Deciphering the Molecular Mechanisms Underlying Active Scaffolding

The extracellular signal-regulated kinases 1and 2 (ERK1/2) signaling pathway plays an essential role in several critical steps of embryonic development, tumor progression and controls critical cellular function such as apoptosis, motility and differentiation. Kinases and phosphatases of this pathway have been extensively studied and targeted therapeutically. However, the mechanisms that determine the signal specificity and orchestrate diverse biological outcomes of ERK1/2 signaling are still poorly understood, limiting clinical efforts. Scaffold proteins are key players in the ERK1/2 signaling pathway that are thought to integrate incoming signals and deliver signaling specificity, and yet their role in signal propagation and the mechanisms of their action are still unknown. The long-term goal of our research is to understand how scaffold proteins are involved in the biological processes regulated by the ERK1/2 cascade for developing new therapeutic strategies. The objective of this particular application is to determine the mechanisms underlying the ability of the key scaffold protein, Shoc2, to regulate ERK1/2-mediated cell motility. Shoc2 is essential for embryonic development and a critical regulator of the ERK1/2 activity in tumor cells. We have shown that depletion of Shoc2 in zebrafish results in a decreased number of cells in the bloodstream and specific defects in angiogenesis. We also found that Shoc2 integrates endocytic and ubiquitin machineries to regulate ERK1/2 signaling. Based on our preliminary data, the central hypothesis of this proposal is that Shoc2 creates a signaling hub that regulates ERK’s developmental signals in a spatio-temporal manner. Once the function of Shoc2 in regulating hematopoiesis is known, this knowledge will improve our understanding of the mechanisms underlying congenital disorders and cancer progression at the molecular level, which will ultimately aid in developing therapeutic agents that can target this pathway when deregulated. Our hypothesis will be tested by pursuing three specific aims: Aim 1 will determine the signaling events mediated through the Shoc2 scaffold complexes that control embryonic development. Aim 2 will determine the spatiotemporal organization and dynamics of Shoc2 scaffold complexes. Aim 3 will reveal the mechanisms by which the protein of the Ubiquitin Proteasomal System, HUWE1, controls Shoc2 function and assembly of the Shoc2 scaffold complexes. Our studies will employ state-of-the-art microscopy, genetic, molecular, and cellular techniques. Our approach is innovative, because Shoc2 is critical protein, but mechanisms regulating its function from the perspective of its therapeutic targeting have never been studied. The proposed research is expected to provide a mechanistic understanding of how Shoc2 is involved in determining the specificity of ERK1/2 signaling outcomes and has significance for uncovering the mechanisms and their physiological significance for developmental disorders and cancer progression, thus contributing to the advancement of novel therapeutic strategies and biomarkers.