Journal of Cell Science htteoiia oki rpryctdadalfrhrdsrbtoso h oko dpainare adaptation or work the terms. of License distributions Commons further provided all Creative medium and same any cited the in Attribution properly to reproduction Commons is subject and work Creative original the distribution the of use, which that terms non-commercial the (http://creativecommons.org/licenses/by-nc-sa/3.0), unrestricted under License permits distributed Alike article Share Access Non-Commercial Open an is This factor Nuclear Introduction words: Key gene of patterns the analyzing NF- By noncanonical the p65. of component of key the accumulation NIK, of nuclear NF- level of the the increased expression APPL markedly altered reduced NF- on APPL1 found overexpressed depletion we detectable the level, depletion NF- not molecular of its or in of was overproduction subunit regulator and function APPL1 p65 TRAF2 upon enhanced the positive its Although transcription of overexpression complex. distribution a for spatial IKK its required as proper the the since was APPL1 regulate of to APPL1 upstream of appeared APPL1 function of role pathway, and recruitment novel process in endosomal this a acting endosomes, for uncover thereby NF- necessary canonical factors, we were activate nuclear proteins Here, to and known pathways. proteins protein signaling signaling adaptor receptors, different NF- membrane of in binds activity transcriptional that and protein trafficking adaptor endosomal multifunctional a is APPL1 Summary 10.1242/jcs.105171 doi: 4090–4102 125, ß Science Cell of Poland Journal Warsaw, 2012 May 02-109 3 Street, Accepted Trojdena Ks. 4 ( Biology, correspondence Cell for and *Author Miaczynska* Molecular of Marta Institute and International Pyrzynska Beata Hupalowska, Anna NF- basal regulates APPL1 4090 ope,wihlast hshrlto n erdto fthe of degradation and In phosphorylation (IKK) I receptor. kinase to kappa-B the leads of which of inhibitor complex, the domain recruit cytoplasmic adaptors the these turn, to TRAFs) as (such proinflammatory factor (IL-1 to necrosis response tumor as in such minutes stimuli, within occurs perform and rapid and 2011). kinetics al., et different (Shih functions with physiological signaling operate nonredundant noncanonical or NF- which canonical 2009). via cascades, activated Karin, be and can dimers Vallabhapurapu 2006; (Scheidereit, k regulation primary NF- The the 2009). of Karin, Karin, and and (Ghosh Vallabhapurapu expression 2002; gene transactivate to heterodimers mammalian The 2008). NF- Ghosh, and Hayden NF- (Hayden 2004; proliferation and Ghosh, survival and cell responses, immune the in NF- of machineries noncanonical and triggered canonical NIK the between of link levels novel high a as turn, APPL1 NF- In places basal cIAP1. This regulates p65. and APPL1 of activation. TRAF3 that activation propose TRAF2, the we containing affects Collectively, which complex p65. degradative of the translocation nuclear with association its reducing NIK k iesi h yols yI by cytoplasm the in dimers B 02 ulse yTeCmayo ilgssLtd Biologists of Company The by Published 2012. B h ciaino h aoia NF- canonical the of activation The a k k b aiyicue iemmes eAp5 -e,RelB, c-Rel, RelA/p65, NF- members: and (p105/p50) five B1 includes family B niio (Naude inhibitor .Ctkn tmlto ed otercuteto adaptors of recruitment the to leads stimulation Cytokine ). k aha prtstruhtesqetaino NF- of sequestration the through operates pathway B PL,NF- APPL1, k (NF- B ´ [email protected] ta. 01 aatadShuih 2011). Scheurich, and Wajant 2011; al., et k k aha,NK TRAF2 NIK, pathway, B )i e rncito atrinvolved factor transcription key a is B) k k 2(10p2 htfr oo or homo- form that (p100/p52) B2 ne aa u o TNF not but basal under B a k (TNF k aha sgenerally is pathway B ) niioyproteins inhibitory B a ritrekn1 interleukin or ) k inln.AP1snrie ihTA2t nueNF- induce to TRAF2 with synergized APPL1 signaling. B a siuae odtos PL a on odrcl neatwt RF,an TRAF2, with interact directly to found was APPL1 conditions. -stimulated k B b k ciiyb stabilizing by activity B rncitoa euain otyb ouaigteatvt of and activity the signaling modulating nuclear by in mostly regulation, also transcriptional participate endocytosis regulate 1997; al., et 2008a). (Malinin al., cells et in Zarnegar accumulates protein NIK when NIK–IKK2axis a through activated be could noted dimers p65-containing was that It studies them. between several interconnections for 2002), evidence provided Baltimore, and (Pomerantz independently noncanonical and canonical NF- the Although NIK 2008b). al., of et targeting (Zarnegar proteasomal is and complex protein degradative polyubiquitination a controlling of form NIK which inhibitor cIAP2, cellular and of (cIAP1) and 1 stability apoptosis TRAF2 TRAF3, the by conditions regulated negatively resting Under NF- NIK. noncanonical the active that to event central leads its The 2011). Sun, to 2006; nucleus Baltimore, the and processing (Hoffmann to dimers p100 RelB–p52 p100 of translocation IKK1-dependent and