Multiple ErbB-2/Neu Phosphorylation Sites Mediate Transformation through Distinct Effector Proteins

Abstract

Amplification of the type I receptor tyrosine kinase ErbB-2 (HER2/Neu) is observed in 20-30% of human mammary carcinomas, correlating with a poor clinical prognosis. We have previously demonstrated that four (Tyr 1144, Tyr1201, Tyr1226/1227, or Tyr 1253) of the five known Neu/ErbB-2 autophosphorylation sites can independently mediate transforming signals. The transforming potential of at least two of these autophosphorylation sites (Tyr1144 and Tyr 1226/1227) has been further correlated with their ability to associate with Grb2 and Shc adapter proteins, respectively. To confirm the specificity of these interactions, we have created a series of second site mutants in these phosphorylation sites. The results showed that Grb2 recruitment to site 1144 is absolutely required for transforming signal from this autophosphorylation site, whereas association of Shc-mediated transformation is dependent on conservation of the NPXY motif spanning Tyr 1227. A stretch of amino acid identity around tyrosines 1201 (ENPEYLTP)and 1253 (ENPEYLDL) exists, and mutation of key residues within this motif reveals distinct requirements for an intact protein tyrosine-binding protein (NPXY). We show that DOK-R, a protein tyrosine-binding site-containing protein implicated in Ras signaling, interacts with Neu/ErbB-2 at Tyr 1253 as do two unidentified proteins, p150 and p34, the latter correlating with transformation. Together these data argue that ErbB-2/Neu is capable of mediating transformation through distinct effector pathways.