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.
Hei
Y J
YJ
Division of Pharmacology and Toxicology, Faculty of Pharmaceutical Sciences, University of British Columbia, Vancouver, Canada.
Pelech
S L
SL
Chen
X
X
Diamond
J
J
McNeill
J H
JH
D000595:Amino Acid Sequence; D000818:Animals; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D006801:Humans; D007328:Insulin / Q000494:pharmacology*; D008297:Male; D008969:Molecular Sequence Data; D009132:Muscles / Q000201:enzymology*; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D019893:Ribosomal Protein S6 Kinases; D012270:Ribosomes / Q000201:enzymology*; D013379:Substrate Specificity
D007328:Insulin; D017346:Protein Serine-Threonine Kinases; D019893:Ribosomal Protein S6 Kinases