ABSENCE OF AUTOPHOSPHORYLATION SITE Y882 IN THE P185(NEU) ONCOGENE PRODUCT CORRELATES WITH A REDUCTION OF TRANSFORMING POTENTIAL

Autophosphorylation of type I receptor tyrosine kinases (RTKs) compris es one step in the signaling events mediated by erbB receptors such as p185(neu) and EGFR. Previous analysis of p185(neu) has indicated that there are at least five tyrosine autophosphorylation sites, Y882, Y10 28, E1143, Y1226/7 and Y1253, of which Y882 might be important because of its location in the kinase activity domain. We have specifically a nalysed the effect of a Y882F (phenylalanine substituted for tyrosine at position 882) mutation in the enzymatic active domain. We also dele ted the carboxyl terminal 122 amino acids which contained three other autophosphorylation sites (TAPstop) and combined mutants of that delet ion with Y882F (Y882F/APstop). Both in vitro and in vivo transformatio n assays showed that substitution of tyrosine(882) by phenylalanine si gnificantly decreased the transforming potential of activated, oncogen ic p185(neu), although no significant difference in the total phosphot yrosine levels of the mutant proteins were observed. To analyse mitoge nic signaling in response to ligand, the intracellular domains of p185 (neu) and Y882F were fused with the extracellular domain of the EGF re ceptor. The proliferation of cells expressing these chimeric receptors was EGF-dependent, and cells expressing EGFR/Y882F chimeric receptors were less responsive to EGF stimulation than those expressing EGFR/ne u receptors. In vitro kinase assays demonstrated that abolishing the a utophosphorylation site Y882 diminished the enzymatic tyrosine kinase activity of p185(neu). These studies, taken together with the phenotyp ic inhibition observed with cells expressing Y882F, suggest that the t yrosine(882). residue may be important for p185(neu)-mediated transfor mation by affecting the enzymatic kinase function of the p185(neu) rec eptor.