Purification and characterisation of the insulin-stimulated protein kinase from rabbit skeletal muscle; close similarity to S6 kinase II.
The insulin-stimulated protein kinase (ISPK) was purified over 50,000-fold from extracts of rabbit skeletal muscle by a procedure involving chromatography on phosphocellulose, fractionation with ammonium sulphate, and further chromatography on DEAE-cellulose, phenyl-Superose, Mono S and Mono Q. About 10 micrograms enzyme was isolated from 800 g muscle (one rabbit) in four days with an overall recovery of 5%. The purified enzyme showed a single protein-staining band of apparent molecular mass 91 kDa when analysed by SDS/polyacrylamide gel electrophoresis. The ISPK comigrated during SDS/polyacrylamide gel electrophoresis with the enzyme S6 kinase II from Xenopus eggs, and was recognised in immunoblotting and immunoprecipitation experiments by antibodies raised against S6 kinase II. The substrate specificities of ISPK and S6 kinase II were also very similar and like S6 kinase II, ISPK that had been inactivated by protein phosphatase 2A could be reactivated by incubation with mitogen-activated protein kinase and MgATP. ISPK was distinct from an insulin-stimulated 70-kDa S6 kinase from rat liver in both substrate specificity and immunological cross reactivity. It is concluded that ISPK is closely related in structure to S6 kinase II and may be a mammalian equivalent of this enzyme. The possibility that ISPK is involved in mediating a number of the actions of insulin is discussed.
Lavoinne
A
A
Department of Biochemistry, University of Dundee, Scotland.
Erikson
E
E
Maller
J L
JL
Price
D J
DJ
Avruch
J
J
Cohen
P
P
D000595:Amino Acid Sequence; D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D007328:Insulin / Q000494:pharmacology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009132:Muscles / Q000201:enzymology*; D009865:Oocytes / Q000201:enzymology; D011494:Protein Kinases / Q000302:isolation & purification / Q000378:metabolism*; D011817:Rabbits; D019893:Ribosomal Protein S6 Kinases; D013379:Substrate Specificity; D014981:Xenopus
D007328:Insulin; D011494:Protein Kinases; D019893:Ribosomal Protein S6 Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases