POTENT SHC TYROSINE PHOSPHORYLATION BY EPIDERMAL GROWTH-FACTOR AT LOWRECEPTOR DENSITY OR IN THE ABSENCE OF RECEPTOR AUTOPHOSPHORYLATION SITES

The importance of epidermal growth factor (EGF) receptor expression le vel and autophosphorylation sites in src homology and collagen protein (SHC) tyrosine phosphorylation has been studied. In contrast to EGF-i nduced tyrosine phosphorylation of the GTPase-activating protein for r as (rasGAP) and phospholipase C-gamma 1 (PLC-gamma 1), SHC tyrosine ph osphorylation occurs at a very low receptor density in parental NIH3T3 mouse fibroblasts expressing less than 1 x 10(4) EGF receptors per ce ll. In transfected NlH3T3 cells expressing human EGF receptors (simila r to 4 x 10(5) receptors per cell), maximal levels of SHC and PLC-gamm a 1 tyrosine phosphorylation occur when approximately 4 x 10(4) recept ors or more are occupied by ligand. At lower levels of receptor occupa ncy only SHC posphorylation was significant. Also, EGF treatment of mo use keratinocytes, which represent a physiological target of EGF, expr ess a low number of EGF receptors (similar to 2 x 10(4) receptors per cell), and stringently require EGF to grow, results in intense SHC tyr osine phosphorylation, compared to rasGAP or PLC-gamma 1. SHC is also efficiently tyrosine phosphorylated by an EGF receptor deletion mutant (Dc214) that is devoid of autophosphorylation sites, but which remain s mitogenically responsive to EGF. The EGF receptor mutant Dc214 is ab le to activate the ras guanine nucleotide exchanger and phosphorylate mitogen-activated protein kinase (MAPK), presumable as a result of com plex formation between tyrosine phosphorylated SHC and GRB2. These res ults indicate that potent EGF-induced SHC tyrosine phosphorylation can be triggered in cells having relatively few receptors. Also, our data show that EGF receptors are able to phosphorylate SHC, activate the e xchange of guanine nucleotide on ras and phosphorylate MAPK by a mecha nism that does not require receptor autophosphorylation sites and, the refore, the src homology 2 (SH2):phosphotyrosine-dependent interaction of SHC or GRB2 with the EGF receptor.