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)

Platelet activation triggers integrin alpha(IIb)beta(3)-dependent signals and the induction of tyrosine phosphorylation of the cytoskeletal protein alpha-actinin. We have previously reported that alpha-actinin is phosphorylated by the focal adhesion kinase (FAK). In this study, a phosphatase of 68 kDa that dephosphorylated alpha-actinin in vitro was isolated from platelet lysates by three sequential chromatography steps. The phosphatase was identified as SHP-1 by electrospray tandem mass spectrometry. alpha-Actinin was dephosphorylated in vitro by recombinant SHP-1 and by SHP-1 immunoprecipitated from unstimulated or thrombin-stimulated platelet lysates. SHP-1 immunoprecipitated from lysates of platelets adherent to fibrinogen, however, failed to dephosphorylate alpha-actinin. In contrast, the activity of SHP-1 against a synthetic substrate was not affected by the mode of platelet activation. The robust and sustained phosphorylation of alpha-actinin detected in platelets adherent to fibrinogen thus correlates with a decrease in the activity of SHP-1 toward it. Tyrosine phosphorylation of alpha-actinin is seen in vanadate-treated COS-7 cells that are co-transfected with alpha-actinin and wild type FAK. Triple transfection of the cells with cDNAs encoding for alpha-actinin, FAK, and wild type SHP-1 abolished the phosphorylation of alpha-actinin. The phosphorylation of FAK, however, was barely affected by the expression of wild type SHP-1. Both alpha-actinin and FAK were phosphorylated in cells co-expressing alpha-actinin, FAK, and a catalytic domain mutant (C453S) of SHP-1. These findings establish that SHP-1 can dephosphorylate alpha-actinin in vitro and in vivo and suggest that SHP-1 may regulate the tethering of receptors to the cytoskeleton and/or the extent of cross-linking of actin filaments in cells such as platelets.
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PMID:The protein-tyrosine phosphatase SHP-1 regulates the phosphorylation of alpha-actinin. 1507 Sep

Stable platelet aggregation, adhesion, and spreading during hemostasis are promoted by outside-in alphaIIbbeta3 signals that feature rapid activation of c-Src and Syk, delayed activation of FAK, and cytoskeletal reorganization. To evaluate these alphaIIbbeta3-tyrosine kinase interactions at nanometer proximity in living cells, we monitored bioluminescence resonance energy transfer between GFP and Renilla luciferase chimeras and bimolecular fluorescence complementation between YFP half-molecule chimeras. These techniques revealed that alphaIIbbeta3 interacts with c-Src at the periphery of nonadherent CHO cells. After plating cells on fibrinogen, complexes of alphaIIbbeta3-c-Src, alphaIIbbeta3-Syk, and c-Src-Syk are observed in membrane ruffles and focal complexes, and the interactions involving Syk require Src activity. In contrast, FAK interacts with alphaIIbbeta3 and c-Src, but not with Syk, in focal complexes and adhesions. All of these interactions require the integrin beta3 cytoplasmic tail. Thus, alphaIIbbeta3 interacts proximally, if not directly, with tyrosine kinases in a coordinated, selective, and dynamic manner during sequential phases of alphaIIbbeta3 signaling to the actin cytoskeleton.
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PMID:Proximal, selective, and dynamic interactions between integrin alphaIIbbeta3 and protein tyrosine kinases in living cells. 1512 37

N-Formyl-methionyl-leucyl-phenylalanine (fMLP) is a potent activator of neutrophil degranulation. The intracellular signaling mechanisms involved in the potentiating effect of fibrinogen on fMLP-induced primary granule release from human neutrophils were investigated. Fibrinogen caused a significant leftward shift of the concentration-response curve of fMLP-induced elastase release. An antibody against Mac-1 (CD11b/CD18) prevented the potentiating effect of fibrinogen, suggesting that soluble fibrinogen potentiates fMLP-induced degranulating effect by a mechanism mediated by the integrin Mac-1. Fibrinogen enhanced fMLP-induced tyrosine phosphorylation in human neutrophils and markedly enhanced the phosphorylation of mitogen-activated protein kinases (MAPK) caused by fMLP. However, U0126, an inhibitor of p44/42 MAPK activation, or SB-203580, an inhibitor of p38 MAPK, did not alter the effect of fibrinogen on fMLP-induced elastase release. Wortmannin, a phosphatidylinositol 3-kinase (PI3K) kinase inhibitor, and genistein, a nonspecific tyrosine kinase inhibitor, strongly inhibited fMLP-induced elastase release both in the presence and in the absence of fibrinogen. An Akt/PKB inhibitor failed to alter the potentiating effect of fibrinogen, suggesting that the effect of fibrinogen is mediated by Akt-independent pathways. Go6976, an inhibitor of classical PKC isoforms, caused a significant inhibition of fMLP-induced elastase release in the presence or absence of fibrinogen, while nonselective inhibitors of PKC, Ro 31-8220, GF-109203X, and staurosporine, caused potentiation of fMLP-induced elastase release. We conclude that fibrinogen potentiation of primary granule release induced by fMLP is mediated by the integrin CD11b/CD18 through pathways dependent on PI3K and tyrosine kinases, but other regulatory mechanisms may be also involved.
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PMID:Primary granule release from human neutrophils is potentiated by soluble fibrinogen through a mechanism depending on multiple intracellular signaling pathways. 1522 6

