Coordinate interactions of Csk, Src, and Syk kinases with [alpha]IIb[beta]3 initiate integrin signaling to the cytoskeleton.
Integrins regulate cell adhesion and motility through tyrosine kinases, but initiation of this process is poorly understood. We find here that Src associates constitutively with integrin alphaIIbbeta3 in platelets. Platelet adhesion to fibrinogen caused a rapid increase in alphaIIbbeta3-associated Src activity, and active Src localized to filopodia and cell edges. Csk, which negatively regulates Src by phosphorylating Tyr-529, was also constitutively associated with alphaIIbbeta3. However, fibrinogen binding caused Csk to dissociate from alphaIIbbeta3, concomitant with dephosphorylation of Src Tyr-529 and phosphorylation of Src activation loop Tyr-418. In contrast to the behavior of Src and Csk, Syk was associated with alphaIIbbeta3 only after fibrinogen binding. Platelets multiply deficient in Src, Hck, Fgr, and Lyn, or normal platelets treated with Src kinase inhibitors failed to spread on fibrinogen. Inhibition of Src kinases blocked Syk activation and inhibited phosphorylation of Syk substrates (Vav1, Vav3, SLP-76) implicated in cytoskeletal regulation. Syk-deficient platelets exhibited Src activation upon adhesion to fibrinogen, but no spreading or phosphorylation of Vav1, Vav3, and SLP-76. These studies establish that platelet spreading on fibrinogen requires sequential activation of Src and Syk in proximity to alphaIIbbeta3, thus providing a paradigm for initiation of integrin signaling to the actin cytoskeleton.
Obergfell
Achim
A
Division of Vascular Biology, Department of Cell Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.
Eto
Koji
K
Mocsai
Attila
A
Buensuceso
Charito
C
Moores
Sheri L
SL
Brugge
Joan S
JS
Lowell
Clifford A
CA
Shattil
Sanford J
SJ
D000818:Animals; D001792:Blood Platelets / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D015153:Blotting, Western; D002678:Chimera; D003599:Cytoskeleton / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004792:Enzyme Precursors / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D005340:Fibrinogen / Q000378:metabolism; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D051379:Mice; D018345:Mice, Knockout; D009154:Mutation; D015539:Platelet Activation / Q000187:drug effects; D010973:Platelet Adhesiveness / Q000187:drug effects; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000378:metabolism*; D011485:Protein Binding; D011505:Protein-Tyrosine Kinases / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D016392:Proto-Oncogene Proteins pp60(c-src); D015398:Signal Transduction / Q000187:drug effects; D013379:Substrate Specificity; D000072377:Syk Kinase; D019061:src-Family Kinases / Q000037:antagonists & inhibitors / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*
D004791:Enzyme Inhibitors; D004792:Enzyme Precursors; D047908:Intracellular Signaling Peptides and Proteins; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D005340:Fibrinogen; C086603:Matk protein, mouse; D011505:Protein-Tyrosine Kinases; C085377:MATK protein, human; D016392:Proto-Oncogene Proteins pp60(c-src); C000606896:SYK protein, human; D000072377:Syk Kinase; C000606897:Syk protein, mouse; D019061:src-Family Kinases