SH2-Balpha is an insulin-receptor adapter protein and substrate that interacts with the activation loop of the insulin-receptor kinase

We identified SH2-Balpha as an insulin-receptor-binding protein based on interaction screening in yeast hybrid systems and co-precipitation in cells. SH2-Balpha contains pleckstrin-homology ('PH') and Src homology 2 (SH2) domains and is closely related to APS (adapter protein with a PH domain and an SH2 domain) and lnk, adapter proteins first identified in lymphocytes. SH2-Balpha is ubiquitously expressed and is present in rat epididymal adipose tissue, liver and skeletal muscle, physiological sites of Insulin action. On SDS/PAGE, SH2-Balpha migrates at a molecular mass of 98 kDa, although the predicted size of SH2-Balpha is 79.6 kDa. Insulin causes an electrophoretic mobility shift. SH2-Balpha can be immunoprecipitated using anti-(Insulin Receptor) antibody from insulin-stimulated cells. Anti-phosphotyrosine antibody or the growth factor receptor-binding protein 2 (Grb2) SH2 domain precipitate SH2-Balpha after Insulin stimulation, suggesting that SH2-Balpha is tyrosine-phosphorylated and may be a substrate for the Insulin Receptor. The SH2-Balpha SH2 domain did not interact with insulin-receptor substrate (IRS) proteins or epidermal-growth-factor receptor. Mutation of the juxtamembrane and C-terminus of the Insulin Receptor did not abolish the interaction with the SH2 domain. This was further confirmed using a panel of activation-loop single point mutants where mutation of Tyr1158, Tyr1162 and Tyr1163 abolished interaction. Thus SH2-Balpha is a likely component in the insulin-signalling pathway and may function as an alternative signalling protein by interacting with the activation loop of the insulin-receptor cytoplasmic domain.