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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)

We previously purified a protein factor, named REKS (Ras-dependent Extracellular Signal-regulated Kinase (ERK)/mitogen-activated protein kinase Kinase (MEK) Stimulator), from Xenopus eggs by use of a cell-free assay system in which recombinant GTP gamma S (guanosine 5'-(3-O-thio)triphosphate)-Ki-Ras activates recombinant MEK. By use of this assay system, we purified here bovine REKS to near homogeneity from the cytosol fraction of bovine brain by successive chromatographies of Mono S, Mono Q, GTP gamma S-glutathione S-transferase-Ha-Ras-coupled glutathione-agarose, and Mono Q columns. It was composed of three proteins with masses of about 95, 32, and 30 kDa as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The 95-, 32-, and 30-kDa proteins were identified by immunoblot analysis to be B-Raf protein kinase, 14-3-3 protein, and 14-3-3 protein, respectively. Moreover, the REKS activity was specifically immunoprecipitated by an anti-B-Raf antibody. Bovine REKS was activated by lipid-modified GTP gamma S-Ki-Ras far more effectively than by a lipid-unmodified one. Lipid-modified GDP-Ki-Ras was inactive. Exogenous addition of 14-3-3 proteins stimulated further the REKS activity both in the presence and absence of GTP gamma S-Ki-Ras. These results indicate that at least one of the direct targets of Ras is B-Raf complexed with 14-3-3 proteins in bovine brain.
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PMID:Purification of a Ras-dependent mitogen-activated protein kinase kinase kinase from bovine brain cytosol and its identification as a complex of B-Raf and 14-3-3 proteins. 774 15

Osmotic shock induces a variety of biochemical and physiological responses in vertebrate cells. By analyzing extracts obtained from rat 3Y1 fibroblastic cells exposed to hyper-osmolar media, we have found that mitogen-activated protein kinases (MAPKs) and stress-activated protein kinases (SAPKs, also known as JNKs) are both activated in response to osmotic shock. MAPKK1 (MEK1) was also activated markedly. Furthermore, Raf-1 and MEKK were activated strikingly by the osmotic shock. Activation of Raf-1 and MEKK in response to osmotic shock was detected also in PC12 cells, in which MEKK activation by the osmotic shock was much stronger than that by epidermal growth factor. Activation of SAPKs in PC12 cells by the osmotic shock was also more marked than that by epidermal growth factor. The activated MEKK phosphorylated not only MAPKKs but also XMEK2, which is distantly related to MAPKK. Recombinant wild-type XMEK2, but not kinase-negative XMEK2, was able to phosphorylate and activate recombinant SAPK alpha in vitro. In addition, this activity of XMEK2 was activated by the activated MEKK. These results suggest that the MAPK cascade consisting of Raf-1, MAPKK, and MAPK and the SAPK cascade consisting of MEKK, XMEK2, and SAPK are both activated in response to osmotic shock. Finally, it was found that XMEK2 is a good substrate for SAPK.
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PMID:Activation of protein kinase cascades by osmotic shock. 775 32

In PC12 cells, cAMP stimulates the MAP kinase pathway by an unknown mechanism. Firstly, we examined the role of calcium ion mobilization and of protein kinase C in cAMP-stimulated MAP kinase activation. We show that cAMP stimulates p44mapk independently of these events. Secondly, we studied the role of B-Raf in this process. We observed that NGF, PMA and cAMP induce the phosphorylation of B-Raf as well as an upward shift in its electrophoretic mobility. We show that B-Raf is activated following NGF and PMA treatment of PC12 cells, and that it can phosphorylate and activate MEK-1. However, cAMP inhibits B-Raf autokinase activity as well as its ability to phosphorylate and activate MEK-1. This inhibition is likely to be due to a direct effect since we found that PKA phosphorylates B-Raf in vitro. Further, we show that B-Raf binds to p21ras, but more important, this binding to p21ras is virtually abolished with B-Raf from PC12 cells treated with CPT-cAMP. Hence, these data indicate that the PKA-mediated phosphorylation of B-Raf hampers its interaction with p21ras, which is responsible for the PKA-mediated decrease in B-Raf activity. Finally, our work suggests that in PC12 cells, cAMP stimulates MAP kinase through the activation of an unidentified MEK kinase and/or the inhibition of a MEK phosphatase.
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PMID:Regulation of the MAP kinase cascade in PC12 cells: B-Raf activates MEK-1 (MAP kinase or ERK kinase) and is inhibited by cAMP. 783 30

