首页 | 本学科首页   官方微博 | 高级检索  
相似文献
 共查询到20条相似文献,搜索用时 703 毫秒
1.
2.
Mitogen-activated protein (MAP) kinases compose a family of serine/threonine kinases that function in many signal transduction pathways and affect various cellular phenotypes. Despite the abundance of available data, the exact role of each MAP kinase is not completely defined, in part because of the inability to activate MAP kinase molecules individually and specifically. Based on activating mutations found in the yeast MAP kinase p38/Hog1 (Bell, M., Capone, R., Pashtan, I., Levitzki, A., and Engelberg, D. (2001) J. Biol. Chem. 276, 25351-25358), we designed and constructed single and multiple mutants of human MAP kinase p38alpha. Single (p38D176A, p38F327L, and p38F327S) and subsequent double (p38D176A/F327L and p38D176A/F327S) mutants acquired high intrinsic activity independent of any upstream regulation and reached levels of 10 and 25%, respectively, in reference to the dually phosphorylated wild type p38alpha. The active p38 mutants have retained high specificity toward p38 substrates and were inhibited by the specific p38 inhibitors SB-203580 and PD-169316. We also show that similar mutations can render p38gamma active as well. Based on the available structures of p38 and ERK2, we have analyzed the p38 mutants and identified a hydrophobic core stabilized by three aromatic residues, Tyr-69, Phe-327, and Trp-337, in the vicinity of the L16 loop region. Upon activation, a segment of the L16 loop, including Phe-327, becomes disordered. Structural analysis suggests that the active p38 mutants emulate the conformational changes imposed naturally by dual phosphorylation, namely, destabilization of the hydrophobic core. Essentially, the hydrophobic core is an inherent stabilizer that maintains low basal activity level in unphosphorylated p38.  相似文献   

3.
4.
p38 MAPK isoforms are important in the regulation of a variety of cellular processes. Among the four described p38 isoforms, p38 alpha, beta, and delta are expressed in keratinocytes (Dashti, S. R., Efimova, T., and Eckert, R. L. (2001) J. Biol. Chem. 276, 8059-8063). However, very little is known about how individual p38 isoforms regulate keratinocyte function. In the present study, we use okadaic acid (OA) as a tool to study the role of p38 MAPKs as regulators of keratinocyte differentiation. We demonstrate that OA activates p38 delta but not other p38 isoforms. p38 delta activation is increased as early as 0.5 h after OA addition, and activity is maximal at 8 and 24 h. ERK1 and ERK2 activity are reduced on an identical time course. We show that p38 delta forms a complex with ERK1/2, and overexpression of p38 delta inhibits ERK1/2 activity without reducing ERK1/2 level. Thus, p38 delta may directly suppress ERK1/2 activity. Additional studies show that p38 delta is expressed in the epidermis, suggesting a role for p38 delta in regulating differentiation. To evaluate its function, we show that increased p38 delta activity is associated with increased levels of AP1 and CAATT enhancer binding protein factors, increased binding of these factors to the involucrin (hINV) promoter, and increased expression. Moreover, these responses are maintained in the presence of SB203580, an agent that inhibits p38 alpha and beta, further suggesting a central role for the p38 delta isoform. Dominant-negative p38 also inhibits these responses. These unique observations suggest that p38 delta is the major p38 isoform driving suprabasal hINV gene expression and that p38 delta directly regulates ERK1/2 activity via formation of a p38 delta-ERK1/2 complex.  相似文献   

5.
A signaling cascade that includes protein kinase C (PKC), Ras, and MEKK1 regulates involucrin (hINV) gene expression in epidermal keratinocytes (Efimova, T., LaCelle, P., Welter, J. F., and Eckert, R. L. (1998) J. Biol. Chem. 273, 24387-24395 and Efimova, T., and Eckert, R. L. (2000) J. Biol. Chem. 275, 1601-1607). Because signal transfer downstream of MEKK1 may involve several MAPK kinases (MEKs), it is important to evaluate the regulatory role of each MEK isoform. In the present study we evaluate the role of MEK6 in transmitting this signal. Constitutively active MEK6 (caMEK6) increases hINV promoter activity and increases endogenous hINV levels. The caMEK6-dependent increase in gene expression is inhibited by the p38 MAPK inhibitor, SB203580, and is associated with a marked increase in p38alpha MAPK activity; JNK and ERK kinases are not activated. In addition, hINV gene expression is inhibited by dominant-negative p38alpha and increased when caMEK6 and p38alpha are co-expressed. caMEK6 also activates p38delta, but p38delta inhibits the caMEK6-dependent activation. These results suggest that MEK6 increases hINV gene expression by regulating the balance between activation of p38alpha, which increases gene expression, and p38delta, which decreases gene expression.  相似文献   

