Ato o orsodne([email protected]) correspondence for *Author USA. USA. 68583, NE Lincoln, Biology, Oral amtyaino lkpii srqie o emsm assembly Roberts J. for Brett required is plakophilin of Palmitoylation ARTICLE RESEARCH ß 3782 2014 June 15 Accepted 2014; January 15 Received 3 devastating 1 have can plaque of the these function of or Disruption desmosomal include expression plakophilins. and proteins the plaque of and Desmosomal desmosome, contact. , set filament cell–cell of intermediate a the sites the to recruit with to of proteins interact plaque core proteins coordinately. function transmembrane transmembrane to form tissue ) the and the desmogleins of (i.e. cells the Desmosomal adjacent allow between and systems link cells structures cytoskeletal stress to adhesive filament mechanical these thought intermediate withstand Structurally, the widely 2012). to al., are ability et they the (Brooke tissues and these tissues, provide cardiac in found and junctions epithelial adhesive cell–cell prominent are INTRODUCTION Palmitoylation Plakophilin, Desmosome, WORDS: is KEY data components adhesion. desmosomal these and assembly of desmosome together, for important palmitoylation Taken that and adhesion. demonstrate components desmosomal desmosome endogenous decrease of localization disrupt proper to manner dominant-negative a in act palmitoylated plakophilin become to disrupts unable mutants plakophilin- plakophilin Finally, assembly. prevents desmosome dependent palmitoylation, and plaque desmosomal the prevents into incorporation residue single this of cysteine Mutation domain. repeat armadillo cysteine the desmosomal conserved in present a residue to palmitoylation plakophilin multiple of site the mapped that We borders. cell–cell at assembly demonstrate desmosome disrupts palmitoylation and palmitoylated lipid trafficking we are posttranslational components in Here, role a important poorly function. an is are plays junction that palmitoylation modification this Protein of contact. assembly understood. known, cell–cell the are desmosome regulating of the mechanisms of sites including components individual to proteins, the Although cytoskeleton plaque filament the desmosomal recruit turn, intermediate of in which, desmoplakin, host and plakoglobin plakophilins, a various with in interact found cadherins desmosomal of domains junctions cytoplasmic The tissues. adhesive epithelial prominent are Desmosomes ABSTRACT uaeu ilg,Toa efro nvriy hldlha A 90,USA. 19107, PA, Philadelphia, University, Jefferson Thomas Biology, Cutaneous yG Mahoney G. My plyIsiuefrRsac nCne n lidDsae,Oaa E68198, NE Omaha, Diseases, Allied and Cancer in Research for Institute Eppley of Department Dentistry, of College Center, Medical Nebraska of University The 04 ulse yTeCmayo ilgssLd|Junlo elSine(04 2,38–73doi:10.1242/jcs.149849 3782–3793 127, (2014) Science Cell of Journal | Ltd Biologists of Company The by Published 2014. 2 1 et .Johnson R. Keith , rse .Johnson E. Kristen , nvivo in 2 eateto emtlg and Dermatology of Department hraooi niiinof inhibition Pharmacologic . 1,3 1 n ae .Wh III Wahl K. James and ahenP McGuinn P. Kathleen , 99 aaae l,19;Toa n pih,2001). Speight, and Thomas 1996; al., normal et al., their et (Davies Harada desmosome from types are tumor Altered several detach 1999; in cells inappropriately. observed to been migrate has tumor ability assembly to the tumorigenesis, and acquire neighbors during is to strength disrupted re- believed adhesive is When or barrier 2012). epithelial re- altered the al., until et bed the (Thomason wound established cells the neighboring during into with migrate this adhesiveness and phenotype During their healing. decrease cells wound motile process, epidermal of a phase example, epithelialization For acquire cues. extracellular to keratinocytes response in cells neighboring 1996). al., et 2004; Ruiz al., 1997; et al., (Gerull et function and McGrath integrity desmosome on effects motn oei h euaino ucinasml and assembly junction of regulation the strength. in adhesive an role plays of plakophilins, important palmitoylation cell the specifically that to and suggest components, respect here suggesting desmosomal with presented manner data plakophilin-3, dominant-negative The weakens expressing adhesion. a cells in and A431 act of mutants in that plakophilin-2 of with plakophilin-3 wild-type compared expression adhesion Finally, and support desmosomal cells. to plakophilin-2 A431D plakophilins in mutant mutant in of assembly results inability of desmosome residue the plakophilin- site and and cysteine the plakophilin-2 3 of is this targeting membrane that of decreased plakophilin-3 Mutation and palmitoylation. plakophilin-2 cysteine cultured in conserved single in a residue identified palmitoylated have we indeed Additionally, cells. are components 1999). al., desmosomal et Toyoda 2010; al., (Sugimoto et date Tomatis 1996; to (LYPLA1, The al., identified et been thioesterases acyl have mammals. 23 LYPLAL1) cytosolic are and targets are LYPLA2 Three in protein thioesterases. There of genes deacylation protein palmitoyl 2012). for DHHC responsible as al., enzymes conserved et proteins (Ohno evolutionarily of the with (PATs) identified family proteins have acyltransferases substrate yeast (DHHC) in of Asp-His-His-Cys is studies association Recent Palmitoylation the membranes. 2011). cellular increase Chamberlain, to thought and (Greaves and linkage activity stability localization, thioester protein regulates including Palmitoylation labile features 2011). protein diverse a al., et through Aicart-Ramos in poorly residues (reviewed linked cysteine is (palmitate) specific are acid to fatty 16-carbon tumorigenesis a whereby modification and events during turnover understood. altered migratory mechanisms and in The stability 2011). normal result assembly, al., should desmosome et regulating signals (Roberts to these dynamics responsive also desmosome and are cues, desmosomes that extracellular likely is it Additionally, el oss h blt omdlt deiesrnt between strength adhesive modulate to ability the possess Cells ntecretsuy ehv eosrtdta multiple that demonstrated have we study, current the In posttranslational reversible a is palmitoylation Protein 1, 2 * itn Saowapa Jintana , 1 oetA Svoboda A. Robert , 1 , enovo de

