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Query: EC:2.7.10.2 (focal adhesion kinase)
 44,029 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Rat pheochromocytoma (PC12) cells contain specific plasma membrane receptors for both epidermal growth factor (EGF) and nerve growth factor (NGF). Whereas EGF addition to PC12 cells causes a persistent enhancement of proliferation. NGF addition induces a transient stimulation of growth, followed by growth arrest and neuronal differentiation. Despite these differences in biological response, EGF and NGF share a number of early receptor-mediated responses, which are likely te be related to their effect on cell proliferation. In this paper we show that EGF, but not NGF, is able to stimulate the phosphorylation of membrane proteins. In addition, EGF was able to stimulate phosphorylation of a synthetic peptide (RR-SRC) by PC12 membranes in a concentration-dependent manner. Kinetic analysis of the phosphorylation reaction indicated that EGF increased the Vmax from 13 to 70 pmoles/min/mg protein, while no change was observed in Km. Furthermore, EGF was able to stimulate tyrosine phosphorylation of angiotensin I and II, to the same extent as RR-SRC. In contrast no effects of NGF on peptide phosphorylation by PC12 membranes were observed. Cross-linking experiments demonstrated the presence of receptors for both NGF and EGF in PC12 membranes. These different effects of NGF and EGF on activation of membrane-associated protein-kinase activity demonstrate that NGF might be able to stimulate growth transiently without stimulating protein kinase activity.
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PMID:Epidermal growth factor, but not nerve growth factor, stimulates tyrosine-specific protein-kinase activity in pheochromocytoma (PC12) plasma membranes. 300 Apr 61

The c-kit receptor tyrosine kinase is highly expressed by about 10% of the neurons in the dorsal root ganglia (DRGs) of mouse embryos. We investigated the in vitro effect of stem cell factor (SCF), the ligand for c-kit receptor, on DRGs. Recombinant murine SCF (rmSCF) induced the outgrowth of c-kit-positive neurites from DRGs of normal (+/+) embryos. The effect of SCF was dose dependent and completely abolished by anti-c-kit ACK2 monoclonal antibody (mAb). Some neurites whose outgrowth was induced by nerve growth factor (NGF) were c-kit-positive, but anti-NGF mAb did not inhibit the rmSCF-induced neurite outgrowth. rmSCF did not induce neurite outgrowth from DRGs of W/W embryos that did not express c-kit receptors on the cell surface and of W42/W42 mutant embryos that expressed c-kit receptors without tyrosine kinase activity. rmSCF also had a trophic effect on c-kit-positive neurons in the culture of dissociated DRG cells. Most c-kit-positive neurons appeared to respond to NGF as well, and the SCF-responsive subpopulation represented about 10% of NGF-responsive neurons. rmSCF did not support the survival of DRG neurons from embryos of W/W and W42/W42 genotypes. These results suggest that the stimulus through the c-kit receptor tyrosine kinase has an important role in development of the peripheral nervous system.
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PMID:Stem cell factor induces outgrowth of c-kit-positive neurites and supports the survival of c-kit-positive neurons in dorsal root ganglia of mouse embryos. 750 40

The activated tyrosine kinase oncoprotein BCR-ABL is responsible for pathogenesis of Philadelphia chromosome-positive human leukemias. Because BCR carries a GAP (GTPase-activating protein) activity toward cytoskeleton-related small GTP-binding proteins, we utilized a neuronal PC12 cell system to test morphogenic potentials of BCR-ABL or BCR. We report here unique morphological phenotypes of PC12 cells expressing either BCR-ABL or a BCR mutant which lacks the SH2-binding domain (BCR Delta162-413). Although MAP kinase was not activated in PC12 cells expressing BCR-ABL, they showed incomplete neurite extensions even in the absence of the nerve growth factor (NGF). Overproduction of BCR Delta162-413 in PC12 cells, on the other hand, induced cell rounding in the absence of NGF. Interestingly, those cells could hardly make terminal differentiation in the presence of NGF and continued to grow without changing their round shape, although NGF receptor as well as MAP kinase appeared to be activated. Interestingly, the botulinum C3 toxin induced neurite-like structures in PC12 cells overexpressing BCR Delta162-413 without NGF.
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PMID:BCR-ABL induces neurite-like structures and BCR lacking the SH2-binding domain induces cell rounding in PC12 cells. 898 27

