FIG. 1.

Rheb-R15G cannot restore the GTPase activity and is less sensitive to TSC2 GAP activity. (A) Rheb contains an arginine residue (the residue in boldface type) at the position corresponding to codon 12 of Ras, which has a glycine. Numbers indicate positions of the last residues. (B) Mutation of Arg15 affects Rheb GTP binding and sensitivity to TSC2 GAP. Wild-type and mutant Myc-Rheb were transfected into HEK293 cells in the presence or absence of TSC1/2 as indicated and labeled with [³²P]phosphate. Myc-Rheb was immunoprecipitated, and the bound nucleotides were eluted and resolved on a cellulose plate. The expression levels of Myc-Rheb and TSC1/2 in a parallel experiment (middle) and quantification of Rheb nucleotide binding (bottom) are shown. The ratio of GTP to GDP was calculated by the following formula: (GTP counts/3)/(GDP counts/2). The values are data from three independent experiments. (C) Rheb-R15G is less sensitive to the TSC1/2 complex. HA-S6K was transfected into HEK293 cells in the presence or absence of Myc-Rheb and TSC1/2. Phosphorylation of S6K was determined by phosphospecific antibody against phospho-S6K(T389). WT, wild type.