Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The experiments presented here were designed to examine the contribution of p125 focal adhesion kinase (p125FAK) tyrosine phosphorylation to the activation of the mitogen-activated protein kinase cascade induced by bombesin, lysophosphatidic acid (LPA), and platelet-derived growth factor (PDGF) in Swiss 3T3 cells. We found that tyrosine phosphorylation of p125FAK in response to these growth factors is completely abolished in cells treated with cytochalasin D or in cells that were suspended in serum-free medium for 30 min. In marked contrast, the activation of p42mapk by these factors was independent of the integrity of the actin cytoskeleton and of the interaction of the cells with the extracellular matrix. The protein kinase C inhibitor GF 109203X and down-regulation of protein kinase C by prolonged pretreatment of cells with phorbol esters blocked bombesin-stimulated activation of p42mapk, p90rsk, and MAPK kinase-1 but did not prevent bombesin-induced tyrosine phosphorylation of p125FAK. Furthermore, LPA-induced p42mapk activation involved a pertussis toxin-sensitive guanylate nucleotide-binding protein, whereas tyrosine phosphorylation of p125FAK in response to LPA was not prevented by pretreatment with pertussis toxin. Finally, PDGF induced maximum p42mapk activation at concentrations (30 ng/ml) that failed to induce tyrosine phosphorylation of p125FAK. Thus, our results demonstrate that p42mapk activation in response to bombesin, LPA, and PDGF can be dissociated from p125FAK tyrosine phosphorylation in Swiss 3T3 cells.
Mol Biol Cell 1996 Dec
PMID:Dissociation of mitogen-activated protein kinase activation from p125 focal adhesion kinase tyrosine phosphorylation in Swiss 3T3 cells stimulated by bombesin, lysophosphatidic acid, and platelet-derived growth factor. 897 Jan 51

The focal adhesion kinase (FAK), a protein-tyrosine kinase (PTK), associates with integrin receptors and is activated by cell binding to extracellular matrix proteins, such as fibronectin (FN). FAK autophosphorylation at Tyr-397 promotes Src homology 2 (SH2) domain binding of Src family PTKs, and c-Src phosphorylation of FAK at Tyr-925 creates an SH2 binding site for the Grb2 SH2-SH3 adaptor protein. FN-stimulated Grb2 binding to FAK may facilitate intracellular signaling to targets such as ERK2-mitogen-activated protein kinase. We examined FN-stimulated signaling to ERK2 and found that ERK2 activation was reduced 10-fold in Src- fibroblasts, compared to that of Src- fibroblasts stably reexpressing wild-type c-Src. FN-stimulated FAK phosphotyrosine (P.Tyr) and Grb2 binding to FAK were reduced, whereas the tyrosine phosphorylation of another signaling protein, p130cas, was not detected in the Src- cells. Stable expression of residues 1 to 298 of Src (Src 1-298, which encompass the SH3 and SH2 domains of c-Src) in the Src- cells blocked Grb2 binding to FAK; but surprisingly, Src 1-298 expression also resulted in elevated p130cas P.Tyr levels and a two- to threefold increase in FN-stimulated ERK2 activity compared to levels in Src- cells. Src 1-298 bound to both FAK and p130cas and promoted FAK association with p130cas in vivo. FAK was observed to phosphorylate p130cas in vitro and could thus phosphorylate p130cas upon FN stimulation of the Src 1-298-expressing cells. FAK-induced phosphorylation of p130cas in the Src 1-298 cells promoted the SH2 domain-dependent binding of the Nck adaptor protein to p130cas, which may facilitate signaling to ERK2. These results show that there are additional FN-stimulated pathways to ERK2 that do not involve Grb2 binding to FAK.
Mol Cell Biol 1997 Mar
PMID:Fibronectin-stimulated signaling from a focal adhesion kinase-c-Src complex: involvement of the Grb2, p130cas, and Nck adaptor proteins. 903 97

In the current studies, we investigated the relationship between tyrosine phosphorylation and neurite formation. In SH-SY5Y neuroblastoma cells, the tyrosine kinase inhibitor methyl 2, 5-dihydroxycinnimate blocked neurite formation on laminin. This corresponded with inhibition of paxillin and focal adhesion kinase tyrosine phosphorylation as well as a disruption of actin filament organization and actin polymerization. This suggests that tyrosine phosphorylation helps direct changes in the actin cytoskeleton required for neurite formation.
Brain Res Mol Brain Res 1996 Dec 31
PMID:The tyrosine kinase inhibitor methyl 2,5-dihydroxycinnimate disrupts changes in the actin cytoskeleton required for neurite formation. 903 51

