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)

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

Two major intracellular signals that regulate neuronal function are calcium and cAMP. In many cases, the actions of these two second messengers involve long term changes in gene expression. One well studied target of both calcium and cAMP signaling is the transcription factor cAMP-responsive element-binding protein (CREB). Multiple signaling pathways have been shown to contribute to the regulation of CREB-dependent transcription, including both protein kinase A (PKA)- and mitogen-activated protein (MAP) kinase/extracellular signal-regulated kinase (ERK)-dependent kinase cascades. We have previously described a mechanism by which cAMP and calcium influx may stimulate ERKs in neuronal cells. This pathway involves the PKA-dependent activation of the Ras-related small G-protein, Rap1, and subsequent stimulation of the neuronal Raf isoform, B-Raf. In this study, we examined the contribution of the Rap1-ERK pathway to the control of gene transcription by calcium influx and cAMP. Using the PC12 cell model system, we found that both calcium influx and cAMP stimulated CREB-dependent transcription via a Rap1-ERK pathway, but this regulation occurred through distinct mechanisms. Calcium-mediated phosphorylation of CREB through the PKA-Rap1-ERK pathway. In contrast, cAMP phosphorylated CREB via PKA directly but required a Rap1-ERK pathway to activate a component downstream of CREB phosphorylation and CREB-binding protein recruitment. These data suggest that the Rap1/B-Raf signaling pathway may have an important role in the regulation of CREB-dependent gene expression.
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PMID:Calcium and cAMP signals differentially regulate cAMP-responsive element-binding protein function via a Rap1-extracellular signal-regulated kinase pathway. 1095 Sep 54

Pertussis toxin (PTx), which inactivates G(i/o) type G proteins, is widely used to investigate the involvement of G(i/o) proteins in signal transduction. Activation of extracellular-regulated kinases 1 and 2 (ERK1/2) by G protein-coupled receptors has been described to occur either through a PTx-insensitive pathway involving activation of phospholipase C and protein kinase C (PKC), or through a PTx-sensitive pathway involving G(i)betagamma-mediated activation of Src. Cholecystokinin (CCK) activates ERK1/2 by a PKC-dependent, and thus presumably PTx-insensitive, pathway. However, CCK has recently been shown to induce activation of G(i) proteins in addition to G(q/11). In the present study, PTx partially inhibited CCK-induced ERK1/2 activation in pancreatic AR42J cells, although activation of phospholipase C was not reduced. PTx also inhibited ERK1/2 activation in response to the PKC activator 12-O-tetradecanoylphorbol-13-acetate (TPA) and epidermal growth factor (EGF) as well as activation of c-Raf-1 by EGF and CCK. In contrast, PTx, CCK, and EGF had only minor effects on A-Raf and B-Raf activity. Forskolin, a direct activator of adenylyl cyclase, inhibited CCK- and EGF-induced activation of c-Raf-1 and ERK1/2 in a manner similar to that of PTx. In PTx-treated cells, the cAMP content was increased and forskolin did not further inhibit CCK- and EGF-induced activation of c-Raf-1 or ERK1/2. In conclusion, the present study shows that PTx-sensitivity of receptor-induced ERK1/2 activation could be a consequence of disinhibition of the adenylyl cyclase signaling pathway, which in turn causes inhibition of c-Raf-1 activation rather than indicating involvement of a PTx-sensitive G protein in this signaling pathway.
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PMID:Pertussis toxin inhibits cholecystokinin- and epidermal growth factor-induced mitogen-activated protein kinase activation by disinhibition of the cAMP signaling pathway and inhibition of c-Raf-1. 1095 55

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

Neurotrophins promote multiple actions on neuronal cells including cell survival and differentiation. The best-studied neurotrophin, nerve growth factor (NGF), is a major survival factor in sympathetic and sensory neurons and promotes differentiation in a well-studied model system, PC12 cells. To mediate these actions, NGF binds to the TrkA receptor to trigger intracellular signaling cascades. Two kinases whose activities mediate these processes include the mitogen-activated protein (MAP) kinase (or extracellular signal-regulated kinase [ERK]) and phosphoinositide 3-kinase (PI3-K). To examine potential interactions between the ERK and PI3-K pathways, we studied the requirement of PI3-K for NGF activation of the ERK signaling cascade in dorsal root ganglion cells and PC12 cells. We show that PI3-K is required for TrkA internalization and participates in NGF signaling to ERKs via distinct actions on the small G proteins Ras and Rap1. In PC12 cells, NGF activates Ras and Rap1 to elicit the rapid and sustained activation of ERKs respectively. We show here that Rap1 activation requires both TrkA internalization and PI3-K, whereas Ras activation requires neither TrkA internalization nor PI3-K. Both inhibitors of PI3-K and inhibitors of endocytosis prevent GTP loading of Rap1 and block sustained ERK activation by NGF. PI3-K and endocytosis may also regulate ERK signaling at a second site downstream of Ras, since both rapid ERK activation and the Ras-dependent activation of the MAP kinase kinase kinase B-Raf are blocked by inhibition of either PI3-K or endocytosis. The results of this study suggest that PI3-K may be required for the signals initiated by TrkA internalization and demonstrate that specific endocytic events may distinguish ERK signaling via Rap1 and Ras.
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PMID:Role of phosphoinositide 3-kinase and endocytosis in nerve growth factor-induced extracellular signal-regulated kinase activation via Ras and Rap1. 1102 77