Journal of Cell Science 09 age l,21) hs fot aeietfe several identified have Cravatt, efforts and These of (Martin 2010). landscape al., cells the et cultured identify Yang proteomic in 2009; to large-scale attempt proteins an utilized palmitoylated in have strategies studies screening several Recently, palmitoylated are components Desmosomal RESULTS ARTICLE RESEARCH eersle ySSPG,adidvda emsm n deesjnto opnnswr eetdb muoltaayi.Pp lkpii;Pg, plakophilin; Pkp, analysis. immunoblot by detected 100 E-; were without E-cad, components or desmoglein; junction with Dsg, adherens medium -2; ( and Dsc-2, without desmosome desmoplakin; and individual DP, (+) and plakoglobin; with SDS-PAGE, processed by and resolved samples were two into split palmitoylated. are were components Desmosomal 1. Fig. eosrtdta lkpii- n lkpii- r amtyae nteasneo -P n amtyaini erae nA3 el rw nm in grown cells A431 in decreased is palmitoylation and 2-BP, of absence the 50 in of palmitoylated absence are 50 or desmoglein-2. plakophilin-3 containing anti and presence and negative desmocollin-2 anti-plakophilin the as plakophilin-2 desmoplakin, isoform-specific in plakoglobin, served that or grown plakophilin-3, Aby) demonstrated HRP–streptavidin cells plakophilin-2, (No of by A431 antibody levels detected from the immunoprecipitating were prepared examine no proteins were with labeled lysates Immunoprecipitations and Cell reaction. SDS-PAGE, (C) click by Cu-catalyzed resolved the were using Immunoprecipitates cells label to added lkpii-,paolbnaddsoli- n2B-rae utrs h rtnX10slblt fdsooln2wsucagdfloig2-BP following unchanged was desmocollin-2 of solubility X-100 Triton The mean cultures. the 2-BP-treated show in Data desmoglein-2 and 50 plakoglobin containing plakophilin-3, medium in grown cells m -P B muolt E rtnX10islbe()ad-oul s rcin eepeae rmcnrlA3 utrs(MO rfo A431 from or (DMSO) cultures A431 control from prepared were fractions (s) -soluble and (p) Triton-X-100-insoluble (E) immunoblot. IB, 2-BP. M 6 ..( s.d. n m 5 7OY o 8hus lkpii- n lkpii- eeimnpeiiae I)fo ellsts n R–itnwas HRP–biotin and lysates, cell from (IP) immunoprecipitated were plakophilin-3 and Plakophilin-2 hours. 48 for 17-ODYA M xeiet) * experiments); 3 m -P muoltaayi eemndta hr a infcn nraei h oul rcino plakophilin-2, of fraction soluble the in increase significant a was there that determined analysis Immunoblot 2-BP. M P , 0.05. A 41cl yae eepeae,adAEwspromdt dniyplioltdpoen.Lysates proteins. palmitoylated identify to performed was ABE and prepared, were lysates cell A431 (A) 2 yrxlmn H) fe h ia tetvdnaaoepl-on h atrdproteins captured the pull-down, streptavidin–agarose final the After (HA). hydroxylamine ) clboi xhneasy clboi xhne(B)is (ABE) exchange Acyl-biotin desmosomal assay. that first exchange an using show We components acyl-biotin function. desmosomal to palmitoylated and identify to out assembly of chose effect desmosome set the determine on to We palmitoylation proteins and palmitoylated 2010). indeed palmitoylated were al., proteins potential et as (Yang components desmosomal m -Pfr1 or,adimnbo nlsswspromdto performed was analysis immunoblot and hours, 18 for 2-BP M ora fCl cec 21)17 7239 doi:10.1242/jcs.149849 3782–3793 127, (2014) Science Cell of Journal a -cat, a -; b b -cat, tblnwsue salaigcnrl D ABE (D) control. loading a as used was - b ctnn B 41clswr rw in grown were cells A431 (B) -catenin. treatment. bodies. controls. edium 3783

Journal of Cell Science lkpii-–F Rbrse l,21)wr rw nthe in expressing grown cells were A431 2011) 2-BP. al., of et presence (Roberts plakophilin-3–GFP the in plakophilin-2, components treatment for 2-BP pool by altered soluble 1E). of significantly (Fig. solubility not the The was desmoglein-2. to desmocollin-2 from and shift plakoglobin pool significant a plakophilin-3, was insoluble there X- 2-BP, with the Triton treated 0.5% cells containing In buffer 100. in solubility components the desmosomal and desmosomal examined plakophilin-2 of we of Additionally, palmitoylation 1D). of the (Fig. treatment in plakophilin-3 2-BP reduction that expression demonstrate control a to in of used resulted was that ABE the with 1C). compared (Fig. cells cells in 2-BP-treated in components reduction substantial dsolis2 emgen3addsooln2,wr indeed were desmocollin-2), (plakophilin-2, and cadherins desmosomal proteins desmoglein-3 pulldown as (desmogleins-2, well plaque Streptavidin as plakoglobin), desmosomal 2007). and plakophilin-3 the al., by that with et cysteine detected captured revealed (Wan are are palmitoylated antibodies proteins individual proteins specific previously and Biotin-labeled pulldown the biotin. streptavidin with of residues tagging by followed bonds covalent thioester hydrolyzing selectively by performed ARTICLE RESEARCH 3784 50 A431 from in al., prepared et lysates grown (Davda cell cells of manner analysis non-selective Immunoblot of a 2013). inhibits in palmitoylation treatment protein 2- 2-BP palmitoylation consequence, for global of a cells. as responsible inhibitor and, proteins, A431 are substrate irreversible global in which an inhibiting of acyltransferases, is assembly effect (2-BP) desmosome the bromopalmitate investigate on to palmitoylation sought we assembly desmosome Next, disrupts palmitoylation of Inhibition detected E- heart we components murine junction both addition, adherens in The likely In ABE shown). cadherin, not is palmitoylated. using (data plakophilin-2 it are tissue of Koch desmocollin-2, 1991) 1991; palmitoylation al., for al., et (Collins blot et were desmocollin-2b bands and the two desmocollin-2a Because in 1A). (Fig. detected cells A431 in palmitoylated u o lkpii-,orsuishr oue nplakophilin-2 on focused here plakophilin-3. studies and our plakophilin-1, plakophilin-3 not and but Because plakophilin-2 medium. express control endogenously in cells and in grown A431 cells detected from was prepared whereas plakophilin-3 Plakophilin-2 lysate 17-ODYA, cell or plakophilin-2 by biotin-labeled 1B). labeled no (Fig. specifically chemistry). were peroxidase plakophilin-3 blotting Cu(I)- horseradish (click by and detected (HRP)–streptavidin and were ligation SDS-PAGE proteins cycloaddition by biotin-labeled Staudinger separated were the proteins azide-alkyne Immunoprecipitated by 17-ODYA catalyzed to covalently been was azide added had Biotin (DMSO). 100 that were 17-ODYA lacking containing cells medium Proteins acid medium A431 2009). in palmitic of grown Cravatt, the lysates and from using immunoprecipitated (Martin cells 17-ODYA A431 analog labeled metabolically we on plakophilin of to palmitoylation adhesion. chose and that of assembly We role desmosome suggested cells. the A431 previously investigate in further Proteomics had to palmitoylation unable desmoplakin were controls. 2010) we detect however, al., palmitoylated; negative also was et desmoplakin as (Yang served analysis and palmitoylated et eeaie h oaiaino desmosomal of localization the examined we Next, nodrt ute ofr h amtyaino plakophilins, of palmitoylation the confirm further to order In b ctnnand -catenin m -Pdmntae htteei no is there that demonstrates 2-BP M a ctnnwr on ontbe not to found were -catenin m 7OY ri control in or 17-ODYA M lkpii-–F Pp3GP eegono ls oesisi medium in coverslips glass 50 on containing grown of were localization (Pkp-3/GFP) the borders. alters plakophilin-3–GFP cell 2-BP at by components palmitoylation desmosomal of Inhibition 2. Fig. opnnsi irpe tcl–elbres cl a:10 bar: Scale borders. cell–cell desmosomal at the of disrupted on localization is effect the components little whereas has E-cadherin, palmitoylation of and of localization (M–R) Inhibition (Pkp-2) (S–X). plakophilin-2 (DP) desmoglein-2 (G–L), desmoplakin for (E-Cad) specific E-cadherin antibodies (A–F), with (Dsg2) immunostained and fixed were irpini h oaiaino emsmlcmoet in a endogenous and of components plakophilin-3–GFP that expected, with As desmosomal compared localization cells. revealed 2-BP control of containing of medium the in localization grown microscopy was cells the A431 and components in hours, Immunofluorescence disruption 18 desmosomal other for examined. and 2-BP plakophilin-3–GFP of presence ora fCl cec 21)17 7239 doi:10.1242/jcs.149849 3782–3793 127, (2014) Science Cell of Journal m -Po MOa eaiecnrl(8hus.Cells hours). (18 control negative a as DMSO or 2-BP M 41clsexpressing cells A431 m m.

