Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.11.24 (mitogen-activated protein kinase)
 95,810 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A number of Raf-associated proteins have recently been identified, including members of the 14-3-3 family of phosphoserine-binding proteins. Although both positive and negative regulatory functions have been ascribed for 14-3-3 interactions with Raf-1, the mechanisms by which 14-3-3 binding modulates Raf activity have not been fully established. We report that mutational disruption of 14-3-3 binding to the B-Raf catalytic domain inhibits B-Raf biological activity. Expression of the isolated B-Raf catalytic domain (B-Rafcat) induces PC12 cell differentiation in the absence of nerve growth factor. By contrast, the B-Rafcat 14-3-3 binding mutant, B-Rafcat S728A, was severely compromised for the induction of PC12 cell differentiation. Interestingly, the B-Rafcat 14-3-3 binding mutant retained significant in vitro catalytic activity. In Xenopus oocytes, the analogous full-length B-Raf 14-3-3 binding mutant blocked progesterone-stimulated maturation and the activation of endogenous mitogen-activated protein kinase kinase and mitogen-activated protein kinase. Similarly, the full-length B-Raf 14-3-3 binding mutant inhibited nerve growth factor-stimulated PC12 cell differentiation. We conclude that 14-3-3 interaction with the catalytic domain is not required for kinase activity per se but is essential to couple B-Raf catalytic activity to downstream effector activation.
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PMID:Disruption of the 14-3-3 binding site within the B-Raf kinase domain uncouples catalytic activity from PC12 cell differentiation. 1066 May 30

14-3-3 proteins may function as adapter or scaffold proteins in signal transduction pathways. We reported previously that several 14-3-3 isotypes bind to protein kinase C (PKC)-zeta and facilitate coupling of PKC-zeta to Raf-1 [van der Hoeven, van der Wal, Ruurs, van Dijk and van Blitterswijk (2000) Biochem. J. 345, 297-306], an event that boosts the mitogen-activated protein kinase (ERK) pathway in Rat-1 fibroblasts. The present work investigated whether bound 14-3-3 would affect PKC-zeta activity. Using recombinant 14-3-3 proteins and purified PKC-zeta in a convenient, newly developed in vitro kinase assay, we found that 14-3-3 proteins stimulated PKC-zeta activity in a dose-dependent fashion up to approx. 2.5-fold. Activation of PKC-zeta by 14-3-3 isotypes was unrelated to their mutual affinity, estimated by co-immunoprecipitation from COS cell lysates. Accordingly, PKC-zeta with a defective (point-mutated) 14-3-3-binding site, showed the same 14-3-3-stimulated activity as wild-type PKC-zeta. As 14-13-3 proteins are acidic, we tested several other acidic proteins, which turned out to stimulate PKC-zeta activity in a similar fashion, whereas neutral or basic proteins did not. These effects were not restricted to the atypical PKC-zeta, but were also found for classical PKC. Together, the results suggest that the stimulation of PKC activity by 14-3-3 proteins is non-specific and solely due to the acidic nature of these proteins, quite similar to that known for acidic lipids.
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PMID:Protein kinase C activation by acidic proteins including 14-3-3. 1076 83

The interaction of BAD (Bcl-2/Bcl-X(L)-antagonist, causing cell death) with Bcl-2/Bcl-X(L) is thought to neutralize the anti-apoptotic effects of the latter proteins, and may represent one of the mechanisms by which BAD promotes apoptosis. A variety of survival signals are reported to induce the phosphorylation of BAD at Ser(112) or Ser(136), triggering its dissociation from Bcl-2/Bcl-X(L). Ser(136) is thought to be phosphorylated by protein kinase B (PKB, also called Akt), which is activated when cells are exposed to agonists that stimulate phosphatidylinositol 3-kinase (PI3K). In contrast, Ser(112) is reported to be phosphorylated by mitogen-activated protein (MAP) kinase-activated protein kinase-1 (MAPKAP-K1, also called RSK) and by cAMP-dependent protein kinase (PKA). Here we identify Ser(155) as a third phosphorylation site on BAD. We find that Ser(155) is phosphorylated preferentially by PKA in vitro and is the only residue in BAD that becomes phosphorylated when cells are exposed to cAMP-elevating agents. The phosphorylation of BAD at Ser(155) prevents it from binding to Bcl-X(L) and promotes its interaction with 14-3-3 proteins. We also provide further evidence that MAPKAP-K1 mediates the phosphorylation of Ser(112) in response to agonists that activate the classical MAP kinase pathway. However insulin-like growth factor 1, a potent activator of PI3K and PKB does not increase the phosphorylation of Ser(136) in BAD-transfected HEK-293 cells, and nor is the basal level of Ser(136) phosphorylation suppressed by inhibitors of PI3K.
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PMID:Regulation of BAD by cAMP-dependent protein kinase is mediated via phosphorylation of a novel site, Ser155. 1088 Mar 54

