Advertisement

Luciferase Reporter Assays to Determine YAP/TAZ Activity in Mammalian Cells

  • Sirio DupontEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1893)

Abstract

This chapter describes the luciferase assays that are available to monitor YAP/TAZ activity in cell lines and to study their regulation, including the choice for the normalizer, a description of the main YAP-/TAZ-responsive luciferase reporters used so far by the community, and technical notes and experimental considerations on the most appropriate positive controls. Some specific examples are provided to use luciferase assays as the basis to distinguish between Hippo-mediated and phosphorylation-mediated regulatory events and regulatory events that regulate YAP/TAZ independent of these inputs. Finally, typical experimental protocols are outlined briefly for an easier setup of YAP/TAZ luciferase assays.

Key words

Luciferase YAP/TAZ Hippo LATS Phosphorylation Structure-function analysis 

References

  1. 1.
    Vassilev A, Kaneko KJ, Shu H, Zhao Y, DePamphilis ML (2001) TEAD/TEF transcription factors utilize the activation domain of YAP65, a Src/Yes-associated protein localized in the cytoplasm. Genes Dev 15:1229–1241.  https://doi.org/10.1101/gad.888601 CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Dong J, Feldmann G, Huang J, Wu S, Zhang N, Comerford SA et al (2007) Elucidation of a universal size-control mechanism in Drosophila and mammals. Cell 130:1120–1133.  https://doi.org/10.1016/j.cell.2007.07.019 CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Chen L, Chan SW, Zhang X, Walsh M, Lim CJ, Hong W et al (2010) Structural basis of YAP recognition by TEAD4 in the hippo pathway. Genes Dev 24:290–300.  https://doi.org/10.1101/gad.1865310 CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Mesrouze Y, Bokhovchuk F, Meyerhofer M, Fontana P, Zimmermann C, Martin T, et al (2017) Dissection of the interaction between the intrinsically disordered YAP protein and the transcription factor TEAD. eLife 6.  https://doi.org/10.7554/eLife.25068
  5. 5.
    Zanconato F, Forcato M, Battilana G, Azzolin L, Quaranta E, Bodega B et al (2015) Genome-wide association between YAP/TAZ/TEAD and AP-1 at enhancers drives oncogenic growth. Nat Cell Biol 17:1218–1227.  https://doi.org/10.1038/ncb3216 CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Galli GG, Carrara M, Yuan W-C, Valdes-Quezada C, Gurung B, Pepe-Mooney B et al (2015) YAP drives growth by controlling transcriptional pause release from dynamic enhancers. Mol Cell 60(2):328–337.  https://doi.org/10.1016/j.molcel.2015.09.001 CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Stein C, Bardet AF, Roma G, Bergling S, Clay I, Ruchti A et al (2015) YAP1 exerts its transcriptional control via TEAD-mediated activation of enhancers. PLoS Genet 11:e1005465.  https://doi.org/10.1371/journal.pgen.1005465 CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Mohseni M, Sun J, Lau A, Curtis S, Goldsmith J, Fox VL et al (2014) A genetic screen identifies an LKB1-MARK signalling axis controlling the Hippo-YAP pathway. Nat Cell Biol 16:108–117.  https://doi.org/10.1038/ncb2884 CrossRefPubMedGoogle Scholar
  9. 9.