Interactions of endothelial cells with fibrin(ogen) are implicated in inflammation, angiogenesis, and wound healing. Cross-linking of the fibrinogen alphaC domains with factor XIIIa generates ordered alphaC oligomers mimicking polymeric arrangement of the alphaC domains in fibrin. These oligomers and those prepared with tissue transglutaminase were used to establish a mechanism of the alphaC domain-mediated interaction of fibrin with endothelial cells. Cell adhesion and chemical cross-linking experiments revealed that oligomerization of the alphaC domains by both transglutaminases significantly increases their RGD (arginyl-glycyl-aspartate)-dependent interaction with endothelial alphaVbeta3 and to a lesser extent with alphaVbeta5 and alpha5beta1 integrins. The oligomerization promotes integrin clustering, thereby increasing cell adhesion, spreading, formation of prominent peripheral focal contacts, and integrin-mediated activation of focal adhesion kinase (FAK) and extracellular signal-regulated kinase (ERK) signaling pathways. The enhanced integrin clustering is likely caused by ordered juxtaposition of RGD-containing integrin-binding sites upon oligomerization of the alphaC domains and increased affinity of these domains for integrins. Our findings provide new insights into the mechanism of the alphaC domain-mediated interaction of endothelial cells with fibrin and imply its potential involvement in cell migration. They also suggest a new role for transglutaminases in regulation of integrin-mediated adhesion and signaling via covalent modification of integrin ligands.
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PMID:Transglutaminase-mediated oligomerization of the fibrin(ogen) alphaC domains promotes integrin-dependent cell adhesion and signaling. 1563 40

CD63 is a member of the tetraspanin superfamily of integral membrane proteins. Present on a variety of cells, tetraspanins can form lateral associations with integrins and may act as 'organizers' of multimolecular networks that modulate integrinmediated signaling, cell morphology, motility and migration. In resting platelets, CD63 is present on the membranes of dense granules and lysosomes but relocates to the plasma membrane following platelet activation and exocytosis where it associates with the platelet integrin alphaIIBbeta3-CD9 complex and with the actin cytoskeleton in an alphaIIBbeta 3-dependent manner. D545, a monoclonal antibody directed at the second extracellular loop of CD63,was used to investigate the role of CD63 in platelet adhesion, spreading and tyrosine phosphorylation. Using immunofluorescence microscopy and confocal imaging, we have demonstrated that D545 does not alter adhesion of platelets to immobilized fibrinogen, but instead platelet spreading. In the presence of buffer or non-specific mouse IgG, activated platelets showed fully spread morphology, F-actin reorganization, redistribution of vinculin and extensive tyrosine phosphorylation, all of which were inhibited by D545. D545 also inhibited the phosphorylation of focal adhesion kinase in thrombin-activated adherent platelets. These results suggest that CD63 may modulate alphaIIBbeta3-dependent cytoskeletal reorganization. To identify signaling enzymes associated with CD63 that could affect this pathway, lipid kinase assays were performed on D545 immunoprecipitates. CD63 co-immunoprecipitated with a lipid kinase which, on the basis of enzymatic properties(stimulated by nonionic detergents, inhibited by adenosine), is consistent with PI 4-kinase type II. The CD63-PI 4-kinase complex was not activation-dependent as the constituents were co-purified from both resting and activated platelets. The linkage of CD63 with PI 4-kinase may result in the recruitment of this signaling enzyme to specific membrane locations in the platelet where it influences phosphoinositide-dependent signaling and platelet spreading.
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PMID:CD63 modulates spreading and tyrosine phosphorylation of platelets on immobilized fibrinogen. 1571 48

