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Riquiqui and Minibrain are regulators of the Hippo pathway downstream of Dachsous

Nature Cell Biology volume 15, pages 1176–1185 (2013)Cite this article

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Abstract

The atypical cadherins Fat (Ft) and Dachsous (Ds) control tissue growth through the Salvador–Warts–Hippo (SWH) pathway, and also regulate planar cell polarity and morphogenesis. Ft and Ds engage in reciprocal signalling as both proteins can serve as receptor and ligand for each other. The intracellular domains (ICDs) of Ft and Ds regulate the activity of the key SWH pathway transcriptional co-activator protein Yorkie (Yki). Signalling from the FtICD is well characterized and controls tissue growth by regulating the abundance of the Yki-repressive kinase Warts (Wts). Here we identify two regulators of the Drosophila melanogaster SWH pathway that function downstream of the DsICD: the WD40 repeat protein Riquiqui (Riq) and the DYRK-family kinase Minibrain (Mnb). Ds physically interacts with Riq, which binds to both Mnb and Wts. Riq and Mnb promote Yki-dependent tissue growth by stimulating phosphorylation-dependent inhibition of Wts. Thus, we describe a previously unknown branch of the SWH pathway that controls tissue growth downstream of Ds.

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Figure 1: Riq forms a physical complex with Ds and controls tissue growth.
Figure 2: Mnb physically interacts with Riq and phenocopies its effect on tissue growth.
Figure 3: Riq and Mnb regulate SWH pathway activity.
Figure 4: Riq and Mnb physically interact with Wts.
Figure 5: Mnb phosphorylates Wts and inhibits its activity.
Figure 6: Riq and Mnb function downstream of Ds to promote Yki activity.
Figure 7: Model of signalling from Ds and Ft to the SWH pathway.

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References

  1. Harvey, K. & Tapon, N. The Salvador–Warts–Hippo pathway—an emerging tumour-suppressor network. Nat. Rev. Cancer 7, 182–191 (2007).

    Article  CAS  PubMed  Google Scholar 

  2. Badouel, C., Garg, A. & McNeill, H. Herding Hippos: regulating growth in flies and man. Curr. Opin. Cell Biol. 21, 837–843 (2009).

    Article  CAS  PubMed  Google Scholar 

  3. Halder, G. & Johnson, R. L. Hippo signaling: growth control and beyond. Development 138, 9–22 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Reddy, B. V. & Irvine, K. D. The Fat and Warts signaling pathways: new insights into their regulation, mechanism and conservation. Development 135, 2827–2838 (2008).

    Article  CAS  PubMed  Google Scholar 

  5. Harvey, K. F., Zhang, X. & Thomas, D. M. The Hippo pathway and human cancer. Nat. Rev. Cancer 13, 246–257 (2013).

    Article  CAS  PubMed  Google Scholar 

  6. Bennett, F. C. & Harvey, K. F. Fat cadherin modulates organ size in Drosophilavia the Salvador/Warts/Hippo signaling pathway. Curr. Biol. 16, 2101–2110 (2006).

    Article  CAS  PubMed  Google Scholar 

  7. Willecke, M. et al. The fat cadherin acts through the hippo tumor-suppressor pathway to regulate tissue size. Curr. Biol. 16, 2090–2100 (2006).

    Article  CAS  PubMed  Google Scholar 

  8. Silva, E., Tsatskis, Y., Gardano, L., Tapon, N. & McNeill, H. The tumor-suppressor gene fat controls tissue growth upstream of expanded in the hippo signaling pathway. Curr. Biol. 16, 2081–2089 (2006).

    Article  CAS  PubMed  Google Scholar 

  9. Tyler, D. M. & Baker, N. E. Expanded and fat regulate growth and differentiation in the Drosophila eye through multiple signaling pathways. Dev. Biol. 305, 187–201 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Cho, E. et al. Delineation of a fat tumor suppressor pathway. Nat. Genet. 38, 1142–1150 (2006).

    Article  CAS  PubMed  Google Scholar 

  11. Willecke, M., Hamaratoglu, F., Sansores-Garcia, L., Tao, C. & Halder, G. Boundaries of Dachsous cadherin activity modulate the Hippo signaling pathway to induce cell proliferation. Proc. Natl Acad. Sci. USA 105, 14897–14902 (2008).

