MOB1 Mediated Phospho-recognition in the Core Mammalian Hippo Pathway

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. 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. First identified in Drosophila melanogaster, the Hippo signaling pathway controls organ size through the regulation of cell proliferation, apoptosis, and contact inhibition (1.Harvey K.F. Pfleger C.M. Hariharan I.K. The Drosophila Mst ortholog, hippo, restricts growth and cell proliferation and promotes apoptosis.Cell. 2003; 114: 457-467Abstract Full Text Full Text PDF PubMed Scopus (738) Google Scholar, 2.Jia J. Zhang W. Wang B. Trinko R. Jiang J. The Drosophila Ste20 family kinase dMST functions as a tumor suppressor by restricting cell proliferation and promoting apoptosis.Genes Dev. 2003; 17: 2514-2519Crossref PubMed Scopus (321) Google Scholar, 3.Justice R.W. Zilian O. Woods D.F. Noll M. Bryant P.J. The Drosophila tumor suppressor gene warts encodes a homolog of human myotonic dystrophy kinase and is required for the control of cell shape and proliferation.Genes Dev. 1995; 9: 534-546Crossref PubMed Scopus (727) Google Scholar, 4.Pantalacci S. Tapon N. Leopold P. The Salvador partner Hippo promotes apoptosis and cell-cycle exit in Drosophila.Nat. Cell Biol. 2003; 5: 921-927Crossref PubMed Scopus (449) Google Scholar, 5.Udan R.S. Kango-Singh M. Nolo R. Tao C. Halder G. Hippo promotes proliferation arrest and apoptosis in the Salvador/Warts pathway.Nat. Cell Biol. 2003; 5: 914-920Crossref PubMed Scopus (573) Google Scholar, 6.Wu S. Huang J. Dong J. Pan D. hippo encodes a Ste-20 family protein kinase that restricts cell proliferation and promotes apoptosis in conjunction with salvador and warts.Cell. 2003; 114: 445-456Abstract Full Text Full Text PDF PubMed Scopus (829) Google Scholar, 7.Xu T. Wang W. Zhang S. Stewart R.A. Yu W. Identifying tumor suppressors in genetic mosaics: the Drosophila lats gene encodes a putative protein kinase.Development. 1995; 121: 1053-1063Crossref PubMed Google Scholar). The pathway is molecularly and functionally conserved from flies to mammals; however, in mammals, paralogous proteins with apparent redundant functions exist for several of the pathway members. Consistent with a tumor suppressor function, dysregulation of the Hippo pathway is associated with human cancers (reviewed in 8.Harvey K.F. Zhang X. Thomas D.M. The Hippo pathway and human cancer.Nat. Rev. Cancer. 2013; 13: 246-257Crossref PubMed Scopus (1267) Google Scholar, 9.Johnson R. Halder G. The two faces of Hippo: targeting the Hippo pathway for regenerative medicine and cancer treatment.Nat. Rev. Drug Discov. 2014; 13: 63-79Crossref PubMed Scopus (650) Google Scholar) though the core components themselves are infrequently mutated (10.Tate G. Kishimoto K. Mitsuya T. Biallelic alterations of the large tumor suppressor 1 (LATS1) gene in infiltrative, but not superficial, basal cell carcinomas in a Japanese patient with nevoid basal cell carcinoma syndrome.Med. Mol. Morphol. 2015; 48: 177-182Crossref PubMed Scopus (5) Google Scholar, 11.Tapon N. Harvey K.F. Bell D.W. Wahrer D.C. Schiripo T.A. Haber D. Hariharan I.K. salvador Promotes both cell cycle exit and apoptosis in Drosophila and is mutated in human cancer cell lines.Cell. 2002; 110: 467-478Abstract Full Text Full Text PDF PubMed Scopus (665) Google Scholar, 12.Lai Z.C. Wei X. Shimizu T. Ramos E. Rohrbaugh M. Nikolaidis N. Ho L.L. Li Y. Control of cell proliferation and apoptosis by mob as tumor suppressor, mats.Cell. 2005; 120: 675-685Abstract Full Text Full Text PDF PubMed Scopus (441) Google Scholar), possibly because of a redundancy of function arising from the presence of paralogous proteins. The canonical mammalian Hippo pathway consists of a short kinase in which mammalian protein kinase 1 and and MST2 protein kinases 1 and gene and to suppressor kinases 1 and one kinase protein as with associated protein Salvador homolog kinases 1 and and protein kinases 1 and gene and to suppressor kinases 1 and one kinase protein as with associated protein Salvador homolog kinases 1 and and (gene and in phosphorylate and activate the downstream kinases LATS1 and LATS2 in flies 6.Wu S. Huang J. Dong J. Pan D. hippo encodes a Ste-20 family protein kinase that restricts cell proliferation and promotes apoptosis in conjunction with salvador and warts.Cell. 2003; 114: 445-456Abstract Full Text Full Text PDF PubMed Scopus (829) Google Scholar, M. The kinase the human large tumor suppressor kinase 2005; PubMed Scopus Google Scholar). LATS1 and LATS2 phosphorylate the transcriptional YAP1 and TAZ (gene YAP1 and in to their and J. S. J. K. Pan D. The Hippo signaling pathway regulates cell proliferation and apoptosis by the Drosophila of 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). The core kinase is by as and MOB1 by MOB1A and in both of which to Hippo pathway MOB1 directly to LATS1 and LATS2, and to two (gene and (gene a to the kinase R. of of protein kinase by the Biol. Full Text Full Text PDF PubMed Scopus Google by binding their kinase both in and in R. of protein kinase by phosphorylation and the Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, D. The