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Molecular and Cellular Biology, November 2002, p. 7428-7438, Vol. 22, No. 21
0270-7306/02/$04.00+0     DOI: 10.1128/MCB.22.21.7428-7438.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

The Rapamycin-Binding Domain Governs Substrate Selectivity by the Mammalian Target of Rapamycin

Lloyd P. McMahon,1 Kin M. Choi,1 Tai-An Lin,2 Robert T. Abraham,3 and John C. Lawrence, Jr.1,4*

Departments of Pharmacology,1 Medicine, University of Virginia School of Medicine, Charlottesville, Virginia 22908,4 Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, New Jersey 08543,2 Signal Transduction Program, Burnham Institute, La Jolla, California 920373

Received 30 April 2002/ Accepted 24 July 2002

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 p70S6K. 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 p70S6K, 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.

* Corresponding author. Mailing address: Department of Pharmacology, University of Virginia Health System, P.O. Box 800735, 1300 Jefferson Park Ave., Charlottesville, VA 22908-0735. Phone: (434) 924-1584. Fax: (434) 982-3575. E-mail: jcl3p{at}virginia.edu.

Molecular and Cellular Biology, November 2002, p. 7428-7438, Vol. 22, No. 21
0022-538X/02/$04.00+0     DOI: 10.1128/MCB.22.21.7428-7438.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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