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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)

Using transient transfection of HEK 293 cells, we have studied the activation of Ras, c-Raf, and MAP kinase by G protein-coupled receptors, activated G protein alpha subunit (G alpha), and beta gamma subunits (G beta gamma). The expression of constitutively activated Gs alpha, Gi alpha, and G11 alpha did not have any effect on MAP kinase phosphorylation. In contrast, overexpression of G beta gamma could stimulate the phosphorylation of MAP kinase and enhance the MEK kinase activity of c-Raf. Coexpression of dominant negative Ras inhibited G beta gamma-induced phosphorylation of MAP kinase. Furthermore, the GTP-bound form of Ras was increased by overexpression of G beta gamma. These results strongly suggest that the G beta gamma may play an important role in signaling from G protein-coupled receptors to the MAP kinase pathway, and the activation of Ras and c-Raf may be involved in this signaling cascade in HEK 293 cells.
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PMID:G protein beta gamma subunit activates Ras, Raf, and MAP kinase in HEK 293 cells. 761 78

Many receptors that couple to heterotrimeric guanine-nucleotide binding proteins (G proteins) have been shown to mediate rapid activation of the mitogen-activated protein kinases Erk1 and Erk2. In different cell types, the signaling pathways employed appear to be a function of the available repertoire of receptors, G proteins, and effectors. In HEK-293 cells, stimulation of either alpha1B- or alpha2A-adrenergic receptors (ARs) leads to rapid 5-10-fold increases in Erk1/2 phosphorylation. Phosphorylation of Erk1/2 in response to stimulation of the alpha2A-AR is effectively attenuated by pretreatment with pertussis toxin or by coexpression of a Gbetagamma subunit complex sequestrant peptide (betaARK1ct) and dominant-negative mutants of Ras (N17-Ras), mSOS1 (SOS-Pro), and Raf (DeltaN-Raf). Erk1/2 phosphorylation in response to alpha1B-AR stimulation is also attenuated by coexpression of N17-Ras, SOS-Pro, or DeltaN-Raf, but not by coexpression of betaARK1ct or by pretreatment with pertussis toxin. The alpha1B- and alpha2A-AR signals are both blocked by phospholipase C inhibition, intracellular Ca2+ chelation, and inhibitors of protein-tyrosine kinases. Overexpression of a dominant-negative mutant of c-Src or of the negative regulator of c-Src function, Csk, results in attenuation of the alpha1B-AR- and alpha2A-AR-mediated Erk1/2 signals. Chemical inhibitors of calmodulin, but not of PKC, and overexpression of a dominant-negative mutant of the protein-tyrosine kinase Pyk2 also attenuate mitogen-activated protein kinase phosphorylation after both alpha1B- and alpha2A-AR stimulation. Erk1/2 activation, then, proceeds via a common Ras-, calcium-, and tyrosine kinase-dependent pathway for both Gi- and Gq/11-coupled receptors. These results indicate that in HEK-293 cells, the Gbetagamma subunit-mediated alpha2A-AR- and the Galphaq/11-mediated alpha1B-AR-coupled Erk1/2 activation pathways converge at the level of phospholipase C. These data suggest that calcium-calmodulin plays a central role in the calcium-dependent regulation of tyrosine phosphorylation by G protein-coupled receptors in some systems.
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PMID:Ras-dependent mitogen-activated protein kinase activation by G protein-coupled receptors. Convergence of Gi- and Gq-mediated pathways on calcium/calmodulin, Pyk2, and Src kinase. 923 1

Mitogen-activated protein kinases (MAPKs) are activated by various extracellular stimuli. The signaling pathways from G protein-coupled receptors to extracellular signal-regulated kinase have been partially elucidated, whereas the mechanisms by which G protein-coupled receptors stimulate c-Jun N-terminal kinase (JNK) and p38 MAPK activities remain largely unknown. We have recently demonstrated that the signal from Gq/11-coupled m1 muscarinic acetylcholine receptor to p38 MAPK is mediated by both Galphaq/11 and Gbeta gamma in HEK-293 cells (Yamauchi, J., Nagao, M., Kaziro, Y., and Itoh, H. (1997) J. Biol. Chem. 272, 27771-27777). In the present study, we report that a constitutively activated mutant of Galpha11 (Galpha11 Q209L) activated not only p38 MAPK, but also JNK, and the activation of JNK and p38 MAPK by Galpha11 Q209L was partially inhibited by prolonged treatment with phorbol 12-myristate 13-acetate and calphostin C. In addition, the Galpha11 Q209L-stimulated activation of both kinases was blocked by a specific inhibitor of protein tyrosine kinases (PP2) and Csk (C-terminal Src kinase). Furthermore, we demonstrated that Galpha11 Q209L stimulated Src family kinase activity and induced tyrosine phosphorylation of several proteins in HEK-293 cells. These results suggest that Galphaq/11 stimulates JNK and p38 MAPK activities through protein kinase C- and Src family kinase-dependent signaling pathways.
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PMID:Involvement of protein kinase C and Src family tyrosine kinase in Galphaq/11-induced activation of c-Jun N-terminal kinase and p38 mitogen-activated protein kinase. 972 8

