title: Dual mechanism of signal transducer and activator of transcription 5 activation by the insulin receptor.
abstract: Insulin stimulates signal transducer and activator of transcription 5 (Stat5) activation in insulin receptor (IR)-overexpressing cell lines and in insulin target tissues of mice. Stat5b and insulin receptor substrate 1 (IRS-1) interact with the same autophosphorylation site in the IR [phosphotyrosine (pY) 972] in yeast two-hybrid assays, and the IR phosphorylates Stat5b in vitro. These data suggest that Stat5 proteins might be recruited to, and phosphorylated by, the activated IR in vivo. Nevertheless, insulin activates Janus kinases (JAKs) in IR-overexpressing cell lines and in insulin target tissues. To determine whether Stat5 proteins must be recruited to the pY972LSA motif in the IR for insulin-stimulated activation in mammalian cells, we generated and tested a series of IR mutants. The L973R/A975D mutation abolishes the ability of the IR to induce Stat5 activation, whereas IRS-1 phosphorylation is unaffected. In contrast, the N969A/P970A mutation in the IR has no effect on Stat5 activation but significantly reduces IRS-1 phosphorylation. In coimmunoprecipitation assays, insulin-stimulated Stat5 activation correlates with Stat5 recruitment to the IR. We also find that insulin stimulates tyrosine phosphorylation of JAKs that are constitutively associated with the IR. Expression of dominant-negative (DN) JAKs, the JAK inhibitor suppressor of cytokine signaling 1, or pretreatment with the JAK inhibitor, AG490, reduces, but does not eliminate, insulin-induced Stat5 activation. Expression of the appropriate pair of DN JAKs in each of the singly JAK-deficient cell lines further establishes a component of insulin-stimulated Stat5 activation that is JAK independent. This likely represents phosphorylation of Stat5 proteins by the IR, as we find that IR kinase domain phosphorylates Stat5b in vitro on Y699 as efficiently as JAK2. Increasing the concentration of Stat5 proteins in cells favors the direct phosphorylation of Stat5 by the IR kinase where the DN-JAK inhibition of insulin-stimulated Stat5 activation becomes insignificant. At physiological levels of Stat5 however, we propose that JAKs and the IR both contribute to the insulin-induced phosphorylation of Stat5.
authors.affiliation: Department of Microbiology, Mount Sinai School of Medicine, New York, NY 10029, USA.
authors.forename: Maithao N
authors.initials: MN
authors.lastname: Le
authors.forename: Ronald A
authors.initials: RA
authors.lastname: Kohanski
authors.forename: Lu-Hai
authors.initials: LH
authors.lastname: Wang
authors.forename: Henry B
authors.initials: HB
authors.lastname: Sadowski
mesh_terms: D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D019556:COS Cells; D002352:Carrier Proteins / Q000494:pharmacology; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015870:Gene Expression; D006801:Humans; D007163:Immunosorbent Techniques; D007328:Insulin / Q000494:pharmacology; D055504:Insulin Receptor Substrate Proteins; D047908:Intracellular Signaling Peptides and Proteins; D053613:Janus Kinase 1; D053614:Janus Kinase 2; D008114:Liver Neoplasms, Experimental / Q000378:metabolism; D008156:Luciferases / Q000235:genetics; D008894:Milk Proteins; D016296:Mutagenesis; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000172:deficiency / Q000378:metabolism; D011518:Proto-Oncogene Proteins; D051381:Rats; D011972:Receptor, Insulin / Q000235:genetics / Q000502:physiology*; D012097:Repressor Proteins; D050799:STAT5 Transcription Factor; D000071222:Suppressor of Cytokine Signaling 1 Protein; D050826:Suppressor of Cytokine Signaling Proteins; D015534:Trans-Activators / Q000378:metabolism*; D014162:Transfection; D014407:Tumor Cells, Cultured; D020032:Tyrphostins / Q000494:pharmacology
chemical_list: D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; C526292:IRS1 protein, human; D007328:Insulin; D055504:Insulin Receptor Substrate Proteins; D047908:Intracellular Signaling Peptides and Proteins; C526295:Irs1 protein, mouse; C526296:Irs1 protein, rat; D008894:Milk Proteins; D010750:Phosphoproteins; D011518:Proto-Oncogene Proteins; D012097:Repressor Proteins; C108944:SOCS1 protein, human; D050799:STAT5 Transcription Factor; C494095:STAT5B protein, human; C491589:Socs1 protein, rat; C494097:Stat5b protein, rat; D000071222:Suppressor of Cytokine Signaling 1 Protein; D050826:Suppressor of Cytokine Signaling Proteins; D015534:Trans-Activators; D020032:Tyrphostins; C095512:alpha-cyano-(3,4-dihydroxy)-N-benzylcinnamide; D019000:Phosphotyrosine; D008156:Luciferases; D011505:Protein-Tyrosine Kinases; D011972:Receptor, Insulin; C507904:JAK1 protein, human; C507924:JAK2 protein, human; C507906:Jak1 protein, rat; C507927:Jak2 protein, rat; D053613:Janus Kinase 1; D053614:Janus Kinase 2