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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)

Platelet glycoprotein (GP) VI is a so-far uncharacterized 62-kDa membrane protein, whose deficiency results in selective impairment in collagen-induced platelet aggregation. Our group previously reported a human polyclonal antibody (anti-p62 IgG) that induces activation of normal, but not of GPVI-deficient, platelets in an Fc-independent manner. The F(ab')2 fragments of this antibody (F(ab')2-anti-p62) stimulated tyrosine phosphorylation of numerous proteins, which was not prevented even in the presence of cAMP-increasing agents such as prostacyclin. Pretreatment of platelets with the protein-tyrosine kinase (PTK) inhibitor tyrphostin A47 completely abolished F(ab')2-anti-p62-induced platelet aggregation in parallel with dose-dependent inhibition of protein-tyrosine phosphorylation, indicating an essential requirement of PTK activity for generating GPVI-mediated signaling. We found that two cytosolic PTKs, c-Src and Syk, became rapidly activated in response to F(ab')2-anti-p62 in a way insensitive to elevation of cAMP. In contrast, in the presence of prostacyclin, F(ab')2-anti-p62 did not stimulate tyrosine phosphorylation of the focal adhesion kinase. cAMP-insensitive activation of c-Src and Syk was also observed in collagen but not thrombin-stimulated platelets. Moreover, either F(ab')2-anti-p62 or collagen stimulated cAMP-insensitive tyrosine phosphorylation of phospholipase C-gamma 2. These results indicate that the receptor-mediated activation of several PTKs in platelets is regulated through a cAMP-sensitive or -insensitive mechanism depending on the nature of each stimulus, and also suggest that GPVI engagement is coupled to cAMP-insensitive activation of c-Src and Syk accompanied by tyrosine phosphorylation of numerous substrates including phospholipase C-gamma 2 in a manner similar to collagen stimulation.
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PMID:Cyclic AMP-insensitive activation of c-Src and Syk protein-tyrosine kinases through platelet membrane glycoprotein VI. 749 87

BTK, the gene that is defective in X-linked agammaglobulinemia, encodes a cytoplasmic tyrosine kinase that is critical for B-cell proliferation, or survival. To identify regulatory elements that control the expression of BTK we evaluated the methylation pattern of this gene in cell lines and in freshly isolated cells. An Hpa II site that was specifically demethylated in mature B cells but not in pre-B cells, T cells, neutrophils, or nonhematopoietic cells was identified in the tenth intron of BTK. In a 40 kilobase (kb) segment of DNA spanning the entire coding region of BTK plus 3 kb upstream of the first exon there were no other sites that demonstrated lineage-specific demethylation. The B-cell-specific demethylation site in intron 10, which falls within the SH2 domain, 26 kb distal to the first exon, occurs in a region rich in regulatory elements including two E2 boxes, two AP-2 sites, and a cAMP response element. It is likely that this site plays a role in maintaining BTK transcription in mature B cells.
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PMID:B-cell-specific demethylation of BTK, the defective gene in X-linked agammaglobulinemia. 754 76

Dictyostelium discoideum cells harbor two annexin VII isoforms of 47 and 51 kDa which are present throughout development. In immunofluorescence and cell fractionation studies annexin VII was found in the cytoplasm and on the plasma membrane. In gene disruption mutants lacking both annexin VII isoforms growth, pinocytosis, phagocytosis, chemotaxis and motility were not significantly impaired under routine laboratory conditions, and the cells were able to complete the developmental cycle on bacterial plates. On non-nutrient agar plates development was delayed by three to four hours and a significant number of aggregates was no longer able to form fruiting bodies. Exocytosis as determined by measuring extracellular cAMP phosphodiesterase, alpha-fucosidase and alpha-mannosidase activity was unaltered, the total amounts of these enzymes were however lower in the mutant than in the wild type. The mutant cells were markedly impaired when they were exposed to low Ca2+ concentrations by adding EGTA to the nutrient medium. Under these conditions growth, motility and chemotaxis were severely affected. The Ca2+ concentrations were similar in mutant and wild-type cells both under normal and Ca2+ limiting conditions; however, the distribution was altered under low Ca2+ conditions in SYN-cells. The data suggest that annexin VII is not required for membrane fusion events but rather contributes to proper Ca2+ homeostasis in the cell.
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PMID:The in vivo role of annexin VII (synexin): characterization of an annexin VII-deficient Dictyostelium mutant indicates an involvement in Ca(2+)-regulated processes. 765 24