Journal of Cell Science hto oto el Fg DF.Telna ucaearray punctate cells increased linear 2-BP-treated with the components The in desmosomal of replaced 2D–F). aggregates was larger (Fig. with cells untreated cells in control with present compared of disrupted 2-BP, was that containing localization the desmosome medium however, of punctate in colocalized; pattern grown linear desmoglein-2 cells and a In plakophilin-3–GFP control 2A–C). in DMSO-treated (Fig. in colocalize sites cells contact cell–cell to at pattern found staining were desmoglein-2 ARTICLE RESEARCH usatal fetdb ramn ih2-BP. 2-BP, with of containing treatment not by assembly is medium affected assembly the substantially junction in adherens on whereas grown impacts junctions, and desmosome cells palmitoylation other 2G–L) in that (Fig. 2M–R) for demonstrating altered (Fig. localization not observed E-cadherin was was plakophilin-2 2S–X). (Fig. not Golgi, pattern desmoplakin components, (data the unclear; staining compartment of is similar desmosomal recycling markers A structures endocytic with shown). the these colocalize or not of 2-BP- lysosomes do nature the they intracellular containing The throughout however, aggregates some observed cultures. are Cytoplasmic treated and components signal. structures desmosomal of punctate accumulation between space , Fg ,lf aes.Adn Ca Adding panels). left 3, (Fig. were Cells low 2-BP. a containing of Ca medium in of absence grown concentration and or coverslips glass presence on seeded the in HaCat keratinocytes using experiment calcium-switch a desmosome performed during we palmitoylation assembly, of role the investigate to order In in assembly keratinocytes desmosome HaCat delays palmitoylation of Inhibition sebedsooe fe . or Fg ,rgtpnl) By panels). to right failed Cells 3, result, (Fig. a hours panels). desmosomal as 2.5 recruit and, after middle membrane desmosomes efficiently plasma assemble to the the 3, (Fig. failed to and components 2-BP desmosomes E-cadherin with of pretreated to assembly of components the junction recruitment membrane, adhesive plasma of the redistribution the induced otiigmdu iho ihu 0m -P Ca 2-BP. mM 50 without or with medium containing rcse o muoloecnemcooyatr25hours 2.5 after were cells microcopy Ca and normal mM, immunofluorescence under 1.8 of for concentration final processed a to medium the to eaioye r rw nmdu otiiglwCa low containing medium in grown are keratinocytes 58%cnlec.Clswr rw vrih nlow-Ca in overnight grown were Cells confluence. 75–80% ucinlpoen r agl yooi hnHaCat when cytosolic largely are proteins Junctional ora fCl cec 21)17 7239 doi:10.1242/jcs.149849 3782–3793 127, (2014) Science Cell of Journal 2+ conditions. 2+ nioisidctd cl a:10 bar: Scale indicated. for antibodies processed the were using cells microscopy and immunofluorescence hours 2.5 for medium i.3 niiino amtyaindsut Ca disrupts palmitoylation of Inhibition 3. Fig. ahrn x,pxl.Dt hwthe show mean Data pixels. Pxl, E- cadherin; E-cad, desmoglein-2; Dsg-2, desmocollin-2; Dsc-2, plakophilin; Pkp, cultures. desmoplakin; cell DP, 2-BP-treated lines, in red intensity cultures; fluorescence average control the for lines, intensity cell Blue fluorescence individual treatment. 30 each across for width borders and length segment equal a of measuring by software image using Slidebook5 determined was intensity fluorescence nue emsm sebyi HaCat in assembly desmosome induced ls oesisi eimcnann o Ca low containing medium in coverslips glass keratinocytes. aes nteasne(idepnl)o the or 50 panels) of (middle presence absence the in panels) Ca 2+ 000m Ca mM (0.050 a de 18m ia ocnrto)t the to concentration) final mM (1.8 added was 6 ..( s.d. n 5 0cl odrmeasurements). border cell 30 aa eaioye eegonon grown were keratinocytes HaCat m -P(ih aes overnight. panels) (right 2-BP M 2+ 2+ ni h utr reached culture the until ) otecluemedium culture the to m .Average m. 2+ a added was 2+ 3785 (left 2+ 2+ 2+ - -

Journal of Cell Science lsammrn u ahrwsseii oeet directing the events to to recruitment specific E-cadherin was of rather assembly. disruption desmosome but a membrane to cell plasma due at inhibition Overall, not this 2-BP. localization and was with assembly E-cadherin desmosome treated inhibited cells fact, treatment HaCat 2-BP in In in enhanced 2-BP. contact was cell–cell borders of of presence sites to the recruited was E-cadherin contrast, ARTICLE RESEARCH 3786 cells A431 in plakophilins cell–cell distribution. mutant of of their expression in sites the cytoplasmic to Interestingly, more localize noticeably efficiently are with and not plakophilin-2 contact did extensively (supplementary S1) (C569S) mutant Fig. colocalized plakophilin-3 material and contrast, 5D–F) and (Fig. By (C603S) borders desmoplakin. cell plakophilin-2 S1) endogenous plakophilin Fig. wild-type to material Myc-tagged (supplementary Exogenous localized plakophilin-3 cells. the and 5A–C) these (Fig. of in localization constructs the determined domain. repeat an armadillo in the present in residue loop cysteine unstructured conserved single a are plakophilins on that plakophilin-3palmitoylated show in data construct, These and 4C). plakophilin (Fig. plakophilin-2 palmitoylated tagged were mutant wild-type the of Myc-tagged cells. palmitoylation corresponding whereas of A431 loss results the the residues cysteine in in these generated of stably protein Mutation (C569S). and plakophilin-3 these mutant we (C603S) cysteine test plakophilin-2 Additionally, conserved To this the 2008). of expressed place al., serine in a et with residue plakophilin-2 Myc-tagged Ren be generated we to Palm; predictions, predicted are (CCS residues palmitoylated cysteine conserved these algorithm, web-based a on a and based as addition, cysteine In conserved residue. (Choi this palmitoylated identified potentially 2010) three unstructured 6 al., et (Yang and al. all the et by Yang 5 generated dataset proteomics in in repeats recent a Additionally, armadillo 2005). present residing Weis, between plakophilins 4A), residue sequence the (Fig. ‘loop’ cysteine of isoforms sequence conserved plakophilin acid a amino revealed the of Examination repeat armadillo palmitoylated plakophilin is the domain in cysteine conserved A eas 41clsedgnul sebedsooe,we desmosomes, assemble endogenously cells A431 Because h nrclua lsesi o understood. not is clusters of intracellular nature the The components. desmosomal the of solubility localization than desmosome rather affect plakophilins data mutant These the unaltered. that was suggest proteins these of solubility X-100 the Triton plakophilins, mutant cells expressing cells in desmosomal A431 altered endogenous was components of in localization X-100 the expressed Although Triton 6A,B). containing plakophilins (Fig. buffer wild-type lysis with in soluble compared more be dominant-negative to found this of investigation. under mechanism currently of is with sites The effect at mutants components contact. desmosomal dominant-negative of cell–cell localization and as the C603S to act plakophilin-2 respect that C569S material suggest supplementary data plakophilin-3 These 5; wild-type the S1G–R). (Fig. expressing expressing Fig. plakophilin-3 cells of cells or in with and localization plakophilin-2 disrupted compared the plakophilins also C603S mutant was Additionally, supplementary plakophilin-2 cadherins of S1B,E). 