Mst2 and Lats Kinases Regulate Apoptotic Function of Yes Kinase-associated Protein (YAP)

The Hippo pathway in Drosophila controls the size and shape of organs. In the fly, activation of this pathway conveys growth-inhibitory signals and promotes apoptosis in epithelial cells. We “reconstituted” the Hippo pathway in a human epithelial cell line and showed that, in contrast to flies, the activation of this pathway results in anti-apoptotic signals. We have shown that in human embryonic kidney (HEK) 293 cells, the complex formation between transcriptional co-activators YAPs (Yes kinase-associated proteins) and Lats kinases requires the intact WW domains of YAPs, as well as intact Pro-Pro-AA-Tyr (where AA is any amino acid) motifs in Lats kinases. These kinases cooperate with the upstream Mst2 kinase to phosphorylate YAPs at Ser-127. Overexpression of YAP2 in HEK293 cells promoted apoptosis, whereas the Mst2/Lats1-induced phosphorylation of YAP partially rescued the cells from apoptotic death. Apoptotic signaling of YAP2 was mediated via stabilization of p73, which formed a complex with YAP2. All components of the Hippo pathway that we studied were localized in the cytoplasm, with the exception of YAP, which also localized in the nucleus. The localization of YAP2 in the nucleus was negatively controlled by the Lats1 kinase. Our apoptotic “readout” of the Hippo pathway in embryonic kidney cells represents a useful experimental system for the identification of the putative upstream receptor, membrane protein, or extracellular factor that initiates an entire signaling cascade and ultimately controls the size of organs. The Hippo pathway in Drosophila controls the size and shape of organs. In the fly, activation of this pathway conveys growth-inhibitory signals and promotes apoptosis in epithelial cells. We “reconstituted” the Hippo pathway in a human epithelial cell line and showed that, in contrast to flies, the activation of this pathway results in anti-apoptotic signals. We have shown that in human embryonic kidney (HEK) 293 cells, the complex formation between transcriptional co-activators YAPs (Yes kinase-associated proteins) and Lats kinases requires the intact WW domains of YAPs, as well as intact Pro-Pro-AA-Tyr (where AA is any amino acid) motifs in Lats kinases. These kinases cooperate with the upstream Mst2 kinase to phosphorylate YAPs at Ser-127. Overexpression of YAP2 in HEK293 cells promoted apoptosis, whereas the Mst2/Lats1-induced phosphorylation of YAP partially rescued the cells from apoptotic death. Apoptotic signaling of YAP2 was mediated via stabilization of p73, which formed a complex with YAP2. All components of the Hippo pathway that we studied were localized in the cytoplasm, with the exception of YAP, which also localized in the nucleus. The localization of YAP2 in the nucleus was negatively controlled by the Lats1 kinase. Our apoptotic “readout” of the Hippo pathway in embryonic kidney cells represents a useful experimental system for the identification of the putative upstream receptor, membrane protein, or extracellular factor that initiates an entire signaling cascade and ultimately controls the size of organs. Metazoans have evolved several well conserved signaling pathways that regulate cell proliferation and growth to create organs, and ultimately organisms, of reproducible size and shape (1Leevers S.J. McNeill H. Curr. Opin. Cell Biol. 2005; 17: 604-609Crossref PubMed Scopus (64) Google Scholar). One such pathway, known as the Hippo (Hpo) 2The abbreviations used are: Hpo, Hippo; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GFP, green fluorescent protein; GST, glutathione S-transferase; HA, hemagglutinin; HEK, human embryonic kidney; Lats, large tumor suppressor kinase; Mst, mammalian ste20-like protein kinase; PARP, poly(ADP-ribose) polymerase; RNAi, RNA interference; Sav, Salvador; Wts, Warts; WW45, WW domain-containing protein of 45-kDa molecular mass; YAP, Yes kinase-associated protein; Yki, Yorkie. pathway, was originally identified in Drosophila melanogaster (2Ryoo H.D. Steller H. Nat. Cell Biol. 2003; 5: 853-855Crossref PubMed Scopus (18) Google Scholar, 3O'Neill E.E. Matallanas D. Kolch W. Cancer Res. 2005; 65: 5485-5487Crossref PubMed Scopus (46) Google Scholar, 4Vidal M. Cagan R.L. Curr. Opin. Genet. Dev. 2006; 16: 10-16Crossref PubMed Scopus (119) Google Scholar, 5Pan D. Genes Dev. 2007; 21: 886-897Crossref PubMed Scopus (520) Google Scholar, 6Harvey K. Tapon N. Nat. Rev. Cancer. 2007; 7: 182-191Crossref PubMed Scopus (524) Google Scholar, 7Saucedo L.J. Edgar B.A. Nat. Rev. Mol. Cell Biol. 2007; 8: 613-621Crossref PubMed Scopus (288) Google Scholar, 8Dong J. Feldmann G. Huang J. Wu S. Zhang N. Comerford S.A. Gayyed M.F. Anders R.A. Maitra A. Pan D. Cell. 2007; 130: 1120-1133Abstract Full Text Full Text PDF PubMed Scopus (1793) Google Scholar, 9Meignin C. Alvarez-Garcia I. Davis I. Palacios I.M. Curr. Biol. 2007; 17: 1871-1878Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 10Bandura J.L. Edgar B.A. Dev. Cell. 2008; 14: 315-316Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar, 11Matallanas D. Romano D. Hamilton G. Kolch W. O'Neill E. Cell Cycle. 2008; 7: 879-884Crossref PubMed Scopus (34) Google Scholar). Flies with “loss-of-function” mutations in components of the Hpo pathway develop significantly overgrown organs. Conversely, activation of this pathway results in reduced proliferation of cells and increased sensitivity to developmentally regulated apoptosis (12Huang J. Wu S. Barrera J. Matthews K. Pan D. Cell. 2005; 122: 421-434Abstract Full Text Full Text PDF PubMed Scopus (1363) Google Scholar). Several proteins constitute the core of the Hpo pathway; these include a scaffolding protein, two serine/threonine kinases, and one transcriptional co-activator (13Edgar B.A. Cell. 2006; 124: 267-273Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar). The most well characterized upstream signaling component of the pathway is Salvador (Sav), a gene product that contains two protein-protein interaction modules known as WW domains (14Bork P. Sudol M. Trends Biochem. Sci. 1994; 19: 531-533Abstract Full Text PDF PubMed Scopus (348) Google Scholar) and is believed to act as a scaffolding protein for Hpo and Warts (Wts) (15Tapon N. Harvey K.F. Bell D.W. Wahrer D.C. Schiripo T.A. Haber D.A. Hariharan I.K. Cell. 2002; 110: 467-478Abstract Full Text Full Text PDF PubMed Scopus (674) Google Scholar, 16Pantalacci S. Tapon N. Leopold P. Nat. Cell Biol. 2003; 5: 921-927Crossref PubMed Scopus (452) Google Scholar). Hpo is a serine/threonine protein kinase that phosphorylates and activates Wts (17Udan R.S. Kango-Singh M. Nolo R. Tao C. Halder G. Nat. Cell Biol. 2003; 5: 914-920Crossref PubMed Scopus (580) Google Scholar, 18Harvey K.F. Pfleger C.M. Hariharan I.K. Cell. 2003; 114: 457-467Abstract Full Text Full Text PDF PubMed Scopus (744) Google Scholar). At the amino-terminal portion of the protein, Wts has five PPXY motifs (where X represents any amino acid), which are binding sites to class I WW domains (19Sudol M. Hunter T. Cell. 2000; 103: 1001-1004Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar). Hpo-activated Wts binds and phosphorylates Yki, subsequently preventing Yki from stimulating the transcription of diap1 and CycE genes, which ultimately results in reduced cell proliferation (12Huang J. Wu S. Barrera J. Matthews K. Pan D. Cell. 2005; 122: 421-434Abstract Full Text Full