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. 1990 Oct;87(20):7944–7948. doi: 10.1073/pnas.87.20.7944

Insulin activates a 70-kDa S6 kinase through serine/threonine-specific phosphorylation of the enzyme polypeptide.

D J Price 1, J R Gunsalus 1, J Avruch 1
PMCID: PMC54868  PMID: 2122450

Abstract

The dominant insulin-stimulated ribosomal protein S6 kinase activity was purified to near homogeneity from insulin-treated 32P-labeled rat H4 hepatoma cells and found to copurify with a 70-kDa 32P-labeled polypeptide. The dominant S6 kinase purified from livers of cycloheximide-treated rats is also a 70-kDa polypeptide. Antiserum raised against rat liver S6 kinase specifically immunoprecipitates the purified 32P-labeled H4 hepatoma insulin-stimulated S6 kinase. This antiserum also specifically precipitates insulin-stimulated S6 kinase activity directly from cytosolic extracts of H4 cells. Immune complexes prepared from the cytosol of 32P-labeled H4 cells contain several 32P-labeled polypeptides; only a 70-kDA 32P-labeled peptide, however, is specifically displaced by preadsorption of the antiserum with nonradioactive rat liver S6 kinase. Insulin treatment increases the 32P content of the immunoprecipitated 70-kDa S6 kinase polypeptide 3- to 4-fold over basal levels; 32P-labeled serine, some 32P-labeled threonine, but no 32P-labeled tyrosine are detected after partial acid hydrolysis. Tryptic peptide maps indicate that the insulin-stimulated S6 kinase purified from 32P-labeled H4 cells is phosphorylated at multiple sites distinct from those which participate in autophosphorylation in vitro. Autophosphorylation of rat liver S6 kinase in vitro does not modify S6 kinase activity. The S6 kinases purified from liver of cycloheximide-treated rat and H4 hepatoma insulin-stimulated enzyme are each completely deactivated by incubation with protein phosphatase type 2A in both autophosphorylating and 40S S6 phosphorylating activities. The phosphatase 2A-deactivated 70-kDa S6 kinase is neither reactivated nor phosphorylated by partially purified insulin-stimulated microtubule-associated protein 2 kinase, in experiments where Xenopus S6 kinase II undergoes phosphorylation and partial reactivation. Thus insulin activates the 70-kDa S6 kinase by promoting phosphorylation of specific serine/threonine residues on the enzyme polypeptide, probably through activating an as-yet-unidentified serine/threonine protein kinase distinct from microtubule-associated protein 2 kinase.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

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