Abstract
The molecular events that lead from the interaction of insulin with its receptor to the activation of protein serine/threonine kinases are still unknown. In this study, we have examined the role of GTP-binding proteins in this signaling pathway using differentiated 3T3-L1 adipocytes permeabilized with alpha-toxin from Staphylococcus aureus. Addition of GTP gamma S (guanosine 5'-O-(3-thiotriphosphate)) or insulin to such permeabilized cells markedly increases protein kinase activities in cell lysates using the microtubule-associated protein-2 kinase substrate peptide KRELVE-PLTPSGEAPNQALLR, which contains the threonine 669 phosphorylation site on the epidermal growth factor receptor. Similar stimulations of protein kinase activity by these agents are observed using the peptide KRRRLASLAA, which is selectively phosphorylated by ribosomal protein S6 kinases. The effects of insulin and GTP gamma S are not additive. Importantly, the GTP-binding protein antagonist GDP beta S (guanosine 5'-O-(2-thiodiphosphate)) inhibits the activation of the protein kinase activities by insulin in permeabilized 3T3-L1 adipocytes. These data are consistent with the hypothesis that activation of Ras or other GTP-binding proteins is a key element of the signaling mechanism whereby insulin receptor tyrosine kinase activates the microtubule-associated protein-2 kinase cascade.
Highlights
We show here that protein kinasecsapable of phospho- depicted in Fig. lA, little radioactivity was released from cells rylating substrate peptidesfor the MAP-2 and ribosomal S6 incubated with bufferin the absence of a-toxin
Triton X-100, which completely disrupts the cell membrane, insulin action on these protein kinases is par- resulted in release of slightly larger amounts of radioactivity tially inhibited by GDPDS which antagonizes the action of than addition of a-toxin
The native nucleotides GTP and GDP had no significant effect on protein kinase activity, whereas the analog GDPPS, which antagonizes GTP activation of GTP-binding proteins (60),reduced the basal activity.Treatment of 3T3-Ll adipocytes with a-toxin itself caused a variable increase in peptide phosphorylation activity in these studies, but insulin, GTPyS and GMP-PNP furtherstimulated MAP-2 kinase peptide phosphorylation in 17 independent experiments
Summary
The effects of insulin serine/threonine kinases are the GTP-binding proteins. Direct activation or inhibition of GTP-binding pro- heat-stable inhibitor of protein kinase A (59) was added to a final teins by G T P analogs can be achieved in cells permeabilized concentration of 5 pg/ml.
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