Lactogens [prolactin (Prl) and growth hormone] stimulate phosphorylation of the 40S ribosomal protein, S6, in Nb2 cells by mechanisms that do not involve participation of cAMP or protein kinase A, protein kinase C, or cGMP-dependent protein kinase. However, inhibition of tyrosine kinase (TK) abrogates Prl-mediated macromolecular biosynthesis. Inasmuch as lactogen signaling may involve sequential activation of protein kinases, the effect of Prl on the well-characterized mitogen-activated protein kinase (MAPK) and S6 kinase (S6K), the enzyme responsible for S6 phosphorylation in vivo, and their relationship to Nb2 macromolecular biosynthesis and mitogenesis were investigated. The results show that MAPK stimulation is transient (peak activity, 30 min) and precedes that of S6K, which reaches a maximum at 1.5-2 h, and slowly returns towards control levels at 6 h. Both staurosporine which inhibits GH receptor-associated kinase (JAK2) and genistein (GEN), an inhibitor of membrane-associated and cytoplasmic TKs, abrogate Prl-stimulated TK, MAPK, and S6K. Rapamycin (RAP), a specific inhibitor of p70S6K, completely blocks S6K but does not affect TK and MAPK. TK and MAPK activity correlates with Prl-stimulated anabolism, i.e., protein and DNA synthesis and mitogenesis. Thus, concentrations of STR and GEN which abrogate TK and MAPK inhibit anabolism virtually 100%. However, RAP, which inhibits S6K (ca. 100%) but not TK or MAPK, only delays Prl-mediated anabolism. These results indicate that Prl signaling in Nb2 cells involves a protein kinase cascade and that regulation of receptor-associated kinase, TK, and MAPK correlates with anabolism. The role of S6K (and S6 phosphorylation) appears to be ancillary.
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PMID:Stimulation of receptor-associated kinase, tyrosine kinase, and MAP kinase is required for prolactin-mediated macromolecular biosynthesis and mitogenesis in Nb2 lymphoma. 784 Jun 14

Actin reorganization is an early response to many extracellular factors. In Swiss 3T3 fibroblasts, the Ras-related GTP-binding proteins Rho and Rac act as key signal transducers in these responses: Rho is required for growth factor-induced formation of stress fibres and focal adhesions, whereas membrane ruffling is regulated by Rac proteins. Several proteins that act as GTPase activating proteins (GAPs) for Rho-related proteins have been identified, and these could act either as targets or down-regulators of Rho or Rac in cells. In vitro, the GAP domain of p190 has a striking preference for Rho as a substrate, and when microinjected into Swiss 3T3 cells it inhibits stress fibre formation but not membrane ruffling induced by growth factors. BcrGAP acts on Rac but not Rho in vitro, and specifically inhibits membrane ruffling in vivo. Finally, RhoGAP acts preferentially on the Rho-related protein G25K/Cdc42Hs in vitro, but can inhibit Rho-mediated responses in vivo. These results suggest that p190, Bcr and RhoGAP play specific roles in signalling pathways through different Rho family members. The mechanisms underlying Rho-regulated stress fibre formation have been investigated further by analysing the role of other signals known to be activated by lysophosphatidic acid (LPA). Neither activation of PK-C, increased intracellular Ca2+, decreased cAMP levels or Ras activation appear to mediate stress fibre formation. However, LPA stimulates tyrosine phosphorylation of a number of proteins, including the focal adhesion kinase, pp125FAK, and genistein, a tyrosine kinase inhibitor, prevents this increase in tyrosine phosphorylation.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Signal transduction through the GTP-binding proteins Rac and Rho. 788 87

