ISOLATION AND CHARACTERIZATION OF E3B1, AN EPS8 BINDING-PROTEIN THAT REGULATES CELL-GROWTH

Eps8, a substrate of receptor tyrosine kinases, is an SH3 domain conta ining protein that plays an important role in mitogenic signaling. To determine the cellular function of eps8, we used the SH3 domain of eps 8 to screen a human fibroblast M426 expression library and identified, a full-length cDNA clone of 3.2 kb. We designated this clone e3B1 for eps8 SH3 domain binding protein 1. Northern analysis revealed that ex pression of e3B1 mRNA was ubiquitious in human tissues. The e3B1 gene encodes a SH3 domain containing protein. We show that anti-e3B1 antibo dies detect three cytosolic protein species of 65, 68 and 72 kDa in ce ll lysate isolated from asynchronously growing NIH3T3 cells. E3B1 bind s to the SH3 domain of eps8 and Abi in vitro. We also demonstrated tha t e3B1 associates with eps8 in vivo. Phosphatase digestion and phospho amino acid analysis revealed that p65(e3B1) is a phosphoserine contain ing protein and p72(e3B1) and p68(e3B1) are hyperserine-phosphorylated form of p65(e3B1). We further determined that the p65(e3B1) was the m ost abundant in serum-starved NIH/EGFR cells. Time course studies init iated by the addition of epidermal growth factor (EGF) revealed that t he p72(e3B1) started to accumulate at 4 h, peaked at 8 h and remained high until 24 h. Finally, we demonstrate that NIH/EGFR fibroblasts ove rexpressing e3B1 grow more slowly relative to matched controls.