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)

To investigate the effects of metal ion binding to the alpha-PDGFR kinase insert domain, a PCR product representing amino acid residues 691-795 (104 amino acids) was bacterially expressed and purified. Secondary structure prediction and circular dichroism spectroscopy indicated this domain to be a mixed alpha + beta protein with a large coil/turn contribution. This 16 kDa, soluble, nonphosphorylated domain bound to 45Ca2+ and 65Zn2+ through a common shared site. Of the unlabeled divalent and trivalent metal ions tested, Ho3+ = Zn2+ > Ni2+ > Ca2+ = Mn2+ > Mg2+, Ba2+ in competing for 45Ca2+ binding to this domain. In the presence of Ca2+ ions, the conformation of the KI domain changed significantly, and this changed conformation was resistant to subtilisin proteolysis. However, in the presence of Zn2+ ions, the conformation of the KI domain changed only slightly. Nevertheless, Zn2+ ions were more effective in rendering the KI domain resistant to proteolysis as compared to that shown by Ca2+ ions. In vitro binding studies using purified baculovirus-expressed alpha-PDGFR showed a marked increase in binding the p85 N-SH2 domain in the presence of Ca2+ or Zn2+ ions (KD = 0.5 microM), suggesting that metal ion binding enhances association of the p85 N-SH2 domain with the receptor. To confirm this, association of the alpha-PDGFR with the p85 N-SH2 domain was tested in the presence of the KI domain. The nonphosphorylated KI domain was effective in competing with the alpha-PDGFR for the binding of the p85 N-SH2 domain. This effect was more pronounced in the presence of Ca2+ ions. Microinjection of this domain into Xenopus oocytes delayed maturation in the presence of insulin but not progesterone. This suggests that the KI domain has a correctly folded three-dimensional structure compatible with biological activity. Together these findings indicate that the recombinant alpha-PDGFR KI domain binds the p85 N-SH2 domain and this binding is modulated by the presence of a novel divalent metal ion binding site within its structure.
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PMID:A divalent metal ion binding site in the kinase insert domain of the alpha-platelet-derived growth factor receptor regulates its association with SH2 domains. 785 21

In situ hybridization analysis of Xenopus laevis embryos reveals that mRNA encoding the platelet-derived growth factor alpha receptor (PDGFR alpha) is expressed in cephalic neural crest masses prior to migration from the future neural tube and during their migration into the visceral arches. The analysis of fluorescently labeled neural crest tissue transplanted to unlabeled host embryos demonstrates that neural crest cells are the only detectable source of PDGFR alpha mRNA within visceral arches. Transcripts encoding PDGF A are present in neural ectoderm, otic vesicle and pharyngeal endoderm. Their location suggests that PDGF A provides a signal, first from the neural epithelium and later from the otic vesicle and pharyngeal endoderm, to cephalic neural crest cells during their migration in the arch region.
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PMID:Localization of PDGF A and PDGFR alpha mRNA in Xenopus embryos suggests signalling from neural ectoderm and pharyngeal endoderm to neural crest cells. 789

Signals transmitted from mesenchyme to epithelia or vice versa constitute the basis of reciprocal epithelial-mesenchymal interactions. As a first step toward understanding epithelial-mesenchymal interactions on the ocular surface where the transit amplifying cell-containing corneal epithelium is anatomically separated from the stem cell-containing limbal epithelium, we sought to characterize the expression patterns of cytokines and their receptors by primary epithelial and early-passaged fibroblast cultures of human cornea and limbus. Northern hybridization with oligonucleotide and cDNA probes to a total of 25 cytokines and 12 of their receptors revealed that the positively expressed cytokines could be divided into the following four patterns. Type I: TGF-alpha, IL-1 beta, and PDGF-B were expressed exclusively by epithelial cells but their respective receptors EGFR and IL-1R were predominantly and PDGFR-beta was exclusively expressed by fibroblasts. Type II: IGF-I, TGF-beta 1, -beta 2, LIF, and bFGF, and their receptors were expressed by both epithelial cells and fibroblasts. FGFR-1 (flg) and FGFR-2 (bek) were expressed more by fibroblasts and bFGF was expressed more by corneal than limbal epithelial cells. Type III: keratinocyte growth factor (KGF) and hepatocyte growth factor (HGF) were expressed exclusively by fibroblasts and their respective receptors, KGFR and c-met, were predominantly expressed by epithelial cells. Combined with RT-PCR, the quantity of KGF and KGFR transcripts was highest in limbal fibroblasts and epithelial cells, respectively. In contrast, the quantity of HGF and HGFR (c-met) transcripts was highest in corneal fibroblasts and epithelial cells, respectively. Type IV: M-CSF and IL-8 were expressed by fibroblasts and/or epithelial cells but their receptors were not expressed by epithelial cells nor fibroblasts, but by immune or inflammatory cells. In addition to these potential paracrine actions, autocrine actions mediated by TGF-alpha/EGFR, IL-1 beta/IL1-R, and bFGF/FGFR-1 were more expressed by corneal than limbal epithelial cells. Immunofluorescence staining on human corneoscleral cryosections confirmed that EGFR and bFGF were not expressed by the limbal basal epithelium, but expressed strongly by the corneal epithelium, a pattern consistent with Northern hybridization. These results indicate that ocular surface epithelial cells and fibroblasts can express a myriad of cytokines, among which the first three patterns constitute the network of potential epithelial-mesenchymal cytokine dialogues. The difference of certain cytokine expression between corneal and limbal regions suggests that this network participates in normal epithelial growth and differentiation, and plays an important role in wound healing.
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PMID:Three patterns of cytokine expression potentially involved in epithelial-fibroblast interactions of human ocular surface. 789 1

The tyrosine phosphorylation sites in the human alpha PDGF receptor (alpha PDGFR) required for association with PI-3 kinase have been identified as tyrosines 731 and 742. Mutation of either tyrosine substantially reduced PDGF-induced PI-3 kinase activity but did not impair the receptor-mediated mitogenic response. We sought to determine whether PDGF-induced PI-3 kinase activity could be further ablated so as to exclude a low threshold requirement for PDGFR signal transduction. Thus, we mutated both tyrosine 731 and 742 and expressed the double mutant (Y731F/Y742F) in 32D hematopoietic cells. In such transfectants, PDGF induced no detectable receptor-associated or anti-P-Tyr recoverable PI-3 kinase activity. Under the same conditions, neither mobility shift of raf-1 nor tyrosine phosphorylation of either PLC gamma or MAP kinase was impaired. 32D transfectants expressing the double mutant showed wild-type alpha PDGFR levels of mitogenic and chemotactic responses to PDGF. To examine the effect of the double mutation in cells that normally respond to PDGF, we generated chimeras in which the cytoplasmic domains of wild-type alpha PDGFR, Y731F, and Y731F/Y742F were linked to the extracellular domain of colony-stimulating factor-1 (CSF-1) receptor (fms). After introduction of the chimeric receptors into mouse NIH/3T3 fibroblasts, the ability of CSF-1 to stimulate growth of these transfectants was examined. Our data show that all these chimeric receptors exhibited similar abilities to mediate CSF-1-stimulated cell growth. These findings lead us to conclude that PDGF-induced PI-3 kinase activity is not required for PDGF-stimulated mitogenic pathway in both NIH/3T3 fibroblasts and 32D hematopoietic cells.
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PMID:Biological function of PDGF-induced PI-3 kinase activity: its role in alpha PDGF receptor-mediated mitogenic signaling. 792 90

