DETECTION OF SRC HOMOLOGY 3-BINDING PROTEINS, INCLUDING PAXILLIN, IN NORMAL AND V-SRC-TRANSFORMED BALB C 3T3 CELLS/

The Src homology 3 (SH3) domain, located in the amino-terminal, noncat alytic half of pp60src, is highly conserved among members of the Src f amily of tyrosine kinases. SH3 domains have also been identified in a variety of proteins otherwise unrelated to protein-tyrosine kinases. T he presence of SH3 domains in proteins with diverse functions suggests this domain may be important for directing protein-protein interactio ns necessary for protein function or cellular localization. To explore possible interactions between the SH3 domain and cellular proteins, w e have established conditions for the isolation of proteins that bind in solution to the Src SH3 domain. A 67-amino acid fragment of c-Src c ontaining either the entire glutathione S-transferase-SH3 domain (GST- SH3) or the SH3 domain from the neuronal form of c-Src (GST-SH3+) was expressed as a glutathione S-transferase fusion protein. The GST fusio n proteins were incubated with lysates from [S-35]methionine-labeled B alb/c 3T3 cells or v-Src-transformed Balb/c 3T3 cells. We found that G STSH3, but not wild-type GST, specifically interacted with multiple ce llular proteins, whereas GST-SH3+ only weakly associated with a small subset of these proteins. The majority of the SH3-binding proteins wer e found in particulate and detergent-insoluble cell fractions. Anti-ph osphotyrosine immunoblots of the SH3-binding proteins revealed that se veral of the SH3-binding proteins are phosphorylated on tyrosine in v- Src-transformed cells. In addition, a number of the SH3-binding protei ns were phosphorylated on serine and/or threonine in in vitro kinase a ssays, suggesting that one or more of the SH3-binding proteins has kin ase activity. We identified paxillin, a vinculin-binding protein, as o ne of the Src SH3-binding proteins. This finding strongly supports the hypothesis that SH3 domains may be involved in subcellular localizati on of proteins to cytoskeleton and/or cellular membranes.