The relationship between receptor number and agonist-induced intracellular responses has been well studied in receptors coupled to adenylate cyclase; however, for receptors coupled to phospholipase C (PLC), very little is known about the effect of receptor number on receptor-mediated processes. To explore this issue, we investigated the effect of the number of receptors for gastrin-releasing peptide (GRP) on ligand affinity and on the ability to activate intracellular messengers [PLC, tyrosine phosphorylation of p125 focal adhesion kinase (p125FAK)] and cause receptor modulation (internalization, desensitization, down-regulation) and ligand degradation. Three BALB 3T3 cell lines were made that stably expressed the gastrin-releasing peptide receptor (GRP-R) with receptor numbers varying by 280-fold (GRP-R-Low, GRP-R-Med, and GRP-R-Hi). Each cell line had the same affinity for agonist. The efficacy for bombesin to increase [3H]inositol phosphates but not tyrosine phosphorylation of p125FAK correlated well with receptor number. In contrast, the EC50 value for [3H]inositol phosphate generation for bombesin was the same in each cell line. Receptor number did not alter internalization. In the absence of protease inhibitors, there was an inverse correlation between receptor number and receptor down-regulation and desensitization. However, with protease inhibitors present, GRP-R-Med and GRP-R-Hi down-regulated significantly less than the GRP-R-Low. Similarly, GRP-R-Low desensitized significantly more than GRP-R-Med or GRP-R-Hi. GRP-R-Hi caused significantly greater ligand degradation than GRP-R-Low, and protease inhibitors completely inhibited degradation by GRP-R-Low and inhibited degradation by 70% for GRP-R-Hi. In conclusion, we show that for the PLC-coupled GRP-R, receptor number had little or no effect on binding affinity, potency for activating PLC, tyrosine phosphorylation of p125FAK, or extent of receptor internalization. In contrast, receptor number had an effect on ligand degradation, down-regulation, desensitization, and efficacy of PLC activation without altering the efficacy of tyrosine phosphorylation of p125FAK. These results demonstrate that the effect of receptor number differs for the different functions mediated by the GRP receptor and differs from that reported for adenylate cyclase-coupled receptors such as receptors mediating the action of adrenergic agents, secretin, and opioids.
Mol Pharmacol 1997 May
PMID:Effect of gastrin-releasing peptide receptor number on receptor affinity, coupling, degradation, and modulation. 914 10

Alveolar epithelial type II cells are the progenitor cells for restoring the alveolar epithelial barrier after acute lung injury. During repair of lung injury, the alveolar epithelial type II cells reepithelialize denuded air spaces, a process that involves breaking and reforming cell adhesions. A novel technique of mechanical separation of cultured alveolar epithelial cells from in vitro matrix was used to examine the intracellular signals that result when alveolar epithelial cell adhesions are broken. The results show that the tyrosine phosphorylation levels of focal adhesion kinase, paxillin, and pp60(src) decreased immediately after mechanical separation of the cells. Levels returned to nearly normal by 24 h after mechanical separation. Paxillin and pp60(scr) coprecipitated with focal adhesion kinase regardless of their phosphorylation state. Interestingly, the tyrosine phosphorylation level of the mitogen-activated protein kinase, p42(erk2), increased 15 min after mechanical separation. Preincubation of cell monolayers with phenylarsine oxide, a protein tyrosine phosphatase inhibitor, blocked the decrease in tyrosine phosphorylation levels of focal adhesion kinase, paxillin and pp60(src). Phenylarsine oxide incubation also prevented readhesion of mechanically separated cells at 24 h, but genistein, a tyrosine kinase inhibitor, had no effect. We conclude that protein tyrosine phosphatases are activated immediately after cultured alveolar epithelial cells are mechanically separated from in vitro matrix, and their activation is required for alveolar epithelial cell readhesion.
Am J Respir Cell Mol Biol 1997 May
PMID:Protein tyrosine phosphatases mediate cell readhesion in alveolar epithelial cells mechanically separated from in vitro matrix. 916 Aug 44