We examined downstream signaling events that followed the exposure of PC12 cells to extracellular ATP and UTP, and we compared the effects of these P2 receptor agonists with those of growth factors and other stimuli. Based on early findings, we focused particular attention on the mitogen-activated protein (MAP) kinase pathway. ATP and/or UTP produced increases in tyrosine phosphorylation of multiple proteins, including p42 MAP (ERK2) kinase, related adhesion focal tyrosine kinase (RAFTK) (PYK2, CAKbeta), focal adhesion kinase (FAK), Shc, and protein kinase Cdelta (PKCdelta). MAP (ERK2) kinase activity (quantified by substrate phosphorylation) was increased by UTP, ATP, phorbol 12-myristate 13-acetate, ionomycin, and growth factors. UTP and ATP were equipotent (EC50 approximately 25 microM) in stimulating MAP kinase activity, suggesting that these effects were mediated via the Gi-linked P2Y2 (P2U) receptor. Consistent with this, the UTP- and ATP-promoted activation of MAP kinase was diminished in pertussis toxin-treated cells. Treatment of cells with pertussis toxin also reduced both the UTP-dependent increases in intracellular calcium ion concentration ([Ca2+]i) and the tyrosine phosphorylation of RAFTK. Similarly, when [Ca2+]i elevation was prevented using BAPTA and EGTA, the activation of MAP kinase by UTP and ionomycin was blocked, and the tyrosine phosphorylation of RAFTK was reduced. The UTP-promoted increase in MAP kinase activity was partially reduced in cells in which PKC was down-regulated, suggesting that both PKC-dependent and PKC-independent pathways were involved. PKCdelta, which increases MAP kinase activity in some systems, became tyrosine-phosphorylated within 15 s of exposure of cells to ATP or UTP; but epidermal growth factor, nerve growth factor, and insulin had little effect. UTP also promoted the association of Shc with Grb2. These results suggest that the P2Y2 receptor-initiated activation of MAP kinase was dependent on the elevation of [Ca2+]i, involved the recruitment of Shc and Grb2, and was mediated by RAFTK and PKC.
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PMID:Activation of P2Y2 receptors by UTP and ATP stimulates mitogen-activated kinase activity through a pathway that involves related adhesion focal tyrosine kinase and protein kinase C. 944 69

The activation of phosphatidylinositol (PtdIns) 3-kinase is considered to be a key event occurring after stimulation of cells with growth factors. The proto-oncogenic protein kinase B (PKB; also known as RAC protein kinase or Akt) has recently been shown to be a downstream target of PtdIns 3-kinase and may be involved in cell survival. We therefore asked whether stimulation of neuronal cells with nerve growth factor (NGF), on which certain types of neurons are dependent for survival, causes activation of PKB. Stimulation of serum-starved PC12 rat pheochromocytoma cells with NGF caused an increase of up to 14-fold in PKB activity. This activation was detected within 1 min of stimulation and occurred at NGF concentrations that are consistent with TrkA-mediated signaling. PKB activation was accompanied by a decrease in electrophoretic mobility of the kinase, which is characteristic of phosphorylation. Both PKB activation and mobility changes were prevented by wortmannin, indicating the upstream involvement of PtdIns 3-kinase in these events. Analyses employing isoform-specific antibodies for immunoprecipitation suggested that all three isoforms of PKB (alpha, beta and gamma) are activated in response to NGF. G-protein-coupled-receptor agonists, lysophosphatidic acid (lyso-PtdH) and thrombin, which induce rapid neurite retraction, neither stimulated PKB activity, nor affected NGF-induced or insulin-induced kinase activation. Wortmannin treatment did not prevent neurite retraction induced by lyso-PtdH or thrombin. These data suggest that PtdIns 3-kinase and PKB are not involved in cytoskeletal changes mediated by the small GTPase Rho.
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PMID:Nerve growth factor promotes activation of the alpha, beta and gamma isoforms of protein kinase B in PC12 pheochromocytoma cells. 949 84

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

The small GTPase RhoA plays a critical role in signaling pathways activated by serum-derived factors, such as lysophosphatidic acid (LPA), including the formation of stress fibers in fibroblasts and neurite retraction and rounding of soma in neuronal cells. Previously, we have shown that ectopic expression of v-Crk, an SH2/SH3 domain-containing adapter proteins, in PC12 cells potentiates nerve growth factor (NGF)-induced neurite outgrowth and promotes the survival of cells when NGF is withdrawn. In the present study we show that, when cultured in 15% serum or lysophosphatidic acid-containing medium, the majority of v-Crk-expressing PC12 cells (v-CrkPC12 cells) display a flattened phenotype with broad lamellipodia and are refractory to NGF-induced neurite outgrowth unless serum is withdrawn. v-Crk-mediated cell flattening is inhibited by treatment of cells with C3 toxin or by mutation in the Crk SH2 or SH3 domain. Transient cotransfection of 293T cells with expression plasmids for p160ROCK (Rho-associated coiled-coil-containing kinase) and v-Crk, but not SH2 or SH3 mutants of v-Crk, results in hyperactivation of p160ROCK. Moreover, the level of phosphatidylinositol-4,5-bisphosphate is increased in v-CrkPC12 cells compared to the levels in mutant v-Crk-expressing cells or wild-type cells, consistent with PI(4)P5 kinase being a downstream target for Rho. Expression of v-Crk in PC12 cells does not result in activation of Rac- or Cdc42-dependent kinases PAK and S6 kinase, demonstrating specificity for Rho. In contrast to native PC12 cells, in which focal adhesions and actin stress fibers are not observed, immunohistochemical analysis of v-CrkPC12 cells reveals focal adhesion complexes which are formed at the periphery of the cell and are connected to actin cables. The formation of focal adhesions correlates with a concomitant upregulation in the expression of focal adhesion proteins FAK, paxillin, alpha3-integrin, and a higher-molecular-weight form of beta1-integrin. Our results indicate that v-Crk activates the Rho-signaling pathway and serves as a scaffolding protein during the assembly of focal adhesions in PC12 cells.
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PMID:Activation of Rho-dependent cell spreading and focal adhesion biogenesis by the v-Crk adaptor protein. 956 23

