This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The long-term goal of this research is to identify and characterize molecular mechanisms through ubiquitylation regulates tumorigenesis. The central hypothesis of the proposal is that specific E3 ligase complexes mediate the ubiquitylation of the molecular scaffold Kinase Suppressor of Ras 1 (KSR1) to control its half-life, and signaling. KSR1 is a scaffold for the Raf/MEK/ERK kinase cascade. We observed that KSR1 interacts with the components of ubiquitin E3 ligases including CUL4, DDB1, VPRBP (DCAF1), EDD1, and BRUCE (BIRC6). Overexpression of CUL4A promotes ubiquitylation of KSR1. The kinase DYRK2, which interacts with the DDB1-VPRBP complex, phosphorylates katanin making it a substrate for ubiquitylation by the ligase. Notably, the consensus sequence for substrate phosphorylation by DYRK2 is found surrounding Thr274 and Ser392 in KSR1. These data suggest that the ability of KSR1 to affect Ras-induced tumorigenesis is regulated by its ubiquitylation by specific E3 ligases. We will test this hypothesis by characterizing the interaction of KSR1 with E3 ligase components and determining whether the ubiquitylation of KSR1 affects cell transformation and tumorigenesis by oncogenic Ras. These studies should reveal novel mechanisms underlying cellular control of proliferation and transformation. Such knowledge should open new avenues for exploration and understanding of the molecular mechanisms affecting cancer susceptibility in humans.