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)

Growth factor-dependent kinases, such as phosphatidylinositol 3-kinase (PI 3-kinase) and Raf kinases, have been implicated in the suppression of apoptosis. We have recently established Rat-1 fibroblast cell lines overexpressing B-Raf, leading to activation of the MEK/Erk mitogen-activated protein kinase pathway. Overexpression of B-Raf confers resistance to apoptosis induced by growth factor withdrawal or PI 3-kinase inhibition. This is accompanied by constitutive activation of Erk without effects on the PI 3-kinase/Akt pathway. The activity of MEK is essential for cell survival mediated by B-Raf overexpression, since either treatment with the specific MEK inhibitor PD98059 or expression of a dominant inhibitory MEK mutant blocks the antiapoptotic activity of B-Raf. Activation of MEK is not only necessary but also sufficient for cell survival because overexpression of constitutively activated MEK, Ras, or Raf-1, like B-Raf, prevents apoptosis after growth factor deprivation. Overexpression of B-Raf did not interfere with the release of cytochrome c from mitochondria after growth factor deprivation. However, the addition of cytochrome c to cytosols of cells overexpressing B-Raf failed to induce caspase activation. It thus appears that the B-Raf/MEK/Erk pathway confers protection against apoptosis at the level of cytosolic caspase activation, downstream of the release of cytochrome c from mitochondria.
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PMID:B-Raf inhibits programmed cell death downstream of cytochrome c release from mitochondria by activating the MEK/Erk pathway. 1040 22

Mitogenic signaling involves protein kinases that phosphorylate the mitogen-activated protein kinase (MAPK) activator, MEK. In rats, basal hepatic MEK kinase activity is low in vivo in both adult rats and late gestation fetal rats, and is markedly stimulated by intraperitoneal administration of epidermal growth factor (EGF). The level of stimulated MEK phosphorylating activity is approximately 15 times higher in fetal liver than in adult liver. To identify regulated forms of the two categories of MEK kinase, Raf and MEKK, Western immunoblotting, immunoprecipitation kinase assays and immunodepletion studies were performed. Western immunoblotting confirmed that Raf-1, A-Raf, B-Raf, MEKK1 and MEKK2 were present at similar levels in E19 and adult liver. However, specific immunoprecipitation kinase assays did not detect any kinases that could account for marked EGF sensitivity or the higher level of activity in E19 fetuses. Immunodepletion studies produced a marked reduction in immunoreactive Raf/MEKK content and activity, but a minimal decrease in the ability of chromatography fractions to phosphorylate and activate recombinant MEK-1. Our results indicate that hepatic, EGF-sensitive MEK kinase activity may reside with a previously unidentified and physiologically relevant form of Raf and/or MEKK.
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PMID:Hepatic epidermal growth factor-regulated mitogen-activated protein kinase kinase kinase activity in the rat: lack of identity with known forms of raf and MEKK. 1064 42

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

We have shown previously in the developing cerebral cortex that estrogen elicits the rapid and sustained activation of multiple signaling proteins within the mitogen-activated protein (MAP) kinase cascade, including B-Raf and extracellular signal-regulated kinase (ERK). Using estrogen receptor (ER)-alpha gene-disrupted (ERKO) mice, we addressed the role of ER-alpha in mediating this action of estrogen in the brain. 17beta-Estradiol increased B-Raf activity and MEK (MAP kinase/ERK kinase)-dependent ERK phosphorylation in cerebral cortical explants derived from both ERKO and their wild-type littermates. The ERK response was stronger in ERKO-derived cultures but, unlike that of wild-type cultures, was not blocked by the estrogen receptor antagonist ICI 182,780. Surprisingly, both the ER-alpha selective ligand 16alpha-iodo-17beta-estradiol and the ER-beta selective ligand genistein failed to elicit ERK phosphorylation, suggesting that a different mechanism or receptor may mediate estrogen-induced ERK phosphorylation in the cerebral cortex. Interestingly, the transcriptionally inactive stereoisomer 17alpha-estradiol did elicit a strong induction of ERK phosphorylation, which, together with the inability of the ER-alpha- and ER-beta-selective ligands to elicit ERK phosphorylation, and of ICI 182,780 to block the actions of estradiol in ERKO cultures, supports the hypothesis that a novel, estradiol-sensitive and ICI-insensitive estrogen receptor may mediate 17beta-estradiol-induced activation of ERK in the brain.
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PMID:Estrogen-induced activation of the mitogen-activated protein kinase cascade in the cerebral cortex of estrogen receptor-alpha knock-out mice. 1068 71

In melanocytes and melanoma cells, cAMP activates extracellular signal-regulated kinases (ERKs) and MEK-1 by an unknown mechanism. We demonstrate that B-Raf is activated by cAMP in melanocytes. A dominant-negative mutant of B-Raf, but not of Raf-1, blocked the cAMP-induced activation of ERK, indicating that B-Raf is the MEK-1 upstream regulator mediating this cAMP effect. Studies using Clostridium sordelii lethal toxin and Clostridium difficile toxin B have suggested that Rap-1 or Ras might transduce cAMP action. We show that Ras, but not Rap-1, is activated cell-specifically and mediates the cAMP-dependent activation of ERKs, while Rap-1 is not involved in this process in melanocytes. Our results suggest a novel, cell-specific mechanism involving Ras small GTPase and B-Raf kinase as mediators of ERK activation by cAMP. Also, in melanocytes, Ras or ERK activation by cAMP is not mediated through protein kinase A activation. Neither the Ras exchange factor, Son of sevenless (SOS), nor the cAMP-responsive Rap-1 exchange factor, Epac, participate in the cAMP-dependent activation of Ras. These findings suggest the existence of a melanocyte-specific Ras exchange factor directly regulated by cAMP.
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PMID:Ras mediates the cAMP-dependent activation of extracellular signal-regulated kinases (ERKs) in melanocytes. 1085 35

The Raf oncoprotein plays critical roles in the transmission of mitogenic signals from cytokine receptors to the nucleus. There are three Raf family members: A-Raf, B-Raf and Raf-1. Conditionally active forms of the Raf proteins were created by ligating N-terminal truncated activated forms to the estrogen-receptor (ER) hormone-binding domain resulting in beta-estradiol-inducible constructs. We introduced these chimeric deltaRaf:ER oncoproteins into the murine FDC-P1 hematopoietic cell line. Two different types of cells were recovered after drug selection in medium containing either cytokine or beta-estradiol: (1) cytokine-dependent cells that expressed the deltaRaf:ER oncoproteins; and (2) Raf-responsive cells that grew in response to the deltaRaf:ER oncoprotein. Depending upon the particular deltaRaf:ER oncoprotein, cytokine-dependent cells were recovered 10(3) to 10(5) times more frequently than Raf-responsive cells. To determine whether BCL2 could synergize with the deltaRaf:ER oncoproteins and increase the frequency of cytokine-independent cells, cytokine-dependent deltaRaf:ER-expressing cells were infected with either a BCL2 containing retrovirus or an empty retroviral vector. BCL2 overexpression, by itself, did not relieve cytokine dependency of the parental cell line. However, BCL2 overexpression increased the frequency of Raf-responsive cells approximately five- to 100-fold. Cytokine-dependent deltaRaf:ER-infected cells entered the G1 phase of the cell cycle after cytokine withdrawal and entered S phase only after cytokine addition. Raf-responsive deltaRaf:ER cells entered the G1 phase of the cell cycle after estrogen deprivation and re-entered the cell cycle after addition of either IL-3 or the estrogen receptor antagonist tamoxifen which activates the deltaRaf:ER constructs. Expression of the BCL2 oncoprotein often delayed the exit from the S and G2/M phases demonstrating the protective effects BCL2 provided to these Raf and BCL2 infected cells. The deltaRaf:ER cells expressed the deltaRaf:ER proteins and downstream MEK and ERK activities after beta-estradiol treatment. Raf-responsive cells that were also infected with BCL2 expressed higher levels of BCL2 than the cells that were not infected with BCL2. Thus BCL2 can synergize with the activated Raf in the abrogation of cytokine dependency of certain hematopoietic cells. These cells will be useful in furthering our understanding of the roles of the Raf and BCL2 oncoproteins in hematopoietic cell growth, cell cycle progression and prevention of apoptosis.
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PMID:Synergy between Raf and BCL2 in abrogating the cytokine dependency of hematopoietic cells. 1086 73