Antibodies raised against the 51C/SHIP2 inositol polyphosphate 5'-phosphatase were used to examine the effects of growth factors and insulin on the metabolism of this protein. Immunoblot analysis revealed that the 51C/SHIP2 protein was widely expressed in fibroblast and nonhematopoietic tumor cell lines, unlike the SHIP protein, which was found only in cell lines of hematopoietic origin. The 51C/SHIP2 antiserum precipitated a protein of approximately 145 kDa along with an activity which hydrolyzed phosphatidylinositol 3,4, 5-trisphosphate to phosphatidylinositol 3,4-bisphosphate. Tyrosine phosphorylation of the 51C/SHIP2 protein occurred in response to treatment of cells with epidermal growth (EGF), platelet-derived growth factor (PDGF), nerve growth factor (NGF), insulin-like growth factor-1 (IGF-1), or insulin. EGF and PDGF induced transient tyrosine phosphorylation of 51C/SHIP2, with maximal tyrosine phosphorylation occurring at 5-10 min following treatment and returning to near basal levels within 20 min. In contrast, treatment of cells with NGF, IGF-1, or insulin resulted in prolonged tyrosine phosphorylation of 51C/SHIP2 protein, with 40-80% maximal phosphorylation sustained for up to 2 h following agonist treatment. The kinetics of activation of the Akt/PKB protein kinase by the various factors correlated well with the kinetics of tyrosine phosphorylation of 51C/SHIP2. EGF, NGF, and PDGF stimulated the association of 51C/SHIP2 protein with the Shc adapter protein; however, no Shc could be detected in 51C/SHIP2-immune precipitates from cells treated with IGF-1 or insulin. The data suggest that 51C/SHIP2 may play a significant role in regulation of phosphatidylinositol 3'-kinase signaling by growth factors and insulin.
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PMID:Growth factors and insulin stimulate tyrosine phosphorylation of the 51C/SHIP2 protein. 966 Aug 33

Stem cell factor (SCF) stimulates mast cell adhesion and, because SCF is produced normally in tissues, it may be a major factor responsible for the adhesion of mast cells to connective tissue matrix. We found that the morphology of rat peritoneal mast cells (RPMC) altered after the addition of recombinant murine SCF (rmSCF) in vitro. The ability of rmSCF to enhance morphological alteration was dose dependent and completely abolished by anti-c-kit ACK2 monoclonal antibody. Exposure of RPMC to transforming growth factor-beta 1, wortmannin, genistein, herbimycin A, staurosporine, indomethacin and cytochalasin D before the addition of rmSCF antagonized rmSCF-induced morphological alteration. However, nordihydroguiaretic acid had no effect. Many RPMC appeared to respond also to nerve growth factor (NGF) but the total number of cells with altered morphology was much greater when the culture was stimulated by rmSCF than by NGF. We suggest that morphological alterations of mast cells by rmSCF is an important step for the participation in adhesion to tissue under resident physiological conditions.
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PMID:Morphological alterations in rat peritoneal mast cells by stem cell factor. 974 47

Janus kinases (JAKs) are cytoplasmic tyrosine kinases critical for signaling by growth hormone (GH) and many other ligands that bind to members of the cytokine receptor superfamily. SH2-Bbeta was previously identified as a JAK2-interacting protein that is tyrosyl phosphorylated in response to GH and other cytokines that activate JAK2. In this study, we examined whether SH2-Bbeta alters the activity of JAK2. SH2-Bbeta, when coexpressed with JAK2, significantly increased the tyrosyl phosphorylation of JAK2 and multiple other cellular proteins and stimulated the kinase activity of JAK2 by approximately 20-fold. Coexpression of SH2-Bbeta with JAK2 dramatically increased tyrosyl phosphorylation of signal transducer and activator of transcription (Stat)5B and Stat3, physiological substrates of JAK2. SH2-Bbeta(R555E) with a defective Src homology 2 domain was unable to stimulate JAK2 and JAK2-mediated tyrosyl phosphorylation of Stat5B and Stat3. More importantly, SH2-Bbeta enhanced GH-induced tyrosyl phosphorylation of endogenous JAK2 and ligand-induced tyrosyl phosphorylation of Stat5B by endogenous JAK2. In contrast, SH2-Bbeta did not potentiate the activation of other tyrosine kinases including the receptors for platelet-derived growth factor, epidermal growth factor, or nerve growth factor (TrkA), tyrosine kinases that also bind SH2-Bbeta. These data demonstrate that SH2-Bbeta is a potent cytoplasmic activator of JAK2 and is thereby expected to be an important cellular regulator of signaling by GH and other hormones and cytokines that activate JAK2.
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PMID:Identification of SH2-bbeta as a potent cytoplasmic activator of the tyrosine kinase Janus kinase 2. 1037 87


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