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:2.7.10.1 (ERK)
95,504 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Using both confocal immunofluorescence microscopy and biochemical approaches, we have examined the role of beta-arrestins in the activation and targeting of extracellular signal-regulated kinase 2 (ERK2) following stimulation of angiotensin II type 1a receptors (AT1aR). In HEK-293 cells expressing hemagglutinin-tagged AT1aR, angiotensin stimulation triggered beta-arrestin-2 binding to the receptor and internalization of AT1aR-beta-arrestin complexes. Using red fluorescent protein-tagged ERK2 to track the subcellular distribution of ERK2, we found that angiotensin treatment caused the redistribution of activated ERK2 into endosomal vesicles that also contained AT1aR-beta-arrestin complexes. This targeting of ERK2 reflects the formation of multiprotein complexes containing AT1aR, beta-arrestin-2, and the component kinases of the ERK cascade, cRaf-1, MEK1, and ERK2. Myc-tagged cRaf-1, MEK1, and green fluorescent protein-tagged ERK2 coprecipitated with Flag-tagged beta-arrestin-2 from transfected COS-7 cells. Coprecipitation of cRaf-1 with beta-arrestin-2 was independent of MEK1 and ERK2, whereas the coprecipitation of MEK1 and ERK2 with beta-arrestin-2 was significantly enhanced in the presence of overexpressed cRaf-1, suggesting that binding of cRaf-1 to beta-arrestin facilitates the assembly of a cRaf-1, MEK1, ERK2 complex. The phosphorylation of ERK2 in beta-arrestin complexes was markedly enhanced by coexpression of cRaf-1, and this effect is blocked by expression of a catalytically inactive dominant inhibitory mutant of MEK1. Stimulation with angiotensin increased the binding of both cRaf-1 and ERK2 to beta-arrestin-2, and the association of beta-arrestin-2, cRaf-1, and ERK2 with AT1aR. These data suggest that beta-arrestins function both as scaffolds to enhance cRaf-1 and MEK-dependent activation of ERK2, and as targeting proteins that direct activated ERK to specific subcellular locations.
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PMID:Activation and targeting of extracellular signal-regulated kinases by beta-arrestin scaffolds. 1122 59

The mitogen-activated protein kinase (MAP kinase) signalling cascade activated by fibroblast growth factors (FGF1 and FGF2) was analysed in a model system, Xenopus oocytes, expressing fibroblast growth factor receptors (FGFR1 and FGFR4). Stimulation of FGFR1 by FGF1 or FGF2 and FGFR4 by FGF1 induced a sustained phosphorylation of extracellular signal-regulated protein kinase 2 (ERK2) and meiosis reinitiation. In contrast, FGFR4 stimulation by FGF2 induced an early transient activation of ERK2 and no meiosis reinitiation. FGFR4 transduction cascades were differently activated by FGF1 and FGF2. Early phosphorylation of ERK2 was blocked by the dominant negative form of growth factor-bound protein 2 (Grb2) and Ras, for FGF1-FGFR4 and FGF2-FGFR4. The phosphatidylinositol 3-kinase (PI3 kinase) inhibitors wortmannin and LY294002 only prevented the early ERK2 phosphorylation triggered by FGF2-FGFR4 but not by FGF1-FGFR4. ERK2 phosphorylation triggered by FGFR4 depended on the Grb2/Ras pathway and also involved PI3 kinase in a time-dependent manner.
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PMID:Fibroblast growth factors 1 and 2 differently activate MAP kinase in Xenopus oocytes expressing fibroblast growth factor receptors 1 and 4. 1133 93

Vaccinia virus (VV) triggers a mitogenic signal at an early stage of infection. VV-induced proto-oncogene c-fos mRNA with kinetics paralleling that stimulated by serum. The VV virokine, or vaccinia virus growth factor (VGF), was not crucial for c-fos induction because it was observed upon infection with the virokine-minus mutant VV (VGF(-)). Furthermore, c-fos expression did not require infectious virus particles, as it occurred even with UV-inactivated VV and was equally induced by the different multiplicities of infection, i.e. 1.0, 5.0, and 25.0. c-fos expression was preceded by VV-induced DNA binding activity and was mediated via the cis-acting elements serum response element (SRE), activating protein-1 (AP-1), and cAMP-response element (CRE). VV activated the protein kinases p42MAPK/ERK2 and p44MAPK/ERK1 and the transcription factor ATF1 in a time-dependent manner with kinetics that paralleled those of VV-stimulated DNA-protein complex formation. The mitogenic signal transmission pathways leading to c-fos activation upon VV infection were apparently mediated by the protein kinases MEK, ERK, and PKA. This assumption was based on the findings that: 1) c-fos transcript was down-regulated; 2) the SRE, AP-1, and CRE binding activities were significantly reduced; and 3) the activation of p42MAPK/ERK2, p44MAPK/ERK1, and ATF1 were drastically affected when the viral infections were carried out in the presence of specific protein kinase inhibitor. Moreover, the mutant VV (VGF(-)) was also able to activate ERK1/2. It is noteworthy that virus multiplication was equally affected by the same kinase inhibitors. Taken together, our data provide evidence that the early mitogenic signal triggered upon VV infection relies upon the activation of the protein kinases MEK, ERK, and PKA, which are needed for both signal transduction and virus multiplication.
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PMID:A mitogenic signal triggered at an early stage of vaccinia virus infection: implication of MEK/ERK and protein kinase A in virus multiplication. 1145 35