Text PDF PubMed Scopus (1363) Google Scholar). Wts, Hpo, and Sav are negative regulators of Yki, as the Yki overexpression phenotype resembles that of the functional loss of these three proteins (12Huang J. Wu S. Barrera J. Matthews K. Pan D. Cell. 2005; 122: 421-434Abstract Full Text Full Text PDF PubMed Scopus (1363) Google Scholar, 20Jia J. Zhang W. Wang B. Trinko R. Jiang J. Genes Dev. 2003; 17: 2514-2519Crossref PubMed Scopus (321) Google Scholar). The human orthologs of the Hpo pathway are well conserved (Fig. 1A). The most upstream is WW45 (WW domain-containing protein, 45-kDa molecular mass), the ortholog of Sav, which also contains two WW domains (15Tapon N. Harvey K.F. Bell D.W. Wahrer D.C. Schiripo T.A. Haber D.A. Hariharan I.K. Cell. 2002; 110: 467-478Abstract Full Text Full Text PDF PubMed Scopus (674) Google Scholar). From this point, the pathway bifurcates. The remaining downstream components of the Hpo pathway in humans are represented by two closely related paralogs. Mst1 and Mst2 (mammalian Ste20-like protein kinases) are the mammalian orthologs of Hpo and are closely related serine/threonine kinases known to phosphorylate large tumor suppressor kinases, Lats1 and Lats2 (two serine/threonine kinases that are orthologs of Wts) (21Graves J.D. Gotoh Y. Draves K.E. Ambrose D. Han D.K. Wright M. Chernoff J. Clark E.A. Krebs E.G. EMBO J. 1998; 17: 2224-2234Crossref PubMed Scopus (325) Google Scholar, 22St John M.A. Tao W. Fei X. Fukumoto R. Carcangiu M.L. Brownstein D.G. Parlow A.F. McGrath J. Xu T. Nat. Genet. 1999; 21: 182-186Crossref PubMed Scopus (376) Google Scholar, 23Deng Y. Pang A. Wang J.H. J. Biol. Chem. 2003; 278: 11760-11767Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 24McPherson J.P. Tamblyn L. Elia A. Migon E. Shehabeldin A. Matysiak-Zablocki E. Lemmers B. Salmena L. Hakem A. Fish J. Kassam F. Squire J. Bruneau B.G. Hande M.P. Hakem R. EMBO J. 2004; 23: 3677-3688Crossref PubMed Scopus (172) Google Scholar, 25Bothos J. Tuttle R.L. Ottey M. Luca F.C. Halazonetis T.D. Cancer Res. 2005; 65: 6568-6575Crossref PubMed Scopus (112) Google Scholar, 26Takahashi Y. Miyoshi Y. Takahata C. Irahara N. Taguchi T. Tamaki Y. Noguchi S. Clin. Cancer Res. 2005; 11: 1380-1385Crossref PubMed Scopus (260) Google Scholar). Phosphorylated Lats1 and Lats2 display enhanced kinase activity (27Wu S. Huang J. Dong J. Pan D. Cell. 2003; 114: 445-456Abstract Full Text Full Text PDF PubMed Scopus (836) Google Scholar). Similar to Wts, Lats1 and Lats2 have PPXY sequence motifs at their amino-terminal regions. The human ortholog of Drosophila Yki, YAP, also exists as two splice isoforms: YAP1, which contains one WW domain, and YAP2, which contains two WW domains (28Sudol M. Bork P. Einbond A. Kastury K. Druck T. Negrini M. Huebner K. Lehman D. J. Biol. Chem. 1995; 270: 14733-14741Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar, 29Komuro A. Nagai M. Navin N.E. Sudol M. J. Biol. Chem. 2003; 278: 33334-33341Abstract Full Text Full Text PDF PubMed Scopus (386) Google Scholar). YAP is a transcriptional co-activator in which function depends on the presence of an intact WW domain(s) (29Komuro A. Nagai M. Navin N.E. Sudol M. J. Biol. Chem. 2003; 278: 33334-33341Abstract Full Text Full Text PDF PubMed Scopus (386) Google Scholar, 30Yagi R. Chen L.F. Shigesada K. Murakami Y. Ito Y. EMBO J. 1999; 18: 2551-2562Crossref PubMed Scopus (453) Google Scholar). We decided to study the YAP signaling function in the context of the Hpo pathway in human cells for several reasons. The first thing to catch our attention was the apparent paradox of YAP functioning either as an oncogene (31Zender L. Spector M.S. Xue W. Flemming P. Cordon-Cardo C. Silke J. Fan S.T. Luk J.M. Wigler M. Hannon G.J. Mu D. Lucito R. Powers S. Lowe S.W. Cell. 2006; 125: 1253-1267Abstract Full Text Full Text PDF PubMed Scopus (915) Google Scholar, 32Overholtzer M. Zhang J. Smolen G.A. Muir B. Li W. Sgroi D.C. Deng C.X. Brugge J.S. Haber D.A. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 12405-12410Crossref PubMed Scopus (745) Google Scholar, 33Zhang J. Smolen G.A. Haber D.A. Cancer Res. 2008; 68: 2789-2794Crossref PubMed Scopus (212) Google Scholar, 34Zhao B. Wei X. Li W. Udan R.S. Yang Q. Kim J. Xie J. Ikenoue T. Yu J. Li L. Zheng P. Ye K. Chinnaiyan A. Halder G. Lai Z.C. Guan K.L. Genes Dev. 2007; 21: 2747-2761Crossref PubMed Scopus (2153) Google Scholar) or a promoter of apoptosis (35Strano S. Munarriz E. Rossi M. Castagnoli L. Shaul Y. Sacchi A. Oren M. Sudol M. Cesareni G. Blandino G. J. Biol. Chem. 2001; 276: 15164-15173Abstract Full Text Full Text PDF PubMed Scopus (358) Google Scholar, 36Strano S. Monti O. Pediconi N. Baccarini A. Fontemaggi G. Lapi E. Mantovani F. Damalas A. Citro G. Sacchi A. Del Sal G. Levrero M. Blandino G. Mol. Cell. 2005; 18: 447-459Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar, 37Levy D. Adamovich Y. Reuven N. Shaul Y. Cell Death Differ. 2006; 14: 743-751Crossref PubMed Scopus (173) Google Scholar, 38Danovi S.A. Rossi M. Gudmundsdottir K. Yuan M. Melino G. Basu S. Cell Death Differ. 2008; 15: 217-219Crossref PubMed Scopus (78) Google Scholar, 39Levy D. Adamovich Y. Reuven N. Shaul Y. Mol. Cell. 2008; 29: 350-361Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar). Secondly, the Hpo pathway is an ideal pathway for studying signaling mediated by WW domains because all components of the main pathway contain either WW domains or their cognate ligand motifs, PPXY (14Bork P. Sudol M. Trends Biochem. Sci. 1994; 19: 531-533Abstract Full Text PDF PubMed Scopus (348) Google Scholar, 40Chen H.I. Sudol M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7819-7823Crossref PubMed Scopus (489) Google Scholar) or PPXF (41Espanel X. Sudol M. J. Biol. Chem. 1999; 274: 17284-17289Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar) (Fig. 1A). Thirdly, the protein module known as the WW domain and its founding protein, YAP, are the main focus of research in our laboratory. Using our expertise and reagents, we have concentrated on the critical role of the WW domain in mediating multiple, yet specific protein-protein interactions that occur in the Hpo pathway. We have shown that in the human epithelial cell line HEK293, the complex formation of YAP1 and YAP2 with Lats1 and Lats2 requires intact WW domain(s) in YAPs as well as intact PPXY motifs in Lats kinases. These kinases cooperate with the Mst2 kinase to phosphorylate YAP at Ser-127. Overexpression of YAP2 in HEK293 cells promotes apoptosis of HEK293 cells, whereas the Mst2/Lats1-induced phosphorylation of YAP partially rescues cells from apoptosis. The apoptotic signaling of YAP2 is mediated via stabilization of p73, which forms a complex with both of the WW domains of YAP2. Our data provide evidence of the proapoptotic function of YAP and contradict that of Yki, the fly homolog of YAP, which has been shown to act as an oncogene (12Huang J. Wu S. Barrera J. Matthews K. Pan D. Cell. 2005; 122: 421-434Abstract Full Text Full Text PDF PubMed Scopus (1363) Google Scholar). Cell Culture and Transfections—HEK293 and NIH3T3 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS). Cells were transiently transfected using Lipofectamine (Invitrogen) according to the manufacturer's instructions. Plasmids—The KpnI-XbaI fragments were excised from p2xFlag-CMV2 YAP2s (wild type (WT), first WW mutant, second WW mutant (29Komuro A. Nagai M. Navin N.E. Sudol M. J. Biol. Chem. 2003; 278: 