    Article  CAS  PubMed  Google Scholar 

  12. Rogulja, D., Rauskolb, C. & Irvine, K. D. Morphogen control of wing growth through the Fat signaling pathway. Dev. Cell 15, 309–321 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Sopko, R. & McNeill, H. The skinny on fat: an enormous cadherin that regulates cell adhesion, tissue growth, and planar cell polarity. Curr. Opin. Cell Biol. 21, 717–723 (2009).

    Article  CAS  PubMed  Google Scholar 

  14. Bosveld, F. et al. Mechanical control of morphogenesis by Fat/Dachsous/Four-jointed planar cell polarity pathway. Science 336, 724–727 (2012).

    Article  CAS  PubMed  Google Scholar 

  15. Lawrence, P. A., Struhl, G. & Casal, J. Do the protocadherins Fat and Dachsous link up to determine both planar cell polarity and the dimensions of organs? Nat. Cell Biol. 10, 1379–1382 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Clark, H. F. et al. Dachsous encodes a member of the cadherin superfamily that controls imaginal disc morphogenesis in Drosophila. Genes Dev. 9, 1530–1542 (1995).

    Article  CAS  PubMed  Google Scholar 

  17. Casal, J., Lawrence, P. A. & Struhl, G. Two separate molecular systems, Dachsous/Fat and starry night/frizzled, act independently to confer planar cell polarity. Development 133, 4561–4572 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Matakatsu, H. & Blair, S. S. Separating planar cell polarity and Hippo pathway activities of the protocadherins Fat and Dachsous. Development 139, 1498–1508 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Thomas, C. & Strutt, D. The roles of the cadherins Fat and Dachsous in planar polarity specification in Drosophila. Dev. Dyn. 241, 27–39 (2012).

    Article  CAS  PubMed  Google Scholar 

  20. Brittle, A., Thomas, C. & Strutt, D. Planar polarity specification through asymmetric subcellular localization of Fat and Dachsous. Curr. Biol. 22, 907–914 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Grusche, F. A., Richardson, H. E. & Harvey, K. F. Upstream regulation of the hippo size control pathway. Curr. Biol. 20, R574–R582 (2010).

    Article  CAS  PubMed  Google Scholar 

  22. Ishikawa, H. O., Takeuchi, H., Haltiwanger, R. S. & Irvine, K. D. Four-jointed is a Golgi kinase that phosphorylates a subset of cadherin domains. Science 321, 401–404 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Simon, M. A., Xu, A., Ishikawa, H. O. & Irvine, K. D. Modulation of fat: Dachsous binding by the cadherin domain kinase four-jointed. Curr. Biol. 20, 811–817 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Brittle, A. L., Repiso, A., Casal, J., Lawrence, P. A. & Strutt, D. Four-jointed modulates growth and planar polarity by reducing the affinity of dachsous for fat. Curr. Biol. 20, 803–810 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Rauskolb, C., Pan, G., Reddy, B. V., Oh, H. & Irvine, K. D. Zyxin links fat signaling to the hippo pathway. PLoS Biol. 9, e1000624 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Matakatsu, H. & Blair, S. S. The DHHC palmitoyltransferase approximated regulates Fat signaling and Dachs localization and activity. Curr. Biol. 18, 1390–1395 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Huang, J., Wu, S., Barrera, J., Matthews, K. & Pan, D. The Hippo signaling pathway coordinately regulates cell proliferation and apoptosis by inactivating Yorkie, the Drosophila homolog of YAP. Cell 122, 421–434 (2005).

    Article  CAS  PubMed  Google Scholar 

  28. Kyriakakis, P., Tipping, M., Abed, L. & Veraksa, A. Tandem affinity purification in Drosophila: the advantages of the GS-TAP system. Fly (Austin) 2, 229–235 (2008).

    Article  Google Scholar 

  29. Nissen, R. M., Amsterdam, A. & Hopkins, N. A zebrafish screen for craniofacial mutants identifies wdr68 as a highly conserved gene required for endothelin-1 expression. BMC Dev. Biol. 6, 28 (2006).

    Article  PubMed  PubMed Central  Google Scholar 

  30. Skurat, A. V. & Dietrich, A. D. Phosphorylation of Ser640 in muscle glycogen synthase by DYRK family protein kinases. J. Biol. Chem. 279, 2490–2498 (2004).

