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)

By applying Western blot analysis using anti-phosphotyrosine antibodies, primary human dermal fibroblasts were examined after having been cultured on type I collagen-coated surfaces or in free-floating type I collagen gels. In both systems cells showed enhanced tyrosine phosphorylation of a M(r) 120,000 protein (pp120) and of a M(r) 42,000 protein (pp42). Phosphorylation was apparent 6 h at the latest after initiation of the culture and was only slightly induced on polylysine or on plastic. In contrast to pp42, pp120 was rapidly dephosphorylated in cells suspended by trypsinization or released from collagen gels by collagenase treatment, but regained phosphorylation in cells cultured in/on type I collagen. Two human sarcoma cell lines (HT-1080 and RD) exhibited identical tyrosine phosphorylation of pp120 but not of pp42. pp120 is identical with pp125FAK, a novel tyrosine kinase localized in focal adhesions, as proved by immunological cross-reactivity with anti-pp125FAK antibodies. Our results suggest that tyrosine phosphorylation is involved in signal transduction triggered by two- and three-dimensional type I collagen-fibroblast contact.
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PMID:Three-dimensional contact with type I collagen mediates tyrosine phosphorylation in primary human fibroblasts. 812 56

The kinases and regulatory proteins that convey signals initiated by transforming growth factor-beta (TGF-beta) to the nucleus are poorly characterized. To study the role of the extracellular signal-regulated kinase (ERK) pathway in this process, we transiently transfected NIH 3T3 fibroblasts with TGF-beta-responsive luciferase reporter genes and expression vectors designed to interrupt this kinase cascade. Mitogen-activated protein (MAP) kinase phosphatase-1 and a dominant negative MAP/ERK kinase 1 mutant reduced stimulation of plasminogen activator inhibitor-1 (PAI-1) promoter activity by TGF-beta1 from 11.5- to 4-fold and 4.9-fold, respectively. Similar results were observed with the type I collagen promoters. TGF-beta1 increased ERK1 activity 4.5-fold at 5 min and 3. 1-fold at 3 h, while Jun kinase and p38 activity were not affected. Cotransfection of a dominant negative mutant of the small G protein, Rac, but not dominant negative Ras, Cdc42, or Rho mutants, reduced the effects of TGF-beta1 on the PAI-1 promoter by approximately half. In support of a role for Rac in signaling by TGF-beta, GTP binding to Rac was increased 3.7-fold following exposure of NIH 3T3 cells to TGF-beta1 for 3 min. These findings indicate that TGF-beta1 modulates gene expression partly through ERK and Rac in NIH 3T3 cells.
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PMID:Extracellular signal-regulated kinase and the small GTP-binding protein, Rac, contribute to the effects of transforming growth factor-beta1 on gene expression. 866 31

Interaction of type I collagen (COL(I)) with alpha2beta1 integrin causes differentiation and transforming growth factor (TGF)-beta receptor down-regulation in osteoblastic cells (Takeuchi, Y., Nakayama, K., and Matsumoto, T. (1996) J. Biol. Chem. 271, 3938-3644). The TGF-beta receptor down-regulation enables cells to escape from the inhibition of differentiation by TGF-beta. To clarify how the cell-matrix interaction regulates these phenotypic changes, signaling pathways were examined in murine MC3T3-E1 cells. Attachment of cells to COL(I) stimulated tyrosine phosphorylation of focal adhesion kinase (FAK) and extracellular signal-regulated kinase (ERK), a mitogen-activated protein kinase (MAPK), and enhanced MAPK activity. Inhibition of tyrosine kinase by herbimycin A, destruction of focal adhesion by cytochalasin D, or overexpression of antisense FAK mRNA prevented the activation of ERK/MAPK and the increase in alkaline phosphatase (ALP) activity. Transient expression of a MAPK-specific phosphatase, CL100, also suppressed the elevation of ALP activity. In addition, introduction of a constitutively active MAPK kinase enhanced ALP activity in the absence of collagen production. TGF-beta receptor down-regulation was abrogated by treatments that inactivate FAK, whereas the expression of CL100 had no effect. These results demonstrate that COL(I)-alpha2beta1 integrin interaction facilitates differentiation and down-regulates TGF-beta receptors via the activation of FAK and its diverse downstream signals. These signaling pathways may play an important role in the sequential differentiation of osteoblasts during bone formation.
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PMID:Differentiation and transforming growth factor-beta receptor down-regulation by collagen-alpha2beta1 integrin interaction is mediated by focal adhesion kinase and its downstream signals in murine osteoblastic cells. 936 Oct 11