6.
Human involucrin (hINV) mRNA level and promoter activity increase when keratinocytes are treated with the differentiating agent, 12-O-tetradecanoylphorbol-13-acetate (TPA). This response is mediated via a p38 mitogen-activated protein kinase-dependent pathway that targets activator protein 1 (Efimova, T., LaCelle, P. T. , Welter, J. F., and Eckert, R. L. (1998) J. Biol. Chem. 273, 24387-24395). In the present study we examine the role of various PKC isoforms in this regulation. Transfection of expression plasmids encoding the novel PKC isoforms delta, epsilon, and eta increase hINV promoter activity. In contrast, neither conventional PKC isoforms (alpha, beta, and gamma) nor the atypical isoform (zeta) regulate promoter activity. Consistent with these observations, promoter activity is inhibited by the PKCdelta-selective inhibitor, rottlerin, but not by Go-6976, an inhibitor of conventional PKC isoforms, and novel PKC isoform-dependent promoter activation is inhibited by dominant-negative PKCdelta. This regulation appears to be physiologically important, as transfection of keratinocytes with PKCdelta, -epsilon, or -eta increases expression of the endogenous hINV gene. Synergistic promoter activation (>/=100-fold) is observed when PKCepsilon- or -eta-transfected cells are treated with TPA. In contrast, the PKCdelta-dependent response is more complex as either activation or inhibition is observed, depending upon PKCdelta concentration.  相似文献   

7.
Hypoxic/ischemic trauma is a primary factor in the pathology of a multitude of disease states. The effects of hypoxia on the stress- and mitogen-activated protein kinase signaling pathways were studied in PC12 cells. Exposure to moderate hypoxia (5% O(2)) progressively stimulated phosphorylation and activation of p38gamma in particular, and also p38alpha, two stress-activated protein kinases. In contrast, hypoxia had no effect on enzyme activity of p38beta, p38beta(2), p38delta, or on c-Jun N-terminal kinase, another stress-activated protein kinase. Prolonged hypoxia also induced phosphorylation and activation of p42/p44 mitogen-activated protein kinase, although this activation was modest compared with nerve growth factor- and ultraviolet light-induced activation. Hypoxia also dramatically down-regulated immunoreactivity of cyclin D1, a gene that is known to be regulated negatively by p38 at the level of gene expression (Lavoie, J. N., L'Allemain, G., Brunet, A., Muller, R., and Pouyssegur, J. (1996) J. Biol. Chem. 271, 20608-20616). This effect was partially blocked by SB203580, an inhibitor of p38alpha but not p38gamma. Overexpression of a kinase-inactive form of p38gamma was also able to reverse in part the effect of hypoxia on cyclin D1 levels, suggesting that p38alpha and p38gamma converge to regulate cyclin D1 during hypoxia. These studies demonstrate that an extremely typical physiological stress (hypoxia) causes selective activation of specific p38 signaling elements; and they also identify a downstream target of these pathways.  相似文献   

8.
The p38 alpha mitogen-activated protein kinase has been implicated in the cellular response to genotoxic agents. Here we show that another p38 family member is also activated in response to cisplatin exposure in human melanoma cells. We identified this isoform as p38gamma based on its recognition by specific antibodies and because, in contrast to p38alpha, its activity was not affected by SB203580. We also found that etoposide caused a much more discrete phosphorylation of both p38alpha and p38gamma than either cisplatin or UV treatment. These results indicate that genotoxic stresses activate several p38 isoforms whose implication in the cellular response might depend on the type of DNA damage.  相似文献   

9.
We and others previously showed that p38 mitogen-activated protein kinase is indispensable for myogenic differentiation. However, it is less clear which of the four p38 isoforms in the mouse genome participates in this process. Using C2C12 myogenic cells as a model, we showed here that p38alpha, beta, and gamma are expressed with distinct expression patterns during differentiation. Knockdown of any of them by small interfering RNA inhibits myogenic differentiation, which suggests that the functions of the three p38 isoforms are not completely redundant. To further elucidate the unique role of each p38 isoform in myogenic differentiation, we individually knocked down one p38 isoform at a time in C2C12 cells, and we compared the whole-genome gene expression profiles by microarrays. We found that some genes are coregulated by all three p38 isoforms, whereas others are uniquely regulated by one particular p38 isoform. Furthermore, several novel p38 target genes (i.e., E2F2, cyclin D3, and WISP1) are found to be required for myogenin expression, which provides a molecular basis to explain why different p38 isoforms are required for myogenic differentiation.  相似文献   

