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Originally published In Press as doi:10.1074/jbc.M306553200 on July 17, 2003

J. Biol. Chem., Vol. 278, Issue 41, 39921-39930, October 10, 2003
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Identification of Dominant Negative Mutants of Rheb GTPase and Their Use to Implicate the Involvement of Human Rheb in the Activation of p70S6K*

Angel P. Tabancay, Jr. {ddagger} §, Chia-Ling Gau {ddagger} ¶, Iara M. P. Machado {ddagger} ||, Erik J. Uhlmann **, David H. Gutmann **, Lea Guo {ddagger} and Fuyuhiko Tamanoi {ddagger} {ddagger}{ddagger}

From the {ddagger}Department of Microbiology, Immunology, and Molecular Genetics, Jonsson Comprehensive Cancer Center, Molecular Biology Institute, University of California, Los Angeles, California 90095-1489 and the **Department of Neurology, Washington University School of Medicine, St. Louis, Missouri 63110

Rheb GTPases represent a unique family of the Ras superfamily of G-proteins. Studies on Rheb in Schizosaccharomyces pombe and Drosophila have shown that this small GTPase is essential and is involved in cell growth and cell cycle progression. The Drosophila studies also raised the possibility that Rheb is involved in the TOR/S6K signaling pathway. In this paper, we first report identification of dominant negative mutants of S. pombe Rheb (SpRheb). Screens of a randomly mutagenized SpRheb library yielded a mutant, SpRhebD60V, whose expression in S. pombe results in growth inhibition, G1 arrest, and induction of fnx1+, a gene whose expression is induced by the disruption of Rheb. Alteration of the Asp-60 residue to all possible amino acids by site-directed mutagenesis led to the identification of two particularly strong dominant negative mutants, D60I and D60K. Characterization of these dominant negative mutant proteins revealed that D60V and D60I exhibit preferential binding of GDP, while D60K lost the ability to bind both GTP and GDP. A possible use of the dominant negative mutants in the study of mammalian Rheb was explored by introducing dominant negative mutations into human Rheb. We show that transient expression of the wild type Rheb1 or Rheb2 causes activation of p70S6K, while expression of Rheb1D60K mutant results in inhibition of basal level activity of p70S6K. In addition, Rheb1D60K and Rheb1D60V mutants blocked nutrient- or serum-induced activation of p70S6K. This provides critical evidence that Rheb plays a role in the mTOR/S6K pathway in mammalian cells.

Received for publication, June 20, 2003

* This work was supported by National Institutes of Health Grants CA41996 and CA32737 (to F. T.), a Tuberous Sclerosis Alliance grant (to D. H. G.), and National Institutes of Health Grant F32-CA94665 (to E. J. U). Flow cytometry performed in the UCLA Flow Cytometry Core Facility was supported by National Institutes of Health Grants CA16042 and AI28697. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Supported by the Eugene Cota Robles Fellowship.

Supported by the Pauley Fellowship.

|| Supported by CNPq (Brasilia, DF, Brazil) and Universidade Federal do Parana (Curitiba, PR, Brazil). Present address: Departamento de Farmacia, Universidade Federal do Parana, Curitiba, Parana, Brazil 80210-170.

{ddagger}{ddagger} To whom correspondence should be addressed: Dept. of Microbiology, Immunology & Molecular Genetics, 1602 Molecular Sciences Bldg., UCLA, Los Angeles, CA 90095-1489. Tel.: 310-206-7318; Fax: 310-206-5231; E-mail: fuyut{at}microbio.ucla.edu.


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