Mst2andLatsKinasesRegulateApoptoticFunctionofYes Kinase ... · first thing to catch our attention was the apparent paradox of YAP functioning either as an oncogene (31–34) or a

Mst2 and Lats Kinases Regulate Apoptotic Function of YesKinase-associated Protein (YAP)*□S

Received for publication, June 6, 2008, and in revised form, July 16, 2008 Published, JBC Papers in Press, July 17, 2008, DOI 10.1074/jbc.M804380200

Tsutomu Oka‡, Virginia Mazack‡, and Marius Sudol‡§1

From the ‡Laboratory of Signal Transduction and Proteomic Profiling, Weis Center for Research, Geisinger Clinic,Danville, Pennsylvania 17822 and the §Department of Medicine, Mount Sinai School of Medicine, New York, New York 10029

TheHippo pathway inDrosophila controls the size and shapeof 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 cellline and showed that, in contrast to flies, the activation of thispathway results in anti-apoptotic signals.Wehave shown that inhuman embryonic kidney (HEK) 293 cells, the complex forma-tion between transcriptional co-activators YAPs (Yes kinase-as-sociated proteins) and Lats kinases requires the intact WWdomains ofYAPs, aswell as intact Pro-Pro-AA-Tyr (whereAA isany amino acid) motifs in Lats kinases. These kinases cooperatewith the upstream Mst2 kinase to phosphorylate YAPs at Ser-127. Overexpression of YAP2 in HEK293 cells promoted apo-ptosis, whereas the Mst2/Lats1-induced phosphorylation ofYAP partially rescued the cells from apoptotic death. Apoptoticsignaling of YAP2 was mediated via stabilization of p73, whichformed a complex with YAP2. All components of the Hippopathway that we studied were localized in the cytoplasm, withthe exception of YAP, which also localized in the nucleus. Thelocalization of YAP2 in the nucleus was negatively controlled bythe Lats1 kinase. Our apoptotic “readout” of theHippo pathwayin embryonic kidney cells represents a useful experimental sys-tem for the identification of the putative upstream receptor,membrane protein, or extracellular factor that initiates anentire signaling cascade and ultimately controls the size oforgans.

Metazoans have evolved several well conserved signalingpathways that regulate cell proliferation and growth to createorgans, and ultimately organisms, of reproducible size andshape (1). One such pathway, known as theHippo (Hpo)2 path-

way, was originally identified in Drosophila melanogaster(2–11). Flies with “loss-of-function” mutations in componentsof the Hpo pathway develop significantly overgrown organs.Conversely, activation of this pathway results in reduced pro-liferation of cells and increased sensitivity to developmentallyregulated apoptosis (12). Several proteins constitute the core ofthe Hpo pathway; these include a scaffolding protein, two ser-ine/threonine kinases, and one transcriptional co-activator(13). The most well characterized upstream signaling compo-nent of the pathway is Salvador (Sav), a gene product that con-tains two protein-protein interaction modules known as WWdomains (14) and is believed to act as a scaffolding protein forHpo and Warts (Wts) (15, 16). Hpo is a serine/threonine pro-tein kinase that phosphorylates and activates Wts (17, 18). Atthe amino-terminal portion of the protein, Wts has five PPXYmotifs (where X represents any amino acid), which are bindingsites to class IWWdomains (19). Hpo-activatedWts binds andphosphorylates Yki, subsequently preventing Yki from stimu-lating the transcription of diap1 and CycE genes, which ulti-mately results in reduced cell proliferation (12). Wts, Hpo, andSav are negative regulators of Yki, as the Yki overexpressionphenotype resembles that of the functional loss of these threeproteins (12, 20).The human orthologs of theHpo pathway are well conserved

(Fig. 1A). Themost upstream isWW45 (WWdomain-contain-ing protein, 45-kDamolecularmass), the ortholog of Sav, whichalso contains twoWWdomains (15). From this point, the path-way bifurcates. The remaining downstream components of theHpo pathway in humans are represented by two closely relatedparalogs. Mst1 and Mst2 (mammalian Ste20-like proteinkinases) are the mammalian orthologs of Hpo and are closelyrelated serine/threonine kinases known to phosphorylate largetumor suppressor kinases, Lats1 and Lats2 (two serine/threo-nine kinases that are orthologs of Wts) (21–26). Phosphoryl-ated Lats1 and Lats2 display enhanced kinase activity (27). Sim-ilar toWts, Lats1 and Lats2 have PPXY sequencemotifs at theiramino-terminal regions. The human ortholog of DrosophilaYki, YAP, also exists as two splice isoforms: YAP1, which con-tains one WW domain, and YAP2, which contains two WWdomains (28, 29). YAP is a transcriptional co-activator in whichfunction depends on the presence of an intact WW domain(s)(29, 30).We decided to study the YAP signaling function in the con-

text of the Hpo pathway in human cells for several reasons. The

* This work was supported by two Breast Cancer Coalition grants from thePennsylvania Department of Health (to M. S.). The costs of publication ofthis article were defrayed in part by the payment of page charges. Thisarticle must therefore be hereby marked “advertisement” in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

□S The on-line version of this article (available at http://www.jbc.org) containssupplemental Fig. 1.

The recombinant plasmids reported in this paper have been deposited in the Add-gene plasmid repository (www.addgene.org).

1 To whom correspondence should be addressed: 100 N. Academy Ave., Dan-ville, PA 17822-2608. Fax: 570-271-6701; E-mail: [email protected].

2 The abbreviations used are: Hpo, Hippo; DMEM, Dulbecco’s modifiedEagle’s medium; FBS, fetal bovine serum; GAPDH, glyceraldehyde 3-phos-phate dehydrogenase; GFP, green fluorescent protein; GST, glutathioneS-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 molecularmass; YAP, Yes kinase-associated protein; Yki, Yorkie.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 283, NO. 41, pp. 27534 –27546, October 10, 2008© 2008 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

27534 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 283 • NUMBER 41 • OCTOBER 10, 2008

by guest on October 24, 2020

http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/cgi/content/full/M804380200/DC1

http://www.jbc.org/

first thing to catch our attention was the apparent paradox ofYAP functioning either as an oncogene (31–34) or a promoterof apoptosis (35–39). Secondly, the Hpo pathway is an idealpathway for studying signaling mediated by WW domainsbecause all components of the main pathway contain eitherWW domains or their cognate ligand motifs, PPXY (14, 40) orPPXF (41) (Fig. 1A). Thirdly, the protein module known as theWWdomain and its founding protein, YAP, are themain focusof research in our laboratory. Using our expertise and reagents,we have concentrated on the critical role of theWWdomain inmediating multiple, yet specific protein-protein interactionsthat occur in the Hpo pathway.We have shown that in the human epithelial cell line

HEK293, the complex formation of YAP1 and YAP2 with Lats1and Lats2 requires intact WW domain(s) in YAPs as well asintact PPXY motifs in Lats kinases. These kinases cooperatewith the Mst2 kinase to phosphorylate YAP at Ser-127. Over-expression of YAP2 in HEK293 cells promotes apoptosis ofHEK293 cells, whereas the Mst2/Lats1-induced phosphoryla-tion of YAP partially rescues cells from apoptosis. The apopto-tic signaling of YAP2 is mediated via stabilization of p73, whichforms a complex with both of the WW domains of YAP2. Ourdata provide evidence of the proapoptotic function of YAP andcontradict that of Yki, the fly homolog of YAP, which has beenshown to act as an oncogene (12).

EXPERIMENTAL PROCEDURES

Cell Culture and Transfections—HEK293 and NIH3T3 cellswere cultured inDulbecco’smodified Eagle’smedium (DMEM)supplemented with 10% fetal bovine serum (FBS). Cells weretransiently 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 (29)) and were ligated into thepcDNA4HIS-MAX vector (Invitrogen), pcDNA4/TO/myc-Hisvector (Invitrogen), pEGFP-C3 vector (Clontech), and pDsRed-Monomer-C1 vector (Clontech). All mutants were generatedusing aQuikChange site-directedmutagenesis kit (Stratagene).PCR was performed to amplify the cDNA of Lats1 using threeprimer sets: 5�-agaagaagaggtaccaatgaagaggagtgaaaagccagaagg-atatag-3�; 5�-aacattttcataactcttttcactctcatc-3� and 5�-cccatctgt-tcctccatacgagtcaatcag-3�; 5�-gcgtgcagctctccgctctaatggcttcag-3�and 5�-tccacggcaagatagcatggatttcagtaa-3�; 5�-agaagaagatctaga-aacatatactagatcgcgatttttaatctctg-3�. The amplified fragmentswere inserted into p2xFlag-CMV2 vector using KpnI, HindIII,BamHI, andXbaI sites to generate full-length Lats1. The result-ing full-length Lats1 was also inserted into pEGFP-C3 vector(Clontech) usingKpnI-SmaI sites. The deletionmutant of Lats1that encodes the amino-terminal portion of Lats1 was con-structed using the HindIII site. pME18SFL-WW45 andpcDNA3-FLAG-Lats2 were kind gifts from Dr. Sumio Suganoand Dr. Tadashi Yamamoto, respectively (Institute of MedicalScience, University of Tokyo). pcDNA3.1-FLAG-Mst2WTandD164Amutant were fromDr. Herman Sillje (Max Planck Insti-tute of Biochemistry, Martinsried, Germany). p2xFlag-CMV2WW45 was constructed by recloning the cDNA frompME18SFL-WW45.