    Article  CAS  PubMed  Google Scholar 

  31. Tejedor, F. et al. minibrain: a new protein kinase family involved in postembryonic neurogenesis in Drosophila. Neuron 14, 287–301 (1995).

    Article  CAS  PubMed  Google Scholar 

  32. Ritterhoff, S. et al. The WD40-repeat protein Han11 functions as a scaffold protein to control HIPK2 and MEKK1 kinase functions. EMBO J. 29, 3750–3761 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Poon, C. L., Zhang, X., Lin, J. I., Manning, S. A. & Harvey, K. F. Homeodomain-interacting protein kinase regulates hippo pathway-dependent tissue growth. Curr. Biol. 22, 1587–1594 (2012).

    Article  CAS  PubMed  Google Scholar 

  34. Chen, J. & Verheyen, E. M. Homeodomain-interacting protein kinase regulates yorkie activity to promote tissue growth. Curr. Biol. 22, 1582–1586 (2012).

    Article  CAS  PubMed  Google Scholar 

  35. Himpel, S. et al. Specificity determinants of substrate recognition by the protein kinase DYRK1A. J. Biol. Chem. 275, 2431–2438 (2000).

    Article  CAS  PubMed  Google Scholar 

  36. Daub, H. et al. Kinase-selective enrichment enables quantitative phosphoproteomics of the kinome across the cell cycle. Mol. Cell 31, 438–448 (2008).

    Article  CAS  PubMed  Google Scholar 

  37. Dephoure, N. et al. A quantitative atlas of mitotic phosphorylation. Proc. Natl Acad. Sci. USA 105, 10762–10767 (2008).

    Article  CAS  PubMed  Google Scholar 

  38. Bodenmiller, B. et al. PhosphoPep–a phosphoproteome resource for systems biology research in Drosophila Kc167 cells. Mol. Syst. Biol. 3, 139 (2007).

    Article  PubMed  PubMed Central  Google Scholar 

  39. Oh, H. & Irvine, K. D. In vivo regulation of Yorkie phosphorylation and localization. Development 135, 1081–1088 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Dong, J. et al. Elucidation of a universal size-control mechanism in Drosophila and mammals. Cell 130, 1120–1133 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Matakatsu, H. & Blair, S. S. Separating the adhesive and signaling functions of the Fat and Dachsous protocadherins. Development 133, 2315–2324 (2006).

    Article  CAS  PubMed  Google Scholar 

  42. Mao, Y. et al. Planar polarization of the atypical myosin Dachs orients cell divisions in Drosophila. Genes Dev. 25, 131–136 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Hamaratoglu, F. et al. The tumour-suppressor genes NF2/Merlin and Expanded act through Hippo signalling to regulate cell proliferation and apoptosis. Nat. Cell Biol. 8, 27–36 (2006).

    Article  CAS  PubMed  Google Scholar 

  44. Genevet, A., Wehr, M. C., Brain, R., Thompson, B. J. & Tapon, N. Kibra is a regulator of the Salvador/Warts/Hippo signaling network. Dev. Cell 18, 300–308 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Baumgartner, R., Poernbacher, I., Buser, N., Hafen, E. & Stocker, H. The WW domain protein kibra acts upstream of Hippo in Drosophila. Dev. Cell 18, 309–316 (2010).

    Article  CAS  PubMed  Google Scholar 

  46. Yu, J. et al. Kibra functions as a tumor suppressor protein that regulates Hippo signaling in conjunction with Merlin and expanded. Dev. Cell 18, 288–299 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Poon, C. L., Lin, J. I., Zhang, X. & Harvey, K. F. The Sterile 20-like Kinase Tao-1 controls tissue growth by regulating the Salvador–Warts–Hippo pathway. Dev. Cell 21, 896–906 (2011).

    Article  CAS  PubMed  Google Scholar 

  48. Boggiano, J. C., Vanderzalm, P. J. & Fehon, R. G. Tao-1 phosphorylates Hippo/MST kinases to regulate the Hippo-Salvador-Warts tumor suppressor pathway. Dev. Cell 21, 888–895 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Grzeschik, N. A., Parsons, L. M., Allott, M. L., Harvey, K. F. & Richardson, H. E. Lgl, aPKC, and Crumbs regulate the Salvador/Warts/Hippo pathway through two distinct mechanisms. Curr. Biol. 20, 573–581 (2010).