in p52 derivative subsequent results and what phosphorylation, (NIK) NF- kinase the of accumulation inducing and stabilization cell- induces upon This B 2008). lymphotoxin hours as (BAFF-R), such of receptors, (LT receptor of range factor group the selected activating the in of operates engagement pathway noncanonical activate and 2009). nucleus Karin, and the (Vallabhapurapu genes to target of translocate transcription to the dimers p65 allows This rwn ubro tde hwta aypoen nw to known proteins many that show studies of number growing A NF- canonical rapid the to contrast In b k ) n eetratvtro NF- of activator receptor and R), ahaswr is huh otasuesignals transduce to thought first were pathways B k ciiyb ouaigtesaiiyo NIK, of stability the modulating by activity B k k agtgnsta noectkns tthe At cytokines. encode that genes target B aha.Ipraty ntecanonical the in Importantly, pathway. B k nteasneo yoiestimulation, cytokine of absence the in B k inln stesaiiainof stabilization the is signaling B k RN)(u n Ley, and (Sun (RANK) B k ciain n both and activation, B eerhArticle Research k inln,the signaling, B k aha,by pathway, B b receptor k k B- B Journal of Cell Science cfodfredsmlsgaigpoen Maznk tal., et (Miaczynska proteins signaling dynamic early endosomal a of as for subpopulation 1 acts domain, APPL1 scaffold a motif endosomes, to APPL homology zipper termed localization endosomes, leucine its pleckstrin an and Through (APPL1). domain, is containing binding proteins al., et phosphotyrosine endocytic protein Pyrzynska multifunctional 2009; adaptor such al., Among et (Borlido 2009). factors nuclear various nusiuae el n a rgesvl eue with reduced progressively TNF was TNF by regulation of and times cells prolonged strongest unstimulated the was TRAF2 and in APPL1 TNF between interaction the on that with depends cells HEK293T TNF TRAF2-transfected interaction treated determine we APPL1–TRAF2 To stimulation, 2011). the Scheurich, and Wajant whether 2007; al., et (Chung the association containing with the 1C). for region (Fig. responsible APPL1 its experiments is with 87–271) that experiments (a.a. converse determined motifs finger pull-down we zinc performing TRAF2 GST of in By fragments the TRAF2 C- 1C). associate the full-length identified (Fig. to as able the was we binding which with 429–709) TRAF2 Next, (a.a. for domain in PTB responsible 1B). down terminal (Fig. APPL1 pull and of to TRAF2 PH able fragment the was translated containing bacteria 274–709; from vitro (a.a. purified GST domains) to verified PTB fragment fused further C-terminal APPL1 We the in as of 1A). direct, (Fig. is TRAF2 binding cells confirmed APPL1–TRAF2 endothelial that as of primary proteins, in endogenous presence an also two demonstrating the the versa, between vice association detected and APPL1 of We of immunoprecipitates we lysates cells. cells from with mammalian test HEK293T co-immunoprecipitations To in interact 2005). reciprocal associate al., to might performed et proteins (Rual shown system these two-hybrid previously whether yeast was a in protein APPL1 TRAF2 TRAF2 The with interacts directly APPL1 Results NF- of that machineries noncanonical between and link canonical a NF- found as the selected serve may of APPL1 triggers that induction indicates This and We turn genes. target nucleus in the which to stimulation. translocation NIK, p65 stabilizes cytokine APPL1 of overexpression without NF- the conditions of regulator 2009). al., APPL1 et (Rashid 2009; signaling that Wnt canonical showed of the expression al., increase recently to able is the We and et of histone activity 2004). the with al., modulates (Banach-Orlowska interactions et Miaczynska (HDACs) by was e.g. APPL1 deacetylases transport, regulation, endosomal in participate transcriptional and and shuttling nucleocytoplasmic of endocytosis undergo to reported in and number adiponectin role from for Apart a its receptors 2009). Dong, and and (Deepa with hormone) DCC stimulating follicle receptor associates 1 receptor netrin factor LKB1, APPL1 TrkA, growth nerve al., (including and et addition, receptors Zhou transmembrane 1999; In Akt al., et Mitsuuchi 2009). 2007; kinases al., 5-phosphatase et inositol (Erdmann serine/threonine OCRL and interacting (PI3K) the an 3-kinase as phosphatidylinositol reported for was APPL1 2009). partner al., et Zoncu 2004; ee erpr htAP1fntosa oe positive novel a as functions APPL1 that report we Here, RF soeo h e atr eitn TNF mediating factors key the of one is TRAF2 a o .,2ad6h nGTpl-onasy edetected we assays pull-down GST In h. 6 and 2 0.5, for a rvddtefrtidcto htAPPL1– that indication first the provided k inln htoeae ne basal under operates that signaling B a ramn Fg D.Ti negative This 1D). (Fig. treatment b ctnnRpi–DCcomplex, -catenin–Reptin–HDAC b ctnnTFtre ee in genes target -catenin/TCF k Bactivation. a signaling k B a RF neato a o eipratfrTNF for important be not NF- may interaction TRAF2 ne oto ftehuman the gene reporter of NF- luciferase of control a activation using the under transcriptional