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Tyrosine Phosphorylation of Phosphoinositide-Dependent Kinase 1 by the Insulin Receptor Is Necessary for Insulin Metabolic Signaling

Francesca Fiory,¹ Anna Teresa Alberobello,¹ Claudia Miele,¹ Francesco Oriente,¹ Iolanda Esposito,¹ Vincenzo Corbo,¹ Menotti Ruvo,² Barbara Tizzano,² Thomas E. Rasmussen,^3, Steen Gammeltoft,³ Pietro Formisano,¹ and Francesco Beguinot^1*

Dipartimento di Biologia e Patologia Cellulare e Molecolare & Istituto di Endocrinologia ed Oncologia Sperimentale del CNR, Università degli Studi di Napoli Federico II, Naples, Italy,¹ Istituto di Biostrutture e Bioimmagini del CNR, Naples, Italy,² Department of Clinical Biochemistry, Glostrup Hospital, DK 2600 Glostrup, Denmark³

Received 31 July 2005/ Accepted 19 September 2005

In L6 myoblasts, insulin receptors with deletion of the C-terminal 43 amino acids (IR₄₃) exhibited normal autophosphorylation and IRS-1/2 tyrosine phosphorylation. The L6 cells expressing IR₄₃ (L6_IR43) 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 Thr₃₀₈. Insulin induced the phosphatidylinositol 3 kinase-dependent coprecipitation of PDK-1 with wild-type IR (IR_WT), but not IR₄₃. Based on overlay blotting, PDK-1 directly bound IR_WT, but not IR₄₃. Insulin-activated IR_WT, and not IR₄₃, 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. TyrPhe 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 Thr₃₀₈, and on glucose uptake. Upon insulin exposure, PDK-1 membrane persistence was significantly reduced in L6_IR43 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.

Present address: Mammalian Cell Technology, Novo Nordisk A/S, DK 2880 Bagsvaerd, Denmark.

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