    Article  CAS  PubMed  Google Scholar 

  50. Robinson, B. S., Huang, J., Hong, Y. & Moberg, K. H. Crumbs regulates Salvador/Warts/Hippo signaling in Drosophila via the FERM-domain protein expanded. Curr. Biol. 20, 582–590 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Ling, C. et al. The apical transmembrane protein Crumbs functions as a tumor suppressor that regulates Hippo signaling by binding to expanded. Proc. Natl Acad. Sci. USA 107, 10532–10537 (2010).

    Article  CAS  PubMed  Google Scholar 

  52. Chen, C. L. et al. The apical-basal cell polarity determinant Crumbs regulates Hippo signaling in Drosophila. Proc. Natl Acad. Sci. USA 107, 15810–15815 (2010).

    Article  CAS  PubMed  Google Scholar 

  53. Tschop, K. et al. A kinase shRNA screen links LATS2 and the pRB tumor suppressor. Genes Dev. 25, 814–830 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Baek, K. H. et al. Down’s syndrome suppression of tumour growth and the role of the calcineurin inhibitor DSCR1. Nature 459, 1126–1130 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Litovchick, L., Florens, L. A., Swanson, S. K., Washburn, M. P. & DeCaprio, J. A. DYRK1A protein kinase promotes quiescence and senescence through DREAM complex assembly. Genes Dev. 25, 801–813 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Ionescu, A. et al. DYRK1A kinase inhibitors with emphasis on cancer. Mini Rev. Med. Chem. 12, 1315–1329 (2012).

    CAS  PubMed  Google Scholar 

  57. Malinge, S. et al. Increased dosage of the chromosome 21 ortholog Dyrk1a promotes megakaryoblastic leukemia in a murine model of Down syndrome. J. Clin. Invest. 122, 948–962 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Gao, J. et al. Mirk/Dyrk1B mediates G0/G1 to S phase cell cycle progression and cell survival involving MAPK/ERK signaling in human cancer cells. Cancer Cell Int. 13, 2 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Davis, S. J. et al. Functional analysis of genes in regions commonly amplified in high-grade serous and endometrioid ovarian cancer. Clin. Cancer Res. 19, 1411–1421 (2013).

    Article  CAS  PubMed  Google Scholar 

  60. Fotaki, V. et al. Dyrk1A haploinsufficiency affects viability and causes developmental delay and abnormal brain morphology in mice. Mol. Cell Biol. 22, 6636–6647 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Moller, R. S. et al. Truncation of the Down syndrome candidate gene DYRK1A in two unrelated patients with microcephaly. Am. J. Hum. Gen. 82, 1165–1170 (2008).

    Article  Google Scholar 

  62. van Bon, B. W. et al. Intragenic deletion in DYRK1A leads to mental retardation and primary microcephaly. Clin. Genet. 79, 296–299 (2011).

    Article  CAS  PubMed  Google Scholar 

  63. Courcet, J. B. et al. The DYRK1A gene is a cause of syndromic intellectual disability with severe microcephaly and epilepsy. J. Med. Genet. 49, 731–736 (2012).

    Article  CAS  PubMed  Google Scholar 

  64. Milton, C. C., Zhang, X., Albanese, N. O. & Harvey, K. F. Differential requirement of Salvador–Warts–Hippo pathway members for organ size control in Drosophila melanogaster. Development 137, 735–743 (2010).

    Article  CAS  PubMed  Google Scholar 

  65. Harvey, K. F., Pfleger, C. M. & Hariharan, I. K. The Drosophila Mst ortholog, hippo, restricts growth and cell proliferation and promotes apoptosis. Cell 114, 457–467 (2003).

    Article  CAS  PubMed  Google Scholar 

  66. Chiu, J. C., Ko, H. W. & Edery, I. NEMO/NLK phosphorylates PERIOD to initiate a time-delay phosphorylation circuit that sets circadian clock speed. Cell 145, 357–370 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  67. Pantalacci, S., Tapon, N. & Leopold, P. The Salvador partner Hippo promotes apoptosis and cell-cycle exit in Drosophila. Nat. Cell Biol. 5, 921–927 (2003).