Saccharomyces cerevisiae FUS3/DAC2 protein kinase, a homolog of mammalian mitogen-activated protein (MAP) kinase, inactivates a G1 cyclin encoded by the CLN3 gene to arrest cell division in the G1 phase and activates a transcriptional factor STE12 in response to mating pheromone during sexual conjugation. To elucidate the role of the FUS3/DAC2 gene product in the mating process, I constructed and characterized dac2 cln3 double mutants. Here, I show that FUS3/DAC2 is required for completion of cell fusion even in the dac2 cln3 double mutants in which the pheromone response is restored, suggesting that FUS3/DAC2 plays a positive role in cell fusion during conjugation. In addition, the cdc dac2 and cdc37 ste double mutants were constructed and investigated for their phenotypes to clarify the relationship between FUS3/DAC2, STE7 or STE11 and CDC gene products (CDC28, 36, 37 and 39). The results indicate that FUS3/DAC2 may act upstream of CDC28 and provide evidence that the G1 arrest and morphological changes conferred by the cdc37 mutation may require FUS3/DAC2 (MAP kinase), STE7(MEK) and STE11 (MEK kinase).
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PMID:Yeast homolog of mammalian mitogen-activated protein kinase, FUS3/DAC2 kinase, is required both for cell fusion and for G1 arrest of the cell cycle and morphological changes by the cdc37 mutation. 784 75

We have used the two-hybrid system of Fields and Song to identify protein-protein interactions that occur in the pheromone response pathway of the yeast Saccharomyces cerevisiae. Pathway components Ste4p, Ste5p, Ste7p, Ste11p, Ste12p, Ste20p, Fus3p and Kss1p were tested in all pairwise combinations. All of the interactions we detected involved at least one member of the MAP kinase cascade that is a central element of the response pathway. Ste5p, a protein of unknown biochemical function, interacted with protein kinases that operate at each step of the MAP kinase cascade, specifically with Ste11p (an MEKK), Ste7p (an MEK), and Fus3p (a MAP kinase). This finding suggests that one role of Ste5p is to serve as a scaffold to facilitate interactions among members of the kinase cascade. In this role as facilitator, Ste5p may make both signal propagation and signal attenuation more efficient. Ste5p may also help minimize cross-talk with other MAP kinase cascades and thus ensure the integrity of the pheromone response pathway. We also found that both Ste11p and Ste7p interact with Fus3p and Kss1p. Finally, we detected an interaction between one of the MAP kinases, Kss1p, and a presumptive target, the transcription factor Ste12p. We failed to detect interactions of Ste4p or Ste20p with any other component of the response pathway.
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PMID:Protein-protein interactions in the yeast pheromone response pathway: Ste5p interacts with all members of the MAP kinase cascade. 785 59

Activation of the mitogen activated protein kinase (MAPK) plays essential roles in many signal transduction pathways. MAPK has been demonstrated to phosphorylate and regulate numerous cellular proteins, including growth factor receptor, transcription factors, cytoskeletal proteins, phospholipase and other protein kinases. Activation of MAPK requires phosphorylation of both threonine and tyrosine residues, which are catalysed by a single protein kinase known as MAPK kinase or MEK. MEK itself is activated by phosphorylation on two conserved serine residues. Three distinct mammalian Ser/Thr kinases, including Raf, Mos and MEKK (for MEK kinase), have been demonstrated to phosphorylate and activate MEK. The MAP kinase cascade is highly conserved in all eukaryotes and involved in numerous cellular responses. Activation of MAPK is a transient event that is tightly regulated by both kinases and phosphatases. A growth factor induced dual specific phosphatase is likely to play an important role in MAPK regulation.
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PMID:The mitogen activated protein kinase signal transduction pathway: from the cell surface to the nucleus. 785 62

The MAP kinase cascade is regulated by many hormones and growth factors and its activation leads to changes in properties of cytoplasmic, membrane-associated, and nuclear proteins. The MAP kinases themselves are activated by MEKS. MEKs lie at a point of convergence for multiple upstream signals, mediated by distinct protein kinases, Raf, MEK kinase, and Mos, all of which have MEK kinase activity. Additional inputs that stimulate the MAP kinase pathway are the activation of protein kinase C and the yeast protein kinase STE20. Mechanisms of regulation of some of the upstream components of this cascade have not yet been fully elucidated.
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PMID:Regulation of the MAP kinase cascade. 787 3

