JournalofMolecularCellBiology(2022),14(6),mjac037 https://doi.org/10.1093/jmcb/mjac037 PublishedonlineJune15,2022
Article
The complex of Fas-associated factor 1 with Hsp70 stabilizes the adherens junction integrity by
suppressing RhoA activation
Soonhwa Song
1,†, Joon Kyu Park
2,†, Sang Chul Shin
2,3,†, Jae-Jin Lee
1, Seung Kon Hong
2,
In-Kang Song
1, Bokyung Kim
1, Eun Joo Song
1,* , Kong-Joo Lee
1,* , and Eunice EunKyeong Kim
2,*
1GraduateSchoolofPharmaceuticalSciences,CollegeofPharmacy,EwhaWomansUniversity,Seoul03760,RepublicofKorea
2BiomedicalResearchInstitute,KoreaInstituteofScienceandTechnology(KIST),Seoul02792,RepublicofKorea
3Presentaddress:TechnologySupportCenter,KoreaInstituteofScienceandTechnology(KIST),Seoul02792,RepublicofKorea
† Theseauthorscontributedequallytothiswork.
*Correspondenceto:EunJooSong,E-mail:[email protected];Kong-JooLee,E-mail:[email protected];EuniceEunKyeongKim,E-mail:[email protected] EditedbyHaianFu
Fas-associatedfactor1(FAF1)isascaffoldingproteinthatplaysmultiplefunctions,anddysregulationofFAF1isassociatedwith manytypesofdiseasessuchascancers.FAF1containsmultipleubiquitin-relateddomains(UBA,UBL1,UBL2,UAS,andUBX),each domaininteractingwithaspecificpartner.Inparticular,theinteractionofUBL1withheatshockprotein70(Hsp70)isassociated withtumorformation,althoughthemolecularunderstandingremainsunknown.Inthisstudy,thestructuralanalysisrevealedthat His160ofFAF1isimportantforitsinteractionwithHsp70.TheassociationofHsp70withFAF1isrequiredfortheinteractionwith IQGAP1.FAF1negativelyregulatesRhoAactivationbyFAF1–Hsp70complexformation,whichtheninteractswithIQGAP1.These stepsplayakeyroleinmaintainingthestabilityofcell-to-celljunction.WeconcludethatFAF1playsacriticalroleinthestructure andfunctionofadherensjunctionduringtissuehomeostasisandmorphogenesisbysuppressingRhoAactivation,whichinducesthe activationofRho-associatedproteinkinase,phosphorylationofmyosinlightchain,formationofactinstressfiber,anddisruption ofadherensjunction.Inaddition,depletionofFAF1increasedcollectiveinvasionina3Dspheroidcellculture.Theseresultsprovide insightintohowtheFAF1–Hsp70complexactsasanovelregulatoroftheadherensjunctionintegrity.Thecomplexcanbeapotential therapeutictargettoinhibittumorigenesisandmetastasis.
Keywords:humanFas-associatedfactor1(FAF1),heatshockprotein70(Hsp70),adherensjunction,RhoAactivation,IQGAP1, X-raycrystallography,FAF1–Hsp70complex
Introduction
Fas-associatedfactor 1 (FAF1) wasinitially identified as a Fas-associatedproteinfactorpotentiatingFas-mediatedapop- tosis (Chu et al., 1995). FAF1 is a scaffolding protein that servesasaubiquitinreceptorandcontainsmultipleubiquitin- relateddomains,includingtheubiquitin-associated(UBA)do- mainandthreedomainswithubiquitin-likefolds,UBL1,UBL2, and ubiquitin-regulatory X(UBX) (Songet al.,2005). FAF1 is involved in diversecell functions by interactingwith various
ReceivedJanuary12,2022.RevisedMarch21,2022.AcceptedJune13,2022.