R- cells, a line of mouse embryo fibroblasts with a targeted disruption of the insulin-like growth factor I (IGF-I) receptor genes, are refractory to transformation by several viral and cellular oncogenes. Using colony formation in soft agar as a measure of full transformation, we report here that R- cells can be transformed by v-src, although they still cannot be transformed by the activated c-src527 (mutation at tyrosine 527 to phenylalanine), which readily transforms mouse embryo cells with a wild-type number of IGF-I receptors (W cells). Although v-src is a more potent inducer of tyrosine phosphorylation than c-src527, the extent of phosphorylation of either insulin receptor substrate 1 or Shc, two of the major substrates of the IGF-I receptor, does not seem sufficiently different to explain the qualitative difference in soft agar growth. v-src, however, is considerably more efficient than c-src527 in its ability to tyrosyl phosphorylate, in R- cells, the focal adhesion kinase, Stat1, and p130cas. These results indicate that v-src, but not c-src527, can bypass the requirement for a functional IGF-I receptor in the full transformation of mouse embryo fibroblasts and suggest that qualitative and quantitative differences between the two oncogenes can be used to identify some of the signals relevant to the mechanism(s) of transformation.
Mol Cell Biol 1997 Jul
PMID:Insulin-like growth factor I receptor signaling in transformation by src oncogenes. 919 8

Genistein (5,7,4'-trihydroxyisoflavone), an isoflavinoid found in soy beans, has been identified as potentially causal for the low incidence of metastatic prostate cancer (PCa) in certain countries. Although genistein-induced PCa cell adhesion has been identified as a possible causative mechanism, direct growth inhibition by genistein has been reported and also could be causal. If in vivo growth inhibition was significant, then growth inhibition should occur at concentrations attained with dietary consumption, the mechanism of growth inhibition should be relevant to PCa, and genistein (a broad-spectrum in vitro protein-tyrosine kinase inhibitor) should have relatively specific kinase inhibitory effects in vivo. These considerations were investigated by measuring growth inhibitory activity in a variety of PCa cell lines. Growth inhibitory effects were shown not to occur with concentrations below the low micromolar range (i.e., 3 logs above that attained in serum). In-depth mechanistic studies with the PC3-M metastatic variant cell line demonstrated that growth inhibition was independent of genistein's estrogenic effects. Genistein was shown to decrease the viability of nonadherent cells, suggesting a lack of dependence on cell adhesion for growth inhibition. However, important molecular and kinetic differences between genistein's effects on growth in adherent versus nonadherent cells were identified. Specific suppression of focal adhesion kinase activity (without global decreases in phosphotyrosine) was shown to precede induction of apoptosis, which was responsible for growth inhibition in adherent cells. These findings do not support an in vivo growth inhibitory role by genistein consumed in quantities associated with a soy-based diet. They do, however, identify genistein as a potential therapeutic agent for PCa and as a tool with which to study the control of apoptosis in PCa.
Mol Pharmacol 1997 Feb
PMID:Genistein-induced apoptosis of prostate cancer cells is preceded by a specific decrease in focal adhesion kinase activity. 920 23

Cell attachment to fibronectin stimulates the integrin-dependent interaction of p85-associated phosphatidylinositol (PI) 3-kinase with integrin-dependent focal adhesion kinase (FAK) as well as activation of the Ras/mitogen-activated protein (MAP) kinase pathway. However, it is not known if this PI 3-kinase-FAK interaction increases the synthesis of the 3-phosphorylated phosphoinositides (3-PPIs) or what role, if any, is played by activated PI 3-kinase in integrin signaling. We demonstrate here the integrin-dependent accumulation of the PI 3-kinase products, PI 3,4-bisphosphate [PI(3,4)P2] and PI(3,4,5)P3, as well as activation of AKT kinase, a serine/threonine kinase that can be stimulated by binding of PI(3,4)P2. The PI 3-kinase inhibitors wortmannin and LY294002 significantly decreased the integrin-induced accumulation of the 3-PPIs and activation of AKT kinase, without having significant effects on the levels of PI(4,5)P2 or tyrosine phosphorylation of paxillin. These inhibitors also reduced cell adhesion/spreading onto fibronectin but had no effect on attachment to polylysine. Interestingly, integrin-mediated Erk-2, Mek-1, and Raf-1 activation, but not Ras-GTP loading, was inhibited at least 80% by wortmannin and LY294002. In support of the pharmacologic results, fibronectin activation of Erk-2 and AKT kinases was completely inhibited by overexpression of a dominant interfering p85 subunit of PI 3-kinase. We conclude that integrin-mediated adhesion to fibronectin results in the accumulation of the PI 3-kinase products PI(3,4)P2 and PI(3,4,5)P3 as well as the PI 3-kinase-dependent activation of the kinases Raf-1, Mek-1, Erk-2, and AKT and that PI 3-kinase may function upstream of Raf-1 but downstream of Ras in integrin activation of Erk-2 MAP and AKT kinases.
Mol Cell Biol 1997 Aug
PMID:Phosphatidylinositol 3-kinase is required for integrin-stimulated AKT and Raf-1/mitogen-activated protein kinase pathway activation. 923 99

