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.