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.
Highlights
Ʈ To whom correspondence should be addressed: Parke-Davis Pharmaceutical Research Div., Warner Lambert Co., 2800 Plymouth Rd., Ann Arbor, MI 48105
The abnormally enlarged cell shape of NIH-3T3-IR cells expressing CAP compared with normal cells was consistent with increased number and thickness of actin stress fibers along with the formation of vinculincontaining focal adhesions
The induction of actin stress fibers and focal adhesion formation in NIH-3T3-IR-CAP cells was independent of the type of integrin receptors engaged with extracellular matrix
Summary
Ʈ To whom correspondence should be addressed: Parke-Davis Pharmaceutical Research Div., Warner Lambert Co., 2800 Plymouth Rd., Ann Arbor, MI 48105. We reported recently that insulin markedly stimulated the tyrosine phosphorylation of the c-Cbl proto-oncogene product in 3T3-L1 adipocytes, but not in 3T3-L1 fibroblasts or in any other cell lines expressing high levels of functional insulin receptors and c-Cbl [16] To explore this specificity, c-Cbl was used as a bait in search for adipose-specific signaling proteins. CAP contains a unique structure with three adjacent SH3 domains in the COOH terminus and a region sharing significant sequence similarity with the porcine peptide hormone sorbin, termed the sorbin homology domain [17, 18] Both CAP mRNA and proteins were expressed in 3T3-L1 adipocytes but not in 3T3-L1 or NIH-3T3 fibroblasts. Our results suggest that CAP may play an important role in protein-protein associations involved in cell architecture changes
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