Site-specific monoubiquitination activates Ras by impeding GTPase-activating protein function

摘要

KRas has recently been shown to be activated by monoubiquitination (mUb). Similar to oncogenic mutations, mUb of Ras at position 147 activates Ras by causing a defect in GTPase activating protein (GAP) function. To characterize the mechanism by whichmUb impairs GAP-mediated downregulation of Ras, we made various modifications at position 147 of Ras and examined the impact on Ras sensitivity to GAP function. Whereas small modifications (iodo-acetamide and glutathione) at position 147 of Ras do not affect GAP-mediated hydrolysis, ligation of Ras to Ub~(G76C) (native linker), Ub~(X77c) (one residue longer), and PDZ2 (with a native ubiquitin linker) was defective in GAP-mediated GTP hydrolysis. However, restoration of GAP activity was observed for Rasmodified with the PDZ2 domain containing a shorter and stiffer linker region than ubiquitin. Therefore, the properties of the linker region dictate whether modification affects GAP-mediated hydrolysis, and our data indicate that the GAP defect requires a minimum linker length of 7 to 8 residues.