattering, whereas treatment of TFG2 cells with the PI3 kinase inhibitors LY294002 or wortmannin did not inhibit PE-induced scattering. Blocking JNK activation with a dominant negative mutant of JNK or blocking AP1 activation with a dominant negative mutant of c-jun (TAM67) significantly inhibited PE-induced cell scattering. These data indicate that PE-induced scattering of TFG2 cells is mediated by complex mechanisms, including activation of p42/44 MAPK, p38 MAPK, and JNK. Cell spreading has been reported to play important roles in wound repair, tumor invasion, and metastasis. Therefore, catecholamines acting via the (alpha1)AR may modulate these physiological and pathological processes.
Any process that results in a change in state or activity of a cell (in terms of movement, secretion, enzyme production, gene expression, etc.) as a result of a ionizing radiation stimulus. Ionizing radiation is radiation with sufficient energy to remove electrons from atoms and may arise from spontaneous decay of unstable isotopes, resulting in alpha and beta particles and gamma rays. Ionizing radiation also includes X-rays.
Cellular senescence--the permanent arrest of cycling in normally proliferating cells such as fibroblasts--contributes both to age-related loss of mammalian tissue homeostasis and acts as a tumour suppressor mechanism. The pathways leading to establishment of senescence are proving to be more complex than was previously envisaged. Combining in-silico interactome analysis and functional target gene inhibition, stochastic modelling and live cell microscopy, we show here that there exists a dynamic feedback loop that is triggered by a DNA damage response (DDR) and, which after a delay of several days, locks the cell into an actively maintained state of 'deep' cellular senescence. The essential feature of the loop is that long-term activation of the checkpoint gene CDKN1A (p21) induces mitochondrial dysfunction and production of reactive oxygen species (ROS) through serial signalling through GADD45-MAPK14(p38MAPK)-GRB2-TGFBR2-TGFbeta. These ROS in turn replenish short-lived DNA damage foci and maintain an ongoing DDR. We show that this loop is both necessary and sufficient for the stability of growth arrest during the establishment of the senescent phenotype.
Any process that results in a change in state or activity of a cell (in terms of movement, secretion, enzyme production, gene expression, etc.) as a result of a vascular endothelial growth factor stimulus.
Proteomic analysis identified HSP27 phosphorylation as a major change in protein phosphorylation stimulated by Vascular Endothelial Growth Factor (VEGF) in Human Umbilical Vein Endothelial Cells (HUVEC). VEGF-induced HSP27 phosphorylation at serines 15, 78 and 82, but whereas HSP27 phosphorylation induced by H2O2 and TNFalpha was completely blocked by the p38 kinase inhibitor, SB203580, VEGF-stimulated serine 82 phosphorylation was resistant to SB203580 and small interfering(si)RNA-mediated knockdown of p38 kinase and MAPKAPK2. The PKC inhibitor, GF109203X, partially reduced VEGF-induced HSP27 serine 82 phosphorylation, and SB203580 plus GF109203X abolished phosphorylation. VEGF activated Protein Kinase D (PKD) via PKC, and siRNAs targeted to PKD1 and PKD2 inhibited VEGF-induced HSP27 serine 82 phosphorylation. Furthermore recombinant PKD selectively phosphorylated HSP27 at serine 82 in vitro, and PKD2 activated by VEGF in HUVECs also phosphorylated HSP27 selectively at this site. Knockdown of HSP27 and PKDs markedly inhibited VEGF-induced HUVEC migration and tubulogenesis, whereas inhibition of the p38 kinase pathway using either SB203580 or siRNAs against p38alpha or MAPKAPK2, had no significant effect on the chemotactic response to VEGF. These findings identify a novel pathway for VEGF-induced HSP27 serine 82 phosphorylation via PKC-mediated PKD activation and direct phosphorylation of HSP27 by PKD, and show that PKDs and HSP27 play major roles in the angiogenic response to VEGF.
The directed movement of a motile cell or organism, or the directed growth of a cell guided by a specific chemical concentration gradient. Movement may be towards a higher concentration (positive chemotaxis) or towards a lower concentration (negative chemotaxis).
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 was assessed using Boyden microchambers. Eotaxin (10(-11) to 10(-7) mol/L) induced concentration-dependent phosphorylation of ERK2 and p38. Phosphorylation was detectable after 30 seconds, peaked at about 1 minute, and returned to baseline after 2 to 5 minutes. Phosphorylation of JNK above baseline could not be detected. The kinase activity of ERK2 and p38 paralleled phosphorylation. PD980 59, an inhibitor of the ERK2-activating enzyme MEK (MAP ERK kinase), blocked phosphorylation of ERK2 in a concentration-dependent manner. The functional relevance of ERK2 and p38 was studied using PD98 059 and the p38 inhibitor SB202 190. PD98 059 and SB202 190 both caused inhibition of eotaxin-induced ECP release and chemotaxis. We conclude that eotaxin induces a rapid concentration-dependent activation of ERK2 and p38 in eosinophils and that the activation of these MAP kinases is required for eotaxin-stimulated degranulation and directed locomotion. (Blood. 2000;95:1911-1917)
The process in which a chondroblast acquires specialized structural and/or functional features of a chondrocyte. A chondrocyte is a polymorphic cell that forms cartilage.
A cell cycle checkpoint that regulates progression through the cell cycle in response to DNA damage. A DNA damage checkpoint may blocks cell cycle progression (in G1, G2 or metaphase) or slow the rate at which S phase proceeds.
The removal of one or more electrons from a fatty acid, with or without the concomitant removal of a proton or protons, by reaction with an electron-accepting substance, by addition of oxygen or by removal of hydrogen.
The chemical reactions and pathways involving glucose, the aldohexose gluco-hexose. D-glucose is dextrorotatory and is sometimes known as dextrose; it is an important source of energy for living organisms and is found free as well as combined in homo- and hetero-oligosaccharides and polysaccharides.
A series of reactions in which a signal is passed on to downstream proteins within the cell by sequential protein phosphorylation and activation of the cascade components.
The p38 family of mitogen-activated protein kinases is composed of several isoforms. Mxi2 is a splicing variant of p38alpha that harbors a unique carboxy-terminus. Here we show that this sole divergence results in remarkable differences between Mxi2 and p38alpha. Mxi2 is distinctively activated by stress stimuli and potently activated by mitogens. Mxi2 affinity for bona fide p38 substrates is remarkably diminished and Mxi2 activity is largely unaffected by the phosphatase CL100. Also, Mxi2 sensitivity to inhibition by SB203580 is greatly reduced. Interestingly, we show that the p38 C-terminus is involved in conferring sensitivity to this compound. Overall, our results point to the p38 carboxy-terminus as a key determinant of the biochemical properties of this family of kinases.
A series of molecular signals initiated by the binding of a lipopolysaccharide (LPS) to a receptor on the surface of a target cell, and ending with regulation of a downstream cellular process, e.g. transcription. Lipopolysaccharides are major components of the outer membrane of Gram-negative bacteria, making them prime targets for recognition by the immune system.
The process in which a relatively unspecialized monocyte acquires the specialized features of an osteoclast. An osteoclast is a specialized phagocytic cell associated with the absorption and removal of the mineralized matrix of bone tissue.
Proteomic analysis identified HSP27 phosphorylation as a major change in protein phosphorylation stimulated by Vascular Endothelial Growth Factor (VEGF) in Human Umbilical Vein Endothelial Cells (HUVEC). VEGF-induced HSP27 phosphorylation at serines 15, 78 and 82, but whereas HSP27 phosphorylation induced by H2O2 and TNFalpha was completely blocked by the p38 kinase inhibitor, SB203580, VEGF-stimulated serine 82 phosphorylation was resistant to SB203580 and small interfering(si)RNA-mediated knockdown of p38 kinase and MAPKAPK2. The PKC inhibitor, GF109203X, partially reduced VEGF-induced HSP27 serine 82 phosphorylation, and SB203580 plus GF109203X abolished phosphorylation. VEGF activated Protein Kinase D (PKD) via PKC, and siRNAs targeted to PKD1 and PKD2 inhibited VEGF-induced HSP27 serine 82 phosphorylation. Furthermore recombinant PKD selectively phosphorylated HSP27 at serine 82 in vitro, and PKD2 activated by VEGF in HUVECs also phosphorylated HSP27 selectively at this site. Knockdown of HSP27 and PKDs markedly inhibited VEGF-induced HUVEC migration and tubulogenesis, whereas inhibition of the p38 kinase pathway using either SB203580 or siRNAs against p38alpha or MAPKAPK2, had no significant effect on the chemotactic response to VEGF. These findings identify a novel pathway for VEGF-induced HSP27 serine 82 phosphorylation via PKC-mediated PKD activation and direct phosphorylation of HSP27 by PKD, and show that PKDs and HSP27 play major roles in the angiogenic response to VEGF.