We are investigating novel, non-transcriptionally mediated mechanisms that may contribute to the differentiative effects of oestrogen in developing forebrain neurons. Recent findings in the cerebral cortex document that 17 alpha- and 17 beta-oestradiol elicit rapid and sustained activation of the Ras-Raf-MAP kinase cascade, a major growth factor signalling pathway. Using oestrogen receptor (ER) alpha knockout (ERKO) mice, we addressed the identity of the receptor mediating activation of the MAP kinase cascade. 17 beta-oestradiol increased B-Raf activity and MEK-dependent ERK phosphorylation in explants of wild-type and ERKO cerebral cortex. Although neither the ER alpha-selective ligand, 16 alpha-iodo-17 beta-oestradiol (16 alpha-IE2) nor the ER beta-selective ligand, genistein, elicited ERK phosphorylation, as little as 0.1 nM 17 beta-oestradiol did so. Moreover, 16 alpha-IE2 acted as an inhibitory modulator of ERK activation, and the ER antagonist ICI 182 780 blocked oestradiol action only in wild-type cultures. These data suggest that neither ER alpha nor ER beta mediate activation of the MAP kinase cascade. A putative, novel, oestradiol-sensitive and ICI 182 780-insensitive receptor, designated ER-X may, rather, be involved. Association of ER-X with flotillin, the neuronal homologue of the caveolar protein, caveolin, places ER-X within plasma membrane caveolae and supports the hypothesis that a membrane-associated ER may mediate rapid oestrogen activation of the MAP kinase cascade.
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PMID:Novel sites and mechanisms of oestrogen action in the brain. 1096 2

Ras-induced cell transformation is mediated through distinct downstream signaling pathways, including Raf, Ral-GEFs-, and phosphatidylinositol 3-kinase (PI 3-kinase)-dependent pathways. In some cell types, strong activation of the Ras-Raf-MEK-extracellular signal-regulated kinase (ERK) cascade leads to cell cycle arrest rather than cell division. We previously reported that constitutive activation of this pathway induces sustained proliferation of primary cultures of postmitotic chicken neuroretina (NR) cells. We used this model system to investigate the respective contributions of Ras downstream signaling pathways in Ras-induced cell proliferation. Three RasV12 mutants (S35, G37, and C40) which differ by their ability to bind to Ras effectors (Raf, Ral-GEFs, and the p110 subunit of PI 3-kinase, respectively) were able to induce sustained NR cell proliferation, although none of these mutants was reported to transform NIH 3T3 cells. Furthermore, they all repressed the promoter of QR1, a neuroretina growth arrest-specific gene. Overexpression of B-Raf or activated versions of Ras effectors Rlf-CAAX and p110-CAAX also induced NR cell division. The mitogenic effect of the RasC40-PI 3-kinase pathway appears to involve Rac and RhoA GTPases but not the antiapoptotic Akt (protein kinase B) signaling. Division induced by RasG37-Rlf appears to be independent of Ral GTPase activation and presumably requires an unidentified mechanism. Activation of either Ras downstream pathway resulted in ERK activation, and coexpression of a dominant negative MEK mutant or mKsr-1 kinase domain strongly inhibited proliferation induced by the three Ras mutants or by their effectors. Similar effects were observed with dominant negative mutants of Rac and Rho. Thus, both the Raf-MEK-ERK and Rac-Rho pathways are absolutely required for Ras-induced NR cell division. Activation of these two pathways by the three distinct Ras downstream effectors possibly relies on an autocrine or paracrine loop, implicating endogenous Ras, since the mitogenic effect of each Ras effector mutant was inhibited by RasN17.
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PMID:Induction of postmitotic neuroretina cell proliferation by distinct Ras downstream signaling pathways. 1098 23