5B,E; desmosomal Fig. punctate Fig. expressing localization (compare cytosolic material cells C569S to the plakophilin-3 redistributed in in partially structures disruption is example, For desmoplakin components. noticeable desmosomal expressed endogenously in results etiuainadtec-eietto fteMyc-tagged examined sucrose-gradient was the caveolin-1 by of and and desmoglein-2 co-sedimentation separated with wild-type the plakophilins expressing were and cells centrifugation plakophilins Cell A431 centrifugation. from mutant sucrose-gradient prepared by Myc-tagged lysates with components of associate ability raft to the mutants lipid plakophilin examined dynamics and We desmosome plakophilins 2014). on wild-type effects al., and has 2011), et rafts al., (Stahley lipid et Resnik of 2012; disruption are al., the et components lipid (Brennan in components desmosomal enriched rafts microdomains membrane that Recent with associated domains. demonstrated the indeed raft increase have lipid to with studies proteins proposed modified is of residues association cysteine of Palmitoylation domains raft lipid association with their enhances plakophilins the of Palmitoylation diinlypaohln2C0Sadpaohln3C6Swere C569S plakophilin-3 and C603S plakophilin-2 Additionally ora fCl cec 21)17 7239 doi:10.1242/jcs.149849 3782–3793 127, (2014) Science Cell of Journal A hydroxylamine. HA, palmitoylated. are plakophilin-3 and plakophilin-2 wild-type whereas palmitoylated, are not C569S that plakophilin-3 demonstrates and ABE C603S (C) shown plakophilin-2 is control. Tubulin loading another. a one as to expressed levels all similar were at proteins plakophilins, Myc-tagged endogenous the the and of those levels to at similar expressed were proteins plakophilin-3. Exogenous or plakophilin-2 antibodies Myc, with against blotted were Lysates Pkp-3 C569S). (myc C569S and plakophilin-3 Pkp-3) Myc-tagged (myc plakophilin-3 Myc- wild-type C603S), tagged Pkp-2 (myc C603S Myc-tagged plakophilin-2 Pkp-2), (myc Myc-tagged plakophilin-2 expressing wild-type cells control A431 from cells, prepared A431 lysates cell (IB) of Immunoblot analysis (B) residue. the cysteine surrounding conserved sequences the shows in respectively) similarity Pkp-3, and Pkp-2 (Pkp-1, (NP_009114) plakophilin-3 and (NP_001005242) plakophilin-2 (NP_001005337), plakophilin-1 is plakophilins in cysteine palmitoylated. conserved A 4. Fig. A euneainetof alignment Sequence (A)

Journal of Cell Science EERHARTICLE RESEARCH oan,btohritrcin r ieyt eivle in involved cadherins). lipid desmosomal be A431 (e.g. in raft to rafts present with components interactions likely desmosomal lipid direct endogenous possibly are other domains, with raft wild-type interactions lipid association to the other recruitment decreased of but in that domains, the of results with palmitoylation of lack compared plakophilins that an suggest fractions data indicating with These raft association plakophilins. 5, decreased lipid a and showed 4 C569S) the Pkp-3 fractions (Pkp- and mutants C603S in Plakophilin plakophilins 2 components. present raft Wild-type lipid be 6C–E). desmoglein-2 with association (Fig. to lysates, preparations found our our 4 were fractions of in in 5 caveolin-1 component and, and raft lipid rafts, the with lipid partitioned to previously with shown was associate Desmoglein-2 analysis. immunoblot by bar: Scale Q. and N K, H, E, B, panels in 10 Desmoplakin shown M,P). is (Dsg-2; colocalization (A,D), desmoglein-2 (DP) tag and epitope G,J) Myc and (Dsc-2; fixed the desmocollin-2 coverslips, recognizing glass C603S antibodies on plakophilin-2 grown using were or immunostained P–R) M–O) and and J–L D–F, G–I C603S; (Pkp-2 A–C, WT; (Pkp-2 plakophilin-2 type components. localization desmosomal the endogenous disrupts of plakophilin-2 Palmitoylation-defective 5. Fig. m m. 41clsepesn wild- expressing cells A431 noaTio--0-noul rcin(eltfato)following fraction) (pellet fraction of Triton-X-100-insoluble a recruitment 7G–I). incorporated into (Fig. becomes the protein impaired fusion and was plakophilin-2–ER membrane contact, Wild-type to plasma punctate cell–cell the contrast to of to In desmoplakin sites 7E). localize desmoplakin to (Fig. the to failed efficiently membrane contact of plakophilin-2-C603S–ER in cell–cell protein, plasma wild-type recruitment of resulted the sites the medium the at structures to culture and the plakophilin-2–ER 7D) 7B). (Fig. (Fig. to of membrane plasma 4OHT was localization the desmoplakin of to and recruited 7A) Addition be not (Fig. plakophilin-2–ER cytoplasm and can 4OHT, to the cytosolic of in that (4OHT) domain absence localized the 4-hydroxytamoxifen protein was In ligand-binding of medium. carboxyl fusion addition culture receptor the its the plakophilin-2–ER by estrogen at a ‘activated’ in fused the resulted plakophilin-2 to of cell–cell terminus of adherens plakophilin Expression sites at endogenous components assemble an contact. desmosomal cells of assemble not A431D expression cells) do the and of 2005). lack (A431DE Wahl, but assembly junctions 1997; E-cadherin plakophilin-dependent al., expressing examine et (Lewis to desmosomes that us system culture cell allowed a characterized laboratory our Previously, in assembly desmosome cells support A431D to fail mutants Plakophilin ntepeetsuy ehv eosrtdta several that demonstrated have we palmitoylated study, are components present desmosomal the In DISCUSSION palmitoylated be to of strength unable to adhesion. respect desmosomal are with proteins that mutants that dominant-negative suggest as plakophilin data function isoforms these expressing plakophilin S1), Fig. Taken cells the is material plakophilin supplementary cells. A431 localization 5; desmoplakin A431 in the decreased (Fig. that parental disrupted in of observation of also resulted the expression that with cells with together fact, A431 compared In in adhesion mutants cultures. adhesion plakophilin palmitoylation intercellular al., these Myc-tagged increase et the to Wolf in 2004; failed of al., mutants expression intercellular et palmitoylation contrast, exogenous Setzer increase By 2013; that can al., 2013). A431 shown et isoforms (Roberts parental previously plakophilin adhesion have of of increased Others that expression 8). conferred with fragments (Fig. compared cell cells plakophilin-3 Myc- adhesion wild-type of and expressing intercellular number plakophilin-2 cells the A431 tagged expected, in intercellular As decrease increased adhesion. indicates 2002). stress a al., mechanical et mild assay, the following Huen 1998; using this al., et mutants In (Calautti of assay plakophilin Myc-tagged strength adhesion Myc-tagged dispase plakophilin-2, adhesive and Myc-tagged intercellular plakophilin-3 expressing relative cells the A431 examined adhesion We cell–cell disrupts plakophilins initiate mutant of to Expression 7K). protein (Fig. fusion cells mutant fraction, A431DE in the plakophilin–ER Triton-X-100-soluble assembly of desmosome mutant the inability the in the indicating However, remained protein 4OHT. fusion of addition the amtyae rtisi U4 rsaecls(age al., potential et (Yang of as cells screen prostate proteomics DU145 identified large-scale in a proteins were in palmitoylated potential proteins a components palmitoylated as several Previously, well dynamics. desmosomal as junction regulating modification of mechanism posttranslational novel a ora fCl cec 21)17 7239 doi:10.1242/jcs.149849 3782–3793 127, (2014) Science Cell of Journal nvivo in uncovering , 3787