In response to bradykinin, phosphorylated MAP kinases (ERK-1 and ERK-2) were abundantly increased in HEK 293 cells, which overexpress the rat B2 kinin receptor. In a similar way des-Arg9-bradykinin stimulation of B1 kinin receptor-overexpressing HEK 293 cells caused activation of the same species of MAP kinase. Furthermore, nuclear translocation of transcription factor AP-1 was also found in the cells after stimulation with either agonist. PD98059, a MAP kinase kinase (MEK-1) inhibitor, blocked the agonist-induced AP-1 translocation as well as the phosphorylation of the MAP kinases. This communication provides the first evidence for both B1 and B2 kinin receptors mediating the MAP kinase signaling pathway to activate AP-1.
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PMID:Agonist stimulation of B1 and B2 kinin receptors causes activation of the MAP kinase signaling pathway, resulting in the translocation of AP-1 in HEK 293 cells. 975 66

Promiscuous coupling between G protein-coupled receptors and multiple species of heterotrimeric G proteins provides a potential mechanism for expanding the diversity of G protein-coupled receptor signaling. We have examined the mechanism and functional consequences of dual Gs/Gi protein coupling of the beta3-adrenergic receptor (beta3AR) in 3T3-F442A adipocytes. The beta3AR selective agonist disodium (R, R)-5-[2[[2-(3-chlorophenyl)-2-hydroxyethyl]-amino]propyl]-1, 3-benzodioxole-2,2-dicarboxylate (CL316,243) stimulated a dose-dependent increase in cAMP production in adipocyte plasma membrane preparations, and pretreatment of cells with pertussis toxin resulted in a further 2-fold increase in cAMP production by CL316,243. CL316,243 (5 microM) stimulated the incorporation of 8-azido-[32P]GTP into Galphas (1.57 +/- 0.12; n = 3) and Galphai (1. 68 +/- 0.13; n = 4) in adipocyte plasma membranes, directly demonstrating that beta3AR stimulation results in Gi-GTP exchange. The beta3AR-stimulated increase in 8-azido-[32P]GTP labeling of Galphai was equivalent to that obtained with the A1-adenosine receptor agonist N6-cyclopentyladenosine (1.56 +/- 0.07; n = 4), whereas inclusion of unlabeled GTP (100 microM) eliminated all binding. Stimulation of the beta3AR in 3T3-F442A adipocytes led to a 2-3-fold activation of mitogen-activated protein (MAP) kinase, as measured by extracellular signal-regulated kinase-1 and -2 (ERK1/2) phosphorylation. Pretreatment of cells with pertussis toxin (PTX) eliminated MAP kinase activation by beta3AR, demonstrating that this response required receptor coupling to Gi. Expression of the human beta3AR in HEK-293 cells reconstituted the PTX-sensitive stimulation of MAP kinase, demonstrating that this phenomenon is not exclusive to adipocytes or to the rodent beta3AR. ERK1/2 activation by the beta3AR was insensitive to the cAMP-dependent protein kinase inhibitor H-89 but was abolished by genistein and AG1478. These data indicate that constitutive beta3AR coupling to Gi proteins serves both to restrain Gs-mediated activation of adenylyl cyclase and to initiate additional signal transduction pathways, including the ERK1/2 MAP kinase cascade.
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PMID:The beta3-adrenergic receptor activates mitogen-activated protein kinase in adipocytes through a Gi-dependent mechanism. 1020 24

