Modulation of the protein kinase activity of mTOR.
mTOR is a founding member of a family of protein kinases having catalytic domains homologous to those in phosphatidylinositol 3-OH kinase. mTOR participates in the control by insulin of the phosphorylation of lipin, which is required for adipocyte differentiation, and the two translational regulators, p70S6K and PHAS-I. The phosphorylation of mTOR, itself, is stimulated by insulin in Ser2448, a site that is also phosphorylated by protein kinase B (PKB) in vitro and in response to activation of PKB activity in vivo. Ser2448 is located in a short stretch of amino acids not found in the two TOR proteins in yeast. A mutant mTOR lacking this stretch exhibited increased activity, and binding of the antibody, mTAb-1, to this region markedly increased mTOR activity. In contrast, rapamycin-FKBP12 inhibited mTOR activity towards both PHAS-I and p70S6K, although this complex inhibited the phosphorylation of some sites more than that of others. Mutating Ser2035 to Ile in the FKBP12-rapamycin binding domain rendered mTOR resistant to inhibition by rapamycin. Unexpectedly, this mutation markedly decreased the ability of mTOR to phosphorylate certain sites in both PHAS-I and p70S6K. The results support the hypotheses that rapamycin disrupts substrate recognition instead of directly inhibiting phosphotransferase activity and that mTOR activity in cells is controlled by the phosphorylation of an inhibitory regulatory domain containing the mTAb-1 epitope.
Lawrence
J C
JC
Department of Pharmacology, University of Virginia School of Medicine, 1300 Jefferson Park Avenue, Charlottesville, VA 22908-0735, USA. JCL3p@virginia.edu
Lin
T A
TA
McMahon
L P
LP
Choi
K M
KM
D048868:Adaptor Proteins, Signal Transducing; D017667:Adipocytes / Q000201:enzymology / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D002352:Carrier Proteins / Q000378:metabolism*; D018797:Cell Cycle Proteins; D039642:Eukaryotic Initiation Factors; D007328:Insulin / Q000378:metabolism; D051379:Mice; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D058570:TOR Serine-Threonine Kinases
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; C087000:EIF4EBP1 protein, human; C499527:Eif4ebp1 protein, mouse; D039642:Eukaryotic Initiation Factors; D007328:Insulin; D010750:Phosphoproteins; D011494:Protein Kinases; C546842:MTOR protein, human; C546843:mTOR protein, mouse; D058570:TOR Serine-Threonine Kinases