The dynamic balance between polymerization and depolymerization of microtubules is critical for cells to enter and exit mitosis, and drugs that disrupt this balance, such as taxol, colchicine, and nocodazole, arrest the cell cycle in mitosis. Although the Raf/MEK/MAPK pathway can be activated by these drugs, its role in mitosis has not been addressed. Here, we characterize activation of Raf/MEK/MAPK by nocodazole when mitosis is induced. We find that at early time points (up to 3 h) in nocodazole induction, Raf/MEK/MAPK is activated, and inhibition of MAPK activation by a MEK inhibitor, PD98059 or U0126, reduces the number of cells entering mitosis by creating a block at G(2). At later time points and in mitosis, activation of MEK/MAPK is severely inhibited, even though Raf-1 activity remains high and can be further increased by growth factor. This inhibition is reversed when cells are released from metaphase and enter G(0)/G(1) phase. In addition, we find that binding of Raf-1 to 14-3-3 is progressively induced by nocodazole, reaching a maximum in mitosis, and that this binding is necessary to maintain mitotic Raf-1 activity. Our present study indicates that activation of the Raf/MEK/MAPK pathway is necessary for the G(2)/M progression.
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PMID:Raf-1/MEK/MAPK pathway is necessary for the G2/M transition induced by nocodazole. 1088 85

Cyclic AMP can either activate or inhibit the mitogen-activated protein kinase (MAPK) pathway in different cell types; MAPK activation has been observed in B-Raf-expressing cells and has been attributed to Rap1 activation with subsequent B-Raf activation, whereas MAPK inhibition has been observed in cells lacking B-Raf and has been attributed to cAMP-dependent protein kinase (protein kinase A)-mediated phosphorylation and inhibition of Raf-1 kinase. We found that cAMP stimulated MAPK activity in CHO-K1 and PC12 cells but inhibited MAPK activity in C6 and NB2A cells. In all four cell types, cAMP activated Rap1, and the 95- and 68-kDa isoforms of B-Raf were expressed. cAMP activation or inhibition of MAPK correlated with activation or inhibition of endogenous and transfected B-Raf kinase. Although all cell types expressed similar amounts of 14-3-3 proteins, approximately 5-fold less 14-3-3 was associated with B-Raf in cells in which cAMP was inhibitory than in cells in which cAMP was stimulatory. We found that the cell type-specific inhibition of B-Raf could be completely prevented by overexpression of 14-3-3 isoforms, whereas expression of a dominant negative 14-3-3 mutant resulted in partial loss of B-Raf activity. Our data suggest that 14-3-3 bound to B-Raf protects the enzyme from protein kinase A-mediated inhibition; the amount of 14-3-3 associated with B-Raf may explain the tissue-specific effects of cAMP on B-Raf kinase activity.
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PMID:Cell type-specific regulation of B-Raf kinase by cAMP and 14-3-3 proteins. 1093 30

The activation of cell cycle checkpoints in response to genotoxic stressors is essential for the maintenance of genomic integrity. Although most prior studies of cell cycle effects of UV irradiation have used UVC, this UV range does not penetrate the earth's atmosphere. Thus, we have investigated the mechanisms of ultraviolet B (UVB) irradiation-induced cell cycle arrest in a biologically relevant target cell type, the early stage human melanoma cell line, WM35. Irradiation of WM35 cells with UVB resulted in arrests throughout the cell cycle: at the G1/S transition, in S phase and in G2. G1 arrest was accompanied by increased association of p21 with cyclin E/cdk2 and cyclin A/cdk2, increased binding of p27 to cyclin E/cdk2 and inhibition of these kinases. A loss of Cdc25A expression was associated with an increased inhibitory phosphotyrosine content of cyclin E- and cyclin A-associated cdk2 and may also contribute to G1 arrest following UVB irradiation. The association of Cdc25A with 14-3-3 was increased by UVB. Reduced cyclin D1 protein and increased binding of p21 and p27 to cyclin D1/cdk4 complexes were also observed. The loss of cyclin D1 could not be attributed to inhibition of either MAPK or PI3K/PKB pathways, since both were activated by UVB. Cdc25B levels fell and the remaining protein showed an increased association with 14-3-3 in response to UVB. Losses in cyclin B1 expression and an increased binding of p21 to cyclin B1/cdk1 complexes also contributed to inhibition of this kinase activity, and G2/M arrest. Oncogene (2000) 19, 4480 - 4490.
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PMID:UVB induced cell cycle checkpoints in an early stage human melanoma line, WM35. 1100 21

We have found that the expression of five 14-3-3 protein isoforms is induced during the retinoic acid (RA)-mediated differentiation of mouse embryonal carcinoma F9 cells. The induced expression of the 14-3-3 proteins is presumed to have a role in enhancing the mitogen-activated protein kinase (MAPK) activity during RA-mediated F9 cell differentiation, because using genetically engineered budding yeast we showed that these isoforms enhanced the signaling in the MAPK cascade mainly through the interaction with Raf-1. Then we assessed the role of increased MAPK activity in F9 cell differentiation by interfering with signaling in the MAPK cascade in F9 cells. The exogenous expression of dominant-negative MEK1 efficiently abrogated RA-mediated induction of the cytokeratins EndoA and EndoC in the F9 cells. These results suggest that the 14-3-3 proteins play a role in the efficient induction of the cytokeratins during F9 cell differentiation through their signal enhancing activity in the MAPK cascade.
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PMID:14-3-3 protein family members have a regulatory role in retinoic acid-mediated induction of cytokeratins in F9 cells. 1101 Aug 14

