Ndr/Lats kinases bind specific Mob-family coactivators through a conserved and modular interface

Abstract Ndr/Lats kinases bind Mob coactivator proteins to form complexes that are essential and deeply conserved components of “Hippo” signaling pathways, which control cell proliferation and morphogenesis in eukaryotes. All Ndr/Lats kinases have a characteristic N-terminal region (NTR) that binds a specific Mob co-factor: Lats kinases associate with Mob1 proteins, and Ndr kinases associate with Mob2 proteins. To better understand the functional significance of Mob protein association with Ndr/Lats kinases and selective binding of Ndr and Lats to distinct Mob co-factors, we solved crystal structures of Saccharomyces cerevisiae Cbk1(NTR)-Mob2 and Dbf2(NTR)-Mob1 and experimentally assessed determinants of Mob cofactor binding and specificity. This significantly refines the previously determined structure of Cbk1 kinase bound to Mob2, presently the only crystallographic model of a full length Ndr/Lats kinase complexed with a Mob cofactor. Our analysis indicates that the Ndr/Lats NTR-Mob interface provides a distinctive kinase regulation mechanism, in which Mob co-factor organizes the Ndr/Lats NTR to interact with the AGC kinase C-terminal hydrophobic motif (HM) activation segment. The Mob-organized NTR appears to mediate HM association with an allosteric site on the kinase N-lobe. We also found that Cbk1 and Dbf2 associated highly specifically with Mob2 and Mob1, respectively. Alteration of specific positions in the Cbk1 NTR allows association of non-cognate Mob co-factor, indicating that cofactor specificity is restricted by discrete sites rather than broadly distributed. Overall, our analysis provides a new picture of the functional role of Mob association and indicates that the Ndr/Lats(NTR)-Mob interface overall is largely a common structural platform that mediates kinase-cofactor binding.