human suppressor LATS1 is by human MOB1 the PubMed Scopus Google Scholar, R.S. D. The MST1 and tumor suppressors control human by regulating kinase Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, Y. R. D. M. M. The of LATS2 kinase regulation in PubMed Scopus Google Scholar). to as a of the and LATS MOB1 is a substrate of MST1 and and phosphorylation the ability of MOB1 to activate LATS1 and LATS2 M. J. phosphorylation by MST1 and MST2 cell Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, Y. M. Pan D. X. for of the core kinase in Hippo Dev. 2015; PubMed Scopus Google Scholar). to function as a Hippo pathway is the ability of MOB1 to directly to MST1 and MST2 key the underlying of that yeast Mob1 in a to the Mitotic Exit Network Nud1 D. R.W. M. of the yeast Hippo pathway by of signaling 2013; PubMed Scopus Google Scholar). identified a short phosphopeptide Nud1 as for for peptide with peptide arrays that yeast Mob1 a for the The for phosphopeptide recognition and in human MOB1 was by a crystal of human MOB1 to The revealed for binding residues and in human MOB1, D. R.W. M. of the yeast Hippo pathway by of signaling 2013; PubMed Scopus Google Scholar). by a the of MOB1 and mammalian Hippo pathway that MOB1 MST1 and MST2 in a G. T. of the mammalian Hippo pathway of 2013; PubMed Scopus Google Scholar) the conserved and by D. R.W. M. of the yeast Hippo pathway by of signaling 2013; PubMed Scopus Google Scholar). on by the crystal of human in complex with a peptide to residues to of MST2. to the generality of the from D. R.W. M. of the yeast Hippo pathway by of signaling 2013; PubMed Scopus Google Scholar, G. T. of the mammalian Hippo pathway of 2013; PubMed Scopus Google Scholar), the revealed a binding in which the of MST2 the of a residues to of MST2 a on of MOB1 function that to be is the of human MOB1 for in The MOB1 sequences in human MST2 and in yeast Nud1 the however, are with the of for yeast Mob1 in a peptide D. R.W. M. of the yeast Hippo pathway by of signaling 2013; PubMed Scopus Google Scholar). the phosphopeptide-binding of MOB1 with the substrate phosphorylation of the upstream MST1 and MST2 targeting the and in for the specificity of sites and by MOB1 in a is the phosphopeptide binding function of MOB1 a of the other human MOB we to the of substrate phosphorylation by MST1 and MST2. peptide binding analyses, and we the required for phosphopeptide binding to the redundancy in a cellular of several phosphorylation sites on MST1 the by Y. M. Pan D. X. for of the core kinase in Hippo Dev. 2015; PubMed Scopus Google Scholar). we demonstrate that all but one of the human MOB proteins the ability to a MST1 thus providing a for to the function of other MOB family members that functions. MST1 kinase to protein MST2 kinase MOB1A and MOB1A and yeast Mob1 in E. as and proteins a MOB and in on and by from the with was from the protein by protein was and by size a and the was MST1 and for and and MST1 and for binding from sequences for and for are in of MOB1A with MST1 and by a of the MOB1A MST peptide by with of protein of with a of and all protein in with of peptide complex and peptide complex with the one was the the W. of in PubMed Scopus Google Scholar, Y. J. of a human Mob1 cell cycle 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). phosphopeptide structures by the MOB1A core as a was in to the of MOB1A K. The for protein Biol. PubMed Scopus Google Scholar). was in P. K. for Biol. 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MST1 and MST2 in their and both and sites are conserved We that the paralogous sequences from MST2 associated with in a We that the and sites are conserved in the Drosophila MST1 and MST2 ortholog, though the the to was to and to human in in a that binding with human as we not the Drosophila MOB1 protein in that the two conserved phosphorylation sites in MST1 are of interactions with MOB1 We to the specificity for binding of peptide arrays and by in the human and peptide revealed a for and of binding for the peptide the and peptide sequences identified binding to the phosphorylation to the phosphorylation to a for the binding we to of the peptide and to of the peptide with all of the binding from the is in in in to the for the a for residues to was core of was on the from peptide to other sequences in the of MST1 and to MOB1A The binding affinities for to the and be by to one in the Consistent with their binding human MOB1 to the MST1 peptide to the peptide yeast and the MST1 peptide sequences that with in peptide arrays to the phosphorylation we for the MST1 and MST2 kinases a cell and R. Wang T. D. R. kinase of the in PubMed Scopus Google Scholar). to the phosphorylation cell in in cell Cell with kinases by to MST1 and MST2 kinase with and the was for the kinase control for and on and by and that in the MST1 MST2 to the kinase identified MST1 and MST2 phosphorylation sites sequences a for the the a for residues in the 1 was the MOB1 phosphopeptide-binding consensus. a of was the binding and MST1 and MST2 kinase the was by the kinase but not for by on we that MST1 a ability to the and which binding sites for the for the binding