Platelet-derived growth factor (PDGF) stimulates mitogenesis and exerts other biologic activities in glomerular mesangial cells. The precise mechanism of PDGF-induced mitogenesis in these cells is not clear. The activation of a signal transducing enzyme, phosphatidylinositol 3 kinase (PI 3 kinase) is associated with mitogenesis. Activation of PI 3 kinase results from stimulation of tyrosine kinase and G-protein-coupled classes of receptors. The synthesis of D3 phosphorylated inositides, the products of this enzymatic reaction, in non-nucleated cells such as blood platelets is dependent upon protein kinase C activation and G-proteins. We studied the activation of PI 3 kinase in response to PDGF in human glomerular mesangial cells. Using a PI 3 kinase 85 kD subunit specific antibody, we detected mesangial cell PI 3 kinase protein as 110 and 85 kD heterodimer. PDGF stimulated PI 3 kinase activity in antiphosphotyrosine immunoprecipitates in a dose-dependent manner showing maximum activation at 12 ng/ml. The antiphosphotyrosine associated PI 3 kinase activity showed biphasic kinetics with a fast peak within two minutes followed by a second peak at 10 minutes. Antiphosphotyrosine and PI 3 kinase immunoprecipitation studies indicated the association of the 85 kD PI 3 kinase subunit with PDGFR. Direct immunoprecipitation with PDGFR beta antibody showed the association of PI 3 kinase activity with the PDGF-receptor. The isoquinoline sulfonyl piperazine compound H7 at concentrations that inhibit PDGF-stimulated PKC activity had no effect on PDGF-stimulated PI 3 kinase activity in antiphospotyrosine immunoprecipitates. These data indicate that PI3 kinase activation is insensitive to PKC. Treatment of mesangial cells with pertussis toxin at concentrations that partially inhibited PDGF-induced DNA synthesis in human mesangial cells did not inhibit PDGF-induced PI 3 kinase activation. These data indicate that PDGF activates PI 3 kinase in mesangial cells and that pertussis toxin-sensitive G-proteins are not involved in PI 3 kinase activation. The data further dissociate activation of PI 3 kinase from mitogenesis in human mesangial cells.
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PMID:PDGF-mediated activation of phosphatidylinositol 3 kinase in human mesangial cells. 793 47

Ligand stimulation of the platelet-derived growth factor (PDGF) beta-receptor leads to activation of its intrinsic tyrosine kinase and autophosphorylation of the intracellular part of the receptor. The autophosphorylated tyrosine residues mediate interactions with downstream signal transduction molecules and thereby initiate different signalling pathways. A pathway leading to activation of the GTP-binding protein Ras involves the adaptor molecule GRB2. Here we show that Tyr-716, a novel autophosphorylation site in the PDGF beta-receptor kinase insert, mediates direct binding of GRB2 in vitro and in vivo. In a panel of mutant PDGF beta-receptors, in which Tyr-716 and the previously known autophosphorylation sites were individually mutated, only PDGFR beta Y716F failed to bind GRB2. Furthermore, a synthetic phosphorylated peptide containing Tyr-716 bound GRB2, and this peptide specifically interrupted the interaction between GRB2 and the wild-type receptor. In addition, the Y716(P) peptide significantly decreased the amount of GTP bound to Ras in response to PDGF in permeabilized fibroblasts as well as in porcine aortic endothelial cells expressing transfected PDGF beta-receptors. The mutant PDGFR beta Y716F still mediated activation of mitogen-activated protein kinases and an increased DNA synthesis in response to PDGF, indicating that multiple signal transduction pathways transduce mitogenic signals from the activated PDGF beta-receptor.
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PMID:Tyr-716 in the platelet-derived growth factor beta-receptor kinase insert is involved in GRB2 binding and Ras activation. 793 91

The murine myeloid progenitor cell line 32D was recently shown to undergo monocytic differentiation when protein kinase C-delta (PKC-delta) was overexpressed and activated by 12-O-tetradecanoylphorbol-13-acetate (TPA) (H. Mischak, J.H. Pierce, J. Goodnight, M.G. Kazanietz, P.M. Blumberg, and J.F. Mushinski, J. Biol. Chem. 268:20110-20115, 1993). Tyrosine phosphorylation of PKC-delta occurred when PKC-delta-transfected 32D cells were stimulated by TPA (W. Li, H. Mischak, J.-C. Yu, L.-M. Wang, J.F. Mushinski, M.A. Heidaran, and J.H. Pierce, J. Biol. Chem. 269:2349-2352, 1994). In order to elucidate the role played by PKC-delta in response to activation of a receptor tyrosine kinase, we transfected platelet-derived growth factor beta receptor (PDGF-beta R) alone (32D/PDGF-beta R) or together with PKC-delta (32D/PDGF-beta R/PKC-delta) into 32D cells. NIH 3T3 cells which endogenously express both PDGF-alpha R and PDGF-beta R were also transfected with PKC-delta (NIH 3T3/PKC-delta). Like TPA treatment, PDGF-BB stimulation caused striking phosphorylation of PKC-delta in vivo and translocation of some PKC-delta from the cytosol fraction to the membrane fraction in both cell systems. Some of the phosphorylation induced by PDGF-BB treatment was found to be on a tyrosine residue(s). Tyrosine-phosphorylated PKC-delta was observed only for the membrane fraction after stimulation with PDGF-BB or TPA. The enzymatic activity of PKC-delta in the membrane fraction also increased after stimulation with TPA or PDGF, providing a positive correlation between PKC-delta tyrosine phosphorylation and its activation. Overnight treatment of 32D/PDGF-beta R/PKC-delta cells with PDGF-BB induced monocytic differentiation as judged by an increase in expression of cell surface macrophage differentiation markers. PDGF-BB had much weaker effects on 32D/PDGF-beta R cell differentiation, suggesting that increased PKC-delta expression enhanced monocytic differentiation. These results indicate that PKC-delta is a downstream molecule in the PDGFR signaling pathway and may play a pivotal role in PDGF-beta R-mediated cell differentiation.
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PMID:Stimulation of the platelet-derived growth factor beta receptor signaling pathway activates protein kinase C-delta. 793 92