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partnersineachdomain.TheN-terminalUBAdomaininteracts with Lys48-linked polyubiquitinated proteins, and this inter- actionisrequiredforFAF1-mediatedapoptosisandstressre- sponse(Songetal.,2005,2009).TheC-terminalUBXdomain interactswithvalosin-containingprotein(VCP,AAAATPasep97) complexedwithNpl4–Ufd1heterodimer,anditplaysarolein promoting ERAD substratedegradation in a VCP–Npl4–Ufd1- dependentmanner(Songet al.,2005; Leeetal.,2013).The linkerregionbetweenUBL2andUASinteractswithinterferon regulatorfactor3(IRF3),akeytranscriptionfactorofIFNβsig- nalingresponsibleforthehost’sinnateimmuneresponse.FAF1 inhibitsIRF3-mediatedIFNβproductionbyreducingtheinter- action between IRF3and IPO5/importin-β3and by inhibiting nucleartranslocationofIRF3(Songetal.,2016).FAF1playsa keyroleasanegativeregulatorofvirus-inducedIFNβproduction.
UBL1domainisassociatedwithheatshockprotein70(Hsp70),
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andapreviousreportshowedthatFAF1regulatesthechaperone activityofHsp70,butnotthesubstrateofHsp70 (Kimetal., 2005).
FAF1isalsoapro-apoptoticproteinandactsasatumorsup- pressorbyreducingHsp70,whichisoverexpressedinvarious cancercells(Kimet al.,2005; Lee etal.,2012).FAF1 mRNA levelwassignificantlydownregulatedingastriccancerwithdis- tantmetastasis(Bjørling-Poulsenetal.,2003).DecreasedFAF1 expressionlevels in the advancedstage (IV) of both gastric andovariancancerhavebeenreported(Bjørling-Poulsenetal., 2003;Kanget al.,2014).Recently,whole-exome sequencing analysesofhereditarycolorectalcancershowedthatFAF1vari- ants induce resistanceto the apoptosis ofcolorectal cancer cellsandincreasetheactivityofβ-cateninandNF-κB(Bonjoch etal.,2020).FAF1inhibitstumorgrowthbyregulatingHsp70 degradation,whichhasbeenidentifiedasthemolecularmech- anismunderlyinglowFAF1expressioninhumancervicalcancer tissues(Lee et al.,2012).FAF1 is knowntopromote β-TrCP- mediateddegradationofcytosolicβ-cateninandsubsequently reducebreastcancermetastasis(Xieetal.,2017).Theseresults implicatethecorrelationbetweenFAF1expressionandcarcino- genesis.However,furthermolecularstudiesare necessaryto understandthespecificroleofFAF1duringcancerprogression.
The integrity of the cellular barrier is important for main- tainingthephysiologicalenvironmentoftheunderlyingtissue.
Adherensjunctionsregulatecell-to-celladhesion,andtheirin- hibitionpromotescancermetastasisbyfacilitatingcancercell migrationfrom the primary site to secondary sites(Farahani et al., 2014; Arnold et al.,2017; Cerutti and Ridley, 2017). Adherensjunctions,composedofcadherinsandcytosolicpro- teins,areconnectedtotheactomyosincytoskeleton.Therefore, thestructure and functions ofadherensjunctions are tightly regulated by actomyosin dynamics. The Rho family of small GTPasesincludingRhoA, Rac1,and Cdc42are crucialregula- tors of actin dynamics,cell–substratum adhesion, and cell–