cells, examined the we HEK293T on First serum-grown APPL1 TRAF2. NF- with NF- of the the interaction effect regulate in its may via TRAF2 APPL1 activity that of hypothesized role we pathway, well-established a NF- Considering of activation the NF- enhances the regulates positively APPL1 ciaino NF- of activation NF- function is APPL1 viability the cell that of argues regulation This the 2D). in (Fig. cells of viability n vrxrsincnitnl eosrtdta PL can NF- APPL1 dose- the depletion that APPL1 regulate demonstrated a of positively consistently effects the in The overexpression over 2C). induction and gene (Fig. 5-fold control maximal reporter vector the empty the with APPL1 manner, activated dependent of potently domain APPL1 NF- expression of PTB effects responsible the the domain, mediating the for PTB in crucial the by is that binding, TRAF2 as confirm for data siRNA-resistant well These of 2B). as overexpression (Fig. by APPL1, NF- rescued of mouse was activity effect transcriptional significantly This basal siRNAs independent the two reduced with APPL1 of depletion ta. 96.Cepeso fbt PL n TRAF2 and APPL1 both of Coexpression (Takeuchi NF- 1996). reported potentiated previously al., as induction, et gene reporter fold low at 11-7082 BAY specifically inhibitor IKK was of (1.5–2 increase use that 2D). concentrations the (Fig. APPL1-driven confirmed by viability abolished we cell this potentiated 2004), Importantly, APPL1 al., of cell et overexpression reduces APPL1 (Miaczynska of silencing proliferation that observation previous a with xeiet PL vrxrsincudntoecm the suggesting TRAF2, overcome NF- of APPL1-induced knockdown not that by could caused inhibition overexpression reporter APPL1 experiment, activation NF- TRAF2-stimulated and of of basal Knockdown the APPL1. both inhibited for APPL1 depleted but TRAF2 overexpressing dominant-negative NF- was any had APPL1 NF- mutants with the overexpression. the on synergize effects S1A). of inhibitory Fig. to none material ability However, (supplementary NF- diminished their the proportionally wild-type stimulate and the to to comparison mutants TRAF2 and in al., three reduced C209A et significantly all was (Habelhah at of reporter TRAF2-RmZm domains) and ability finger C57A; RING The zinc 2004). the fourth and the within within C54A H51A C212A C49A, H51A, at C49A, mutated RING and the at used: dR) on mutated (deltaRING, domain (TRAF2-Rm were domain mutants point RING mutants quaternary N-terminal two TRAF2 NF- the in of characterized APPL1 deletion activity with previously ligase synergize E3 to Three the ability in their deficient for mutants TRAF2 the NF- analyzed activate APPL1 that TRAF2 indicating vector, and expression APPL1 of amounts high efrhrtse hte PL n RF yegz nthe in synergize TRAF2 and APPL1 whether tested further We odtriei PL srqie o RF oatvt the activate to TRAF2 for required is APPL1 if determine To k k aha,w esrdterpre ciiyi cells in activity reporter the measured we pathway, B signaling. B k PL otosbslNF- basal controls APPL1 splmnaymtra i.SB.I converse a In S1B). Fig. material (supplementary B k aha.I aall efudta APPL1 that found we parallel, In pathway. B k k ciain ihtemxmmosre for observed maximum the with activation, B .TA2oeepeso eutdi pt 20- to up in resulted overexpression TRAF2 B. m )wihd o e fetteoverall the affect yet not do which M) k k ciaini eitdvaTRAF2 via mediated is activation B yegsial Fg E.W also We 2E). (Fig. synergistically B k inln aha.I agreement In pathway. signaling B k ciainidcdb APPL1 by induced activation B k rmtr efudthat found We promoter. B k nue yTRAF2 by induced B k k aha and pathway B B-dependent. k ciiy4091 activity B k k activation. B Fg 2A). (Fig. B a -stimulated k Bin k k k B B B Journal of Cell Science E23 el eetasetdwt F-agdTA2(ullnt ..151 etpnl raa 751 –7,8–7,rgtpnl 4hpirt lys to prior h 24 to panel) of right 10% shows 87–271, Input antibody. 1–271, anti-GFP 87–501, with TNF detected with a.a. was or APPL1 to panel; bound ( GFP–TRAF2 left 429–709). lysate. 1–501, (a.a. cell GST–APPL1 a.a. or alone length ( GST (full with wi TRAF2. incubated blotting TRAF2 translated were western GFP-tagged lysates by vitro with analyzed in were transfected complexes total were protein of The cells GST. 5% HEK293T with ( shows fused immunoprecipitation. 274–709) Input for (a.a. antibody. used APPL1 monoclonal lysate or alone cell TRAF2 GST total with m of incubated and was 10% Rabbit TRAF2 represents antibody. translated monoclonal Input vitro mouse respectively. anti-TRAF2 controls, or negative antiserum as anti-APPL1 rabbit used either were with coimmunoprecipitation to subjected were cells HUVEC PL n RF r eesr o h ulatvto fNF- of activation full the both for that necessary are indicate TRAF2 data and APPL1 These controls. S1C). loading as Fig. serve material panel) (supplementary (lower staining S Ponceau and level GAPDH lysate. cell total of 10% shows Input panel). (upper TRAF2. with interacts APPL1 1. Fig. 4092 ptemo h K ope,smlryt RF Wjn and on acts effect (Wajant no APPL1 TRAF2 had depletion that to APPL1 similarly Consistently, arguing 2011). complex, Scheurich, 2F), IKK (Fig. the of IKK2 upstream the of on mutant APPL1 defective of effect stimulatory The NF- 1997). NF- al., inhibit et to (Mercurio reported IKK2 of previously mutant kinase-deficient (IKK2-K44M), the used we acts APPL1 pathway ofrhraayea hc tpo h NF- the of step which at analyze further To k eotrwsaoihduo oxrsino h kinase- the of coexpression upon abolished was reporter B a o .,2 rlf nrae.Cl yae eesbetdt uldw xeiet ihGTaoeo S–PL aa 7–0)adpoesda nB in as processed and 274–709) (a.a. GST–APPL1 or alone GST with experiments pull-down to subjected were lysates Cell untreated. left or h 6 2, 0.5, for ora fCl cec 2 (17) 125 Science Cell of Journal D PL–RF neato srgltdb TNF by regulated is interaction APPL1–TRAF2 ) ( A noeosAP1adTA2itrc nHK9TadHVCcls ellstsfo nrnfce E23 and HEK293T untransfected from lysates Cell cells. HUVEC and HEK293T in interact TRAF2 and APPL1 Endogenous ) k k activation B a signaling B E23 el eetasetdwt RF xrsinvco n,atr2 ,wr stimulated were h, 24 after and, vector expression TRAF2 with transfected were cells HEK293T . k B. C ciainadfntosusra fteIKcomplex. IKK the of upstream regulates functions and positively activation APPL1 that NF- indicate the data assay reporter Cumulatively, the S1D). Fig. material (supplementary p65 component emdAP nooe,det t neatoswt Rab5 with endosomes, interactions early its of to subpopulation due a endosomes, to APPL recruited termed is APPL1 its NF- for the necessary in is function APPL1 of recruitment Endosomal h NF- the h icfne eino RF id otePBdmi fAPPL1. of domain PTB the to binds TRAF2 of region finger zinc The ) k k ciainidcdb vrxrsino nNF- an of overexpression by induced activation B aha,i eurdfrTA2mdae NF- TRAF2-mediated for required is pathway, B k pathway B B PL n RF neatdrcl.A in An directly. interact TRAF2 and APPL1 ) ueIgG ouse s Cell is. hanti- th k k tal B B Journal of Cell Science ornfce ihrpre etr n ifrn obntoso PL n K2K4 xrsinvcos sidctd(l ausaein are values (all indicated as vectors, expression IKK2-K44M and APPL1 of combinations different and vectors reporter with cotransfected obntoso PL n RF xrsinvcos sidctd(l ausaein are values (all indicated as vectors, expression TRAF2 and APPL1 of combinations ( ocnrtoso A 178 rDS,a oto,fr4 .Cl iblt a esrda ecie nteMtrasadMtos n xrse sOD as expressed and Methods, and Materials the in described as measured was viability Cell h. 48 for control, a as DMSO, or 11-7082 BAY of concentrations anti-HA and 1 anti-APPL1 or with ( 0.5 detected were as PTB/APPL1 well and as mAPPL1 vectors panel). (bottom blotting western ( by respectively. analyzed antibodies, in were GAPDH are and values TRAF2 (all mAPPL1, indicated APPL1, as (mAPPL1), ( HA–APPL1 control. ( NF- mouse loading siRNA the non-targeting length a on or depletion as #2 its GAPDH and of with #1 effect panel) siRNA inhibitory APPL1 (lower the immunoblotting with overcomes by domain confirmed PTB was its level or ( APPL1 APPL1 siRNA of the non-targeting of or Knockdown #2 Methods. and and #1 Materials siRNA APPL1 the and vectors reporter NF- with the cotransfected were of cells activity enhances APPL1 2. Fig. E D PL n RF c yegsial oidc h NF- the induce to synergistically act TRAF2 and APPL1 ) PL-nue nraei elvaiiyi eedn nNF- on dependent is viability cell in increase APPL1-induced ) C m oersos feto PL vrxrsino h NF- the on overexpression APPL1 of effect Dose–response ) fAP1epeso etr PL n AD laigcnrl eeswr nlzdb etr ltig(otmpanel). (bottom blotting western by analyzed were levels control) (loading GAPDH and APPL1 vector. expression APPL1 of g k aha n cssnritclywt TRAF2. with synergistically acts and pathway B w ln ri obnto ihvcosecdn h Atge T fhmnAP1(T/PL)o full- or (PTB/APPL1) APPL1 human of PTB HA-tagged the encoding vectors with combination in or alone ) k eotr E23 el eectasetdwt eotrvcos oehrwt h different the with together vectors, reporter with cotransfected were cells HEK293T reporter. B k .HK9Tclswr rnfce ihAP1epeso etradtetdwt different with treated and vector expression APPL1 with transfected were cells HEK293T B. m fDA.Telcfrs sa a efre si uprpnl.Telvl fPTB/ of levels The panel). (upper A in as performed was assay luciferase The DNA). of g k eotr E23 el eectasetdwt eotrvcostogether vectors reporter with cotransfected were cells HEK293T reporter. B m k fDA.