Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.10.2 (focal adhesion kinase)
 44,029 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Chemoattractant-stimulated polymorphonuclear leukocytes (PMNs) that are adherent to extracellular matrix proteins exhibit a massive, sustained respiratory burst that requires cell spreading. However, the signaling pathways culminating in PMN spreading are not well characterized. Studies showing that protein tyrosine phosphorylation increases with PMN spreading suggest that phosphorylation is critical for this process. In the present study, we observed increased tyrosine phosphorylation of both focal adhesion kinase and Syk in FMLP-activated PMNs that had been plated onto fibrinogen; an increase in Syk activity, but not focal adhesion kinase activity, was apparent. The time course of Syk phosphorylation correlated with the initiation of cell spreading and H2O2 release. Pretreatment of PMNs with piceatannol, a Syk-selective inhibitor, blocked Syk activity, cell spreading, and H2O2 release, indicating that Syk activity was required for the activation of adherent PMNs. Paxillin is a cytoskeletally associated protein that is also tyrosine phosphorylated during PMN spreading and H2O2 release. Paxillin phosphorylation is kinetically slower than Syk phosphorylation and is inhibited with piceatannol, suggesting that paxillin is a substrate for Syk. An analysis of Syk immunoprecipitates indicated that Syk and paxillin associate during PMN spreading. This interaction is not mediated by the src kinases Lyn and Fgr, since neither kinase coprecipitated with Syk. Syk from FMLP-activated, adherent PMNs phosphorylated paxillin-glutathione S-transferase, suggesting that paxillin is a substrate for Syk in vivo. These results indicate that PMN spreading and H2O2 release require a Syk-dependent signaling pathway leading to paxillin phosphorylation.
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PMID:Syk activation is required for spreading and H2O2 release in adherent human neutrophils. 959 Feb 68

Integrin alphaIIb beta3 mediates platelet aggregation and "outside-in" signaling. It is regulated by changes in receptor conformation and affinity and/or by lateral diffusion and receptor clustering. To document the relative contributions of conformation and clustering to alphaIIb beta3 function, alphaIIb was fused at its cytoplasmic tail to one or two FKBP12 repeats (FKBP). These modified alphaIIb subunits were expressed with beta3 in CHO cells, and the heterodimers could be clustered into morphologically detectable oligomers upon addition of AP1510, a membrane-permeable, bivalent FKBP ligand. Integrin clustering by AP1510 caused binding of fibrinogen and a multivalent (but not monovalent) fibrinogen-mimetic antibody. However, ligand binding due to clustering was only 25-50% of that observed when alphaIIb beta3 affinity was increased by an activating antibody or an activating mutation. The effects of integrin clustering and affinity modulation were additive, and clustering promoted irreversible ligand binding. Clustering of alphaIIb beta3 also promoted cell adhesion to fibrinogen or von Willebrand factor, but not as effectively as affinity modulation. However, clustering was sufficient to trigger fibrinogen-independent tyrosine phosphorylation of pp72(Syk) and fibrinogen-dependent phosphorylation of pp125(FAK), even in non-adherent cells. Thus, receptor clustering and affinity modulation play complementary roles in alphaIIb beta3 function. Affinity modulation is the predominant regulator of ligand binding and cell adhesion, but clustering increases these responses further and triggers protein tyrosine phosphorylation, even in the absence of affinity modulation. Both affinity modulation and clustering may be needed for optimal function of alphaIIb beta3 in platelets.
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PMID:Complementary roles for receptor clustering and conformational change in the adhesive and signaling functions of integrin alphaIIb beta3. 964 59

