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

Abstract

In L6 myoblasts, insulin receptors with deletion of the C-terminal 43 amino acids (IR ⌬43 ) exhibited normal autophosphorylation and IRS-1/2 tyrosine phosphorylation. The L6 cells expressing IR ⌬43 (L6 IR⌬43 ) 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 kinasedependent coprecipitation of PDK-1 with wild-type IR (IR WT ), but not IR ⌬43 . Based on overlay blotting, PDK-1 directly bound IR WT , but not IR ⌬43 . Insulin-activated IR WT , and not IR ⌬43 , 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. Tyr3Phe 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 IR⌬43 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.The interaction of insulin with its cell surface receptors leads to the activation of phosphatidylinositol 3-kinase (PI 3-kinase, or PI 3-K) and generation of phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P 3 ] at the inner surface of the cell membrane (19). PtdIns(3,4,5)P 3 generation leads to the activation of a group of AGC family protein kinases, including different protein kinase B (PKB)/Akt isoforms, p70 ribosomal S6 kinase, serum-and glucocorticoid-induced protein kinase, and atypical protein kinase C (1,9,11,12,24,28,33). These kinases play key roles in the regulation of cell metabolism, proliferation, and survival by insulin, as well as other growth factors (4). How the AGC kinases are activated following insulin stimulation of PI 3-kinase has been extensively investigated (41). PKB/Akt activation requires phosphorylation at two highly conserved Ser/Thr residues by 3-phosphoinositidedependent protein kinase 1 (PDK-1) (22) and by a distinct 3-phosphoinositide-dependent protein kinase 2 (PDK-2), recently identified as the mammalian target of rapamycin (21, 10, 36). Thus, PDKs represent master regulators of AGC kinase signal transduction (38,40,44). PDK-1 is composed of a Cterminal PH domain and a catalytic domain similar to the catalytic domains of PKA, PKB, and PKC (3). After stimulation by insulin, PI 3-kinase-generated lipids bind the PH domain of PDK-1, promoting its translocation to the membrane, together with that of other PH dom...

Differential Pathway Coupling of the Activated Insulin Receptor Drives Signaling Selectivity by XMetA, an Allosteric Partial Agonist Antibody

“…We observed that for both IR-A and IR-B the kinase regulatory domain was most sensitive to insulin, and the C-terminal region was least sensitive to insulin. Mutation and deletion studies regarding the C-terminal phosphotyrosines' contributions to insulin signaling support the notion that the C-terminal tyrosines have a moderate role in activating metabolic insulin signaling by acting as a binding substrate to the Akt-activating PDK-1 enzyme (Fiory et al, 2005), but have little if any effect on mitogenic insulin signaling McClain et al, 1988;Takata et al, 1991). In the CHO-hIR-A cells, the dose response of all three sites to XMetA was similar; XMetA was able to only partially stimulate receptor autophosphorylation to a level about 20% that of insulin.…”

Selective Disruption of Insulin-like Growth Factor-1 (IGF-1) Signaling via Phosphoinositide-dependent Kinase-1 Prevents the Protective Effect of IGF-1 on Human Cancer Cell Death

“…It has been previously reported (19) that the region corresponding to the insulin receptor (IR) C terminus is critical for the interaction with PDK1. With this background, peptides corresponding to the C-terminal residues 1295-1337 of IGF-1R (C43), 1316 -1337 (C22), and similar peptides featuring tyrosine replacement with phenylalanine (C43F and C22F) were synthesized (Fig.…”

“…There is also evidence that PDK1 undergoes tyrosine phosphorylation in response to several growth factors. Studies with the tyrosine phosphatase inhibitor pervanadate and insulin indicate that full activation of PDK1 requires phosphorylation at Tyr 373/376 (17)(18)(19). Consistent with its role in transformation, PDK1 is highly expressed in a large number of invasive human breast cancer cell lines and in ovarian cancers (20,21).…”

Changes in the Expression of Insulin Signaling Pathway Molecules in Endometria from Polycystic Ovary Syndrome Women with or without Hyperinsulinemia

“…In the signaling pathway, the binding of insulin to its receptor provokes autophosphorylation in tyrosine residues and phosphorylation on IRS (IRS-1 and -2) (19,20). When the PI3K pathway is activated (1,21), phosphorylation of Akt is achieved in T308 and S473 residues (19,22,23).…”

“…When the PI3K pathway is activated (1,21), phosphorylation of Akt is achieved in T308 and S473 residues (19,22,23). One of the several substrates that may be influenced by activated Akt is AS160, a Rab-GAP protein, which is phosphorylated in T642.…”