Cellular senescence--the permanent arrest of cycling in normally proliferating cells such as fibroblasts--contributes both to age-related loss of mammalian tissue homeostasis and acts as a tumour suppressor mechanism. The pathways leading to establishment of senescence are proving to be more complex than was previously envisaged. Combining in-silico interactome analysis and functional target gene inhibition, stochastic modelling and live cell microscopy, we show here that there exists a dynamic feedback loop that is triggered by a DNA damage response (DDR) and, which after a delay of several days, locks the cell into an actively maintained state of 'deep' cellular senescence. The essential feature of the loop is that long-term activation of the checkpoint gene CDKN1A (p21) induces mitochondrial dysfunction and production of reactive oxygen species (ROS) through serial signalling through GADD45-MAPK14(p38MAPK)-GRB2-TGFBR2-TGFbeta. These ROS in turn replenish short-lived DNA damage foci and maintain an ongoing DDR. We show that this loop is both necessary and sufficient for the stability of growth arrest during the establishment of the senescent phenotype.
Any process that results in a change in state or activity of an organism (in terms of movement, secretion, enzyme production, gene expression, etc.) as a result of a muramyl dipeptide stimulus. Muramyl dipeptide is derived from peptidoglycan.
Any process that results in a change in state or activity of a cell or an organism (in terms of movement, secretion, enzyme production, gene expression, etc.) as a result of a disturbance in organismal or cellular homeostasis, usually, but not necessarily, exogenous (e.g. temperature, humidity, ionizing radiation).
Production of interleukin-1 and tumour necrosis factor from stimulated human monocytes is inhibited by a new series of pyridinyl-imidazole compounds. Using radiolabelled and radio-photoaffinity-labelled chemical probes, the target of these compounds was identified as a pair of closely related mitogen-activated protein kinase homologues, termed CSBPs. Binding of the pyridinyl-imidazole compounds inhibited CSBP kinase activity and could be directly correlated with their ability to inhibit cytokine production, suggesting that the CSBPs are critical for cytokine production.
The cellular process in which a signal is conveyed to trigger a change in the activity or state of a cell. Signal transduction begins with reception of a signal (e.g. a ligand binding to a receptor or receptor activation by a stimulus such as light), or for signal transduction in the absence of ligand, signal-withdrawal or the activity of a constitutively active receptor. Signal transduction ends with regulation of a downstream cellular process, e.g. regulation of transcription or regulation of a metabolic process. Signal transduction covers signaling from receptors located on the surface of the cell and signaling via molecules located within the cell. For signaling between cells, signal transduction is restricted to events at and within the receiving cell.
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 was assessed using Boyden microchambers. Eotaxin (10(-11) to 10(-7) mol/L) induced concentration-dependent phosphorylation of ERK2 and p38. Phosphorylation was detectable after 30 seconds, peaked at about 1 minute, and returned to baseline after 2 to 5 minutes. Phosphorylation of JNK above baseline could not be detected. The kinase activity of ERK2 and p38 paralleled phosphorylation. PD980 59, an inhibitor of the ERK2-activating enzyme MEK (MAP ERK kinase), blocked phosphorylation of ERK2 in a concentration-dependent manner. The functional relevance of ERK2 and p38 was studied using PD98 059 and the p38 inhibitor SB202 190. PD98 059 and SB202 190 both caused inhibition of eotaxin-induced ECP release and chemotaxis. We conclude that eotaxin induces a rapid concentration-dependent activation of ERK2 and p38 in eosinophils and that the activation of these MAP kinases is required for eotaxin-stimulated degranulation and directed locomotion. (Blood. 2000;95:1911-1917)
Cellular senescence--the permanent arrest of cycling in normally proliferating cells such as fibroblasts--contributes both to age-related loss of mammalian tissue homeostasis and acts as a tumour suppressor mechanism. The pathways leading to establishment of senescence are proving to be more complex than was previously envisaged. Combining in-silico interactome analysis and functional target gene inhibition, stochastic modelling and live cell microscopy, we show here that there exists a dynamic feedback loop that is triggered by a DNA damage response (DDR) and, which after a delay of several days, locks the cell into an actively maintained state of 'deep' cellular senescence. The essential feature of the loop is that long-term activation of the checkpoint gene CDKN1A (p21) induces mitochondrial dysfunction and production of reactive oxygen species (ROS) through serial signalling through GADD45-MAPK14(p38MAPK)-GRB2-TGFBR2-TGFbeta. These ROS in turn replenish short-lived DNA damage foci and maintain an ongoing DDR. We show that this loop is both necessary and sufficient for the stability of growth arrest during the establishment of the senescent phenotype.
The developmental sequence of events leading to the formation of adult muscle that occurs in the anima. In vertebrate skeletal muscle the main events are: the fusion of myoblasts to form myotubes that increase in size by further fusion to them of myoblasts, the formation of myofibrils within their cytoplasm and the establishment of functional neuromuscular junctions with motor neurons. At this stage they can be regarded as mature muscle fibers.
Cellular senescence--the permanent arrest of cycling in normally proliferating cells such as fibroblasts--contributes both to age-related loss of mammalian tissue homeostasis and acts as a tumour suppressor mechanism. The pathways leading to establishment of senescence are proving to be more complex than was previously envisaged. Combining in-silico interactome analysis and functional target gene inhibition, stochastic modelling and live cell microscopy, we show here that there exists a dynamic feedback loop that is triggered by a DNA damage response (DDR) and, which after a delay of several days, locks the cell into an actively maintained state of 'deep' cellular senescence. The essential feature of the loop is that long-term activation of the checkpoint gene CDKN1A (p21) induces mitochondrial dysfunction and production of reactive oxygen species (ROS) through serial signalling through GADD45-MAPK14(p38MAPK)-GRB2-TGFBR2-TGFbeta. These ROS in turn replenish short-lived DNA damage foci and maintain an ongoing DDR. We show that this loop is both necessary and sufficient for the stability of growth arrest during the establishment of the senescent phenotype.
The process in which a relatively unspecialized cell acquires specialized features of a striated muscle cell; striated muscle fibers are divided by transverse bands into striations, and cardiac and voluntary muscle are types of striated muscle.
Any series of molecular signals initiated by the binding of an extracellular ligand to a vascular endothelial growth factor receptor (VEGFR) located on the surface of the receiving cell, and ending with regulation of a downstream cellular process, e.g. transcription.
Proteomic analysis identified HSP27 phosphorylation as a major change in protein phosphorylation stimulated by Vascular Endothelial Growth Factor (VEGF) in Human Umbilical Vein Endothelial Cells (HUVEC). VEGF-induced HSP27 phosphorylation at serines 15, 78 and 82, but whereas HSP27 phosphorylation induced by H2O2 and TNFalpha was completely blocked by the p38 kinase inhibitor, SB203580, VEGF-stimulated serine 82 phosphorylation was resistant to SB203580 and small interfering(si)RNA-mediated knockdown of p38 kinase and MAPKAPK2. The PKC inhibitor, GF109203X, partially reduced VEGF-induced HSP27 serine 82 phosphorylation, and SB203580 plus GF109203X abolished phosphorylation. VEGF activated Protein Kinase D (PKD) via PKC, and siRNAs targeted to PKD1 and PKD2 inhibited VEGF-induced HSP27 serine 82 phosphorylation. Furthermore recombinant PKD selectively phosphorylated HSP27 at serine 82 in vitro, and PKD2 activated by VEGF in HUVECs also phosphorylated HSP27 selectively at this site. Knockdown of HSP27 and PKDs markedly inhibited VEGF-induced HUVEC migration and tubulogenesis, whereas inhibition of the p38 kinase pathway using either SB203580 or siRNAs against p38alpha or MAPKAPK2, had no significant effect on the chemotactic response to VEGF. These findings identify a novel pathway for VEGF-induced HSP27 serine 82 phosphorylation via PKC-mediated PKD activation and direct phosphorylation of HSP27 by PKD, and show that PKDs and HSP27 play major roles in the angiogenic response to VEGF.
The p38 family of mitogen-activated protein kinases is composed of several isoforms. Mxi2 is a splicing variant of p38alpha that harbors a unique carboxy-terminus. Here we show that this sole divergence results in remarkable differences between Mxi2 and p38alpha. Mxi2 is distinctively activated by stress stimuli and potently activated by mitogens. Mxi2 affinity for bona fide p38 substrates is remarkably diminished and Mxi2 activity is largely unaffected by the phosphatase CL100. Also, Mxi2 sensitivity to inhibition by SB203580 is greatly reduced. Interestingly, we show that the p38 C-terminus is involved in conferring sensitivity to this compound. Overall, our results point to the p38 carboxy-terminus as a key determinant of the biochemical properties of this family of kinases.