We have previously reported that non-activated platelets can be induced by morphological changes from the recombinant fusion protein of GST-rhodostomin [GST-RHO(RGD)], a member of disintegrin with an arginine-glycine-aspartic acid (RGD) motif. In this study, we further characterized the factors involved in platelet shape changes induced by rhodostomin. From less to full-spreading, four cell spreading indexes, p1, p2, s1 and s2, were designated to the platelet shape based on the scanning electron micrographs. Results of peptide competition and antibody blocking confirmed that interaction between the RGD of rhodostomin and the alpha(IIb)beta3 integrins of platelets was required for induction of a higher percentage of s2 cells. When platelets were pretreated with calphostin C, herbimycin A and cytochalasin B, respectively, the percentage of p1 and p2 cells on rhodostomin-coated plates was increased and, concomitantly, the percentage of s1 and s2 cells was decreased. Biochemical analyses indicated that the focal adhesion kinase (FAK or pp125FAK) in platelets that adhered to GST-RHO(RGD) was phosphorylated in contrast to little or no phosphorylation of FAK in cells adhered to fibrinogen or non-activated cells. Furthermore, the degree of FAK phosphorylation was consistently correlated with morphological changes in platelets treated with various drugs. Taking all the results together, we suggested that rhodostomin could directly bind to integrins of platelets and then trigger signal transduction leading to FAK phosphorylation and actin polymerization and finally resulting in platelet full-spreading.
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PMID:Full-spreading platelets induced by the recombinant rhodostomin are via binding to integrins and correlated with FAK phosphorylation. 969 Jul 77

The function and the outside-in signaling pathways of alphaIIbbeta3 were examined in relation to cell adhesion using a megakaryoblastic leukemia cell line, CMK. After 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment, the cells adhered to the culture plate and underwent megakaryocytic differentiation with expression of alphaIIbbeta3. Binding of soluble fibrinogen to the cells via alphaIIbbeta3 was dependent on cell adhesion. Cell detaching reduced the affinity of this integrin for soluble fibrinogen, although its surface expression was almost unchanged. In contrast, detached cells became tightly adherent to the fibrinogen-coated plate (solid-phase fibrinogen). The same ligand, fibrinogen, present either in soluble or solid-phase form, triggered differential signaling pathways mediated by alphaIIbbeta3. By the stimulation with soluble fibrinogen, Syk was tyrosine-phosphorylated but FAK was dephosphorylated, whereas solid-phase fibrinogen promptly caused tyrosine phosphorylation of FAK followed by delayed phosphorylation of Syk. In addition, the binding of soluble fibrinogen to the cells adherent to fibrinogen-coated plate resulted in tyrosine phosphorylation of integrin beta3 and a complex formation of integrin beta3 with Syk. This implies the cooperation of both soluble and solid-phase fibrinogen-mediated signaling pathways.
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PMID:Outside-In signaling of soluble and solid-phase fibrinogen through integrin alphaIIbbeta3 is different and cooperative with each other in a megakaryoblastic leukemia cell line, CMK. 969 16

Platelet membrane glycoprotein IIIa (GPIIIa) is the most polymorphic integrin subunit in man, with at least seven recognized allelic isoforms present in the human gene pool. Whether these allelic variants of the GPIIb-IIIa complex differ in the ability to interact with the adhesive ligand fibrinogen (Fg) is still unknown. Since the Pena and Penb allelic forms of GPIIIa are distinguished by a single Arg143Gln amino acid substitution within the RGD binding domain of GPIIIa and anti-Pena human alloantibodies have been shown to bind GPIIb-IIIa on the platelet surface and inhibit ADP-induced platelet aggregation, we expressed both forms of this integrin in Chinese hamster ovary (CHO) cells and examined the relative adhesive properties. Both allelic forms of GPIIb-IIIa were expressed on the cell surface and were recognized by a well-characterized panel of murine and human monoclonal and polyclonal antibodies. Like Pena, the Penb form of GPIIb-IIIa could undergo conformational changes in response to RGD peptide binding, and could be induced by activating antibodies to bind Fg and the Fg mimetic antibody P1-55. The binding affinity for Fg of the Pena form of the GPIIb-IIIa complex was not significantly different from that of the Penb form, nor was its ability to signal to focal adhesion kinase, suggesting that Arg143Gln polymorphism has little or no effect on integrin function. Examination of the functional consequences of other integrin polymorphisms may be necessary to determine whether they constitute a risk factor for thrombosis or hemorrhage.
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PMID:Adhesive and signaling properties of a naturally occurring allele of glycoprotein IIIa with an amino acid substitution within the ligand binding domain-the Pena/Penb platelet alloantigenic epitopes. 978 62