In PC12 cells, epidermal growth factor (EGF) transiently stimulates the mitogen-activated protein (MAP) kinases, ERK1 and ERK2, and provokes cellular proliferation. In contrast, nerve growth factor (NGF) stimulation leads to the sustained activation of the MAPKs and subsequently to neuronal differentiation. It has been shown that both the magnitude and longevity of MAPK activation governs the nature of the cellular response. The activations of MAPKs are dependent upon two distinct small G-proteins, Ras and Rap1, that link the growth factor receptors to the MAPK cascade by activating c-Raf and B-Raf, respectively. We found that Ras was transiently stimulated upon both EGF and NGF treatment of PC12 cells. However, EGF transiently activated Rap1, whereas NGF stimulated prolonged Rap1 activation. The activation of the ERKs was due almost exclusively (>90%) to the action of B-Raf. The transient activation of the MAPKs by EGF was a consequence of the formation of a short lived complex assembling on the EGF receptor itself, composed of Crk, C3G, Rap1, and B-Raf. In contrast, NGF stimulation of the cells resulted in the phosphorylation of FRS2. FRS2 scaffolded the assembly of a stable complex of Crk, C3G, Rap1, and B-Raf resulting in the prolonged activation of the MAPKs. Together, these data provide a signaling link between growth factor receptors and MAPK activation and a mechanistic explanation of the differential MAPK kinetics exhibited by these growth factors.
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PMID:Identification of the mechanisms regulating the differential activation of the mapk cascade by epidermal growth factor and nerve growth factor in PC12 cells. 1127 45

Thrombopoietin (TPO) regulates growth and differentiation of megakaryocytes. We previously showed that extracellular signal-regulated kinases (ERKs) are required for TPO-mediated full megakaryocytic maturation in both normal progenitors and a megakaryoblastic cell line (UT7) expressing the TPO receptor (Mpl). In these cells, intensity and duration of TPO-induced ERK signal are controlled by several regions of the cytoplasmic domain of Mpl. In this study, we explored the signaling pathways involved in this control. We show that the small GTPases Ras and Rap1 contribute together to TPO-induced ERK activation in UT7-Mpl cells and that they do so by activating different Raf kinases as downstream effectors: a Ras-Raf-1 pathway is required to initiate ERK activation while Rap1 sustains this signal through B-Raf. Indeed, (i) in cells expressing wild-type or mutant Mpl, TPO-induced Ras and Rap1 activation correlates with early and sustained phases of ERK signal, respectively; (ii) interfering mutants of Ras and Rap1 both inhibit ERK kinase activity and ERK-dependent Elk1 transcriptional activation in response to TPO; (iii) the kinetics of activation of Raf-1 and B-Raf by TPO follow those of Ras and Rap1, respectively; (iv) RasV12-mediated Elk1 activation was modulated by the wild type or interfering mutants of Raf-1 but not those of B-Raf; (v) Elk1 activation mediated by a constitutively active mutant of Rap1 (Rap1V12) is potentiated by B-Raf and inhibited by an interfering mutant of this kinase. UT7-Mpl cells represent the second cellular model in which Ras and Rap1 act in concert to modulate the duration of ERK signal in response to a growth factor and thereby the differentiation program. This is also, to our knowledge, the first evidence suggesting that Rap1 may play an active role in megakaryocytic maturation.
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PMID:Thrombopoietin-mediated sustained activation of extracellular signal-regulated kinase in UT7-Mpl cells requires both Ras-Raf-1- and Rap1-B-Raf-dependent pathways. 1128 46

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 Raf serine/threonine kinase plays an essential role to relay intracellular signals from the protooncogene Ras to activation of the mitogen-activated protein kinase (MAPK) cascade. The Raf kinase family consists of C-Raf (Raf-1), B-Raf, and A-Raf. Extensive efforts have been made in the last decade to study Raf regulation; however, precise molecular mechanism for Raf activation is still not fully understood. In this report, we discuss the current model of Raf regulation. Here we also report our recent findings that phosphorylation of Thr598 and Ser601, which lie between kinase subdomains VII and VIII, is essential for B-Raf activation by Ras. Substitution of these residues to alanine (B-RafAA) abolished Ras-induced B-Raf activation, without altering the association of B-Raf with other signaling proteins. Phosphopeptide mapping and immunoblotting with phosphospecific antibodies, which selectively recognize Thr598 and Ser601, phosphorylated B-Raf, confirmed that Thr598 and Ser601 are in vivo phosphorylation sites induced by Ras. Further, replacement of these two sites with acidic residues (B-RafED) renders B-Raf constitutively active. Consistent with these data, B-RafAA and B-RafED exhibited diminished and enhanced ability, respectively, to stimulate extracellular signal-regulated kinase (ERK) and Elk-dependent transcription. Moreover, functional studies revealed that B-RafED was able to promote NIH3T3 cell transformation and PC12 cell differentiation. Because Thr598 and Ser601 are conserved in all Raf family members, from Caenorhabditis elegans to mammals, we propose that phosphorylation of these two residues may be a general mechanism for Raf activation.
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PMID:Regulation of the Raf kinase by phosphorylation. 1129 29


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