( DNA). of g eotratvto.HK9Tclswr ornfce ihreporter with cotransfected were cells HEK293T activation. reporter B PL otosbslNF- basal controls APPL1 ( w A .Telcfrs eotratvt a esrda ecie in described as measured was activity reporter luciferase The ). PL ncdw niisteNF- the inhibits knockdown APPL1 ) F PL csusra fIK.HK9Tclswere cells HEK293T IKK2. of upstream acts APPL1 ) k ciiy4093 activity B k aha.HEK293T pathway. B m fDNA). of g B Overexpression ) 450 . Journal of Cell Science salln acr,SB3(ratcne)adDD (colon DLD1 I and and cancer) APPL1 of (breast Levels SKBR3 cancer). cancer), lung (small data nucleus. the these to NF- together, translocation overexpression the p65 Taken activates APPL1 APPL1 S3). of that indicate level Fig. the material did with staining (supplementary correlate nuclear further upon p65 directly was increased of level and not p65 the in 4C) although of (Fig. cytoplasm overexpression, APPL1 pool its of the nuclear depletion in upon The reduced sequestered cells. HEK293T predominantly control was with revealed p65 agreement microscopy In that immunofluorescence nuclear assays. again data, reporter 4B), the biochemical (Fig. the the cells of knockdown control results in the APPL1 than confirming p65 upon less contained full Conversely, fraction in S1). are results Fig. overexpression These APPL1 NF- 4A). of the (Fig. effects on immunofluorescence nucleus stimulatory the the with by in agreement increase cells level strong a and p65 in HEK293T of resulted fractionation overexpression from APPL1 that experiments cell revealed localization Fractionation by nuclear microscopy. the further p65 monitored To NF- nucleus. of and NF- of of the regulation APPL1 the to activation in overexpressed p65 canonical APPL1 of of the role the translocation in confirm the events is final pathway the nucleus of the to One translocation p65 triggers APPL1 or or endogenous untreated in of endosomes localization APPL unequivocal to TNF TRAF2 an overexpressed in detect failed enriched analyses microscopy fractions to However, cellular 3B). of (Fig. endosomes blotting could early western TRAF2 NF- by of pool detected the small be in A membranes. function endosomal on its APPL1 for important is k APPL1 of localization 3A). (Fig. components could signaling mutants these 2007). of k al., none assay, et NF- reporter the Zhu stimulate the 2004; in tested al., When et Rab21 (Miaczynska or (APPL1-G320A) Rab5 APPL1-A318D) bind to and unable mutants, (APPL1-K280E/Y283C/G319R described membranes, previously endosomal three used to We NF- binding the or in stimulate Rab5 deficient can with thus associate to and tested unable We Rab21 mutants, 2007). al., point et APPL1 (Zhu Rab21 whether and 2004) al., et (Miaczynska 4094 PL n RF neato nooa ebae na in membranes endosomal on stable manner. that interact a dynamic possible TRAF2 transient, still not is is and it however TRAF2 APPL1 panel). endosomes, that lower APPL 3C, of indicate (Fig. punctate component data vacuoles in the these of concentrated Cumulatively, vicinity although be the TNF S2), in to structures of Fig. seemed large material independently TRAF2 APPL1-positive supplementary overexpressed TRAF2, 3C; conditions, for (Fig. these treatment negative (Miaczynska Under were APPL1 al., 2004). as vacuoles et such and al., (Stenmark effectors fusion et endosomes Rab5 enlarged containing membrane of 1994) enhances formation overexpressed the which of causes presence the mutant, and in APPL1 even Rab5-Q79L of observed Colocalization not shown). was not TRAF2 data S2; Fig. material aha,w etdwehrTA2mgtb rsn with present be might TRAF2 whether tested we pathway, B some of sequestration unproductive to due possibly activity, B efrhrvldtdteefnigi acrcl ie H1299 lines cell cancer in finding these validated further We endosomal and binding Rab that determined Having a tetdHK9To eacls(i.3;supplementary 3C; (Fig. cells HeLa or HEK293T -treated ora fCl cec 2 (17) 125 Science Cell of Journal k uieaerpre Fg ;splmnaymaterial supplementary 2; (Fig. reporter luciferase B k ciiy nta,te eue h aa NF- basal the reduced they Instead, activity. B k ciiya h idtp rti does. protein wild-type the as activity B k B a nteecl ie ifrby differ lines cell these in k aha yincreasing by pathway B k ciiywe activity B k B a gis PL rd n A(re) cl as 20 bars: Scale (green). HA antibodies and with (red) stained with and APPL1 combination fixed against were in cells or HeLa panel), panel) (bottom (upper Rab5-Q79L alone after HA–TRAF2 hours with Twenty-four transfection endosomes. with APPL1-positive blotting enlarged ( the western control). to by (loading recruited analyzed -GAPDH were and total cells -TRAF2 and HeLa anti-APPL1, (PNS) from supernatant (TCL) postnuclear lysate fractions, cell (C) cytoplasmic and (lower (EE) antibody anti-APPL1 APPL1 with ( of immunoblotting panel). levels by overexpression tested The were panel). variants (upper luciferase the made of were measurements activity and (myc-APPL1-G320A), myc-APPL1- Rab21 and or (myc-APPL1-K280E,Y283C,G319R A318D) Rab5 bind to unable oehrwt . n 1 and 0.5 with together k i.3 nooa erimn fAP1i eesr o t ucinin function its for necessary is APPL1 NF- of the recruitment Endosomal 3. Fig. aeilFg 4,) hs aaageta lhuhi hs cell nuclear these in the although that argue (supplementary and data cells APPL1 These activity SKBR3 S4D,E). and Fig. of reporter H1299 material in overexpression the p65 of weekly accumulation However, (supplementary only p65 S4B,C). enhanced of Fig. accumulation nuclear NF- material the reduced other of the each activity with these with the in correlate APPL1 inhibited of not cells Depletion do S4A). Fig. they material although (supplementary two-fold, to up ciain