Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.12.2 (MEK)
18,161 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A constitutively active fragment of rat MEK kinase 1 (MEKK1) consisting of only its catalytic domain (MEKK-C) expressed in bacteria quantitatively activates recombinant mitogen-activated protein (MAP) kinase/extracellular signal-regulated protein kinase (ERK) kinases 1 and 2 (MEK1 and MEK2) in vitro. Activation of MEK1 by MEKK-C is accompanied by phosphorylation of S218 and S222, which are also phosphorylated by the protein kinases c-Mos and Raf-1. MEKK1 has been implicated in regulation of a parallel but distinct cascade that leads to phosphorylation of N-terminal sites on c-Jun; thus, its role in the MAP kinase pathway has been questioned. However, in addition to its capacity to phosphorylate MEK1 in vitro, MEKK-C interacts with MEK1 in the two-hybrid system, and expression of mouse MEKK1 or MEKK-C in mammalian cells causes constitutive activation of both MEK1 and MEK2. Neither cotransfected nor endogenous ERK2 is highly activated by MEKK1 compared to its stimulation by epidermal growth factor in spite of significant activation of endogenous MEK. Thus, other as yet undefined mechanisms may be involved in determining information flow through the MAP kinase and related pathways.
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PMID:MEKK1 phosphorylates MEK1 and MEK2 but does not cause activation of mitogen-activated protein kinase. 762 24

Ras proteins function through the formation of specific complexes with Raf-1, B-raf, PI-3 kinase and RalGDS. These interactions all require Ras-GTP with an intact effector binding domain (Switch I region). We have examined the requirements of the Switch II region (amino acids 60-72) for the production of stable interactions between Ras and its downstream effectors. A point mutation at position 65 or 64 combined with additional mutations at either position 65 or 71 rendered nucleotide-free Ras protein unable to stably interact with Ras specific guanine nucleotide exchange factors. Ha-Ras containing point mutations at positions 65 and 71 possessed a twofold higher affinity for B-raf and consequently MEK1. The point mutation at 64, in combination with additional point mutations at either position 65 or 71, resulted in a protein which failed to interact with either PI-3 kinase or neurofibromin, though these Ras mutants effectively bound both Raf-1 and B-raf. An activated form of Ras, Q61L-Ras, associated with all effector proteins independent of the bound guanine nucleotide. Q61L-Ras-GDP was almost as effective as wild type Ras-GMPPNP in the in vitro activation of MEK1 and MAP kinase. Competitive studies with the catalytic domain if neurofibromin, NF1-GRD, demonstrated that its interaction with Ras-GMPPNP is mutually exclusive with both Raf-1 and B-raf. These data suggest that rasGAP and neurofibromin are unable to downregulate Ras-GTP complexed to Raf-1 or B-raf.
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PMID:Different structural requirements within the switch II region of the Ras protein for interactions with specific downstream targets. 763 Jun 28

Mitogen-activated protein kinases (MAPKs) play a pivotal role in the mitogenic signal transduction pathway and are essential components of the MAPK cascade, which includes MEK (also known as MAP kinase kinase), Raf-1, and Ras. In this study, we examined whether constitutive activation of the MAPK cascade was associated with the carcinogenesis of human renal cell carcinomas in a series of 25 tumors and in corresponding normal kidneys. Constitutive activation of MAPKs in tumor tissue, as determined by the appearance of phosphorylated forms, was found in 12 cases (48%), and this activation was confirmed by a direct in vitro kinase assay of immunoprecipitate using myelin basic protein as the substrate. The phosphorylation of MEK and of Raf-1, as monitored by a mobility shift in SDS-PAGE, which is reportedly associated with the activation of these kinases, occurred in 9 of 18 cases (50%) and in 6 of 11 cases (55%) respectively. The activation of MAPKs was correlated with MEK activation (P = 0.0045) and with Raf-1 activation (P = 0.067). Furthermore, overexpression of MEK was found in 13 of 25 cases (52%) by Western blot analysis, and this overexpression was associated significantly with MAPK activation (P = 0.034). No mutations were noted in H-,K-, or N-ras genes by PCR direct sequencing in any of the 25 tumor samples. Of the patients studied, 8 of 18 (44%) stage pT2 patients and four of six (67%) stage pT3 patients showed MAPK activation. The single stage pT1 patient did not evidence MAPK activation. Furthermore, one of seven (14%) grade 1 patients, 9 of 13 (69%) grade 2 patients, and two of five (40%) grade 3 patients showed MAPK activation (grade 1 versus grades 2 and 3, P = 0.046). Our results suggest that constitutive activation of MAPKs may be associated with the carcinogenesis of human RCCs.
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PMID:Constitutive activation of mitogen-activated protein (MAP) kinases in human renal cell carcinoma. 766 95

