title: MOB1 Mediated Phospho-recognition in the Core Mammalian Hippo Pathway.
abstract: The Hippo tumor suppressor pathway regulates organ size and tissue homoeostasis in response to diverse signaling inputs. The core of the pathway consists of a short kinase cascade: MST1 and MST2 phosphorylate and activate LATS1 and LATS2, which in turn phosphorylate and inactivate key transcriptional coactivators, YAP1 and TAZ (gene WWTR1). The MOB1 adapter protein regulates both phosphorylation reactions firstly by concurrently binding to the upstream MST and downstream LATS kinases to enable the trans phosphorylation reaction, and secondly by allosterically activating the catalytic function of LATS1 and LATS2 to directly stimulate phosphorylation of YAP and TAZ. Studies of yeast Mob1 and human MOB1 revealed that the ability to recognize phosphopeptide sequences in their interactors, Nud1 and MST2 respectively, was critical to their roles in regulating the Mitotic Exit Network in yeast and the Hippo pathway in metazoans. However, the underlying rules of phosphopeptide recognition by human MOB1, the implications of binding specificity for Hippo pathway signaling, and the generality of phosphopeptide binding function to other human MOB family members remained elusive.Employing proteomics, peptide arrays and biochemical analyses, we systematically examine the phosphopeptide binding specificity of MOB1 and find it to be highly complementary to the substrate phosphorylation specificity of MST1 and MST2. We demonstrate that autophosphorylation of MST1 and MST2 on several threonine residues provides multiple MOB1 binding sites with varying binding affinities which in turn contribute to a redundancy of MST1-MOB1 protein interactions in cells. The crystal structures of MOB1A in complex with two favored phosphopeptide sites in MST1 allow for a full description of the MOB1A phosphopeptide-binding consensus. Lastly, we show that the phosphopeptide binding properties of MOB1A are conserved in all but one of the seven MOB family members in humans, thus providing a starting point for uncovering their elusive cellular functions.
authors.affiliation: From the ‡Lunenfeld-Tanenbaum Research Institute, Sinai Health System, Toronto, Ontario, Canada, M5G 1X5.
authors.forename: Amber L
authors.identifier: 0000-0003-0057-6686
authors.initials: AL
authors.lastname: Couzens
authors.affiliation: From the ‡Lunenfeld-Tanenbaum Research Institute, Sinai Health System, Toronto, Ontario, Canada, M5G 1X5.|§Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada, M5S 1A8.
authors.forename: Shawn
authors.initials: S
authors.lastname: Xiong
authors.affiliation: From the ‡Lunenfeld-Tanenbaum Research Institute, Sinai Health System, Toronto, Ontario, Canada, M5G 1X5.
authors.forename: James D R
authors.initials: JDR
authors.lastname: Knight
authors.affiliation: From the ‡Lunenfeld-Tanenbaum Research Institute, Sinai Health System, Toronto, Ontario, Canada, M5G 1X5.
authors.forename: Daniel Y
authors.initials: DY
authors.lastname: Mao
authors.affiliation: From the ‡Lunenfeld-Tanenbaum Research Institute, Sinai Health System, Toronto, Ontario, Canada, M5G 1X5.|¶New address: The Institute of Cancer Research, Divisions of Structural Biology and Cancer Biology, London, UK, SW7 3RP.
authors.forename: Sebastian
authors.initials: S
authors.lastname: Guettler
authors.affiliation: ‖Structural Genomics Consortium, University of Oxford, Old Road Campus Research Building, Roosevelt Drive, Oxford OX3 7DQ, U.K.
authors.forename: Sarah
authors.initials: S
authors.lastname: Picaud
authors.affiliation: **NE-CAT APS, Building 436E, Argonne National Lab, 9700 S. Cass Avenue, Argonne, Illinois 60439.
authors.forename: Igor
authors.initials: I
authors.lastname: Kurinov
authors.affiliation: ‖Structural Genomics Consortium, University of Oxford, Old Road Campus Research Building, Roosevelt Drive, Oxford OX3 7DQ, U.K.|‡‡Ludwig Institute for Cancer Research, University of Oxford, Oxford OX3 7DQ, U.K.
authors.forename: Panagis
authors.initials: P
authors.lastname: Filippakopoulos
authors.affiliation: From the ‡Lunenfeld-Tanenbaum Research Institute, Sinai Health System, Toronto, Ontario, Canada, M5G 1X5, sicheri@lunenfeld.ca gingras@lunenfeld.ca.|§Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada, M5S 1A8.|§§Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada, M5S 1A8.
authors.forename: Frank
authors.initials: F
authors.lastname: Sicheri
authors.affiliation: From the ‡Lunenfeld-Tanenbaum Research Institute, Sinai Health System, Toronto, Ontario, Canada, M5G 1X5, sicheri@lunenfeld.ca gingras@lunenfeld.ca.|§§Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada, M5S 1A8.
authors.forename: Anne-Claude
authors.identifier: 0000-0002-6090-4437
authors.initials: AC
authors.lastname: Gingras
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D010748:Phosphopeptides / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D000090742:Serine-Threonine Kinase 3; D015398:Signal Transduction; D000099317:Hippo Kinases
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D047908:Intracellular Signaling Peptides and Proteins; D010748:Phosphopeptides; D017346:Protein Serine-Threonine Kinases; D011994:Recombinant Proteins; D000090742:Serine-Threonine Kinase 3; C496255:MOB1B protein, human; C095566:STK4 protein, human; D000099317:Hippo Kinases; C097835:STK3 protein, human