Autophosphorylation of the two C-terminal tyrosine residues Tyr(1316) and Tyr(1322) modulates the activity of the insulin receptor kinase in vitro

Previously, several studies have demonstrated that autophosphorylation of t he C-terminal tyrosine residues (Tyr(1316) and Tyr(1322)) affects the Signa ling properties of the insulin receptor in vivo. To assess the biochemical consequences of the C-terminal phosphorylation in vitro, we have constructe d, purified and characterized 45 kDa soluble insulin receptor kinase domain s (IRKD), either with (IRKD) or without (IRKD-Y2F) the two C-terminal tyros ine phosphorylation sites, respectively. According to HPLC phosphopeptide m apping, autophosphorylation of the three tyrosines in the activation loop o f the IRKD-Y2F kinase (Tyr(1146), Tyr(1150), and Tyr(1151)) was not affecte d by the mutation, In addition, the Y2F mutation did not significantly chan ge the K-m values for exogenous substrates, However, the mutation in IRKD-Y 2F resulted in a decrease in the maximum velocities of the phosphotransfera se reaction in substrate phosphorylation reactions. Moreover, the exchange of the tyrosines in IRKD-Y2F led to an increase in the apparent K-m values for ATP, suggesting a cross-talk of the C-terminus and the catalytic domain of the enzyme. In addition, as judged by size exclusion chromatography, co nformational changes of the enzyme following autophosphorylation were aboli shed by the removal of the two C-terminal tyrosines, These data suggest a r egulatory role of the two C-terminal phosphorylation sites in the phosphotr ansferase activity of the insulin receptor, (C) 2000 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reser ved.