Abstract A023: Structural basis for regulation of MAPK signaling by DUSP5 and DUSP6

Abstract The Ras/mitogen-activated protein kinase (MAPK) cascade plays an essential role in several critical cellular processes, such as cell growth and proliferation, and hyperactivation of this pathway is frequently observed in cancer. DUSP5 and DUSP6 are dual specificity phosphatases (capable of dephosphorylating phospho-tyrosines as well as phospho-serines/threonines) that act as key regulators of MAPK signaling that inactivate ERK by dephosphorylating its activation loop and sequestering ERK in an inactive state. DUSP5 and DUSP6 have been reported to function as tumor suppressors in some Ras-driven cancers, including skin cancer and pancreatic cancer, while promoting tumorigenesis in other contexts, such as thyroid cancer or glioblastoma. Despite the importance of these phosphatases in modulating ERK signaling, the structural basis for their interaction with ERK and their dual specificity remain poorly understood. It has also been reported that ERK binding allosterically activates DUSP6, but not DUSP5, and the mechanism underlying this is unclear. While structures of individual domains of these proteins have been solved, there are no structures of full-length DUSP5 or DUSP6, or of an ERK/DUSP complex. To gain insight into the molecular determinants of ERK dephosphorylation by these phosphatases, we have purified full-length DUSP5 and DUSP6, as well as the phosphatase domains of both proteins. We have also generated full-length phosphorylated ERK2 (pERK2). Through in vitro dephosphorylation assays, we have observed that full-length DUSP5 and DUSP6 dephosphorylate ERK2 much more rapidly than the isolated phosphatase domains, indicating an essential role for their N-terminal regulatory domains in this process. Using biolayer interferometry, we show that catalytically inactive mutants of full-length DUSP5 and DUSP6 bind very tightly to pERK2 with low nanomolar affinity. We have reconstituted complexes of pERK2 bound to catalytically inactive full-length DUSP5 or DUSP6, and we are pursuing structures of these complexes using x-ray crystallography and cryo-electron microscopy. These studies will improve our understanding of the regulation of ERK and its phosphatases DUSP5 and DUSP6 and shed light on the similarities and differences between these complexes. These structures could also potentially reveal the structural features underlying the dual specificity of DUSP5 and DUSP6. Citation Format: Jennifer E. Kung, Jawahar Sudhamsu. Structural basis for regulation of MAPK signaling by DUSP5 and DUSP6 [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr A023.