CD47 (integrin-associated protein) serves as a receptor for thrombospondin-1 (TSP-1) and Src homology 2 domain-containing protein tyrosine phosphatase substrate-1 (SHPS-1), and the TSP-1/CD47 interaction has been believed to augment integrin-mediated platelet function. Here, employing SHPS-1-immunoglobulin (Ig) as a ligand, we have newly demonstrated that CD47 acts as an inhibitory receptor for platelet function. The binding of SHPS-1-Ig was solely mediated by CD47, because CD47-deficient platelets failed to bind murine SHPS-1-Ig. The human SHPS-1/CD47 interaction inhibited the platelet aggregation induced by several kinds of agonists at a low concentration. Moreover, human SHPS-1 expressed on the cell surface as well as soluble SHPS-1-Ig markedly inhibited the platelet spreading on, but not initial adhesion to, immobilized fibrinogen. Again, neither murine SHPS-1 expressed on the cell surface nor murine SHPS-1-Ig inhibited the spreading of CD47-deficient platelets. We further investigated the tyrosine phosphorylation of signaling proteins during platelet spreading on immobilized fibrinogen. Unexpectedly, SHPS-1 inhibited alpha(IIb)beta(3)-mediated platelet spreading without disturbing focal adhesion kinase (FAK) tyrosine phosphorylation. Further examination revealed that SHPS-1 inhibited the tyrosine phosphorylation of alpha-actinin, a downstream effector of FAK, but not of cortactin. Thus, it is likely that the SHPS-1/CD47 interaction inhibits alpha(IIb)beta(3)-mediated outside-in signaling by interfering with the downstream pathway of FAK. Taken together, our data suggest that SHPS-1 negatively regulates platelet function via CD47, especially alpha(IIb)beta(3)-mediated outside-in signaling.
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PMID:SHPS-1 negatively regulates integrin alphaIIbbeta3 function through CD47 without disturbing FAK phosphorylation. 1584 60

The main pharmacokinetic characteristics of a plasma-derived, pasteurised fibrinogen concentrate were assessed in an open, multicentre, non-controlled study in five patients with congenital afibrinogenaemia or severe congenital hypofibrinogenaemia. Plasma samples were assayed for fibrinogen content in laboratories of the participating clinical centres (CCs) and additionally in a central laboratory at Aventis Behring (ABL). The values of the pharmacokinetic variables, using the fibrinogen determination at ABL, yielded a somewhat shorter terminal half-life compared with that determined at the CCs, with median (range) values of 2.7 days (2.5-3.7 days) versus 3.6 days (3.0-5.3 days), respectively. Fibrinogen clearance rate was clearly lower at the ABL with values of 0.91 ml/h/kg (0.84-1.22 ml/h/kg) compared with 1.65 ml/h/kg (0.82-2.55 ml/h/kg) at the CCs. The distribution volume at steady state (V-ss) of 89 ml/kg (81-116 ml/kg) was also smaller at the ABL than at the CCs (101 ml/kg [84-139 ml/kg]). Response, in vivo recovery and area under the curve did not differ noticeably between the laboratories. The normalisation or near normalisation of pre-infusion pathological coagulation tests indicated a good haemostatic efficacy of the tested fibrinogen concentrate, which was also generally well tolerated and not associated with any serious adverse reactions.
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PMID:Pharmacokinetic properties of a pasteurised fibrinogen concentrate. 1591 41

A number of RGD-type integrins rely on a synergistic site in addition to the canonical RGD site for ligand binding and signaling, although it is still unclear whether these two recognition sites function independently, synergistically, or competitively. Experimental evidence has suggested that fibrinogen binding to the RGD-type integrin alphaIIbbeta3 occurs exclusively through the synergistic gamma(400-411) sequence, thus questioning the functional role of the RGD recognition site. Here we have investigated the respective role of the fibrinogen gamma(400-411) sequence and the RGD motif in the molecular events leading to ligand-induced alphaIIbbeta3-dependent Chinese hamster ovary (CHO) cell or platelet spreading, by using intact fibrinogen and well characterized plasmin-generated fibrinogen fragments containing either the RGD motif (fragment C) or the gamma(400-411) sequence (fragment D), and CHO cells expressing resting wild type (alphaIIbbeta3wt), constitutively active (alphaIIbbeta3T562N), or non-functional (alphaIIbbeta3D119Y) receptors. Our data provide evidence that the gamma(400-411) site by itself is able to initiate alphaIIbbeta3 clustering and recruitment of intracellular proteins to early focal complexes, mediating cell attachment, FAK phosphorylation, and Rac1 activation, while the RGD motif subsequently acts as a molecular switch on the beta3 subunit to trigger cell spreading. More importantly, we show that the premier functional role of the RGD site is not to reinforce cell attachment but, rather, to imprint a conformational change on the beta3 subunit leading to maximal RhoA activation and actin cytoskeleton organization in CHO cells as well as in platelets. Finally, alphaIIbbeta3-dependent RhoA stimulation and cell spreading, but not cell attachment, are Src-dependent and phosphoinositide 3-kinase-independent and are inhibited by the Src antagonist PP2.
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PMID:A new functional role of the fibrinogen RGD motif as the molecular switch that selectively triggers integrin alphaIIbbeta3-dependent RhoA activation during cell spreading. 1595 23