Journal of Cell Science EERHARTICLE RESEARCH dsoli-,dsoli- n emcli-)and desmocollin-2) and 3788 desmoglein-3 (desmoglein-2, dynamics. role desmosome the that in determine show to palmitoylation to and of out palmitoylated set are we proteins sets, positives’ desmosomal data ‘false proteomic of large incidence high with a associated for potential the Given 2010). 6. Fig. edmntae htbt h emsmlcadherins desmosomal the both that demonstrated We e etpg o legend. for page next See osre ytiepeeti h rail eetdomain a repeat to armadillo the plakophilin-3 in and present and plakophilin-2 site cysteine Plakophilin-2 in the mapped conserved palmitoylated. palmitoylation We cysteines. not of eight contain the are each plakophilin-3 whereas desmoplakin palmitoylated, E-cadherin, and constituents are junction proteins adherens plaque desmosomal ora fCl cec 21)17 7239 doi:10.1242/jcs.149849 3782–3793 127, (2014) Science Cell of Journal a -catenin, b -catenin

Journal of Cell Science lkpii- 63 n y-agdpaohln3C6Swr on obe to found were C569S Myc-tagged plakophilin-3 system. Myc-tagged Imaging and Odyssey C603S signal Li-Cor plakophilin-2 the a and using performed, quantified and was was desmoglein-2 analysis plakoglobin, Immunoblot tag, (B) using epitope desmocollin-2/3. performed Myc was the analysis against plakophilin-3 (IB) antibodies or Immunoblot WT) C569S). (Pkp-3 (Pkp-3 plakophilin-3 C569S plakophilin-2 wild-type WT), C603S), from (Pkp-2 (Pkp-2 prepared plakophilin-2 C603S were wild-type (S) expressing fractions cells plakophilin. soluble A431 wild-type and of (P) and that Triton-X-100-insoluble solubility with (A) X-100 compared Triton association altered raft display lipid mutants Plakophilin 6. Fig. ARTICLE RESEARCH R o empai D)lclz ocl odr AC.Adto f10n OTt h eimfr1 or eut npaohln2E oaiainto localization effic plakophilin-2–ER not in did 10 results proteins. C603S/ER) bar: hours fusion (Pkp-2 Scale plakophilin-2 18 plakophilin-2-C603S–ER (G–I). the for protein, recruited of medium wild-type expression efficiently the the equivalent not to demonstrates to was 4OHT plakophilin-2-C603S–ER contrast desmoplakin or nM In and plakophilin-2–ER 100 (D–F). borders of desmoplakin cell Addition of to cells. (A–C). recruitment localize A431DE borders the in cell and assembly to borders desmosome localize cell initiate (DP) efficiently desmoplakin to nor fails ER) C603S Plakophilin-2 7. Fig. family of disruption catenin and neurons, be hippocampal p120 to cultured shown in been another has palmitoylated Delta-catenin palmitoylated. that also is Recent demonstrated member 2005). Weis, and have (Choi 6 studies and 5 repeats armadillo between plakophilins. wild-type mean the the of show that data with compared B,E, C603S are 5 For signal plakophilin-2 and 4 of desmoglein-2 fractions ratios corresponding in The the reduced graphically. to present shown C569S signal is plakophilin-3 desmoglein-2 5 and the and to 4 signal fractions Myc in of ratio wild-type The (Cav-1), and (E) caveolin-1 plakophilin-2 plakophilin-3. component wild-type 5 raft and and lipid desmoglein-2 4 the endogenous Fractions in antibodies. enriched indicated are the (underlined) with performed immunoblot was and to collected analysis subjected were fractions were 1-ml Lysates centrifugation, (D). sucrose-density C569S plakophilin-3 plakophilin-3 wild-type Myc-tagged Myc-tagged and and and (C) plakophilin-2 C603S were wild-type plakophilin-2 lysates Myc-tagged Myc-tagged Cell expressing cells (C,D) A431 the plakophilin-3. from by or prepared altered plakophilin-2 not mutant was of (PG), (Dsc-2) expression plakoglobin desmocollin-2/3 endogenous and of (Dsg-2) solubility desmoglein-2 and X-100 plakophilin-2 Triton wild-type The Myc-tagged plakophilin-3. with compared soluble more S n rtnX10islbeple P rcin eepeae n muolte ihatbde gis lkpii-.Dt eecletdu collected were Data plakophilin-2. Triton-X-100-sol against hours. antibodies 18 with mean for immunoblotted the 4OHT and show nM prepared Data 100 were imaging. containing fractions medium near-infrared in (P) Odyssey grown pellet were Triton-X-100-insoluble plakophilin-2-C603S–ER and or (S) plakophilin-2–ER expressing cells A431DE (K) 6 ..[ s.d. n 5 BE] * (B,E)]; 3 P , 0.05. 6 ..( s.d. n 5 ) ** 3); P , 0.01. eutn nteadto f1-abnpliaet specific al., et to (Fukata domain palmitate PATs DHHC of family 16-carbon Cys-rich A conserved linkage. of the thioester a containing addition through chains side the cysteine in resulting possibilities. these address in to studies that role required Further a be disassembly. will plays indicate or possibility also stability the proteins maturation, importance data address desmosome desmosomal particular not of do palmitoylation these of but that is assembly, together, plakophilins desmosome during cell– the Taken global of disrupt palmitoylation simply adhesion. not junction does cell 2-BP HaCat adherens in that incubation suggesting and 2-BP cells, following localization disrupted not were E-cadherin calcium- assembly a in assay. assembly desmosome 2- of switch inhibition by an palmitoylation in global resulted Preventing desmosome BP initiate cells. to A431D ability in plasma their assembly to the with interfered plakophilins to and localization the membrane their of disrupted the inability palmitoylated, the from become in resulted distinct plakophilin-3 plakophilins. are the and in DHHC5 cysteines identified PAT have These the we that 2014). by cysteine palmitoylated al., conserved are et that (Brigidi cysteines C961) two and identified (C960 authors with association these its Interestingly, decreases N-cadherin. delta-catenin of palmitoylation the h eesbentr fti atclrmdlto ae tan it makes modulation 2009). particular al., this et Zeidman of 2001; nature Schmidt, and reversible LYPLA1L) Veit The 2012; and al., LYPLA2 catalyzed et (LYPLA1, is (Tian thioesterases palmitate of cytosolic palmitate removal of the by addition whereas the proteins, for responsible substrate are to 2002) al., et Roth 2004; amtyaini eesbepstasainlmodification posttranslational reversible a is Palmitoylation plakophilin-2 in residue cysteine conserved the of Mutation m ora fCl cec 21)17 7239 doi:10.1242/jcs.149849 3782–3793 127, (2014) Science Cell of Journal .()Imnbo I)aayi fA3D el expressing cells A431DE of analysis (IB) Immunoblot (J) m. nteasneo OT ete lkpii-–R(Pkp-2/ plakophilin-2–ER neither 4OHT, of absence the In b tblni nldda odn control loading a as included is -tubulin igLi-Cor sing iently 3789 uble

