Abstract

The insulin-signaling pathway leading to the activation of Akt/protein kinase B has been well characterized except for a single step, the phosphorylation of Akt at Ser-473. Double-stranded DNA-dependent protein kinase (DNA-PK), ataxia telangiectasia mutated (ATM) gene product, integrin-linked kinase (ILK), protein kinase Calpha (PKCalpha), and mammalian target of rapamycin (mTOR), when complexed to rapamycin-insensitive companion of mTOR (RICTOR), have all been identified as playing a critical role in Akt Ser-473 phosphorylation. However, the apparently disparate results reported in these studies are difficult to evaluate, given that different stimuli and cell types were examined and that all of the candidate proteins have never been systematically studied in a single system. Additionally, none of these studies were performed in a classical insulin-responsive cell type or tissue such as muscle or fat. We therefore examined each of these candidates in 3T3-L1 adipocytes. In vitro kinase assays, using different subcellular fractions of 3T3-L1 adipocytes, revealed that phosphatidylinositol 3,4,5-trisphosphate-stimulated Ser-473 phosphorylation correlated well with the amount of DNA-PK, mTOR, and RICTOR but did not correlate with levels of ATM, ILK, and PKCalpha. PKCalpha was completely absent from compartments with Ser-473 phosphorylation activity. Although purified DNA-PK could phosphorylate a peptide derived from Akt that contains amino acid Ser-473, it could not phosphorylate full-length Akt2. Vesicles immunoprecipitated from low density microsomes using antibodies directed against mTOR or RICTOR had phosphatidylinositol 3,4,5-trisphosphate-stimulated Ser-473 activity that was sensitive to wortmannin but not staurosporine. In contrast, immunopurified low density microsome vesicles containing ILK could not phosphorylate Akt on Ser-473 in vitro. Small interference RNA knockdown of RICTOR, but not DNA-PK, ATM, or ILK, suppressed insulin-activated Ser-473 phosphorylation and, to a lesser extent, Thr-308 phosphorylation in 3T3-L1 adipocytes. Based on our cell-free kinase and small interference RNA results, we conclude that mTOR complexed to RICTOR is the Ser-473 kinase in 3T3-L1 adipocytes.

Highlights

  • Understood, the early insulin signaling pathway has been well characterized except for one step, the phosphorylation of Akt at Ser-473

  • Utilizing the cell-free system as well as in vivo small interference RNA (siRNA) knockdown studies, we addressed whether integrin-linked kinase (ILK), PKC␣, DNA-PK, ataxia telangiectasia mutated (ATM), or mammalian target of rapamycin (mTOR) was involved in the insulin-stimulated phosphorylation of Akt at Ser-473 in 3T3-L1 adipocytes

  • Subcellular Localizations of DNA-PK and mTOR1⁄7RICTOR Correlate with hydrophobic motif domain protein kinase (HMD-PK) Activity—ILK, PKC␣, DNA-PK, ATM, and mTOR have all been proposed to play a major role in Akt Ser-473 phosphorylation based on experiments carried out on a variety of different cell types and stimuli [24]

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Summary

Introduction

Understood, the early insulin signaling pathway has been well characterized except for one step, the phosphorylation of Akt at Ser-473. Utilizing the cell-free system as well as in vivo siRNA knockdown studies, we addressed whether ILK, PKC␣, DNA-PK, ATM, or mTOR was involved in the insulin-stimulated phosphorylation of Akt at Ser-473 in 3T3-L1 adipocytes.

Results
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