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

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 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 Cbk1NTR-Mob2 and Dbf2NTR-Mob1 and experimentally assessed determinants of Mob cofactor binding and specificity. This allowed significant refinement of 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/LatsNTR-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 specifically with Mob2 and Mob1, respectively. Alteration of residues 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/LatsNTR-Mob interface is largely a common structural platform that mediates kinase-cofactor binding.