Cripto-1 (CR-1) is a novel protein that contains a modified EGF-like motif and that does not directly bind to any of the known erb B type-1 receptor tyrosine kinase receptors. To more clearly define the biological effects of CR-1 and to more adequately compare the structure-function relationships of CR-1 with other members of the EGF family of growth factors, we have expressed a modified, full-length recombinant human CR-1 protein (rhCR-1) in E. coli and have devised a procedure for the solubilization, refolding and purification of a biologically active form of this protein. We have generated the mature form of hCR-1 from computer assisted predictions of potential signal peptide cleavage sites. Expression of the modified rhCR-1 protein in E. coli was limited to the inclusion bodies. The rhCR-1 protein was found to be expressed at high levels in bacterial cells when fused to a histidine-tag sequence. Refolding of rhCR-1 was found to be difficult because of the large number of cysteine residues in the protein which results in protein aggregation. By chemically modifying the cysteine residues in the rhCR-1 protein with 3-trimethylammoniopropyl methanethiosulfonate, additional positive charges have been introduced into the protein by this disulfiding reagent. This modification facilitates solubilization of the protein when rhCR-1 is denatured. The solubilized, denatured protein was then purified by CM cation exchange and C4 reverse phase HPLC chromatography and refolded in a redox buffer. The refolded, modified rhCR-1 protein was found to be biologically active by its ability to inhibit beta-casein expression, to stimulate the tyrosine phosphorylation of Shc and the activation of MAPK and by its capacity to facilitate branching growth of mouse mammary epithelial cells in type I collagen gels.
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PMID:Purification and characterization of a recombinant human cripto-1 protein. 957 42

Tenascin-C is an extracellular matrix glycoprotein, the expression of which is upregulated in remodeling arteries. In previous studies we showed that the presence of tenascin-C alters vascular smooth muscle cell shape and amplifies their proliferative response by promoting growth factor receptor clustering and phosphorylation. Moreover, we demonstrated that denatured type I collagen induces smooth muscle cell tenascin-C protein production via beta3 integrins. In the present study, we examine the pathway by which beta3 integrins stimulate expression of tenascin-C, and define a promoter sequence that is critical for its induction. On native collagen, A10 smooth muscle cells adopt a stellate morphology and produce low levels of tenascin-C mRNA and protein, whereas on denatured collagen they spread extensively and produce high levels of tenascin-C mRNA and protein, which is incorporated into an elaborate extracellular matrix. Increased tenascin-C synthesis on denatured collagen is associated with elevated protein tyrosine phosphorylation, including activation of extracellular signal-regulated kinases 1 and 2 (ERK1 and ERK2). beta3 integrin function-blocking antibodies attenuate ERK1/2 activation and tenascin-C protein synthesis. Consistent with these findings, treatment with the specific MEK inhibitor, PD 98059, results in suppression of tenascin-C protein synthesis. To investigate whether beta3 integrin-dependent activation of ERK1/2 regulates the tenascin-C promoter, we transfected A10 cells with a full-length (approx. 4 kb) mouse tenascin-C gene promoter-chloramphenicol acetyltransferse reporter construct and showed that, relative to native collagen, its activity is increased on denatured collagen. Next, to identify regions of the promoter involved, we examined a series of tenascin-C promoter constructs with 5' deletions and showed that denatured collagen-dependent promoter activity was retained by a 122-base pair element, located -43 to -165 bp upstream of the RNA start site. Activation of this element was suppressed either by blocking beta3 integrins, or by preventing ERK1/2 activation. These observations demonstrate that smooth muscle cell binding to beta3 integrins activates the mitogen activated protein kinase pathway, which is required for the induction of tenascin-C gene expression via a potential extracellular matrix response element in the tenascin-C gene promoter. Our data suggest a mechanism by which remodeling of type I collagen modulates tenascin-C gene expression via a beta3 integrin-mediated signaling pathway, and as such represents a paradigm for vascular development and disease whereby smooth muscle cells respond to perturbations in extracellular matrix composition by altering their phenotype and patterns of gene expression.
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PMID:Induction of vascular smooth muscle cell tenascin-C gene expression by denatured type I collagen is dependent upon a beta3 integrin-mediated mitogen-activated protein kinase pathway and a 122-base pair promoter element. 991 56