    Article  CAS  PubMed  Google Scholar 

  68. Callus, B. A., Verhagen, A. M. & Vaux, D. L. Association of mammalian sterile twenty kinases, Mst1 and Mst2, with hSalvador via C-terminal coiled-coil domains, leads to its stabilization and phosphorylation. FEBS J. 273, 4264–4276 (2006).

    Article  CAS  PubMed  Google Scholar 

  69. Harvey, K. F. et al. FOXO-regulated transcription restricts overgrowth of Tsc mutant organs. J. Cell Biol. 180, 691–696 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We thank C. House for assistance with mass spectrometry, J. Lin and K. Hannan for expertise with kinase assays and S. Blair (University of Madison, Wisconsin, USA), I. Edery (Rutgers University, USA), C. House (Peter MacCallum Cancer Centre, Australia), K. Irvine (Rutgers University, USA), D. Pan (Johns Hopkins University, USA), M. Simon (Stanford University, USA), D. Strutt, University of Sheffield, UK), N. Tapon (Cancer Research UK, UK), F. Tejedor (Universidad Miguel Hernandez, Spain), K. Yu (Korea Research Institute of Bioscience and Biotechnology), the Developmental Studies Hybridoma Bank, the Vienna Drosophila RNAi Center, the Australian Drosophila Research Support Facility (www.ozdros.com), the National Institute of Genetics and the Bloomington Stock Centre for fly stocks, plasmids and antibodies. K.F.H. is a Sylvia and Charles Viertel Senior Medical Research Fellow. This research was supported by a Project Grant from the National Health and Medical Research Council of Australia, and by NIH grants GM097727 and CA156734 and NSF grant 0640700 to A.V. Mass spectrometry was performed at the Taplin Facility, Harvard Medical School, USA and Bio21, University of Melbourne, Australia.

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Authors and Affiliations

  1. Cell Growth and Proliferation Laboratory, Peter MacCallum Cancer Centre, 7 St Andrews Place, East Melbourne, Victoria 3002, Australia

    Joffrey L. Degoutin, Claire C. Milton, Eefang Yu & Kieran F. Harvey

  2. Sir Peter MacCallum Department of Oncology, University of Melbourne, Parkville, Victoria 3010, Australia

    Joffrey L. Degoutin, Claire C. Milton, Eefang Yu & Kieran F. Harvey

  3. Department of Pathology, University of Melbourne, Parkville, Victoria 3010, Australia

    Claire C. Milton & Kieran F. Harvey

  4. Department of Biology, University of Massachusetts Boston, Boston, Massachusetts 02125, USA

    Marla Tipping, Liu Yang & Alexey Veraksa

  5. Division and Morphogenesis Team, Polarity, Institut Curie, CNRS UMR 3215, INSERM U934, 26 Rue d’Ulm, 75248 Paris Cedex 05, France

    Floris Bosveld & Yohanns Bellaiche

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Contributions

J.L.D. and C.C.M. performed Drosophila genetic experiments. J.L.D. and E.Y. carried out biochemistry and molecular biology experiments. M.T., L.Y. and A.V. performed affinity purification and mass spectrometry. F.B. and Y.B. analysed Dachs localization in the pupal notum. J.L.D. and K.F.H. designed experiments, analysed data and wrote the manuscript.

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Correspondence to Kieran F. Harvey.

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Integrated supplementary information

Supplementary Figure 1 Dachsous promotes localization of Riquiqui to apical junctions and cell membranes.

(a-c”) X-Z cross-sections of D. melanogaster third instar larval wing imaginal discs expressing the following elements: (a, a’, a”) rn-Gal4, UAS-riq; (b, b’, b”) rn-Gal4, UAS-riq and UAS-ds; (c, c’, c”) rn-Gal4, UAS-riq and UAS-dsRi. Riq is in white, E-cadherin marks the adherens junction in green. Arrows indicate Riq protein that is present at apical junctions in Ds-expressing tissues. Magnified images are inset in (a) (a’) (b) and (b’). Merged images are on the right. Riq was junctionally localised immediately apical to E cadherin. Riq was largely absent from apical junctions of tissues where Ds was depleted by RNAi. (d and e) D. melanogaster S2 cells expressing either Riq (d) or Riq and DsΔECD (e). Riq is in red, DsΔECD is green, Direct interference contrast (DIC) images are grey. Arrows indicate Riq protein that is present at cell membranes in DsΔECD-expressing cells. Scale bar represents 10 μm in (a-c”) and 5 μm in (d) and (e).

