A Role for CAP, a Novel, Multifunctional Src Homology 3 Domain-containing Protein in Formation of Actin Stress Fibers and Focal Adhesions*
- From the ‡Department of Physiology, University of Michigan School of Medicine, Ann Arbor, Michigan 48109, the§Department of Cell Biology, Parke-Davis Pharmaceutical Research Division, Warner Lambert Company, Ann Arbor, Michigan 48105, and the ¶Department of Pharmacology, University of Wisconsin, Madison, Wisconsin 53706
Abstract
c-Cbl-associated protein, CAP, was originally cloned from a 3T3-L1 adipocyte cDNA expression library using full-length c-Cbl as a bait. CAP contains a unique structure, with three adjacent Src homology-3 (SH3) domains in the COOH terminus and a region sharing significant sequence similarity with the peptide hormone sorbin. Expression of CAP in NIH-3T3 cells overexpressing the insulin receptor induced the formation of stress fibers and focal adhesions. This effect of CAP expression on the organization of the actin-based cytoskeleton was independent of the type of integrin receptors engaged with extracellular matrix, whereas membrane ruffling and decreased actin stress fibers induced by insulin were not affected by expression of CAP. Immunofluorescence microscopy demonstrated that CAP colocalized with actin stress fibers. Moreover, CAP interacted with the focal adhesion kinase, p125FAK, both in vitro and in vivo through one of the SH3 domains of CAP. The increased formation of stress fibers and focal adhesions in CAP-expressing cells was correlated with decreased tyrosine phosphorylation of p125FAK in growing cells or upon integrin-mediated cell adhesion. These results suggest that CAP may mediate signals for the formation of stress fibers and focal adhesions.
Footnotes
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↵* 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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↵‖ To whom correspondence should be addressed: Parke-Davis Pharmaceutical Research Div., Warner Lambert Co., 2800 Plymouth Rd., Ann Arbor, MI 48105. Tel.: 313-996-3960; Fax: 313-996-5668.
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↵1 The abbreviations used are: SH2 and SH3, Src homology 2 and 3, respectively; CAP, c-Cbl-associated protein; IR, insulin receptor; p125FAK, focal adhesion kinase; PAGE, polyacrylamide gel electrophoresis; TRITC, tetramethylrhodamine isothiocyanate; GST, glutathione S- transferase; DMEM, Dulbecco’s modified Eagle’s medium; PBS, phosphate-buffered saline.
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- Received October 22, 1997.
- Revision received November 21, 1997.
- The American Society for Biochemistry and Molecular Biology, Inc.
