Tyrosine Phosphorylation of the β3 Cytoplasmic Domain Mediates Integrin-Cytoskeletal Interactions*
- Alison L. Jenkins‡§,
- Lisa Nannizzi-Alaimo‡§,
- Debra Silver¶,
- James R. Sellers¶,
- Mark H. Ginsberg‖,
- Debbie A. Law‡ and
- David R. Phillips‡**
- From ‡COR Therapeutics, Inc., South San Francisco, California 94080, the ¶Section of Cellular and Molecular Motility, Laboratory of Molecular Cardiology, NHLBI, National Institutes of Health, Bethesda, Maryland 20892-1762, and the‖Scripps Research Institute, La Jolla, California 92037
Abstract
Tyrosine phosphorylation of the β3 subunit of the major platelet integrin αIIbβ3 has been shown to occur during thrombin-induced platelet aggregation (1). We now show that a wide variety of platelet stimuli induced β3 tyrosine phosphorylation, but that this phosphorylation occurred only following platelet aggregation. Several lines of evidence suggest that the β3 cytoplasmic domain tyrosine residues and/or their phosphorylation function to mediate interactions between β3 integrins and cytoskeletal proteins. First, phospho-β3 was retained preferentially in a Triton X-100 insoluble cytoskeletal fraction of thrombin-aggregated platelets. Second, in vitro experiments show that the cytoskeletal protein, myosin, associated in a phosphotyrosine-dependent manner with a diphosphorylated peptide corresponding to residues 740–762 of β3. Third, mutation of both tyrosines in the β3 cytoplasmic domain to phenylalanines markedly reduced β3-dependent fibrin clot retraction. Thus, our data indicate that platelet aggregation is both necessary and sufficient for β3 tyrosine phosphorylation, and this phosphorylation results in the physical linkage of αIIbβ3 to the cytoskeleton. We hypothesize that this linkage may involve direct binding of the phosphorylated integrin to the contractile protein myosin in order to mediate transmission of force to the fibrin clot during the process of clot retraction.
Footnotes
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↵* This work was supported in part by National Institutes of Health Grant HL 48728 (to M. H. G).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵§ These authors contributed equally to this work.
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↵** To whom correspondence should be addressed: COR Therapeutics, Inc., 256 E. Grand Ave., South San Francisco, CA 94080. Tel.: 650-244-6884; Fax: 650-244-9270; E-mail: david_phillips{at}corr.com.
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↵1 The abbreviations used are: PAGE, polyacrylamide gel electrophoresis; HBB, HEPES blot buffer; CHO, Chinese hamster ovary; FACS, fluorescence-activated cell sorter; ITAM, immune receptor tyrosine-based activation motif.
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- Received October 21, 1997.
- Revision received February 6, 1998.
- The American Society for Biochemistry and Molecular Biology, Inc.
