Characterization of the Interaction between Rheb and a Truncated Construct of Tuberous Sclerosis Complex 2 (TSC2) and the Potential Effect of Heparin on TSC2-Stimulated GTP Hydrolysis

Abstract

Rheb is a member of the Ras superfamily of proteins, a known regulator of cell growth, proliferation, and signaling. Interactions involving Rheb and the TSC2 protein regulate the cycling of Rheb between its nucleotide-bound states and, thereby, cell growth. The C-terminus of TSC2 contains a “perceived” conserved GAP (GTPase-activating protein)-binding domain. Rheb binds to TSC2 and this binding facilitates TSC2-stimulated Rheb GTP hydrolysis to regulate cell signaling. At present, little is known about the molecular features of the Rheb-TSC2 protein interaction. We present biochemical and biophysical data on the interaction of Rheb with TSC2, using a shorter, 218 amino acid GTPase derivative of TSC2 (TSC2-218) that has been used to characterize the GTP hydrolysis rate of activated Rheb.Heparan sulfate is a derivative of heparin, a sulfonated polysaccharide known to interact with diverse proteins and, thus, biologically important. TSC2-218 contains a polybasic region and preliminary studies indicate that heparan sulfate may be capable of binding to TSC2-218, but not Rheb. We present evidence that the binding of heparan sulfate to TSC2-218 does not interfere with TSC2-218-Rheb binding and may enhance the ability of TSC2-218 to stimulate Rheb GTP hydrolysis. NMR spectroscopy was used to outline TSC2 residues that might be important for interactions with Rheb and define conformational changes induced in TSC2-218 by the presence of heparan sulfate as defined by chemical shift changes. This work highlights molecular features of the Rheb-TSC2 interaction that may contribute to our understanding of new ways to regulate abnormal cell signaling activity facilitated by the reduced GTP-GDP cycling of Rheb in the presence of disease-causing mutants of TSC2.