Antibodies—HAantibody (Y-11), Lats1 antibody (N-18), p73antibody (C-17), and cyclin E antibody (H-145) were purchasedfrom Santa Cruz Biotechnology. Anti-poly(ADP-ribose) poly-merase (PARP) was obtained from Roche Applied Science.Phospho-YAP (Ser-127) antibody was from Cell Signaling.GAPDH antibody was from Abcam. Active caspase-3 antibodyand FLAG-M2 antibody were from Sigma. Polyclonal antibodyagainst humanYAPwas generated in rabbits. The animals wereinjected with purified antigen that contained GST fused toamino acids 302–450 of human YAP1 (GenBank NP 00609728) expressed in bacteria using pGEX-2TK vector. GST-YAPfusion protein was purified to homogeneity using glutathione-Sepharose chromatography. Immune sera were partiallydepleted of antibodies reacting with GST by affinity chroma-tography on GST-Sepharose. The sera were then affinity-puri-fied on GST-YAP antigen coupled to Sepharose, concentratedby ammonium sulfate precipitation, and dialyzed against phos-phate-buffered saline solution (42). The high titer of the prep-aration was represented by positive Western blotting dataobtained at a 1:20,000 dilution of the antibody.Immunoprecipitation—HEK293 cells were transfected with

expression vectors that encoded the protein of interest usingLipofectamine (Invitrogen). 24 h later, cells were lysed withmodified 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) andimmunoprecipitated 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 ofHEK293 cell lysates. The immunoprecipitates were washedwith the modified radioimmune precipitation assay buffer, andbound proteins were separated by SDS-PAGE followed byimmunoblotting.Establishment of Cell Lines That Express YAP in an Inducible

System—HEK293 cells were transfected with pcDNA6/TR vec-tor (Invitrogen), which encodes the Tet repressor under thecontrol of the human cytomegalovirus promoter. Cells thatfailed to express this repressor were removed by adding blasti-cidin to a final concentration of 5 �g/ml. After the selection,cells were transfected with YAP cDNAs that were cloned intopcDNA4/TO/myc-His vector (Invitrogen). Selection was per-formed again by adding both blasticidin (5 �g/ml) and zeocin(400 �g/ml). Single-cell isolation was never performed whencells were selected. After the establishment of the cell lines, theconcentration of zeocin was reduced to 200 �g/ml, and that ofblasticidin remained at 5 �g/ml. Tetracycline was added to themedium to a final concentration of 1 �g/ml to induce theexpression of YAPs. The same protocol was used for NIH3T3cells, except higher concentrations of blasticidin (10 �g/ml)were used in the selection of transformants.Cell Counting Assay—293 cells were distributed in DMEM

containing 1% FBS, blasticidin (5 �g/ml), and zeocin (200�g/ml) at an initial density of about 2–3%. 12 h later, tetracy-cline (1 �g/ml) was added to the medium to induce the expres-sion of YAPs. 96 h later, floating cells were removed, andattached cells were trypsinized and counted. For NIH3T3 cells,

Biological Assay for the Mammalian Hippo Pathway

OCTOBER 10, 2008 • VOLUME 283 • NUMBER 41 JOURNAL OF BIOLOGICAL CHEMISTRY 27535


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

more FBS and blasticidin wereadded to themediumup to 2.5% and10 �g/ml, respectively.RNAi Treatment—The RNAi oli-

gos forp73 andYAPwere purchasedfrom Dharmacon and Santa CruzBiotechnology, respectively. Lucif-erase-specific RNAi oligo was usedas a control. The target sequence forp73 is CCAUCCUGUACAACU-UCAUGU. HEK293 cells thatexpress YAP2 WT in an induciblesystem or normal HEK293 cellswere plated at �25% confluency.Transfections of the RNAi oligoswere conducted by using Lipo-fectamine RNAiMAX (Invitrogen).Cells were incubated for 48 h, andthe amount of endogenous p73 andYAP was determined by Westernblotting.

RESULTS

YAP Binds Preferentially to BothLats1 and Lats2—Because all com-ponents of the Hpo pathway inhuman contain either a PPXY/PPXFor cognateWWdomain, the poten-tial exists formulticomponent com-plexes. To determine whetherWW45, Mst2, Lats1, and Lats2interact physically with YAP, a co-precipitation assay was performed.Each of the four proteins was fusedto a FLAG tag and transiently co-transfectedwithYAP2 intoHEK293cells. Immunoprecipitation usingFLAG antibody was performedprior to immunoblotting with YAPantibody (Fig. 1B). As expected,YAP2 was co-precipitated withLats1 and Lats2. No signal was seenfor WW45. A weak and somewhatdiffused band of YAP immunoreac-tivity was visible in the Mst2 lane.Similar results were obtained whenYAP1 isoform was substituted forYAP2 (data not shown). The associ-ation between YAP and Lats1 wasalso detected by testing endogenousproteins (Fig. 1C).Binding between Lats1 and YAP2

Requires Intact PPXY Motifs andWW Domains—Because Lats1 con-tains PPXY motifs and YAP2 con-tains WW domains, we hypothe-sized that the Lats1-YAP2interaction is mediated by a PPXY-

FIGURE 1. YAP binds to Lats1 and Lats2 through the WW domain-PPXY complex. A, diagram comparing thecomponents of the Hippo pathway in Drosophila with their human orthologs. The percentage of sequencesimilarity between individual orthologs was derived from DDBJ-ClustalW comparisons. The number of WWdomains and PPXY/PPXF motifs present in individual proteins is also shown. Note that recently an isoform of Ykithat contains one WW domain was identified. B, YAP2 forms a complex with Lats1 and Lats2. HEK293 cells weretransiently co-transfected with YAP2 and various constructs expressing FLAG-tagged orthologs of the Hippo-Salvador pathway (p2xFlag-CMV2 vector as a control, WW45, Mst2, Lats1, and Lats2). Cell lysates were immu-noprecipitated with FLAG antibodies, resolved on SDS-PAGE, and immunoblotted with YAP antibody (upperpanel). The middle and lower panels show the expression of transfected proteins. C, endogenous YAP binds toendogenous Lats1 in HEK293 cells. HEK293 cell lysates were incubated with protein A-Sepharose conjugatedwith either control IgG or YAP antibody. The immunoprecipitates (IP) were washed, and bound proteinswere separated by SDS-PAGE followed by immunoblotting (IB). D, intact PPXY motifs in Lats1 are requiredfor binding to YAP2. Two PPPY sequences in Lats1 were mutated to PPPA. WT or single (PY1* or PY2*) ordouble (PY1*&2*) mutants of Lats1 in the FLAG tag vector were transiently co-transfected with YAP2 (inpcDNA4/HisMax) into HEK293 cells. Cells were lysed and analyzed as described in B. E, intact WW domainsin YAP2 are required for binding to Lats1. Two WW domains in YAP2 were mutated to render them inactivein terms of ligand binding. WT or single (1st WW* or 2nd WW*) or double (1&2 WW*) mutant of YAP2 inpcDNA4/HisMax vector were co-transfected with FLAG-Lats1 or FLAG vector alone. Cells were lysed andanalyzed as described in B.




http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

WWdomain complex. To test this hypothesis, wemutated twoPPXY sequences in Lats1 (first, PPXY amino acids 373–376,and second, PPXY amino acids 556–559) by substituting theterminal Tyr for Ala. Such a mutation has been known to abro-gate binding to WW domains (29, 43). The mutants of thePPXY cores (named PY1* and PY2*) and double mutant(PY1*&2*) were constructed. All threemutants, as well as Lats1WY,were fused to FLAG tags and transiently co-expressedwithYAP2 in HEK293 cells followed by immunoprecipitation andimmunoblotting (Fig. 1D). Lats1 WT bound strongly to YAP2(Fig. 1D, upper panel). As anticipated, binding became weakerwhen either of the single mutants (PY1* or PY2*) was analyzed.When both PPXY motifs in Lats1 were mutated (PY1* andPY2*), the complex with YAP2 was abrogated. These data sug-gest 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

YAP2mediate the complex with Lats1. To test this query, pointmutations were introduced into the WW domains of YAP2.Two highly conserved amino acids in each of the two WWdomains, the second signature Trp and the carboxyl-terminalPro, were mutated to Ala. TheWQDP sequence of amino acids199–202 in the first WW domain of YAP2 was changed toAQDA, whereas the WLDP sequence of amino acids 258–261in the second WW domain of YAP2 was changed to ALDA.Such a substitution in WW domains renders the mutateddomains inactive in terms of ligand binding (29). The mutantswere named 1st WW* and 2nd WW*, respectively. A doublemutant was named 1&2WW*. All three YAP2mutants and theWT were co-transfected with FLAG-Lats1 into HEK293 cellsfollowed by immunoprecipitation and immunoblotting (Fig.1E). Relatively strong binding was detected in the case of WTYAP2 (Fig. 1E, upper panel); however, this binding was weak-ened when the first WW domain of YAP2 was mutated. Thesecond WW domain mutant seemed to have no influence onbinding. Finally, when both WW domains of YAP2 weremutated, the bindingwas barely detectable. Similar resultswereobtained for Lats2 (data not shown). These results suggest thatboth WW domains in YAP2 play a crucial role in binding toLats1 and Lats2 and that the first WW domain plays a moredominant role in the complex. Interestingly, although the firstWW domain of YAP2 corresponds to the only WW domain ofYAP1, the binding ability of YAP1 to Lats1 and Lats2 was abro-gated when its WW domain was mutated (data not shown). Insum, the results presented in Fig. 1, D and E, suggest that thebinding between YAP2 and Lats1 (Lats2) is mediated by theWW 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 phosphorylatesYKI, we decided to investigate whether Lats1 and Lats2 phos-phorylate YAP in mammalian cells. We employed a gel shiftassay, to detect YAP phosphorylation. (For the analysis of phos-phorylation sites we used YAP1 because of the battery ofmutants that were available in our laboratory.) The band ofYAP1 was shifted upward when Mst2 and Lats1 were trans-fected together (Fig. 2A, upper panel, compare third and firstlanes). This mobility shift was abrogated by phosphatase treat-ment (Fig. 2A, fourth lane), which suggests that this shift is due

to protein phosphorylation. A small difference between the firstand second lanes (Fig. 2A) in terms of shift of YAP1 implies thatthere are endogenous kinases that phosphorylate YAP1 inHEK293 cells.To confirm that Lats1 is the genuine kinase that phospho-

rylates YAP1, YAP1 together with various combinations ofMst2, Lats1, and Lats2 was transfected into HEK293 cells. Thecell lysates were resolved by SDS-PAGE followed by probingwith YAP antibody (Fig. 2C, upper panel). YAP1 and vectoralone were co-transfected and used as a control (Fig. 2C, upperpanel, first lane). No shift was detected in the presence of Lats1alone or Mst2 alone (Fig. 2C, upper panel, second and thirdlanes), yet when both Lats1 andMst2 were present, the band ofYAP1 shifted slightly upward (Fig. 2C, upper panel, fourth lane)suggesting that YAP1 is phosphorylated when both Lats1 andMst2 are present. Mst2 kinase-deadmutant (D164A) could notshift the band of YAP1when co-transfectedwith Lats1 (Fig. 2C,upper panel, fifth lane). This result confirms a previous obser-vation showing that the kinase activity of Lats1 is enhancedwhen Mst2 phosphorylates Lats1 (44). The amino-terminalportion of Lats1 (amino acids 1–587), which lacks the kinasecatalytic region but still contains two PPXY sequences, was alsonot able to shift the band of YAP1 (Fig. 2C, upper panel, sixthlane). This finding further suggested that the kinase activity ofLats1 is indispensable for shifting the protein band of YAP1upward, implying phosphorylation of YAP1. Lats2 exhibitsactivity similar to that of Lats1, as Lats2 can be substituted forLats1 (Fig. 2C, upper panel, seventh lane). These results suggestthat Mst2 kinase activates Lats1 or Lats2 kinases, which in turnphosphorylate YAP1.Ser-127 in YAP1 Is the Target Site Phosphorylated by Lats1—

YAP1 is likely to be phosphorylated by activated Lats1 andLats2. We evaluated the potential phosphorylation sites ofYAP1 with the help of the Netphos 2.0 program (TechnicalUniversity of Denmark). Ten candidate sites of phosphoryla-tion by serine kinaseswere identifiedwithin theYAP1 sequencebased on a relative score given by the program. In addition, weincluded Ser-127, a site that was previously shown to be a targetof Akt kinase (45), and Thr-110, which together with Ser-109,were reported as phosphorylation sites of YAP isolated fromthe nuclei of HeLa cells (46). These 12 sites were individuallymutated to alanine (Fig. 2B). Each of the 12 YAP1 mutants wastransfected into HEK293 cells together with either control vec-tor or FLAG-Lats1 and FLAG-Mst2. Shift assay was performedas shown in Fig. 2A.With the exception of S127A, everymutantwas 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 detectedwhenYAP1was transfectedwithMst2 and Lats1 (Lats2). Thesedata correlated with the results of the mobility shift assay. Fur-thermore, the protein band of YAP1 S127A mutant no longershifted upward even though it was co-transfected with FLAG-Lats1 and FLAG-Mst2 (Fig. 2D). This suggests that Ser-127 is acommon phosphorylation site for Lats1 andAkt kinases. In Fig.2, we used YAP1 to conduct a gel shift assay. The YAP2 proteinalso has a Ser-127 site, and the protein bandwas shifted upward asYAP1 was in the presence of Lats1 andMst2 (data not shown). Inour experimental setting, we could not confirm Ser-109 and Thr-




http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

110 of YAP1 as sites phosphorylated by Lats1 kinase. Recent invitro studies have shown that Lats1 kinase canphosphorylateYAPon several serine residues in addition to Ser-127 (47).

Expression of YAP2 Results inSerum-dependent Detachment ofCells—To study the biological func-tion of YAP, HEK293 cells individu-ally expressing YAP1 WT, YAP2WT, YAP2 1&2 WW*, YAP2S127A, and control vector wereestablished in an inducible system.The induction of these proteins hadno influence on cell detachmentwhen cells were maintained inDMEM containing 10% FCS. How-ever, when the concentration ofserum was reduced to 1%, the effectof YAP2 overexpression on celldetachment was detectable. Theinduction of YAP2 WT expressionin HEK293 cells cultured in DMEMcontaining 1% FCS caused signifi-cant detachment of the cells fromthe plate compared with controls inwhich expression of YAP2 was notinduced. The number of attachedcells was counted following threewashes to remove floating cells. Theratio of attached cells with inducedYAP2WT to attached cells withoutinduced YAP2 WT was 0.661 (Fig.3A, second bar), i.e. the number ofattached cells decreased by 33.9% inYAP2 induced cultures. A relativelymodest decrease (15.9%) wasobserved when the double mutantof YAP2 1&2 WW* was induced(Fig. 3A, third bar), suggesting thatthe WW domains of YAP2 play akey role in reducing the number ofattached cells. Interestingly, theeffect of YAP2 S127A mutant over-expression on cell detachment wasmore dramatic than that of YAP2WT (Fig. 3A, fourth bar). As thismutant is known to preferentiallylocalize in the nucleus (29, 45), it islikely that the function of YAP2 inregulating cell attachment isdependent on its nuclear targets.Wenoted that the overexpression ofYAP1 was not as efficient as that ofYAP2 in terms of reducing cellnumber (Fig. 3A, fifth bar). Thepresence of an additional WWdomain in YAP2 may account forthe observed difference in the celldetachment “readout.”

To study the biological function of YAP under more physio-logical conditions, we reduced the level of endogenous YAP inHEK293 cells by RNAi and measured the rate of cell growth

FIGURE 2. Lats1 cooperates with Mst2 to phosphorylate YAP. A, co-expression of Mst2 and Lats1 resulted ina mobility shift of YAP1. YAP1 was transiently expressed in HEK293 cells with either FLAG vector or the combi-nation of FLAG-Mst2 and FLAG-Lats1. Cell lysates were treated with 20 units of Calf Intestine Alkaline Phospha-tase (CIAP) (Promega) for 5 min in 37 °C and resolved on SDS-PAGE. Upper panel, Immunoblot (IB) with YAPantibody. Lower panel, immunoblot with FLAG antibody. B, list of serines and one threonine residue in YAP,which were predicted as potential phosphorylation sites using the Netphos 2.0 server (Technical University ofDenmark). Ser-109 and Thr-110 were shown as in vivo phosphorylation sites of YAP isolated from the nucleusof HeLa cells (45). Ser-127 was previously shown as an Akt kinase phosphorylation site (44). C, activated Lats1 isable to phosphorylate YAP. YAP1 was transiently co-expressed in HEK293 cells with FLAG-tagged Lats1, Lats2,Mst2, and their mutants or the control vector. At 24 h post-transfection, cell lysates were resolved on SDS-PAGEfollowed by immunoblotting with polyclonal antibody against YAP (upper panel), phospho-YAP (Ser-127) antibody(middle panel), or FLAG antibody (lower panel). D, Ser-127 is also the target of Lats1 kinase. Mutation of YAP1-serine127 to alanine reduced the shift. The faint horizontal line in the upper panel is used to visualize a shift in the molecularweightofYAPduetophosphorylation. Upper panel, immunoblotwithYAPantibody. Middle panel, immunoblotwithphospho-YAP (Ser-127) antibody. Lower panel, immunoblot probed with FLAG antibody.




