Absence of autophosphorylation site Y882 in the p185^neu oncogene product correlates with a reduction of transforming potential

Abstract

Autophosphorylation of type I receptor tyrosine kinases (RTKs) comprises 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, Y1028, Y1143, Y1226/7 and Y1253, of which Y882 might be important because of its location in the kinase activity domain. We have specifically analysed the effect of a Y882F (phenylalanine substituted for tyrosine at position 882) mutation in the enzymatic active domain. We also deleted the carboxyl terminal 122 amino acids which contained three other autophosphorylation sites (TAPstop) and combined mutants of that deletion with Y882F (Y882F/APstop). Both in vitro and in vivo transformation assays showed that substitution of tyrosine⁸⁸² by phenylalanine significantly decreased the transforming potential of activated, oncogenic p185^neu, although no significant difference in the total phosphotyrosine levels of the mutant proteins were observed. To analyse mitogenic signaling in response to ligand, the intracellular domains of p185^neu and Y882F were fused with the extracellular domain of the EGF receptor. 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/neu receptors. In vitro kinase assays demonstrated that abolishing the autophosphorylation site Y882 diminished the enzymatic tyrosine kinase activity of p185^neu. These studies, taken together with the phenotypic inhibition observed with cells expressing Y882F, suggest that the tyrosine⁸⁸² residue may be important for p185^neu-mediated transformation by affecting the enzymatic kinase function of the p185^neu receptor.