Supplementary Figure 2 UAS-riq RNAi transgenes decrease Riq expression.

(a) A wing imaginal disc from a third instar D. melanogaster larva expressing UAS-riqi1 under the control of hh-Gal4. Riq expression (grey in a) was assessed using an antibody directed against DCAF7 (human homologue of Riq). Cubitus interruptus (Ci) expression (red in a’) marked the anterior compartment of wing imaginal discs. A merged image is shown in (a”). (b) The level of riq mRNA relative to the control actin5C mRNA, assessed by QRT-PCR, in wing imaginal discs harbouring the following transgenes: hh-Gal4 and UAS-EYFP; or hh-Gal4 and UAS-riqi1. Data is presented as mean±SEM,n = 3, ** = p<0.01. Scale bar represents 50 μm in (a). Statistics source data for Supplementary Fig. S2b is available in Supplementary Table S1.

Supplementary Figure 3 Riquiqui and Minibrain regulate SWH pathway activity.

Wing imaginal discs from third instar D. melanogaster larvae harbouring the following elements: (a-a”) en-Gal4, UAS-EYFP and th-lacZ; (b-b”) en-Gal4, UAS-riq and th-lacZ; (c-c”) en-Gal4, UAS-mnb and th-lacZ; (d,d’) hh-Gal4, UAS-EYFP and ex-lacZ; (e,e’) hh-Gal4, UAS-riq and ex-lacZ; (f,f’) hh-Gal4, UAS-mnb and ex-lacZ; (g,g’) hh-Gal4, UAS-riq, UAS-mnb and ex-lacZ. Yki activity (grey) was reported by th-lacZ (a–c) or ex-lacZ (d–g). All transgenes were expressed in the posterior compartment of wing imaginal discs; Ci expression (red) marks the anterior compartment. Merged images are shown in (a”–c” and in d’–g’). (h) An adult female fly expressing both riq and mnb under control of the rn-Gal4 driver. Unlike driving expression of riq and mnb alone, driving expression of both transgenes together caused severe folding and crumpling of wings, which prevented quantification of their size. (e) Quantification of LacZ expression driven by ex-lacZ, ban-lacZ or th-lacZ in wing imaginal discs displayed in Fig. 3 and Supplementary Fig. S3a–c. Anterior is to the left. For each disc fluorescence intensity was measured by ImageJ in two boxes of defined size in both the anterior (ant.) and posterior (post.) compartments. These values were averaged and plotted for each experiment. Scale bar represents 50 μm.

Supplementary Figure 4 Riquiqui and Minibrain affect wing growth independently of Dachs.

Wing imaginal discs from third instar D. melanogaster larvae containing clones of UAS-riqi1 (a) or UAS-mnbi1 (b) were stained for Dachs (grey in a and b). Clones were positively marked by co-expression of GFP (a’ and b’). Merged images are shown in (a” and b”). X-Z sections through the wing pouch are shown below planar sections of wing discs in a–b”. Dashed lines represent the section used to obtain X-Z images. No changes in Dachs expression or localization were observed. (d–e”) All images were recorded in pupal scutellum tissue (right-sided hemi-scutellum) at 20 hour after pupa formation. Anterior is to the right, the midline is to the top. Yellow circles mark the macrocheatae. Spatial information on the Fj-Ds gradient is shown in c as in ref. 1. Flip out clones of UAS-riqi and UAS-mnbi were generated and marked in red (d” and e” respectively). White boxes are magnified in d’ and e’. Yellow dots mark the mutant cells abutting wt cells (d’ and e’). In these Flip-Out clones no clear reduction of D:GFP levels was observed when compared with the surrounding tissue. There is also no clear repolarization of D:GFP near clone boundaries. (f) Wing area from adult female flies expressing UAS-EYFP as control, UAS-riqi, UAS-dachs RNAi (di), UAS-riqi concomitantly with UAS-di, under the control of rn-Gal4 (n = 20 for each genotype). Representative images of each genotype are shown. Data is presented as mean±SEM, *** = p<0.001. These results suggest Dachs and Riq control tissue growth by acting in parallel. Scale bars represents 50 μm in (a) and (b), 10 μm in (c), (d) and (e), and 200 μm in (f).

