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Purification and characterization of an insulin-stimulated kinase

University of British Columbia

The rapid effects of insulin action appear to be mediated at least in part by the activation of a number of discreteprotein serine/threonine kinases. The cellular targets of some of these kinases include key metabolic enzymes such as acetyl-CoA carboxylase (ACC) and the ribosomal S6. While, the S6 kinases have now been purified and characterized, very little is known about the nature of the proteinkinase(s) able to phosphorylate ACC particularly on the insulin-directed site. Previous studies demonstrated that amyelin basic protein-kinase (MBP-kinase) from sea-star is able to phosphorylate the insulin-directed site on ACC. Thus, it was interesting to find out whether a mammalian homolog of this kinase existed. Rapid chromatography of supernatant fractions from rat adipose tissue by MonoQ ion exchange using fast protein liquid chromatography revealed several peaks of insulin-stimulated protein-serine/threonine kinase activity towards myelin basic protein. Progress in the purification and characterization of these kinases was initially impeded by the rapid decay in protein kinase activity of these extracts. Thus, a major concern initially was stabilization of the insulin-stimulated protein serine/threonine kinase activity which would enable subsequent purification and characterization of the kinases. Stabilization was achieved by the use of phosphatase inhibitors, particularly in combination with a rapid procedure which included ammonium sulphate precipitation.This enabled storage of the activated kinases (with apparently very little loss in activity) and further purification. After first examining the chromatographic properties using individual column techniques, the insulin-activated serine/threonine kinases were purified by a procedure which involved ammonium sulphate precipitation, and sequential chromatography on polylysine-agarose, MonoQ, heparin-agarose and a final MonoQ. The purified enzyme showed only two major silver-stained polypeptides (with subunit sizes of 200 and 44 kDa) as judged by SDS-PAGE. From analysis of Western blots performed with several different anti-kinase antibodies, the 44 kDa polypeptide was found to be immunologically related to a sea-star MBP-kinaseas well as to a family of mammalian mitogen-activated kinases designated ERKs. It is concluded that the 44 kDa MBP-kinase in rat adipose tissue is a member of the family of mitogen-activated kinases. However, the precise relationship to these kinases as well as the regulatory properties of this insulin-activated kinase await further characterization including molecular cloning of the rat adipose enzyme.

Thesis/Dissertation

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2008-09-17

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http://hdl.handle.net/2429/2120

Biochemistry and Molecular Biology

University of British Columbia

UBCV

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