10.
Ca2+/calmodulin-dependent protein kinase (CaMK)II is highly expressed in the CNS and mediates activity-dependent neuronal plasticity. Four CaMKII isoforms, alpha, beta, gamma and delta, have a large number of splicing variants. Here we identified isoforms of CaMKII in the rat substantia nigra (SN). Northern blot and RT-PCR analyses revealed that the gamma and delta isoform mRNAs with several splicing variants were predominantly expressed in SN. Immunoblot analysis indicated that the major isoforms were gammaA, gammaC, delta1 and delta3. An immunohistochemical study also confirmed the preferential localization of gamma and delta isoforms in SN dopaminergic neurons. In dopaminergic neurons, immunoreactivity against anti-CaMKIIdelta1-4 antibody was detected in both nucleus and cytoplasm, in contrast to the predominant expression of gamma isoforms in the cytoplasm. Furthermore, we showed expression of brain-derived neurotrophic factor (BDNF) mRNAs with exons II and IV in SN. Taken together with our previous observations, the results suggest that the CaMKIIdelta3 isoform is involved in the expression of BDNF in the SN.  相似文献   

11.
We have investigated the ability of the mitogen-activated protein kinase (MAPK) kinase MKK6 to activate different members of the p38 subfamily of MAPKs and found that some MKK6 mutants can efficiently activate p38alpha but not p38gamma. In contrast, a constitutively active MKK6 mutant activated both p38 MAPK isoforms to similar extents. The same results were obtained upon co-expression in Xenopus oocytes and in vitro using either MKK6 immunoprecipitates from transfected cells or bacterially produced recombinant proteins. We also found that the preferential activation of p38alpha by MKK6 correlated with more efficient binding of MKK6 to p38alpha than to p38gamma. Furthermore, increasing concentrations of constitutively active MKK6 differentially activated either p38alpha alone (low MKK6 activity) or both p38alpha and p38gamma (high MKK6 activity), both in vitro and in injected oocytes. The determinants for selectivity are located at the carboxyl-terminal lobe of p38 MAPKs but do not correspond to the activation loop or common docking sequences. We also showed that different stimuli can induce different levels of endogenous MKK6 activity that correlate with differential activation of p38 MAPKs. Our results suggest that the level of MKK6 activity triggered by a given stimulus may determine the pattern of downstream p38 MAPK activation in the particular response.  相似文献   

12.
The quinazolinone and pyridol-pyrimidine classes of p38 MAP kinase inhibitors have a previously unseen degree of specificity for p38 over other MAP kinases. Comparison of the crystal structures of p38 bound to four different compounds shows that binding of the more specific molecules is characterized by a peptide flip between Met109 and Gly110. Gly110 is a residue specific to the alpha, beta and gamma isoforms of p38. The delta isoform and the other MAP kinases have bulkier residues in this position. These residues would likely make the peptide flip energetically unfavorable, thus explaining the selectivity of binding. To test this hypothesis, we constructed G110A and G110D mutants of p38 and measured the potency of several compounds against them. The results confirm that the selectivity of quinazolinones and pyridol-pyrimidines results from the presence of a glycine in position 110. This unique mode of binding may be exploited in the design of new p38 inhibitors.  相似文献   

13.
14.
A novel branch of the Ras family, Rit, was recently identified. Rit exhibits a distinct C-terminus and effector domain, and does not activate mitogen-activated protein kinase (MAPK) but can cooperate with Raf to transform fibroblasts. Here, we found that when overexpressed, activated mutants of Rit transform NIH 3T3 cells efficiently, and stimulate p38gamma but not MAPK, p38alpha, p38gamma, p38delta, or ERK5. Furthermore, we provide evidence that p38gamma activation is required for the ability of Rit to stimulate gene expression and cellular transformation. These findings suggest that this unique GTPase stimulates proliferative pathways distinct from those regulated by other Ras family members.  相似文献   