It is increasingly evident that the stromal cells are involved in key metastatic processes of melanoma and some malignant solid tumors. (-)-Epigallocatechin-3-gallate (EGCG), a polyphenolic compound from green tea, has been shown to have anti-tumor activity, inhibiting adhesion, migration, and proliferation of tumor cells. However, little attention has been paid on its effects on stromal cells. In the present study, we determined the effects of EGCG on stromal fibroblasts. We showed that fibroblast adhesion to collagen, fibronectin, and fibrinogen were inhibited by EGCG. One of the possible mechanisms is binding of EGCG to fibronectin and fibrinogen but not to collagen. We then focused how EGCG affected fibroblast adhesion to collagen. EGCG treatment attenuated the antibody binding to fibroblast's integrin alpha2beta1, indicating EGCG may affect the expression and affinity of integrin alpha2beta1. Moreover, intracellular H2O2 level was decreased by EGCG treatment, suggesting that the tonic maintenance of intracellular H2O2 may be required for cell adhesion to collagen. In parallel, collagen-induced FAK phosphorylation, actin cytoskeleton reorganization in fibroblasts, migration and matrix metalloproteinase(s) (MMPs) activity were also affected by EGCG. Tubular networks formed by melanoma cells grown on three-dimensional Matrigel were also disrupted when fibroblasts were treated with EGCG in a non-contact coculture system. Taken together, we provided here the first evidence that EGCG is an effective inhibitor on behaviors of the stromal fibroblasts, affecting their adhesion and migration. The inhibitory activity of EGCG may contribute to its anti-tumor activity. The findings and concepts disclosed here may provide important basis for a further experiment towards understanding tumor-stroma interaction.
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PMID:(-)-Epigallocatechin-3-gallate, a polyphenolic compound from green tea, inhibits fibroblast adhesion and migration through multiple mechanisms. 1605 24

Previous studies have implicated the immunoglobulin (Ig)-immunoreceptor tyrosine-based inhibitory motif (ITIM) superfamily member platelet endothelial cell adhesion molecule-1 (PECAM-1) in the regulation of integrin function. While PECAM-1 has been demonstrated to play a role as an inhibitory coreceptor of immunoreceptor tyrosine-based activation motif (ITAM)-associated Fcgamma receptor IIa (FcgammaRIIa) and glycoprotein VI (GPVI)/FcR gamma-chain signaling pathways in platelets, its physiologic role in integrin alpha(IIb)beta3-mediated platelet function is unclear. In this study, we investigate the functional importance of PECAM-1 in murine platelets. Using PECAM-1-deficient mice, we show that the platelets have impaired "outside-in" integrin alpha(IIb)beta3 signaling with impaired platelet spreading on fibrinogen, failure to retract fibrin clots in vitro, and reduced tyrosine phosphorylation of focal adhesion kinase p125 (125FAK) following integrin alpha(IIb)beta3-mediated platelet aggregation. This functional integrin alpha(IIb)beta3 defect could not be attributed to altered expression of integrin alpha(IIb)beta3. PECAM-1-/- platelets displayed normal platelet alpha granule secretion, normal platelet aggregation to protease-activated receptor-4 (PAR-4), adenosine diphosphate (ADP), and calcium ionophore, and static platelet adhesion. In addition, PECAM-1-/- platelets displayed normal "inside-out" integrin alpha(IIb)beta3 signaling properties as demonstrated by normal agonist-induced binding of soluble fluoroscein isothiocyanate (FITC)-fibrinogen, JON/A antibody binding, and increases in cytosolic-free calcium and inositol (1,4,5)P3 triphosphate (IP3) levels. This study provides direct evidence that PECAM-1 is essential for normal integrin alpha(IIb)beta3-mediated platelet function and that disruption of PECAM-1 induced a moderate "outsidein" integrin alpha(IIb)beta3 signaling defect.
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PMID:The Ig-ITIM superfamily member PECAM-1 regulates the "outside-in" signaling properties of integrin alpha(IIb)beta3 in platelets. 1608 92


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