specificity of MOB1A for phosphopeptide we the crystal structures of human MOB1A to human MST1 and and for a and for and The binding revealed by structures are in with the binding for a yeast Nud1 phosphopeptide in complex with human as as a MST2 phosphopeptide in complex with human Y. M. Pan D. X. for of the core kinase in Hippo Dev. 2015; PubMed Scopus Google Scholar, D. R.W. M. of the yeast Hippo pathway by of signaling 2013; PubMed Scopus Google Scholar). However, we that the in to from the and by the MST1 and to the MOB1A identified by D. R.W. M. of the yeast Hippo pathway by of signaling 2013; PubMed Scopus Google Scholar), with the of the threonine directly by and The for be by the size of the that the The for was because the to However, we that the of the to the for interactions with MOB1A C. Zhang R. M. P. S. of a for kinase 2014; Full Text Full Text PDF PubMed Scopus Google Scholar). The 1 of and on and in by and on The of in the peptide the The of and in and to a by and the of the on the of the 1 to binding the for residues The and which the of specificity the crystal of MOB1 to phosphopeptide from MST2 Y. M. Pan D. X. for of the core kinase in Hippo Dev. 2015; PubMed Scopus Google Scholar). peptide residues the in residues and and was to to Y. M. Pan D. X. for of the core kinase in Hippo Dev. 2015; PubMed Scopus Google Scholar) the MST1 We that the to the was for by the residues to the in MST2 to of residues to which a on from the phosphopeptide binding of we that the peptide in MST1 to the was to to in peptide the of all human MOB proteins of the residues to and in that ability to phosphopeptide sequences of we that all human MOB proteins with the of for the MST1 peptide in with from for the proteins to for binding was of the phosphopeptide binding of MOB protein and it in the of a cellular we with the of the which phosphopeptide binding function in protein as as a in which the residues and mutated to in cellular with by controls of with a for that both MST1 and MST2. The with MST1 and MST2 with was on the residues and that human MOB proteins the to with MST1 and and that by the by D. R.W. M. of the yeast Hippo pathway by of signaling 2013; PubMed Scopus Google Scholar). we to the of multiple phosphorylation sites on MST1 that with MOB1 a of the two favored in sites identified to in to with in a a point to the in to not binding that all with MOB1 in we and sites in in the with to and cell We a with and interactions with MST1 by the MST1 protein with in the MST1 in which all identified binding sites and mutated to a in with the that it was a to the protein we that the MST1 and and a and a in binding in to though not as as the of in a cellular a redundancy of sites the of MOB1 with and that phosphorylation sites in a as the contribute to binding in with peptide sequences that peptide recognition on MOB1 we the the of MST1 and MST2 kinases to MOB1, which in the Hippo pathway by the MST1 and MST2 kinases in to their the LATS family of MST1 and MST2 kinases by upstream on sites in their in of MST1 kinase sites of and Biol. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). we show that the substrate phosphorylation for MST1 and MST2 kinases is with the MOB1 phosphopeptide binding that of MST1 and MST2 sites that are by the phosphopeptide binding of The structures of MOB1A and MST1 and are in full with the binding by D. R.W. M. of the yeast Hippo pathway by of signaling 2013; PubMed Scopus Google Scholar), and by Y. M. Pan D. X. for of the core kinase in Hippo Dev. 2015; PubMed Scopus Google Scholar), in which the and the peptide not identified multiple peptide sites on MST1 that with in a revealed a of residues that with in a phosphorylation of the of Y. M. Pan D. X. for of the core kinase in Hippo Dev. 2015; PubMed Scopus Google Scholar), with the that to a phosphopeptide binding The binding of MOB1 redundancy to the binding of MOB1 to MST1 in phosphopeptide binding sites of is that the redundancy of in MST1 provides a to the of a which be to short Hippo pathway function and the on YAP1 and functions. is that multiple phosphorylation sites in MST1 and MST2 allow multiple MOB1 proteins to MST1 kinase concurrently to the in multiple in protein PubMed Scopus Google which in Hippo pathway signaling to the downstream LATS1 and LATS2 is that the presence of multiple with binding to MOB1, the cell to the of signaling through the Hippo The rules that the of phosphopeptide recognition by MOB1 to of function in a and Lastly, the that the phosphopeptide binding function of MOB1 is to the function of other MOB proteins. We of the seven human MOB all but one of residues the and that with a ability to in show that with MST1 and MST2 in a on not that the other MOB proteins in a but that roles in Hippo pathway of the phosphopeptide binding ability and specificity of MOB family in the signaling functions that in a cellular to the the for The of and for and are in the the for of The associated with the of the MST1 and MST2 the associated and associated with the of by in through partner as a and the and We for with and for for with with

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