33334-33341Abstract Full Text Full Text PDF PubMed Scopus (386) Google Scholar)) and were ligated into the pcDNA4HIS-MAX vector (Invitrogen), pcDNA4/TO/myc-His vector (Invitrogen), pEGFP-C3 vector (Clontech), and pDsRed-Monomer-C1 vector (Clontech). All mutants were generated using a QuikChange site-directed mutagenesis kit (Stratagene). PCR was performed to amplify the cDNA of Lats1 using three primer sets: 5′-agaagaagaggtaccaatgaagaggagtgaaaagccagaaggatatag-3′; 5′-aacattttcataactcttttcactctcatc-3′ and 5′-cccatctgttcctccatacgagtcaatcag-3′; 5′-gcgtgcagctctccgctctaatggcttcag-3′ and 5′-tccacggcaagatagcatggatttcagtaa-3′; 5′-agaagaagatctagaaacatatactagatcgcgatttttaatctctg-3′. The amplified fragments were inserted into p2xFlag-CMV2 vector using KpnI, HindIII, BamHI, and XbaI sites to generate full-length Lats1. The resulting full-length Lats1 was also inserted into pEGFP-C3 vector (Clontech) using KpnI-SmaI sites. The deletion mutant of Lats1 that encodes the amino-terminal portion of Lats1 was constructed using the HindIII site. pME18SFL-WW45 and pcDNA3-FLAG-Lats2 were kind gifts from Dr. Sumio Sugano and Dr. Tadashi Yamamoto, respectively (Institute of Medical Science, University of Tokyo). pcDNA3.1-FLAG-Mst2 WT and D164A mutant were from Dr. Herman Sillje (Max Planck Institute of Biochemistry, Martinsried, Germany). p2xFlag-CMV2 WW45 was constructed by recloning the cDNA from pME18SFL-WW45. Antibodies—HA antibody (Y-11), Lats1 antibody (N-18), p73 antibody (C-17), and cyclin E antibody (H-145) were purchased from Santa Cruz Biotechnology. Anti-poly(ADP-ribose) polymerase (PARP) was obtained from Roche Applied Science. Phospho-YAP (Ser-127) antibody was from Cell Signaling. GAPDH antibody was from Abcam. Active caspase-3 antibody and FLAG-M2 antibody were from Sigma. Polyclonal antibody against human YAP was generated in rabbits. The animals were injected with purified antigen that contained GST fused to amino acids 302–450 of human YAP1 (GenBank NP 006097 28) expressed in bacteria using pGEX-2TK vector. GST-YAP fusion protein was purified to homogeneity using glutathione-Sepharose chromatography. Immune sera were partially depleted of antibodies reacting with GST by affinity chromatography on GST-Sepharose. The sera were then affinity-purified on GST-YAP antigen coupled to Sepharose, concentrated by ammonium sulfate precipitation, and dialyzed against phosphate-buffered saline solution (42Sudol M. Oncogene. 1994; 9: 2145-2152PubMed Google Scholar). The high titer of the preparation was represented by positive Western blotting data obtained at a 1:20,000 dilution of the antibody. Immunoprecipitation—HEK293 cells were transfected with expression vectors that encoded the protein of interest using Lipofectamine (Invitrogen). 24 h later, cells were lysed with modified radioimmune precipitation assay buffer (50 mm Tris-HCl (pH 7.45), 5 mm EDTA, 300 mm NaCl, 1% glycerol, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS) and immunoprecipitated using anti-FLAG M2 affinity gel (Sigma). For the immunoprecipitation of YAP, protein A-Sepharose (Sigma) was incubated with anti-YAP antibody or control IgG (Santa Cruz Biotechnology) followed by the addition of HEK293 cell lysates. The immunoprecipitates were washed with the modified radioimmune precipitation assay buffer, and bound proteins were separated by SDS-PAGE followed by immunoblotting. Establishment of Cell Lines That Express YAP in an Inducible System—HEK293 cells were transfected with pcDNA6/TR vector (Invitrogen), which encodes the Tet repressor under the control of the human cytomegalovirus promoter. Cells that failed to express this repressor were removed by adding blasticidin to a final concentration of 5 μg/ml. After the selection, cells were transfected with YAP cDNAs that were cloned into pcDNA4/TO/myc-His vector (Invitrogen). Selection was performed again by adding both blasticidin (5 μg/ml) and zeocin (400 μg/ml). Single-cell isolation was never performed when cells were selected. After the establishment of the cell lines, the concentration of zeocin was reduced to 200 μg/ml, and that of blasticidin remained at 5 μg/ml. Tetracycline was added to the medium to a final concentration of 1 μg/ml to induce the expression of YAPs. The same protocol was used for NIH3T3 cells, except higher concentrations of blasticidin (10 μg/ml) were used in the selection of transformants. Cell Counting Assay—293 cells were distributed in DMEM containing 1% FBS, blasticidin (5 μg/ml), and zeocin (200 μg/ml) at an initial density of about 2–3%. 12 h later, tetracycline (1 μg/ml) was added to the medium to induce the expression of YAPs. 96 h later, floating cells were removed, and attached cells were trypsinized and counted. For NIH3T3 cells, more FBS and blasticidin were added to the medium up to 2.5% and 10 μg/ml, respectively. RNAi Treatment—The RNAi oligos for p73 and YAP were purchased from Dharmacon and Santa Cruz Biotechnology, respectively. Luciferase-specific RNAi oligo was used as a control. The target sequence for p73 is CCAUCCUGUACAACUUCAUGU. HEK293 cells that express YAP2 WT in an inducible system or normal HEK293 cells were plated at ∼25% confluency. Transfections of the RNAi oligos were conducted by using Lipofectamine RNAiMAX (Invitrogen). Cells were incubated for 48 h, and the amount of endogenous p73 and YAP was determined by Western blotting. YAP Binds Preferentially to Both Lats1 and Lats2—Because all components of the Hpo pathway in human contain either a PPXY/PPXF or cognate WW domain, the potential exists for multicomponent complexes. To determine whether WW45, Mst2, Lats1, and Lats2 interact physically with YAP, a co-precipitation assay was performed. Each of the four proteins was fused to a FLAG tag and transiently co-transfected with YAP2 into HEK293 cells. Immunoprecipitation using FLAG antibody was performed prior to immunoblotting with YAP antibody (Fig. 1B). As expected, YAP2 was co-precipitated with Lats1 and Lats2. No signal was seen for WW45. A weak and somewhat diffused band of YAP immunoreactivity was visible in the Mst2 lane. Similar results were obtained when YAP1 isoform was substituted for YAP2 (data not shown). The association between YAP and Lats1 was also detected by testing endogenous proteins (Fig. 1C). Binding between Lats1 and YAP2 Requires Intact PPXY Motifs and WW Domains—Because Lats1 contains PPXY motifs and YAP2 contains WW domains, we hypothesized that the Lats1-YAP2 interaction is mediated by a PPXY-WW domain complex. To test this hypothesis, we mutated two PPXY sequences in Lats1 (first, PPXY amino acids 373–376, and second, PPXY amino acids 556–559) by substituting the terminal Tyr for Ala. Such a mutation has been known to abrogate binding to WW domains (29Komuro A. Nagai M. Navin N.E. Sudol M. J. Biol. Chem. 2003; 278: 33334-33341Abstract Full Text Full Text PDF PubMed Scopus (386) Google Scholar, 43Chen H.I. Einbond A. Kwak S.J. Linn H. Koepf E. Peterson S. Kelly J.W. Sudol M. J. Biol. Chem. 1997; 272: 17070-17077Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). The mutants of the PPXY cores (named PY1* and PY2*) and double mutant (PY1*&2*) were constructed. All three mutants, as well as Lats1 WY, were fused to FLAG tags and transiently co-expressed with YAP2 in HEK293 cells followed by immunoprecipitation and immunoblotting (Fig. 1D). Lats1 WT bound strongly to YAP2 (Fig. 1D, upper panel). As anticipated, binding became weaker when either of the single mutants (PY1* or PY2*) was analyzed. When both PPXY motifs in Lats1 were mutated (PY1* and PY2*), the complex with YAP2 was abrogated. These data suggest that both PPXY sequences in Lats1 are necessary for full-strength association with YAP2. We then asked whether either one or both WW domains in YAP2 mediate the complex with Lats1. To test this query, point mutations were introduced into the WW domains of YAP2. Two highly conserved amino acids in each of the two WW domains, the second signature Trp and the carboxyl-terminal Pro, were mutated to Ala. The WQDP sequence of amino acids 199–202 in the first WW domain of YAP2 was changed to AQDA, whereas the WLDP sequence of amino acids 258–261 in the second WW domain of YAP2 was changed to ALDA. Such a substitution in WW domains renders the mutated domains inactive in terms of ligand binding (29Komuro A. Nagai M. Navin N.E. Sudol M. J. Biol. Chem. 2003; 278: 33334-33341Abstract Full Text Full Text PDF PubMed Scopus (386) Google Scholar). The mutants were named 1st WW* and 2nd WW*, respectively. A double mutant was named 1&2 WW*. All three YAP2 mutants and the WT were co-transfected with FLAG-Lats1 into HEK293 cells followed by immunoprecipitation and immunoblotting (Fig. 1E). Relatively strong binding was detected in the case of WT YAP2 (Fig. 1E, upper panel); however, this binding was weakened when the first WW domain of YAP2 was mutated. The second WW domain mutant seemed to have no influence on binding. Finally, when both WW domains of YAP2 were mutated, the binding was barely detectable. Similar results were obtained for Lats2 (data not shown). These results suggest that both WW domains in YAP2 play a crucial role in binding to Lats1 and Lats2 and that the first WW domain plays a more dominant role in the complex. Interestingly, although the first WW domain of YAP2 corresponds to the only WW domain of YAP1, the binding ability of YAP1 to Lats1 and Lats2 was abrogated when its WW domain was mutated (data not shown). In sum, the results presented in Fig. 1, D and E, suggest that the binding between YAP2 and Lats1 (Lats2) is mediated by the WW domains of YAP2 and the PPXY motifs of Lats1 (Lats2). Lats1 and Lats2 Cooperate with Mst2 to Phosphorylate YAP—Because Drosophila Wts interacts with and phosphorylates YKI, we decided to investigate whether Lats1 and Lats2 phosphorylate YAP in mammalian cells. We employed a gel shift assay, to detect YAP phosphorylation. (For the analysis of phosphorylation sites we used YAP1 because of the battery of mutants that were available in our laboratory.) The band of YAP1 was shifted upward when Mst2 and Lats1 were transfected together (Fig. 2A, upper panel, compare third and first lanes). This mobility shift was abrogated by phosphatase treatment (Fig. 2A, fourth lane), which suggests that this shift is due to protein phosphorylation. A small difference between the first and second lanes (Fig. 2A) in terms of shift of YAP1 implies that there are endogenous kinases that phosphorylate YAP1 in HEK293 cells. To confirm that Lats1 is the genuine kinase that phosphorylates YAP1, YAP1 together with various combinations of Mst2, Lats1, and Lats2 was transfected into HEK293 cells. The cell lysates were resolved by SDS-PAGE followed by probing with YAP antibody (Fig. 2C, upper panel). YAP1 and vector alone were co-transfected and used as a control (Fig. 2C, upper panel, first lane). No shift was detected in the presence of Lats1 alone or Mst2 alone (Fig. 2C, upper panel, second and third lanes), yet when both Lats1 and Mst2 were present, the band of YAP1 shifted slightly upward (Fig. 2C, upper panel, fourth lane) suggesting that YAP1 is phosphorylated when both Lats1 and Mst2 are present. Mst2 kinase-dead mutant (D164A) could not shift the band of YAP1 when co-transfected with Lats1 (Fig. 2C, upper panel, fifth lane). This result confirms a previous observation showing that the kinase activity of Lats1 is enhanced when Mst2 phosphorylates Lats1 (44Chan E.H. Nousiainen M. Chalamalasetty R.B. Schafer A. Nigg E.A. Sillje H.H. Oncogene. 