cell adhesion. Rho activation leads to the assembly of con- tractileactin–myosinfilaments(stressfibers) andassociated focaladhesion complexes,while Rac1activationinducesthe assemblyofameshworkofactinfilamentsatthecellperiphery toproducelamellipodiaandmembraneruffles,andCdc42ac- tivationinducesactin-richsurfaceprotrusionscalledfilopodia (Hall,1998).RhoGTPasesfunctionasmolecularswitchesfora cyclebetweenGTP-boundactivestateandGDP-boundinactive state.Theyareactivatedbyguaninenucleotideexchangefactors (GEFs), whereas GTPase-activating proteins (GAPs) inactivate RhoGTPases.Amongthese,RhoAdirectlyenhancesstressfiber formationbyactingonitseffector,Rho-associatedproteinki- nase(ROCK).ActivatedROCKphosphorylatesmyosinlightchain (MLC)andincreasesthecontractilityofstressfibers(Ceruttiand Ridley,2017;Dybergetal.,2017).RhoAhasoncogenicproper- tiesandisupregulatedinmanycancers,includingbreast,lung, ovarian,andgastriccancers(Casteeletal.,2012;Jeongetal., 2016;SvensmarkandBrakebusch,2019).IQGAP1isascaffold proteininvolvedintheassemblyofadherensjunctions.IQGAP1
interactswithsignalingandstructuralmolecules,therebyregu- latingmanybiologicalprocesses(Tanosetal.,2018).Inepithe- lialcells,IQGAP1localizesatthesitesofcell–cellcontactand colocalizeswithp190RhoGAPandRhoA,inactivatingRhoAand suppressing airwaysmooth musclecontraction(Bhattacharya etal.,2014).Inthepresentstudy,wedeterminedthecrystal structureofFAF1 UBL1/Hsp70complexandfoundthecritical residuesforthisinteraction.Weidentifiedadditionalinteracting proteins ofFAF1 suchas nonmuscle myosinheavy chain IIA (NMIIA),IQGAP1, p190RhoGAP,and actin.Weconfirmedthat the associationofFAF1 UBL1 withHsp70 isrequired forthe interactionwithIQGAP1 andp190RhoGAP.These interactions regulateFAF1-mediatedRhoAactivationtomaintainthestruc- tureofadherensjunctionduringmorphogenesis.Inaddition, depletionofFAF1increasedcollectiveinvasionintranswelland 3Dspheroidcellculture.ThesefindingssuggestthatFAF1neg- ativelyregulatesRhoAactivationbytheinteractionoftheFAF1–
Hsp70complexwithIQGAP1,therebymaintainingthestability ofcell-to-celljunction.Therefore,FAF1maintainsthestructure and functionofadherensjunctionduringtissue homeostasis andmorphogenesisbyregulatingRhoAactivation,stressfiber formation,andadherensjunctionassembly.
Results
StructuralanalysisoftheFAF1–Hsp70complex
FAF1 plays a key role as a tumor suppressor by strongly interacting with Hsp70 (Kim et al.,2005; Lee et al., 2012). To understand the molecular mechanism, we examined the structuralbasisofthecomplexofFAF1andHsp70.Basedonan earlierreport(Kimetal.,2005),thenucleotide-bindingdomain ofHsp70(Hsp70NBD,aa1–382)andvariousconstructsofFAF1 were prepared(Figure 1A),and crystallization attempts were made. Crystals ofFAF1 (aa 100–171) and FAF1 (aa 91–171) complexed with Hsp70 NBD were obtained. To distinguish thetwoFAF1fragments,theyarenamedFAF1UBL1andFAF1 L-UBL1, respectively. Diffractiondata werecollectedto 1.2Å and 2.2 Å resolution, respectively. The crystal structure of FAF1 UBL1 (aa 100–171) alone was determined using the multi-wavelength anomalous diffraction (MAD) technique, and the FAF1 UBL1–Hsp70 NBD complex structure was determinedusingFAF1 UBL1aloneand Hsp70NBDstructure (PDB code: 1S3X) (Sriram et al.,1997). Table 1summarizes statistics on the data collection and refinement. FAF1 UBL1 adopts a tightly-packed globular structure comprising four β-strands, two η-helices, and one α-helix resembling the β-grasp fold of ubiquitin (Figure 1B), despite low sequence identity (18.6%). In the FAF1 L-UBL1–Hsp70 NBD complex structure, the UBL1 domain of FAF1 retained its globular fold, whereas the residues 91Arg–Arg96 of FAF1 adopted a β-structure (Figure 1C). FAF1 binds to Hsp70 in two parts.
