title: Tyrosine phosphorylation of phosphoinositide-dependent kinase 1 by the insulin receptor is necessary for insulin metabolic signaling.
abstract: In L6 myoblasts, insulin receptors with deletion of the C-terminal 43 amino acids (IR(Delta43)) exhibited normal autophosphorylation and IRS-1/2 tyrosine phosphorylation. The L6 cells expressing IR(Delta43) (L6(IRDelta43)) also showed no insulin effect on glucose uptake and glycogen synthase, accompanied by a >80% decrease in insulin induction of 3-phosphoinositide-dependent protein kinase 1 (PDK-1) activity and tyrosine phosphorylation and of protein kinase B (PKB) phosphorylation at Thr(308). Insulin induced the phosphatidylinositol 3 kinase-dependent coprecipitation of PDK-1 with wild-type IR (IR(WT)), but not IR(Delta43). Based on overlay blotting, PDK-1 directly bound IR(WT), but not IR(Delta43). Insulin-activated IR(WT), and not IR(Delta43), phosphorylated PDK-1 at tyrosines 9, 373, and 376. The IR C-terminal 43-amino-acid peptide (C-terminal peptide) inhibited in vitro PDK-1 tyrosine phosphorylation by the IR. Tyr-->Phe substitution prevented this inhibitory action. In the L6(hIR) cells, the C-terminal peptide coprecipitated with PDK-1 in an insulin-stimulated fashion. This peptide simultaneously impaired the insulin effect on PDK-1 coprecipitation with IR(WT), on PDK-1 tyrosine phosphorylation, on PKB phosphorylation at Thr(308), and on glucose uptake. Upon insulin exposure, PDK-1 membrane persistence was significantly reduced in L6(IRDelta43) compared to control cells. In L6 cells expressing IR(WT), the C-terminal peptide also impaired insulin-dependent PDK-1 membrane persistence. Thus, PDK-1 directly binds to the insulin receptor, followed by PDK-1 activation and insulin metabolic effects.
authors.affiliation: Dipartimento di Biologia e Patologia Cellulare e Molecolare, Università di Napoli Federico II, Italy.
authors.forename: Francesca
authors.initials: F
authors.lastname: Fiory
authors.forename: Anna Teresa
authors.initials: AT
authors.lastname: Alberobello
authors.forename: Claudia
authors.initials: C
authors.lastname: Miele
authors.forename: Francesco
authors.initials: F
authors.lastname: Oriente
authors.forename: Iolanda
authors.initials: I
authors.lastname: Esposito
authors.forename: Vincenzo
authors.initials: V
authors.lastname: Corbo
authors.forename: Menotti
authors.initials: M
authors.lastname: Ruvo
authors.forename: Barbara
authors.initials: B
authors.lastname: Tizzano
authors.forename: Thomas E
authors.initials: TE
authors.lastname: Rasmussen
authors.forename: Steen
authors.initials: S
authors.lastname: Gammeltoft
authors.forename: Pietro
authors.initials: P
authors.lastname: Formisano
authors.forename: Francesco
authors.initials: F
authors.lastname: Beguinot
mesh_terms: D064413:3-Phosphoinositide-Dependent Protein Kinases; D000818:Animals; D002478:Cells, Cultured; D005947:Glucose / Q000378:metabolism; D006801:Humans; D007328:Insulin / Q000378:metabolism* / Q000494:pharmacology; D055504:Insulin Receptor Substrate Proteins; D047908:Intracellular Signaling Peptides and Proteins; D051379:Mice; D032446:Myoblasts / Q000187:drug effects / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051381:Rats; D011972:Receptor, Insulin / Q000235:genetics / Q000378:metabolism*; D017384:Sequence Deletion; D015398:Signal Transduction; D014443:Tyrosine / Q000378:metabolism*
chemical_list: C526292:IRS1 protein, human; C526297:IRS2 protein, human; D007328:Insulin; D055504:Insulin Receptor Substrate Proteins; D047908:Intracellular Signaling Peptides and Proteins; C526295:Irs1 protein, mouse; C526296:Irs1 protein, rat; C526298:Irs2 protein, mouse; C526299:Irs2 protein, rat; D010750:Phosphoproteins; D014443:Tyrosine; D011972:Receptor, Insulin; D064413:3-Phosphoinositide-Dependent Protein Kinases; C578562:PDPK1 protein, human; D017346:Protein Serine-Threonine Kinases; D005947:Glucose