Brain subjected to acute ischemic attack caused by an arterial blockage needs immediate arterial recanalization. However, restoration of cerebral blood flow can cause tissue injury, which is termed reperfusion injury. It is important to inhibit reperfusion injury to achieve greater brain protection. Because oxidative stress has been shown to activate mitogen-activated protein kinases (MAPKs), and because oxidative stress contributes to reperfusion injury, MAPK may be a potential target to inhibit reperfusion injury after brain ischemia. Here, we demonstrate that reperfusion after forebrain ischemia dramatically increases phosphorylation level of extracellular signal-regulated kinase 2 (ERK2) in the gerbil hippocampus. In addition, i.v. administration of U0126 (100-200 mg/kg), a specific inhibitor of MEK (MAPK/ERK kinase), protects the hippocampus against forebrain ischemia. Moreover, treatment with U0126 at 3 h after ischemia significantly reduces infarct volume after transient (3 h) focal cerebral ischemia in mice. This protection is accompanied by reduced phosphorylation level of ERK2, substrates for MEK, in the damaged brain areas. Furthermore, U0126 protects mouse primary cultured cortical neurons against oxygen deprivation for 9 h as well as nitric oxide toxicity. These results provide further evidence for the role of MEK/ERK activation in brain injury resulting from ischemia/reperfusion, and indicate that MEK inhibition may increase the resistance of tissue to ischemic injury.
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PMID:Intravenous administration of MEK inhibitor U0126 affords brain protection against forebrain ischemia and focal cerebral ischemia. 1157 56

We have recently shown that the platelet integrin alpha(IIb)beta(3) is activated by von Willebrand factor (vWF) binding to its platelet receptor, glycoprotein Ib-IX (GPIb-IX), via the protein kinase G (PKG) signaling pathway. Here we show that GPIb-IX-mediated activation of integrin alpha(IIb)beta(3) is inhibited by dominant negative mutants of Raf-1 and MEK1 in a reconstituted integrin activation model in Chinese hamster ovary (CHO) cells and that the integrin-dependent platelet aggregation induced by either vWF or low dose thrombin is inhibited by MEK inhibitors PD98059 and U0126. Thus, mitogen-activated protein kinase (MAPK) pathway is important in GPIb-IX-dependent activation of platelet integrin alpha(IIb)beta(3). Furthermore, vWF binding to GPIb-IX induces phosphorylation of Thr-202/Tyr-204 of extracellular signal-regulated kinase 2 (ERK2). GPIb-IX-induced ERK2 phosphorylation is inhibited by PKG inhibitors and enhanced by overexpression of recombinant PKG. PKG activators also induce ERK phosphorylation, indicating that activation of MAPK pathway is downstream from PKG. Thus, our data delineate a novel integrin activation pathway in which ligand binding to GPIb-IX activates PKG that stimulates MAPK pathway, leading to integrin activation.
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PMID:A mitogen-activated protein kinase-dependent signaling pathway in the activation of platelet integrin alpha IIbbeta3. 1152 89