Mixed lineage kinases (MLKs) form a family of serin/threonine protein kinases with multiple protein/protein interaction domains (SH3, Cdc42 Rac interactive binding sequence, leucine zipper, and proline rich region), the physiological roles of which are largely unknown. We show that overexpression of wild type MLK3 leads to morphological transformation of NIH 3T3 fibroblasts and growth in soft agar. Consistent with this transforming potential, we demonstrate that MLK3 strongly induces transcription from a reporter construct that is driven by a composite AP-1-/Ets-1-enhancer element in HEK 293 cells. In the same cell system, MLK3 preferentially activates the c-Jun NH2-terminal kinase/stress-activated protein kinase (JNK/SAPK) mitogen-activated protein kinase cascade and to a lesser degree the extracellular signal-regulated kinase (ERK) pathway. Activation of the latter can be further enhanced by coexpression of wild type MEK1 and is blocked by the synthetic MEK inhibitor PD 098059 or a kinase-dead MEK1 mutant. Immunoprecipitated MLK3 catalyses the phosphorylation of MEK1 in vitro, but this phosphorylation leads only to a marginal activation. In support of these data, we also show that MEK1 is highly phosphorylated in vivo on Ser 217/221 in MLK3-transformed fibroblasts, whereas activating ERK phosphorylations are barely detectable. Nevertheless, MLK3-transformed NIH 3T3 fibroblasts are partially reverted when activation of MEK is specifically blocked with PD 098059. Our combined data show that although MLK3 is primarily an activator of the JNK/SAPK pathway, overexpression of the wild type protein leads to a transformed phenotype in NIH 3T3 cells that can be partially reversed by a synthetic MEK inhibitor. We conclude that the ERK pathway is necessary for MLK3-mediated transformation.
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PMID:The JNK/SAPK activator mixed lineage kinase 3 (MLK3) transforms NIH 3T3 cells in a MEK-dependent fashion. 1023 8

G protein-coupled receptors (GPCRs) initiate Ras-dependent activation of the Erk 1/2 mitogen-activated protein kinase cascade by stimulating recruitment of Ras guanine nucleotide exchange factors to the plasma membrane. Both integrin-based focal adhesion complexes and receptor tyrosine kinases have been proposed as scaffolds upon which the GPCR-induced Ras activation complex may assemble. Using specific inhibitors of focal adhesion complex assembly and receptor tyrosine kinase activation, we have determined the relative contribution of each to activation of the Erk 1/2 cascade following stimulation of endogenous GPCRs in three different cell types. The tetrapeptide RGDS, which inhibits integrin dimerization, and cytochalasin D, which depolymerizes the actin cytoskeleton, disrupt the assembly of focal adhesions. In PC12 rat pheochromocytoma cells, both agents block lysophosphatidic acid (LPA)- and bradykinin-stimulated Erk 1/2 phosphorylation, suggesting that intact focal adhesion complexes are required for GPCR-induced mitogen-activated protein kinase activation in these cells. In Rat 1 fibroblasts, Erk 1/2 activation via LPA and thrombin receptors is completely insensitive to both agents. Conversely, the epidermal growth factor receptor-specific tyrphostin AG1478 inhibits GPCR-mediated Erk 1/2 activation in Rat 1 cells but has no effect in PC12 cells. In HEK-293 human embryonic kidney cells, LPA and thrombin receptor-mediated Erk 1/2 activation is partially sensitive to both the RGDS peptide and tyrphostin AG1478, suggesting that both focal adhesion and receptor tyrosine kinase scaffolds are employed in these cells. The dependence of GPCR-mediated Erk 1/2 activation on intact focal adhesions correlates with expression of the calcium-regulated focal adhesion kinase, Pyk2. In all three cell types, GPCR-stimulated Erk 1/2 activation is significantly inhibited by the Src kinase inhibitors, herbimycin A and 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo-D-3,4-pyrimidine (PP1), suggesting that Src family nonreceptor tyrosine kinases represent a point of convergence for signals originating from either scaffold.
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PMID:Pleiotropic coupling of G protein-coupled receptors to the mitogen-activated protein kinase cascade. Role of focal adhesions and receptor tyrosine kinases. 1031 9

