EC:2.7.11.13 - FACTA Search

Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.11.13 (protein kinase C)
49,245 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

FAK is a focal adhesion kinase that is phosphorylated on tyrosine in activated platelets. Induction of FAK phosphorylation requires both fibrinogen binding to integrin alpha IIb beta 3 and post-occupancy events during agonist-induced platelet aggregation or platelet spreading on a fibrinogen matrix. To identify the signaling pathways necessary for tyrosine phosphorylation of FAK, we have examined the conditions that stimulate or inhibit this phosphorylation in platelets in which fibrinogen binding to alpha IIb beta 3 and platelet aggregation were induced directly with an anti-beta 3 Fab fragment (anti-LIBS6). Apyrase was added to prevent effects of the endogenous platelet agonist, ADP. Under these conditions, neither fibrinogen binding nor primary platelet aggregation was sufficient to induce FAK phosphorylation, suggesting that a second "costimulatory" event was required. Indeed, when epinephrine was added with fibrinogen and anti-LIBS6, large platelet aggregates formed and FAK phosphorylation occurred. This response was prevented by blockade of cyclooxygenase with indomethacin or thromboxane A2 receptors with SQ 30,741. A stable thromboxane A2 analogue (U46619) could substitute for epinephrine as the costimulus. Epinephrine costimulation of FAK phosphorylation was also prevented by chelation of intracellular Ca2+ with BAPTA or selective inhibition of protein kinase C (PKC) with bisindolylmaleimide, indicating that Ca2+ and PKC are necessary for FAK phosphorylation under these conditions. Epinephrine also promoted FAK phosphorylation and adhesive spreading of apyrase-treated platelets on a fibrinogen matrix. Cytochalasin D, an inhibitor of actin polymerization, blocked FAK phosphorylation under all these conditions. Thus, tyrosine phosphorylation of FAK in platelets requires coordinated signaling through occupied integrin and agonist receptors. These separate pathways may converge to increase free Ca2+ and activate PKC and thus promote the cytoskeletal reorganization required for activation of FAK.
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PMID:Tyrosine phosphorylation of pp125FAK in platelets requires coordinated signaling through integrin and agonist receptors. 751 81

Lysophosphatidic acid (LPA) and bombesin rapidly stimulate the formation of focal adhesions and actin stress fibres in serum-starved Swiss 3T3 fibroblasts, a process regulated by the small GTP binding protein Rho. To investigate further the signalling pathways leading to these responses, we have tested the roles of three intracellular signals known to be induced by LPA: activation of protein kinase C (PK-C), Ca2+ mobilization and decreased cAMP levels. Neither PK-C activation nor increased [Ca2+]i, alone or in combination, induced stress fibre formation, and in fact activators of PK-C inhibited this response to LPA and bombesin. The G(i)-mediated decrease in cAMP was not required for the response to LPA, and increased cAMP levels did not prevent stress fibre formation. In contrast, the tyrosine kinase inhibitor genistein inhibited the formation of stress fibres induced by both extracellular factors and microinjected Rho protein. Genistein also inhibited the Rho-dependent clustering of phosphotyrosine-containing proteins at focal adhesions, and the increased tyrosine phosphorylation of several proteins including pp125FAK, induced by LPA and bombesin. This suggests a model where Rho-induced activation of a tyrosine kinase is required for the formation of stress fibres.
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PMID:Signal transduction pathways regulating Rho-mediated stress fibre formation: requirement for a tyrosine kinase. 751 76

