THE USE OF THE YEAST 2-HYBRID SYSTEM TO EVALUATE ERBB-3 INTERACTIONS WITH SH2 DOMAIN-CONTAINING PROTEINS

Several mutations in the tyrosine kinase domain of ErbB-3 have been po stulated to render this enzyme catalytically inactive. To test which a mino acid mutations in ErbB-3 might be critical for kinase inactivatio n, we used a yeast two hybrid assay of protein-protein interaction. We monitored restoration of ErbB-3 kinase activity by investigating the ability of wild type or mutant ErbB-3 to associate with the SH2 contai ning proteins Syp and Phosphatidyl-inositol-3-kinase (PI3K). Our resul ts demonstrate that changing individual amino acids to tyrosine kinase consensus sequences did not increase the interaction of ErbB-3 with S yp or PI3K. Mutation of the consensus Asp832 of rat ErbB-3 to Asn obse rved in human and bovine ErbB-3 significantly increased the interactio n of ErbB-3 and Syp and PI3K II or 26 fold respectively. A double muta nt (Asp832Asn, Asp757 His) exhibited a 96 or 350 fold increase in the ability to bind PI3K and Syp. (C) 1998 Academic Press.