Abstract IA15: Membrane targeting of Ras

Ras proteins are targeted to membranes by virtue of post-translational modifications of their C-terminal hypervariable regions (HVRs). Because Ras proteins signal only when associated with cellular membranes, the Ras trafficking pathway is considered an attractive area for anti-cancer drug discovery. The three mammalian Ras genes, hras, nras and kras, give rise to four Ras proteins because the transcript of the kras locus is alternatively spliced to encode K-Ras4A and K-Ras4B. The kras locus is most often mutated in human cancer and when mutated gives rise to oncogenic forms of both K-Ras4A and K-Ras4B. The four Ras protein differ only in their membrane targeting HVRs. Each Ras protein ends in a CAAX sequence, which signals for a three-step modification that includes farnesylation, AAX proteolysis and carboxyl methylation. N-Ras, H-Ras and K-Ras4A are further modified by palmitoylation of one or two cysteines upstream of the CAAX sequence. Palmitoylation of N-Ras and H-Ras occurs on the cytoplasmic face of the Golgi apparatus. Whereas farensylation affords relatively weak membrane affinity, palmitoylation of the farnesylated protein provides stable association with membranes. Palmitoylation is readily reversible and a palmitoylation/depalmitoylation cycle drives a cycle of trafficking to and from the Golgi. K-Ras4B is unique in utilizing a polybasic sequence upstream of the CAAX motif to associate with the plasma membrane through an electrostatic interaction. We have shown that K-Ras4B can be phosphorylated by PKC at serine 181 in the HVR and this modification partially neutralizes the electrostatic charge such that K-Ras4B loses affinity for the plasma membrane and is relocated on endomembranes in a process we have called the farnesyl-electrostatic switch. Because phosphorylation of K-Ras4B is associated with diminished cell viability we have explored the possibility that induction of phosphorylation of oncogenic K-Ras4B may be one way to target K-Ras transformed cells. Other aspects of K-Ras biology that will be discussed are the relative expression of K-Ras4A and K-Ras4B and the unique membrane trafficking of K-Ras4A. A K-Ras4B membrane release assay suitable for siRNA and small compound screening will also be discussed. Citation Format: Frederick Tsai, Nicole Fehernbacher, Pamela Sung, Helen Court, Mark R. Philips. Membrane targeting of Ras. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr IA15. doi: 10.1158/1557-3125.RASONC14-IA15