Enhancement of tyrosyl phosphorylation and protein expression of eps8 by v-Src

Two eps8 isoforms, p97(eps8) and p68(eps8), were previously identified as s ubstrates for receptor tyrosine kinases. Analysis of eps8 phosphotyrosine c ontent in v-Src transformed cells (IV5) revealed that both isoforms were hi ghly tyrosyl phosphorylated and their readiness to be phosphorylated by Src in vitro further indicated that they were putative Src substrates as well. Indeed, the enhancement of tyrosyl phosphorylation of p97(eps8) detected i n cells coexpressing both p97(eps8) and active Src relative to that in cell s expressing p97(eps8) alone supported our hypothesis. The existence of com mon phosphotryptic peptides between in vitro P-32-labeled p97(eps8) and p68 (eps8) indicated that these two proteins shared the same Src-mediated sites . Further in vitro binding assays demonstrated that p68(eps8) was the major eps8 isoforms that could be precipitated by bacterial fusion protein conta ining Src SH3. Interestingly, both p68(eps8) and p97(eps8) were preferentia lly expressed in v-Src transformed cells and the presence of p68(eps8) appe ared to depend on Src. Since p97(eps8) has been implicated in mitogenesis a nd tumorigenesis, its readiness to be phosphorylated and induced by v-Src m ight attribute to v-Src-mediated transformation. (C) 1999 Elsevier Science B.V. All rights reserved.