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)

Agents which increase the intracellular cyclic GMP (cGMP) concentration and cGMP analogs inhibit cell growth in several different cell types, but it is not known which of the intracellular target proteins of cGMP is (are) responsible for the growth-suppressive effects of cGMP. Using baby hamster kidney (BHK) cells, which are deficient in cGMP-dependent protein kinase (G-kinase), we show that 8-(4-chlorophenylthio)guanosine-3', 5'-cyclic monophosphate and 8-bromoguanosine-3',5'-cyclic monophosphate inhibit cell growth in cells stably transfected with a G-kinase Ibeta expression vector but not in untransfected cells or in cells transfected with a catalytically inactive G-kinase. We found that the cGMP analogs inhibited epidermal growth factor (EGF)-induced activation of mitogen-activated protein (MAP) kinase and nuclear translocation of MAP kinase in G-kinase-expressing cells but not in G-kinase-deficient cells. Ras activation by EGF was not impaired in G-kinase-expressing cells treated with cGMP analogs. We show that activation of G-kinase inhibited c-Raf kinase activation and that G-kinase phosphorylated c-Raf kinase on Ser43, both in vitro and in vivo; phosphorylation of c-Raf kinase on Ser43 uncouples the Ras-Raf kinase interaction. A mutant c-Raf kinase with an Ala substitution for Ser43 was insensitive to inhibition by cGMP and G-kinase, and expression of this mutant kinase protected cells from inhibition of EGF-induced MAP kinase activity by cGMP and G-kinase, suggesting that Ser43 in c-Raf is the major target for regulation by G-kinase. Similarly, B-Raf kinase was not inhibited by G-kinase; the Ser43 phosphorylation site of c-Raf is not conserved in B-Raf. Activation of G-kinase induced MAP kinase phosphatase 1 expression, but this occurred later than the inhibition of MAP kinase activation. Thus, in BHK cells, inhibition of cell growth by cGMP analogs is strictly dependent on G-kinase and G-kinase activation inhibits the Ras/MAP kinase pathway (i) by phosphorylating c-Raf kinase on Ser43 and thereby inhibiting its activation and (ii) by inducing MAP kinase phosphatase 1 expression.
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PMID:Cyclic-GMP-dependent protein kinase inhibits the Ras/Mitogen-activated protein kinase pathway. 981 86

Signalling by the epidermal growth factor (EGF) receptor (EGFR) has been studied intensively, but for most cell types the analysis is complicated by the fact that EGFR not only homodimerizes but can also form heterodimers with other EGFR family members. Heterodimerization is a particular problem in the study of EGFR mutants, where the true phenotype of the mutants is confounded by the contribution of the heterodimer partner to signal transduction. We have made use of the murine hemopoietic cell line BaF/3, which does not express EGFR family members, to express wild-type (WT) EGFR, three kinase-defective EGFR mutants (V741G, Y740F, and K721R), or a C-terminally truncated EGFR (CT957) and have measured their responses to EGF. We found that under the appropriate conditions EGF can stimulate cell proliferation of BaF/3 cells expressing WT or CT957 EGFRs but not that of cells expressing the kinase-defective mutants. However, EGF promotes the survival of BaF/3 cells expressing either of the kinase-defective receptors (V741G and Y740F), indicating that these receptors can still transmit a survival signal. Analysis of the early signalling events by the WT, V741G, and Y740F mutant EGF receptors indicated that EGF stimulates comparable levels of Shc phosphorylation, Shc-GRB-2 association, and activation of Ras, B-Raf, and Erk-1. Blocking the mitogen-activated protein kinase (MAPK) signalling pathway with the specific inhibitor PD98059 abrogates completely the EGF-dependent survival of cells expressing the kinase-defective EGFR mutants but has no effect on the EGF-dependent proliferation mediated by WT and CT957 EGFRs. Similarly, the Src family kinase inhibitor PP1 abrogates EGF-dependent survival without affecting proliferation. However blocking phosphatidylinositol-3-kinase or JAK-2 kinase with specific inhibitors does arrest growth factor-dependent cell proliferation. Thus, EGFR-mediated mitogenic signalling in BaF/3 cells requires an intact EGFR tyrosine kinase activity and appears to depend on the activation of both the JAK-2 and PI-3 kinase pathways. Activation of the Src family of kinases or of the Ras/MAPK pathway can, however, be initiated by a kinase-impaired EGFR and is linked to survival.
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PMID:Activation of the Ras/mitogen-activated protein kinase pathway by kinase-defective epidermal growth factor receptors results in cell survival but not proliferation. 981 6

