Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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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)

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.
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PMID:Thrombin-mediated focal adhesion plaque reorganization in endothelium: role of protein phosphorylation. 937 19

The Ras-dependent activation of Erk kinases by G protein-coupled receptors (GPCRs) is thought to involve tyrosine phosphorylation of docking proteins that serve as scaffolds for the plasma membrane recruitment of Ras guanine nucleotide exchange factors, such as the Grb2-mSos complex. We have investigated the role of two GPCR-regulated tyrosine phosphoproteins, p125(FAK) (FAK) and Shc, in the Ras-dependent activation of Erk kinases by endogenously expressed GPCRs in Rat 1a fibroblasts. Several lines of evidence suggest that tyrosine phosphorylation of FAK and Shc are independently regulated. The GPCRs for lysophosphatidic acid (LPA), thrombin, and bombesin mediate equivalent increases in FAK tyrosine phosphorylation and FAK-Grb2 association. In contrast, only LPA and thrombin receptors significantly stimulate Shc tyrosine phosphorylation and Shc-Grb2 complex formation. Tyrosine phosphorylation of FAK is pertussis toxin-insensitive, can be mimicked by calcium ionophore, and is inhibited by treatment with cytochalasin D, which depolymerizes the actin cytoskeleton. In contrast, tyrosine phosphorylation of Shc is inhibited by pertussis toxin treatment, is not induced by calcium ionophore, and is insensitive to cytochalasin D. In each case, the rapid stimulation of Erk 1/2 correlates with tyrosine phosphorylation of Shc but not of FAK. The dissociation of FAK-Grb2 complex formation from receptor-mediated activation of Erk 1/2 indicates that recruitment of Grb2-mSos to the plasma membrane is not sufficient to mediate rapid Erk activation. Using four mechanistically distinct inhibitors of clathrin-mediated endocytosis, concanavalin A, hypertonic medium, depletion of intracellular potassium, and monodansylcadaverine, we find that GPCR-mediated Erk 1/2 activation is also endocytosis-dependent. Thus, we propose that an additional step involving vesicle-mediated endocytosis is required for the rapid, Ras-dependent activation of Erk kinases in fibroblasts.
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PMID:G protein-coupled receptors mediate two functionally distinct pathways of tyrosine phosphorylation in rat 1a fibroblasts. Shc phosphorylation and receptor endocytosis correlate with activation of Erk kinases. 939 6

Recent studies show that tyrosine phosphorylation by a number of neuropeptides may be an important intracellular pathway in mediating changes in cell function, particularly related to growth. Neuromedin B (NMB), a mammalian bombesin related peptide, functions through a distinct receptor, the neuromedin B receptor (NMB-R), of which little is known about its cellular basis of action. In the present study we explored the ability of NMB-R activation to cause tyrosine phosphorylation of focal adhesion kinase (p125(FAK)), an important substrate for tyrosine phosphorylation by other neuropeptides. NMB caused rapid increases in p125(FAK) phosphorylation which reached maximum at 2 min in both rat C6 glioblastoma cells which possess native NMB-Rs and rat neuromedin B receptor (rNMR-R) transfected BALB 3T3 cells. NMB had a half-maximal effect was at 0.4 nM and was 30-fold more potent than gastrin-releasing peptide (GRP). The stoichiometric relationships between increased p125(FAK) tyrosine phosphorylation and other cellular processes was similar in both C6 cells and rNMB-R transfected cells. TPA (1 microM) caused 45% and the calcium ionophore, A23187, 11% of maximal tyrosine phosphorylation of p125(FAK) seen with NMB. A23187 potentiated the effect of TPA. Pretreatment with the selective PKC inhibitor, GF109203X, inhibited TPA-induced p125(FAK) tyrosine phosphorylation, but it had no effect on the NMB stimulation. Pretreatment with thapsigargin completely inhibited NMB-stimulated increases in [Ca2+]i, but had no effect on NMB-stimulation of p125(FAK) phosphorylation either alone or with GF109203X. The tyrosine kinase inhibitor, tyrphostin A25, inhibited NMB-induced phosphorylation of p125(FAK) by 52%. However, tyrphostin A25 did not inhibit NMB-stimulated increases in [3H]inositol phosphates. Cytochalasin D, an agent which disrupts actin microfilaments, inhibited BN- and TPA-induced tyrosine phosphorylation of p125(FAK) completely. In contrast, colchicine, an agent which disrupts microtubules, had no effect. Pretreatment with Clostridium botulinum C3 exoenzyme which inactivates the small GTP-binding protein rho p21, also inhibited tyrosine phosphorylation of p125(FAK) by 55%. These results demonstrate that activation of NMB-R can cause rapid tyrosine phosphorylation of p125(FAK). NMB-induced tyrosine phosphorylation of p125(FAK) is independent of NMB-induced changes in [Ca2+]i or PKC. The integrity of the actin cytoskeleton but not of microtubules is necessary for NMB-stimulated phosphorylation of p125(FAK). The ras-related small GTP-binding protein rho p21 is at least partially involved in mediating NMB-induced tyrosine phosphorylation of p125(FAK). These results suggest that similar to some other neuropeptides, activation of this pathway may be an important mechanism in mediating cellular changes by this receptor such as growth.
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PMID:Neuromedin B receptor activation causes tyrosine phosphorylation of p125FAK by a phospholipase C independent mechanism which requires p21rho and integrity of the actin cytoskeleton. 940 68