E23 el eectasetdwt eotrvectors reporter with cotransfected were cells HEK293T activation. B k B pathway. B RF slclzdo nooa ebae.Eryendosomal Early membranes. endosomal on localized is TRAF2 ) ( A m PL nooa oaiaini eesr o NF- for necessary is localization endosomal APPL1 ) futge idtp PL rAP1mutants APPL1 or APPL1 wild-type untagged of g k eotr concomitant reporter, B m m. C RF snot is TRAF2 ) Journal of Cell Science PL ee a ofre yimnbotn rgtpnl ihGPHlvla odn control. loading a as level GAPDH with panel) (right immunoblotting by confirmed was level APPL1 hs eut eosrt htAP1de o lyarl nthe in role a play conditions. basal not under does mainly TNF APPL1 of that regulation demonstrate results these (supplementary APPL1 of depletion o-agtn iN ( siRNA non-targeting ( siRNA non-targeting or ( #1 siRNA APPL1 with transfected I nlzdb etr ltigwt niAP1 p5 GPHad-ai-/ niois(pe ae) n h nest ftep5bn a quantified was band p65 the of intensity ( (N) the nuclear shown. and and is panel), experiments (C) (upper independent cytoplasmic antibodies (TCL), four -lamin-A/C lysate from and cell result -GAPDH representative total transfection -p65, A anti-APPL1, after panel). with hours Forty-eight blotting control. western a by as analyzed vector empty or vector APPL1-expressing PL epromdR-C nlssadmaue the measured and analysis RT-PCR performed we NF- APPL1 which determine To NF- of expression the modulate levels APPL1 overe TNF upon p65 of distribution the in TNF of NF- basal findings of types. control these cell pathway the different to the together in APPL1 activation of potentiate taken contribution NF- to a Nevertheless, basal confirm sufficient further. the not for activity are necessary levels is increased APPL1 lines p65. of accumulation nuclear induces APPL1 4. Fig. C k B muoloecnemcocp ofrsihbto ftasoaino 6 otencesuo PL ncdw.Fryegthusatrtransfec after hours Forty-eight knockdown. APPL1 upon nucleus the to p65 of translocation of inhibition confirms microscopy Immunofluorescence ) ncnrs,w ol o eetayrl o PL nteregulation the in APPL1 for role any detect not could we contrast, In a a siuae el splmnaymtra i.SAB.Similarly, S5A,B). Fig. material (supplementary cells -stimulated erdto pnTNF upon degradation a siuae NF- -stimulated a w rAP1sRA # r#) E23 el eefxdadsandwt nip5atbd lf ae) cl a:20 bar: Scale panel). (left antibody anti-p65 with stained and fixed were cells HEK293T #2), or (#1 siRNAs APPL1 or ) inln n prtsi h NF- the in operates and signaling k ciiy eddntosreaychanges any observe not did We activity. B k agtgnsmyb euae by regulated be may genes target B a tmlto a o hne upon changed not was stimulation peso rdpeino PL in APPL1 of depletion or xpression aeilFg 5) Collectively, S5C). 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B nodrt e ute ehnsi nihsit o APPL1 how into insights mechanistic further get to order In inhibition through act could APPL1 whether tested further We ora fCl cec 2 (17) 125 Science Cell of Journal k ciainocr i I.Consistently, NIK. via occurs activation B k pathway B k ciain oevr fractionation Moreover, activation. B k ciaini h tblzto of stabilization the is activation B k ciainb luciferase by activation B k aha nAPPL1- in pathway B ikdt NF- to linked ihIA I-Rascae iae obokits block to kinase) Moreover, 2000). al., et associated NF- (Burns and IL-1R of complex IKK (IL-1R stimulation a prevent in and and found IRAK phosphorylation Yamakami is 2006; Tollip al., with Additionally, et (Toll- 2004). 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B B a k n rvnigits preventing and ciainupon activation B k ,inducing B, b -arrestins a .In k B Journal of Cell Science dpnci eetr,AP1mdae ciaino the (Mao AMPK of with and activation interacting LKB1 By Akt, mediates 2007). including al., APPL1 kinases, et downstream receptors, Tang 2007; adiponectin al., et to (Hattori or NF- 2000) al., the et Ouchi activate 2005; Spurlock, shown and (Ajuwon was NF- adipocytes adiponectin type, suppress cell either the to on Depending 2003). al., et ta. 06 at ta. 07.Mroe,AP1directly APPL1 Moreover, al., 2007). et Mitsuuchi 2009; al., al., et et (Cheng LKB1 and Saito Akt with 2006; interacts al., et k aha nsnva rcricfibroblasts cardiac or synovial in pathway B k ciiy iei nohla el and cells endothelial in like activity, B ope La ta. 04 ige l,20) RF exhibits TRAF2 2005). constant al., degradative et its NF- Qing of in TRAF2–TRAF3–cIAP1/2 2004; roles because diverse al., APPL1 low et the the (Liao very conditions complex kept with by resting is non- Under NIK in association cues. of induced caused level This intrinsic be protein can by stimulation. be pathway cells stabilization cytokine can noncanonical immune of the pathway absence that NIK less noncanonical argues the in the is via overexpression, show cells we pathway HEK293T Here activated cells. in noncanonical non-immune that in the particularly investigated, stimulation, cytokine manner. that stabilization, type-dependent NIK indicate cell and/or a NF- data activation in of Akt our possibly via regulation and e.g. the levels, reports to different These contribute IKK can 1999). 