Activated by cell stresses such as DNA damage, heat shock, osmotic shock, anisomycin and sodium arsenite, as well as pro-inflammatory stimuli such as bacterial lipopolysaccharide (LPS) and interleukin-1. Activation occurs through dual phosphorylation of Thr-180 and Tyr-182 by either of two dual specificity kinases, MAP2K3/MKK3 or MAP2K6/MKK6, and potentially also MAP2K4/MKK4, as well as by TAB1-mediated autophosphorylation. MAPK14 phosphorylated on both Thr-180 and Tyr-182 is 10-20-fold more active than MAPK14 phosphorylated only on Thr-180, whereas MAPK14 phosphorylated on Tyr-182 alone is inactive. Whereas Thr-180 is necessary for catalysis, Tyr-182 may be required for auto-activation and substrate recognition. Phosphorylated at Tyr-323 by ZAP70 in an alternative activation pathway in response to TCR signaling in T-cells. This alternative pathway is inhibited by GADD45A. Inhibited by dual specificity phosphatases, such as DUSP1, DUSP10, and DUSP16. Specifically inhibited by the binding of pyridinyl-imidazole compounds, which are cytokine-suppressive anti-inflammatory drugs (CSAID).
Signaling-responsive MAP kinases (MAPKs) are key in mediating immune responses and are activated through the phosphorylation of a Thr-X-Tyr motif by upstream MAPK kinases. Here we show that T cells stimulated through the T cell receptor (TCR) used an alternative mechanism in which p38 was phosphorylated on Tyr323 and subsequently autophosphorylated residues Thr180 and Tyr182. This required the TCR-proximal tyrosine kinase Zap70 but not the adaptor protein LAT, which was required for activation of extracellular signal-regulated protein kinase MAPKs. TCR activation of p38 lacking Tyr323 was diminished, and blocking of p38 activity prevented p38 dual phosphorylation in normal T cells but not in B cells. Thus, phosphorylation of Tyr323 dependent on the tyrosine kinase Lck and mediated by Zap70 serves as an important mechanism for TCR activation of p38 in T cells.
One of the major families of the mitogen-activated kinases (MAPK), p38, has been shown to transduce extracellular stress stimuli into cellular responses. Among them, p38 alpha is the best characterized isoform and many biological phenomena, especially in the inflammatory responses, were attributed to the specific inhibitor-sensitive isoforms, namely p38 alpha and p38 beta. However, the roles played by each member are still unclear. Here, we report the identification of a new splice variant of p38 alpha, Exip (for exon skip), by RT-PCR using mRNA derived from a renal tumor cell line, OS-RC-2. Exip is predicted to encode a 307-amino-acid protein and the absence of exons 10, 11, and 11' results in the shift of the reading frame at the exon 9-12 junction to produce a unique 53-amino-acid C-terminus. The expression of mRNA was barely observed in cultured cells tested, but substantial amounts of mRNA were detected in mouse tissues. Unlike p38 alpha, Exip lost a common docking domain well conserved in major MAPK families for their specific interactions with upstream kinases or downstream substrates. Even though Exip is not phosphorylated at conserved TGY motif by p38-activating treatments, such as an osmotic shock or coexpression with a constitutive active form of MKK6 in HeLa cells, Exip can induce an earlier onset of apoptosis in HeLa cells. These results indicate that Exip has unique properties as a member of p38 alpha and may play role(s) in the signal transduction pathway(s) different from those of p38 alpha.
Two new classes of diphenylether inhibitors of p38alpha MAP kinase are described. Both chemical classes are based on a common diphenylether core that is identified by simulated fragment annealing as one of the most favored chemotypes within a prominent hydrophobic pocket of the p38alpha ATP-binding site. In the fully elaborated molecules, the diphenylether moiety acts as an anchor occupying the deep pocket, while polar extensions make specific interactions with either the adenine binding site or the phosphate binding site of ATP. The synthesis, crystallographic analysis, and biological activity of these p38alpha inhibitors are discussed.
The p38 mitogen-activated protein (MAP) kinase signal transduction pathway is activated by proinflammatory cytokines and environmental stress. The detection of p38 MAP kinase in the nucleus of activated cells suggests that p38 MAP kinase can mediate signaling to the nucleus. To test this hypothesis, we constructed expression vectors for activated MKK3 and MKK6, two MAP kinase kinases that phosphorylate and activate p38 MAP kinase. Expression of activated MKK3 and MKK6 in cultured cells caused a selective increase in p38 MAP kinase activity. Cotransfection experiments demonstrated that p38 MAP kinase activation causes increased reporter gene expression mediated by the transcription factors ATF2 and Elk-1. These data demonstrate that the nucleus is one target of the p38 MAP kinase signal transduction pathway.
J. Biol. Chem. 274, 19949-19956 (1999)[PubMed:10391943]
A group of dual specificity protein phosphatases negatively regulates members of the mitogen-activated protein kinase (MAPK) superfamily, which consists of three major subfamilies, MAPK/extracellular signal-regulated kinase (ERK), stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK), and p38. Nine members of this group of dual specificity phosphatases have previously been cloned. They show distinct substrate specificities for MAPKs, different tissue distribution and subcellular localization, and different modes of inducibility of their expression by extracellular stimuli. Here we have cloned and characterized a novel dual specificity phosphatase, which we have designated MKP-5. MKP-5 is a protein of 482 amino acids with a calculated molecular mass of 52.6 kDa and consists of 150 N-terminal amino acids of unknown function, two Cdc25 homology 2 regions in the middle, and a C-terminal catalytic domain. MKP-5 binds to p38 and SAPK/JNK, but not to MAPK/ERK, and inactivates p38 and SAPK/JNK, but not MAPK/ERK. p38 is a preferred substrate. The subcellular localization of MKP-5 is unique; it is present evenly in both the cytoplasm and the nucleus. MKP-5 mRNA is widely expressed in various tissues and organs, and its expression in cultured cells is elevated by stress stimuli. These results suggest that MKP-5 is a novel type of dual specificity phosphatase specific for p38 and SAPK/JNK.
Bioorg. Med. Chem. Lett. 13, 277-280 (2003)[PubMed:12482439]
The development of potent, orally bioavailable (in rat) and selective dihydroquinazolinone inhibitors of p38alpha MAP kinase is described. These analogues are hybrids of a pyridinylimidazole p38alpha inhibitor reported by Merck Research Laboratories and VX-745. Optimization of the C-5 phenyl and the C-7 piperidinyl substituents led to the identification of 15i which gave excellent suppression of TNF-alpha production in LPS-stimulated whole blood (IC(50)=10nM) and good oral exposure in rats (F=68%, AUCn PO=0.58 microM h).
The RING finger ubiquitin ligase Siah2 controls the stability of various substrates involved in stress and hypoxia responses, including the PHD3, which controls the stability of HIF-1alpha. In the present study we determined the role of Siah2 phosphorylation in the regulation of its activity toward PHD3. We show that Siah2 is subject to phosphorylation by p38 MAPK, which increases Siah2-mediated degradation of PHD3. Consistent with these findings, MKK3/MKK6 double-deficient cells, which cannot activate p38 kinases, exhibit impaired Siah2-dependent degradation of PHD3. Phosphopeptide mapping identified T24 and S29 as the primary phospho-acceptor sites. Phospho-mutant forms of Siah2 (S29A or T24A/S29A) exhibit impaired degradation of PHD3, particularly after hypoxia. Conversely, a phospho-mimic form of Siah2 (T24E/S29D) exhibits stronger degradation of PHD3, compared with wild type Siah2. Whereas phospho-mutant Siah2 exhibits weaker association with PHD3, phospho-mimic Siah2 associates as well as wild type and is localized within the perinuclear region, suggesting that phosphorylation of Siah2 affects its subcellular localization and, consequently, the degree of its association with PHD3. In all, our findings reveal the phosphorylation of Siah2 by p38 and the implications of such phosphorylation for Siah2 activity toward PHD3.
Phosphorylation of mitogen-activated protein kinases (MAPKs) on specific tyrosine and threonine sites by MAP kinase kinases (MAPKKs) is thought to be the sole activation mechanism. Here, we report an unexpected activation mechanism for p38alpha MAPK that does not involve the prototypic kinase cascade. Rather it depends on interaction of p38alpha with TAB1 [transforming growth factor-beta-activated protein kinase 1 (TAK1)-binding protein 1] leading to autophosphorylation and activation of p38alpha. We detected formation of a TRAF6-TAB1-p38alpha complex and showed stimulus-specific TAB1-dependent and TAB1-independent p38alpha activation. These findings suggest that alternative activation pathways contribute to the biological responses of p38alpha to various stimuli.
