Purification and characterization of a novel ribosomal S6 kinase from skeletal muscle of insulin-treated rats.

The predominant 40 S ribosomal protein S6 kinase in skeletal muscle extracts from insulin-treated rats was purified over 10,000-fold to near homogeneity with approximately 4.5% recovery of starting activity. This S6 kinase was resolved from the catalytic subunit of cAMP-dependent protein kinase only by the seventh and final column chromatography step. The purified S6 kinase migrated as a tight doublet of approximately 31 kDa 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. p70S6K and p90rsk. It was inhibited by [ethylenebis(oxyethylenenitrilo)]tetraacetic acid and beta-glycerophosphate at concentrations routinely used to stabilize p70S6K and p90rsk. In addition to S6, phosvitin was also a substrate, whereas myelin basic protein, casein, protamine, and histones 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 intermediary component in a cascade of insulin-activated protein kinases.