Raf is a key serine-threonine protein kinase which participates in the transmission of growth, anti-apoptotic and differentiation messages. These signals can be initiated after receptor ligation and are transmitted to members of the MAP kinase cascade that subsequently activate transcription factors controlling gene expression. Raf is a member of a multigene family which includes: Raf-1, A-Raf and B-Raf. The roles that individual Raf kinases play in the regulation of normal and malignant hematopoietic cell growth are not clear. The following studies show that all three Raf kinases are functionally present in certain human hematopoietic cells, and their aberrant expression can result in abrogation of cytokine dependency. Cytokine-dependent TF-1 cells were infected with retroviruses encoding amino-terminal deleted (delta) A-Raf, B-Raf and Raf-1 proteins. These Raf proteins were conditionally inducible as they were fused to the hormone-binding domain of the estrogen receptor (ER). A hierarchy in the abilities of Raf-containing retroviruses to abrogate cytokine dependency was observed as deltaA-Raf:ER was 20- to 200-fold more efficient than either deltaRaf-1:ER or deltaB-Raf:ER, respectively. This result was unexpected as A-Raf is an intrinsically weaker kinase than either Raf-1 or B-Raf. The activated Raf proteins induced downstream MEK and MAP (ERK1 and ERK2) kinase activities in the cells which proliferated in response to Raf activation. Furthermore, a functional MEK signaling pathway was necessary as treatment of the cells with a MEK1-inhibitor suppressed Raf-mediated proliferation. To determine whether the regulatory phosphorylation residues contained in the modified Raf oncoproteins were necessary for transformation, they were altered by site-directed mutagenesis. Substitution of the regulatory phosphorylation tyrosine residues with phenylalanine in either A-Raf or Raf-1 reduced the capacity of these oncoproteins to abrogate cytokine dependency. In contrast, changing the critical aspartic acid residues of B-Raf to either tyrosine or phenylalanine increased the frequency of estradiol-responsive cells. Thus, the amino acids present in the regulatory residues modulated the capability of Raf proteins to abrogate the cytokine dependency of TF-1 cells. Differences in the levels of Raf and downstream kinase activities were observed between cytokine-dependent and estradiol-responsive deltaRaf:ER-infected cells as estradiol-responsive cells usually expressed more Raf and MEK activity than GM-CSF-dependent, deltaRaf:ER-infected cells. Abrogation of cytokine dependency by the activated deltaRaf:ER proteins was associated with autocrine growth factor synthesis which was sufficient to promote the growth of uninfected TF-1 cells. In summary, these observations indicate that the aberrant expression of certain activated deltaRaf:ER oncoproteins can alter the cytokine dependency of human hematopoietic TF-1 cells. These cells will be useful in evaluating the roles of the individual Raf oncoproteins in signal transduction, cell cycle progression, autocrine transformation, regulation of apoptosis and differentiation. Moreover, these Raf-infected cells may be important in evaluating the efficacy of novel anticancer drugs designed to inhibit Raf and downstream signal transduction molecules.
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PMID:Differential abilities of activated Raf oncoproteins to abrogate cytokine dependency, prevent apoptosis and induce autocrine growth factor synthesis in human hematopoietic cells. 984 21

Evidence suggests that membrane depolarization is able to promote neuronal survival through a sustained, although moderate, increase in the intracellular calcium. We have used the PC12 cell line to study the possible intracellular pathways that can be activated by calcium influx. Previously, we observed that membrane depolarization-induced calcium influx was able to activate the extracellular-regulated kinase (ERK)/mitogen-activated protein kinase pathway and most of this activation was calmodulin-dependent. We demonstrated that a part of the ERK activation is due to the phosphorylation of the epidermal growth factor receptor. Here, we show that both the epidermal growth factor receptor phosphorylation and the Shc-Grb2-Ras activation are not calmodulin-modulated. Moreover, dominant negative mutant Ha-ras (Asn-17) prevents the activation on ERKs by membrane depolarization, suggesting that Ras and calmodulin are both necessaries to activate ERKs by membrane depolarization. We failed to observe any significant induction and/or modulation of the A-Raf, B-Raf or c-Raf-1 kinase activities, thus suggesting the existence of a MEK kinase different from the classical Raf kinases that directly or indirectly can be modulated by Ca2+/calmodulin.
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PMID:Calcium influx activates extracellular-regulated kinase/mitogen-activated protein kinase pathway through a calmodulin-sensitive mechanism in PC12 cells. 986 13