Novel potent trisubstituted pyridazine inhibitors of p38 MAP (mitogen activated protein) kinase are described that have activity in both cell-based assays of cytokine release and animal models of rheumatoid arthritis. They demonstrated potent inhibition of LPS-induced TNF-alpha production in mice and exhibited good efficacy in the rat collagen induced arthritis model.
The p38 family of mitogen-activated protein kinases is composed of several isoforms. Mxi2 is a splicing variant of p38alpha that harbors a unique carboxy-terminus. Here we show that this sole divergence results in remarkable differences between Mxi2 and p38alpha. Mxi2 is distinctively activated by stress stimuli and potently activated by mitogens. Mxi2 affinity for bona fide p38 substrates is remarkably diminished and Mxi2 activity is largely unaffected by the phosphatase CL100. Also, Mxi2 sensitivity to inhibition by SB203580 is greatly reduced. Interestingly, we show that the p38 C-terminus is involved in conferring sensitivity to this compound. Overall, our results point to the p38 carboxy-terminus as a key determinant of the biochemical properties of this family of kinases.
Mitogen-activated protein (MAP) kinase phosphatase 1 (MKP-1/CL100) is an inducible nuclear dual specificity protein phosphatase that can dephosphorylate and inactivate both mitogen- and stress-activated protein kinases in vitro and in vivo. However, the molecular mechanism responsible for the substrate selectivity of MKP-1 is unknown. In addition, it has been suggested that the signal transducers and activators of transcription 1 (STAT1) transcription factor is a physiological non-MAP kinase substrate for MKP-1. We have used the yeast two-hybrid assay to demonstrate that MKP-1 is able to interact selectively with the extracellular signal-regulated kinase 1/2 (ERK1/2), p38alpha, and c-Jun NH(2)-terminal kinase (JNK) MAP kinase isoforms. Furthermore, this binding is accompanied by catalytic activation of recombinant MKP-1 protein in vitro, and these end points show an absolute correlation with MKP-1 substrate selectivity in vivo. In contrast, MKP-1 does not interact with STAT1. Recombinant STAT1 does not cause catalytic activation of MKP-1; nor does MKP-1 block tyrosine phosphorylation of STAT1 in vivo. Both binding and catalytic activation of MKP-1 are abrogated by mutation of a conserved docking site in ERK2, p38alpha, and JNK1 MAP kinases. Within MKP-1, MAP kinase binding is mediated by the amino-terminal noncatalytic domain of the protein. However, mutation of a conserved cluster of positively charged residues within this domain abolishes the binding and activation of MKP-1 by ERK2 and p38alpha but not JNK1, indicating that there are distinct binding determinants for these MAP kinase isoforms. We conclude that the substrate selectivity of MKP-1 is determined by specific protein-protein interactions coupled with catalytic activation of the phosphatase and that these interactions are restricted to members of the MAP kinase family of enzymes.
J. Biol. Chem. 273, 1741-1748 (1998)[PubMed:9430721]
The cellular response to treatment with proinflammatory cytokines or exposure to environmental stress is mediated, in part, by the p38 group of mitogen-activated protein (MAP) kinases. We report the molecular cloning of a novel isoform of p38 MAP kinase, p38 beta 2. This p38 MAP kinase, like p38 alpha, is inhibited by the pyridinyl imidazole drug SB203580. The p38 MAP kinase kinase MKK6 is identified as a common activator of p38 alpha, p38 beta 2, and p38 gamma MAP kinase isoforms, while MKK3 activates only p38 alpha and p38 gamma MAP kinase isoforms. The MKK3 and MKK6 signal transduction pathways are therefore coupled to distinct, but overlapping, groups of p38 MAP kinases.
Mitogen-activated protein (MAP) kinase p38 alpha is activated in response to environmental stress and cytokines, and plays a significant role in inflammatory responses. For these reasons, it is an important target for the treatment of a wide range of inflammatory and autoimmune diseases. The crystals of p38 alpha that we obtained by published procedures were usually small, quite mosaic, and difficult to reproduce and thus posed a difficulty for the intensive high-resolution studies required for a structure-guided drug discovery approach. Based on crystallographic and biochemical evidences, we prepared a single point mutation of a surface cysteine (C162S) and found that it prevents aggregation and improves the homogeneity and stability of the enzyme. This mutation also facilitates the crystallization process and increases the diffracting power of p38 alpha crystals. Surprisingly, we found that the mutation induces a change in the conformation of a nearby surface loop resulting in stronger lattice interactions, consistent with the improved crystal quality. The mutant protein, because of its improved stability and strengthened lattice interactions, thus provides a significantly improved reagent for use in structure-based drug design for this important disease target.
The p38 MAP kinase plays a crucial role in regulating the production of proinflammatory cytokines, such as tumor necrosis factor and interleukin-1. Blocking this kinase may offer an effective therapy for treating many inflammatory diseases. Here we report a new allosteric binding site for a diaryl urea class of highly potent and selective inhibitors against human p38 MAP kinase. The formation of this binding site requires a large conformational change not observed previously for any of the protein Ser/Thr kinases. This change is in the highly conserved Asp-Phe-Gly motif within the active site of the kinase. Solution studies demonstrate that this class of compounds has slow binding kinetics, consistent with the requirement for conformational change. Improving interactions in this allosteric pocket, as well as establishing binding interactions in the ATP pocket, enhanced the affinity of the inhibitors by 12,000-fold. One of the most potent compounds in this series, BIRB 796, has picomolar affinity for the kinase and low nanomolar inhibitory activity in cell culture.
Inhibition of the biosynthesis of proinflammatory cytokines such as tumor necrosis factor and interleukin-1 via p38 has been an approach toward the development of a disease modifying agent for the treatment of chronic inflammation and autoimmune diseases. The development of a new core structure of p38 inhibitors, 3-(4-fluorophenyl)-2-(pyridin-4-yl)-1H-pyrrolo[3,2-b] pyridine, is described. X-ray crystallographic data of the lead bound to the active site of p38 was used to guide the optimization of the series. Specific focus was placed on modulating the physical properties of the core while maintaining potent inhibition of p38. These efforts identified 42c as a potent inhibitor of p38, which also possessed the required physical properties worthy of advanced studies.
The p38 MAP kinase (MAPK) is phosphorylated and activated by upstream MAPK kinases. T cells have an alternative pathway in which T cell receptor-activated tyrosine kinase Zap70 phosphorylates p38 on Tyr323. Mice lacking Gadd45alpha, a small p38-binding molecule, develop a lupus-like autoimmune disease. Here we show that resting T cells but not B cells from Gadd45a(-/-) mice had spontaneously increased p38 activity in the absence of 'upstream' MAPK kinase activation. The p38 from resting Gadd45a(-/-) T cells was spontaneously phosphorylated on Tyr323, and its activity was specifically inhibited by recombinant Gadd45alpha in vitro. Thus, constitutive activation of T cell p38 through the alternative pathway is prevented by Gadd45alpha, the absence of which results in p38 activation, T cell hyperproliferation and autoimmunity.
Mitogen-activated protein kinases (MAPKs) are inactivated via dephosphorylation of either the threonine or tyrosine residue or both in the P-loop catalyzed by protein phosphatases which include serine/threonine phosphatases, tyrosine phosphatases, and dual specificity phosphatases. Nine members of the dual specificity phosphatases specific for MAPKs, termed MKPs, have been reported. Each member has its own substrate specificity, tissue distribution, and subcellular localization. In this study, we have cloned and characterized a novel MKP, designated MKP-7. MKP-7 is most similar to hVH5, a member of previously known MKPs, in the primary structure. MKP-7 is predominantly localized in the cytoplasm when expressed in cultured cells, whereas hVH5 is both in the nucleus and the cytoplasm. MKP-7 binds to and inactivates p38 MAPK and JNK/SAPK, but not ERK. Furthermore, we have found that MKPs have the substrate specificity toward the isoforms of the p38 family (alpha, beta, gamma, and delta). MKP-7 binds to and inactivates p38 alpha and -beta, but not gamma or delta. MKP-5 and CL100/MKP-1 also bind to p38 alpha and -beta, but not gamma or delta. Finally, we propose a tentative classification of MKPs based on the sequence characteristics of their MAPK-docking site.
J. Biol. Chem. 270, 7420-7426 (1995)[PubMed:7535770]
Protein kinases activated by dual phosphorylation on Tyr and Thr (MAP kinases) can be grouped into two major classes: ERK and JNK. The ERK group regulates multiple targets in response to growth factors via a Ras-dependent mechanism. In contrast, JNK activates the transcription factor c-Jun in response to pro-inflammatory cytokines and exposure of cells to several forms of environmental stress. Recently, a novel mammalian protein kinase (p38) that shares sequence similarity with mitogen-activated protein (MAP) kinases was identified. Here, we demonstrate that p38, like JNK, is activated by treatment of cells with pro-inflammatory cytokines and environmental stress. The mechanism of p38 activation is mediated by dual phosphorylation on Thr-180 and Tyr-182. Immunofluorescence microscopy demonstrated that p38 MAP kinase is present in both the nucleus and cytoplasm of activated cells. Together, these data establish that p38 is a member of the mammalian MAP kinase group.
