Activation of protein kinases by insulin and non-hydrolyzable GTP analogs in permeabilized 3T3-L1 adipocytes.
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.
Department of Biochemistry and Molecular Biology, University of Massachusetts Medical Center, Worcester 01605.
J K
JK
Klarlund
N
N
Khalaf
L
L
Kozma
M P
MP
Czech
D016475:3T3 Cells; D000273:Adipose Tissue / Q000201:enzymology*; D000595:Amino Acid Sequence; D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002463:Cell Membrane Permeability; D004768:Enterotoxins; D004789:Enzyme Activation; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006153:Guanosine Diphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives* / Q000494:pharmacology*; D006165:Guanylyl Imidodiphosphate / Q000494:pharmacology; D007328:Insulin / Q000494:pharmacology*; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D010455:Peptides / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism*; D013211:Staphylococcus aureus; D013379:Substrate Specificity; D013873:Thionucleotides / Q000494:pharmacology
D004768:Enterotoxins; D007328:Insulin; D010455:Peptides; D013873:Thionucleotides; D006153:Guanosine Diphosphate; D006165:Guanylyl Imidodiphosphate; C026066:enterotoxin A, Staphylococcal; D016244:Guanosine 5'-O-(3-Thiotriphosphate); C023427:guanosine 5'-O-(2-thiodiphosphate); D006160:Guanosine Triphosphate; D011494:Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases