Identification of Insulin Receptor Tyrosine Residues Autophosphorylated in Vitro

摘要: To identify the autophosphorylation sites on human insulin receptor (IR), partially purified IR was incubated in vitro presence of and manganese [gamma-32P]ATP so as to achieve near-maximal activation histone 2b kinase activity. Approximately 70% all beta subunit [32P]phosphotyrosine resides two tryptic peptide segments identified by microsequencing precursor (Ullrich, A., Bell, J. R., Chen, E.-Y., Herrera, Petruzelli, L. M., Dull, T. J., Gray, Coussens, L., Liao, Y.-C., Tsubokawa, Mason, Seeburg, P. H., Grunfeld, C., Rosen, O. Ramachandran, (1985) Nature 313, 756-761) 1144-1152 (tyrosine at 1146, 1150, 1151, designated 5) 1315-1329 1316, 1322, 8), which were recovered approximately equal amounts. Half remaining unidentified residues reside another Mr 4000-5000. Assignment specific required subdigestion Edman degradation 32P peptides covalently coupled solid supports. Peptide 5 triple double phosphorylated forms a molar ratio about 2:1. Tyr-1146 contained both 5; form, phenylthiohydantoin-[32P]phosphotyrosine Tyr-1150 Tyr-1151, 1:2. Thus, is presumably mixture Tyr-P-1146/1150 Tyr-P-1146/1151, predominantly latter. 8 only form. We conclude that leads phosphorylation least 6 13 tyrosine intracellular extension. Five these tyrosines are clustered domains; one domain structurally unique C-terminal tail contains Tyr-1316 -1322 phosphorylated. The second located segment region homologous major site pp60 v-src Tyr-1146, fully phosphorylated, -1151; although majority subunits exhibit 1150 up 20-25% remains unphosphorylated complete activation.