PURIFICATION AND CHARACTERIZATION OF A NOVEL RIBOSOMAL S6 KINASE FROMSKELETAL-MUSCLE OF INSULIN-TREATED RATS

The predominant 40 S ribosomal protein S6 kinase in skeletal muscle ex tracts from insulin-treated rats was purified over 10,000-fold to near homogeneity with similar to 4.5% recovery of starting activity. This S6 kinase was resolved from the catalytic subunit of cAMP-dependent pr otein kinase only by the seventh and final column chromatography step. The purified S6 kinase migrated as a tight doublet of similar to 31 k Da on an SDS-polyacrylamide gel, and it was eluted from gel filtration columns with a similar apparent M(r), which indicated that the enzyme exists as a monomer. This S6 kinase was immunologically distinct from the other known insulin-activated S6 kinases, i.e. p70(S6K) and p90(r sk). It was inhibited by [ethylenebis(oxyethylenenitrilo)]tetraacetic acid and beta-glycerophosphate at concentrations routinely used to sta bilize p70(S6K) and p90(rsk). In addition to S6, phosvitin was also a substrate, whereas myelin basic protein, casein, protamine, and histon es were poorly phosphorylated if at all by the purified S6 kinase. The purified enzyme was inactivated upon incubation with serine/threonine -specific protein phosphatase 2A, which indicated that it may be an in termediary component in a cascade of insulin-activated protein kinases .