Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.11.26 (GSK)
 6,788 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Solid-phase ELISAs for the determination of EGF receptor (EGF-R) and pp60c-src tyrosine protein kinase activity are described. The methods were developed and optimized using purified recombinant EGF-R intracellular domain (ICD) and pp60c-src tyrosine protein kinases. A standardized assay that utilizes poly (GluNa-Tyr)4:1 as substrate and a monoclonal antiphosphotyrosine antibody for detection is described. Assay conditions for both enzymes were optimized with respect to substrate and ELISA plate-coating condition, divalent metal ion preferences, enzyme concentration, apparent kinetic constants for ATP, and reaction linearity. Following standardization, a number of reference tyrosine protein kinase inhibitors were tested in the ELISAs and compared to results obtained using solution-phase radioactive tyrosine protein kinase assays, which are based on the transfer of 32P from [gamma-32P]ATP to synthetic substrate. To enable a comprehensive comparison, IC50 values obtained in the ELISA were compared with values obtained in radioactive assays using both the holo-EGF-R and EGF-R ICD kinases. No substantial qualitative differences between these assays were seen. For many routine tyrosine protein kinase assays, semiquantitative or qualitative measurement of TPK activity is adequate. For such purposes, the ELISAs would be an attractive alternative to radioactive assays.
 ...
PMID:Development of solid-phase enzyme-linked immunosorbent assays for the determination of epidermal growth factor receptor and pp60c-src tyrosine protein kinase activity. 138 73

Spleen TPK-IIB is an acidophilic protein tyrosine kinase, devoid of autophosphorylation activity, whose phosphorylation of the src-peptide NEYTA is crucially specified by Glu-2[(1991) J. Biol. Chem. 266, 17798-17803]. We show that phosphothreonine, phosphotyrosine and phosphoserine are, in this order, specificity determinants even more effective than glutamic acid if they are replacing Glu-2, to give the phosphopeptides NTpYTA, NYpYTA, NSpYTA, respectively. Non-phosphorylated threonine, tyrosine and serine are conversely ineffective. Consequently also the heptapeptide GEGTYGV reproducing the phosphoacceptor and inhibitory site of p34cdc2 is not appreciably affected by TPK-IIB, unless its threonyl residue is previously phosphorylated, the phosphoderivative GEGTpYGV being readily phosphorylated at its tyrosyl residue. Such a behaviour is unique for TPK-IIB among the protein tyrosine kinases tested (lyn-TPK, fgr-TPK and EGF-receptor, besides TPK-IIB). These data provide the first evidence that, in some instances, the targeting by protein tyrosine kinases can be specifically determined by the previous phosphorylation of the peptide substrate, thus extending the concept of 'hierarchal phosphorylation' [(1991) J. Biol. Chem. 266, 14139-14142] to tyrosyl residues as well.
 ...
PMID:Phosphorylated residues as specificity determinants for an acidophilic protein tyrosine kinase. A study with src and cdc2 derived phosphopeptides. 768 79

Activation of EGF receptors is closely involved in vascular proliferative diseases. The signaling mechanisms of EGF ligands, including betacellulin (BTC) and amphiregulin (AR), are poorly understood. We examined how BTC and AR induced DNA synthesis activity in primary cultures of human thoracic aortic smooth muscle cells (HTASMCs). BTC induced phosphorylation of all four EGF receptors present on HTASMCs: ErbB1, ErbB2, ErbB3, and ErbB4. BTC rapidly induced the phosphorylation of Akt, GSK3alpha/beta, and two FoxO factors, FKHR and AFX, in a dose- and time-dependent manner. BTC increased nuclear beta-catenin accumulation. BTC increased cyclin D1 mRNA, cyclin D1 protein, and DNA synthesis activity. Pretreatment with the phosphatidylinositol 3'-kinase (PI 3'-kinase) inhibitor wortmannin suppressed BTC-induced cyclin D1 mRNA and protein and DNA synthesis activity. In contrast, AR, a specific ErbB1 ligand, induced sustained ERK1/2 and Elk1 phosphorylation, increased cyclin D1 mRNA and protein, and increased DNA synthesis activity. AR did not produce any changes in Akt phosphorylation. Pretreatment with PD98059 suppressed AR-induced cyclin D1 mRNA and protein. Thus, the PI 3'-kinase/Akt/GSK/FoxO/beta-catenin pathway could be the major signaling cascade for BTC-induced upregulation of cyclin D1 protein, whereas a sustained ERK/Elk1 activation could be the major signaling cascade for AR-induced upregulation of cyclin D1 protein in HTASMCs. Moreover, immunohistochemical staining revealed that that BTC, ErbB1, and ErbB4 are upregulated in the plaques of human atherosclerotic coronary arteries. Taken together, BTC and AR could be potent growth factors in proliferative vascular diseases.
 ...
PMID:Betacellulin and amphiregulin induce upregulation of cyclin D1 and DNA synthesis activity through differential signaling pathways in vascular smooth muscle cells. 1286 89

