The rapamycin-binding domain governs substrate selectivity by the mammalian target of rapamycin.
The mammalian target of rapamycin (mTOR) is a Ser/Thr (S/T) protein kinase, which controls mRNA translation initiation by modulating phosphorylation of the translational regulators PHAS-I and p70(S6K). Here we show that in vitro mTOR is able to phosphorylate these two regulators at comparable rates. Both (S/T)P sites, such as Thr36, Thr45, and Thr69 in PHAS-I and the h(S/T)h site (where h is a hydrophobic amino acid) Thr389 in p70(S6K), were phosphorylated. Rapamycin-FKBP12 inhibited mTOR activity. Surprisingly, the extent of inhibition depended on the substrate. Moreover, mutating Ser2035 in the rapamycin-binding domain (FRB) not only decreased rapamycin sensitivity as expected but also dramatically affected the sites phosphorylated by mTOR. The results demonstrate that mutations in Ser2035 are not silent with respect to mTOR activity and implicate the FRB in substrate recognition. The findings also impose new limitations on interpreting results from experiments in which rapamycin and/or rapamycin-resistant forms of mTOR are used to investigate mTOR function in cells.
McMahon
Lloyd P
LP
Department of Pharmacology, University of Virginia School of Medicine, Charlottesville, Virginia 22908, USA.
Choi
Kin M
KM
Lin
Tai-An
TA
Abraham
Robert T
RT
Lawrence
John C
JC
D048868:Adaptor Proteins, Signal Transducing; D000903:Antibiotics, Antineoplastic / Q000494:pharmacology*; D001665:Binding Sites; D002352:Carrier Proteins / Q000378:metabolism; D018797:Cell Cycle Proteins; D002460:Cell Line; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D006801:Humans; D007700:Kinetics; D008954:Models, Biological; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D011494:Protein Kinases / Q000378:metabolism / Q000502:physiology*; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000378:metabolism; D038762:Ribosomal Protein S6 Kinases, 70-kDa / Q000378:metabolism; D012694:Serine / Q000737:chemistry / Q000378:metabolism; D020123:Sirolimus / Q000494:pharmacology*; D013379:Substrate Specificity; D058570:TOR Serine-Threonine Kinases; D013912:Threonine / Q000737:chemistry; D013997:Time Factors; D014162:Transfection
D048868:Adaptor Proteins, Signal Transducing; D000903:Antibiotics, Antineoplastic; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D018076:DNA, Complementary; C087000:EIF4EBP1 protein, human; D010750:Phosphoproteins; D011994:Recombinant Proteins; D013912:Threonine; D012694:Serine; D011494:Protein Kinases; C546842:MTOR protein, human; D038762:Ribosomal Protein S6 Kinases, 70-kDa; D058570:TOR Serine-Threonine Kinases; D020123:Sirolimus