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)

The transformed phenotype of v-Ras- or Bacillus cereus phosphatidylcholine-hydrolyzing phospholipase C (PC-PLC)-expressing NIH 3T3 cells is reverted by expressing a kinase-defective mutant of protein kinase C lambda (lambdaPKC). We report here that extracellular signal-regulated kinase (ERK)-1 and -2 are constitutively activated in v-Ras- and PC-PLC-transformed cells in the absence of added growth factors. Interestingly, the activated ERKs were exclusively localized to the cell nucleus. Consistently, the transactivating potential of the C-terminal domain of Elk-1, which is activated upon ERK-mediated phosphorylation, was strongly induced in serum-starved cells expressing v-Ras or PC-PLC. Reversion of v-Ras- or PC-PLC-induced transformation by expression of dominant negative lambdaPKC abolished the nuclear ERK activation suggesting lambdaPKC as a novel, direct or indirect, activator of mitogen-activated protein kinase/ERK kinase in response to activated Ras or elevated levels of phosphatidylcholine-derived diacylglycerol. Transient transfection experiments confirmed that lambdaPKC acts downstream of Ras but upstream of mitogen-activated protein kinase/ERK kinase. We found both the v-Ras- and PC-PLC-transformed cells to be insensitive to stimulation with platelet-derived growth factor (PDGF). No detectable receptor level, autophosphorylation, or superinduction of DNA synthesis could be observed in response to treatment with PDGF. Reversion of the transformed cell lines by expression of dominant negative lambdaPKC restored the receptor level and the ability to respond to PDGF in terms of receptor autophosphorylation, ERK activation, and induction of DNA synthesis.
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PMID:Reversion of Ras- and phosphatidylcholine-hydrolyzing phospholipase C-mediated transformation of NIH 3T3 cells by a dominant interfering mutant of protein kinase C lambda is accompanied by the loss of constitutive nuclear mitogen-activated protein kinase/extracellular signal-regulated kinase activity. 911 Oct 71

In normal embryos, mRNA encoding platelet-derived growth factor A (PDGF A) and the platelet-derived growth factor receptor alpha (PDGFR alpha) are found within and adjacent to the site of vertebral development, the sclerotome. These patterns of expression are consistent with PDGF action on the developing sclerotome and dermis. Homozygous Patch (Ph) mutant mouse embryos lack the receptor gene (Pdgfra) due to an extensive deletion at that locus. Consistent with the spatial pattern of Pdgfra expression, striking deformities are found in the spine and ribcage of Ph/Ph embryos. In particular, we show that late-gestation Ph/Ph embryos have occult spina bifida involving the entire spinal column. We have analyzed the progression of the axial defects in homozygous Patch embryos in detail. By late gestation it appears that the components of the vertebrae are present, yet the neural arches of the spine are misshapen. We propose that PDGF A is required for proper positioning of the neural arch condensation at all axial levels. Furthermore, since the neural tube appears to close normally, we suggest that spina bifida in the Ph homozygote is caused primarily by a somitic mesoderm abnormality rather than a neural tube defect.
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PMID:Spina bifida occulta in homozygous Patch mouse embryos. 914