Integrins and other adhesion receptors are essential components for outside-in and inside-out signaling through the cell membrane. The platelet glycoprotein IIb-IIIa (also known as fibrinogen receptor or integrin alpha IIb beta 3) is activated by platelet agonists, inhibited by cyclic-nucleotide-elevating agents, and is involved in the activation of protein tyrosine kinases including the 125-kDa focal adhesion kinase (pp125FAK). However, the molecular details of glycoprotein IIb-IIIa regulation are not well understood. Here we report that in ADP-activated human platelets cAMP- and cGMP-dependent protein-kinase-mediated phosphorylation of the focal adhesion vasodilator-stimulated phosphoprotein (VASP) at Ser157 correlates well with glycoprotein IIb-IIIa inhibition. Human platelets contain similar concentrations of glycoprotein IIb-IIIa complexes (fibrinogen binding sites) and VASP. Using gel-filtered platelets, cAMP-elevating agents [e.g. prostaglandin E1 and the forskolin analog 6-(3-dimethylaminopropionyl)forskolin (NKH 477)] caused VASP Ser157 phosphorylation and inhibited glycoprotein IIb-IIIa activation up to 70-100%. NO-generating, cGMP-elevating agents [e.g. 3-morpholinosydnonimine hydrochloride (SIN1) and sodium nitroprusside] stimulated VASP Ser157 phosphorylation and inhibited glycoprotein IIb-IIIa activation up to a maximal extent of 30-50%. The effects of cAMP- and cGMP-elevating agents on VASP phosphorylation and fibrinogen binding were reversible and could be mimicked by membrane-permeant selective activators of platelet cAMP- or cGMP-dependent protein kinase, respectively. Using threshold concentrations, the nitrovasodilator SIN 1 potentiated the effects of the forskolin analog NKH 477 with respect to inhibition of platelet aggregation, VASP phosphorylation and glycoprotein IIb-IIIa inhibition. It is proposed that the inhibition of glycoprotein IIb-IIIa induced by cyclic nucleotide involves cAMP-and cGMP-dependent protein-kinase-mediated VASP phosphorylation at Ser157.
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PMID:Phosphorylation of focal adhesion vasodilator-stimulated phosphoprotein at Ser157 in intact human platelets correlates with fibrinogen receptor inhibition. 792 40

Signal transduction of two mitogens for pancreatic beta-cells, GH and PRL, was investigated using the differentiated insulin-secreting cell line, INS-1. Addition of human GH (hGH) or ovine PRL in a serum-substitute medium increased growth, insulin content, and nutrient metabolism evaluated by tetrazolium salt reduction. hGH, bovine GH (bGH), and PRL also stimulated [3H]thymidine incorporation in a dose-dependent manner (1 pM - 1 nM). hGH induced cytosolic Ca2+ ([Ca2+]i) rises, which were transient, dependent on the presence of extracellular Ca2+, blocked by verapamil, calciseptine, and the hyperpolarizing agent diazoxide, suggesting that hGH stimulates Ca(2+)-influx through L-type Ca(2+)-channels. Similar effects on [Ca2+]i were observed with bGH or PRL. hGH caused membrane depolarization in a small proportion of the cells ( < 25%) as detected by cell-attached patch-clamp analysis. However, hGH failed to stimulate acute insulin secretion. hGH, bGH, and PRL promoted tyrosine phosphorylation of JAK2 tyrosine kinase. Verapamil inhibited neither [3H]thymidine incorporation nor JAK2 phosphorylation stimulated by hGH, whereas a tyrosine kinase inhibitor, lavendustin A, blocked the mitogenic effect. Involvement of cAMP is suggested because Rp-cyclic adenosine-3', 5'-monophosphorothioate, a competitive inhibitor of protein kinase A, abolished hGH-induced [Ca2+]i rises and DNA synthesis. cAMP appears to play a permissive role, although hGH failed to raise cellular cAMP levels. These results support the idea that activation of JAK2 is a major signaling event, whereas the [CA2+]i rise is not a prerequisite, for the mitogenic effects of GH and PRL in insulin-secreting cells.
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PMID:Postreceptor signalling of growth hormone and prolactin and their effects in the differentiated insulin-secreting cell line, INS-1. 861 23

The binding of CD40 ligand on activated T cells to CD40 on resting B cells induces the expression of costimulatory molecules B7-1 (CD80) and B7-2 (CD86). The induction of B7 molecules by CD40 ligand-CD40 interaction represents a critical step in rendering B cells competent for antigen presentation. The CBA/N mouse has a defect in CD40 signalling which has been attributed to a mutation in Bruton's tyrosine kinase. We have compared the ability of murine CD40 ligand to induce B7-1 and B7-2 expression on B cells isolated from CBA/N and wild-type CBA/J mice. We find that the CBA/N defect partially impairs both B7-1 and B7-2 induction via CD40. Subsequent experiments investigated the roles of different second messenger systems in B7-1 and B7-2 induction in normal B cells. In M12 B lymphomas either CD40 cross-linking or cAMP treatment can induce B7 molecules. Here we report that treatment with dibutyryl-cAMP also induces B7 molecules in normal B cells provided that they have been preactivated by CD40 cross-linking. We also find that PMA and ionomycin treatment of B cells induces B7-2 but not B7-1 expression. Our data therefore show roles for BTK, cAMP and PMA/ionomycin in B7 induction, as well as providing further evidence for differential regulation of B7-1 and B7-2 induction in B cells.
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PMID:Induction of costimulatory molecules B7-1 and B7-2 in murine B cells. the CBA/N mouse reveals a role for Bruton's tyrosine kinase in CD40-mediated B7 induction. 870 Jan 70