    Sansores-Garcia L, Bossuyt W, Wada K-I, Yonemura S, Tao C, Sasaki H et al (2011) Modulating F-actin organization induces organ growth by affecting the Hippo pathway. EMBO J 30:2325–2335.  https://doi.org/10.1038/emboj.2011.157 CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Liu-Chittenden Y, Huang B, Shim JS, Chen Q, Lee S-J, Anders RA et al (2012) Genetic and pharmacological disruption of the TEAD-YAP complex suppresses the oncogenic activity of YAP. Genes Dev 26:1300–1305.  https://doi.org/10.1101/gad.192856.112 CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Ribeiro PS, Josué F, Wepf A, Wehr MC, Rinner O, Kelly G et al (2010) Combined functional genomic and proteomic approaches identify a PP2A complex as a negative regulator of Hippo signaling. Mol Cell 39:521–534.  https://doi.org/10.1016/j.molcel.2010.08.002 CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Heidary Arash E, Shiban A, Song S, Attisano L (2017) MARK4 inhibits Hippo signaling to promote proliferation and migration of breast cancer cells. EMBO Rep 18:420–436.  https://doi.org/10.15252/embr.201642455 CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Enzo E, Santinon G, Pocaterra A, Aragona M, Bresolin S, Forcato M et al (2015) Aerobic glycolysis tunes YAP/TAZ transcriptional activity. EMBO J 34:1349–1370.  https://doi.org/10.15252/embj.201490379 CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    McLean BG, Lee KS, Simpson PC, Farrance IKG (2003) Basal and alpha1-adrenergic-induced activity of minimal rat betaMHC promoters in cardiac myocytes requires multiple TEF-1 but not NFAT binding sites. J Mol Cell Cardiol 35:461–471CrossRefGoogle Scholar
  15. 15.
    Wang Z, Wu Y, Wang H, Zhang Y, Mei L, Fang X et al (2014) Interplay of mevalonate and Hippo pathways regulates RHAMM transcription via YAP to modulate breast cancer cell motility. Proc Natl Acad Sci 111:E89–E98.  https://doi.org/10.1073/pnas.1319190110 CrossRefPubMedGoogle Scholar
  16. 16.
    Dupont S, Morsut L, Aragona M, Enzo E, Giulitti S, Cordenonsi M et al (2011) Role of YAP/TAZ in mechanotransduction. Nature 474:179–183.  https://doi.org/10.1038/nature10137 CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Mahoney WM, Hong J-H, Yaffe MB, Farrance IKG (2005) The transcriptional co-activator TAZ interacts differentially with transcriptional enhancer factor-1 (TEF-1) family members. Biochem J 388:217–225.  https://doi.org/10.1042/BJ20041434 CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Couzens AL, Knight JDR, Kean MJ, Teo G, Weiss A, Dunham WH et al (2013) Protein interaction network of the mammalian Hippo pathway reveals mechanisms of kinase-phosphatase interactions. Sci Signal 6:rs15–rs15.  https://doi.org/10.1126/scisignal.2004712 CrossRefPubMedGoogle Scholar
  19. 19.
    Zhao B, Ye X, Yu J, Li L, Li W, Li S et al (2008) TEAD mediates YAP-dependent gene induction and growth control. Genes Dev 22:1962–1971.  https://doi.org/10.1101/gad.1664408 CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Kanai F, Marignani PA, Sarbassova D, Yagi R, Hall RA, Donowitz M et al (2000) TAZ: a novel transcriptional co-activator regulated by interactions with 14-3-3 and PDZ domain proteins. EMBO J 19:6778–6791.  https://doi.org/10.1093/emboj/19.24.6778 CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Hong J-H, Hwang ES, McManus MT, Amsterdam A, Tian Y, Kalmukova R et al (2005) TAZ, a transcriptional modulator of mesenchymal stem cell differentiation. Science 309:1074–1078.  https://doi.org/10.1126/science.1110955 CrossRefPubMedGoogle Scholar
  22. 22.
    Murakami M, Nakagawa M, Olson EN, Nakagawa O (2005) A WW domain protein TAZ is a critical coactivator for TBX5, a transcription factor implicated in Holt-Oram syndrome. Proc Natl Acad Sci U S A 102:18034–18039.  https://doi.org/10.1073/pnas.0509109102 CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Zhao B, Wei X, Li W, Udan RS, Yang Q, Kim J et al (2007) Inactivation of YAP oncoprotein by the Hippo pathway is involved in cell contact inhibition and tissue growth control. Genes Dev 21:2747–2761.  https://doi.org/10.1101/gad.1602907 CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Wada K-I, Itoga K, Okano T, Yonemura S, Sasaki H (2011) Hippo pathway regulation by cell morphology and stress fibers. Development 138:3907–3914.  