Investigating the Regulation of Ras Protein Prenylation

Abstract

Ras family small GTPases undergo prenylation for proper localization to the plasma membrane, where they carry out their signaling function. Prevalent mutations in these proteins that render them persistently active have attracted a lot of attention due to their association with disease and developmental syndromes, such as cancer and RASopathies. Protein farnesyltransferase (FTase) catalyzes the incorporation of a farnesyl moiety to the Cys residue in the C-terminal CAAX motif of Ras, stimulating proper membrane association. Small GTP-binding protein GDP-dissociation stimulator (SmgGDS) proteins are chaperones involved in binding and trafficking small GTPases. Recent data suggest that SmgGDS proteins also regulate prenylation of small GTPases in vivo in a substrate-selective manner. In addition to the CAAX motif, many Ras family members contain a polybasic region (PBR) upstream of the CAAX that is proposed to increase binding affinity to different protein interactors, including FTase and SmgGDS-607, and play a role in protein trafficking. To investigate the role of SmgGDS-607 in Ras prenylation, we developed a model for regulation of farnesylation that highlights the importance of the PBR and CAAX motif on recognition and processing of full-length Ras proteins. Here we demonstrate that SmgGDS-607 differentially regulates farnesylation of several small GTPases. For some proteins, such as DiRas1, SmgGDS-607 inhibits farnesylation by sequestering the substrate and limiting modification catalyzed by FTase. The competitive binding affinities of the small GTPase for SmgGDS-607 and FTase dictate the extent of inhibition. Additionally, we demonstrate a novel function for SmgGDS-607, increasing the rate of farnesylation of HRas by enhancing product release from FTase. Since the cell contains multiple small GTPases regulated by SmgGDS, these differential interactions can lead to large changes in prenylation status. We systematically studied the role of the PBR and CAAX motif in modulating substrate recognition by both FTase and SmgGDS-607 through use of a set of Ras chimeric mutants in kinetic and binding experiments. This work demonstrates that SmgGDS-607 binds to a broad range of small GTPases and does not require a PBR for recognition, although the presence of a PBR influences the strength of the SmgGDS:Ras interaction. These studies demonstrate that the PBR enhances catalytic efficiency, as higher values for both prenylation turnover (kcat) and substrate selectivity (kcat/KM) are observed for substrates containing PBRs. Furthermore, the PBR plays an important role in facilitating product release. However, the CAAX identity also contributes to the extent of enhancement of kcat and the single-turnover rate. Thus, the synergistic effects of the PBR and CAAX motif ultimately dictate whether SmgGDS-607 will inhibit or enhance prenylation. Together, this work provides mechanistic insight into the regulation of protein farnesylation of small GTPases, elucidating the role of the PBR and CAAX in farnesylation kinetics and demonstrating that SmgGDS-607 has multiple modes of substrate recognition, which can be of great importance for the development of novel anti-cancer therapeutics.