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Papers In Press, published online ahead of print October 11, 2005
Department of Cell Biology and Physiology, Washington University, School of Medicine, St. Louis, MO 63110-1093
Corresponding Author: mike{at}cellbiology.wustl.edu
The insulin-signaling pathway leading to the activation of Akt/PKB has been well-characterized except for a single step, the phosphorylation of Akt at Ser473. Double-stranded DNA-dependent protein kinase (DNA-PK). ataxia telangiectasia mutated (ATM) gene product, Integrin-linked kinase (ILK), Protein Kinase Ca (PKCa), 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-Ser473 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 (PIP3)-stimulated Ser473 phosphorylation correlated well with the amount of DNA-PK, mTOR, and RICTOR but did not correlate with levels of ATM, ILK, and PKCa. PKCa was completely absent from compartments with Ser473 phosphorylation activity. Although purified DNA-PK could phosphorylate a peptide derived from Akt that contains amino acid Ser473, it could not phosphorylate full-length Akt2. Vesicles immunoprecipitated from low-density microsomes (LDM) using antibodies directed against mTOR or RICTOR had PIP3-stimulated Ser473 activity that was sensitive to wortmannin but not staurosporine. In contrast, immunopurified LDM vesicles containing ILK could not phosphorylate Akt on Ser473 in vitro. Small interference RNA (siRNA)-knockdown of RICTOR, but not DNA-PK, ATM, or ILK, suppressed insulin-activated Ser473 phosphorylation and to a lesser extent Thr308 phosphorylation in 3T3-L1 adipocytes. Based on our cell-free kinase and siRNA results, we conclude mTOR complexed to RICTOR is the Ser473 kinase in 3T3-L1 adipocytes.
J. Biol. Chem, 10.1074/jbc.M508361200
Submitted on July 29, 2005
Revised on September 27, 2005
Accepted on October 11, 2005
mTOR/RICTOR is the Ser473 kinase for Akt/PKB in 3T3-L1 adipocytes
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