EGF receptor (EGFR) overexpression correlates with metastasis in a variety of carcinomas, but the underlying mechanisms are poorly understood. We demonstrated that EGF disrupted cell-cell adhesion and caused epithelial-to-mesenchymal transition (EMT) in human tumor cells overexpressing EGFR, and also induced caveolin-dependent endocytosis of E-cadherin, a cell-cell adhesion protein. Chronic EGF treatment resulted in transcriptional downregulation of caveolin-1 and induction of the transcriptional repressor Snail, correlating with downregulation of E-cadherin expression. Caveolin-1 downregulation enhanced beta-catenin-TCF/LEF-1 transcriptional activity in a GSK-3beta-independent manner. Antisense RNA-mediated reduction of caveolin-1 expression in EGFR-overexpressing tumor cells recapitulated these EGF-induced effects and enhanced invasion into collagen gels. We propose that EGF-induced negative regulation of caveolin-1 plays a central role in the complex cellular changes leading to metastasis.
 ...
PMID:Downregulation of caveolin-1 function by EGF leads to the loss of E-cadherin, increased transcriptional activity of beta-catenin, and enhanced tumor cell invasion. 1470 41

Wound keratinocytes form long cellular extensions that facilitate their migration from the wound edge into provisional matrix. We have previously shown that similar extensions can be induced by a long-term exposure to EGF or rapidly by staurosporine in cultured cells. This morphological change depends on the activity of glycogen synthase kinase-3 (GSK-3). Here, we have characterized the cytoskeletal changes involved in formation of these extended lamellipodia (E-lam) in human HaCaT keratinocytes. E-lams contained actin filaments, stable microtubules and keratin intermediate filaments. E-lam formation was prevented by cytochalasin D, colchicine and low concentrations of taxol and nocodazole, suggesting that actin and microtubule organization and dynamics are essential for E-lam formation. Staurosporine induced recruitment of filamentous actin (F-actin), cortactin, filamin, Arp2/3 complex, Rac1 GTPase and phospholipase C-gamma1 (PLC-gamma1) to lamellipodia. Treatment of cells with the GSK-3 inhibitors SB-415286 and LiCl(2) inhibited E-lam formation and prevented the accumulation of Rac1 and Arp2/3 complex at lamellipodia. The formation of E-lams was dependent on fibronectin-binding integrins and normally regulated Rac1, and expression of either dominant-negative or constitutively active forms of Rac1 prevented E-lam formation. Overexpression of either RhoA or Cdc42 GTPases suppressed E-lam formation. We conclude that extended lamellipodia formation in keratinocytes requires actin and tubulin assembly at the leading edge, and this process is regulated by Rac1 downstream of GSK-3.
 ...
PMID:Glycogen synthase kinase-3 regulates cytoskeleton and translocation of Rac1 in long cellular extensions of human keratinocytes. 1472 58

Beta-catenin is a critical regulator of cell behavior during embryogenesis and neoplastic processes. It also plays a crucial role in repair by modulating dermal fibroblast activity during the proliferative phase of cutaneous wound healing. We hypothesize that growth factors liberated during the initial phase of wound healing convey signals to induce activation of beta-catenin-mediated TCF-dependent signaling during the proliferative phase. Dermal fibroblasts were isolated and cultured from mice containing a beta-galactosidase reporter responsive to beta-catenin-TCF transactivation (TCF-beta-gal). Cells were stimulated with growth factors present at the initial phase of wound healing. EGF and TGF-beta1 significantly increased beta-catenin protein levels and transcriptional activity, whereas beta-catenin mRNA expression was unaffected. This increase was attributed to inactivation of GSK-3beta, a kinase important for beta-catenin destabilization. Subcutaneous injection of EGF or TGF-beta1 before wounding of TCF-beta-gal mice resulted in larger scars and fibroblasts within these wounds that strongly stained for beta-galactosidase, indicating significant beta-catenin transcriptional activity in vivo. Thus, beta-catenin-mediated signaling is activated downstream of growth factors released during the initial phase of wound repair, and may act during the proliferative phase of wound healing to integrate signals from initial phase factors into the expression of genes important during the later, remodeling phase.
 ...
PMID:Growth factors regulate beta-catenin-mediated TCF-dependent transcriptional activation in fibroblasts during the proliferative phase of wound healing. 1472 64

Accumulation of beta-catenin, which leads to enhanced TCF/LEF-1 driven transcription and thereby contributes to tumor development, can result from mutation of beta-catenin itself, inactivation of the adenomatous polyposis coli (APC) protein, or Wnt pathway inhibition of the GSK-3beta kinase that together with APC promotes beta-catenin degradation. Nevertheless, emerging evidence shows that the activation of beta-catenin can occur independently of Wnt signaling to GSK-3beta. In response to EGF, tumor cells overexpressing EGF receptor display GSK-3beta-independent activation of beta-catenin, which may result from a combination of effects-EGF-stimulated, caveolin-1-dependent internalization of E-cadherin, resulting in release of beta-catenin from cell-cell contacts, and EGF-induced downregulation of caveolin-1, relieving the inhibition of signaling molecules sequestered by caveolin-1 at caveolae.
 ...
PMID:Wnt-independent beta-catenin transactivation in tumor development. 1510 3

