Identification and characterization of a cytoskeleton-associated, epidermal growth factor sensitive pp60c-src substrate

Abstract

In studies aimed at identifying and characterizing pp60c-src substrates that participate in the enhanced mitogenic response to epidermal growth factor (EGF) observed in murine C3H10T1/2 fibroblasts overexpressing c-src, we have identified a 75-kDa protein (p75) whose properties are consistent with those expected of such a substrate. We present evidence to show that p75 is immunologically related to a recently described, cytoskeleton-associated, pp60v-src substrate [Wu et al. (1991). Mol. Cell. Biol., 11, 5113-5124), and that its phosphotyrosine content is increased cooperatively by c-src overexpression and EGF stimulation. p75 is rapidly (within 2 min) phosphorylated on tyrosine upon EGF treatment and undergoes a second, prolonged phase of tyrosyl phosphorylation from 7 to 21 h after EGF addition, suggesting that tyrosyl phosphorylation of p75 is important for late as well as early events following EGF receptor activation. Enhanced tyrosyl phosphorylation of p75 is also seen when cells overexpressing c-src are treated with platelet-derived growth factor (PDGF), but significantly less phosphorylation is observed with insulin and fibroblast growth factor (FGF). Both basal and EGF-induced tyrosyl phosphorylation of p75 are reduced in cells overexpressing mutated forms of c-src (unmyristylated, or kinase deficient) as compared with wild-type c-src overexpressers, indicating the dependence of the enhanced tyrosyl phosphorylation on membrane-associated, enzymatically active pp60c-src. In cellular fractionation experiments p75 partitions with the cytosol, while immunofluorescence studies reveal a striking colocalization with pp60c-src at the plasma membrane and in the perinuclear region. Partial co-staining of p75 and actin occurs at the cell's periphery. These data provide evidence for p75 being a direct substrate of pp60c-src. The possible role of p75 in the enhanced response to EGF seen in c-src overexpressers is discussed.