Figure1Dshowsthetwointerfaceshighlightedonthemolecular surfaceofHsp70bycoloringtheresiduesofHsp70within4.0Å ofFAF1.ThedetailedinteractionsareshowninFigure1EandF,
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Songetal.,J.Mol.CellBiol.(2022),14(6),mjac037
Figure1 CrystalstructuresofFAF1UBLandFAF1L-UBL1complexedwithHsp70NBD.(A)DomainstructuresofFAF1andHsp70andthe constructs preparedinthisstudy. (B)RibbonpresentationofFAF1UBL1alone(aa 100–171).Thesecondarystructuresareindicated.
(C)RibbonrepresentationofthecomplexofFAF1L-UBL1(aa91–100)andHsp70NBD(aa1–362)showninmagentaandsilver,respec- tively,withtheboundadenosinediphosphate(ADP)andphosphate(Pi)showninthestickmodel.SubdomainsofHsp70areindicated.
(D)ThemolecularsurfaceofHsp70within4.0Å fromFAF1iscolored.ResiduesincontactwiththelinkerregionofFAF1(aa91–99)are coloredinpink,whileresiduesincontactwiththeUBL1domain(aa100–171)areinmagenta.(EandF)InteractionsbetweenFAF1L-UBL1 andHsp70NBDareshowninthestickmodel.Hydrogenbondsareindicatedasdashedlines.
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Table1 Statisticsonthedatacollectionandrefinement.
FAF1UBL1 FAF1L-UBL1complexedto
Hsp70NBDwithADP+Pi
Native SeMetMAD Native
Peak Edge Remote
Datacollection
Beamline PLSBL-5C
Wavelength,Å 1.0000 0.9794 0.9796 0.9717 1.0000
Spacegroup P42212 P3121
Celldimensions
a,Å 65.02 65.02 103.33
b,Å 65.02 65.02 103.33
c,Å 33.56 33.55 127.09
α,° 90.00 90.00 90.00
β,° 90.00 90.00 90.00
γ,° 90.00 90.00 120.00
aResolution,Å 50.0–1.2 50.0–1.6 50.0–2.2
(1.28–1.20) (1.66–1.60) (2.28–2.20)
Totalreflections 659957 321179 321309 32847 1609110
Uniquereflections 23153 18815 18830 18813 40388
aCompleteness,% 99.7(99.6) 99.3(99.0) 99.2(98.8) 99.2(98.8) 99.9(99.9)
aI/σ(I) 49.6(6.9) 37.9(6.8) 37.3(6.9) 37.8(7.1) 24.4(5.2)
bRmerge,% 5.0(21.6) 8.3(20.7) 6.5(19.6) 6.4(19.7) 9.8(32.2)
Refinement
Resolution,Å 50.0–1.2 50.0–2.2
cRcryst/Rfree,% 22.2/24.3 17.4/20.6
No.ofproteinatoms 591 3638
No.ofwateratoms 169 454
RMSdeviation
Bondlength,Å 0.005 0.007
Bondangle,° 0.771 0.876
Ramachandrananalysis
Favoredregion,% 98.63 96.92
Allowedregion,% 1.37 3.08
PDBcode 7FGN 7FGM
aValues in parentheses are for the outer most resolution shell.
bR merge= hi| I ( h ,i ) −< I ( h ) > |/ hiI ( h ,i ) , where I ( h ,i ) is the intensity of the i thmeasurement of reflection h and < I ( h ) > is the mean value of I ( h ,i ) for all i measurements.
cR freewas calculated from randomly selected 5% set of reflections not included in the calculation of the R value.
