Journal of Biological Chemistry
Volume 272, Issue 38, 19 September 1997, Pages 23696-23702
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CELL BIOLOGY AND METABOLISM
Insulin Receptor Substrate Proteins Create a Link between the Tyrosine Phosphorylation Cascade and the Ca2+-ATPases in Muscle and Heart*

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Following phosphorylation by the insulin receptor kinase, the insulin receptor substrates (IRS)-1 and IRS-2 bind to and activate several Src homology 2 (SH2) domain proteins. To identify novel proteins that interact with IRS proteins in muscle, a human skeletal muscle cDNA expression library was created in the λEXlox system and probed with baculovirus-produced and tyrosine-phosphorylated human IRS-1. One clone of the 10 clones which was positive through three rounds of screening represented the C terminus of the human homologue of the adult fast twitch skeletal muscle Ca2+-ATPase (SERCA1) including the cytoplasmic tail and part of transmembrane region 10. Western blot analysis of extracts of rat muscle demonstrated co-immunoprecipitation of both IRS-1 and IRS-2 with the skeletal muscle Ca2+-ATPase (SERCA1) and the cardiac muscle isoform (SERCA2). In both cases, injection of insulin stimulated a 2- to 6-fold increase in association of which was maximal within 5 min. In primary cultures of aortic smooth muscle cells and C2C12 cells, the insulin-stimulated interaction between IRS proteins and SERCA1 and -2 was dose-dependent with a maximum induction at 100 nm insulin. This interaction was confirmed in a “pull down” experiment using a glutathioneS-transferase fusion protein containing the C terminus of the human SERCA isoform and phosphorylated IRS-1 in vitroand could be blocked by a FLVRES-like domain peptide present in the human SERCA sequence. Affinity chromatography of phosphopeptide libraries using the glutathione S-transferase fusion protein of the C terminus of SERCA1 indicated a consensus sequence for binding of XpYGSS; this is identical to potential tyrosine phosphorylation sites at position 431 of human IRS-1 and at position 500 of human IRS-2. In streptozotocin diabetic rats the interaction between IRS proteins and SERCA1 in skeletal muscle and SERCA2 in cardiac muscle was significantly reduced. Taken together, these results indicate that the IRS proteins bind to the Ca2+-ATPase of the sarcoplasmic reticulum in an insulin-regulated fashion, thus creating a potential link between the tyrosine phosphorylation cascade and effects of insulin on calcium.

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*

This work was supported in part by National Institutes of Health Grants DK 33201 (to C. R. K.), the Joslin's Diabetes and Endocrinology Research Center Grant P30 DK36836, and a grant from the Bristol-Myers Squibb Co.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Supported by a postdoctoral fellowship of the Deutsche *Forschungsgemeinschaft.

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Current address: Dept. of Cellular and Molecular Physiology, College of Medicine, Pennsylvania State University, Hershey, PA 17033.

Howard Hughes Medical Institute Physician Postdoctoral Fellow.