To determine the capability of alpha PDGFR to couple with chemotactic signaling, we established 32D cells expressing wild type alpha PDGFR (alpha RWT), a kinase-defective mutant of alpha PDGFR (alpha R627R), or wild type beta PDGFR (beta RWT). Using a modified Boyden chamber, we showed that PDGF induced significant cell migration of 32D cells expressing alpha RWT or beta RWT, but not of those expressing alpha R627R. Furthermore, the cell migration was largely reduced in each case when the same concentration of PDGF was present in both chambers, suggesting that cell migration observed in 32D expressing alpha RWT is mainly due to alpha PDGFR-mediated chemotaxis. Consistent with these results, PDGF-AA induced significant cell migration of NIH3T3 fibroblasts, which was markedly blocked by the presence of excess neutralizing polyclonal antibody to PDGF-AA. These results provide evidence that alpha PDGFR kinase activity is essential for mediating ligand-induced chemotactic and chemokinetic responses in two different cell types.
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PMID:The alpha PDGFR tyrosine kinase mediates locomotion of two different cell types through chemotaxis and chemokinesis. 798 May 24

Macrophage colony stimulating factor (CSF-1 or M-CSF) is involved in haemopoiesis and probably in mouse gestation. Sexual steroids induce its production by uterine glandular epithelial cells and its receptor (product of the protooncogene C-FMS) is expressed on placental trophoblastic cells. We measured M-CSF serum levels in 119 pregnant women and in eight women undergoing ovarian hyperstimulation for in vitro fertilization. M-CSF increased early (4-8 weeks) and progressively during gestation. Its rapid elevation during the course of ovarian hyperstimulation suggests that its synthesis is probably induced by sexual steroids. This locally produced M-CSF could play a role in human pregnancy and in the pathogenesis of thrombocytopenias observed during pregnancy.
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PMID:Elevation of serum M-CSF concentrations during pregnancy and ovarian hyperstimulation. 804 54

Chemotaxis is an important component of wound healing, development, immunity and metastasis, yet the signalling pathways that mediate chemotaxis are poorly understood. Platelet-derived growth factor (PDGF) acts both as a mitogen and a chemoattractant. Upon stimulation, the tyrosine kinase PDGF receptor-beta (PDGFR-beta) autophosphorylates and forms a complex that includes SII2(Src homology 2)-domain-containing proteins such as the phosphatidylinositol-specific phospholipase C-gamma, Ras-GTPase-activating protein (GAP), and phosphatidylinositol-3-OH kinase. Specific tyrosine-to-phenylalanine substitutions in the PDGFR-beta can prevent binding of one SH2-domain-containing protein without affecting binding of other receptor-associated proteins. Here we use phospholipase C-gamma and PDGFR-beta mutants to map specific tyrosines involved in both positive and negative regulation of chemotaxis towards the PDGF-BB homodimer. Our results indicate that a delicate balance of migration-promoting (phospholipase C-gamma and phosphatidylinositol-3-OH kinase) and migration-suppressing (GAP) activities are recruited by the PDGFR-beta to drive chemotaxis towards PDGF-BB.
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PMID:Regulation of chemotaxis by the platelet-derived growth factor receptor-beta. 810 7


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