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