The mitogen-activated protein kinases (MAP kinases) play a central role in signaling pathways initiated by extracellular stimuli such as growth factors, cytokines, and various forms of environmental stress. Full activation of the MAP kinases requires dual phosphorylation of the Thr and Tyr residues in the TXY motif of the activation loop by MAP kinase kinases. Interestingly, down-regulation of MAP kinase activity can be initiated by multiple Ser/Thr phosphatases, Tyr-specific phosphatases, and dual-specificity phosphatases. This would inevitable lead to the formation of monophosphorylated MAP kinases. However, in much of the literature investigating MAP kinase signaling, there has been the implicit assumption that the monophosphorylated forms are inactive. Thus, the significance for the need of multiple phosphatases in regulating MAP kinase activity is not clear, and the biological functions of these monophosphorylated MAP kinases are currently unknown. We have prepared extracellular signal-regulated protein kinase 2 (ERK2) in all phosphorylated forms and kinetically characterized them using two proteins (the myelin basic protein and Elk-1) and ATP as substrates. Our results revealed that a single phosphorylation in the activation loop of ERK2 produces an intermediate activity state. Thus, the catalytic efficiencies of the monophosphorylated ERK2/pY and ERK2/pT (ERK2 phosphorylated on Tyr-185 and Thr-183, respectively) are approximately 2-3 orders of magnitude higher than that of the unphosphorylated ERK2 and are only 1-2 orders of magnitude lower than that of the fully active bisphosphorylated ERK2/pTpY. This raises the possibility that the monophosphorylated ERK2s may have distinct biological roles in vivo. Different phosphorylation states in the activation loop could be linked to graded effects on a single ERK2 function. Alternatively, they could be linked to distinct ERK2 functions. Although less active than the bisphosphorylated species, the monophosphorylated ERK2s may differentially phosphorylate pathway components.
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PMID:The activity of the extracellular signal-regulated kinase 2 is regulated by differential phosphorylation in the activation loop. 1183 61

The activities of many protein kinases are regulated by phosphorylation. The phosphorylated protein kinases thus represent an important class of substrates for protein phosphatases. However, our ability to study the phosphatase-catalyzed substrate dephosphorylation has been limited in many cases by the difficulty in preparing sufficient amount of stoichiometrically phosphorylated kinases. We have applied the kinetic theory of substrate reaction during irreversible modification of enzyme activity to the study of phosphatase-catalyzed regulation of kinase activity. As an example, we measured the effect of the hematopoietic protein-tyrosine phosphatase (HePTP) on the reaction catalyzed by the fully activated, bisphosphorylated extracellular signal-regulated protein kinase 2 (ERK2/pTpY). Because only a catalytic amount of ERK2/pTpY is required, this method alleviates the need for large quantities of phospho-ERK2. Kinetic analysis of the ERK2/pTpY-catalyzed substrate reaction in the presence of HePTP leads to the determination of the rate constants for the HePTP-catalyzed dephosphorylation of free ERK2/pTpY and ERK2/pTpY*substrate(s) complexes. The data indicate that ERK2/pTpY is a highly efficient substrate for HePTP (k(cat)/K(m) = 3.05 x 10(6) M(-1) s(-1)). The data also show that binding of ATP to ERK2/pTpY has no effect on ERK2/pTpY dephosphorylation by HePTP. In contrast, binding of an Elk-1 peptide substrate to ERK2/pTpY completely blocks the HePTP action. This result indicates that phosphorylation of Tyr185 is important for ERK2 substrate recognition and that binding of the Elk-1 peptide substrate to ERK2/pTpY blocks the accessibility of pTyr185 to HePTP for dephosphorylation. Collectively, the results establish that the kinetic theory of irreversible enzyme modification can be applied to study the phosphatase catalyzed regulation of kinase activity.
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PMID:A kinetic approach for the study of protein phosphatase-catalyzed regulation of protein kinase activity. 1205 17

Utilizing mutants of extracellular signal-regulated kinase 2 (ERK2) that are defective for intrinsic mitogen-activated protein kinase or ERK kinase (MEK) binding, we have identified a convergent signaling pathway that facilitates regulated MEK-ERK association and ERK activation. ERK2-delta19-25 mutants defective in MEK binding could be phosphorylated in response to mitogens; however, signaling from the Raf-MEK pathway alone was insufficient to stimulate their phosphorylation in COS-1 cells. Phosphorylation of ERK2-delta19-25 but not of wild-type ERK2 in response to Ras V12 was greatly inhibited by dominant-negative Rac. Activated forms of Rac and Cdc42 could enhance the association of wild-type ERK2 with MEK1 but not with MEK2 in serum-starved adherent cells. This effect was p21-activated kinase (PAK) dependent and required the putative PAK phosphorylation sites T292 and S298 of MEK1. In detached cells placed in suspension, ERK2 was complexed with MEK2 but not with MEK1. However, upon replating of cells onto a fibronectin matrix, there was a substantial induction of MEK1-ERK2 association and ERK activation, both of which could be inhibited by dominant-negative PAK1. These data show that Rac facilitates the assembly of a mitogen-activated protein kinase signaling complex required for ERK activation and that this facilitative signaling pathway is active during adhesion to the extracellular matrix. These findings reveal a novel mechanism by which adhesion and growth factor signals are integrated during ERK activation.
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PMID:Rac-PAK signaling stimulates extracellular signal-regulated kinase (ERK) activation by regulating formation of MEK1-ERK complexes. 1216 97