Two new classes of diphenylether inhibitors of p38alpha MAP kinase are described. Both chemical classes are based on a common diphenylether core that is identified by simulated fragment annealing as one of the most favored chemotypes within a prominent hydrophobic pocket of the p38alpha ATP-binding site. In the fully elaborated molecules, the diphenylether moiety acts as an anchor occupying the deep pocket, while polar extensions make specific interactions with either the adenine binding site or the phosphate binding site of ATP. The synthesis, crystallographic analysis, and biological activity of these p38alpha inhibitors are discussed.
Signaling-responsive MAP kinases (MAPKs) are key in mediating immune responses and are activated through the phosphorylation of a Thr-X-Tyr motif by upstream MAPK kinases. Here we show that T cells stimulated through the T cell receptor (TCR) used an alternative mechanism in which p38 was phosphorylated on Tyr323 and subsequently autophosphorylated residues Thr180 and Tyr182. This required the TCR-proximal tyrosine kinase Zap70 but not the adaptor protein LAT, which was required for activation of extracellular signal-regulated protein kinase MAPKs. TCR activation of p38 lacking Tyr323 was diminished, and blocking of p38 activity prevented p38 dual phosphorylation in normal T cells but not in B cells. Thus, phosphorylation of Tyr323 dependent on the tyrosine kinase Lck and mediated by Zap70 serves as an important mechanism for TCR activation of p38 in T cells.
Inhibition of the biosynthesis of proinflammatory cytokines such as tumor necrosis factor and interleukin-1 via p38 has been an approach toward the development of a disease modifying agent for the treatment of chronic inflammation and autoimmune diseases. The development of a new core structure of p38 inhibitors, 3-(4-fluorophenyl)-2-(pyridin-4-yl)-1H-pyrrolo[3,2-b] pyridine, is described. X-ray crystallographic data of the lead bound to the active site of p38 was used to guide the optimization of the series. Specific focus was placed on modulating the physical properties of the core while maintaining potent inhibition of p38. These efforts identified 42c as a potent inhibitor of p38, which also possessed the required physical properties worthy of advanced studies.
Mitogen-activated protein (MAP) kinase phosphatase 1 (MKP-1/CL100) is an inducible nuclear dual specificity protein phosphatase that can dephosphorylate and inactivate both mitogen- and stress-activated protein kinases in vitro and in vivo. However, the molecular mechanism responsible for the substrate selectivity of MKP-1 is unknown. In addition, it has been suggested that the signal transducers and activators of transcription 1 (STAT1) transcription factor is a physiological non-MAP kinase substrate for MKP-1. We have used the yeast two-hybrid assay to demonstrate that MKP-1 is able to interact selectively with the extracellular signal-regulated kinase 1/2 (ERK1/2), p38alpha, and c-Jun NH(2)-terminal kinase (JNK) MAP kinase isoforms. Furthermore, this binding is accompanied by catalytic activation of recombinant MKP-1 protein in vitro, and these end points show an absolute correlation with MKP-1 substrate selectivity in vivo. In contrast, MKP-1 does not interact with STAT1. Recombinant STAT1 does not cause catalytic activation of MKP-1; nor does MKP-1 block tyrosine phosphorylation of STAT1 in vivo. Both binding and catalytic activation of MKP-1 are abrogated by mutation of a conserved docking site in ERK2, p38alpha, and JNK1 MAP kinases. Within MKP-1, MAP kinase binding is mediated by the amino-terminal noncatalytic domain of the protein. However, mutation of a conserved cluster of positively charged residues within this domain abolishes the binding and activation of MKP-1 by ERK2 and p38alpha but not JNK1, indicating that there are distinct binding determinants for these MAP kinase isoforms. We conclude that the substrate selectivity of MKP-1 is determined by specific protein-protein interactions coupled with catalytic activation of the phosphatase and that these interactions are restricted to members of the MAP kinase family of enzymes.
Mitogen-activated protein kinases (MAPKs) are inactivated via dephosphorylation of either the threonine or tyrosine residue or both in the P-loop catalyzed by protein phosphatases which include serine/threonine phosphatases, tyrosine phosphatases, and dual specificity phosphatases. Nine members of the dual specificity phosphatases specific for MAPKs, termed MKPs, have been reported. Each member has its own substrate specificity, tissue distribution, and subcellular localization. In this study, we have cloned and characterized a novel MKP, designated MKP-7. MKP-7 is most similar to hVH5, a member of previously known MKPs, in the primary structure. MKP-7 is predominantly localized in the cytoplasm when expressed in cultured cells, whereas hVH5 is both in the nucleus and the cytoplasm. MKP-7 binds to and inactivates p38 MAPK and JNK/SAPK, but not ERK. Furthermore, we have found that MKPs have the substrate specificity toward the isoforms of the p38 family (alpha, beta, gamma, and delta). MKP-7 binds to and inactivates p38 alpha and -beta, but not gamma or delta. MKP-5 and CL100/MKP-1 also bind to p38 alpha and -beta, but not gamma or delta. Finally, we propose a tentative classification of MKPs based on the sequence characteristics of their MAPK-docking site.
The p38 MAP kinase (MAPK) is phosphorylated and activated by upstream MAPK kinases. T cells have an alternative pathway in which T cell receptor-activated tyrosine kinase Zap70 phosphorylates p38 on Tyr323. Mice lacking Gadd45alpha, a small p38-binding molecule, develop a lupus-like autoimmune disease. Here we show that resting T cells but not B cells from Gadd45a(-/-) mice had spontaneously increased p38 activity in the absence of 'upstream' MAPK kinase activation. The p38 from resting Gadd45a(-/-) T cells was spontaneously phosphorylated on Tyr323, and its activity was specifically inhibited by recombinant Gadd45alpha in vitro. Thus, constitutive activation of T cell p38 through the alternative pathway is prevented by Gadd45alpha, the absence of which results in p38 activation, T cell hyperproliferation and autoimmunity.
One of the major families of the mitogen-activated kinases (MAPK), p38, has been shown to transduce extracellular stress stimuli into cellular responses. Among them, p38 alpha is the best characterized isoform and many biological phenomena, especially in the inflammatory responses, were attributed to the specific inhibitor-sensitive isoforms, namely p38 alpha and p38 beta. However, the roles played by each member are still unclear. Here, we report the identification of a new splice variant of p38 alpha, Exip (for exon skip), by RT-PCR using mRNA derived from a renal tumor cell line, OS-RC-2. Exip is predicted to encode a 307-amino-acid protein and the absence of exons 10, 11, and 11' results in the shift of the reading frame at the exon 9-12 junction to produce a unique 53-amino-acid C-terminus. The expression of mRNA was barely observed in cultured cells tested, but substantial amounts of mRNA were detected in mouse tissues. Unlike p38 alpha, Exip lost a common docking domain well conserved in major MAPK families for their specific interactions with upstream kinases or downstream substrates. Even though Exip is not phosphorylated at conserved TGY motif by p38-activating treatments, such as an osmotic shock or coexpression with a constitutive active form of MKK6 in HeLa cells, Exip can induce an earlier onset of apoptosis in HeLa cells. These results indicate that Exip has unique properties as a member of p38 alpha and may play role(s) in the signal transduction pathway(s) different from those of p38 alpha.
The p38 family of mitogen-activated protein kinases is composed of several isoforms. Mxi2 is a splicing variant of p38alpha that harbors a unique carboxy-terminus. Here we show that this sole divergence results in remarkable differences between Mxi2 and p38alpha. Mxi2 is distinctively activated by stress stimuli and potently activated by mitogens. Mxi2 affinity for bona fide p38 substrates is remarkably diminished and Mxi2 activity is largely unaffected by the phosphatase CL100. Also, Mxi2 sensitivity to inhibition by SB203580 is greatly reduced. Interestingly, we show that the p38 C-terminus is involved in conferring sensitivity to this compound. Overall, our results point to the p38 carboxy-terminus as a key determinant of the biochemical properties of this family of kinases.
Phosphorylation of mitogen-activated protein kinases (MAPKs) on specific tyrosine and threonine sites by MAP kinase kinases (MAPKKs) is thought to be the sole activation mechanism. Here, we report an unexpected activation mechanism for p38alpha MAPK that does not involve the prototypic kinase cascade. Rather it depends on interaction of p38alpha with TAB1 [transforming growth factor-beta-activated protein kinase 1 (TAK1)-binding protein 1] leading to autophosphorylation and activation of p38alpha. We detected formation of a TRAF6-TAB1-p38alpha complex and showed stimulus-specific TAB1-dependent and TAB1-independent p38alpha activation. These findings suggest that alternative activation pathways contribute to the biological responses of p38alpha to various stimuli.