Mouse Swiss 3T3 fibroblasts cultured in serum-free medium lose their actin stress fibres and vinculin-containing focal adhesions, a process that can be reversed by the addition of serum, lysophosphatidic acid (LPA) or bombesin, and is mediated by rhoA (A. J. Ridley and A. Hall (1992) Cell 70, 389-399). We have shown that the addition of serum to these cells induces the recruitment of the cytoskeletal proteins talin, vinculin and paxillin, and the protein kinases pp125FAK and PKC-delta, to newly formed focal adhesions, and that alpha-actinin is distributed along the actin stress fibres associated with these structures. The newly formed focal adhesions stained heavily with an antibody to phosphotyrosine. A similar response was elicited by 100 ng/ml LPA. The effect of serum was rapid, with focal staining for paxillin largely restricted to cell margins seen within 2 minutes of serum addition, and preceding the assembly of actin filaments. Phosphotyrosine staining differed in that it was predominantly punctate and was widely distributed throughout the cell. By 5 minutes, the paxillin and phosphotyrosine staining was concentrated at the ends of actin filaments largely at the cell margins. The structures stained ranged from circular to oval, but by 10 minutes they more closely resembled the elongated focal adhesions found in cultured fibroblasts. Within 10 minutes, the addition of serum or LPA induced a marked increase in the levels of pp125FAK and paxillin immune-precipitated by an anti-phosphotyrosine antibody. The results suggest that both pp125FAK and paxillin undergo changes in tyrosine phosphorylation upon activation of rhoA, and that these changes are associated with the assembly of focal adhesions and actin stress fibres. The observation that formation of focal adhesions can be induced by the tyrosine phosphatase inhibitor vanadyl hydroperoxide is consistent with the direct involvement of tyrosine phosphorylation in the assembly process. The localisation of PKC-delta to newly formed focal adhesions suggests that serine/threonine phosphorylation may also be important in this regard.
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PMID:The RhoA-dependent assembly of focal adhesions in Swiss 3T3 cells is associated with increased tyrosine phosphorylation and the recruitment of both pp125FAK and protein kinase C-delta to focal adhesions. 752 52

Our previous work demonstrated that 12(S)-HETE, a lipoxygenase metabolite of arachidonic acid, promoted B16 amelanotic melanoma (B16a) cell spreading on fibronectin. In the current study, we investigated the biochemical mechanisms of the 12(S)-HETE induced response. 12(S)-HETE treatment resulted in a time-dependent increase in B16a cell spreading on fibronectin, which was blocked by either calphostin C or by genistein but not by H8. Two hours following cell plating, both spontaneous and 12(S)-HETE promoted cell spreading reached their maximum (nearly 100%). Spontaneous cell spreading was inhibited by the select 12-lipoxygenase inhibitor, BHPP, whose inhibitory effect could be overcome by increasing doses of exogenous 12(S)-HETE. 12(S)-HETE-treated B16a cells plated on either fibronectin or cultured on their own extracellular matrix demonstrated increased vinculin and tyrosine-phosphorylated proteins, which were colocalized at focal adhesions. The increase in vinculin localization to focal adhesions appeared to be a post-transcriptional process, since 12(S)-HETE treatment did not alter the overall protein level of vinculin in tumor cells, but resulted in a specific enrichment of vinculin to focal adhesions. Pretreatment of B16a cells with either calphostin C or genistein abolished 12(S)-HETE-increased formation of vinculin- and phosphotyrosine-containing focal adhesions. Immunoblotting using antiphosphotyrosine antibody 4G10 demonstrated, following 12(S)-HETE stimulation, an increased tyrosine phosphorylation of several proteins in focal adhesions; most prominently, a approximately 155 kd protein, a 120-130 kd protein cluster, a 76 kd protein, and a 42/44 kd complex. Immunoprecipitation with anti-phosphotyrosine antibody PY20 revealed increased tyrosine phosphorylation, post 12(S)-HETE stimulation, of proteins migrating at 120, 76, and 42/44 kd, of which the 120 kd protein co-migrated with pp125FAK. Immunoprecipitation with anti-FAK antibody BC-3 followed by immunoblotting with anti-phosphotyrosine antibody RC20H demonstrated a time-dependent hyperphosphorylation of pp125FAK. The present study suggests that 12(S)-HETE promoted melanoma cell spreading on fibronectin involves tyrosine phosphorylation of pp125FAK and protein kinase C- and tyrosine kinase-dependent focal adhesion formation.
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PMID:Melanoma cell spreading on fibronectin induced by 12(S)-HETE involves both protein kinase C- and protein tyrosine kinase-dependent focal adhesion formation and tyrosine phosphorylation of focal adhesion kinase (pp125FAK). 759 7