c-Mil is the avian homologue of the mammalian serine/threonine kinase c-Raf-1. c-Mil/Raf is a mediator of signal transduction leading to gene expression via the c-Jun DNA-binding site, AP-1. Here we show that c-Mil immunopurified from MC29-virus-transformed quail fibroblasts phosphorylates c-Jun in vitro near its N terminus (Ser-63 and -73). Furthermore, the viral oncogene product Gag-Mil of the avian wild-type retrovirus MH2 phosphorylates c-Jun in vitro. A contribution by other known kinases phosphorylating c-Jun, such as the mitogen-activated protein kinases (MAPKs) and the c-Jun N-terminal kinases, was excluded by control reactions. c-Raf-1 and c-Jun directly interact in vitro as shown by various immobilized glutathione S-transferase-Raf fusion proteins which specify the cysteine-rich region of c-Mil/Raf as the major N-terminal binding site. An additional minor binding site is located in the C-terminal region. The biological relevance of these results is demonstrated by coimmunoprecipitation of c-Jun and c-Mil from 32P-labeled MC29- and MH2-transformed fibroblasts as well as normal quail embryo fibroblasts, whereby c-Jun was identified by tryptic phosphopeptide analysis. The complexed c-Jun exhibits a decreased electrophoretic mobility corresponding to a more highly phosphorylated state. Cell fractionation analyses indicate that the c-Mil/c-Jun complex is located in the cytoplasm. The data demonstrate that c-Jun can be a direct target of the protein kinase c-Mil/Raf, suggesting an alternative pathway, which leads to c-Jun phosphorylation independent of the MAPKs and MAPK-related proteins.
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PMID:Direct interaction and N-terminal phosphorylation of c-Jun by c-Mil/Raf. 787 94

Tumor necrosis factor alpha (TNF alpha) is bound by two cell surface receptors, CD120a (p55) and CD120b (p75), that belong to the TNF/nerve growth factor receptor family and whose signaling is initiated by receptor multimerization in the plane of the plasma membrane. The initial signaling events activated by receptor crosslinking are unknown, although activation of the mitogen-activated protein kinase (MAPK) cascade occurs shortly after ligand binding to CD120a. In this study, we investigated the upstream kinases that mediate the activation of the 42-kDa MAPK p42mapk/erk2 following crosslinking of CD120a in mouse macrophages. Exposure of mouse macrophages to TNF alpha stimulated a time-dependent increase in the activity of MAPK/ERK kinase (MEK) that temporally preceded peak activation of p42mapk/erk2. MEKs, dual-specificity threonine/tyrosine kinases, act as a convergence point for several signaling pathways including Ras/Raf, MEK kinase (MEKK), and Mos. Incubation of macrophages with TNF alpha was found to transiently stimulate a MEKK that peaked in activity within 30 sec of exposure and progressively declined toward basal levels by 5 min. By contrast, under these conditions, activation of either c-Raf-1 or Raf-B was not detected. These data suggest that the activation of the MAPK cascade in response to TNF alpha is mediated by the sequential activation of a MEKK and a MEK in a c-Raf-1- and Raf-B-independent fashion.
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PMID:Tumor necrosis factor alpha rapidly activates the mitogen-activated protein kinase (MAPK) cascade in a MAPK kinase kinase-dependent, c-Raf-1-independent fashion in mouse macrophages. 787 28

Nerve growth factor (NGF) activates the mitogen-activated protein (MAP) kinase cascade through a p21ras-dependent signal transduction pathway in PC12 cells. The linkage between p21ras and MEK1 was investigated to identify those elements which participate in the regulation of MEK1 activity. We have screened for MEK activators using a coupled assay in which the MAP kinase cascade has been reconstituted in vitro. We report that we have detected a single NGF-stimulated MEK-activating activity which has been identified as B-Raf. PC12 cells express both B-Raf and c-Raf1; however, the MEK-activating activity was found only in fractions containing B-Raf. c-Raf1-containing fractions did not exhibit a MEK-activating activity. Gel filtration analysis revealed that the B-Raf eluted with an apparent M(r) of 250,000 to 300,000, indicating that it is present within a stable complex with other unidentified proteins. Immunoprecipitation with B-Raf-specific antisera quantitatively precipitated all MEK activator activity from these fractions. We also demonstrate that B-Raf, as well as c-Raf1, directly interacted with activated p21ras immobilized on silica beads. NGF treatment of the cells had no effect on the ability of B-Raf or c-Raf1 to bind to activated p21ras. These data indicate that this interaction was not dependent upon the activation state of these enzymes; however, MEK kinase activity was found to be associated with p21ras following incubation with NGF-treated samples at levels higher than those obtained from unstimulated cells. These data provide direct evidence that NGF-stimulated B-Raf is responsible for the activation of the MAP kinase cascade in PC12 cells, whereas c-Raf1 activity was not found to function within this pathway.
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PMID:The mitogen-activated protein kinase cascade is activated by B-Raf in response to nerve growth factor through interaction with p21ras. 793 11


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