Previous studies demonstrated that corneal epithelial cells isolated without basal lamina respond to extracellular matrix (ECM) in an actin dependent manner; the basal cell surface flattens and the actin cortical mat reorganizes. We hypothesize that the actin reorganization is initiated by intracellular signaling mechanisms that includes tyrosine phoshporylation and activation of the Rho, MAP kinase, and PI3 kinase signal transduction pathways. Our goals were to develop a morphological assay to test this hypothesis by answering the following questions: 1) Do the actin bundle formations in the cortical mat have the same configuration in response to different ECM molecules? 2) What is the minimum time ECM molecules need to be in contact with the tissue for the actin to reorganize? 3) Will blocking tyrosine phosphorylation inhibit reorganization of the actin? 4) Are known signal transduction proteins phosphorylated in response to soluble matrix molecules? The actin cortical mat demonstrated distinct bundle configurations in the presence of different ECM molecules. Soluble fibronectin accumulated at the basal cell surfaces 75-fold over 30 min in a clustered pattern. The cells need contact with ECM for a minimum of 10 min to reform the actin bundles at 2 hr. In contrast, two substances that bind to heptahelical receptors to stimulate the Rho pathway, bombesin and lysophosphatidic acid, reorganized the actin bundles in 15-30 min. Focal adhesion kinase, p190 Rho-GAP, tensin, and paxillin were tyrosine phosphorylated in response to soluble fibronectin, type I collagen, or laminin 1. Erk-1, erk-2, and PI3 kinase were activated after 1 hr stimulation by type I collagen. Herbimycin A blocked actin reorganization induced by ECM molecules. In conclusion, we have developed two morphological assays to examine the response of corneal epithelial cells to ECM molecules. In addition, actin bundle reorganization involved tyrosine phosphorylation, MAP kinase, and PI3 kinase activation.
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PMID:ECM-stimulated actin bundle formation in embryonic corneal epithelia is tyrosine phosphorylation dependent. 1009 66

Insulin and insulin-like growth factor (IGF-1) are mitogenic for fibroblasts and smooth muscle cells. IGF-1 increases in inflamed and fibrotic tissues and induces proliferation of rat hepatic stellate cells (HSC). This study evaluates the potential roles of these hormones in the development of liver fibrosis. Insulin and IGF-1 receptor expression was evaluated by immunohistochemistry in both cultured human HSC and human liver tissue. Phosphorylation of both 70-kd S6 kinase and extracellular-regulated kinase (ERK), cell proliferation, type I collagen gene expression, and accumulation in HSC culture media were evaluated by Western blot, immunohistochemistry for bromodeoxyuridine (BrdU), Northern blot, and enzyme-linked immunosorbent assay, respectively. Insulin and IGF-1 receptors were detected in HSC in vitro and in liver sections from patients with chronic active hepatitis. Insulin and IGF-1 induced 70-kd S6 kinase phosphorylation in HSC, whereas IGF-1 only induced ERK phosphorylation. Insulin and IGF-1 stimulated HSC proliferation in a dose-dependent fashion, with IGF-1 being four to five times more potent than insulin. Cell exposure to specific inhibitors showed that both phosphatidylinositol 3-kinase (PI3-K) and ERK are involved in IGF-1-induced mitogenesis, whereas insulin stimulated mitogenesis through a PI3-K-dependent ERK-independent pathway. IGF-1 increased type I collagen gene expression and accumulation in HSC culture media through a PI3-K- and ERK-dependent mechanism. In conclusion, insulin and IGF-1, which stimulate HSC mitogenesis and collagen synthesis, may act in concert to promote liver fibrosis in vivo by a differential activation of PI3-K- and ERK1-dependent pathways.
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PMID:Insulin and insulin-like growth factor-1 stimulate proliferation and type I collagen accumulation by human hepatic stellate cells: differential effects on signal transduction pathways. 1034 17

Downstream signaling triggered by the binding of fibroblast growth factor-2 (FGF2) to its tyrosine-kinase receptors involves the activation of mitogen-activated protein kinase kinase (MEK) with consequent phosphorylation of extracellular signal-regulated kinases (ERKs). Here we demonstrate that FGF2 induces ERK1/2 activation in bovine aortic endothelial (BAE) cells and that the continuous presence of the growth factor is required for sustained ERK1/2 phosphorylation. This is prevented by the MEK inhibitors PD 098059 and U0126, which also inhibit FGF2-mediated upregulation of urokinase-type plasminogen activator (uPA) and in vitro formation of capillary-like structures in three-dimensional type I collagen gel. Various FGF2 mutants originated by deletion or substitution of basic amino acid residues in the amino terminus or in the carboxyl terminus of FGF2 retained the capacity to induce a long-lasting activation of ERK1/2 in BAE cells. Among them, K128Q/R129Q-FGF2 was also able to stimulate uPA production and morphogenesis whereas R129Q/K134Q-FGF2 caused uPA upregulation only. In contrast, K27, 30Q/R31Q-FGF2, K128Q/K138Q-FGF2 and R118,129Q/K119,128Q-FGF2 exerted a significant uPA-inducing and morphogenic activity in an ERK1/2-dependent manner only in the presence of heparin. Furthermore, no uPA upregulation and morphogenesis was observed in BAE cells treated with the deletion mutant (delta)27-32-FGF2 even in the presence of soluble heparin. Thus, mutational analysis of FGF2 dissociates the capacity of the growth factor to induce a persistent activation of ERK1/2 from its ability to stimulate uPA upregulation and/or in vitro angiogenesis. In conclusion, the data indicate that ERK1/2 phosphorylation is a key step in the signal transduction pathway switched on by FGF2 in endothelial cells. Nevertheless, a sustained ERK1/2 activation is not sufficient to trigger uPA upregulation and morphogenesis. FGF2 mutants may represent useful tools to dissect the signal transduction pathway(s) mediating the complex response elicited by an angiogenic stimulus in endothelial cells.
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PMID:Role of endothelial cell extracellular signal-regulated kinase1/2 in urokinase-type plasminogen activator upregulation and in vitro angiogenesis by fibroblast growth factor-2. 1039 15

