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Published online 3 May 2004. doi:10.1083/jcb.200402064
The Rockefeller University Press, 0021-9525 $8.00
JCB, Volume 165, Number 3, 305-311
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Article

 Proximal, selective, and dynamic interactions between integrin {alpha}IIbß3 and protein tyrosine kinases in living cells

Maddalena de Virgilio1, William B. Kiosses1, and Sanford J. Shattil1,2

1 Department of Cell Biology, The Scripps Research Institute, La Jolla, CA 92037
2 Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA 92037

Address correspondence to S.J. Shattil, Department of Cell Biology, The Scripps Research Institute, 10550 North Torrey Pines Rd., VB-5, La Jolla, CA 92037. Tel.: (858) 784-7148. Fax: (858) 784-7422. email: shattil{at}scripps.edu

Stable platelet aggregation, adhesion, and spreading during hemostasis are promoted by outside-in {alpha}IIbß3 signals that feature rapid activation of c-Src and Syk, delayed activation of FAK, and cytoskeletal reorganization. To evaluate these {alpha}IIbß3–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 {alpha}IIbß3 interacts with c-Src at the periphery of nonadherent CHO cells. After plating cells on fibrinogen, complexes of {alpha}IIbß3–c-Src, {alpha}IIbß3–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 {alpha}IIbß3 and c-Src, but not with Syk, in focal complexes and adhesions. All of these interactions require the integrin ß3 cytoplasmic tail. Thus, {alpha}IIbß3 interacts proximally, if not directly, with tyrosine kinases in a coordinated, selective, and dynamic manner during sequential phases of {alpha}IIbß3 signaling to the actin cytoskeleton.

Key Words: signaling; Src; Syk; FAK; bioluminescence

The online version of this article includes supplemental material.

Abbreviations used in this paper: BiFC, bimolecular fluorescence complementation; BRET, bioluminescence resonance energy transfer; BSA, bovine serum albumin; Rluc, Renilla luciferase.


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