In this study we examine signaling pathways linking the M(1) subtype of muscarinic acetylcholine receptor (M(1) mAChR) to activation of extracellular signal-regulated kinases (ERK) 1 and 2 in neuronal PC12D cells. We first show that activation of ERK1/2 by the M(1) mAChR agonist carbachol takes place primarily via a Ras-independent pathway that depends largely upon Rap1, another small GTP-binding protein in the Ras family. Rap1 in turn activates B-Raf, an upstream activator of ERK1/2. Consistent with these results, carbachol was found to activate Rap1 more potently than Ras. Similar to other small GTP-binding proteins, activation of Rap1 requires a guanine nucleotide exchange factor (GEF) to promote its conversion from the GDP- to GTP-bound form. Using specific antibodies, we show that a recently identified Rap1 GEF, calcium- and diacylglycerol-regulated guanine nucleotide exchange factor I (CalDAG-GEFI), is expressed in PC12D cells and that carbachol stimulates the formation of a complex containing CalDAG-GEFI, Rap1, and activated B-Raf. Finally, we show that expression of CalDAG-GEFI antisense RNA largely blocks carbachol-stimulated activation of hemagglutinin (HA)1-tagged B-Raf and formation of the CalDAG-GEFI/Rap1/HA1-tagged B-Raf complex. Together, these data define a novel signaling pathway for M(1) mAChR, where increases in Ca(2+) and diacylglycerol stimulate the sequential activation of CalDAG-GEFI, Rap1, and B-Raf, resulting in the activation of MEK and ERK1/2.
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PMID:A CalDAG-GEFI/Rap1/B-Raf cassette couples M(1) muscarinic acetylcholine receptors to the activation of ERK1/2. 1129 31

The PI3K/Akt and Raf/MEK/ERK signal transduction cascades are pivotal in transmitting signals from membrane receptors to downstream targets that regulate apoptosis, gene expression, and cell growth. The abilities of activated PI3K, Akt, Raf, and MEK proteins to abrogate the cytokine dependence of three different hematopoietic cell lines were determined. Activated PI3K or Akt expression by themselves did not efficiently annul cytokine dependence. Raf and MEK could abrogate the cytokine dependence of murine FDC-PI and human TF-1 cells; however, the frequency of transformation was dependent on the particular oncogene examined, as more factor-independent cells were isolated after infection with activated retroviruses encoding A-Raf or Raf-1 than were with MEK1 or B-Raf. Cytokine-independent deltaRaf-1-infected cells formed tumors on injection into immunocompromised mice, whereas cytokine-dependent cell lines did not, demonstrating the oncogenic effects of activation of the Raf/MEK/ERK pathway. Overexpression of the antiapoptotic Bcl-2 protein synergized with activation of the Raf/MEK/ERK cascade and increased the efficiency of transformation of FDC-PI and TF-1 cells. In contrast to the results observed with FDC-P1 and TF-I cells, the activated Raf genes did not relieve the cytokine dependence of murine FL5.12 cells. The abilities of the Raf and PI3K pathways to interact and annul the cytokine dependence of FL5.12 cells were determined. The combination of Raf and either PI3K or Akt expression relieved cytokine dependence of some FL5.12 cells, and the efficiency of transformation could be enhanced further by Bcl-2 or Bcl-XL overexpression. Thus, the antiapoptotic PI3K/Akt and Bcl-2/Bcl-XL proteins can interact with the growth-promoting Raf/MEK/ERK pathway and annul the cytokine dependence of certain hematopoietic cells.
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PMID:Interactions between the PI3K and Raf signaling pathways can result in the transformation of hematopoietic cells. 1153 Oct 15


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