USING A PROTEOMIC APPROACH TO STUDY THE DISTINCT AND OVERLAPPING FUNCTIONS OF RAS

Abstract

The RAS gene family members are the most frequently mutated genes in human cancers and encode four different but highly homologous protein isoforms, HRas, NRas, KRas4A, and KRas4B. The Ras GTPases play important roles in cellular proliferation, survival, and differentiation through various downstream effector pathways. Because of their homology and shared downstream effector proteins and upstream activators, it has remained unclear the degree of overlapping or distinct functional roles they play in cellular processes. RAS isoforms have been found to be preferentially activated in specific types of cancers suggesting distinct mechanistic modes of activation of the Ras isoforms. In order to help explain the shared and unique signaling functions of the Ras isoforms, I began the project with a quantitative proteomic approach to identify the unique and shared interacting proteins of the four Ras isoforms. This method identified many known and novel interacting proteins. Among the many new interacting proteins, I identified and validated CARM1 and Chk1 as HRas-specific interacting proteins and PIP4K2C and IPO7 as KRas-specific. Additionally, RADIL was identified as a previously unknown effector protein of Ras. Targeting the isoform-specific interacting proteins could selectively target cells transformed with the corresponding Ras isoform. Surprisingly, such selectivity was not observed. This interesting finding was rationalized by noticing that the proteomic data suggest that different isoforms of Ras could interact with each other. Using a FRET-based approach, HRas and KRas were found to form heterodimers in cells. The heterodimer formation could explain the lack of Ras isoform specificity when targeting isoform-specific interactors. Thus, by identifying isoform-specific interacting proteins and Ras heterodimers, the Ras interactome analysis provides novel insights into understanding the overlapping roles of Ras isoforms.