Abstract IA07: KRAS4b’s unique diffusion behavior is defined by plasma membrane and effector interactions

Abstract

Abstract RAS proteins are GTP-dependent switches that control and regulate signaling pathways involved in cell fate and are frequently mutated in cancer. RAS association with the plasma membrane, or with certain endomembrane compartments, is a required step for its activity. Why precisely this is so remains an open question. One possibility is that RAS is merely required for recruitment of RAF and other effectors to the membrane where it can associate with key regulatory molecules that lead to signaling activation. However, four isoforms exist in humans (HRAS, NRAS, and two splice variants, KRAS4b and KRAS4a) and their differences lie within 22 amino acids in the C-terminal hypervariable region (HVR, aa 167-189). These differences in the domain responsible for membrane association result in the recruitment and organization of RAS into distinct membrane nanodomains, and it is thought that this results in differential signaling behavior from RAS isoforms. Isoform-specific RAS nanodomains are thought to both be highly dynamic, differentially composed of a variety of lipids and proteins, and supported by interactions with cytoskeletal structures. How these domains are regulated, and how they influence isoform-specific RAS signaling, remains unclear. Here, we report the molecular mobility of RAS variants in the plasma membrane of living cancer cells using single-molecule tracking methods. Detailed analysis of tracks revealed that KRAS4b molecules exhibit confined mobility with three diffusive states in the active plasma membrane of living cells. This diffusion characteristic was unique to KRAS4b and influenced by both the hypervariable region and globular domain of the protein, compared to all the other Ras isoforms. Importantly, the occupancy of each diffusive states was altered for the oncogenic mutant of KRAS4b, suggesting that the diffusive states we observe are related to signaling events. Our working hypothesis is that the HVR of KRAS4b may be directly involved in assembling the membrane lipid and protein nanodomain necessary for KRAS4b signaling activity. In addition, using two-color single-molecule tracking studies, we are beginning to characterize the interactions of KRAS4b with its major effector RAF. From these studies, we are learning about the kinetics of RAS/RAF interactions in the membrane of living cells. Understanding the underlying principle of KRAS4b functionality on cell membranes is useful for developing novel therapeutic strategies for targeting oncogenic KRAS4b. Citation Format: Debanjan Goswami, De Chen, John Columbus, Yue Yang, Felice Lightstone, Thomas Turbyville. KRAS4b’s unique diffusion behavior is defined by plasma membrane and effector interactions [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr IA07.