J. Biol. Chem. 273, 1741-1748 (1998)[PubMed:9430721]
The cellular response to treatment with proinflammatory cytokines or exposure to environmental stress is mediated, in part, by the p38 group of mitogen-activated protein (MAP) kinases. We report the molecular cloning of a novel isoform of p38 MAP kinase, p38 beta 2. This p38 MAP kinase, like p38 alpha, is inhibited by the pyridinyl imidazole drug SB203580. The p38 MAP kinase kinase MKK6 is identified as a common activator of p38 alpha, p38 beta 2, and p38 gamma MAP kinase isoforms, while MKK3 activates only p38 alpha and p38 gamma MAP kinase isoforms. The MKK3 and MKK6 signal transduction pathways are therefore coupled to distinct, but overlapping, groups of p38 MAP kinases.
The RING finger ubiquitin ligase Siah2 controls the stability of various substrates involved in stress and hypoxia responses, including the PHD3, which controls the stability of HIF-1alpha. In the present study we determined the role of Siah2 phosphorylation in the regulation of its activity toward PHD3. We show that Siah2 is subject to phosphorylation by p38 MAPK, which increases Siah2-mediated degradation of PHD3. Consistent with these findings, MKK3/MKK6 double-deficient cells, which cannot activate p38 kinases, exhibit impaired Siah2-dependent degradation of PHD3. Phosphopeptide mapping identified T24 and S29 as the primary phospho-acceptor sites. Phospho-mutant forms of Siah2 (S29A or T24A/S29A) exhibit impaired degradation of PHD3, particularly after hypoxia. Conversely, a phospho-mimic form of Siah2 (T24E/S29D) exhibits stronger degradation of PHD3, compared with wild type Siah2. Whereas phospho-mutant Siah2 exhibits weaker association with PHD3, phospho-mimic Siah2 associates as well as wild type and is localized within the perinuclear region, suggesting that phosphorylation of Siah2 affects its subcellular localization and, consequently, the degree of its association with PHD3. In all, our findings reveal the phosphorylation of Siah2 by p38 and the implications of such phosphorylation for Siah2 activity toward PHD3.
Bioorg. Med. Chem. Lett. 13, 277-280 (2003)[PubMed:12482439]
The development of potent, orally bioavailable (in rat) and selective dihydroquinazolinone inhibitors of p38alpha MAP kinase is described. These analogues are hybrids of a pyridinylimidazole p38alpha inhibitor reported by Merck Research Laboratories and VX-745. Optimization of the C-5 phenyl and the C-7 piperidinyl substituents led to the identification of 15i which gave excellent suppression of TNF-alpha production in LPS-stimulated whole blood (IC(50)=10nM) and good oral exposure in rats (F=68%, AUCn PO=0.58 microM h).
The p38 mitogen-activated protein (MAP) kinase signal transduction pathway is activated by proinflammatory cytokines and environmental stress. The detection of p38 MAP kinase in the nucleus of activated cells suggests that p38 MAP kinase can mediate signaling to the nucleus. To test this hypothesis, we constructed expression vectors for activated MKK3 and MKK6, two MAP kinase kinases that phosphorylate and activate p38 MAP kinase. Expression of activated MKK3 and MKK6 in cultured cells caused a selective increase in p38 MAP kinase activity. Cotransfection experiments demonstrated that p38 MAP kinase activation causes increased reporter gene expression mediated by the transcription factors ATF2 and Elk-1. These data demonstrate that the nucleus is one target of the p38 MAP kinase signal transduction pathway.
J. Biol. Chem. 274, 19949-19956 (1999)[PubMed:10391943]
A group of dual specificity protein phosphatases negatively regulates members of the mitogen-activated protein kinase (MAPK) superfamily, which consists of three major subfamilies, MAPK/extracellular signal-regulated kinase (ERK), stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK), and p38. Nine members of this group of dual specificity phosphatases have previously been cloned. They show distinct substrate specificities for MAPKs, different tissue distribution and subcellular localization, and different modes of inducibility of their expression by extracellular stimuli. Here we have cloned and characterized a novel dual specificity phosphatase, which we have designated MKP-5. MKP-5 is a protein of 482 amino acids with a calculated molecular mass of 52.6 kDa and consists of 150 N-terminal amino acids of unknown function, two Cdc25 homology 2 regions in the middle, and a C-terminal catalytic domain. MKP-5 binds to p38 and SAPK/JNK, but not to MAPK/ERK, and inactivates p38 and SAPK/JNK, but not MAPK/ERK. p38 is a preferred substrate. The subcellular localization of MKP-5 is unique; it is present evenly in both the cytoplasm and the nucleus. MKP-5 mRNA is widely expressed in various tissues and organs, and its expression in cultured cells is elevated by stress stimuli. These results suggest that MKP-5 is a novel type of dual specificity phosphatase specific for p38 and SAPK/JNK.
Mitogen-activated protein (MAP) kinase p38 alpha is activated in response to environmental stress and cytokines, and plays a significant role in inflammatory responses. For these reasons, it is an important target for the treatment of a wide range of inflammatory and autoimmune diseases. The crystals of p38 alpha that we obtained by published procedures were usually small, quite mosaic, and difficult to reproduce and thus posed a difficulty for the intensive high-resolution studies required for a structure-guided drug discovery approach. Based on crystallographic and biochemical evidences, we prepared a single point mutation of a surface cysteine (C162S) and found that it prevents aggregation and improves the homogeneity and stability of the enzyme. This mutation also facilitates the crystallization process and increases the diffracting power of p38 alpha crystals. Surprisingly, we found that the mutation induces a change in the conformation of a nearby surface loop resulting in stronger lattice interactions, consistent with the improved crystal quality. The mutant protein, because of its improved stability and strengthened lattice interactions, thus provides a significantly improved reagent for use in structure-based drug design for this important disease target.
Novel potent trisubstituted pyridazine inhibitors of p38 MAP (mitogen activated protein) kinase are described that have activity in both cell-based assays of cytokine release and animal models of rheumatoid arthritis. They demonstrated potent inhibition of LPS-induced TNF-alpha production in mice and exhibited good efficacy in the rat collagen induced arthritis model.
The p38 MAP kinase plays a crucial role in regulating the production of proinflammatory cytokines, such as tumor necrosis factor and interleukin-1. Blocking this kinase may offer an effective therapy for treating many inflammatory diseases. Here we report a new allosteric binding site for a diaryl urea class of highly potent and selective inhibitors against human p38 MAP kinase. The formation of this binding site requires a large conformational change not observed previously for any of the protein Ser/Thr kinases. This change is in the highly conserved Asp-Phe-Gly motif within the active site of the kinase. Solution studies demonstrate that this class of compounds has slow binding kinetics, consistent with the requirement for conformational change. Improving interactions in this allosteric pocket, as well as establishing binding interactions in the ATP pocket, enhanced the affinity of the inhibitors by 12,000-fold. One of the most potent compounds in this series, BIRB 796, has picomolar affinity for the kinase and low nanomolar inhibitory activity in cell culture.
J. Biol. Chem. 270, 7420-7426 (1995)[PubMed:7535770]
Protein kinases activated by dual phosphorylation on Tyr and Thr (MAP kinases) can be grouped into two major classes: ERK and JNK. The ERK group regulates multiple targets in response to growth factors via a Ras-dependent mechanism. In contrast, JNK activates the transcription factor c-Jun in response to pro-inflammatory cytokines and exposure of cells to several forms of environmental stress. Recently, a novel mammalian protein kinase (p38) that shares sequence similarity with mitogen-activated protein (MAP) kinases was identified. Here, we demonstrate that p38, like JNK, is activated by treatment of cells with pro-inflammatory cytokines and environmental stress. The mechanism of p38 activation is mediated by dual phosphorylation on Thr-180 and Tyr-182. Immunofluorescence microscopy demonstrated that p38 MAP kinase is present in both the nucleus and cytoplasm of activated cells. Together, these data establish that p38 is a member of the mammalian MAP kinase group.
J. Biol. Chem. 270, 7420-7426 (1995)[PubMed:7535770]
Protein kinases activated by dual phosphorylation on Tyr and Thr (MAP kinases) can be grouped into two major classes: ERK and JNK. The ERK group regulates multiple targets in response to growth factors via a Ras-dependent mechanism. In contrast, JNK activates the transcription factor c-Jun in response to pro-inflammatory cytokines and exposure of cells to several forms of environmental stress. Recently, a novel mammalian protein kinase (p38) that shares sequence similarity with mitogen-activated protein (MAP) kinases was identified. Here, we demonstrate that p38, like JNK, is activated by treatment of cells with pro-inflammatory cytokines and environmental stress. The mechanism of p38 activation is mediated by dual phosphorylation on Thr-180 and Tyr-182. Immunofluorescence microscopy demonstrated that p38 MAP kinase is present in both the nucleus and cytoplasm of activated cells. Together, these data establish that p38 is a member of the mammalian MAP kinase group.