PD 089828, a novel protein tyrosine kinase inhibitor of a new structural class, the 6-aryl-pyrido-[2,3-d]pyrimidines, was identified by screening a compound library with assays that measured protein tyrosine kinase activity. PD 089828 was found to inhibit human full-length fibroblast growth factor (FGF) receptor-1 (FGFR-1), platelet-derived growth factor (PDGF) receptor beta subunit (PDGFR-beta), Src nonreceptor tyrosine kinase (c-Src) and epidermal growth factor (EGF) receptor (EGFR) tyrosine kinases with half-maximal inhibitory potencies (IC50 values) of 0.15 +/- 0.02 (n = 4), 0.18 +/- 0.04 (n = 3), 1.76 +/- 0.28 (n = 4) and 5.47 +/- 0.78 (n = 6) microM, respectively. PD 089828 was further characterized as an ATP competitive inhibitor of the growth factor receptor tyrosine kinases (FGFR-1, PDGFR-beta and EGFR) but a noncompetitive inhibitor of c-Src tyrosine kinase with respect to ATP. In addition, PD 089828 inhibited PDGF- and EGF-stimulated receptor autophosphorylation in vascular SMC (VSMC) and basic FGF-mediated tyrosine phosphorylation in A121 cells with IC50 values similar to the potencies observed for inhibition of receptor tyrosine kinase activity. The inhibition of PDGF receptor autophosphorylation in VSMC by PD 089828 occurred rapidly, with maximal effects reached within 5 min of drug exposure. Inhibition after single exposure was long lasting but also rapidly reversible, occurring within 5 min after drug removal. The PDGF-induced association of downstream signaling proteins, including phosphoinositide-3-kinase (PI-3K), growth factor receptor binding protein-2 (GRB2), SH-2 domain and collagen like (Shc) and phospholipase Cgamma (PLCgamma), with VSMC PDGF receptors was also blocked as a result of the inhibition of PDGF-stimulated receptor autophosphorylation by PD 089828. PD 089828 also inhibited the PDGF-induced tyrosine phosphorylation of the 44- and 42-kDa mitogen-activated protein kinase isoforms. Moreover, the effects of PD 089828 were demonstrated in functional assays in which PDGF-stimulated DNA synthesis, PDGF-directed migration and serum-stimulated growth of VSMC were all inhibited to the same extent as PDGF receptor autophosphorylation (IC50 = 0.8, 4.5 and 1.8 microM, respectively). These results highlight the biological characteristics of PD 089828 as a novel, broadly active protein tyrosine kinase inhibitor with long-lasting but reversible cellular effects. The potential therapeutic use of these broadly acting, nonselective inhibitors as antiproliferative and antimigratory agents could extend to such diseases as cancer, atherosclerosis and restenosis in which redundancies in growth-signaling pathways are known to exist.
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PMID:Inhibition of growth factor-mediated tyrosine phosphorylation in vascular smooth muscle by PD 089828, a new synthetic protein tyrosine kinase inhibitor. 919 Aug 82

Mutations in the gene for human fibroblast growth factor receptor 3 (hFGFR3) cause a variety of skeletal dysplasias, including the most common genetic form of dwarfism, achondroplasia (ACH). Evidence indicates that these phenotypes are not due to simple haploinsufficiency of FGFR3 but are more likely related to a role in negatively regulating skeletal growth. The effects of one of these mutations on FGFR3 signaling were examined by constructing chimeric receptors composed of the extracellular domain of human platelet-derived growth factor receptor beta (hPDGFR beta) and the transmembrane and intracellular domains of hFGFR3 or of an ACH (G375C) mutant. Following stable transfection in PC12 cells, which lack platelet-derived growth factor (PDGF) receptors, all clonal cell lines, with either type of chimera, showed strong neurite outgrowth in the presence of PDGF but not in its absence. Antiphosphotyrosine immunoblots showed ligand-dependent autophosphorylation, and both receptor types stimulated strong phosphorylation of mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase, an event associated with the differentiative response of these cells. In addition, ligand-dependent phosphorylation of phospholipase Cgamma and Shc was also observed. All of these responses were comparable to those observed from ligand activation, such as by nerve growth factor, of the native PC12 cells used to prepare the stable transfectants. The cells with the chimera bearing the ACH mutation were more rapidly responsive to ligand with less sustained MAPK activation, indicative of a preactivated or primed condition and consistent with the view that these mutations weaken ligand control of FGFR3 function. However, the full effect of the mutation likely depends in part on structural features of the extracellular domain. Although FGFR3 has been suggested to act as a negative regulator of long-bone growth in chrondrocytes, it produces differentiative signals similar to those of FGFR1, to which only positive effects have been ascribed, in PC12 cells. Therefore, its regulatory effects on bone growth likely result from cellular contexts and not the induction of a unique FGFR3 signaling pathway.
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PMID:Chimeras of the native form or achondroplasia mutant (G375C) of human fibroblast growth factor receptor 3 induce ligand-dependent differentiation of PC12 cells. 919 52

Smooth muscle cell (SMC) proliferation is dependent on both anchorage to the extracellular matrix by integrins and the presence of growth factors. Integrins and growth factor receptors transduce signals that seem to converge on the extracellular signal-regulated (ERK) pathway, but the molecular basis for this interaction is not known. SMC proliferation has previously been shown to be supported by culture on fibronectin (FN), whereas cells cultured on laminin (LN) are growth inhibited. In the present study, we examined the mitogenic response to platelet-derived growth factor BB (PDGF-BB) in baboon SMCs cultured on FN vs. LN. Induction of DNA synthesis and the activity of ERK and the ERK activating kinase MKK-1 were reduced only slightly after stimulation with PDGF-BB in cells cultured on LN vs. those cultured on FN. We tested the possibility that endogenous FN secretion contributes to the ability of the cells to respond to PDGF stimulation during culture on LN. Inhibition of interactions between FN and integrin alpha 5 beta 1 by the competitive GRGDSP-peptide or anti-alpha 5 integrin antibody restricted cell spreading, reduced cell-surface staining for alpha 5 beta 1 and FN fibrils, and inhibited PDGF-BB-induced DNA synthesis. These results showed that SMC growth on LN required a provisional FN matrix. Although disruption of interactions between alpha 5 beta 1 and FN by the GRGDSP-peptide prevented PDGF-BB-induced DNA synthesis, neither ERK activity nor translocation of ERKs into the nucleus was inhibited. These results show that integrins regulate SMC growth through pathways that function in parallel with, but distinct from, growth factor-mediated ERK signaling.
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PMID:Disruption of integrin alpha 5 beta 1 signaling does not impair PDGF-BB-mediated stimulation of the extracellular signal-regulated kinase pathway in smooth muscle cells. 920 31