Journal of Cell Science EERHARTICLE RESEARCH fdsooa opnnsi h ln ftemmrn to 3790 membrane the electron of by seen plane plaque the electron-dense characteristic in the packing generate components the in that desmosomal the assist of might likely into increases ultimately, Palmitoylation and, is plaques. rafts components lipid desmosomal into It desmosomal proteins these palmitoylation. of the incorporation plakophilin was of assay by this palmitoylation The in affected fractions centrifugation. raft sucrose-gradient not with in desmoglein-2 of markers association association decreased raft that show mutants lipid palmitoylated plakophilin with that become shown to Stahley have unable 2011; we are al., Here, et 2014). Resnik the 2012; al., al., associated et et in are (Brennan rafts. role components lipid desmosomal with studies a 2006). that Recent play microdomains. demonstrated membrane (Resh, have to to proteins membrane thought of targeting is plasma cellular palmitoylation with the Furthermore, association including increased in membranes, results proteins substrate palmitoylation- in events PAT of assembly. the sequence desmosome dependent the of be understand will to components Identification desmosomal necessary the of components. as the modification well for desmosomal identify as responsible plakophilins to other the laboratory of the our palmitoylation cells. for in desmosome epithelial responsible underway in regulating PATs are strength of adhesive efforts even capable Currently, or is stability that assembly, mechanism attractive the of Quantification mean (F) the assay. show dispase Data the assays. in dispase counted of examples fragments representative sheet and depict cell A–E D) (E). (Pkp-3; Myc- C569S B), plakophilin-3 plakophilin-3 (Pkp-2; Myc-tagged Myc-tagged plakophilin-2 (C), Myc-tagged C603S expressing plakophilin-2 cells tagged cells A431 A431 parental in in and adhesion (A) desmosomal of strength relative the disrupts examine mutants palmitoylation adhesion. plakophilin desmosomal of Expression 8. Fig. eea rushv eosrtdta h amtyainof palmitoylation the that demonstrated have groups Several ips deinasy eepromdto performed were assays adhesion Dispase 6 ..( s.d. n 5 ) * 3); P , 0.05. h blt fteedsooa opnnst neatwt h the initia with interact and and to cytoskeleton components desmosomal plakophilin-2 these of ability Like the o Palmitoylation 2000). shown). not (data al al., is plakophilin-1 et plakophilin-3, (Hatzfeld edge keratinocytes leading cytoskeleton actin the the near to Plakophilin localize 2011). cells recently al., between et filaments (Roberts We cells desmoso actin to that epithelial cells. proximity determined migrating A431 domain we in repeat and in assembly into desmosome armadillo localization insertion characterized the membrane and in disrupts localization substitution proper h cysteine-to-serine we for Here, complexes. needed desmosomal are h the repeats in sequences that suggest amtyain(y2B ramn)peet desmosome prevents treatment) Ca 2-BP global following of Inhibition assembly (by future. the these in on palmitoylation addressed palmitoylation be of of should effect processes degradation The or affects other components. disassembly and effect desmosomal desmosome the manner examine on possibly ordered to palmitoylation unable an of were studies (and in Our to assembly. membrane proteins junction these the plakophilins of with ability interact the the affects components) desmosomal of lipid palmitoylation of role the dynamics. on desmosome light on shed rafts additional should of components palmitoylation desmosomal the of Characterization microscopy. TC(aass A.A3 n aa eaioye Buape al., et (Boukamp from keratinocytes obtained HaCat was and line A431 cell VA). carcinoma (Manassas, cell ATCC squamous cervical A431 The culture Cell METHODS AND MATERIALS n hoseae epnil o h diinadrmvlof removal and addition the for responsible thioesterases thereby and and microdomain components membrane assembly. desmosomal ordered desmosome of affect highly association is this Palmitoylation the 2014). with al., influence et to al., Stahley 2011; et likely al., (Brennan et assembly Resnik raft desmosome 2012; lipid 4-OHT for that required upon demonstrated is have cells association studies A431DE Recent 7). in (Fig. assembly addition desmosome unable is initiate plakophilin-2-C603S–ER to specifically, More palmitoylation 3). of 2, inhibition (Figs by unaffected is localization cadherin lkpii- eut nisrecr its (Bonne the in to results recruitment for plakophilin-3 head sufficient expression not and N-terminal membrane, is plasma alone the exp domain head by the terminal mediated Interestingly, is domain. components th desmosomal that shown has analysis plakophilin-3 Previous of membrane. is with or association it interaction the strengthens identified, protein–protein simply been alters palmitoylation have of whether domain partners unclear repeat binding armadillo no plakophilin Because the plakophilin association. membrane and influences plakophilin-2 the do the compartment subcellular which at palmitoylated. become and components desmosomal reside PATs compartment membrane the specific which in know to and important be identified will It dynamics. desmosomal be to respect with must characterized activity their components desmosomal from palmitate ae ntedt rsne ee epooethat propose we here, presented data the on Based eea neetn usin eant eadesd h PATs The addressed. be to remain questions interesting Several diinly ti urnl nnw o amtyainof palmitoylation how unknown currently is it Additionally, ´ ora fCl cec 21)17 7239 doi:10.1242/jcs.149849 3782–3793 127, (2014) Science Cell of Journal ta. 03 oet ta. 01.Tkntgte,teedata these together, Taken 2011). al., et Roberts 2003; al., et edsooeassembly. desmosome te 2+ diini aa el,weesE- whereas cells, HaCat in addition a oana ela h armadillo the as well as domain ead imn otepam membrane plasma the to uitment eso ftepaohln3N- plakophilin-3 the of ression hni soeepesdi HaCat in overexpressed is it when oplioltdi utrdcells cultured in palmitoylated so neato fpaohln3with plakophilin-3 of interaction e h lkpiismgtinfluence might plakophilins the f 1hspeiul ensonto shown been previously has -1 fteamdlorpa oanof domain repeat armadillo the of e ntaeasml nclose in assembly initiate mes 3amdlorpa domains repeat armadillo -3 v eosrtdta single a that demonstrated ave

Journal of Cell Science 59)wr eeae sn h QuikChange the using pkp-3 and generated C603S (pkp-2 were mutations point C569S) Plakophilin site. restriction h diino . MCaCl mM 1.8 of addition the ta. 07.Rtoial netdcl ouain eeruieygrown routinely 500 Sobolik-Delmaire were containing populations 2011; DMEM cell al., infected in infection et Retrovirally 2007). retroviral (Roberts al., and previously et particles described retroviral been 1997; al., of has et Generation (Lewis previously 2005). described UT). Wahl, supplemented were Logan, cells Laboratories, MO) A431DE (HyClone Louis, and (FBS) A431D St serum bovine PA) (Sigma, fetal Pennsylvania, DMEM 10% of with in University grown Jensen, Pamela routinely from were gift kind (a 1988) ARTICLE RESEARCH ecie rvosy(a ta. 07.Clswr avse nlysis in harvested Millipore, (EMD were BB Empigen Cells 0.5% or 2007). X-100 Triton al., 1% containing et buffer as (Wan prepared were previously lysates and described confluence 80–90% to grown were cells A431 exchange Acyl-biotin an add and cDNA standard each by from generated codon was stop al., 1997) the ligand- et al., remove receptor et Roberts to (Feil estrogen procedures 2009; variant the al., T2 to domain et fused binding (Hall (pkp-2-ER) previously Plakophilin-2 described 2011). as were (NP_009114) plakophilin-3 cDNA and (NP_001005242) plakophilin-2a Human mM constructs 2 cDNA of SDS, Generation resolved 2% and buffer of 8.0, sample ml pH Laemmli 1 SDS-PAGE. in in Tris-HCl by prepared pellet were the mM Lysates of (10 EDTA). resuspension to buffer prior for SDS-containing buffer centrifugation lysis for by shaking X-100 14,000 with collected Triton at ice was on material minutes incubated 15 insoluble 0.5% and The 8.0, EDTA) minutes. pH mM 1 15 into Tris-HCl 2 scraped mM with and were (10 X-100 monolayers flask buffer Triton T25 Triton-X-100-containing cell a of washing in ml grown first 1 -insoluble Cells and by saline. (s) buffered isolated Triton-X-100-soluble phosphate were (Sobolik-Delmaire 2000). al., fractions previously et (p) described Wahl 2006; as al., prepared et were lysates Cell extraction Detergent The E0646). 