Abstract A27: Increasing proximity triggers Mst2 autophosphorylation

Abstract

Abstract The Hippo pathway limits cell number and differentiation by blocking the transcriptional activity of YAP. The fidelity of signal transduction relies on the coordinated activity of a core kinase cassette that includes the kinases Mst1/2 and Lats1/2 and the accessory proteins Mob1 and hSalvador. Mst1/2 contains a kinase domain, linker region, and a C-terminal coiled-coil domain termed SARAH and is responsible for the phosphorylation of downstream components. Activation of Mst1/2 requires phosphorylation of the activation loop, a process believed to be a consequence of trans-autophosphorylation that is promoted by SARAH-domain mediated homodimerization. We aimed to understand what mechanistically triggers Mst1/2 autophosphorylation. Using purified proteins, we show that the kinase domain of Mst2 is sufficient for autophosphorylation but full-length Mst2 undergoes autophosphorylation faster than variants lacking the SARAH domain. This increased autophosphorylation could arise from either a specific contribution of a SARAH-domain mediated homodimer (allostery) or the increased proximity of kinase domains following homodimerization (effective local concentration). We investigated both possibilities using a series of biochemical assays and revealed that autophosphorylation of Mst2 variants lacking a SARAH domain could be stimulated by either chemically induced dimerization or increased localization following recruitment to the surface of a lipid-vesicle. Our results suggest that SARAH-domain mediated homodimerization is not the only event that promotes autophosphorylation of Mst1/2 and, instead, supports a more inclusive model that relies on colocalization. Any cellular event that increases the proximity of Mst1/2 could activate the kinase such as SARAH-domain mediated homodimerization, higher-order complex formation with hSalvador, or membrane recruitment. Citation Format: Thao Tran, Jennifer M. Kavran. Increasing proximity triggers Mst2 autophosphorylation [abstract]. In: Proceedings of the AACR Special Conference on the Hippo Pathway: Signaling, Cancer, and Beyond; 2019 May 8-11; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(8_Suppl):Abstract nr A27.