PMID- 16221682
OWN - NLM
STAT- MEDLINE
DCOM- 20060306
LR  - 20211203
IS  - 0021-9258 (Print)
IS  - 0021-9258 (Linking)
VI  - 280
IP  - 49
DP  - 2005 Dec 9
TI  - mTOR.RICTOR is the Ser473 kinase for Akt/protein kinase B in 3T3-L1 adipocytes.
PG  - 40406-16
AB  - 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.
FAU - Hresko, Richard C
AU  - Hresko RC
AD  - Department of Cell Biology and Physiology, Washington University School of 
      Medicine, St. Louis, Missouri 63110, USA.
FAU - Mueckler, Mike
AU  - Mueckler M
LA  - eng
GR  - R01 DK067229/DK/NIDDK NIH HHS/United States
PT  - Journal Article
PT  - Research Support, N.I.H., Extramural
PT  - Research Support, Non-U.S. Gov't
DEP - 20051011
PL  - United States
TA  - J Biol Chem
JT  - The Journal of biological chemistry
JID - 2985121R
RN  - 0 (Carrier Proteins)
RN  - 0 (Insulin)
RN  - 0 (Phosphatidylinositol Phosphates)
RN  - 0 (RICTOR protein, human)
RN  - 0 (RNA, Small Interfering)
RN  - 0 (Rapamycin-Insensitive Companion of mTOR Protein)
RN  - 0 (phosphatidylinositol 3,4,5-triphosphate)
RN  - 452VLY9402 (Serine)
RN  - EC 2.7.- (Protein Kinases)
RN  - EC 2.7.1.1 (MTOR protein, human)
RN  - EC 2.7.1.1 (mTOR protein, mouse)
RN  - EC 2.7.11.1 (DNA-Activated Protein Kinase)
RN  - EC 2.7.11.1 (Proto-Oncogene Proteins c-akt)
RN  - EC 2.7.11.1 (TOR Serine-Threonine Kinases)
SB  - IM
MH  - 3T3-L1 Cells
MH  - Adipocytes/*enzymology/ultrastructure
MH  - Animals
MH  - Carrier Proteins/analysis/*metabolism
MH  - DNA-Activated Protein Kinase/analysis/metabolism
MH  - Insulin/pharmacology
MH  - Mice
MH  - Microsomes/enzymology
MH  - Phosphatidylinositol Phosphates/pharmacology
MH  - Phosphorylation
MH  - Protein Kinases/analysis/*metabolism
MH  - Proto-Oncogene Proteins c-akt/chemistry/*metabolism
MH  - RNA, Small Interfering/pharmacology
MH  - Rapamycin-Insensitive Companion of mTOR Protein
MH  - Serine/*metabolism
MH  - Subcellular Fractions/enzymology
MH  - TOR Serine-Threonine Kinases
EDAT- 2005/10/14 09:00
MHDA- 2006/03/07 09:00
CRDT- 2005/10/14 09:00
PHST- 2005/10/14 09:00 [pubmed]
PHST- 2006/03/07 09:00 [medline]
PHST- 2005/10/14 09:00 [entrez]
AID - S0021-9258(20)59002-9 [pii]
AID - 10.1074/jbc.M508361200 [doi]
PST - ppublish
SO  - J Biol Chem. 2005 Dec 9;280(49):40406-16. doi: 10.1074/jbc.M508361200. Epub 2005 
      Oct 11.