http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

(Fig. 3B). Interestingly, the cells inwhich the level of YAP was reducedgrew significantly faster comparedwith the control cells (31- versus21-fold increase after 5 days of cul-ture). This result suggests that therelative level of YAP in HEK293cells can regulate the balancebetween cell growth and celldetachment (apoptosis).Effect of YAP2 onReducedAttach-

ment of Cells Is Rescued byMst2 andLats1—Because YAP2 S127A mu-tant had a more significant influ-ence on the number of detachedcells than YAP2 WT (Fig. 3A), wepostulated that Lats1, Lats2, andMst2 could regulate this processby phosphorylating YAP at Ser-127. To investigate the effects ofLats1, Lats2, and Mst2 on cellattachment, these proteins weretransiently expressed as FLAG-tagged proteins in HEK293 cellsthat expressed an inducible YAP2WT. After the transfection of theupstream kinases Lats1, Lats2, andMst2, we induced expression ofYAP2 WT. The number of cellsdecreased by 28.7% when theexpression of YAP2 WT wasinduced (Fig. 3C, first bar). Theeffect of YAP2 was partially dimin-ished by Lats1 or Mst2 (Fig. 3C,second and third bars, respec-tively) and was completely abol-ished by Mst2 and Lats1 co-trans-fected prior to the induction ofYAP2 WT (Fig. 3C, fourth bar).Lats2 fully replaced Lats1 in thisassay (Fig. 3C, seventh bar), suggest-ing that Lats2 has the same functionas Lats1. The role of Lats1 andMst2 kinases as upstream factorsof YAP was further confirmed bythe observation that both theMst2kinase-dead mutant (D164A) andthe Lats1 amino-terminal portion(which lacks kinase activity) werebarely able to diminish YAP2function (Fig. 3C, fifth and sixthbars, respectively). The data pre-sented in Figs. 2 and 3 suggest thatupon activation by Mst2 kinase,Lats1 and Lats2 kinases phospho-rylate YAP at Ser-127 and inacti-vate its function as a stimulator ofcell detachment.

FIGURE 3. Induction of YAP2 expression results in reduced cell attachment, which is rescued by Mst2 andLats1/Lats2. A, the expression of YAP2 leads to significant detachment of cells. The expressions of controlvector, YAP2 WT, YAP2 1&2 WW*, YAP2 S127A, and YAP1 WT were individually induced in HEK293 cells andmaintained in DMEM containing 1% FBS for 96 h. After the removal of detached cells, attached cells weretrypsinized and their numbers counted. The ratios of number of induced cells to number of noninduced cells ineach protein are shown. The lower panel indicates immunoblotting (IB) with YAP antibody to verify the expres-sion of induced proteins. B, reduced levels of endogenous YAP in HEK293 cells promoted cell growth. ControlRNAi oligo or YAP-specific RNAi oligo was transfected to HEK293 cells. 96 h after the transfection, cells weretransferred into new plates. 24 h later, the medium was changed to DMEM containing 1% FCS and cells weremaintained for 5 days. The graph shows that the number of cells increased in the 5-day interval. Expression ofYAP and GAPDH was monitored by immunoblotting. C, Lats1 and Mst2 cooperate to abolish the effects of YAP2expression. The indicated FLAG-tagged proteins were transfected into HEK293 cells that express YAP2 WT in aninducible system. At 24 h post-transfection, the cells were distributed into new plates, and the expression of YAP2WT was induced by tetracycline. 96 h post-induction, cells were trypsinized and analyzed as described in A. Theexpression of induced YAP2 WT and transfected FLAG-tagged proteins was monitored by immunoblotting.




http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

Expression of YAP2 inHEK293Cells Promotes Apoptosis—Asshown in the cell counting assay described in Fig. 3, when weobserved YAP2 WT induced in HEK293 cells, the 96-h timepoint exhibited the most significant change in the ratio ofattached versus floating cells. However, at the 48-h time pointthe number of floating cells was minimal. Therefore, the cellsbegan to detach from the plate during the 48–96-h time period.Knowing that YAP has been implicated in the apoptotic path-way by stabilizing p73 (35–37), we decided to examine whetherapoptosis could be detected in cells overexpressing YAP2. Weassayed the total population of cells that included both theattached cells and floating cells. It is known that the 113-kDapoly(ADP-ribose) polymerase (PARP) is cleaved during apo-ptosis into 89- and 24-kDa fragments, serving as amarker of celldeath. In the absence of YAP2 WT, the cleavage fragment ofPARP was barely detectable (Fig. 4A, upper panel, third lane).However, in the presence of YAP2 WT, the 89-kDa cleavagefragment was clearly detectable (Fig. 4A, upper panel, fourthlane), suggesting that expression of YAP2 promotes apoptosisin HEK293 cells. YAP2 S127A was also capable of generatingthe cleaved fragment of PARP (Fig. 4A, upper panel, eighthlane). However, when the WW domains of YAP2 weremutated, the cleaved band of PARP was barely detectable (Fig.4A, upper panel, sixth lane). This result suggests that WWdomains of YAP2 play a crucial role in promoting apoptosis.Interestingly, YAP1 failed to generate a cleaved fragment ofPARP (Fig. 4A, upper panel, 10th lane).

To rule out the possibility that the YAP-induced apoptosis isanHEK293 cell line-specific phenomenon,we used another cellline, NIH3T3, in which we expressed YAP1 WT, YAP2 WT,YAP2 1&2WW*, and YAP2 S127Amutant and control vector,each in an inducible fashion. The cell counting assay and theapoptotic assay with PARP antibodies were performed in thesame manner as for HEK293 cells. The data showed thatNIH3T3 cells behaved similarly to HEK293 (Fig. 4B), althoughthe observed effects of YAP2 WT expression in NIH3T3 cellswere not as strong as those in HEK293 cells. The proapoptotictendency of cells with overexpressed YAPwas common in bothcells. The fact that YAP2 S127A, a mutant that localizes in thenucleus, was a strong proapoptotic factor in both cells suggeststhat the nuclear localization of YAP2 is crucial for its proapo-ptotic function.To determine when the apoptotic markers are detected after

the induction of YAP2 WT, we analyzed the time course of itseffects in HEK293 cells (Fig. 4C). There was almost no appear-ance of PARPor active caspase-3 at the 24-h timepoint after theYAP2 WT induction. However, at the 48-h time point, a weaksignal of PARP cleavage was detected, and active caspase-3 wasclearly detected in cell lysates with YAP2WT. At that point wedid not observe cell detachment. PARP cleavage was clearlydetectable, and cell detachment began to occur at 72 h andbecame robust at the 96-h time point.The up-regulation of cyclin E was shown previously as a

downstream effect of YAP overexpression, which leads togrowth promotion and neoplastic transformation (12, 31).Therefore, we decided to determine the levels of cyclin E inour system. In contrast to previous observations, YAP2 WT

overexpression did not up-regulate cyclin E in HEK293 cells(Fig. 4C).Because the results shown in Fig. 4A demonstrate a correla-

tion with the data presented in Fig. 3A, we considered that thecell counting assay might measure the degree of apoptosis inHEK293 cells. YAP2 WT and YAP2 S127A are competent interms of promoting apoptosis, whereas YAP2 1&2 WW* andYAP1 lack this ability. Next, we investigated the effects of Lats1andMst2 on PARP cleavage, because the effect of YAP2WTonreduced attachment of cellswas rescued byLats1 andMst2 (Fig.3C). As anticipated, the ability of YAP2 WT to promote PARPcleavage was completely abrogated when both Lats1 and Mst2were present (Fig. 4D, upper panel, compare fourth and secondlanes).Both WW Domains in YAP2 Are Required to Bind to p73—