Aggregation of the FcepsilonRI, a member of the immune receptor family, induces the activation of proteintyrosine kinases and results in tyrosine phosphorylation of proteins that are involved in downstream signaling pathways. Here we report that Pyk2, another member of the focal adhesion kinase family, was present in the RBL-2H3 mast cell line and was rapidly tyrosine-phosphorylated and activated after FcepsilonRI aggregation. Tyrosine phosphorylation of Pyk2 was also induced by the calcium ionophore A23187, by phorbol myristate acetate, or by stimulation of G-protein-coupled receptors. Adherence of cells to fibronectin dramatically enhanced the induced tyrosine phosphorylation of Pyk2. Although Src family kinases are activated by FcepsilonRI stimulation and tyrosine-phosphorylate the receptor subunits, the activation and tyrosine phosphorylation of Pyk2 were downstream of Syk. In contrast, tyrosine phosphorylation of Pyk2 by stimulation of G-protein-coupled receptors was independent of Syk. Therefore, the FcepsilonRI-induced tyrosine phosphorylation of Pyk2 is downstream of Syk and may play a role in cell secretion.
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PMID:Activation of protein-tyrosine kinase Pyk2 is downstream of Syk in FcepsilonRI signaling. 940 54

Sik, the mouse homologue of the breast tumor kinase Brk, is expressed in differentiating cells of the gastrointestinal tract and skin. We examined expression and activity of Sik in primary mouse keratinocytes and a mouse embryonic keratinocyte cell line (EMK). Calcium-induced differentiation of these cells has been shown to be accompanied by the activation of tyrosine kinases and rapid phosphorylation of a 65-kDa GTPase-activating protein (GAP)-associated protein (GAP-A.p65). We demonstrate that Sik is activated within 2 min after calcium addition in primary keratinocytes and EMK cells. In EMK cells, Sik binds GAP-A.p65, and this interaction is mediated by the Sik Src homology 2 domain. Although Sik directly complexes with GAP-A.p65, overexpression of wild-type or kinase defective Sik in EMK cells does not lead to detectable changes in GAP-A.p65 phosphorylation. These data suggest that Sik is not responsible for phosphorylation of GAP-A.p65. GAP-A. p65 may act as an adapter protein, bringing Sik into proximity of an unidentified substrate. Overexpression of Sik in EMK cells results in increased expression of filaggrin during differentiation, supporting a role for Sik in differentiation.
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PMID:A role for the epithelial-cell-specific tyrosine kinase Sik during keratinocyte differentiation. 940 38

The glycoprotein hormone, erythropoietin is the principal regulator of the production of circulating erythrocytes by controlling proliferation, differentiation and survival of its target erythroid progenitor cells. The receptor for erythropoietin is a type I cytokine receptor lacking intrinsic tyrosine kinase activity. It mediates tyrosine phosphorylation through its association with nonreceptor tyrosine kinases such as JAK2 and initiates a cascade of signalling events in response to erythropoietin. Significant progress has been made in identifying signalling pathways triggered by erythropoietin. However, the exact signalling mechanisms mediating the known physiological effects of erythropoietin in erythroid progenitor cells are poorly understood. There are many open questions including the role of Ca2+ in erythropoietin induced signal transduction. Although the results concerning the effect of erythropoietin on [Ca2+]i in various erythroid cells are conflicting, [Ca2+]i-increasing agents mimic the effect of erythropoietin on c-myb expression and activate the program of haemoglobin synthesis in murine erythroleukemia cells. An attempt is made in this review to survey recent data on the erythropoietin-induced signal transduction with respect to the different physiological effects of this hormone.
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PMID:Signalling mechanisms in erythropoiesis: the enigmatic role of calcium. 941 12

