PARTIAL ACTIVATION OF THE INSULIN-RECEPTOR KINASE DOMAIN BY JUXTAMEMBRANE AUTOPHOSPHORYLATION

Increased enzymatic activity of receptor tyrosine kinases occurs after trans-phosphorylation of one or two tyrosines in the activation loop, located near the catalytic cleft. Partial activation of the insulin r eceptor's kinase domain was observed at dilute concentrations of kinas e, suggesting that cis-autophosphorylation was occurring. Autophosphor ylation during partial activation mapped to the juxtamembrane (JM) tyr osines and not to activation loop tyrosines, Furthermore, a double JM Tyr-to-Phe mutant kinase (JMY2F) did not undergo partial activation bu t catalyzed substrate phosphorylation at a very low rate. Steady-state kinetics of peptide phosphorylation were determined with and without JM autophosphorylation, The JMY2F mutant was used to prevent concurren t cis-autophosphorylation and therefore to approximate the basal state apoenzyme in the kinetic analysis. Partial activation was dominated b y a decreased Michaelis constant for peptide substrate, from K-M,K-PEP greater than or equal to 2.5 mM in the basal state to 0.2 mM in the p artially activated state; the KM,ATP remained virtually unchanged at s imilar to 1 mM, and k(cat) increased from 180 to 600 min(-1). Tne high K-M,K-PEP suggests weak binding of peptide substrates to the apoenzym e, This was confirmed by K-i > 1 mM for peptide substrates used as inh ibitors of JM autophosphorylation. The absence of comparably large cha nges in k(cat) and K-M,K-ATP suggests that the JM region is primarily a strong barrier to the peptide entry step of trans-phosphorylation re actions. The JM region therefore functions as an intrasteric inhibitor in the basal state of the insulin receptor's kinase domain.