Our data indicate that YAP2 promotes apoptosis of HEK293cells in a serum-dependent fashion. This proapoptotic functionwas decreased when both Lats1 and Mst2 were co-expressedwith YAP2 and phosphorylated at Ser-127.To elucidate the downstream signaling factors of YAP,which

are implicated in apoptotic function, we focused on p73, amember of the p53 family. It was shown that YAP interactsfunctionally with p73 and increases the stability and accumula-tion of p73 in response to DNA damage (35–37). However, theprevious study did not determine whether theWWdomains ofYAP2 played a role in p73 stabilization. YAP1 WT and YAP2WT, as well as S127A, S127D and WW domain mutants, weretransiently expressed in HEK293 cells together with HA-p73.The cell lysates were immunoprecipitated with FLAG antibodyfollowed by immunoblotting usingHA antibody (Fig. 5A). OnlyYAP2 WT, YAP2 S127A, and YAP2 S127D formed a complexwith p73, whereas all other YAPWWdomainmutants failed toprecipitate HA-p73. Surprisingly, YAP1 WT also failed to pre-cipitate p73. This result indicates that YAP2, with intact WWdomains, is a cognate partner of p73.Functional WW Domains of YAP2 Are Required for Stabili-

zation of p73—The fact that YAP2 with intact WW domainscould bindp73promptedus to further study the effects of YAP2onp73.As p73was shown to be stabilized byYAP in response toDNA damage (35–37), we decided to investigate whether sta-bilization of p73 by YAP2 takes place in our own cellular sys-tem. As before, we challenged HEK293 cells with low serum(1%). YAP2WTwas expressed as an inducible protein, and cellswere transfected with HA-p73. Expression of YAP2 wasinduced, and the levels ofHA-p73weremonitored as a functionof time (Fig. 5B, upper panel). After 72 h, the expression ofHA-p73 showed a 2.21-fold difference between YAP2-inducedand noninduced cells (Fig. 5B, upper panel, compare sixth lanewith fifth lane). This difference became larger (2.75-fold) at the96-h time point (Fig. 5A, upper panel, compare eighth lanewithseventh lane). In the absence of YAP2, the expression level ofHA-p73 decreased, suggesting a stabilizing effect of YAP2 onp73.As we already knew that the WW domains of YAP2 are

indispensable in promoting apoptosis (Fig. 4A) and bindingto p73 (Fig. 5A), we looked into the role of WW domains tostabilize p73. The induced expression of YAP at 96 h repre-sented the ideal time point to visualize stabilization of p73 by




http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

FIGURE 4. YAP2 promotes apoptosis. A, YAP2 WT and YAP2 S127A generate a cleaved fragment of PARP. The expressions of control vector, YAP2 WT, YAP21&2 WW*, YAP2 S127A, and YAP1 WT were individually induced in HEK293 cells and maintained in DMEM containing 1% FBS for 96 h. Crude cell extracts wereresolved on SDS-PAGE and immunoblotted (IB) with PARP antibody (upper panel), YAP antibody (middle panel), and GAPDH antibody (lower panel). B, inductionof YAP2 expression results in reduced cell attachment in NIH3T3 cells. The expression of each construct, control vector, YAP2 WT, YAP2 1&2 WW*, YAP2 S127A,and YAP1 WT, was individually induced in NIH3T3 cells maintained in DMEM containing 2.5% FBS for 96 h. After the removal of detached cells, attached cellswere trypsinized and their numbers counted. The ratios of number of induced cells to number of noninduced cells for each induced protein are shown. Thelower panels show Western blots that verify the expression of the indicated proteins. C, time course of the effects of YAP2 WT induction in HEK293 cells. Theexpression of YAP2 WT was induced in HEK293 cells and maintained in DMEM containing 1% FBS for the indicated periods of time. After the removal ofdetached cells, attached cells were trypsinized and their numbers counted. The ratios of the number of induced cells to number of noninduced cells are shown.The lower panels show immunoblots verifying the expression of the indicated proteins. D, the combination of Lats1 and Mst2 spoils the ability of YAP2 togenerate cleaved fragment of PARP. Either FLAG control vector or FLAG-Lats1 with Flag-Mst2 were transfected into HEK293 cells capable of expressing YAP2WT in an inducible system. 24 h after transfection, the cells were distributed into new plates, and the expression of YAP2 WT was induced by tetracycline. 96 hpost-induction, cells were trypsinized and analyzed as described in A. The cleaved fragment of PARP was monitored (upper panel). The expression of inducedYAP2 WT, transfected FLAG-tagged proteins, and GAPDH was also checked by immunoblotting.




http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

YAP2 WT (Fig. 5B). As we expected, the amount of p73 wasincreased when YAP2 WT or YAP2 S127A were induced,whereas YAP2 1&2WW* and YAP1WT failed to stabilize p73

(Fig. 5C). As shown previously (37),we also used cycloheximide treat-ment of cells to enhance the detec-tion of protein stability and showedthat the amount of HA-p73 clearlyincreased in the presence of YAP2or YAP2 S127A (Fig. 5D). Thesedata further support our results(Fig. 5C).To investigate whether p73 is

the major molecule to take overthe proapoptosis function ofYAP2, we conducted RNAi treat-ment in HEK293 cells and evalu-ated its effect on cell detachmentin the presence of YAP2WT. Afterthe p73-directed RNAi treatment,the expression of endogenous p73was completely abrogated (Fig. 5E).The p73-specific RNAi oligo or con-trol RNAi oligo was transfected intoHEK293 cells followed by the induc-tion of YAP2 WT expression. After96 h of culture inDMEMcontaining1% FCS, the endogenous p73 wasnot detectable in either case (datanot shown). This could possibly bedue to the short half-life of endoge-nous p73. The cells were counted asdescribed for Fig. 3. When controlRNAi was transfected, the ratio ofattached cells with induced YAP2WT to attached cells withoutinduced YAP2 WT was 0.525 (Fig.5F). However, when p73 RNAi wastransfected, the ratio was increasedto 0.735 (Fig. 5F), indicating that theproapoptotic function of YAP2 waspartially impaired when p73 wasremoved. This result supports therole of YAP2 in promoting apopto-sis via p73. In sum, YAP2 binds top73 viaWWdomains, leading to thestabilization of p73 and promotionof apoptosis.Lats1 Controls the Localization of

YAP2 in HEK293 Cells—YAP is atranscriptional co-activator, and itsprotein product is detected both inthe cytoplasm and the nucleus (29).The WW domain of YAP interactswith several transcription factors,and its localization in the nucleuscorrelates with its co-activating rolein transcription (30, 48, 49). The

S127A YAP mutant was shown to enter the nucleus and stim-ulate transcription more efficiently than the WT YAP (29).because Lats1 forms a complex with YAP through a PPPY

FIGURE 5. YAP2 is able to stabilize p73. A, both WW domains of YAP2 are required to bind to p73. HA-p73 withthe indicated YAPs was transfected into HEK293 cells. Lysates were immunoprecipitated (IP) with FLAG anti-bodies, resolved on SDS-PAGE, and immunoblotted (IB) with HA antibodies (upper panel). The expression levelsof total HA-p73 (middle panel) and FLAG-tagged proteins (lower panel) were also monitored. B, time course ofdegradation of HA-p73 in the presence of YAP2. HEK293 cells that express YAP2 WT in an inducible system weretransfected with HA-p73. 24 h later, the cells were plated in fresh DMEM containing 1% FBS, blasticidin (5�g/ml), and zeocin (200 �g/ml). Tetracycline (1 �g/ml) was added to the medium to induce the expression ofYAP2 WT. At 72 h and 96 h after induction, the cells were harvested followed by immunoblotting usingantibodies of HA (upper panel), YAP (middle panel), or GAPDH (lower panel). C, the WW domains of YAP2 arerequired to stabilize the expression of p73. HEK293 cells that express control vector, YAP2 WT, YAP2 1&2 WW*,YAP2 S127A, or YAP1 WT in an inducible system were transfected with HA-p73 followed by analysis asdescribed in B. Tetracycline was added to the medium for 96 h. D, YAP2 stabilizes p73. HA-p73 with theindicated YAPs was transfected into HEK293 cells, and each plate was divided into two. 24 h later, one plate washarvested, and 300 �g/ml of cycloheximide (CHX; Sigma) was added to the other plate and harvested afteradditional 24 h. Cell lysates were immunoblotted using antibodies to HA (upper panel), YAP (middle panel), orGAPDH (lower panel). E, endogenous p73 was removed by p73-specific RNAi. Control RNAi oligo or p73-specificRNAi oligo was transfected to HEK293 cells that express YAP2 WT in an inducible system, and the expression ofendogenous p73 was monitored by p73 antibody. F, removal of endogenous p73 weakens proapoptotic functionof YAP2. 48 h after the transfection of RNAi oligos in E, cells were scattered into fresh plates and maintained in DMEMcontaining 1% FCS. Expression of YAP2 WT was induced by adding tetracycline. 96 h later, the numbers of cells werecounted, and expression of YAP2 WT was monitored as described in the legend for Fig. 3.




