A ROLE FOR TYROSINE PHOSPHORYLATION IN BOTH ACTIVATION AND INHIBITIONOF THE INSULIN-RECEPTOR TYROSINE KINASE IN-VIVO

Upon insulin binding, a conformational change in the insulin receptor (IR) leads to IR beta-subunit autophosphorylation, an increase in IR b eta-subunit exogenous tyrosine kinase activity, and the rapid endocyto sis of the ligand-receptor complex into endosomes. Previous work has s hown that upon internalization, rat hepatic endosomal IRs manifest inc reased autophosphorylating and exogenous tyrosine kinase activity comp ared to IRs located at the plasma membrane. As this period of enhanced activity is associated with reduced endosomal IR beta-subunit phospho tyrosine content, it has been proposed that partial dephosphorylation of the internalized IR beta-subunit by an endosomally located phosphot yrosine phosphatase(s) [PTPase(s)] mediates this effect. To test wheth er endosomal PTPase activity was required for internalization-dependen t augmentation of IR tyrosine kinase activity, the present study used the peroxovanadium PTPase inhibitor, bis-peroxo(1,10-phenanthroline)ox ovanadate anion [bpV(phen)], to block IR dephosphorylation within this subcellular compartment. Rats were pretreated with bpV(phen) before r eceiving insulin (1.5 mu g/100 g BW). bpV(phen) inhibited the dephosph orylation of P-32-labeled hepatic endosomal IR by similar to 97% at 15 min post-bpV(phen) injection and prevented a decrease in IR beta-subu nit phosphotyrosine content after IR internalization. Fifteen-minute b pV(phen) pretreatment produced a significant reduction (75%; P < 0.001 ) in maximal insulin-stimulated endosomal IR exogenous kinase activity and decreased IR autophosphorylating activity by 4.3-fold in this sub cellular fraction. In conclusion, these findings suggest that an hepat ic endosomal PTPase(s) regulates internalization-dependent increases i n IR exogenous tyrosine kinase activity.