The Janus kinase family of protein tyrosine kinases constitutes a novel type of signal transduction pathway activated in response to a wide variety of polypeptide ligands and has four known members: JAK1, JAK2, JAK3, and Tyk2. In this study, we examined the ability of insulin to stimulate JAK2 tyrosine phosphorylation in insulin-sensitive tissues of the intact rat using immunoprecipitation and immunoblotting. The results demonstrate that after an infusion of insulin, JAK2 is rapidly tyrosine phosphorylated (and the kinase is activated) in the liver, adipose tissue, skeletal muscle, heart, and isolated adipocytes. The presence of phosphorylated JAK2 was detectable after an infusion of insulin that increased serum insulin to physiological postprandial levels (40-70 microunits/ml). Co-immunoprecipitation with anti-insulin receptor antibody, anti JAK2 antibody, and anti-IRS-1 antibody showed that JAK2 interacts with the insulin receptor and IRS-1 to form stable complexes following stimulation by insulin. In two animal models of insulin resistance the regulation of JAK2 tyrosine phosphorylation after insulin infusion paralleled the phosphorylation of the insulin receptor and of IRS-1. In conclusion, our data indicate that after physiological stimulation by insulin in the intact animal, JAK2 associates with the insulin receptor and is tyrosine phosphorylated in insulin sensitive tissues in a time- and dose-dependent fashion.271362210022104