Changes in the concentration of extracellular calcium can affect the balance between proliferation and differentiation in several cell types, including keratinocytes, breast epithelial cells, and fibroblasts. This report demonstrates that elevation of extracellular calcium stimulates proliferation-associated signaling pathways in rat fibroblasts and implicates calcium-sensing receptors (CaR) as mediators of this response. Rat-1 fibroblasts express CaR mRNA and protein and respond to known agonists of the CaR with increased IP3 production and release of intracellular calcium. Agonists of the CaR can stimulate increased c-SRC kinase activity and increased extracellular signal-regulated kinase 1/mitogen-activated protein kinase activity. Both of the increases in SRC activity and mitogen-activated protein kinase activation are blocked in the presence of a nonfunctional mutant of the CaR, R796W. Proliferation of wild-type Rat-1 cells is sensitive to changes in extracellular calcium, but expression of the nonfunctional CaR mutant or inhibition of the calcium-dependent increase in SRC kinase activity block the proliferative response to calcium. These results provide evidence of a novel signal transduction pathway modulating the response of fibroblasts to extracellular calcium and imply that calcium-sensing receptors may play a role in regulating cell growth in response to extracellular calcium, in addition to their well known function in systemic calcium homeostasis.
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PMID:Functional calcium-sensing receptors in rat fibroblasts are required for activation of SRC kinase and mitogen-activated protein kinase in response to extracellular calcium. 942 77

Pyk2 is a recently described cytoplasmic tyrosine kinase that is related to focal adhesion kinase (FAK) and can be activated by a variety of stimuli that elevate intracellular calcium. In this report, we showed that Pyk2 and FAK tyrosine phosphorylation are regulated differentially by integrin-mediated cell adhesion and soluble factors both in rat aortic smooth muscle cells, which express endogenous Pyk2 and FAK, and in transfected Chinese hamster ovary cells. We also found that Pyk2 is diffusely present throughout the cytoplasm, while FAK is localized in focal contacts as expected, suggesting that the different localization may account for their differential regulation. By analyzing a chimeric protein contain N-terminal and kinase domains of Pyk2 and C-terminal domain of FAK, we provided evidence that the distinctive C-terminal domains of Pyk2 and FAK were responsible for their differential regulation by integrins and soluble stimuli as well as their subcellular localization. Finally, we correlated FAK, Pyk2, and the chimeric protein binding to talin, but not paxillin, with their regulation by integrins and focal contact localization. These results demonstrate that the distinctive C-terminal domain of Pyk2 and FAK confer their differential regulation by different subcellular localization and association with the cytoskeletal protein talin.
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PMID:Differential regulation of Pyk2 and focal adhesion kinase (FAK). The C-terminal domain of FAK confers response to cell adhesion. 944 86

The possible role of tyrosine kinase in the regulation of fowl sperm motility was investigated by using a stable analogue of erbstatin, methyl 2,5-dihydroxycinnamate (2,5-MeC), a specific inhibitor of tyrosine kinase. This inhibited the motility of intact spermatozoa at 30 degrees C in a dose-dependent manner. In contrast, the motility of demembranated spermatozoa was not inhibited by the same concentrations of 2,5-MeC. At 40 degrees C, both intact and demembranated spermatozoa were almost immotile with or without 2,5-MeC. Additionally, intact spermatozoa, stimulated by the addition of Ca2+ or calyculin A, a specific inhibitor of protein phosphatases, lost their motility with the subsequent addition of 2,5-MeC at 40 degrees C. However, unlike the motility, the ATP concentrations of spermatozoa were maintained in about 30-35 nmol ATP/10(9) cells during these incubation periods. The activity of tyrosine kinase of spermatozoa at 30 degrees C, estimated by measuring the phosphorylation of a synthetic peptide substrate, RR-SRC, was 0.17 pmol/min per milligram of protein. This activity was lower than that of fowl testes or chick brain but higher than that of chick liver. These results suggest that tyrosine kinase activity, which is not retained in the axoneme and/or accessory cytoskeletal components, may be involved in the maintenance of flagellar movement of fowl spermatozoa at 30 degrees C.
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PMID:Effects of tyrosine kinase inhibitor on the motility and ATP concentrations of fowl spermatozoa. 944 62

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


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