Insulin stimulates serine and tyrosine phosphorylation in the juxtamembrane region of the insulin receptor

Insulin-stimulated autophosphorylation of the cytoplasmic juxtamembrane region of the human insulin receptor was examined by Tricine/SDS-PAGE. Various mutant receptor molecules were used to identify two tryptic phosphopeptides associated with the juxtamembrane region which accounts for 15% of the autophosphorylation of partially purified insulin receptor. These phosphopeptides were immunoprecipitated with an antipeptide antibody against the juxtamembrane sequence and were phosphorylated exclusively on tyrosine. Substitution of both Tyr⁹⁶⁰ and Tyr⁹⁵³ with alanine eliminated insulin-stimulated phosphorylation of the juxtamembrane region without affecting tyrosine autophosphorylation in the C terminus or regulatory regions. Monosubstitution of Tyr⁹⁶⁰ with phenylalanine or alanine reduced phosphorylation in the juxtamembrane region by more than 50%, and manual Edman degradation indicated that Tyr⁹⁶⁰ was phosphorylated in wild-type receptor. In vivo, phosphorylation of the juxtamembrane region accounts for one-third of the insulin receptor phosphorylation and contains both phosphoserine and phosphotyrosine. Deletion of Tyr⁹⁶⁰ and 11 adjacent amino acids eliminated insulin-stimulated phosphorylation of the juxtamembrane region. Substitution of Tyr⁹⁶⁰ reduced this phosphorylation by more than 50%. The insulin receptor also undergoes serine phosphorylation outside of the juxtamembrane region which depends on the presence of Tyr¹¹⁵¹. Together with our previous studies, this report suggests that phosphorylation of Tyr⁹⁶⁰ may play an important role in signal transduction by the insulin receptor.