IDENTIFICATION OF SITES ON EPIDERMAL GROWTH-FACTOR RECEPTORS WHICH ARE PHOSPHORYLATED BY PP60(SRC) IN-VITRO

The Epidermal Growth Factor Receptor (EGF-R) becomes constitutively ty rosine phosphorylated on two novel sites in v-Src transformed cells, a nd these phosphorylations are associated with enhanced signaling activ ity [1]. To determine whether Src could directly phosphorylate these s ites, we have examined the ability of the Src kinase to phosphorylate both wild-type and kinase-defective EGF-Rs in vitro. Although purified Src could phosphorylate EGF-Rs, the pattern of phosphorylation sites was not identical to what was previously found in vivo [1]: Src in vit ro directly phosphorylated EGF-Rs on one autophosphorylation site (Tyr 1173) which was not a site of Src-induced in vivo phosphorylation, su ggesting the in vivo inaccessibility of this site. One Src-specific in vitro phosphorylation site (Tyr 703) appeared to correspond to one of the in vivo Src-induced sites (sPY2), but the other Src-specific in v ivo site (sPY1) was not significantly phosphorylated in vitro, raising the possibility of a Src-induced tyrosine kinase cascade. The ability of Src to phosphorylate the EGF-R is consistent with the suggestion t hat the receptor can function as a kinase substrate independent of its intrinsic enzymatic activity, as implied by recent studies on signali ng by kinase-defective EGF-Rs.