title: Site-specific monoubiquitination activates Ras by impeding GTPase-activating protein function.
abstract: 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 which mUb 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 (iodoacetamide 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 Ras modified 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.
authors.affiliation: Department of Biochemistry and Biophysics; University of North Carolina; Chapel Hill, NC USA.
authors.forename: G Aaron
authors.initials: GA
authors.lastname: Hobbs
authors.affiliation: Department of Biochemistry and Biophysics; University of North Carolina; Chapel Hill, NC USA.
authors.forename: Harsha P
authors.initials: HP
authors.lastname: Gunawardena
authors.affiliation: Department of Biochemistry and Biophysics; University of North Carolina; Chapel Hill, NC USA.
authors.forename: Rachael
authors.initials: R
authors.lastname: Baker
authors.affiliation: Department of Biochemistry and Biophysics; University of North Carolina; Chapel Hill, NC USA; Lineberger Comprehensive Cancer Center; University of North Carolina; Chapel Hill, NC USA.
authors.forename: Sharon L
authors.initials: SL
authors.lastname: Campbell
mesh_terms: D020690:GTPase-Activating Proteins / Q000378:metabolism*; D005978:Glutathione / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D009154:Mutation; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D054875:Ubiquitination; D018631:ras Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D020690:GTPase-Activating Proteins; D018631:ras Proteins; D005978:Glutathione
keywords: GTP hydrolysis; GTPase Activating Proteins; Post-translational modifications; glutathiolation; monoubiquitination
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