Tuberous sclerosis complex (TSC) is a genetic disease caused by a mutation in 
either the tsc1 or tsc2 tumor suppressor gene. Recent studies have demonstrated 
that TSC2 displays GAP (GTPase-activating protein) activity specifically towards 
the small G protein Rheb and inhibits its ability to stimulate the mTOR 
signaling pathway. Rheb and TSC2 comprise a unique pair of GTPase and GAP, 
because Rheb has high basal GTP levels and TSC2 does not have the catalytic 
arginine finger found in Ras-GAP. To investigate the function of TSC2 and Rheb 
in mTOR signaling, we analyzed the TSC2-stimulated Rheb GTPase activity. We 
found that Arg15, a residue equivalent to Gly12 in Ras, is important for Rheb to 
function as a substrate for TSC2 GAP. In addition, we identified asparagine 
residues essential for TSC2 GAP activity. We demonstrated a novel catalytic 
mechanism of the TSC2 GAP and Rheb that TSC2 uses a catalytic "asparagine thumb" 
instead of the arginine finger found in Ras-GAP. Furthermore, we discovered that 
farnesylation and membrane localization of Rheb is not essential for Rheb to 
stimulate S6 kinase (S6K) phosphorylation. Analysis of TSC1 binding defective 
mutants of TSC2 shows that TSC1 is not required for the TSC2 GAP activity but 
may function as a regulatory component in the TSC1/TSC2 complex. Our data 
further demonstrate that GAP activity is essential for the cellular function of 
TSC2 to inhibit S6K phosphorylation.