Tyrosine phosphorylation of pp125FAK in platelets requires coordinated signaling through integrin and agonist receptors.
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.
Shattil
S J
SJ
Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia.
Haimovich
B
B
Cunningham
M
M
Lipfert
L
L
Parsons
J T
JT
Ginsberg
M H
MH
Brugge
J S
JS
D019796:15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; D000199:Actins / Q000097:blood; D000527:Alprostadil / Q000494:pharmacology*; D001081:Apyrase / Q000494:pharmacology; D001792:Blood Platelets / Q000378:metabolism*; D002118:Calcium / Q000097:blood*; D015815:Cell Adhesion Molecules / Q000097:blood*; D003593:Cytoplasm / Q000378:metabolism; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D004837:Epinephrine / Q000494:pharmacology; D005340:Fibrinogen / Q000378:metabolism; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000494:pharmacology; D066298:In Vitro Techniques; D007211:Indoles / Q000494:pharmacology; D007213:Indomethacin / Q000494:pharmacology; D008301:Maleimides / Q000494:pharmacology; D008954:Models, Biological; D010766:Phosphorylation; D019000:Phosphotyrosine; D010980:Platelet Membrane Glycoproteins / Q000187:drug effects / Q000378:metabolism*; D011450:Prostaglandin Endoperoxides, Synthetic / Q000494:pharmacology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000097:blood; D011505:Protein-Tyrosine Kinases / Q000097:blood*; D015398:Signal Transduction; D013928:Thromboxane A2 / Q000031:analogs & derivatives / Q000037:antagonists & inhibitors / Q000494:pharmacology; D014443:Tyrosine / Q000031:analogs & derivatives / Q000097:blood; D014665:Vasodilator Agents / Q000494:pharmacology
D000199:Actins; D015815:Cell Adhesion Molecules; D007140:Immunoglobulin Fab Fragments; D007211:Indoles; D008301:Maleimides; D010980:Platelet Membrane Glycoproteins; D011450:Prostaglandin Endoperoxides, Synthetic; D014665:Vasodilator Agents; C054215:SQ 30741; D019000:Phosphotyrosine; D014443:Tyrosine; D004533:Egtazic Acid; D013928:Thromboxane A2; D019796:15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; D005340:Fibrinogen; D011505:Protein-Tyrosine Kinases; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496050:PTK2 protein, human; D011493:Protein Kinase C; D001081:Apyrase; D000527:Alprostadil; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; C070515:bisindolylmaleimide I; D002118:Calcium; D007213:Indomethacin; D004837:Epinephrine