In the endothelial cell line EAhy 926, 1-oleoyl-lysophosphatidic acid (LPA) stimulated the tyrosine phosphorylation of the pp42 isoform of mitogen-activated protein (MAP) kinase. Maximum phosphorylation was observed within 5 min of LPA addition, but the response was sustained for up to 120 min. Re-addition of LPA after 60 min stimulated a further sustained increase in the tyrosine phosphorylation of MAP kinase. In cells pretreated with phorbol 12-myristate 13-acetate (PMA; 24 h) or preincubated with the protein kinase C inhibitor Ro-318220, LPA-induced tyrosine phosphorylation of pp42 MAP kinase was substantially reduced at 2 min but potentiated at 60 min. Ro-318220 in combination with either PMA or pertussis toxin pretreatment abolished the LPA response at all time points, suggesting an involvement of protein kinase C in the pertussis toxin-sensitive part of the pathway. Agents which raised intracellular cyclic AMP levels did not affect the initial phase of LPA-stimulated MAP kinase activation, but abolished the late phase. However, this effect was prevented by Ro-318220, implicating a greater role for protein kinase C than protein kinase A in the regulation of sustained MAP kinase responses. LPA stimulated an increase in the tyrosine phosphorylation of focal adhesion kinase pp125 (pp125FAK) in EAhy 926 cells which was both protein kinase C- and pertussis toxin-independent. These results are discussed in terms of the pathways regulating both MAP kinase and pp125FAK in response to LPA in the EAhy 926 endothelial cells line.
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PMID:Regulation of lysophosphatidic acid-stimulated tyrosine phosphorylation of mitogen-activated protein kinase by protein kinase C- and pertussis toxin-dependent pathways in the endothelial cell line EAhy 926. 774 5

The aggregation of the high affinity IgE receptor (Fc epsilon RI) in adherent rat basophilic leukemia (RBL-2H3) cells induces the tyrosine phosphorylation of several proteins. We examined whether focal adhesion-associated tyrosine kinase, pp125FAK, is one of these proteins. Anti-pp125FAK monoclonal antibody immunoblotted and precipitated a 115-kDa tyrosine-phosphorylated protein. In the absence of Fc epsilon RI aggregation, pp125FAK was tyrosine-phosphorylated only in adherent cells. Aggregating Fc epsilon RI in adherent cells markedly enhanced tyrosine phosphorylation of pp125FAK. This increase was detectable within 1 min of Fc epsilon RI aggregation and was maximal by 15 min. In contrast, in nonadherent cells Fc epsilon RI aggregation did not induce tyrosine phosphorylation of pp125FAK. The enhanced influx of calcium by calcium ionophore or the activation of protein kinase C by phorbol myristate acetate induced tyrosine phosphorylation of pp125FAK only in adherent cells. Thus, Fc epsilon RI-induced tyrosine phosphorylation of pp125FAK could be mediated by the activation of protein kinase C and/or the induction of calcium influx. The data indicate that cell adherence is essential for Fc epsilon RI-induced tyrosine phosphorylation of pp125FAK.
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PMID:Tyrosine phosphorylation of pp125FAK by the aggregation of high affinity immunoglobulin E receptors requires cell adherence. 846 10

Platelet adhesion to immobilized fibrinogen stimulates the induction of tyrosine phosphorylation of multiple proteins. However, platelet spreading and tyrosine phosphorylation of three proteins, the focal adhesion kinase pp125FAK and proteins of 101 and 105 kD (pp101 and pp105), require a second adenosine diphosphate (ADP)-dependent costimulatory event. In this study we show that protein kinase C (PKC) inhibitors prevented the induction of tyrosine phosphorylation of pp125FAK, pp101 and pp105, and abolished spreading. These inhibitory effects were not observed after treatment of the platelets with the intracellular Ca2+ chelator BAPTA-AM. This suggested that in platelets, PKC regulates spreading and related protein tyrosine phosphorylation. In addition, the inhibitory effects of apyrase, an ADP scavenger, on spreading and tyrosine phosphorylation of pp125FAK, pp101, and pp105, were not observed in the presence of phorbol 12-myristate 13-acetate (PMA). These data implied that in fibrinogen-adherent platelets integrin ligation and an agonist receptor occupancy are required for the functional association of PKC and the alpha IIb beta 3-mediated signaling pathways. Taken together these results show that PKC plays a central role in the transduction of intracellular signals downstream from alpha IIb beta 3 that regulate spreading and pp125FAK phosphorylation.
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PMID:Protein kinase C regulates tyrosine phosphorylation of pp125FAK in platelets adherent to fibrinogen. 854 37

Platelets express a single low affinity receptor for immunoglobulin, FcgammaRII, that triggers multiple cellular responses upon interaction with multivalent immune complexes. In this study we show that immobilized IgG is also a potent stimulant of platelet activation triggering adhesion, aggregation, massive dense granule secretion, and thromboxane production. Platelet adhesion to IgG was blocked by the FcgammaRII receptor-specific monoclonal antibody, IV. 3. Pretreatment of the platelets with cytochalasin D to inhibit actin polymerization similarly prevented cell binding to IgG having no effect on platelet binding to fibrinogen. Platelet adhesion to IgG also led to the induction of tyrosine phosphorylation of multiple proteins including pp125(FAK) and p72(SYK). These proteins were also tyrosine-phosphorylated in alphaIIbbeta3-deficient IgG-adherent platelets from patients with Glanzmann's thrombasthenia. These data demonstrate that FcgammaRII mediates pp125(FAK) phosphorylation and platelet adhesion to IgG independent of the integrin alphaIIbbeta3. Treatment of the platelets with bisindolylmaleimide to inhibit protein kinase C prevented phosphorylation of pp125(FAK) as well as several other proteins, but not p72(SYK) phosphorylation. This study establishes that the FcgammaRII receptor mediates pp125(FAK) phosphorylation via protein kinase C.
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PMID:The FcgammaRII receptor triggers pp125FAK phosphorylation in platelets. 866 17

Fluid shear stress regulates endothelial cell function, but the signal transduction mechanisms involved in mechanotransduction remain unclear. Recent findings demonstrate that several intracellular kinases are activated by mechanical forces. In particular, members of the mitogen-activated protein (MAP) kinase family are stimulated by hyperosmolarity, stretch, and stress such as heat shock. We propose a model for mechanotransduction in endothelial cells involving calcium-dependent and calcium-independent protein kinase pathways. The calcium-dependent pathway involves activation of phospholipase C, hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2), increases in intracellular calcium and stimulation of kinases such as calcium-calmodulin and C kinases (PKC). The calcium-independent pathway involves activation of a small GTP-binding protein and stimulation of calcium-independent PKC and MAP kinases. The calcium-dependent pathway mediates the rapid, transient response to fluid shear stress including activation of nitric oxide synthase (NOS) and ion transport. In contrast, the calcium-independent pathway mediates a slower response including the sustained activation of NOS and changes in cell morphology and gene expression. We propose that focal adhesion complexes link the calcium-dependent and calcium-independent pathways by regulating activity of phosphatidylinositol 4-phosphate (PIP) 5-kinase (which regulates PIP2 levels) and p125 focal adhesion kinase (FAK, which phosphorylates paxillin and interacts with cytoskeletal proteins). This model predicts that dynamic interactions between integrin molecules present in focal adhesion complexes and membrane events involved in mechanotransduction will be integrated by calcium-dependent and calcium-independent kinases to generate intracellular signals involved in the endothelial cell response to flow.
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PMID:Protein kinases as mediators of fluid shear stress stimulated signal transduction in endothelial cells: a hypothesis for calcium-dependent and calcium-independent events activated by flow. 866 84

The mechanisms by which stimuli that raise cytosolic free Ca2+ concentrations in neurons can increase protein tyrosine phosphorylation are not known. Using rat hippocampal slices and cortical synaptosomes, we have examined the regulation of two highly related cytoplasmic tyrosine kinases, pp125 focal adhesion kinase (pp125(FAK)) and proline-rich tyrosine kinase 2/cell adhesion kinase beta (PYK2/CAKbeta). Membrane depolarization increased tyrosine phosphorylation of PYK2/CAKbeta and pp125(FAK). These effects were blocked by EGTA or by protein kinase C inhibitors (RO31-8220; GF109203X) and mimicked by ionomycin or phorbol 12-myristate 13-acetate, in the case of pp125(FAK), or their combination in the case of PYK2/CAKbeta. Glutamate and specific agonists of ionotropic (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate and N-methyl-D-aspartate) or metabotropic (trans-1-aminocyclopentane-1,3, -dicarboxylate) glutamate receptors stimulated the phosphorylation of pp125(FAK), but not of PYK2/CAKbeta. Glutamate effects were prevented by GF109203X. Thus, in hippocampal slices, tyrosine phosphorylation of pp125(FAK) and PYK2/CAKbeta are regulated differentially by pathways involving Ca2+ and protein kinase C. pp125(FAK) and PYK2/CAKbeta may provide specific links between neuronal activity, increases in cytosolic Ca2+ and protein tyrosine phosphorylation, which may be important for neuronal survival, and synaptic plasticity.
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PMID:Differential regulation of proline-rich tyrosine kinase 2/cell adhesion kinase beta (PYK2/CAKbeta) and pp125(FAK) by glutamate and depolarization in rat hippocampus. 891 May 43

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