http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

sequence-WW domain association, we suspected that thelocalization of YAP is affected by Lats1.To characterize human orthologs of the Drosophila Hpo

pathway in terms of protein localization, we expressed the indi-vidual proteins WW45, Mst2, Lats1, and YAP2 as GFP-taggedproducts in HEK293 cells and determined the locale of eachprotein by fluorescence microscopy (Fig. 6A). WW45, Mst2,and Lats1 localized in the cytosol. However, GFP-YAP2 wasdetected in both the nucleus and cytosol. We divided cells thatexpress GFP-YAP2 into two categories: “localized in thecytosol” and “localized in the nucleus.” 65.3% of the cells thatexpressed GFP-YAP2 localized in the cytosol, whereas 34.7% ofthem localized in the nucleus. Representative images of proteinlocalizations are shown in Fig. 6A.To know whether Lats1 affects the localization of YAP2 in

cells, pDsRed-YAP2 WT or pDsRed-YAP2 S127A wasco-transfected with GFP-Lats1 WT or GFP-Lats1 PY1*&2*(Fig. 6B). Using fluorescence microscopy, the localizations ofpDsRed-YAP2WT, pDsRed-YAP2 S127A (Fig. 6B, upper row),

and GFP-tagged proteins (Fig. 6B, lower row) were monitored.In the presence of GFP-Lats1 WT, the pDsRed-YAP2 WT sel-dom localized in the nucleus, and the silhouette of the nucleuswas clearly discernible when pDsRed-YAP2 was observed incells. The percentage of cells in which pDsRed-YAP2 WT wasobserved in the nucleus was only 10.7% (Fig. 6, B and C). How-ever, when GFP-Lats1 PY1*&2* was present, the localization ofpDsRed-YAP2WT changed. The percentage of pDsRed-YAP2WT localized in the nucleus increased significantly to 35.3%(Fig. 6, B and C). A similar tendency was observed when YAP2S127A was expressed instead of YAP2 WT. The YAP2 S127Aprotein is likely to localize in the nucleus; 38.3% of YAP2 S127Alocalized in the nucleus in the presence of GFP-Lats1 WT.However, this percentage dramatically increased to 83.0% inthe presence of GFP-Lats1 PY1*&2*, a mutant that lacks theability to associate with YAP2.These results suggest that Lats1 prevents YAP2 from enter-

ing the nucleus and that the binding ability of Lats1 to YAP2controls the localization of YAP2. The regulation of YAP local-ization is critical for its function; the most recent report impli-cates YAP in the control of cell contact inhibition (34). Thisactivity of YAP correlated well with the subcellular localizationof YAP.

DISCUSSION

The Hpo pathway controls the intrinsic size of organs bycoordinating proliferative and apoptotic signals. Thus theelucidation of the molecular mechanism of this pathwayshould help us in understanding the process of normal devel-opment and its numerous pathological aberrations, includ-ing cancer.We reconstituted the core of theDrosophilaHpo pathway in

human epithelial kidney cells, focusing on the role of WWdomain-mediated complexes, which are particularly prevalentin this pathway. By expressing tagged human orthologs of theHpo pathway in HEK293 cells, we documented that Mst2 andLats1/2 kinases act upstream of YAP1/2 transcriptional co-ac-tivators. These two kinases cooperate in phosphorylating YAPs(YAP1 and YAP2) at the Ser-127 site. Akt kinase is known toinduce accumulation of YAP in the cytosol rather than in thenucleus by phosphorylating YAP at Ser-127 (45). Our resultsshow that Lats1 by itself can anchor YAP2 in the cytosol, andthe direct binding between PPXY motifs of Lats1 and WWdomains of YAP2 controls the localization of YAP2. Lats1retains YAP2 in the cytosol in two different ways: by phospho-rylating YAP at Ser-127 and creating the 14-3-3 anchor site andby forming a direct protein-protein complex. The YAPs thatevade contacts with Lats1 enter the nucleus freely and promoteapoptosis of cells by stabilizing p73, a proapoptotic member ofthe p53 family (Fig. 7).The following aspects of this study deserve further comment:

(i) the apparent paradox of YAP signaling as an oncogene orpromoter of apoptosis; (ii) the role of the WW domain in thespecificity of signaling complexes of the Hpo pathway; (iii) themodulating function of isoforms of Mst, Lats, and YAP inthe Hpo pathway output; (iv) the differentiation between apo-ptosis versus anoikis in the cell assay; and (v) the usefulness ofthe apoptotic readout in HEK293 cells for the identification of

FIGURE 6. Lats1 controls the localization of YAP2 in HEK293 cells. A, local-ization of components of the Hpo pathway. WW45, Mst2, Lats1, and YAP2were fused to GFP and expressed in HEK293 cells. The cells were observedunder a fluorescence microscope. Bar, 20 �m. B, effects of Lats1 on the local-ization of YAP2. pDsRed-YAP2 WT and pDsRed-YAP2 S127A were co-trans-fected with GFP-Lats1 WT or GFP-Lats1 PY1*&2* in HEK293 cells. The localiza-tion of pDsRed-YAP2 WT and pDsRed-YAP2 S127A (upper panels) and that ofGFP-tagged proteins (lower panels) was observed. Bar, 20 �m. C, graphic rep-resentation of results shown. The ratios of nuclear localization of pDsRed-YAP2 WT and pDsRed-YAP2 S127A in the presence of GFP-Lats1 WT or GFP-Lats1 PY1*&2* are indicated. This observation was repeated three timesindependently, and about 100 cells were observed in each case.




http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

the upstream factors that initiate the cascade of Hpo pathway,ultimately controlling the size of organs.Recent reports have suggested that human YAP is a candi-

date oncogene because it is a part of the 11q22 amplicondetected in several cancer types (31, 32). Gene expressionstudies have shown that in liver cancer, YAP is co-amplifiedwith a gene that encodes an inhibitor of apoptosis, cIAP1,and both contribute to the development of hepatocellularcarcinoma (31). In MCF-10A mammary epithelial cells, theoverexpression of YAP was shown to cause oncogenic trans-formation (32). These two observations seem to contradictprevious reports of YAP acting as an activator of apoptosis inresponse to DNA damage (35–37). However, we do not con-sider these reports contradictory. The “YAP paradox” is con-sistent with the still emerging, yet widely accepted notionthat signaling proteins often play diverse roles depending onthe cellular context, i.e. the repertoire of signaling molecules

available in given cells or tissues. The comparison of signal-ing proteins with electronic components in which the effectsdepend on their specific placement within the entire electri-cal circuit is an accepted analogy in the biology of signaltransduction systems (50). Therefore, YAP should be consid-ered a facultative oncogene. In liver cells, but not in othercells of the body that contain the same 11q22 amplicon, YAPmay cooperate not only with cIAP1 but also with liver-spe-cific genes to cross the threshold required in hepatocytes foroncogenesis. Similarly, the overexpression of YAP in mam-mary epithelial cells elicits transformation, perhaps becauseYAP collaborates in these cells with the amplified myc (51).Simple overexpression of YAP in certain epithelial or fibro-blastic cells (HEK293, NIH3T3) does not cause oncogenictransformation, unless additional genes are co-expressed oramplified (32, 52). The presence of overexpressed c-myc insystems where YAP caused transformation suggested thatfor YAP to be oncogenic requires the expression of an addi-tional gene that encodes the anti-apoptotic protein (such asc-Myc, cyclin E, or Runx2). Our work supports a model offunctional dichotomy of YAP. If the interaction partners ofYAP (such as c-myc or cyclin E) in given cells or organs areanti-apoptotic, then YAP is oncogenic. If partners of YAP areproapoptotic, such as p73, and cyclin E is not induced byYAP, then YAP is proapoptotic. In HEK293 and NIH3T3cells, YAP interacts with p73 and does not induce cyclin E.Because Drosophila does not have the p73 homolog, Yorkie(dYAP) may be unable to engage in proapototic signalingdirectly. Moreover, cell-specific and varying levels of endo-genously expressed p63 and p73 (members of the p53 familythat interact with YAP) may tip the balance from transfor-mation to apoptosis in mammalian cells (53). The tissue-specific expression of YAP targets, namely the members ofthe TEAD/TEF family of transcription factors, also could beresponsible for the dichotomy of YAP/Yki function as onco-genic or proapoptotic factor (54–57). Intuitively, it seemsthat a protein that has the capability to sense the signalingbackground of cells and mediate appropriately such diverseprocesses as proliferation or apoptosis would be ideal forcontrolling the size of organs and organisms during develop-ment and beyond. YAP and Yki are nuclear effectors ofthe Hpo pathway acting as rheostats for proliferation andapoptosis.TheWWdomain wasmapped in the human proteome using

a domain-peptide interaction screen (58). By using more than69,000 data points and assigning relative strength of bindingbetween 57 WW domains and 1830 proline-rich peptides cor-responding to known proteins, several new complexes andstrings of interactors emerged. Yet none of the map-predictedpathways or currently known signaling pathways is as “rich” inthe WW domain-PPXY/PPXF complexes as the Hpo pathway.All components of the core of the Hpo pathway contain eitherWW domains or their cognate ligand motifs, PPXY or PPXF(Figs. 1A and 7). Two aspects of the WW domain should beconsidered in future studies of the pathway. (i) Because theWW domain complexes can be inhibited with small non-pep-tide or peptoid compounds, the use of such inhibitors in thebiochemical analysis of the pathway should be illuminating