respectively. The firstinterface involves theresidues 91Arg–
Arg96 ofFAF1 adopting a β-structure and binding tightly to thecleft,makingbackbone–backboneinteractionwiththelast β-strandoftheβ-sheetintheHsp70NBD(Figure1E).Thesec- ondinterface involves146Lys–Asp149, Ser158, His160,and 162Pro–Ser166ofFAF1(Figure 1F).Theresidues involved in theintermolecularinteractionswerebothpolarandhydropho- bic. There are three salt bridgesbetween thetwo molecules (FAF1Arg91 with Hsp70Asp186 , FAF1Arg96 with Hsp70Glu218 , and FAF1His160 withHsp70Asp152 ).FAF1His160 withHsp70Asp152 inter- actionisofparticularinterest.ThesidechainsofHsp70Arg155 and FAF1Gln97 make a π–π interaction with imidazole of FAF1His160 (Figure1F).Inadditiontothesedirectprotein–protein interactions, three well-ordered water molecules bridged the twoproteins.Theinterfaceareainthecomplexwas∼2400Å2 , consistingof∼1000 Å2 for thelinker and ∼1400 Å2 for the UBL1 domain of FAF1. The overall structures of FAF1 UBL1 and Hsp70 in the complex were the same as those in the
freeform,e.g.r.m.s.d.valueswere0.65Å forFAF1UBL1and 0.45 Å for Hsp70 NBD (Supplementary Figure S1A and B). However,theloopspanning146Lys–Asp149ofFAF1,whichis involved inthe Hsp70binding, showedashiftof4Å.These differencesareinthesameorderofmagnitudeasthoseseen for the solution structure. The binding mode of the linker region of FAF1 in the complex seen here resembled that of the inter-domain linker in the crystal structureof ATP-bound DnaK, EscherichiacoliHsp70(PDBentry:4B9Q)(Kityket al., 2012, 2018; Supplementary Figure S2A). In this state, the β-domain ofthe substrate-bindingdomain (SBD)ofDnaKoc- cupiesthesamespaceastheUBL1domainofFAF1.However, theFAF1UBL1bindingsiteofHsp70identifiedherewasquite different fromthat seen in the complexstructures ofHsp70 NBD withauxilinJD (Jianget al.,2007),Hsp110/sse1(Polier etal.,2008),Sil1(Yanetal.,2011),Bag1(Sondermannetal., 2001),Bag2(Xuetal.,2008),orBag5(Arakawaetal.,2010) (SupplementaryFigureS2B).
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Songetal.,J.Mol.CellBiol.(2022),14(6),mjac037
Figure2 ITCanalysisofFAF1andHsp70NBD.(A)ITCresultsofFAF1bindingtoHsp70NBD.FAF1(aa1–171),FAF1(aa91–171)[His160Ala], FAF1(aa91–171),andFA1(aa91–171)[His160Ala]weretitratedtoHsp70(aa1–362)withADP.Thetoppanelshowsthethermaleffects associatedwiththeinjection,whilethebottompanelshowsthebindingisothermcorrespondingtothedatainthetoppanelandthebest- fittedcurvewithaone-sitebindingmodel.(B)SummaryofITCmeasurement.
BindingaffinitybetweenFAF1UBL1andHsp70usingITC Next,weexaminedtheinteractionsbetweenHsp70NBDand variousfragmentsofFAF1usingisothermaltitrationcalorimetry (ITC)(Figure2AandB).First,bothFAF1(aa1–171)andFAF1(aa 91–171)boundtoHsp70NBDinthepresenceofADPata1:1 molarratio,asinthecrystalstructure,withbindingaffinities(KD ) of15.9±0.4μMand3.1±0.1μM,respectively(Figure2A), while the corresponding values in the absence ofADP were 56.5 ±1.1 μM and 3.1 ±0.1 μM,respectively (Figure 2B). However,otherconstructstried,includingFAF1(aa1–90),FAF1 (aa99–171),and FAF1(aa172–650),showednodetectable binding. Also, the peptide corresponding to 91Arg–Gln–Ile–
Val–Glu–Arg96 (91 RQIVER96 ) and FAF1 (aa 1–171) missing 91Arg–Arg96exhibitednodetectablebinding.Therefore,these
resultssuggestthat boththe linkerregionand theUBL1(aa 100–171) domain of FAF1 are critical in binding to Hsp70.