We have shown that estrogen elicits a selective enhancement of the growth and differentiation of axons and dendrites (neurites) in the developing CNS. We subsequently demonstrated widespread colocalization of estrogen and neurotrophin receptors (trk) within developing forebrain neurons and reciprocal transcriptional regulation of these receptors by their ligands. Using organotypic explants of the cerebral cortex, we tested the hypothesis that estrogen/neurotrophin receptor coexpression also may result in convergence or cross-coupling of their signaling pathways. Estradiol elicited rapid (within 5-15 min) tyrosine phosphorylation/activation of the mitogen-activated protein (MAP) kinases, ERK1 and ERK2, that persisted for at least 2 hr. This extracellular signal-regulated protein kinase (ERK) activation was inhibited successfully by the MEK1 inhibitor PD98059, but not by the estrogen receptor (ER) antagonist ICI 182,780, and did not appear to result from estradiol-induced activation of trk. Furthermore, we also found that estradiol elicited an increase in B-Raf kinase activity. The latter and subsequent downstream events leading to ERK activation may be a consequence of our documentation of a multimeric complex consisting of, at least, the ER, hsp90, and B-Raf. These novel findings provide an alternative mechanism for some of the estrogen actions in the developing CNS and could explain not only some of the very rapid effects of estrogen but also the ability of estrogen and neurotrophins to regulate the same broad array of cytoskeletal and growth-associated genes involved in neurite growth and differentiation.
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PMID:Estrogen-induced activation of mitogen-activated protein kinase in cerebral cortical explants: convergence of estrogen and neurotrophin signaling pathways. 995 96

TC21 is a member of the Ras superfamily of small GTP-binding proteins and, like Ras, has been implicated in the regulation of growth-stimulating pathways. Point mutations introduced into TC21 based on equivalent H-Ras oncogenic mutations are transforming in cultured cells, and oncogenic mutations in TC21 have been isolated from several human tumours. The mechanism of TC21 signalling in transformation is poorly understood. While activation of the serine/threonine kinases Raf-1 and B-Raf has been implicated in signalling pathways leading to transformation by H-Ras, it has been argued that TC21 does not activate Raf-1 or B-Raf. Since the Raf-signalling pathway is important in transformation by other Ras proteins, we assessed whether the Raf pathway is important to transformation by TC21. Raf-1 and B-Raf are constitutively active in TC21-transformed cells and the ERK/MAPK cascade is required for the maintenance of the transformed state. We demonstrate that oncogenic V23 TC21, like Ras, interacts with Raf-1 and B-Raf (but not with A-Raf), resulting in the translocation of the Raf proteins to the plasma membrane and in their activation. Furthermore, using point mutations in the effector loop of TC21, we show that the interaction of TC21 with Raf-1 is crucial for transformation.
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PMID:Activation of the Raf/MAP kinase cascade by the Ras-related protein TC21 is required for the TC21-mediated transformation of NIH 3T3 cells. 1006 93

Cytokines trigger the rapid assembly of multimolecular signaling complexes that direct the activation of downstream protein kinase cascades. Two protein kinases that have been linked to growth factor-regulated proliferation and survival are mitogen-activated protein/ERK kinase (MEK) and its downstream target Erk, a member of the mitogen-activated protein kinase family. Using complementary pharmacological and genetic approaches, we demonstrate that MEK and Erk activation requires a phosphatidylinositol 3-kinase (PI3-K)-generated signal in an interleukin (IL)-3-dependent myeloid progenitor cell line. Analysis of the upstream pathway leading to MEK activation revealed that inhibition of PI3-K did not block c-Raf activation, whereas MEK activation was effectively blocked under these conditions. Furthermore, agents that elevated cAMP suppressed IL-3-induced c-Raf activation but did not inhibit MEK activation. Because c-Raf activation and MEK activation were inversely affected by PI3-K- and cAMP-dependent pathways, we examined whether IL-3 activated the alternative Raf isoforms A-Raf and B-Raf. Although IL-3 did not activate B-Raf, A-Raf was activated by the cytokine. Moreover, A-Raf activation, like MEK activation, was blocked by inhibition of PI3-K but was insensitive to cAMP. Experiments with dominant negative mutants of the Raf isoforms showed that overexpression of dominant negative c-Raf did not prevent MEK activation. However, dominant negative A-Raf effectively blocked MEK activation, suggesting that activation of the MEK-Erk signaling cascade is mediated through A-Raf. Taken together, these results suggest that IL-3 receptors engage and activate both c-Raf and A-Raf in hemopoietic cells. However, these intermediates are differentially regulated by upstream signaling cascades and selectively coupled to downstream signaling pathways.
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PMID:A phosphatidylinositol 3-kinase-dependent pathway that differentially regulates c-Raf and A-Raf. 1006 54

The cAMP-dependent protein kinase (PKA) exhibits both inhibitory and stimulatory effects upon growth factor signaling mediated by the mitogen-activated protein kinase signaling pathway. PKA has been demonstrated to inhibit Raf-1-mediated cellular proliferation. PKA can both prevent Ras-dependent Raf-1 activation and directly inhibit Raf-1 catalytic activity. In contrast to the inhibitory effect of PKA on Raf-1-dependent processes, PKA potentiates nerve growth factor-stimulated PC12 cell differentiation, a B-Raf mediated process. This potentiation, rather than inhibition, of PC12 cell differentiation is curious in light of the ability of PKA to inhibit Raf-1 catalytic activity. The kinase domains of Raf-1 and B-Raf are highly conserved, and it has been predicted that B-Raf catalytic activity would also be inhibited by PKA. In this study we examined the ability of PKA to regulate the kinase activity of the B-raf proto-oncogene. We report that nerve growth factor-stimulated B-Raf activity is not inhibited by PKA. By contrast, an N-terminally truncated, constitutively active form of B-Raf is inhibited by PKA both in vitro and in transfected PC12 cells. These results suggest that the N-terminal regulatory domain interferes with the ability of PKA to modulate B-Raf catalytic activity and provide an explanation for the observed resistance of B-Raf-dependent processes to PKA inhibition.
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PMID:Nerve growth factor-stimulated B-Raf catalytic activity is refractory to inhibition by cAMP-dependent protein kinase. 1022 75

We demonstrated previously that in bovine tracheal myocytes, pretreatment with either forskolin or histamine significantly reduces both platelet-derived growth factor (PDGF)- and epidermal growth factor- induced Raf-1 activation but fails to inhibit extracellular signal-regulated kinase (ERK) activation substantially, evidence of a Raf-1-independent ERK activation pathway. To identify Raf-1-independent upstream signaling intermediates of mitogen-activated protein kinase/ERK kinase-1 (MEK1), the dual-function kinase required and sufficient for ERK activation in these cells, lysates from forskolin and PDGF-treated bovine tracheal myocytes were resolved using ion exchange chromatography. Kinase activity for MEK1 was assessed by in vitro phosphorylation assay. In all experiments, the major peak of MEK1 phosphorylation activity was detected in fractions 18 through 26 (80 to 160 mM NaCl), with the peak fraction eluting at a NaCl concentration of 140 mM. The ability of these fractions to activate MEK1 was confirmed by examining the phosphorylation of myelin basic protein, a known substrate for ERKs, in the presence of functional MEK1 and ERK1. Fractions containing kinase activity were also probed with antibodies against MEK kinase-1, Raf-1, A-Raf, B-Raf, Mos, and Tpl-2. None of these proteins was detected in fractions containing peak kinase activity, suggesting the presence of a novel PDGF-stimulated, forskolin-insensitive MEK1 kinase. Further separation of fractions holding peak MEK phosphorylation activity by gel filtration suggested an apparent molecular mass of 40 to 45 kD. We conclude that PDGF-induced activation of MEK1 in bovine tracheal myocytes is mediated at least in part by a novel kinase.
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PMID:Partial characterization of a novel mitogen-activated protein kinase/extracellular signal-regulated kinase activator in airway smooth-muscle cells. 1022 75

Estrogen elicits a selective enhancement of the growth and differentiation of axons and dendrites (neurites) in the developing brain. Widespread colocalization of estrogen and neurotrophin receptors (trk) within estrogen and neurotrophin targets, including neurons of the cerebral cortex, sensory ganglia, and PC12 cells, has been shown to result in differential and reciprocal transcriptional regulation of these receptors by their ligands. In addition, estrogen and neurotrophin receptor coexpression leads to convergence or cross-coupling of their signaling pathways, particularly at the level of the mitogen-activated protein (MAP) kinase cascade. 17beta-Estradiol elicits rapid (within 5-15 min) and sustained (at least 2 h) tyrosine phosphorylation and activation of the MAP kinases, extracellular-signal regulated kinase (ERK)1, and ERK2, which is successfully inhibited by the MAP kinase/ERK kinase 1 inhibitor PD98059, but not by the estrogen receptor (ER) antagonist ICI 182,780 and also does not appear to result from estradiol-induced activation of trk. Furthermore, the ability of estradiol to phosphorylate ERK persists even in ER-alpha knockout mice, implicating other estrogen receptors such as ER-beta in these actions of estradiol. The existence of an estrogen receptor-containing, multimeric complex consisting of hsp90, src, and B-Raf also suggests a direct link between the estrogen receptor and the MAP kinase signaling cascade. Collectively, these novel findings, coupled with our growing understanding of additional signaling substrates utilized by estrogen, provide alternative mechanisms for estrogen action in the developing brain which could explain not only some of the very rapid effects of estrogen, but also the ability of estrogen and neurotrophins to regulate the same broad array of cytoskeletal and growth-associated genes involved in neurite growth and differentiation. This review expands the usually restrictive view of estrogen action in the brain beyond the confines of sexual differentiation and reproductive neuroendocrine function. It considers the much broader question of estrogen as a neural growth factor with important influences on the development, survival, plasticity, regeneration, and aging of the mammalian brain and supports the view that the estrogen receptor is not only a ligand-induced transcriptional enhancer but also a mediator of rapid, nongenomic events.
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PMID:Novel mechanisms of estrogen action in the brain: new players in an old story. 1032 86


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