One Ras-dependent protein kinase cascade leading from growth factor receptors to the ERK (extracellular signal-regulated kinases) subgroup of mitogen-activated protein kinases (MAPKs) is dependent on the protein kinase Raf-1, which activates the MEK (MAPK or ERK kinase) dual specificity kinases. A second protein kinase cascade leading to activation of the Jun kinases (JNKs) is dependent on MEKK (MEK kinase). A dual-specificity kinase that activates JNK, named JNKK, was identified that functions between MEKK and JNK. JNKK activated the JNKs but did not activate the ERKs and was unresponsive to Raf-1 in transfected HeLa cells. JNKK also activated another MAPK, p38 (Mpk2; the mammalian homolog of HOG1 from yeast), whose activity is regulated similarly to that of the JNKs.
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PMID:Identification of a dual specificity kinase that activates the Jun kinases and p38-Mpk2. 771 21

Ligation of membrane immunoglobulin M (mIgM) receptor in the Ramos B-cell line induced tyrosine phosphorylation of several intracellular substrates, including the adaptor protein. Shc. Phosphorylated Shc could be seen to associate with Grb2 in a complex which included hSOS. Inasmuch as hSOS is involved in p21ras activation, we also demonstrated that mIgM ligation activated a Ras-dependent kinase cascade in which sequential activation of Raf-1 and MEK-1 culminates in the activation of p42 mitogen-activated protein (MAP) kinase (ERK-2). The tumour promoter and protein kinase C agonist, phorbol 12-myristate 13-acetate (PMA), also activated Raf-1, MEK-1, and MAP kinase in Ramos cells, but did not induce tyrosine phosphorylation of Shc or Shc/Grb2 association. Okadaic acid, another tumour promoter and serine/threonine phosphatase inhibitor, activated p42 MAP kinase without activating Raf-1 or MEK-1, suggesting the existence of a serine/threonine phosphatase which directly regulates MAP kinase activity.
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PMID:The membrane immunoglobulin receptor utilizes a Shc/Grb2/hSOS complex for activation of the mitogen-activated protein kinase cascade in a B-cell line. 771 78

Prostaglandin (PG) F2 alpha activated mitogen-activated protein (MAP) kinase and MAP kinase kinase in NIH-3T3 cells by a mechanism that was completely inhibited by protein kinase inhibitors, staurosporine (20 nM) or H-7 (20 microM), but was insensitive to pretreatment with islet-activating protein (100 ng/ml; 24 h) or 12-O-tetradecanoylphorbol 13-acetate (2.5 microM; 24 h). PGF2 alpha stimulation also led to a significant increase in Ras.GTP complex. Transfection of a cDNA encoding a constitutively active mutant of Gq alpha-subunit (Q209L) mimicked PGF2 alpha-induced MAP kinase activation, increase in Ras.GTP complex, and DNA synthesis in these cells, suggesting that activation of Gq mediates the PGF2 alpha-activation of Ras-MAP kinase pathway and mitogenesis in NIH-3T3 cells. These data provide a new insight into regulatory mechanisms of Ras-MAP kinase pathway through heterotrimeric G-protein-mediated pathways.
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PMID:Prostaglandin F2 alpha stimulates formation of p21ras-GTP complex and mitogen-activated protein kinase in NIH-3T3 cells via Gq-protein-coupled pathway. 772 8

Interleukin 5 (IL-5) regulates the growth and function of eosinophils. The objective of this study was to investigate the intracellular signal transduction mechanism of IL-5 in eosinophils. Purified eosinophils were stimulated with IL-5, and the involvement of various kinases was investigated by immunoblotting, immune complex kinase assay, and in situ denatured/renatured kinase assay. We found that IL-5 induced tyrosine phosphorylation and activation of a number of kinases. Two species of lyn kinases (53 and 56 kD) were present in eosinophils. Both forms were Tyr-phosphorylated and activated rapidly within 1 min. Further, lyn kinase was physically associated with the IL-5 beta receptor in eosinophils. Ras was studied by immunoprecipitation followed by thin-layer chromatography. Ras bound higher quantities of [alpha-32P]guanosine 5'triphosphate upon stimulation with IL-5. Raf-1 kinase showed increased Tyr phosphorylation on immunoblotting and increased activity in the immune complex kinase assay. Two species of MEK (MAP or Erk kinase) (41 and 45 kD) were identified in eosinophils, which underwent autophosphorylation upon stimulation. Microtubule-associated protein (MAP) kinase (p44) was Tyr-phosphorylated on immunoblotting and had increased activity in the immune-complex kinase assay. MAP kinases were also studied after metabolic radiolabeling of the cells with [32P]orthophosphates. IL-5 stimulated phosphorylation of MAP kinases in situ. Thus, we have delineated major components of an important signaling pathway in eosinophils. We believe that one of the signals generated by IL-5 receptor activation is propagated through the lyn-Ras-Raf-1-MEK-MAP kinase pathway.
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PMID:The intracellular signal transduction mechanism of interleukin 5 in eosinophils: the involvement of lyn tyrosine kinase and the Ras-Raf-1-MEK-microtubule-associated protein kinase pathway. 772 58