In epithelial cells, the cell surface glycoprotein E-cadherin is a key molecule in the establishment of cell-cell adhesion. In addition to its contribution to cell adhesion, E-cadherin was found to induce ligand-independent activation of the EGF receptor (EGFR), likely as a result of their co-clustering. As it has also been reported that ligand activation of the overexpressed EGFRs disturb E-cadherin-mediated cell-cell adhesion, we analyzed E-cadherin-EGFR interactions and their consequences in A431 cells and in two colorectal cancer cell lines using immunoblotting and analyzes of several protein kinase activities. Activation of the PI3-K/Akt/GSK-3 signaling pathway upon EGF treatment that we observed in the analyzed cells indicates that EGFRs are functional even in the colorectal cancer cells containing a low density of EGFRs. The transactivation of EGFR by E-cadherin did not occur either in the colorectal cancer cells tested or in A431 cells containing a high density of both EGFRs and E-cadherin on their surface. This observation suggests that high amounts of both molecules on the surface of tumour cells did not predetermine ligand-independent activation of EGFRs.
 ...
PMID:Transactivation of E-cadherin is not involved in the activity of EGF receptor in colorectal carcinoma cells. 1549 39

A potential mechanism underlying ethanol-induced alterations in gene expression is the disruption of transcription factor activity. Growth factor receptors, particularly receptor tyrosine kinases, play an important role in modulating many biological effects of ethanol. We demonstrated here that the expression of epidermal growth factor receptor (EGFR) mediated the effect of ethanol on the activity of transcription factor activator protein-1 (AP-1). Ethanol had little effect on AP-1 activity in the fibroblast cells devoid of EGFR (B82); however, it significantly suppressed AP-1 activity in B82 cells that were stably transfected with either a wild-type EGFR (B82L) or a kinase-deficient receptor (B82M721) in a concentration-dependent manner. EGF activated AP-1 only in B82L cells; the activation was mediated primarily by Akt and ERK. Ethanol inhibited EGF-induced EGFR autophosphorylation, phosphorylation of ERK as well as Akt and its substrate GSK-3beta, and subsequently blocked EGF-stimulated AP-1 activation in B82L cells. On the other hand, ethanol had little effect on EGF-stimulated JNK activation. Phorbol ester 12-O-teradecanoyl-phorbol-13-acetate (TPA) activated AP-1 in B82L and B82M721 cells, but not B82 cells. TPA-induced activation of ERK and PKCdelta was dependent on the expression of EGFR although the intrinsic kinase activity of EGFR was not required. In contrast, TPA-induced phosphorylation of p38 MAPK, JNKs and other PKC isoforms was independent of EGFR. Ethanol selectively inhibited TPA-induced phosphorylation of ERK and PKCdelta, and modestly suppressed TPA-stimulated AP-1 activation in B82L and B82M721 cells. Thus, EGFR plays a critical role in the interaction between ethanol and AP-1.
 ...
PMID:The role of epidermal growth factor receptor in ethanol-mediated inhibition of activator protein-1 transactivation. 1587 57

The ovarian surface epithelium (OSE) is the precursor of common epithelial ovarian carcinomas. In the present study, we examined the molecular mechanisms and possible physiological basis for the propensity of OSE cells to undergo epithelio-mesenchymal transition (EMT) in response to environmental influences. We hypothesized that EMT may be a homeostatic mechanism that permits displaced OSE to assume a stromal phenotype within the ovarian cortex. We report that EGF in conjunction with hydrocortisone is the EMT-inducing factor of OSE as shown by changes to a fibroblast-like morphology and growth pattern. EGF increased cell motility, enhanced the activities of secreted pro-matrix metalloproteinase (MMP)-2 and -9, and enhanced expression and activation of Erk and integrin-linked kinase (ILK). Increased ILK expression correlated with the activation of PKB/Akt, the phosphorylation of GSK-3beta, and the increased expression of cyclin E and cdk2 kinase. EGF withdrawal resulted in a more epithelial morphology and reversal of the EGF-induced activation of signaling pathways and pro-MMP activity. In contrast, treatment of EGF-treated cells with specific inhibitors of phosphatidylinositol 3-kinase, Mek, or ILK inhibited the inhibitor-specific pathways. The inhibitors caused suppression of EGF-induced migration and pro-MMP-2/-9 activities but did not lead to any change in EGF-induced mesenchymal morphology. ILK small interfering RNA inhibited Akt phosphorylation and reduced pro-MMP-2/-9 activities but had no effect on Erk activation or cell morphology. These results indicate that the EGF-induced morphological and functional changes in OSE cells are controlled by distinct signaling mechanisms working in concert. EMT of OSE cells displaced by ovulation likely permits their survival and integration with a fibroblast-like identity within the stroma. Failure to do so may lead to the formation of epithelium-derived inclusion cysts, which are known preferential sites of malignant transformation.
 ...
PMID:Molecular pathways regulating EGF-induced epithelio-mesenchymal transition in human ovarian surface epithelium. 1639 28


1 2 3 Next >>