CD43, one of the most abundant glycoproteins on the T cell surface, has been implicated in selection and maturation of thymocytes and migration, adhesion, and activation of mature T cells. The adapter molecule Cbl has been shown to be a negative regulator of Ras. Furthermore, it may also regulate intracellular signaling through the formation of several multi-molecular complexes. Here we investigated the role of Cbl in the CD43-mediated signaling pathway in human T cells. Unlike T cell receptor signaling, the interaction of the adapter protein Cbl with Vav and phosphatidylinositol 3-kinase, resulting from CD43-specific signals, is independent of Cbl tyrosine phosphorylation, suggesting an alternative mechanism of interaction. CD43 signals induced a Cbl serine phosphorylation-dependent interaction with the tau-isoform of 14-3-3. protein. Protein kinase C-mediated Cbl serine phosphorylation was required for this interaction, because the PKC inhibitor RO-31-8220 prevented it, as well as 14-3-3 dimerization. Moreover, mutation of Cbl serine residues 619, 623, 639, and 642 abolished the interaction between Cbl and 14-3-3. Overexpression of Cbl in Jurkat cells inhibited the CD43-dependent activation of the mitogen-activated protein kinase (MAPK) pathway and AP-1 transcriptional activity, confirming nevertheless a negative role for Cbl in T cell signaling. However, under normal conditions, PKC activation resulting from CD43 engagement was required to activate the MAPK pathway, suggesting that phosphorylation of Cbl on serine residues by PKC and its association with 14-3-3 molecules may play a role in preventing the Cbl inhibitory effect on the Ras-MAPK pathway. These data suggest that by inducing its phosphorylation on serine residues, CD43-mediated signals may regulate the molecular associations and functions of the Cbl adapter protein.
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PMID:Regulation of Cbl molecular interactions by the co-receptor molecule CD43 in human T cells. 1102 37

The 14-3-3 proteins are associated with proto-oncogene and oncogene products. Here, we generated NIH 3T3 cells overexpressing the beta isoform of the 14-3-3 proteins (14-3-3 beta) to examine the function of this isoform in cellular proliferation and oncogenic transformation. Overexpression of 14-3-3 beta in NIH 3T3 cells stimulated cell growth and supported anchorage-independent growth in soft agar medium and tumor formation in nude mice. To elucidate the molecular mechanisms of 14-3-3 beta-mediated NIH 3T3 transformation, we examined the activity of mitogen-activated protein kinase (MAPK) after serum stimulation. Overexpression of 14-3-3 beta augmented MAPK activity after serum stimulation, and MAPK activity correlated well with the amount of 14-3-3 beta expression. The colony-forming ability of NIH 3T3 cells overexpressing 14-3-3 beta in soft agar medium was efficiently abolished by exogenous expression of a dominant-negative mutant of MEK1 and 14-3-3 beta physically interacted with Raf-1 in these cells. These findings indicate that 14-3-3 beta has oncogenic potential, mainly through enhancement of Raf-1 activation and resultant augmentation of signaling in the MAPK cascade.
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PMID:Role of the beta isoform of 14-3-3 proteins in cellular proliferation and oncogenic transformation. 1106 70

The X protein from a chronic strain of hepatitis B virus (HBx) was determined to inhibit Fas-mediated apoptosis and promote cell survival. Fas-mediated apoptosis is the major cause of hepatocyte damage during liver disease. Experiments demonstrated that cell death caused by anti-Fas antibodies was blocked by the expression of HBx in human primary hepatocytes and mouse embryo fibroblasts. This effect was also observed in mouse erythroleukemia cells that lacked p53, indicating that protection against Fas-mediated apoptosis was independent of p53. Components of the signal transduction pathways involved in this protection were studied. The SAPK/JNK pathway has previously been suggested to be a survival pathway for some cells undergoing Fas-mediated apoptosis, and kinase assays showed that SAPK activity was highly up-regulated in cells expressing the HBx protein. Normal mouse fibroblasts expressing HBx were protected from death, whereas identical fibroblasts lacking the SEK1 component from the SAPK pathway succumbed to Fas-mediated apoptosis, whether HBx was present or not. Assays showed that caspase 3 and 8 activities and the release of cytochrome c from mitochondria were inhibited, in the presence of HBx, following stimulation with anti-Fas antibodies. Coprecipitation and confocal immunofluorescence microscopy experiments demonstrated that HBx localizes with a cytoplasmic complex containing MEKK1, SEK1, SAPK, and 14-3-3 proteins. Finally, mutational analysis of HBx demonstrated that a potential binding region for 14-3-3 proteins was essential for induction of SAPK/JNK activity and protection from Fas-mediated apoptosis.
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PMID:X protein of hepatitis B virus inhibits Fas-mediated apoptosis and is associated with up-regulation of the SAPK/JNK pathway. 1109 94


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