Previous work has suggested a role for phosphatidylinositide 3'-kinase (PI3-kinase) in platelet-derived growth factor (PDGF)-induced actin reorganization and chemotaxis. In support of this notion, we show in this report that the PI3-kinase inhibitor wortmannin inhibits chemotaxis of PDGF beta-receptor expressing porcine aortic endothelial (PAE/PDGFR-beta) cells. Treatment with wortmannin resulted in a dose-dependent decrease in chemotaxis with an IC50 value of about 15-20 nM. Higher concentrations of wortmannin also reduced basal random migration of transfected cells in the absence of PDGF. We also investigated the role of Rac in PDGF-induced actin reorganization and cell motility. Overexpression of wt Rac in PAE/PDGFR-beta cells led to an increased cell motility and edge ruffling in response to PDGF-BB, compared to control cells. In PAE/PDGFR-beta cells transfected with inducible V12Rac (a constitutively active Rac mutant), membrane ruffling occurred in the absence of PDGF stimulation and was independent of PI3-kinase activity. On the other hand, PAE/PDGFR-beta cells transfected with inducible N17Rac (a dominant negative Rac mutant) failed to show membrane ruffling in response to PDGF stimulation. Together with previous observations, these data indicate that activation of PI3-kinase is crucial for initiation of PDGF-induced cell motility responses and that Rac has a major role downstream of PI3-kinase, in this pathway.
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PMID:Involvement of phosphatidylinositide 3'-kinase and Rac in platelet-derived growth factor-induced actin reorganization and chemotaxis. 926 Sep 14

Exposure of cultured rat aortic vascular smooth muscle (VSM) cells to the Ca2+ ionophore ionomycin produced an increase in extracellular signal-regulated kinase 1/2 (ERK1/2) activity that was maximal between 2 and 5 minutes but then declined to basal values within 20 minutes of stimulation. Elevation of [Ca2+]i in VSM cells leads to an even more rapid activation of Ca2+/calmodulin-dependent protein kinase II (CaM kinase II); thus, it was postulated that the Ca(2+)-dependent component of ERK1/2 activation was mediated by CaM kinase II. Transient ERK1/2 activation by ionomycin was almost completely abolished by pretreating cells with 30 mumol/L KN-93, a CaM kinase II inhibitor. Treatment of cells with KN-93 did not antagonize the ability of ionomycin to mobilize intracellular Ca2+ but prevented CaM kinase II and ERK1/2 activation with almost identical potencies. Consistent with a role for Ca2+ and calmodulin in intracellular Ca(2+)-induced activation of ERK, cells pretreated with calmodulin inhibitors (W-7 or calmidazolium) exhibited an attenuated ERK response to ionomycin. ERK1/2 activation in response to phorbol esters and platelet-derived growth factor were not significantly affected by KN-93, whereas the response to angiotensin II and thrombin were attenuated by 60% and 40%, respectively. Transient expression of wild-type delta 2 CaM kinase II in COS-7 cells resulted in increased ERK2 activity, whereas coexpression of wild-type and a kinase-negative mutant resulted in a diminution of this response. These data suggest that regulation of cellular responses by Ca(2+)-dependent pathways in VSM cells may be mediated in part by CaM kinase II-dependent activation of ERK1/2.
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PMID:A role for Ca2+/calmodulin-dependent protein kinase II in the mitogen-activated protein kinase signaling cascade of cultured rat aortic vascular smooth muscle cells. 931 39