Stress fibers, composed of actin filaments, converge upon and associate with a number of proteins, including focal adhesion kinase (p125FAK), and integrin receptors to form areas of close contact between cells and the extracellular matrix referred to as focal adhesions. Treatment of mesangial cells with cAMP-elevating agents causes a loss of focal adhesions, fragmentation of stress fibers, and decreased tyrosine phosphorylation of p125FAK. Thrombin reverses these effects of cAMP, and this model can be used to address some of the cellular mechanisms involved in regulating the loss and formation of focal adhesions. This study reports the effects of cAMP and thrombin on mesangial cell shape, distribution of actin, formation of stress fibers, and tyrosine phosphorylation of p125FAK. cAMP-treated cells display a condensed cell body with slender processes that traverse the area formerly covered by the cell. Addition of thrombin to these cells restores actin filaments (stress fibers) and increases tyrosine phosphorylation of p125FAK, and the cells resume a flattened morphology, even in the continued presence of cAMP-elevating agents. Peptides that mimic the tethered ligand portion of the thrombin receptor have the same effects on cell morphology and stress fiber formation as thrombin. In selected experiments, agents that disrupt either stress fibers (cytochalasin D) or microtubules (nocodazole; Sigma Chemical, St. Louis, MO) were used to examine the role of these cytoskeletal elements in thrombin-induced restoration of focal adhesions. Cytochalasin D blocked the ability of thrombin to restore focal adhesions and phosphorylate p125FAK. The effects of nocodazole, an agent that destabilizes microtubules (but which has no known receptor), are very similar to those of thrombin. The findings discussed in this study indicate that thrombin can modulate the formation of focal adhesions. The organization of stress fibers and microtubules is apparently intimately related to the phosphorylation of p125FAK and can be modulated by soluble receptor agonists such as thrombin or via altered polymerization of microtubules.
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PMID:Tyrosine phosphorylation of focal adhesion kinase (p125FAK): regulation by cAMP and thrombin in mesangial cells. 870 7

Incubation of human platelets with EGTA under conditions that dissociate the alphaIIbbeta3-integrin stimulated tyrosine phosphorylation of pp72(syk) (6.8-fold) and of proteins of 62 (2. 2-fold), 68 (2.5-fold) and 130 kDa (1.4-fold). Stimulation of tyrosine phosphorylation of pp72(syk) was associated with an increase of pp72(syk) kinase activity. In contrast to pp72(syk), tyrosine phosphorylation of the focal adhesion kinase pp125(FAK) was not stimulated by EGTA. Preincubation of platelets with the monoclonal antibody P2, which binds to the alphaIIbbeta3 complex and thus stabilizes it, strongly reduced the increase of tyrosine phosphorylation of pp72(syk), p62, and p68 induced by EGTA. The Y2/51 monoclonal antibody, which recognizes only the beta3 glycoprotein, did not inhibit the stimulation of protein tyrosine phosphorylation evoked by EGTA. Stimulation of tyrosine phosphorylation of pp72(syk), p62, p68, and p130 induced by EGTA was not observed in thrombasthenic platelets, which lack the alphaIIbbeta3-integrin. The results indicate that the dissociation of the alphaIIbbeta3 complex in intact platelets activates pp72(syk). The mechanism of activation was found to be insensitive to inhibition by cAMP and cGMP and only partially dependent on cytosolic Ca2+, suggesting a close functional coupling of alphaIIbbeta3-integrin and pp72(syk). Since platelets retain their discoid shape after EGTA treatment, we further conclude that pp72(syk) stimulation alone is not sufficient for platelet activation.
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PMID:Dissociation of the alphaIIbbeta3-integrin by EGTA stimulates the tyrosine kinase pp72(syk) without inducing platelet activation. 890 Jan 25


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