2005; 24: 2076-2086Crossref PubMed Scopus (439) Google Scholar). The amino-terminal portion of Lats1 (amino acids 1–587), which lacks the kinase catalytic region but still contains two PPXY sequences, was also not able to shift the band of YAP1 (Fig. 2C, upper panel, sixth lane). This finding further suggested that the kinase activity of Lats1 is indispensable for shifting the protein band of YAP1 upward, implying phosphorylation of YAP1. Lats2 exhibits activity similar to that of Lats1, as Lats2 can be substituted for Lats1 (Fig. 2C, upper panel, seventh lane). These results suggest that Mst2 kinase activates Lats1 or Lats2 kinases, which in turn phosphorylate YAP1. Ser-127 in YAP1 Is the Target Site Phosphorylated by Lats1—YAP1 is likely to be phosphorylated by activated Lats1 and Lats2. We evaluated the potential phosphorylation sites of YAP1 with the help of the Netphos 2.0 program (Technical University of Denmark). Ten candidate sites of phosphorylation by serine kinases were identified within the YAP1 sequence based on a relative score given by the program. In addition, we included Ser-127, a site that was previously shown to be a target of Akt kinase (45Basu S. Totty N.F. Irwin M.S. Sudol M. Downward J. Mol. Cell. 2003; 11: 11-23Abstract Full Text Full Text PDF PubMed Scopus (654) Google Scholar), and Thr-110, which together with Ser-109, were reported as phosphorylation sites of YAP isolated from the nuclei of HeLa cells (46Beausoleil S.A. Jedrychowski M. Schwartz D. Elias J.E. Villen J. Li J. Cohn M.A. Cantley L.C. Gygi S.P. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 12130-12135Crossref PubMed Scopus (1239) Google Scholar). These 12 sites were individually mutated to alanine (Fig. 2B). Each of the 12 YAP1 mutants was transfected into HEK293 cells together with either control vector or FLAG-Lats1 and FLAG-Mst2. Shift assay was performed as shown in Fig. 2A. With the exception of S127A, every mutant was still phosphorylated by Lats1 and Mst2 (data not shown). We investigated whether Ser-127 was phosphorylated (Fig. 2C, middle panel) and found that strong signals were detected when YAP1 was transfected with Mst2 and Lats1 (Lats2). These data correlated with the results of the mobility shift assay. Furthermore, the protein band of YAP1 S127A mutant no longer shifted upward even though it was co-transfected with FLAG-Lats1 and FLAG-Mst2 (Fig. 2D). This suggests that Ser-127 is a common phosphorylation site for Lats1 and Akt kinases. In Fig. 2, we used YAP1 to conduct a gel shift assay. The YAP2 protein also has a Ser-127 site, and the protein band was shifted upward as YAP1 was in the presence of Lats1 and Mst2 (data not shown). In our experimental setting, we could not confirm Ser-109 and Thr-110 of YAP1 as sites phosphorylated by Lats1 kinase. Recent in vitro studies have shown that Lats1 kinase can phosphorylate YAP on several serine residues in addition to Ser-127 (47Hao Y. Chun A. Cheung K. Rashidi B. Yang X. J. Biol. Chem. 2008; 283: 5496-5509Abstract Full Text Full Text PDF PubMed Scopus (618) Google Scholar). Expression of YAP2 Results in Serum-dependent Detachment of Cells—To study the biological function of YAP, HEK293 cells individually expressing YAP1 WT, YAP2 WT, YAP2 1&2 WW*, YAP2 S127A, and control vector were established in an inducible system. The induction of these proteins ha