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B k w ciiy(rc ta. 04.Aayigthe Analyzing 2004). al., et (Grech activity B k yoeepesdAP1cnfurther can APPL1 overexpressed by B rAP1#) ot-ih or fe rnfcin the transfection, after hours Forty-eight #1). APPL1 or inln,a tatvtstecnnclNF- canonical the activates it as signaling, B w rNK#;efcec fNKkokonwas knockdown NIK of efficiency #1; NIK or k inln eie yp65 by defined signaling B k ciiyi h bec of absence the in activity B a n nuepoesn of processing induce and , m k fDA.Teluciferase The DNA). of g ciiy4099 activity B k B–NF- k k inln via signaling B inln at signaling B k pathway B C k NIK ) B a Journal of Cell Science ieto) 5 5 digestion), pairs: oligonucleotides the TGTCAGGTC-3 using PCR GCTATGGCTGCAGCTAGC-3 by ImaGenes, Germany) BioScience amplified Source Berlin, was the TRAF2 from (obtained human IRAKp961L0764Q Full-length clone the CA). from Jolla, Warsaw). (La Stratagene Research, from obtained Technological Fundamental of Dr pNF- Goethe from (Institute II, gift Biochemistry Lipniacki a of Tomasz was (Institute Rajalingam construct pcDNA3Flag-Strawberry-p65 Krishna Frankfurt). pcDNA3-IKK2-K44M Dr University, from The gift described kind 1994). a (Mercurio pcDNA3-NIK-KDwere 11104) pcDNA3myc-cIAP1-H588A, al., previously plasmid pcDNA3myc-cIAP1, (Addgene 1997). et Mercurio al., Frank et Stenmark Dr were by provided 2004; was construct al., pCMV-Rab5-Q79L et (Miaczynska and myc-APPL1-A318D, myc-APPL1-K280E,Y283C,G319R, myc-APPL1-G320A) 429–709, a.a. pcDNA3HA- APPL1 and pGEX-6P-3-APPL1 274–709, a.a. pcDNA3HA- pGEX-6P-3-APPL1 mAPPL1, pcDNA3HA-APPL1, (pcDNA3-APPL1, plasmids APPL1-encoding Plasmids (611254, IKK2 against I monoclonal CA), mouse Jose, used: San were BD, antibodies following The all In Antibodies h. 48 either siRNA. or contransfection non-targeting by 24 or constant for vector kept after empty was used analyzed amount with performed siRNA was and were and DNA instructions transfections MD) Hilden, experiments All manufacturers’ Frederick, siRNA. 33 (Qiagen, to and and (SABiosciences, according siRNA DNA HiPerFect for of SureFECT nM or transfection 10 esiRNA). simultaneous of delivery concentration for final DNA Fugene6 (at nM delivery or for siRNA (Invitrogen) for WI) Germany) Lipofectamine2000 either Madison, Transient albumin). using (Promega, serum done were bovine was 0.2% cells transfection containing HEK293T medium TNF Switzerland). (serum-free human medium Basel, of ng/ml (Lonza, from 20 with with supplemented kit purchased treated medium SingleQuots were basal EBM-2 EGM-2 cells) as in the grown endothelial supplemented and vein (Sigma), CA) (Carlsbad, umbilical medium Invitrogen RPMI-1640 (human lung in cell HUVEC non-small grown above. (human serum H1299 were MO). calf cells Louis, DMEM fetal St carcinoma) in (Sigma, 10% L-glutamine maintained with mM were supplemented 2 cells and medium) carcinoma) HeLa Eagle’s breast and modified carcinoma) (human (Dulbecco’s colon SKBR3 (human 293T), DLD1 kidney and embryonic (human HEK293T transfection and culture Cell Methods and Materials NF- pathways. basal metabolic coordinating or signaling help other may with activity APPL1 that conceivable 4100 uiiddne nimue(1-0-5)adat-abt(1-0-5)IgGs Belgium). (711-005-152) peptide of ImmunoResearch. 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City, were Real-Time Foster calculations Fast performed Biosystems, 7900HT change NF- (Applied a was the thermocycler using PCR recommendations, for PCR interest Quantitative manufacturer’s designed of SABiosciences). to genes array reverse 84 according (PAHS-025, of PCR detection For employed M-MLV for 96-well instructions. pairs was primer and a manufacturer’s containing and expression, to pathway oligo(dT)23 signaling gene according nonamers, of used estimation random were (Sigma) synthesis transcriptase cDNA For eut r xrse stefl nuto eaiet h aa ee esrdin measured level basal the to relative induction efficiency. fold transfection firefly the the for as The control expressed a are (Promega). as Results activity instructions luciferase Renilla NF- manufacturer’s respective the its with the from luciferase derived lysed dual to activity the and luciferase using according collected assayed were kit were lysates assay Cell cells (Promega). transfection buffer 1.25 lysis after passive transfected total hours DNA a of Forty-eight amount for vector. the constant assays, all kept In was siRNA. or plasmids of combinations k ncdw r5hatrtetetwt TNF or with overexpression treatment APPL1 after upon h h 5 48 or cells knockdown HEK293T from extracted was RNA Total PCR Quantitative 100 37 at of h 2 volume for 10 with final plates treated h, and a 48 transfected After in were serum. well) (Sigma) cells/ 11–7082 (5000 plates BAY 96-well in grown Cells assays viability Cell fractions cytoplasmic and nuclear For (800 centrifuged 2011). trypsinized, al., HeLa were from et cells prepared HEK293T (Urbanska were described PNS and as cytosol cells