15.
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.  相似文献   

16.
The p38 mitogen-activated protein kinase (MAPK) group is represented by four isoforms in mammals (p38alpha, p38beta2, p38gamma and p38delta). These p38 MAPK isoforms appear to mediate distinct functions in vivo due, in part, to differences in substrate phosphorylation by individual p38 MAPKs and also to selective activation by MAPK kinases (MAPKKs). Here we report the identification of two factors that contribute to the specificity of p38 MAPK activation. One mechanism of specificity is the selective formation of functional complexes between MAPKK and different p38 MAPKs. The formation of these complexes requires the presence of a MAPK docking site in the N-terminus of the MAPKK. The second mechanism that confers signaling specificity is the selective recognition of the activation loop (T-loop) of p38 MAPK isoforms. Together, these processes provide a mechanism that enables the selective activation of p38 MAPK in response to activated MAPKK.  相似文献   

17.
p38 mitogen-activated protein (MAP) kinases function in numerous signaling processes and are crucial for normal functions of cells and organisms. Abnormal p38 activity is associated with inflammatory diseases and cancers making the understanding of its activation mechanisms highly important. p38s are commonly activated by phosphorylation, catalyzed by MAP kinase kinases (MKKs). Moreover, it was recently revealed that the p38alpha is also activated via alternative pathways, which are MKK independent. The structural basis of p38 activation, especially in the alternative pathways, is mostly unknown. This lack of structural data hinders the study of p38's biology as well as the development of novel strategies for p38 inhibition. We have recently discovered and optimized a novel set of intrinsically active p38 mutants whose activities are independent of any upstream activation. The high-resolution crystal structures of the intrinsically active p38alpha mutants reveal that local alterations in the L16 loop region promote kinase activation. The L16 loop can be thus regarded as a molecular switch that upon conformational changes promotes activation. We suggest that similar conformational changes in L16 loop also occur in natural activation mechanisms of p38alpha in T-cells. Our biochemical studies reveal novel mechanistic insights into the activation process of p38. In this regard, the results indicate that the activation mechanism of the mutants involves dimerization and subsequent trans autophosphorylation on Thr180 (on the phosphorylation lip). Finally, we suggest a model of in vivo p38alpha activation induced by the L16 switch with auto regulatory characteristics.  相似文献   

18.
19.
Calponin is a basic smooth-muscle-specific protein capable of binding to F-actin, tropomyosin and calmodulin in vitro. Using two-dimensional gel electrophoresis, we show that calponin exists as multiple isoelectric variants in avian and mammalian tissues. During chick embryogenesis, one isoform is expressed in gizzard that shows a pI identical to the most basic adult alpha variant; around 10 d after hatching multiple isoforms then appear. SM 22 [Pearlstone, J. R., Weber, M., Lees-Miller, J. P., Carpenter, M. R. & Smillie, L. B. (1987) J. Biol. Chem. 262, 5985-5991], which has sequence-motifs related to calponin, displays a similar isoform pattern during development; one isoform (alpha) is present in the embryo and three in the adult. In living smooth-muscle strips from chicken gizzard and guinea pig taenia coli, labelled with 32PO4, no phosphate incorporation could be detected in any of the calponin or SM 22 isoforms during either contraction or relaxation. From the additional observation that antibodies against phosphoserine also failed to label calponin and SM 22 in two-dimensional gel immunoblots, we conclude that the multiple isoforms do not arise via differential phosphorylation. These results support the claim [Barany, M., Rokolya, A. & Barany, K. (1991) FEBS Lett. 279, 65-68] that calponin phosphorylation is not involved in smooth muscle regulation in vivo, as has been suggested from in vitro studies [Winder, S. J. & Walsh, M. J. (1990) J. Biol. Chem. 265, 10148-10155]. In vitro translation of porcine and chicken smooth-muscle mRNA produced only a single (alpha) isoform of calponin, suggesting that the adult isoforms do not derive from multiple gene products; in the same assay two polypeptides appeared in the position of SM 22, one corresponding to the alpha isoform and a second more basic spot, not observed in tissue samples. Whereas calponin and SM 22 appear synchronously during smooth muscle differentiation in vivo, SM 22 is not fully down-regulated like calponin, metavinculin and heavy-caldesmon in smooth muscle cells in culture, pointing to a differential regulation of expression of the alpha SM 22 isoform during smooth-muscle phenotype modulation in vitro.  相似文献   

20.
设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号