THE MOLECULAR ROLE OF MOB1 IN LATS1 KINASE ACTIVATION IN HIPPO SIGNALING

The Hippo signaling pathway, a highly conserved pathway between flies and mammals, regulates the rate of cell growth and apoptosis to ultimately control organ size and tissue homeostasis (Saucedo & Edgar, 2007). The pathway was named after an overgrowth phenotype, a fly resembling a hippopotamus, observed in Drosophila upon mutating Hpo (Wu et al., 2003). The action of the Hippo pathway arises from the activity of a set of two kinases. Mst1/2 kinase phosphorylates and activates Lats1/2 kinase in a process that may be stimulated by the effector protein Mob1, which is also a substrate of Mst1/2 (Ni et al., 2015). This study aims to identify a minimal, functional unit of Lats1 more amenable to both purification in vitro and X-ray crystallization. We designed truncations of both the N and C-termini of Lats1. We transiently transfected Mst2, Mob1, Lats1, and YAP in HEK293 cells and validated kinase activity of the truncated Lats1 variants by Western blot and the luciferase reporter assay. We also analyzed complex formation between Mob1 and the truncated Lats1 variants using co-immunoprecipitation from lysate of HEK293 cells transiently transfected with both proteins. We will purify the minimal, functional unit of Lats1 in vitro for use in future biochemical and structural assays. This study also aims to understand the molecular role of Mob1 in the activation of Lats1 kinase. We transiently transfected HEK293 cells with Mst2 and Lats1 with or without Mob1 and monitored Lats1 activation by Western blot. Expression of Mob1 stimulated phosphorylation of Lats1 by Mst2, highlighting a potential role for Mob1 in Lats1 activation and ternary complex formation between Lats1, Mob1, and Mst2. We aim to isolate the ternary complex in vitro and test the phosphospecific nature of assembly of the ternary complex. We will also determine the sequence of molecular events that lead to Lats1 activation by systematically disrupting pairwise interactions between either Mst2 and Mob1 or Lats1 and Mob1 and monitor the effect on Lats1 activation in cells. Several studies have identified mutations in human cancers that affect Lats1/2 expression and kinase activity (Yu, Bachman & Lai, 2013). Thus, the results of this study will provide insight into the mechanisms that regulate Lats1 activation and provide a framework to guide targeted molecular therapies