Structural and Biochemical Analyses of the Core Components of the Hippo Pathway

  • Lisheng Ni
  • Xuelian LuoEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1893)


The Hippo pathway controls organ size and maintains tissue homeostasis through a central MST–LATS kinase cascade. When Hippo signaling is on, activated MST1/2 partner with SAV1 to phosphorylate and activate the LATS1/2–MOB1 complexes, which in turn phosphorylate and inactivate YAP/TAZ transcription co-activators. This process halts the expression of Hippo-responsive genes, thereby inhibiting cell proliferation and promoting apoptosis. Our studies have shown that two core adaptor proteins MOB1 and SAV1 use distinctive mechanisms to enhance Hippo signaling. MOB1 promotes MST-dependent LATS activation through dynamic scaffolding and allosteric regulation. SAV1 promotes MST activation by antagonizing the PP2A phosphatase activity. Here we describe the detailed methods for the purification and crystallization of the MST2–SAV1 and pMOB1–LATS1 complexes, for assaying the SAV1-dependent inhibition of PP2A, and for analyzing LATS1 kinase activation using in vitro reconstitution.

Key words

Hippo pathway Kinase cascade Protein purification X-ray crystallography Kinase assay In vitro reconstitution Autophosphorylation PP2A 



This work was supported by the National Institutes of Health (GM107415 to XL) and the Robert A. Welch Foundation (I-1932 to XL).