https://doi.org/10.1242/dev.070987 CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Zhao B, Li L, Wang L, Wang C-Y, Yu J, Guan K-L (2012) Cell detachment activates the Hippo pathway via cytoskeleton reorganization to induce anoikis. Genes Dev 26:54–68.  https://doi.org/10.1101/gad.173435.111 CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Kim M, Kim M, Lee S, Kuninaka S, Saya H, Lee H et al (2013) cAMP/PKA signalling reinforces the LATS-YAP pathway to fully suppress YAP in response to actin cytoskeletal changes. EMBO J 32:1543–1555.  https://doi.org/10.1038/emboj.2013.102 CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Yu F-X, Zhang Y, Park HW, Jewell JL, Chen Q, Deng Y et al (2013) Protein kinase A activates the Hippo pathway to modulate cell proliferation and differentiation. Genes Dev 27:1223–1232.  https://doi.org/10.1101/gad.219402.113 CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Iglesias-Bartolome R, Torres D, Marone R, Feng X, Martin D, Simaan M et al (2015) Inactivation of a Gα(s)-PKA tumour suppressor pathway in skin stem cells initiates basal-cell carcinogenesis. Nat Cell Biol 17:793–803.  https://doi.org/10.1038/ncb3164 CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Sorrentino G, Ruggeri N, Specchia V, Cordenonsi M, Mano M, Dupont S et al (2014) Metabolic control of YAP and TAZ by the mevalonate pathway. Nat Cell Biol 16:357–366.  https://doi.org/10.1038/ncb2936 CrossRefPubMedGoogle Scholar
  30. 30.
    Piccolo S, Dupont S, Cordenonsi M (2014) The biology of YAP/TAZ: hippo signaling and beyond. Physiol Rev 94:1287–1312.  https://doi.org/10.1152/physrev.00005.2014 CrossRefPubMedGoogle Scholar
  31. 31.
    Liu C-Y, Zha Z-Y, Zhou X, Zhang H, Huang W, Zhao D et al (2010) The hippo tumor pathway promotes TAZ degradation by phosphorylating a phosphodegron and recruiting the SCF{beta}-TrCP E3 ligase. J Biol Chem 285:37159–37169.  https://doi.org/10.1074/jbc.M110.152942 CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Kwan J, Sczaniecka A, Arash EH, Nguyen L, Chen C-C, Ratkovic S et al (2016) DLG5 connects cell polarity and Hippo signaling protein networks by linking PAR-1 with MST1/2. Genes Dev 30:2696–2709.  https://doi.org/10.1101/gad.284539.116 CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Meng Z, Moroishi T, Mottier-Pavie V, Plouffe SW, Hansen CG, Hong AW et al (2015) MAP4K family kinases act in parallel to MST1/2 to activate LATS1/2 in the Hippo pathway. Nat Commun 6:8357.  https://doi.org/10.1038/ncomms9357 CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Aragona M, Panciera T, Manfrin A, Giulitti S, Michielin F, Elvassore N et al (2013) A mechanical checkpoint controls multicellular growth through YAP/TAZ regulation by actin-processing factors. Cell 154:1047–1059.  https://doi.org/10.1016/j.cell.2013.07.042 CrossRefPubMedPubMedCentralGoogle Scholar
  35. 35.
    Azzolin L, Zanconato F, Bresolin S, Forcato M, Basso G, Bicciato S et al (2012) Role of TAZ as mediator of Wnt signaling. Cell 151:1443–1456.  https://doi.org/10.1016/j.cell.2012.11.027 CrossRefPubMedGoogle Scholar
  36. 36.
    Cordenonsi M, Zanconato F, Azzolin L, Forcato M, Rosato A, Frasson C et al (2011) The Hippo transducer TAZ confers cancer stem cell-related traits on breast cancer cells. Cell 147:759–772.  https://doi.org/10.1016/j.cell.2011.09.048 CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

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

Authors and Affiliations

  1. 1.Department of Molecular MedicineSchool of Medicine, University of PadovaPadovaItaly

Personalised recommendations