Abstract 5074: Identification of Ras compartment-specific interacting proteins

Abstract

Abstract Ras proteins are powerful molecular switches that control a wide range of functions, including cell growth, differentiation, apoptosis, cytoskeleton remodeling, etc. In humans, there are three RAS genes encode structurally highly similar Ras proteins that appear to have different functions in the cell. We have previously reported that a given Ras protein can signal from different cell compartments to control distinct functions in yeast. This present project aims to identify proteins that bind Ras in a cell compartment-specific manner in human cells to define how compartmentalized Ras signaling impacts tumor formation. We used the biomolecular fluorescence complementation (BiFC) method to screen a human cDNA library. Sixty two proteins were thus identified that bound the constitutively activated H-Ras (H-RasG12V), but not the GST control. These proteins also bound weakly to H-RasG12V whose effector binding motif has been mutated. To determine if these proteins can alter Ras functions, we first overexpressed them and then tested for their impacts on Ras-induced cell transformation and transcription. Only 25 of the isolated proteins could alter Ras signaling in these assays. Many of these 25 proteins are known to either associate with cellular membrane and/or regulate protein transportation, suggesting that as a group they highlight the importance of regulating Ras trafficking in the control of cell transformation. In particular, a number of isolated proteins may regulate endosome sorting, and we thus focused on two such proteins, CHMP6/VPS20 and VPS4A, for further investigation. We first validated the Ras binding by co-immunoprecipitation; furthermore the microscopy data showed that the binding is readily detectable in endosomes. Moreover, up- or down-regulating of level of either CHMP6/VPS20 or VPS4A dramatically inhibited Ras-induced transformation of NIH-3T3 cells. To determine how these molecules regulate Ras activities, we first examined whether they modulate Ras protein intracellular transport. Cell fractionation using sucrose gradients has shown that VPS4A knock-down could alter both wide-type and constitutively activated Ras membrane localization. In summary, we indentified Ras compartment-specific binding proteins by a cell-based approach. Many of the proteins are trafficking regulating proteins. In particular, two endosome sorting proteins CHMP6/VPS20 and VPS4A have been shown to regulate the oncogenic effects of Ras, possibly by localizing Ras proteins into proper cell compartments. We believe that the study of compartment-specific Ras interacting proteins is indispensable for better understanding the complex roles of Ras in oncogenesis of breast cancer as well as many other cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5074.