  1. 1.
    Harvey K, Tapon N (2007) The Salvador-Warts-Hippo pathway—an emerging tumour-suppressor network. Nat Rev Cancer 7(3):182–191CrossRefGoogle Scholar
  2. 2.
    Pan D (2010) The hippo signaling pathway in development and cancer. Dev Cell 19(4):491–505. CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Johnson R, Halder G (2014) The two faces of Hippo: targeting the Hippo pathway for regenerative medicine and cancer treatment. Nat Rev Drug Discov 13(1):63–79. CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Yu FX, Zhao B, Guan KL (2015) Hippo pathway in organ size control, tissue homeostasis, and cancer. Cell 163(4):811–828. CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Badouel C, Garg A, McNeill H (2009) Herding Hippos: regulating growth in flies and man. Curr Opin Cell Biol 21(6):837–843CrossRefGoogle Scholar
  6. 6.
    Staley BK, Irvine KD (2012) Hippo signaling in Drosophila: recent advances and insights. Dev Dyn 241(1):3–15. CrossRefPubMedGoogle Scholar
  7. 7.
    Barry ER, Camargo FD (2013) The Hippo superhighway: signaling crossroads converging on the Hippo/Yap pathway in stem cells and development. Curr Opin Cell Biol 25(2):247–253. CrossRefPubMedGoogle Scholar
  8. 8.
    Zhao B, Lei QY, Guan KL (2008) The Hippo-YAP pathway: new connections between regulation of organ size and cancer. Curr Opin Cell Biol 20(6):638–646CrossRefGoogle Scholar
  9. 9.
    Harvey KF, Zhang X, Thomas DM (2013) The Hippo pathway and human cancer. Nat Rev Cancer 13(4):246–257. CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Halder G, Johnson RL (2011) Hippo signaling: growth control and beyond. Development 138(1):9–22. CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Yu FX, Guan KL (2013) The Hippo pathway: regulators and regulations. Genes Dev 27(4):355–371. CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Chan EH, Nousiainen M, Chalamalasetty RB, Schafer A, Nigg EA, Sillje HH (2005) The Ste20-like kinase Mst2 activates the human large tumor suppressor kinase Lats1. Oncogene 24(12):2076–2086. CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Praskova M, Xia F, Avruch J (2008) MOBKL1A/MOBKL1B phosphorylation by MST1 and MST2 inhibits cell proliferation. Curr Biol 18(5):311–321. CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Hansen CG, Moroishi T, Guan KL (2015) YAP and TAZ: a nexus for Hippo signaling and beyond. Trends Cell Biol 25(9):499–513. CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Huang J, Wu S, Barrera J, Matthews K, Pan D (2005) The Hippo signaling pathway coordinately regulates cell proliferation and apoptosis by inactivating Yorkie, the Drosophila Homolog of YAP. Cell 122(3):421–434CrossRefGoogle Scholar
  16. 16.
    Zhao B, Wei X, Li W, Udan RS, Yang Q, Kim J, Xie J, Ikenoue T, Yu J, Li L, Zheng P, Ye K, Chinnaiyan A, Halder G, Lai ZC, Guan KL (2007) Inactivation of YAP oncoprotein by the Hippo pathway is involved in cell contact inhibition and tissue growth control. Genes Dev 21(21):2747–2761CrossRefGoogle Scholar
  17. 17.
    Luo X (2010) Snapshots of a hybrid transcription factor in the Hippo pathway. Protein Cell 1(9):811–819. CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Zhao B, Ye X, Yu J, Li L, Li W, Li S, Yu J, Lin JD, Wang CY, Chinnaiyan AM, Lai ZC, Guan KL (2008) TEAD mediates YAP-dependent gene induction and growth control. Genes Dev 22(14):1962–1971CrossRefGoogle Scholar
  19. 19.
    Avruch J, Zhou D, Fitamant J, Bardeesy N, Mou F, Barrufet LR (2012) Protein kinases of the Hippo pathway: regulation and substrates. Semin Cell Dev Biol 23(7):770–784. CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Jin Y, Dong L, Lu Y, Wu W, Hao Q, Zhou Z, Jiang J, Zhao Y, Zhang L (2012) Dimerization and cytoplasmic localization regulate Hippo kinase signaling activity in organ size control. J Biol Chem 287(8):5784–5796. CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Ni L, Li S, Yu J, Min J, Brautigam CA, Tomchick DR, Pan D, Luo X (2013) Structural basis for autoactivation of human Mst2 kinase and its regulation by RASSF5. Structure 21(10):1757–1768. CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Xiong S, Couzens AL, Kean MJ, Mao DY, Guettler S, Kurinov I, Gingras AC, Sicheri F (2017) Regulation of protein interactions by Mps One binder (MOB1) phosphorylation. Mol Cell Proteomics 16(6):1111–1125. CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Couzens AL, Xiong S, Knight JDR, Mao DY, Guettler S, Picaud S, Kurinov I, Filippakopoulos P, Sicheri F, Gingras AC (2017) MOB1 mediated phospho-recognition in the core mammalian hippo pathway. Mol Cell Proteomics 16(6):1098–1110. CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Cairns L, Tran T, Kavran JM (2017) Structural insights into the regulation of hippo signaling. ACS Chem Biol 12:601. CrossRefPubMedGoogle Scholar
  25. 25.
    Bae SJ, Ni L, Osinski A, Tomchick DR, Brautigam CA, Luo X (2017) SAV1 promotes Hippo kinase activation through antagonizing the PP2A phosphatase STRIPAK. eLife 6:e30278. CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Meng Z, Moroishi T, Guan KL (2016) Mechanisms of Hippo pathway regulation. Genes Dev 30(1):1–17. CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Hoa L, Kulaberoglu Y, Gundogdu R, Cook D, Mavis M, Gomez M, Gomez V, Hergovich A (2016) The characterisation of LATS2 kinase regulation in Hippo-YAP signalling. Cell Signal 28(5):488–497. CrossRefPubMedGoogle Scholar
  28. 28.
    Hergovich A (2016) The roles of NDR protein kinases in hippo signalling. Genes 7(5):21. CrossRefPubMedCentralGoogle Scholar
  29. 29.
    Ni L, Zheng Y, Hara M, Pan D, Luo X (2015) Structural basis for Mob1-dependent activation of the core Mst-Lats kinase cascade in Hippo signaling. Genes Dev 29(13):1416–1431. CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Hergovich A (2012) Mammalian Hippo signalling: a kinase network regulated by protein-protein interactions. Biochem Soc Trans 40(1):124–128. CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Hergovich A (2013) Regulation and functions of mammalian LATS/NDR kinases: looking beyond canonical Hippo signalling. Cell Biosci 3(1):32. CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Kim SY, Tachioka Y, Mori T, Hakoshima T (2016) Structural basis for autoinhibition and its relief of MOB1 in the Hippo pathway. Sci Rep 6:28488. CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Praskova M, Khoklatchev A, Ortiz-Vega S, Avruch J (2004) Regulation of the MST1 kinase by autophosphorylation, by the growth inhibitory proteins, RASSF1 and NORE1, and by Ras. Biochem J 381(Pt 2):453–462. CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Cho US, Xu W (2007) Crystal structure of a protein phosphatase 2A heterotrimeric holoenzyme. Nature 445(7123):53–57. CrossRefPubMedGoogle Scholar

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© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Departments of Pharmacology and BiophysicsUniversity of Texas Southwestern Medical CenterDallasUSA

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