Endothelial cell (EC) gap formation and barrier function are subject to dual regulation by (1) axial contractile forces, regulated by myosin light chain kinase activity, and (2) tethering forces, represented by cell-cell and cell-substratum adhesions. We examined whether focal adhesion plaque proteins (vinculin and talin) and focal adhesion kinase, p125FAK (FAK), represent target regulatory sites involved in thrombin-mediated EC barrier dysfunction. Histologically, thrombin produced dramatic rearrangement of EC actin, vinculin, and FAK in parallel with the evolution of gap formation and barrier dysfunction. Vinculin and talin were in vitro substrates for phosphorylation by EC PKC, a key effector enzyme involved in thrombin-induced EC barrier dysfunction. Although vinculin and talin were phosphorylated in situ under basal conditions in 32P-labeled EC, thrombin failed to alter the basal level of phosphorylation of these proteins. Phosphotyrosine immunoblotting showed that neither vinculin nor talin was significantly phosphorylated in situ on tyrosine residues in unstimulated ECs, and this was not further increased after thrombin. In contrast, both thrombin and the thrombin receptor-activating peptide (TRAP) produced an increase in FAK phosphotyrosine levels (corrected for immunoreactive FAK content) present in EC immunoprecipitates. Ionomycin, which produces EC barrier dysfunction in a myosin light chain kinase-independent manner, was used to increase intracellular Ca2+ and evaluate the Ca2+ sensitivity of this observation. In contrast to thrombin, ionomycin effected a dramatic decrease in the phosphotyrosine-to-immunoreactive FAK ratios, suggesting distinct effects of the two agents on FAK phosphorylation and function. These data indicate that modulation of cell tethering via phosphorylation of focal adhesion proteins is complex, agonist-specific, and may be a relevant mechanism of EC barrier dysfunction in permeability models that do not depend on an increase in myosin 20-kD regulatory light chain phosphorylation.
Am J Respir Cell Mol Biol 1997 Oct
PMID:Thrombin-mediated focal adhesion plaque reorganization in endothelium: role of protein phosphorylation. 937 19

Graf is a GTPase-activating protein for Rho that interacts with focal adhesion kinase and co-localizes with the actin cytoskeleton (Hildebrand, J. D., Taylor, J. M. and Parsons, J. T. (1996) Mol. Cell. Biol. 16, 3169-3178). We examined the expression and regulation of Graf as a prelude to understanding the role of Graf in mediating signal transduction in vivo. We demonstrated that Graf is a ubiquitously expressed 95-kDa protein with high levels observed in heart and brain and cells derived from these tissues. Stimulation of PC12 cells with epidermal growth factor or nerve growth factor induced a phosphatase-reversible mobility shift upon gel electrophoresis, indicative of phosphorylation. In vitro, purified mitogen-activated protein (MAP) kinase catalyzed the phosphorylation of Graf on serine 510, suggesting that Graf phosphorylation may be mediated through MAP kinase signaling. In addition, the mutation of serine 510 to alanine inhibited the epidermal growth factor-induced mobility shift of mutant Graf protein in vivo, consistent with serine 510 being the site of in vivo phosphorylation. Based on these data we suggest that phosphorylation of Graf by MAP kinase or related kinases may be a mechanism by which growth factor signaling modulates Rho-mediated cytoskeletal changes in PC12 and perhaps other cells.
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PMID:Characterization of graf, the GTPase-activating protein for rho associated with focal adhesion kinase. Phosphorylation and possible regulation by mitogen-activated protein kinase. 952 7


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