Abstract 2465 Effect of Phosphorylation State and Compensatory Manipulations Affecting of Binding of HSP70 to the Co-Chaperones CHIP and HOP in Renal Cancer Cells

The protein quality control (PQC) system is a regulatory mechanism that aids in cell growth and division, external stimuli response, and stress response. Components of the PQC system, including chaperones and their binding partners, guide substrates to different fates, such as refolding or degradation. If one step in this control mechanism malfunctions, it can be disastrous for the health and homeostasis of the cell, causing uncontrolled protein accumulation and poor quality. Proteins of the PQC system can also be hijacked by cancer cells to promote unrestricted growth, a hallmark of a cancer cell. Chaperones, such as heat shock protein 70 (HSP70), and co-chaperones, such as carboxyl-terminus of heat shock 70 interacting protein (CHIP) and HSP organizing protein (HOP), determine the fate of substrates depending on the level of interaction. CHIP has ubiquitin (UB)-dependent functions, serving as an E3 UB ligase to mark proteins for destruction while HOP aids in refolding of damaged or newly synthesized proteins. Post-translational modifications, such as phosphorylation, can affect protein function and interaction within the PQC system. This work sought to better understand the mechanism of interaction between HSP70 and CHIP and HOP and if there is a way to manipulate the binding preference of HSP70. Cell free crystal structures and biolayer interferometry show that phosphorylated HSP70 decreased binding with CHIP. Cellular based work through NanoBiT luminescence assay confirms this change in interaction in live cells. Additionally, in a phosphorylated state, HSP70 preferentially binds to HOP over CHIP, promoting a pro-folding, pro-proliferation environment in HEK-293 cancer cells, shown through co-immunoprecipitation and proliferation experiments. Accommodating the phosphorylation on HSP70 through amino acid manipulation of CHIP allows for the preferential binding of CHIP over HOP, increased degradation of downstream proteins such as iNOS, and decreased cancer cell proliferation. Additionally, phosphorylated CHIP also enhances the binding of HSP70 to CHIP over HOP, thus encouraging a pro-degradation environment. Promoting a shift in the PQC system to favor CHIP highlights an opportunity to regulate cancer cell proliferation. M. Stewart is funded by the National Science Foundation Graduate Research Fellowship Program. The Schisler Lab is funded by the McAllister Heart Institute at UNC Chapel Hill and by the NIH.