In human neutrophils, both changes in intracellular Ca(2+) concentrations, [Ca(2+)]i, and activation of phosphatidylinositol-3 kinase (PtdIns3K) have been proposed to play a role in regulating cellular function induced by chemoattractants. In this study we have investigated the role of [Ca(2+)]i and its effector molecule calmodulin in human neutrophils. Increased [Ca(2+)]i alone was sufficient to induce phosphorylation of extracellular signal-regulated protein kinase 2 (ERK2), p38 mitogen activated kinase (p38 MAPK), protein kinase B (PKB) and glycogen synthase kinase-3alpha (GSK-3alpha). Inhibition of calmodulin using a calmodulin antagonist N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W7), did not effect N-formyl-methionyl-leucyl-phenylalanine (fMLP) induced ERK, p38 MAPK or GSK-3alpha phosphorylation, but attenuated fMLP induced PKB phosphorylation. PCR analysis of human neutrophil cDNA demonstrated variable expression of members of the Ca(2+)/calmodulin-dependent kinase family. The roles of calmodulin and PtdIns3K in regulating neutrophil effector functions were further compared. Neutrophil migration was abrogated by inhibition of calmodulin, while no effect was observed when PtdIns3K was inhibited. In contrast, production of reactive oxygen species was sensitive to inhibition of both calmodulin and PtdIns3K. Finally, we demonstrated that chemoattractants are unable to modulate neutrophil survival, despite activation of PtdIns3K and elevation [Ca(2+)]i. Taken together, our data indicate critical roles for changes in [Ca(2+)]i and calmodulin activity in regulating neutrophil migration and respiratory burst and suggest that chemoattractant induced PKB phosphorylation may be mediated by a Ca(2+)/calmodulin sensitive pathway in human neutrophils.
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PMID:Role of Ca2+/calmodulin regulated signaling pathways in chemoattractant induced neutrophil effector functions. Comparison with the role of phosphotidylinositol-3 kinase. 1223 May 75

Chemical-induced oxidative stress to a cell can signal many cellular responses which include proliferation, differentiation, hemeostasis, apoptosis or necrosis. To better understand the underlying molecular mechanisms after exposure to chemicals, we investigated the signal transduction pathways, in particular the mitogen-activated protein kinase (MAPK) pathway and the ICE/Ced-3 protease (caspase) pathway, activated by different agents. Butylated hydroxyanisol (BHA) and its metabolite, t-butyl-hydroquinone (tBHQ), both are well known phenolic antioxidants used in food preservatives, strongly activated c-Jun N-terminal kinase 1 (JNK1) and/or extracellular signal-regulated protein kinase 2 (ERK2) in a dose- and time-dependent fashion. Pretreatment with free radical scavengers N-acetyl-L-cysteine (NAC), glutathione (GSH), or vitamin E, inhibited ERK2 activation and, to a much lesser extent, JNK 1 activation by BHA and tBHQ, implicating the role of oxidative stress. Under conditions where JNK1 and ERK2 were activated, BHA also activated transcription factors nuclear factor kappa B (NF-kappaB), activated-protein-1 (AP-1), and anti-oxidant response element (ARE), leading to induction of genes such as c-jun, and c-fos. At relatively high concentrations, BHA and tBHQ stimulated proteolytic activity of ICE/Ced3 cysteine proteases, and caused apoptosis, which was blocked by pretreatment with NAC. Further increase in concentrations lead to rapid cell death predominantly occurred via necrosis. Some naturally occurring phytochemicals, such as phenylethyl isothiocyanate (PEITC), green tea polyphenols (GTP), and sulfarophane, which have been shown to be potent inducers of Phase II enzymes, also differentially regulated the activities of JNK, ERK, or CPP-32, in a time- and dose-dependent manner. Our data, together with the work of others, enable us to propose a model in which low concentrations of these chemicals (e.g., BHA, PEITC) activate MAPKs leading to induction of gene expression (e.g., c-jun, c-fos, GSI) which may protect the cells against toxic insults and enhance cell survival. At relatively high concentrations, these agents activated both MAPKS, and the ICE/Ced-3 caspase pathway, leading to apoptosis. The exact mechanisms by which MAPK and caspases are activated by these agents are currently unknown, but may involve oxidative modification of glutathione (GSH) and/or protein thiols, and/or generation of secondary messengers, ceramide and calcium, which further activate downstream events. Taken together, our results suggest that chemicals including phenolic antioxidants activate MAPK pathways which may lead to the induction of genes producing protection and survival mechanisms, as well as the ICE/Ced-3 protease pathway, leading to apoptosis. The balancing amongst these pathways may dictate the fate of the cells upon exposure to chemicals.
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PMID:Differential activation of MAPK and ICE/Ced-3 protease in chemical-induced apoptosis. The role of oxidative stress in the regulation of mitogen-activated protein kinases (MAPKs) leading to gene expression and survival or activation of caspases leading to apoptosis. 1267 Dec 99


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