Activation of the focal adhesion kinase pp125FAK correlates with its phosphorylation on tyrosine residues and is mediated by multiple receptor-ligand pairs. In platelets, pp125FAK phosphorylation is triggered by alpha IIb beta 3 integrin or Fc gamma RII receptor interaction with immobilized fibrinogen and IgG, respectively. In this study we used platelets as a model system to explore the role of PI 3-kinase relative to pp125FAK phosphorylation. Treatment of the platelets with two PI 3-kinase inhibitors, wortmannin and LY294002, inhibited in a dose-dependent manner alpha IIb beta 3-mediated platelet spreading on fibrinogen having no effect on platelet spreading on IgG. Both inhibitors also completely abolished alpha IIb beta 3-mediated pp125FAK phosphorylation but not pp72syk phosphorylation. Furthermore, Fc gamma RII- and thrombin-induced pp125FAK phosphorylation were not affected by wortmannin and LY294002. Finally, the PI 3-kinase inhibitors' effect on alpha IIb beta 3-mediated spreading and pp125FAK phosphorylation was reversed by phorbol ester treatment. These results establish that the role of PI 3-kinase relative to pp125FAK phosphorylation in platelets is receptor type-specific yet essential for alpha IIb beta 3-mediated cell spreading and pp125FAK phosphorylation.
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PMID:Integrin alpha IIb beta 3-mediated pp125FAK phosphorylation and platelet spreading on fibrinogen are regulated by PI 3-kinase. 999 Mar 7

Inside-out signaling regulates the ligand-binding function of integrins through changes in receptor affinity and/or avidity. For example, alphaIIbbeta3 is in a low-affinity/avidity state in resting platelets, and activation of the receptor by platelet agonists enables fibrinogen to bind. In addition, certain mutations and truncations of the integrin cytoplasmic tails are associated with a high-affinity/avidity receptor. To further evaluate the structural basis of integrin activation, stable Chinese hamster ovary (CHO) cell transfectants were screened for high-affinity/avidity variants of alphaIIbbeta3. One clone (AM-1) expressed constitutively active alphaIIbbeta3, as evidenced by (1) binding of soluble fibrinogen and PAC1, a ligand-mimetic antialphaIIbbeta3 antibody; and (2) fibrinogen-dependent cell aggregation. Sequence analysis and mutant expression in 293 cells proved that a single amino acid substitution in the cysteine-rich, extracellular portion of beta3(T562N) was responsible for receptor activation. In fact, T562N also activated alphaVbeta3, leading to spontaneous binding of soluble fibrinogen to 293 cells. In contrast, neither T562A nor T562Q activated alphaIIbbeta3, suggesting that acquisition of asparagine at residue 562 was the relevant variable. T562N also led to aberrant glycosylation of beta3, but this was not responsible for the receptor activation. The binding of soluble fibrinogen to alphaIIbbeta3(T562N) was not sufficient to trigger tyrosine phosphorylation of pp125(FAK), indicating that additional post-ligand binding events are required to activate this protein tyrosine kinase during integrin signaling. These studies have uncovered a novel gain-of-function mutation in a region of beta3 intermediate between the ligand-binding region and the cytoplasmic tail, and they suggest that this region is involved in integrin structural changes during inside-out signaling.
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PMID:A mutation in the extracellular cysteine-rich repeat region of the beta3 subunit activates integrins alphaIIbbeta3 and alphaVbeta3. 1019 35

Agonists induce inside-out alphaIIbbeta3 signaling resulting in fibrinogen binding and platelet aggregation. These in turn trigger outside-in signaling resulting in further platelet stimulation. Because the Syk tyrosine kinase is activated during both phases of integrin signaling, we evaluated its role in alphaIIbbeta3 function in murine platelets rendered null for Syk by gene targeting and in human platelets incubated with piceatannol, a tyrosine kinase inhibitor reportedly selective for Syk. Both Syk null murine platelets and piceatannol-treated human platelets exhibited a partial, but statistically significant defect in activation of alphaIIbbeta3 by adenine diphosphate (ADP) +/- epinephrine as assessed by fibrinogen binding. Syk null platelets adhered normally to immobilized fibrinogen, and mice with these platelets exhibited normal tail bleeding times. In contrast, piceatannol treatment of human platelets completely inhibited platelet adhesion to immobilized fibrinogen. The discrepancy in extent of integrin dysfunction between murine and human platelet models may be due to lack of specificity of piceatannol, because this compound inhibited the activity of Src and FAK as well as Syk and also reduced tyrosine phosphorylation of multiple platelet proteins. These results provide genetic evidence that Syk plays a role in alphaIIbbeta3 signaling in platelets and pharmacological evidence that, although piceatannol also inhibits alphaIIbbeta3 signaling, it does so by inhibtion of multiple protein tyrosine kinases.
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PMID:Genetic and pharmacological analyses of Syk function in alphaIIbbeta3 signaling in platelets. 1019 44

