INSULIN-RECEPTOR SUBSTRATE PROTEINS CREATE A LINK BETWEEN THE TYROSINE PHOSPHORYLATION CASCADE AND THE CA2-ATPASES IN MUSCLE AND HEART()

Following phosphorylation by tile insulin receptor kinase, the insulin receptor substrates (IRS)-1 and IRS-2 bind to and activate several Sr c homology 2 (SH2) domain proteins. To identify novel proteins that in teract with IRS proteins in muscle, a human skeletal muscle cDNA expre ssion library was created in the lambda EXLox system and probed with b aculovirus-produced and tyrosine-phosphorylated human IRS-I, 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 t witch skeletal muscle Ca2+ ATPase (SERCA1) including the cytoplasmic t ail and part of transmembrane region 10, Western blot analysis of extr acts of rat muscle demonstrated co-immunoprecipitation of both IRS-1 a nd IRS-2 with the skeletal muscle Ca2+-ATPase (SERCA1) and the cardiac muscle isoform (SERCA2), In both cases, injection of insulin stimulat ed 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 cel ls, 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 glutathione S-transferase fusion protein containing the C terminus of the human SERCA. isoform and phosphorylated IRS-I in vitro and could b e blocked by a FLVRES-like domain peptide present in the human SERCA s equence, 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 identica l 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 muscl e and SERCA2 in cardiac muscle was significantly reduced, Taken togeth er, these results indicate that the IRS proteins bind to the Ca2+-ATPa se of the sarcoplasmic reticulin in an insulin-regulated fashion, thus creating a potential link between the tyrosine phosphorylation cascad e and effects of insulin on calcium.