Agonist-elicited receptor sequestration is strikingly different for the alpha(2A)- versus alpha(2B)-adrenergic receptor (alpha(2)-AR) subtypes; the alpha(2B)-AR undergoes rapid and extensive disappearance from the HEK 293 cell surface, whereas the alpha(2A)-AR does not (Daunt, D. A., Hurt, C., Hein, L., Kallio, J., Feng, F., and Kobilka, B. K. (1997) Mol. Pharmacol. 51, 711-720; Eason, M. G., and Liggett, S. B. (1992) J. Biol. Chem. 267, 25473-25479). Since recent reports suggest that endocytosis is required for some G protein-coupled receptors to stimulate the mitogen-activated protein (MAP) kinase cascade (Daaka, Y., Luttrell, L. M., Ahn, S., Della Rocca, G. J., Ferguson, S. S., Caron, M. G., and Lefkowitz, R. J. (1998) J. Biol. Chem. 273, 685-688; Luttrell, L. M., Daaka, Y., Della Rocca, G. J., and Lefkowitz, R. J. (1997) J. Biol. Chem. 272, 31648-31656; Ignatova, E. G., Belcheva, M. M., Bohn, L. M., Neuman, M. C., and Coscia, C. J. (1999) J. Neurosci. 19, 56-63), we evaluated the differential ability of these two subtypes to activate MAP kinase. We observed no correlation between subtype-dependent agonist-elicited receptor redistribution and receptor activation of the MAP kinase cascade. Furthermore, incubation of cells with K(+)-depleted medium eliminated alpha(2B)-AR internalization but did not eliminate MAP kinase activation, suggesting that receptor internalization is not a general prerequisite for activation of the MAP kinase cascade via G(i)-coupled receptors. We also noted that neither dominant negative dynamin (K44A) nor concanavalin A treatment dramatically altered MAP kinase activation or receptor redistribution, indicating that these experimental tools do not universally block G protein-coupled receptor internalization.
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PMID:Stimulation of mitogen-activated protein kinase by G protein-coupled alpha(2)-adrenergic receptors does not require agonist-elicited endocytosis. 1045 69

The A(2A)-adenosine receptor, a prototypical G(s)-coupled receptor, activates mitogen-activated protein (MAP) kinase in a manner independent of cAMP in primary human endothelial cells. In order to delineate signaling pathways that link the receptor to the regulation of MAP kinase, the human A(2A) receptor was heterologously expressed in Chinese hamster ovary (CHO) and HEK293 cells. In both cell lines, A(2A) agonist-mediated cAMP accumulation was accompanied by activation of the small G protein rap1. However, rap1 mediates A(2A) receptor-dependent activation of MAP kinase only in CHO cells, the signaling cascade being composed of G(s), adenylyl cyclase, rap1, and the p68 isoform of B-raf. This isoform was absent in HEK293 cells. Contrary to CHO cells, in HEK293 cells activation of MAP kinase by A(2A) agonists was not mimicked by 8-bromo-cAMP, was independent of Galpha(s), and was associated with activation of p21(ras). Accordingly, overexpression of the inactive S17N mutant of p21(ras) and of a dominant negative version of mSos (the exchange factor of p21(ras)) blocked MAP kinase stimulation by the A(2A) receptor in HEK 293 but not in CHO cells. In spite of the close homology between p21(ras) and rap1, the S17N mutant of rap1 was not dominant negative because (i) overexpression of rap1(S17N) failed to inhibit A(2A) receptor-dependent MAP kinase activation, (ii) rap1(S17N) was recovered in the active form with a GST fusion protein comprising the rap1-binding domain of ralGDS after A(2A) receptor activation, and (iii) A(2A) agonists promoted the association of rap1(S17N) with the 68-kDa isoform of B-raf in CHO cells. We conclude that the A(2A) receptor has the capacity two activate MAP kinase via at least two signaling pathways, which depend on two distinct small G proteins, namely p21(ras) and rap1. Our observations also show that the S17N version of rap1 cannot be assumed a priori to act as a dominant negative interfering mutant.
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PMID:Activation of mitogen-activated protein kinase by the A(2A)-adenosine receptor via a rap1-dependent and via a p21(ras)-dependent pathway. 1046 24

The chemokine receptor CCR-7 is expressed in T, NK, and dendritic cells in a time-ordered and stimulus-dependent manner. Thorough analyses of the pharmacological profiles of the recombinant ligands for CCR-7, MIP-3beta/ELC/CK-beta 11, and SLC/Exodus-2/TCA4/6C-kine, using CCR-7-expressing HEK-293E transfectants determine that ligands both bind with a K(d) in the 100 pM range-10- to 100-fold greater affinities than published K(d) values. High-affinity binding of each ligand is associated with rapid mobilization of intracellular calcium and cell migration as predicted for chemokine GPCRs, and in keeping with more recent evidence, robust activation of mitogen-activated protein kinase (MAPK).
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PMID:Pharmacological and signaling analysis of human chemokine receptor CCR-7 stably expressed in HEK-293 cells: high-affinity binding of recombinant ligands MIP-3beta and SLC stimulates multiple signaling cascades. 1051 40


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