2007). 2011; al., al., number et et (Roberts previously Sobolik-Delmaire catalog described immunofluorescence as MO; and performed analysis were microscopy Louis, immunoblot lysates, (St cell of generation Sigma was from receptor estrogen hybridoma against obtained and antibody IA) Rabbit Studies City, generated. Iowa was Developmental Iowa, supernatant The of University from (The Anti- obtained Green Bank CA). Hybridoma was Cruz, Kathleen hybridoma (Santa (N20) (E7) Biotechnology of anti-caveolin-1 Cruz tubulin gift Santa Rabbit from 1996). kind IL). obtained al., was a Chicago, et Wahl University, was 2007; (Northwestern (NW6) al., et 2006; Antibodies anti-desmoplakin Sobolik-Delmaire al., 2002). Rabbit et 1999; 2009; (Bazzi al., Wahl, previously et al., 2011; described Nieman and as et al., (4A2) were (Hall E-cadherin (7G6) et (6D8), desmocollin-2/3 previously Roberts desmoglein-2 (20B6), described 1993; desmoplakin against as al., et generated Johnson were plakophilin- (8H6) and (11F2) 2 plakophilin-3 for specific antibodies Monoclonal Antibodies pnLR Ieo ta. 02,wihwr rnfce noPhoenix into transfected particles. were retroviral of which based generation 2002), vectors the al., into for cloned et cells previously were (Ireton constructs described LZRS cDNA upon been 2011). vector al., has retroviral et pSPUTK LZRS (Roberts Plakophilin-3–GFP an modified into vector. subcloned a the with subsequently expression in together were cDNAs tag tag plakophilin epitope The c-Myc epitope 1991). Andrews, the and (Falcone encoding of downstream cDNA 2 cDNAs plakophilin the a subcloning by no achieved of have was to terminus Addition CA). shown and changes. Clara, sequenced completely unintended Santa were cDNAs Technologies, modified The (Stratagene/Agilent kit mutagenesis acu-wtheprmns aa el eegoni MMlacking DMEM in grown were Ca cells HaCat experiments, calcium-switch 2+ upeetdwt 0 ilzdFSfra es 8huspirto prior hours 48 least at for FBS dialyzed 10% with supplemented g h noul eltwswse newith once washed was pellet insoluble The . m /lG1 Mdaeh eno,V) For VA). Herndon, (Mediatech, G418 g/ml 2 n 50 and m 6 2-BP. M -y ptp a oteN- the to tag epitope c-Myc TM site-directed Xho b 6 I - okal(im) n h te afwsdltdfvfl ihbuffer with fivefold diluted was ( half hydroxylamine other the lacking and (Sigma)] cocktail 0m E.Tesmlsaeicbtda 4 at incubated containing are buffer lysis samples in The fourfold The diluted NEM. and minutes. chloroform-methanol mM EDTA) 2–3 10 mM for 5 were 7.4, dry pH air HCl Samples to 300 allowed in resuspended was monolayers. was pellet pellet desmoplakin the solubilize cell and most to precipitated for used A431 used was was BB from X-100 Empigen Triton mM but needle. 10 experiments ice 25-gauge 1% containing on ABE a scraping EDTA) 7.4, through by pH mM passed collected 5 and were Tris-HCl and BB, Sigma), mM Empigen (N-ethylmaleimide, 50 0.5% NEM or NaCl, X-100 mM Triton (150 Germany) Darmstadt, ecie rvosy(cun n aoe,2014). as Mahoney, centrifugation and or sucrose-gradient (McGuinn wild-type by previously expressing described analyzed lines and cell A431 plakophilins from mutant prepared were lysates Cell analysis fraction raft Lipid 150 with mixed 75 only) medium, DMSO the 17- 80% of dissolution in facilitate To 2012). ODYA to al., et (Zoltewicz hours grown 48 for (Sigma) were 100 vehicle with DMSO treated or cells Co.) were Chemical Cayman (17-octadecynoic and acid; 17-ODYA A431 dishes culture tissue labeling, 100-mm in metabolic confluence 17-ODYA exchange For acyl-biotin and labeling Metabolic ehnletatdtretmsadtefnlpleswr eupne in resuspended were chloroform- pellets were final Samples the and 240 hour. times 1 three for extracted rocking methanol gentle with temperature X-100, 1 Triton and (PMSF) 0.2% fluoride HPDP–biotin, phenylmethanesulfonyl mM mM 1 hydroxylamine1 hydroxylamine, M 0.7 M containing [0.7 buffer (+HA) with fivefold diluted was half one was 100 pellet the in precipitation, resuspended last chloroform- the sequential following three and, precipitations performing methanol by removed was NEM agitation. rtiswr eetdb ltigwt R-aee tetvdn(Jackson streptavidin HRP-labeled PA) with Grove, blotting West ImmunoResearch, by biotin-labeled detected and were SDS-PAGE proteins by separated were 1 proteins Labeled in PAGE. SDS- prepared to prior boiled were not were Samples 2-mercaptoethanol. Samples containing buffer hour. 1 for temperature totalreactionvolumeof50 a efre sdsrbdpeiul Carne l,20) rel,47 Briefly, 2009). al., et reaction (Charron click Cu-catalyzed previously The described SDS). as 2% 47 performed NaCl, was in mM 150 Immunoprecipitated beads 7.4, Tween-20). the pH HEPES 0.5% from eluted NaCl, were mM mM proteins 150 (10 TBST 8.0, in pH times three Tris-HCl washed were complexes immune and lysates Empigen-BB-soluble from immunoprecipitated were Plakophilins min). 15 (14,000 centrifugation mM by pelleted 2 were BB, proteins insoluble Empigen 0.5% and 8.0, EDTA), pH Tris-HCl mM 0.5% containing (10 detergent buffer prior extraction BB vortexed in Empigen was lysed mixture were Cells the cells. and to DMEM, added being of to ml 15 to added was (Sigma) ufr(%SS 0m rsHlp .,5m DA n samples and EDTA) mM 5 chloroform-methanol 7.4, pH sequential 75 Tris-HCl in mM three 50 resuspended gentle SDS, to was with (2% pellet prior buffer temperature final room hour The 1 precipitations. at mM for incubated 1 0.2 EDTA, PMSF, agitation were mM mM 5 Samples 1 7.4, X-100, cocktail). pH Triton Tris-HCl mM 0.2% 50 HPDP–biotin, NaCl, mM (150 buffer yi ufrcnann .%SSad02 rtnX10 Captured X-100. Triton 0.2% and PAGE. 2 in SDS in beads boiled 0.1% were the proteins containing washing by buffer streptavidin– removed to lysis were added proteins bound were non-specifically and protein streptavidin–agarose on captured of were proteins Biotin-labeled amounts were agarose. concentrations equal Protein PMSF. and mM determined 1 1 X-100, and Triton cocktail 0.2% containing inhibitor buffer protease lysis in SDS 0.1% to diluted were feue rti a de o0.25 to added was protein eluted of lmty]mn}(im)ad0.5 and (Sigma) yl)methyl]amine} 0.25 (Sigma), 0.5 m m f5 MTE ti-2croyty)hshn]hydrochloride [tris-(2-carboxyethyl)phosphine] TCEP mM 50 of l f4 D ufraddltdwt 960 with diluted and buffer SDS 4% of l ora fCl cec 21)17 7239 doi:10.1242/jcs.149849 3782–3793 127, (2014) Science Cell of Journal m f1 MTT {tris[(1-benzyl-1H-1,2,3-triazol-4- TBTA mM 10 of l m f4 D ufr h apewsdvddi w – two in divided was sample The buffer. SDS 4% of l 6 2 aml apebfe n eovdb SDS- by resolved and buffer sample Laemmli m m A.Smlswr nuae troom at incubated were Samples HA). f2 M1-DAsoki MO(or DMSO in stock 17-ODYA mM 20 of l m m ,adteslto a nuae troom at incubated was solution the and l, 0 at-cdfe oiesrmalbumin serum bovine fatty-acid-free 10% l f4 D ufr(%SS 0m Tris- mM 50 SDS, (4% buffer SDS 4% of l m m f1 Mboi zd (Invitrogen), azide biotin mM 10 of l f5 MCuSO mM 50 of l m feuinbfe 5 mM (50 buffer elution of l ˚ vrih ihgentle with overnight C m flow-HPDP–biotin of l 6 6 6 rtaeinhibitor protease rtaeinhibitor protease aml sample Laemmli 4 m f2 SDS 2% of l Sga o a for (Sigma) 3791 g m for M m 6 l

