THE SH3 DOMAIN OF CRK BINDS SPECIFICALLY TO A CONSERVED PROLINE-RICH MOTIF IN EPS15 AND EPS15R

The Crk protein belongs to the family of proteins consisting of mainly Src homology 2 and 3 (SH2 and SH3) domains. These proteins are though t to transduce signals from tyrosine kinases to downstream effecters. In order to understand the specificity and effector function of the SH 3 domain of Crk, we screened an expression library for binding protein s. We isolated Eps15, a substrate of the epidermal growth factor recep tor (EGFR) tyrosine kinase, and Eps15R, a novel protein with high sequ ence homology to the carboxyl-terminal domain of Eps15. Antibodies rai sed against a fragment of the Eps15R gene product immunoprecipitated a protein of 145 kDa, Eps15 and Eps15R bound specifically to the amino- terminal SH3 domain of Crk and coprecipitated equivalently with both c -Crk and v-Crk from cell lysates. The amino acid sequences of Eps15 an d Eps15R featured several proline-rich regions as putative binding mot ifs for SH3 domains. In both Eps15 and Eps15R, we identified one proli ne-rich motif which accounts for their interaction with the Crk SH3 do main. Each binding motif contains the sequence P-X-L-P-X-K, an amino a cid stretch that is highly conserved in all proteins known to interact specifically with the first SH3 domain of Crk. Furthermore, we found that immunoprecipitates of activated EGFR-kinase stably bound in vitro -translated Eps15 only in the presence of in vitro-translated v-Crk. C rk might therefore be involved in Eps15-mediated signal transduction t hrough the EGFR.