FIGURE 7. Schematic representation of the Hpo signaling pathway,which functions to promote survival of cells. All components investi-gated here are shown in solid gray with names in white letters and numbers.Other proteins depicted by symbols without any background and withblack letters and/or numbers represent components shown previously asregulators of the Hpo pathway. K denotes kinase activity, and P in a circledenotes a phosphorylation site. Protruding triangles denote PPXY/PPXFmotifs, and “cut out” triangles represent WW domains that recognizePPXY/PPXF motifs. WW45 acts as a scaffold protein for assembling Mst2and Lats1 kinases. With the apparent help of WW45, Mst2 phosphorylatesLats1, which in turn phosphorylates YAP2 at Ser-127 (the same site onYAP1 is phosphorylated by Akt kinase (44)). Phosphorylated YAP2 isanchored in the cytoplasm by 14-3-3 protein. Apart from phosphorylation,Lats1 by itself can accumulate YAP2 in the cytoplasm. YAP2 free of phos-phorylation and association with Lats1 is able to enter the nucleus andpromote transcription of proapoptotic genes. The YAP2-p73 complex sta-bilizes p73 and prevents its degradation, which is mediated by Itch, acompeting WW domain-containing E3 ubiquitin ligase.




http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

(59). (ii) In addition, Lats1/2 kinases and nuclear targets ofYAPs should be analyzed for potential Y phosphorylation at thePPXY sites leading to a regulatory event that negatively controlsthe WW domain complexes (43, 60).The reason for multiple isoforms of the proteins Mst, Lats,

and YAP in the human pathway, compared with the singleorthologs in theDrosophila pathway, is not clear.Our data indi-cate that YAP2 seems to be amore potent regulator of apoptosisthan YAP1. Most recently, YAP1 (but not YAP2) was shown tobe expressed in stem cells and was implicated in the expan-sion of undifferentiated progenitor cells (61). Perhaps otherdifferences apply to isoforms of Mst and Lats kinases. Smallchanges in the amino acid sequence among the isoforms maytranslate into important changes in their activities. Alterna-tively, the isoforms represent tissue-specific variants that areexpressed mostly as single proteins in a given tissue or organ.Additional work needs to be done on the tissue-specificexpression of the mammalian orthologs of the Hpo pathwayin order to understand the potential regulatory function ofthe isoforms on the output of the pathway. It should be notedthat more isoforms of the Hpo pathway could be identified inthe fly and in human proteomes in the near future. We areaware of the isoform of Yki, which contains oneWWdomainand could be considered a YAP1 ortholog. In mammals,there are additional isoforms of YAP, which either containsmall insertions (29) or large deletions of the transcriptionalactivation domain and are involved in controlling cell deathin neurons (49).Because in our biological readout we observed both cell

detachment and apoptosis, we needed to consider the possibil-ity that what we observed was a specific kind of cell death,known as anoikis. Anoikis is a program of detachment-inducedcell death. To differentiate between anoikis and apoptosis, weseparated the attached cells from the floating cells after 96 h ofYAP2 overexpression and probed for PARP cleavage products.We found a similar level of PARP cleavage in attached and infloating cells (see supplemental Fig. 1). Active caspase-3 wasdetected in both the attached cells and the floating cells whenthe expression of YAP2 WT was induced, whereas it was notdetected in the control, noninduced cells. These resultsstrongly suggest that YAP2 causes true apoptosis and notanoikis. We also preformed a DNA ladder assay on the YAP2-overexpressing cells and confirmed the apoptosis by an alter-native biochemical test (data not shown).The apoptotic readout of the Hpo pathway in HEK293 cells

described here represents a useful experimental system for theidentification of the putative upstream receptor or membraneprotein that initiates an entire signaling cascade and ultimatelycontrols the size of organs. Because HEK293 cells are easy tomaintain and the efficiency of DNA transfection in these cells ishigh, one could design a simple screen for genes that encodemembrane proteins and subsequently affect the apoptoticresponse significantly. Considering the high level of conserva-tion among the known orthologs that constitute the pathway,priority in such a screen should be given to those genes thatencode membrane proteins and are well conserved betweenflies and mammals (62–67).

Acknowledgments—We particularly thank Dr. Tony Hunter fordetailed and constructive comments on the first version of our manu-script.Wealso thank our colleagues Rick Stahl andDr. ThomasHynesfor expert help with the fluorescent microscopy and Dr. Ray Birge foradvice on apoptotic assays. We thank Dr. Tadashi Yamamoto forpcDNA3-FLAG-Lats2,Dr. Sumio Sugano for pME18SFL-WW45 con-structs, Dr. Herman Sillje for pcDNA3.1-FLAG-Mst2WTandD164Amutant constructs, Dr. Giovanni Blandino for pcDNA3HAp73� con-struct, Dr. SubhamBasu for advice on the YAP-phospho-specific anti-body, and Dr. Marc Therrien for valuable discussions.

REFERENCES1. Leevers, S. J., and McNeill, H. (2005) Curr. Opin. Cell Biol. 17, 604–6092. Ryoo, H. D., and Steller, H. (2003) Nat. Cell Biol. 5, 853–8553. O’Neill, E. E., Matallanas, D., and Kolch, W. (2005) Cancer Res. 65,

5485–54874. Vidal, M., and Cagan, R. L. (2006) Curr. Opin. Genet. Dev. 16, 10–165. Pan, D. (2007) Genes Dev. 21, 886–8976. Harvey, K., and Tapon, N. (2007) Nat. Rev. Cancer 7, 182–1917. Saucedo, L. J., and Edgar, B. A. (2007)Nat. Rev. Mol. Cell Biol. 8, 613–6218. Dong, J., Feldmann, G., Huang, J., Wu, S., Zhang, N., Comerford, S. A.,

Gayyed, M. F., Anders, R. A., Maitra, A., and Pan, D. (2007) Cell 130,1120–1133

9. Meignin, C., Alvarez-Garcia, I., Davis, I., and Palacios, I. M. (2007) Curr.Biol. 17, 1871–1878

10. Bandura, J. L., and Edgar, B. A. (2008) Dev. Cell 14, 315–31611. Matallanas, D., Romano, D., Hamilton, G., Kolch, W., and O’Neill, E.

(2008) Cell Cycle 7, 879–88412. Huang, J., Wu, S., Barrera, J., Matthews, K., and Pan, D. (2005) Cell 122,

421–43413. Edgar, B. A. (2006) Cell 124, 267–27314. Bork, P., and Sudol, M. (1994) Trends Biochem. Sci. 19, 531–53315. Tapon, N., Harvey, K. F., Bell, D.W.,Wahrer, D. C., Schiripo, T. A., Haber,

D. A., and Hariharan, I. K. (2002) Cell 110, 467–47816. Pantalacci, S., Tapon, N., and Leopold, P. (2003)Nat. Cell Biol. 5, 921–92717. Udan, R. S., Kango-Singh,M., Nolo, R., Tao, C., andHalder, G. (2003)Nat.

Cell Biol. 5, 914–92018. Harvey, K. F., Pfleger, C.M., andHariharan, I. K. (2003)Cell 114, 457–46719. Sudol, M., and Hunter, T. (2000) Cell 103, 1001–100420. Jia, J., Zhang, W., Wang, B., Trinko, R., and Jiang, J. (2003) Genes Dev. 17,

2514–251921. Graves, J. D., Gotoh, Y., Draves, K. E., Ambrose, D., Han,D. K.,Wright,M.,

Chernoff, J., Clark, E. A., and Krebs, E. G. (1998) EMBO J. 17, 2224–223422. St John, M. A., Tao, W., Fei, X., Fukumoto, R., Carcangiu, M. L., Brown-

stein, D. G., Parlow, A. F., McGrath, J., and Xu, T. (1999) Nat. Genet. 21,182–186

23. Deng, Y., Pang,A., andWang, J. H. (2003) J. Biol. Chem.278, 11760–1176724. McPherson, J. P., Tamblyn, L., Elia, A., Migon, E., Shehabeldin, A., Maty-

siak-Zablocki, E., Lemmers, B., Salmena, L., Hakem,A., Fish, J., Kassam, F.,Squire, J., Bruneau, B. G., Hande,M. P., andHakem, R. (2004) EMBO J. 23,3677–3688

25. Bothos, J., Tuttle, R. L., Ottey,M., Luca, F. C., andHalazonetis, T.D. (2005)Cancer Res. 65, 6568–6575

26. Takahashi, Y., Miyoshi, Y., Takahata, C., Irahara, N., Taguchi, T., Tamaki,Y., and Noguchi, S. (2005) Clin. Cancer Res. 11, 1380–1385

27. Wu, S., Huang, J., Dong, J., and Pan, D. (2003) Cell 114, 445–45628. Sudol,M., Bork, P., Einbond, A., Kastury, K., Druck, T., Negrini,M., Hueb-

ner, K., and Lehman, D. (1995) J. Biol. Chem. 270, 14733–1474129. Komuro, A., Nagai, M., Navin, N. E., and Sudol, M. (2003) J. Biol. Chem.

278, 33334–3334130. Yagi, R., Chen, L. F., Shigesada, K.,Murakami, Y., and Ito, Y. (1999) EMBO

J. 18, 2551–256231. Zender, 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., and Lowe, S. W. (2006) Cell 125, 1253–1267




http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/cgi/content/full/M804380200/DC1

http://www.jbc.org/

32. Overholtzer, M., Zhang, J., Smolen, G. A., Muir, B., Li, W., Sgroi, D. C.,Deng, C. X., Brugge, J. S., and Haber, D. A. (2006) Proc. Natl. Acad. Sci.U. S. A. 103, 12405–12410

33. Zhang, J., Smolen, G. A., and Haber, D. A. (2008) Cancer Res. 68,2789–2794

34. Zhao, 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., andGuan, K. L. (2007) Genes Dev. 21, 2747–2761

35. Strano, S., Munarriz, E., Rossi, M., Castagnoli, L., Shaul, Y., Sacchi, A.,Oren, M., Sudol, M., Cesareni, G., and Blandino, G. (2001) J. Biol. Chem.276, 15164–15173

36. Strano, S., Monti, O., Pediconi, N., Baccarini, A., Fontemaggi, G., Lapi, E.,Mantovani, F., Damalas, A., Citro, G., Sacchi, A., Del Sal, G., Levrero, M.,and Blandino, G. (2005)Mol. Cell 18, 447–459

37. Levy, D., Adamovich, Y., Reuven, N., and Shaul, Y. (2006) Cell DeathDiffer. 14, 743–751

38. Danovi, S. A., Rossi, M., Gudmundsdottir, K., Yuan, M., Melino, G., andBasu, S. (2008) Cell Death Differ. 15, 217–219

39. Levy, D., Adamovich, Y., Reuven, N., and Shaul, Y. (2008) Mol. Cell 29,350–361

40. Chen, H. I., and Sudol, M. (1995) Proc. Natl. Acad. Sci. U. S. A. 92,7819–7823

41. Espanel, X., and Sudol, M. (1999) J. Biol. Chem. 274, 17284–1728942. Sudol, M. (1994) Oncogene. 9, 2145–215243. Chen, H. I., Einbond, A., Kwak, S. J., Linn, H., Koepf, E., Peterson, S., Kelly,

J. W., and Sudol, M. (1997) J. Biol. Chem. 272, 17070–1707744. Chan, E. H., Nousiainen,M., Chalamalasetty, R. B., Schafer, A., Nigg, E. A.,

and Sillje, H. H. (2005) Oncogene 24, 2076–208645. Basu, S., Totty, N. F., Irwin,M. S., Sudol,M., andDownward, J. (2003)Mol.

Cell 11, 11–2346. Beausoleil, S. A., Jedrychowski, M., Schwartz, D., Elias, J. E., Villen, J., Li, J.,

Cohn, M. A., Cantley, L. C., and Gygi, S. P. (2004) Proc. Natl. Acad. Sci.U. S. A. 101, 12130–12135

47. Hao, Y., Chun, A., Cheung, K., Rashidi, B., and Yang, X. (2008) J. Biol.Chem. 283, 5496–5509

48. Vassilev, A., Kaneko, K. J., Shu,H., Zhao, Y., andDePamphilis,M. L. (2001)Genes Dev. 15, 1229–1241

49. Hoshino, M., Qi, M. L., Yoshimura, N., Miyashita, T., Tagawa, K., Wada,Y., Enokido, Y., Marubuchi, S., Harjes, P., Arai, N., Oyanagi, K., Blandino,

G., Sudol, M., Rich, T., Kanazawa, I., Wanker, E. E., Saitoe, M., and Oka-zawa, H. (2006) J. Cell Biol. 172, 589–604

50. Vogelstein, B., and Kinzler, K. W. (2004) Nat. Med. 10, 789–79951. Debnath, J., Muthuswamy, S. K., and Brugge, J. S. (2003) Methods 30,

256–26852. Vitolo, M. I., Anglin, I. E., Mahoney,W.M., Jr., Renoud, K. J., Gartenhaus,

R. B., Bachman, K. E., and Passaniti, A. (2007) Cancer Biol. Ther. 6,856–863

53. Rocco, J. W., Leong, C. O., Kuperwasser, N., DeYoung, M. P., and Ellisen,L. W. (2006) Cancer Cell 9, 45–56

54. Goulev, Y., Fauny, J. D., Gonzalez-Marti, B., Flagiello, D., Silber, J., andZider, A. (2008) Curr. Biol. 18, 435–441

55. Sun, S., Zhao, S., and Wang, Z. (2008) Dev. Dyn. 237, 270–27556. Wu, S., Liu, Y., Zheng, Y., Dong, J., and Pan, D. (2008) Dev. Cell 14,

388–39857. Zhang, L., Ren, F., Zhang, Q., Chen, Y., Wang, B., and Jiang, J. (2008) Dev.

Cell 14, 377–38758. Hu, H., Columbus, J., Zhang, Y., Wu, D., Lian, L., Yang, S., Goodwin, J.,

Luczak, C., Carter, M., Chen, L., James, M., Davis, R., Sudol, M., Rodwell,J., and Herrero, J. J. (2004) Proteomics 4, 643–655

59. Nguyen, J. T., Turck, C. W., Cohen, F. E., Zuckermann, R. N., and Lim,W. A. (1998) Science 282, 2088–2092

60. Sotgia, F., Lee,H., Bedford,M.T., Petrucci, T., Sudol,M., and Lisanti,M. P.(2001) Biochemistry 40, 14585–14592

61. Camargo, F. D., Gokhale, S., Johnnidis, J. B., Fu, D., Bell, G.W., Jaenisch, R.,and Brummelkamp, T. R. (2007) Curr. Biol. 17, 2054–2060

62. O’Neill, E., Rushworth, L., Baccarini, M., and Kolch, W. (2004) Science306, 2267–2270

63. Hamaratoglu, F., Willecke, M., Kango-Singh, M., Nolo, R., Hyun, E., Tao,C., Jafar-Nejad, H., and Halder, G. (2006) Nat. Cell Biol. 8, 27–36

64. Bennett, F. C., and Harvey, K. F. (2006) Curr. Biol. 16, 2101–211065. Silva, E., Tsatskis, Y., Gardano, L., Tapon, N., andMcNeill, H. (2006)Curr.

Biol. 16, 2081–208966. Willecke, M., Hamaratoglu, F., Kango-Singh, M., Udan, R., Chen, C. L.,

Tao, C., Zhang, X., and Halder, G. (2006) Curr. Biol. 16, 2090–210067. Matallanas, D., Romano, D., Yee, K., Meissl, K., Kucerova, L., Piazzolla, D.,

Baccarini, M., Vass, J. K., Kolch, W., and O’Neill, E. (2007) Mol. Cell 27,962–975




http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

Tsutomu Oka, Virginia Mazack and Marius SudolProtein (YAP)

Mst2 and Lats Kinases Regulate Apoptotic Function of Yes Kinase-associated

doi: 10.1074/jbc.M804380200 originally published online July 17, 20082008, 283:27534-27546.J. Biol. Chem.

10.1074/jbc.M804380200Access the most updated version of this article at doi:

Alerts:

When a correction for this article is posted•

When this article is cited•

to choose from all of JBC's e-mail alertsClick here

Supplemental material:

http://www.jbc.org/content/suppl/2008/10/07/M804380200.DC1

http://www.jbc.org/content/283/41/27534.full.html#ref-list-1

This article cites 67 references, 24 of which can be accessed free at


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/lookup/doi/10.1074/jbc.M804380200

http://www.jbc.org/cgi/alerts?alertType=citedby&addAlert=cited_by&cited_by_criteria_resid=jbc;283/41/27534&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/283/41/27534

http://www.jbc.org/cgi/alerts?alertType=correction&addAlert=correction&correction_criteria_value=283/41/27534&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/283/41/27534

http://www.jbc.org/cgi/alerts/etoc

http://www.jbc.org/content/suppl/2008/10/07/M804380200.DC1

http://www.jbc.org/content/283/41/27534.full.html#ref-list-1

http://www.jbc.org/

Documents

Mst2andLatsKinasesRegulateApoptoticFunctionofYes Kinase ... · first thing to catch our attention was the apparent paradox of YAP functioning either as an oncogene (31–34) or a