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PMID: 2965579 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

Two systems in vitro that show insulin-stimulated serine kinase activity towards the insulin receptor.

The Biochemical journal ·Vol. 250 ·No. 2 ·1988-03-01 ·Pages 509-19

Smith DM, King MJ, Sale GJ

Abstract

Two systems in vitro are described that show insulin-stimulated phosphorylation of the insulin receptor on serine residues. In the first system, insulin receptor was purified partially from Fao rat hepatoma cells by direct solubilization of the cells in Triton X-100 and chromatography on wheat-germ-agglutinin-agarose. Phosphorylation of these preparations with [gamma-32P]ATP in the presence or absence of insulin resulted in 32P incorporation exclusively into phosphotyrosine residues. Serine kinase activity towards the insulin receptor was reconstituted by adding extracts of Fao cells. Prior exposure of the cells to insulin stimulated serine kinase activity towards the insulin receptor in extracts 7.2-fold. A receptor serine kinase activity enhanced by treatment of cells with cyclic AMP analogues was also retained in the reconstituted system. In the second system, insulin receptor and insulin-sensitive serine kinase activity towards the insulin receptor were co-purified from human placenta. The protocol involved preparation of membranes, before solubilization and chromatography on wheat-germ-agglutinin-agarose, by using gentle procedures designed not to disrupt a potentially labile association between the insulin receptor and the serine kinase. Serine kinase activity in these preparations towards the insulin receptor was stimulated up to 10-fold by insulin, and the stoicheiometry of serine phosphorylation was estimated to be approx 0.8 mol/mol of insulin receptor for phosphorylations performed in the presence of insulin. Thus a preparation of insulin receptor is described for the first time that is phosphorylated to high stoicheiometry on serine in an insulin-dependent manner. Conditions that facilitate recovery and assay of serine kinase activity are defined and discussed. These systems provide a basis for characterizing the nature of the insulin-sensitive serine kinase that phosphorylates the insulin receptor, and defining its role in insulin action and control of receptor function.

MeSH Terms
Animals Cell Membrane/drug effects,metabolism Cells, Cultured Electrophoresis, Polyacrylamide Gel Female Humans Insulin/pharmacology Liver Neoplasms, Experimental/metabolism Phosphorylation Placenta/drug effects,metabolism Pregnancy Protein Kinases/metabolism Protein Serine-Threonine Kinases Rats Receptor, Insulin/metabolism Serine/metabolism Stimulation, Chemical
Chemicals
Insulin Serine Protein Kinases Receptor, Insulin Protein Serine-Threonine Kinases
Authors & Affiliations
3 authors, click to expand affiliations / ORCID
Smith D M
Department of Biochemistry, School of Biochemical and Physiological Sciences, University of Southampton, U.K.
King M J
Sale G J
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Article Info
Journal
The Biochemical journal
Abbr.
Biochem J
ISSN
0264-6021
DOI
10.1042/bj2500509
Published
1988-03-01
Pages
509-19
Language
English
Region
England
NLM ID
2984726R
PMCID
PMC1148885
Subset
IM
External Links
PubMed source DOI: 10.1042/bj2500509 PMC Full Text View journal details
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