Journal of Cell Science n ** and saswr efre ntilct n h aaaeepesda the as expressed are counted, data were well. the per fragments and fragments of triplicate sheet number in average Cell by performed to inversion. were dish subjected and assays by culture tubes conical stress the 15-ml Cell to mechanical IN). from transferred post- Indianapolis, carefully removed hours Science, were 24 Applied sheets were (Roche and, dispase sheets dishes with six-well incubating cell in the confluence to confluence, grown were Cells assays Dispase ARTICLE RESEARCH 3792 Borgland, M., Mobasser, K., Pitman, D., Beccano-Kelly, Y., Sun, K., S., J. G. Wahl, Brigidi, J., K. Green, W., Medhat, A., Dowling, S., Peltonen, D., Brennan, A. Markham, J., Hornung, D., Breitkreutz, T., R. Petrussevska, P., Boukamp, Bonne Wahl, L., Langbein, A., Ishida-Yamamoto, I. G., M. Rodriguez-Crespo, Mahoney, A., and Getz, H., A. Bazzi, R. Valero, C., Aicart-Ramos, References at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.149849/-/DC1 online available material Supplementary material Supplementary months. 12 Health. after of release Institutes for National PMC [grant the in Diseases from Deposited Skin M.G.M.] and to and Musculoskeletal R01AR056067 K.R.J.]; and number General and Arthritis of J.K.W. of Institute to Institute National P20GM103489 National the number from [grant grants Sciences by Medical supported was project This authors. by Funding other written the primarily from was input manuscript with J.K.W., the and and J.K.W., K.R.J. and B.R.J., experiments K.R.J. The study. site-directed by this the directed for performed constructs were R.A.S. cDNA the and assembled J.S. and analysis. M.G.M. mutagenesis raft and K.P.M. lipid experiments. the acyl-biotin labeling performed performed metabolic K.E.J. and and experiments B.J.R. exchange microscopy. the performed B.J.R. contributions Author in used antibodies the of some of sale studies. the these from royalties receive J.K.W. and K.R.J. interests Competing data when and assumed Student’s was deviation, significance two-tailed standard Statistical using variances. represent bars compared error were experiments, all For analysis Statistical roe .A,Nti,D n esl,D P. D. Kelsell, and D. Nitoiu, A., M. Brooke, aati . aoi . ti,P . azed . eesa .adPaolo and N. Kedersha, M., Hatzfeld, L., P. Stein, S., Cabodi, E., Calautti, ais .L,Ge .M,Ccrn,R . in,W . hra .K., A. Sharma, G., W. Jiang, A., R. Cochrane, M., J. Gee, L., E. Davies, I. W. Weis, and J. H. Choi, E. Shamir, S., A. Raghavan, J., Wilson, S., J. Yount, M., M. Zhang, G., Charron, ad,D,E zon,M . o,C . enne,J . amdr .D., J. Majmudar, L., J. Hernandez, T., C. Tom, A., and M. 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Garrod, 1. plakophilin ,S,Glet . azed . hn . re,K .advnRy F. Roy, van and J. K. Green, X., Chen, M., Hatzfeld, B., Gilbert, S., ´, P , 0.01. 19) yoiepopoyainadscfml iae control kinases family src and phosphorylation Tyrosine (1998). .Ml Biol. Mol. J. 74 129-140. , 19) lnn n euneaayi fdesmosomal of analysis sequence and Cloning (1991). 20) outfursetdtcino rti fatty-acylation protein of detection fluorescent Robust (2009). 18) omlkrtnzto naspontaneously a in keratinization Normal (1988). .A.Ce.Soc. Chem. Am. J. 346 20) tutr fteamdlorpa oanof domain repeat armadillo the of Structure (2005). .Pathol. J. 367-376. , .Cl Biol. Cell J. 20) emgen4i xrse nhighly in expressed is 4 Desmoglein (2006). 21) rfln agt fteirreversible the of targets Profiling (2013). 226 ici.Bohs Acta Biophys. Biochim. 158-171. , C hm Biol. Chem. 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Journal of Cell Science ooa . uioo .adYmsia S. Yamashita, and H. Sugimoto, T., Toyoda, L. J. Daniotti, and A. J. G. Gomez, M. A., Trenchi, Shipston, M., V. and Tomatis, P. Ruth, G., H. Knaus, H., McClafferty, L., Tian, hmsn .A,Coe,N . nel .M,Ci,M,Mri,A J., A. Merrit, M., Chiu, M., D. Ansell, H., N. Cooper, A., H. M. P. Thomason, Speight, and J. G. Thomas, the,S . at,M,Fudz . oa,M,Mthye,A .and L. A. Mattheyses, S. Yamashita, and M., H. Hayashi, Koval, H., Sugimoto, V., Faundez, M., Saito, N., S. Stahley, Wahl, and G. M. Mahoney, A., S. Keim, D., Katafiasz, T., Sobolik-Delmaire, ARTICLE RESEARCH shrci oi n characteri and coli, Escherichia membrane-associated peripheral of deacylation the GAP-43. catalyzes 2 thioesterase expression surface channels. control potassium thioesterases calcium-activated and of transferases protein acyl Distinct ipy.Acta Biophys. desmosomal in changes with correlation adhesiveness. re-epithelialization: wound regulates ada,M .adGro,D R. D. Garrod, and J. M. Hardman, cancer. n euaino yohshlps rmrtliver. rat from lysophospholipase of regulation and raft-dependent. P. membrane A. Kowalczyk, cells. carcinoma cell 109. squamous neck and head in III K., J. rt e.Oa il Med. Biol. Oral Rev. Crit. 20) erae lkpii- xrsinpooe nrae motility increased promotes expression plakophilin-1 Decreased (2007). LSONE PLoS 1437 .Pathol. J. 182-193. , 21) emsm sebyaddssebyare disassembly and assembly Desmosome (2014). 5 e15045. , 227 LSONE PLoS 346-356. , 12 aino yohshlps II. lysophospholipase of zation 21) ieteiec htPKC that evidence Direct (2012). 20) elahso oeue n oral and molecules adhesion Cell (2001). 479-498. , 9 e87809. , .Bo.Chem. Biol. J. 19) uiiain DAcloning, cDNA Purification, (1996). 19) eune xrsinin expression Sequence, (1999). .Bo.Chem. Biol. J. elCmu.Adhes. Commun. Cell 287 21) Acyl-protein (2010). 14718-14725. , 271 7705-7711. , a positively Biochim. (2012). 14 99- , ag . iVzo . icnr . te,H n rea,M R. M. Freeman, and H. Steen, M., Kirchner, D., Vizio, Di W., Yang, Hu M., Glaß, K., Rietscher, A., Wolf, a,J,Rt,A . aly .O n ai,N G. N. Davis, and O. A. Bailey, F., A. Roth, J., Wan, Sauppe M., T. McGranahan-Sadler, A., P. Sacco, K., J. Wahl, al .K,3rd K., J. Wahl, 3rd K., J. Wahl, et .adShit .F. M. Schmidt, and M. Veit, otwc,S . e,S,Cito,V . read .M,Rnaau S., Rangaraju, M., S. Freeland, G., V. Chittoor, S., Lee, J., S. Zoltewicz, eda,R,Jcsn .S n ae,A I. A. Magee, and S. C. Jackson, R., Zeidman, k2sice lkpii ucinfo tblzn elahso opromoting to adhesion cell stabilizing from proliferation. function cell 1 M. plakophilin Hatzfeld, and switches A. Akt2 Mun, A., Wingenfeld, A., Sinz, soito ihtedsooa ahrn n h lsia cadherins: classical identification. and the purification proteins: and domains. cadherins shared R. and desmosomal unique K. of the identification Johnson, with and association J. M. assembly. members. family desmoglein lcpoen ihay-rti hoseae1i vitro. in 1 thioesterase acyl-protein with glycoproteins ouae pteilcl opooyadmigration. and morphology cell L. epithelial Notterpek, modulates and A. D. Zacharias, rtoesaecaatrzto fhmnSayae rtisi ii raft- lipid Biol. in Membr. Mol. proteins S-acylated human membranes. non-raft of and enriched characterization scale Proteome ora fCl cec 21)17 7239 doi:10.1242/jcs.149849 3782–3793 127, (2014) Science Cell of Journal .Cl.Biochem. Cell. J. 20) oefrpaohln1i h ntaino desmosome of initiation the in plakophilin-1 for role A (2005). .Cl Sci. Cell J. 20) eeaino oolnlatbde pcfcfor specific antibodies monoclonal of Generation (2002). 26 32-41. , 96 126 yrd Hybridomics Hybrid. 390-403. , 19) lkgoi oan htdfn its define that domains Plakoglobin (1996). 20) nyai eamtyaino viral of depalmitoylation Enzymatic (2001). 1832-1844. , tlae,S,Shtosi . hig C., Ihling, M., Schutkowski, S., ttelmaier, ¨ o.Cl.Proteomics Cell. Mol. 21) h amtyainsaeo PMP22 of state palmitoylation The (2012). a.Protoc. Nat. 20) rti clthioesterases. acyl Protein (2009). .Cl Sci. Cell J. 21 21) nui inln via signaling Insulin (2013). 2 S Neuro ASN 37-44. , 1573-1584. , Virology 109 20) Palmitoylated (2007). 9 54-70. , ,L . Wheelock, M., L. ´, 1143-1154. , 288 4 409-421. , 89-95. , (2010). 3793

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