Mitogen-activated protein kinase (MAPK) is one of the protein kinases activated during meiotic maturation of Xenopus laevis oocytes. The c-Mosxe protein kinase, which has been shown to be sufficient to promote germinal vesicle breakdown (GVBD) in meiosis I, can directly activate MAP kinase kinase in vitro and leads to the activation of MAPK in vivo. Recently we have shown that constitutively activated MAPK induces metaphase arrest when injected into one blastomere of a two-cell embryo. This arrest mimics the natural arrest of vertebrate unfertilized eggs in second meiotic metaphase due to cytostatic factor and c-Mosxe activity. We show here that microinjection of constitutively activated thiophosphorylated MAPK into resting oocytes is able to activate maturation-promoting factor (MPF) and promote GVBD. These results strongly support the hypothesis that MAPK plays an important role in the pathway that links c-Mosxe to the activation of MPF.
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PMID:Induction of Xenopus oocyte meiotic maturation by MAP kinase. 772 98

Activation of mitogen-activated protein kinase (MAP kinase) plays an important role in the cellular effects of nerve growth factor (NGF). Although the precise pathway by which NGF activates MAP kinase is not clear, several enzymes have been identified that may form a linear phosphorylation cascade, in which MAP kinase is activated by MAP kinase kinase (MEK). A key enzyme that links the ras-GTP complex to MEK is widely believed to be the raf kinase. However, immunoprecipitation experiments in PC-12 cells revealed that raf is not the major NGF-dependent MEK kinase [Zheng, Ohmichi, Saltiel and Guan (1994) Biochemistry 33, 5595-5599]. We have identified a protein kinase from PC-12 cells that catalyses both the phosphorylation and activation of MEK. This activity is stimulated 3-fold in cells treated with NGF. The partial purification on FPLC and characterization of this MEK kinase indicate that it is distinct from raf, MEK, MAP kinase and other previously described NGF-stimulated protein kinases. The activity of this enzyme is unaffected by direct addition to the assay of heparin, staurosporine, K252A and the heat-stable cyclic AMP-dependent kinase peptide inhibitor, but is slightly inhibited by NaF and calcium ions. Comparison of its behaviour on gel permeation and sucrose-density gradients indicates a molecular mass in the region of 50,000 Da. Moreover, isoelectric focusing of the enzyme revealed a pI of approx. 7.3. The kinase activity is specific for ATP as substrate with a Km of 11 microM, and requires Mg2+ as a cofactor. Analysis of the activation of this enzyme in PC-12 cells transfected with a dominant inhibitory mutant of p21ras suggests that this MEK kinase resides downstream of ras in the MAP kinase activation pathway. Moreover, site-directed mutation of the residues on MEK that are phosphorylated by raf does not completely abrogate phosphorylation by the MEK kinase, suggesting that this enzyme may share some phosphorylation sites with raf, but also phosphorylates MEK on other sites.
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PMID:Nerve growth factor stimulates a novel protein kinase in PC-12 cells that phosphorylates and activates mitogen-activated protein kinase kinase (MEK). 773 91

Mitogen-activated protein kinase kinase kinase (MEKK1) is a serine-threonine kinase that regulates sequential protein kinase pathways involving stress-activated protein kinases and mitogen-activated protein kinases. MEKK1 is activated in response to growth factor stimulation of cells and by expression of activated Ras. We demonstrate that the kinase domain of MEKK1 (MEKKCOOH) binds to GST-RasV12 in a GTP-dependent manner. Purified bacterially expressed MEKKCOOH binds to GST-RasV12(GTP gamma S) (GTP gamma S is guanosine 5'-3-O-(thio)triphosphate), demonstrating a direct interaction of the two proteins. A Ras effector domain peptide blocks the binding of MEKKCOOH to GST-RasV12(GTP gamma S). MEKKCOOH complexed with GST-RasV12(GTP gamma S) is capable of phosphorylating MEK1. These findings indicate that MEKK1 directly binds Ras.GTP. Thus, Ras interacts with protein kinases of both the Raf and MEKK families.
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PMID:Direct interaction between Ras and the kinase domain of mitogen-activated protein kinase kinase kinase (MEKK1). 774 23


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