Insulin receptors mediate growth effects in cultured fetal neurons. II. Activation of a protein kinase that phosphorylates ribosomal protein S6.
As an initial attempt to identify early steps in insulin action that may be involved in the growth responses of neurons to insulin, we investigated whether insulin receptor activation increases the phosphorylation of ribosomal protein S6 in cultured fetal neurons and whether activation of a protein kinase is involved in this process. When neurons were incubated for 2 h with 32Pi, the addition of insulin (100 ng/ml) for the final 30 min increased the incorporation of 32Pi into a 32K microsomal protein. The incorporation of 32Pi into the majority of other neuronal proteins was unaltered by the 30-min exposure to insulin. Cytosolic extracts from insulin-treated neurons incubated in the presence of exogenous rat liver 40S ribosomes and [gamma-32P]ATP displayed a 3- to 8-fold increase in the phosphorylation of ribosomal protein S6 compared to extracts from untreated cells. Inclusion of cycloheximide during exposure of the neurons to insulin did not inhibit the increased cytosolic kinase activity. Activation of S6 kinase activity by insulin was dose dependent (seen at insulin concentration as low as 0.1 ng/ml) and reached a maximum after 20 min of incubation. Addition of phosphatidylserine, diolein, and Ca2+ to the in vitro kinase reaction had no effect on the phosphorylation of ribosomal protein S6. Likewise, treatment of neurons with (Bu)2cAMP did not alter the phosphorylation of ribosomal protein S6 by neuronal cytosolic extracts. We conclude that insulin activates a cytosolic protein kinase that phosphorylates ribosomal S6 in neurons and is distinct from protein kinase-C and cAMP-dependent protein kinase. Stimulation of this kinase may play a role in insulin signal transduction in neurons.
Heidenreich
K A
KA
Department of Medicine, University of California-San Diego, La Jolla 92093.
Toledo
S P
SP
D000818:Animals; D001921:Brain / Q000166:cytology; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D002642:Chick Embryo; D004789:Enzyme Activation; D007328:Insulin / Q000494:pharmacology*; D007700:Kinetics; D008099:Liver / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D009474:Neurons / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D010710:Phosphates / Q000378:metabolism; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D051381:Rats; D011972:Receptor, Insulin / Q000502:physiology*; D038601:Ribosomal Protein S6; D019893:Ribosomal Protein S6 Kinases; D012269:Ribosomal Proteins / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism
D007328:Insulin; D009419:Nerve Tissue Proteins; D010710:Phosphates; D010761:Phosphorus Radioisotopes; D038601:Ribosomal Protein S6; D012269:Ribosomal Proteins; D011494:Protein Kinases; D011972:Receptor, Insulin; D019893:Ribosomal Protein S6 Kinases