Phosphorylation of mitogen-activated protein kinases (MAPKs) on specific tyrosine and threonine sites by MAP kinase kinases (MAPKKs) is thought to be the sole activation mechanism. Here, we report an unexpected activation mechanism for p38alpha MAPK that does not involve the prototypic kinase cascade. Rather it depends on interaction of p38alpha with TAB1 [transforming growth factor-beta-activated protein kinase 1 (TAK1)-binding protein 1] leading to autophosphorylation and activation of p38alpha. We detected formation of a TRAF6-TAB1-p38alpha complex and showed stimulus-specific TAB1-dependent and TAB1-independent p38alpha activation. These findings suggest that alternative activation pathways contribute to the biological responses of p38alpha to various stimuli.
Mitogen-activated protein (MAP) kinase phosphatase 1 (MKP-1/CL100) is an inducible nuclear dual specificity protein phosphatase that can dephosphorylate and inactivate both mitogen- and stress-activated protein kinases in vitro and in vivo. However, the molecular mechanism responsible for the substrate selectivity of MKP-1 is unknown. In addition, it has been suggested that the signal transducers and activators of transcription 1 (STAT1) transcription factor is a physiological non-MAP kinase substrate for MKP-1. We have used the yeast two-hybrid assay to demonstrate that MKP-1 is able to interact selectively with the extracellular signal-regulated kinase 1/2 (ERK1/2), p38alpha, and c-Jun NH(2)-terminal kinase (JNK) MAP kinase isoforms. Furthermore, this binding is accompanied by catalytic activation of recombinant MKP-1 protein in vitro, and these end points show an absolute correlation with MKP-1 substrate selectivity in vivo. In contrast, MKP-1 does not interact with STAT1. Recombinant STAT1 does not cause catalytic activation of MKP-1; nor does MKP-1 block tyrosine phosphorylation of STAT1 in vivo. Both binding and catalytic activation of MKP-1 are abrogated by mutation of a conserved docking site in ERK2, p38alpha, and JNK1 MAP kinases. Within MKP-1, MAP kinase binding is mediated by the amino-terminal noncatalytic domain of the protein. However, mutation of a conserved cluster of positively charged residues within this domain abolishes the binding and activation of MKP-1 by ERK2 and p38alpha but not JNK1, indicating that there are distinct binding determinants for these MAP kinase isoforms. We conclude that the substrate selectivity of MKP-1 is determined by specific protein-protein interactions coupled with catalytic activation of the phosphatase and that these interactions are restricted to members of the MAP kinase family of enzymes.
Mitogen-activated protein kinases (MAPKs) are inactivated via dephosphorylation of either the threonine or tyrosine residue or both in the P-loop catalyzed by protein phosphatases which include serine/threonine phosphatases, tyrosine phosphatases, and dual specificity phosphatases. Nine members of the dual specificity phosphatases specific for MAPKs, termed MKPs, have been reported. Each member has its own substrate specificity, tissue distribution, and subcellular localization. In this study, we have cloned and characterized a novel MKP, designated MKP-7. MKP-7 is most similar to hVH5, a member of previously known MKPs, in the primary structure. MKP-7 is predominantly localized in the cytoplasm when expressed in cultured cells, whereas hVH5 is both in the nucleus and the cytoplasm. MKP-7 binds to and inactivates p38 MAPK and JNK/SAPK, but not ERK. Furthermore, we have found that MKPs have the substrate specificity toward the isoforms of the p38 family (alpha, beta, gamma, and delta). MKP-7 binds to and inactivates p38 alpha and -beta, but not gamma or delta. MKP-5 and CL100/MKP-1 also bind to p38 alpha and -beta, but not gamma or delta. Finally, we propose a tentative classification of MKPs based on the sequence characteristics of their MAPK-docking site.
One of the major families of the mitogen-activated kinases (MAPK), p38, has been shown to transduce extracellular stress stimuli into cellular responses. Among them, p38 alpha is the best characterized isoform and many biological phenomena, especially in the inflammatory responses, were attributed to the specific inhibitor-sensitive isoforms, namely p38 alpha and p38 beta. However, the roles played by each member are still unclear. Here, we report the identification of a new splice variant of p38 alpha, Exip (for exon skip), by RT-PCR using mRNA derived from a renal tumor cell line, OS-RC-2. Exip is predicted to encode a 307-amino-acid protein and the absence of exons 10, 11, and 11' results in the shift of the reading frame at the exon 9-12 junction to produce a unique 53-amino-acid C-terminus. The expression of mRNA was barely observed in cultured cells tested, but substantial amounts of mRNA were detected in mouse tissues. Unlike p38 alpha, Exip lost a common docking domain well conserved in major MAPK families for their specific interactions with upstream kinases or downstream substrates. Even though Exip is not phosphorylated at conserved TGY motif by p38-activating treatments, such as an osmotic shock or coexpression with a constitutive active form of MKK6 in HeLa cells, Exip can induce an earlier onset of apoptosis in HeLa cells. These results indicate that Exip has unique properties as a member of p38 alpha and may play role(s) in the signal transduction pathway(s) different from those of p38 alpha.
Novel potent trisubstituted pyridazine inhibitors of p38 MAP (mitogen activated protein) kinase are described that have activity in both cell-based assays of cytokine release and animal models of rheumatoid arthritis. They demonstrated potent inhibition of LPS-induced TNF-alpha production in mice and exhibited good efficacy in the rat collagen induced arthritis model.
Bioorg. Med. Chem. Lett. 13, 277-280 (2003)[PubMed:12482439]
The development of potent, orally bioavailable (in rat) and selective dihydroquinazolinone inhibitors of p38alpha MAP kinase is described. These analogues are hybrids of a pyridinylimidazole p38alpha inhibitor reported by Merck Research Laboratories and VX-745. Optimization of the C-5 phenyl and the C-7 piperidinyl substituents led to the identification of 15i which gave excellent suppression of TNF-alpha production in LPS-stimulated whole blood (IC(50)=10nM) and good oral exposure in rats (F=68%, AUCn PO=0.58 microM h).
Signaling-responsive MAP kinases (MAPKs) are key in mediating immune responses and are activated through the phosphorylation of a Thr-X-Tyr motif by upstream MAPK kinases. Here we show that T cells stimulated through the T cell receptor (TCR) used an alternative mechanism in which p38 was phosphorylated on Tyr323 and subsequently autophosphorylated residues Thr180 and Tyr182. This required the TCR-proximal tyrosine kinase Zap70 but not the adaptor protein LAT, which was required for activation of extracellular signal-regulated protein kinase MAPKs. TCR activation of p38 lacking Tyr323 was diminished, and blocking of p38 activity prevented p38 dual phosphorylation in normal T cells but not in B cells. Thus, phosphorylation of Tyr323 dependent on the tyrosine kinase Lck and mediated by Zap70 serves as an important mechanism for TCR activation of p38 in T cells.
J. Biol. Chem. 273, 1741-1748 (1998)[PubMed:9430721]
The cellular response to treatment with proinflammatory cytokines or exposure to environmental stress is mediated, in part, by the p38 group of mitogen-activated protein (MAP) kinases. We report the molecular cloning of a novel isoform of p38 MAP kinase, p38 beta 2. This p38 MAP kinase, like p38 alpha, is inhibited by the pyridinyl imidazole drug SB203580. The p38 MAP kinase kinase MKK6 is identified as a common activator of p38 alpha, p38 beta 2, and p38 gamma MAP kinase isoforms, while MKK3 activates only p38 alpha and p38 gamma MAP kinase isoforms. The MKK3 and MKK6 signal transduction pathways are therefore coupled to distinct, but overlapping, groups of p38 MAP kinases.
Two new classes of diphenylether inhibitors of p38alpha MAP kinase are described. Both chemical classes are based on a common diphenylether core that is identified by simulated fragment annealing as one of the most favored chemotypes within a prominent hydrophobic pocket of the p38alpha ATP-binding site. In the fully elaborated molecules, the diphenylether moiety acts as an anchor occupying the deep pocket, while polar extensions make specific interactions with either the adenine binding site or the phosphate binding site of ATP. The synthesis, crystallographic analysis, and biological activity of these p38alpha inhibitors are discussed.
The p38 mitogen-activated protein (MAP) kinase signal transduction pathway is activated by proinflammatory cytokines and environmental stress. The detection of p38 MAP kinase in the nucleus of activated cells suggests that p38 MAP kinase can mediate signaling to the nucleus. To test this hypothesis, we constructed expression vectors for activated MKK3 and MKK6, two MAP kinase kinases that phosphorylate and activate p38 MAP kinase. Expression of activated MKK3 and MKK6 in cultured cells caused a selective increase in p38 MAP kinase activity. Cotransfection experiments demonstrated that p38 MAP kinase activation causes increased reporter gene expression mediated by the transcription factors ATF2 and Elk-1. These data demonstrate that the nucleus is one target of the p38 MAP kinase signal transduction pathway.
J. Biol. Chem. 274, 19949-19956 (1999)[PubMed:10391943]
A group of dual specificity protein phosphatases negatively regulates members of the mitogen-activated protein kinase (MAPK) superfamily, which consists of three major subfamilies, MAPK/extracellular signal-regulated kinase (ERK), stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK), and p38. Nine members of this group of dual specificity phosphatases have previously been cloned. They show distinct substrate specificities for MAPKs, different tissue distribution and subcellular localization, and different modes of inducibility of their expression by extracellular stimuli. Here we have cloned and characterized a novel dual specificity phosphatase, which we have designated MKP-5. MKP-5 is a protein of 482 amino acids with a calculated molecular mass of 52.6 kDa and consists of 150 N-terminal amino acids of unknown function, two Cdc25 homology 2 regions in the middle, and a C-terminal catalytic domain. MKP-5 binds to p38 and SAPK/JNK, but not to MAPK/ERK, and inactivates p38 and SAPK/JNK, but not MAPK/ERK. p38 is a preferred substrate. The subcellular localization of MKP-5 is unique; it is present evenly in both the cytoplasm and the nucleus. MKP-5 mRNA is widely expressed in various tissues and organs, and its expression in cultured cells is elevated by stress stimuli. These results suggest that MKP-5 is a novel type of dual specificity phosphatase specific for p38 and SAPK/JNK.
Mitogen-activated protein (MAP) kinase p38 alpha is activated in response to environmental stress and cytokines, and plays a significant role in inflammatory responses. For these reasons, it is an important target for the treatment of a wide range of inflammatory and autoimmune diseases. The crystals of p38 alpha that we obtained by published procedures were usually small, quite mosaic, and difficult to reproduce and thus posed a difficulty for the intensive high-resolution studies required for a structure-guided drug discovery approach. Based on crystallographic and biochemical evidences, we prepared a single point mutation of a surface cysteine (C162S) and found that it prevents aggregation and improves the homogeneity and stability of the enzyme. This mutation also facilitates the crystallization process and increases the diffracting power of p38 alpha crystals. Surprisingly, we found that the mutation induces a change in the conformation of a nearby surface loop resulting in stronger lattice interactions, consistent with the improved crystal quality. The mutant protein, because of its improved stability and strengthened lattice interactions, thus provides a significantly improved reagent for use in structure-based drug design for this important disease target.
The p38 MAP kinase plays a crucial role in regulating the production of proinflammatory cytokines, such as tumor necrosis factor and interleukin-1. Blocking this kinase may offer an effective therapy for treating many inflammatory diseases. Here we report a new allosteric binding site for a diaryl urea class of highly potent and selective inhibitors against human p38 MAP kinase. The formation of this binding site requires a large conformational change not observed previously for any of the protein Ser/Thr kinases. This change is in the highly conserved Asp-Phe-Gly motif within the active site of the kinase. Solution studies demonstrate that this class of compounds has slow binding kinetics, consistent with the requirement for conformational change. Improving interactions in this allosteric pocket, as well as establishing binding interactions in the ATP pocket, enhanced the affinity of the inhibitors by 12,000-fold. One of the most potent compounds in this series, BIRB 796, has picomolar affinity for the kinase and low nanomolar inhibitory activity in cell culture.
The p38 MAP kinase (MAPK) is phosphorylated and activated by upstream MAPK kinases. T cells have an alternative pathway in which T cell receptor-activated tyrosine kinase Zap70 phosphorylates p38 on Tyr323. Mice lacking Gadd45alpha, a small p38-binding molecule, develop a lupus-like autoimmune disease. Here we show that resting T cells but not B cells from Gadd45a(-/-) mice had spontaneously increased p38 activity in the absence of 'upstream' MAPK kinase activation. The p38 from resting Gadd45a(-/-) T cells was spontaneously phosphorylated on Tyr323, and its activity was specifically inhibited by recombinant Gadd45alpha in vitro. Thus, constitutive activation of T cell p38 through the alternative pathway is prevented by Gadd45alpha, the absence of which results in p38 activation, T cell hyperproliferation and autoimmunity.
Inhibition of the biosynthesis of proinflammatory cytokines such as tumor necrosis factor and interleukin-1 via p38 has been an approach toward the development of a disease modifying agent for the treatment of chronic inflammation and autoimmune diseases. The development of a new core structure of p38 inhibitors, 3-(4-fluorophenyl)-2-(pyridin-4-yl)-1H-pyrrolo[3,2-b] pyridine, is described. X-ray crystallographic data of the lead bound to the active site of p38 was used to guide the optimization of the series. Specific focus was placed on modulating the physical properties of the core while maintaining potent inhibition of p38. These efforts identified 42c as a potent inhibitor of p38, which also possessed the required physical properties worthy of advanced studies.
The p38 family of mitogen-activated protein kinases is composed of several isoforms. Mxi2 is a splicing variant of p38alpha that harbors a unique carboxy-terminus. Here we show that this sole divergence results in remarkable differences between Mxi2 and p38alpha. Mxi2 is distinctively activated by stress stimuli and potently activated by mitogens. Mxi2 affinity for bona fide p38 substrates is remarkably diminished and Mxi2 activity is largely unaffected by the phosphatase CL100. Also, Mxi2 sensitivity to inhibition by SB203580 is greatly reduced. Interestingly, we show that the p38 C-terminus is involved in conferring sensitivity to this compound. Overall, our results point to the p38 carboxy-terminus as a key determinant of the biochemical properties of this family of kinases.
The RING finger ubiquitin ligase Siah2 controls the stability of various substrates involved in stress and hypoxia responses, including the PHD3, which controls the stability of HIF-1alpha. In the present study we determined the role of Siah2 phosphorylation in the regulation of its activity toward PHD3. We show that Siah2 is subject to phosphorylation by p38 MAPK, which increases Siah2-mediated degradation of PHD3. Consistent with these findings, MKK3/MKK6 double-deficient cells, which cannot activate p38 kinases, exhibit impaired Siah2-dependent degradation of PHD3. Phosphopeptide mapping identified T24 and S29 as the primary phospho-acceptor sites. Phospho-mutant forms of Siah2 (S29A or T24A/S29A) exhibit impaired degradation of PHD3, particularly after hypoxia. Conversely, a phospho-mimic form of Siah2 (T24E/S29D) exhibits stronger degradation of PHD3, compared with wild type Siah2. Whereas phospho-mutant Siah2 exhibits weaker association with PHD3, phospho-mimic Siah2 associates as well as wild type and is localized within the perinuclear region, suggesting that phosphorylation of Siah2 affects its subcellular localization and, consequently, the degree of its association with PHD3. In all, our findings reveal the phosphorylation of Siah2 by p38 and the implications of such phosphorylation for Siah2 activity toward PHD3.
Protein involved in apoptotic programmed cell death. Apoptosis is characterized by cell morphological changes, including blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation and chromosomal DNA fragmentation, and eventually death. Unlike necrosis, apoptosis produces cell fragments, called apoptotic bodies, that phagocytic cells are able to engulf and quickly remove before the contents of the cell can spill out onto surrounding cells and cause damage. In general, apoptosis confers advantages during an organism's life cycle.
Protein involved in the response to stress, a change in state or activity of a cell or an organism (in terms of movement, secretion, enzyme production, gene expression, etc.) as a result of some stressful conditions. The stress is usually, but not necessarily, exogenous (e.g. temperature, humidity, ionizing radiation, hypertonicity, amino acid deprivation).
Protein involved in the transfer of genetic information from DNA to messenger RNA (mRNA) by DNA-directed RNA polymerase. In the case of some RNA viruses, protein involved in the transfer of genetic information from RNA to messenger RNA (mRNA) by RNA-directed RNA polymerase.
Protein which catalyzes the phosphorylation of serine or threonine residues on target proteins by using ATP as phosphate donor. Such phosphorylation may cause changes in the function of the target protein. Protein kinases share a conserved catalytic core common to both serine/ threonine and tyrosine protein kinases.
A reference proteome is a set of protein sequences derived from a complete proteome which constitutes a defined standard for a particular user community. Reference proteomes are manually defined according to a number of criteria. They cover the proteomes of well- studied model organisms and other proteomes of interest for biomedical and biotechnological research. Reference proteomes have been selected to provide broad coverage of the tree of life, and constitute a representative cross-section of the taxonomic diversity to be found within UniProtKB.
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