Adhesive and locomotive properties of cells have key roles in normal physiology and disease. Cell motility and adhesion require the assembly and organization of actin microfilaments into stress fibers, lamellipodia and filopodia, and the formation of these structures is mediated by signalling through Rho; GTPases. Here we identify c-Cbl (a multi-adaptor proto-oncogene product involved in protein tyrosine kinase signalling) as an important regulator of the actin cytoskeleton. By immunofluorescence microscopy we have determined that c-Cbl co-localizes with the adaptor protein Crk to submembranous actin lamellae in NIH 3T3 fibroblasts and that c-Cbl's actin localization requires specific SH3-binding sequences. Further, we have found that truncation of this SH3-binding domain in c-Cbl profoundly alters the morphology of NIH 3T3 fibroblasts by inhibiting the formation of actin lamellae, lamellipodia and membrane ruffles. The induction of lamellipodia and membrane ruffles are also inhibited during cell spreading and migration, conditions when these structures are normally most prominent. The inhibitory effect of truncated c-Cbl expression on lamellipodia formation can be reversed by mutational inactivation of its divergent SH2 domain, by the co-expression of constitutively active Rac or by the overexpression of c-Cbl. This study therefore identifies a cytoskeletal role for c-Cbl which may involve the regulation of Crk and Rac, and which is dependent on targeting of c-Cbl to actin lamellae and the ability to recruit signalling protein(s) associated with its divergent SH2 domain.

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