Supplementary Figure 5 Riquiqui, Minibrain and Warts physically interact with each other and Minibrain phosphorylates Warts.

(a) S2 cells were transfected with the indicated plasmids and immunoprecipitations performed using anti-HA antibodies. Subsequently, immunoprecipitates and input lysates were subjected to SDS-PAGE and Western blotted to reveal the indicated proteins. (b–d) Western blot analysis of lysates from S2 cells transfected with the indicated plasmids and Western blotted to reveal the indicated proteins. In (b) Riq or Mnb were expressed in S2 cells in the presence or absence of dsRNA specific for Riq or Mnb, respectively. In (c) Mnb did not induce changes in Hpo or Sav mobility. In (d) Mnb, but not Hipk influenced Wts mobility. (e) Kinase assays performed using recombinant GST-Wts1 or GST-CACYBP as substrates and either Mnb or Mnb K386R immunoprecipitated from S2 cells. Immunopurified and recombinant proteins were incubated alone or together in kinase buffer containing γ32P-ATP and subjected to SDS-PAGE (upper panel). Western blotting was used to detect input proteins (lower panels). Mnb phosphorylated GST-Wts1 but not the negative control substrate GST-CACYBP. (f) V5-tagged Wts was expressed in the presence or absence of dsRNA specific for Riq or Mnb in S2 cells. Cells were lysed and lysates were immunoblotted using antibodies to V5 and to Tubulin as a loading control. Wts levels were unchanged when Riq or Mnb were depleted from cells by RNAi.

Supplementary Figure 6 Minibrain phosphorylates six amino acids in the Warts protein, including a DYRK1A consensus site.

(a and b) Wts1 (amino acids 1-318 of Wts) peptides that possessed amino acids that were phosphorylated when incubated with either wild-type Mnb or kinase-dead Mnb (Mnb-KD) immunoprecipitates. Experiments 1 and 2 were performed independently and analyzed by different mass spectrometry facilities. In experiment 1, five Wts amino acids were phosphorylated by wild-type Mnb with high confidence, whereas Mnb-KD induced no observable phosphorylation. In experiment 2, six Wts amino acids were phosphorylated by wild-type Mnb with high confidence, whilst two amino acids were phosphorylated in the presence of Mnb-KD. Amino acids that were phosphorylated by Mnb, but not Mnb-KD, are highlighted; amino acids that were phosphorylated in both independent experiments are red and amino acids that were phosphorylated in only one experiment are blue. (c) A summary of mass spectrometry data outlined in (a) and (b). The amino acid sequence of Wts1 is shown and the degree of conservation with human LATS1, as assessed by CLUSTAL, is indicated below: * indicates perfect conservation, : indicates strongly similar;. indicates weakly similar. As in (a) and (b), amino acids that were phosphorylated in both independent experiments by Mnb, but not Mnb-KD, are highlighted in red and amino acids that were phosphorylated by Mnb in only one experiment are in blue. The Wts sequence that was phosphorylated by Mnb and closely resembles a DYRK1A consensus site (RPXS/TP; ref. 2) is in bold font. Of note, this site was phosphorylated in each experiment and is conserved between Wts and LATS1.

Supplementary Figure 7 Reduction of Riq or Mnb expression does not affect Dachsous expression.

Wing imaginal discs from third instar D. melanogaster larvae containing clones of UAS-riqi1 (a) or UAS-mnbi1 (b) were stained for Ds (grey in a and b). Clones were positively marked by co-expression of GFP (a’ and b’). Merged images are shown in (a” and b”). X-Z sections through the wing pouch are shown below planar sections of wing discs in a–b”. Dashed lines represent the section used to obtain X-Z images. Scale bar represents 50 μm.

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Degoutin, J., Milton, C., Yu, E. et al. Riquiqui and Minibrain are regulators of the Hippo pathway downstream of Dachsous. Nat Cell Biol 15, 1176–1185 (2013). https://doi.org/10.1038/ncb2829

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