PROJECT SUMMARY/ABSTRACT The overall goal of this application is to elucidate the mechanism of Lys63 (K63)-linked polyubiquitination of extracellular signal-regulated kinases ERK1 and ERK2, and to determine the contribution of this novel post-translational modification to tumorigenesis. ERK1 and ERK2, which share high structural and functional similarities, are downstream effectors of a mitogen-activated protein kinase (MAPK) cascade that dictates cellular behavior and cell fate decisions in response to a wide range of intracellular signals. The ERK signaling pathway is the primary mitogenic pathway in mammalian cells. It is aberrantly upregulated in a large fraction of human tumors due to prevalence of mutations in the upstream signaling components, and reactivation of ERK is also the most common mechanism for resistance to drugs that target these upstream components. Developing a therapy that is applicable for the wide range of tumors driven by a hyperactive ERK pathway and that can circumvent drug resistance remains a major challenge in cancer research. To contend with this challenge requires a comprehensive understanding of how ERK is specifically and efficiently activated within the MAPK cascade. The current knowledge of ERK activation is largely limited to their phosphorylation by the upstream kinase MEK. In our preliminary studies, we have found that ERK is conjugated to K63-linked polyubiquitin chains, and that this post-translational modification correlates with ERK activation. Furthermore, we have identified the tripartite-motif protein TRIM15 as a ubiquitin ligase, and the tumor suppressor CYLD as a deubiquitinating enzyme (DUB), that may dynamically regulate ERK ubiquitination. Here we will test the central hypothesis that K63 ubiquitination of ERK is critical for the specific and efficient activation of these kinases and that dysregulation of this post-translational modification contributes to the pathogenesis and therapeutic resistance of tumors. We propose three specific aims. First, we will characterize the role of TRIM15 and CYLD in K63 ubiquitination of ERK and define how their interactions with ERK are regulated by mitogenic stimuli. Second, we will elucidate the function and mechanism of K63 ubiquitination in the activation of ERK. Third, we will determine the role of TRIM15 and CYLD in tumorigenesis and therapeutic resistance. Collectively, these aims will address fundamental issues in ERK biology and oncogenic signaling, and will provide valuable information for the development of effective therapies for the plethora of human tumors that are driven by a hyperactive ERK signaling pathway.