In Chinese hamster embryo fibroblasts (IIC9 cells), platelet-derived growth factor (PDGF) stimulated mitogen-activated protein kinase/extracellular-signal-regulated kinase (MAP kinase/ERK) activity, but not that of c-jun N-terminal kinase (JNK), and induced G1 phase progression. ERK1 activation was biphasic and was sustained throughout the G1 phase of the cell cycle. PDGF induced cyclin D1 protein and mRNA levels in a time-dependent manner. Inhibition of PDGF-induced ERK1 activity by the addition of a selective inhibitor of MEK1 (MAP kinase kinase/ERK kinase 1) activation, PD98059, or transfection with a dominant-negative ERK1 (dnERK-) was correlated with growth arrest. In contrast, growth was unaffected by expression of dominant-negative JNK (dnJNK-). Interestingly, addition of PD98059 or dnERK-, but not dnJNK-, resulted in a dramatic decrease in cyclin D1 protein and mRNA levels, concomitant with a decrease in cyclin D1-cyclin-dependent kinase activity. To investigate the importance of sustained ERK1 activation, ERK1 activity was blocked by the addition of PD98059 throughout G1. Addition of PD98059 up to 4 h after PDGF treatment decreased ERK1 activity to the levels found in growth-arrested IIC9 cells. Loss of cyclin D1 mRNA and protein expression was observed within 1 h after inhibition of the second sustained phase of ERK1 activity. Disruption of sustained ERK1 activity also resulted in G1 growth arrest. These data provide evidence for a role for sustained ERK activity in controlling G1 progression through positive regulation of the continued expression of cyclin D1, a protein known to positively regulate G1 progression.
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PMID:Sustained activation of extracellular-signal-regulated kinase 1 (ERK1) is required for the continued expression of cyclin D1 in G1 phase. 933 51

The vascular endothelial growth factor (VEGF) and the VEGF-C promote growth of blood vessels and lymphatic vessels, respectively. VEGF activates the endothelial VEGF receptors (VEGFR) 1 and 2, and VEGF-C activates VEGFR-3 and VEGFR-2. Both VEGF and VEGF-C are also potent vascular permeability factors. Here we have analyzed the receptor binding and activating properties of several cysteine mutants of VEGF-C including those (Cys156 and Cys165), which in other platelet-derived growth factor/VEGF family members mediate interchain disulfide bonding. Surprisingly, we found that the recombinant mature VEGF-C in which Cys156 was replaced by a Ser residue is a selective agonist of VEGFR-3. This mutant, designated DeltaNDeltaC156S, binds and activates VEGFR-3 but neither binds VEGFR-2 nor activates its autophosphorylation or downstream signaling to the ERK/MAPK pathway. Unlike VEGF-C, DeltaNDeltaC156S neither induces vascular permeability in vivo nor stimulates migration of bovine capillary endothelial cells in culture. These data point out the critical role of VEGFR-2-mediated signal transduction for the vascular permeability activity of VEGF-C and strongly suggest that the redundant biological effects of VEGF and VEGF-C depend on binding and activation of VEGFR-2. The DeltaNDeltaC156S mutant may provide a valuable tool for the analysis of VEGF-C effects mediated selectively via VEGFR-3. The ability of DeltaNDeltaC156S to form homodimers also emphasizes differences in the structural requirements for VEGF and VEGF-C dimerization.
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PMID:A recombinant mutant vascular endothelial growth factor-C that has lost vascular endothelial growth factor receptor-2 binding, activation, and vascular permeability activities. 950 53

Receptor tyrosine kinases are classified into subfamilies, which are believed to function independently, with heterodimerization occurring only within the same subfamily. In this study, we present evidence suggesting a direct interaction between the epidermal growth factor (EGF) receptor (EGFR) and the platelet-derived growth factor beta (PDGFbeta) receptor (PDGFbetaR), members of different receptor tyrosine kinase subfamilies. We find that the addition of EGF to COS-7 cells and to human foreskin Hs27 fibroblasts results in a rapid tyrosine phosphorylation of the PDGFbetaR and results in the recruitment of phosphatidylinositol 3-kinase to the PDGFbetaR. In R1hER cells, which overexpress the EGFR, we find ligand-independent tyrosine phosphorylation of the PDGFbetaR and the constitutive binding of a substantial amount of PI-3 kinase activity to it, mimicking the effect of ligand in untransfected cells. In support of the possibility that this may be a direct interaction, we show that the two receptors can be coimmunoprecipitated from untransfected Hs27 fibroblasts and from COS-7 cells. This association can be reconstituted by introducing the two receptors into 293 EBNA cells. The EGFR/PDGFbetaR association is ligand-independent in all cell lines tested. We also demonstrate that the fraction of PDGFbetaR bound to the EGFR in R1hER cells undergoes an EGF-induced mobility shift on Western blots indicative of phosphorylation. Our findings indicate that direct interactions between receptor tyrosine kinases classified under different subfamilies may be more widespread than previously believed.
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PMID:The epidermal growth factor receptor associates with and recruits phosphatidylinositol 3-kinase to the platelet-derived growth factor beta receptor. 950 92


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