To better understand the means by which cells such as human platelets regulate the binding of the integrin alphaIIbbeta3 to fibrinogen, we have examined agonist-initiated inside-out and outside-in signalling in CHRF-288 cells, a megakaryoblastic cell line that expresses alphaIIbbeta3 and the human thrombin receptor, PAR1. The results show several notable similarities and differences. (1) Activation of PAR1 caused CHRF-288 cells to adhere and spread on immobilized fibrinogen in an alphaIIbbeta3-dependent manner, but did not support the binding of soluble fibrinogen or PAC-1, an antibody specific for activated alphaIIbbeta3. (2) Direct activation of protein kinase C with PMA or disruption of the actin cytoskeleton with low concentrations of cytochalasin D also caused CHRF-288 cells to adhere to fibrinogen. (3) Despite the failure to bind soluble fibrinogen, activation of PAR1 in CHRF-288 cells caused phosphoinositide hydrolysis, arachidonate mobilization and the phosphorylation of p42MAPK, phospholipase A2 and the Rac exchange protein, Vav, all of which occur in platelets. PAR1 activation also caused an increase in cytosolic Ca2+, which, when prevented, blocked adhesion to fibrinogen. (4) Finally, as in platelets, adhesion of CHRF-288 cells to fibrinogen was followed by a burst of integrin-dependent ('outside-in') signalling, marked by FAK phosphorylation and a more prolonged phosphorylation of p42MAPK. However, in contrast to platelets, adhesion to fibrinogen had no effect on Vav phosphorylation. Collectively, these observations show that signalling initiated through PAR1 in CHRF-288 cells can support alphaIIbbeta3 binding to immobilized ligand, but not the full integrin activation needed to bind soluble ligand. This would suggest that there has been an increase in integrin avidity without an accompanying increase in affinity. Such increases in avidity are thought to be due to integrin clustering, which would also explain the results obtained with cytochalasin D. The failure of alphaIIbbeta3 to achieve the high affinity state in CHRF-288 cells was not due to the failure of PAR1 activation to initiate a number of signalling events that normally accompany platelet activation nor did it prevent at least some forms of outside-in signalling. However, at least one marker of outside-in signalling, the augmentation of Vav phosphorylation seen during platelet aggregation, did not occur in CHRF-288 cells.
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PMID:PAR1 activation initiates integrin engagement and outside-in signalling in megakaryoblastic CHRF-288 cells. 1039 38

Integrin-mediated interactions between cytoskeletal proteins and extracellular fibrinogen are required for platelet adhesion. We have previously demonstrated that the major platelet integrin, alpha(IIb)beta(3), becomes incorporated into the actin cytoskeleton of platelets in an activation-dependent, aggregation-independent manner. To determine if regulatory molecules are also associated with these integrin-rich cytoskeletal complexes, we examined actin cytoskeletons for the presence of kinases and phosphoproteins. Western immunoblot analysis revealed that the tyrosine kinases Src, Fyn, and Lyn are specifically associated with actin cytoskeletons of activated, nonaggregated platelets. However, as noted by others, the cytoskeletal association of focal adhesion kinase depends on platelet aggregation. Actin cytoskeletons isolated from (32)P-labeled platelets also contain a number of phosphorylated proteins. Interestingly, an approximately 18-kDa phosphoprotein was uniquely present in activated platelet cytoskeletons. Collectively, our results demonstrate that actin cytoskeletons of activated, nonaggregated platelets contain not only integrins, but also kinases and phosphoproteins that could regulate platelet adhesion and transmembrane communication.
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PMID:Selective association of the tyrosine kinases Src, Fyn, and Lyn with integrin-rich actin cytoskeletons of activated, nonaggregated platelets. 1040 44


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