Our previous studies have shown that Fc gamma receptor (FcgammaR)-mediated uptake of LDL-containing immune complexes (oxLDL-ICs) by human monocyte-derived macrophages leads to not only transformation of macrophages into foam cells but also macrophage activation and release of cytokines. It has been shown that cross-linking of FcgammaR triggers activation of signal transduction pathways that alter gene expression in macrophages. In this study, we determined whether engagement of FcgammaR by oxLDL-ICs leads to activation of mitogen-activated protein (MAP) kinase pathway, a signaling cascade serving many important functions, including the regulation of gene expression, in THP-1 macrophage-like cells. Our results from immunoblotting, using specific anti-phosphorylated MAP kinase antibodies, showed that oxLDL-ICs induced extracellular signal regulated kinase 2 (ERK2) MAP kinase phosphorylation in THP-1 macrophage-like cells in time- and dose-dependent manners. Cholesterol loading before stimulation led to a longer phosphorylation of ERK2. Nuclear translocation of phosphorylated ERK was markedly increased after the stimulation. Moreover, our data showed that oxLDL-IC induction of MAP kinase was prevented by human monomeric IgG1, suggesting that the specific engagement of type I FcgammaR by oxLDL-IC is responsible for the MAP kinase activation. Finally, we showed that human anti-oxLDL autoantibody-containing immune complexes immobilized on type I collagen induced MAP kinase activation in THP-1 cells. These results strongly suggest that oxLDL-IC, which has been detected in atherosclerotic plaques, may play an important role in macrophage activation and atherogenesis.
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PMID:Oxidized LDL-containing immune complexes induce Fc gamma receptor I-mediated mitogen-activated protein kinase activation in THP-1 macrophages. 1039 76

We examined the role of mitogen-activated protein (MAP) kinases in the signal transduction of basic fibroblast growth factor (bFGF)-mediated effects in endothelial cells (ECs). When MSS31 murine endothelial cells were stimulated with bFGF, three MAP kinase homologs, extracellular signal-regulated kinase (ERK) 1/2, c-Jun N-terminal kinase (JNK) 1, and p38 MAP kinase were activated. The inhibition of the ERK1/2 pathway with PD98059, a specific inhibitor of MEK1, or of the p38 MAP kinase pathway with SB203580, a specific inhibitor of p38 MAP kinase, abrogated bFGF-mediated tube formation by MSS31 cells in type I collagen gel. Tube formation in type I collagen gel requires proliferation and migration of these cells, and degradation of the extracellular matrix by these cells. Both PD98059 and SB203580 inhibited bFGF-stimulated DNA synthesis as well as migration of MSS31 cells. Cell migration requires cytoskeleton reorganization and cell adhesion. bFGF induced actin reorganization and vinculin assembly in the focal adhesion plaque, both of which were inhibited by SB203580 but not by PD98059. bFGF induced the expression of the transcription factor ETS-1 in MSS31 cells. ETS-1 is responsible for the expression of proteases as well as integrin beta 3 subunit in ECs, and converts ECs to invasive phenotype. PD98059 inhibited this induction of ETS-1, whereas SB203580 did not. These results indicate that ERK1/2 and p38 MAP kinase are requisite for the signal transduction of bFGF in ECs. The roles of these two MAP kinase homologs are not identical, but these kinases work in a coordinated fashion.
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PMID:Roles of extracellular signal-regulated kinase 1/2 and p38 mitogen-activated protein kinase in the signal transduction of basic fibroblast growth factor in endothelial cells during angiogenesis. 1042 57


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