endosomes, early in enriched Fractions fractions cell of Preparation at of representative are and experiments. parallel three mean in are least performed Values transfections siRNA. independent or four vector or control with transfected cells IAbfe 5 MTi-C H74 5 MNC,05m DA %Triton 1% in EDTA, lysed mM 5 was 0.5 pellet SDS, NaCl, nuclear mM 0.1% 150 the deoxycholate, 7.4, and 0.5% pH buffer, Tris-HCl X-100, sucrose mM the (50 buffer of RIPA top the from harvested ersnaieo tlattreexperiments. three least at of representative 6 nalssbfe 2 MHpsp .,2 MNF MNa mM 1 NaF, mM 20 7.9, pH Hepes mM (20 buffer lysis a in XL2tefloigpiso rmr eeue:NK5 NIK 5 used: and were primers of and GCTGTGTGT-3 pairs CCL2 IL-8, following APPL1, NIK, the of CXCL12 expression the of estimation For (SABiosciences). ohfatoswr etiue o 5mna 20,000 at min 15 for centrifuged were fractions Both 0m C,1 MNC,1 MTi/C H75 MET,05mM 0.5 centrifuged EDTA, and mM X-100) sucrose, 2 M Triton 7.5, (0.7 pH buffer 0.1% Tris/HCl sucrose and mM of 15 2-mercaptoethanol ml Afterwards 6 (1300 NaCl, min. mM of 15 14 mM top further 15 EGTA, for the and KCl, 0.2% on DNase of mM applied to 60 concentration DTT, added were final was mM P40 lysates a 1 Nonidet cell to ice, on EGTA, extracts lysis of mM cell min 1 the 15 After EDTA, cocktail). inhibitor mM protease 1 pyrophosphate, sodium euto nAP1mN ee)wr sdi hs experiments. these in used were level) 70% least mRNA (at silencing APPL1 efficient with in samples reduction Only GAPDH. and ACTB of according level normalized The the replicates. were Data to expression. biological gene used. of change 3 Biosystems) fold estimate (Applied from to were software used Assist duplicate were Data Biosystems) in and method least (Applied (RQ) quantification at Relative Mix obtained were Master values expression Expression Gene TaqMan ATGTCT-3 / o ula rcin.Dt r hw stefl hnet h respective the to change fold the as 1. shown to normalized are Lamin were to Data or which fractions. fractions, controls, cytoplasmic nuclear were and was levels lysates for intensity p65 cell A/C band total System. for The Imaging GAPDH marker. 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Kit Isolation Pure High using 9 9 frad,5 (forward), -TCTTTGCGGATGTCCACGT-3 2 9 g C aaAayi Software Analysis Data PCR i,4 min, 3 , m R rvre,AT 5 ACTB (reverse), eimcnann 0.5% containing medium l sas(4482 n the and (#4448892) Assays 9 g -GATGCCAGTGGCG- 9 -GCTCTCTTGGCAG- 9 9 -GACTTTGGCCAT- -CTTGGCCACAAT- ormv insoluble remove to ˚ )adresuspended and C) 6 9 9 ..fo three from s.d. frad,5 (forward), frad,5 (forward), 3 VO 9 9 (reverse), 4 (reverse) ,1mM 9 -CAG- k B 9 9 9 - - 9 atr,Y,Htoi . kmt,K,Nsiii . uui . asoa .and H. Matsuoka, K., Suzuki, T., Nishikimi, K., Akimoto, S., Hattori, Y., Hattori, R. Brink, and A. Basten, S., Gardam, T., Chan, M., Amesbury, P., A. Grech, aehh . aaah,S,Co .G,Kdy,T,Wtnb,T n oa,Z. Ronai, and T. Watanabe, T., Kadoya, G., S. Cho, S., Takahashi, H., Habelhah, G. Pei, and B. Qu, Y., M. Wang, Karin, and B., S. Luan, Ghosh, Y., Wu, Y., Sun, H., Gao, Q. L. H. Dong, and Wu, S. and S. Deepa, Q. Yin, M., Lu, Y., J. Chung, rmn,K . a,Y,MCe,H . oc,R,Le . aaie S., Paradise, S., Lee, R., Zoncu, J., H. McCrea, Y., Mao, S., K. Erdmann, W., Zhu, G., Sweeney, Y., Wang, S., K. Lam, A., M. Iglesias, K., K. Cheng, hnrskr . oltn .H,Vnaahlm . ese,N . Prabhu, J., N. Webster, K., Venkatachalam, H., W. Boylston, B., Chandrasekar, gatnme NF2-I10] eoie nPCfrimmediate Fund for PMC Research in release. Polish-Norwegian Deposited PNRF-27-AI-1/07]. the number [grant by and (HEALTH-PROT)]; 229676 the No Union GA from (EndoTrack), European LSHG-CT-2006-019050 Fellowship the numbers [grant Research 076469/Z/05/Z]; number Senior [grant a Trust by Wellcome supported was work This Funding (IFTR, Lipniacki construct. University, cIAP1 Tomasz p65 the and the Jastrzebski (Goethe manuscript, for providing Kamil the Warsaw) and for of Pilecka Rajalingam reading and Iwona critical thank project for Krishna We the constructs. NIK on to and advice grateful for Frankfurt) are We (** charts the Acknowledgements on asterisks the The test. with Whitney marked Mann were the controls three by to assessed relative least was significance at statistical The of analysis representative Statistical are and parallel in the experiments. to performed according mean MN) transfections are Minneapolis, Values Systems, estimated recommendations. (R&D was manufacturer’s supernatants ELISA culture Quantikine cell the in protein using IL-8 human of concentration The estimation protein IL-8 un,K,Cawrh,J,Mri,L,Mrio,F,Pupo,C,Mshr,B., Maschera, C., Plumpton, F., S., Martinon, Lippens, L., Martin, V., J., Swoboda, Clatworthy, K., F., Burns, Martinon, M., Kropf, L., Agostini, B., G. Brissoni, I. Mills, and V. Zecchini, J., Borlido, E. M. Spurlock, and M. K. Ajuwon, References at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.105171/-/DC1 online available material Supplementary aahOlwk,M,Plca . ou,A,Prysa .adMaznk,M. 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