Comparison of FAF1 in the two structures, i.e. FAF1 alone andthecomplex,showedalargeshiftatthe146Lys–Asp149 region of FAF1, suggesting that this region is quite flexi- ble (Supplementary Figure S1A and B). As such,we hypoth- esized that the FAF1His160 –Hsp70Asp152 interaction is critical for the interaction between the two proteins, as suggested in the complex structure (Figure 1F). To test the idea, we constructed the FAF1His161Ala mutant in both FAF1 (aa 1–
171) and FAF1 (aa 91–171). To our surprise, both mutants showedno bindingtoHsp70 NBD (Figure 2A), thusconfirm- ing that the interaction is critical forthe binding ofthe two proteins.
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FAF1interactswithproteincomplexesinthecelladherens junction
To investigate the function ofthe FAF1–Hsp70complexin cells, we identified the FAF1-interacting proteins by a pull- downassay.ProteinsdirectlybindingtoGST-FAF1weresepa- rated andsubjected toin-geldigestion withtrypsin followed by peptide sequencing employing nanoUPLC-ESI-q-TOF tan- demmassspectrometry(MS)(equippedatDrugDevelopment ResearchCore Center) (Figure 3A).In additiontothe already reportedinteractingproteins,e.g.Hsp70,VCP,Npl4,andubiq- uitin,wefoundNMIIA,IQGAP1,tubulin,β-actin,andPRKCapop- tosisWT1regulatorprotein(PAWR)asFAF1-interactingproteins (SupplementaryTable S1). NMIIA,a constituent ofactinfila- ment,isimportantforthedynamicsofadherensjunction,asit regulatescellmorphogenesis,tissuehomeostasis,andhealing byregulatingstressfiberformationwithactinfilaments(Heuzé et al., 2019). IQGAP1, a scaffolding protein involved in the assemblyofadherensjunctions,co-localizeswithp190-RhoGAP andRhoA(Bhattacharyaetal.,2014;Xuetal.,2018).Toidentify theFAF1-bindingsiteoftheseproteins,weperformedaGST-pull- downusingthefull-lengthFAF1(aa1–650)andFAF1(aa352–
650)lackingUBA,UBL1,andUBL2.Wefoundthatthebinding toactin,NMIIA,andIQGAP1wasabolishedinFAF1(aa 352–
650),unlikefull-lengthFAF1(Figure3B),suggestingthattheN- terminalregionofFAF1,i.e.FAF1(aa1–351),maybeinvolved in regulating adherens junction.To validate the newly iden- tified interacting proteins, we examined cellular interactions byimmunoprecipitation.LysatesfromHeLaandHEK293Tcells overexpressingFlag-FAF1were immunoprecipitatedwith anti- Flagantibodies.Immunecomplexesweredetectedbywestern blottinganalysisusinganti-IQGAP1,anti-NMIIA,andanti-actin antibodies(Figure3C;SupplementaryFigureS3A).FAF1further interacted withp190B-RhoGAP, knownas IQGAP1-interacting protein(SupplementaryFigureS3B).However,FAF1interacted withp190B-RhoGAPandactinonlywhenthecellswerelysedin amildhypotonicsolutioncontainingoctylβ-D-glucopyranoside but not NP-40 (Supplementary Figure S3B), suggesting that p190B-RhoGAPandactinweaklyinteractwithFAF1.
Tofurtheridentify whichspecificregionofFAF1 isinvolved inrecruitingIQGAP1andNMIIA,weperformedanimmunopre- cipitationassayafterthetransfectionwithFlag-FAF1full-length (aa1–650),FAF1(aa1–201)containingUBAandUBL1domain, andFAF1(aa82–650)deletingUBAdomain.Allthree,thefull- lengthand the twotruncated mutants, commonlycontaining theUBL1domain,interactedwithIQGAP1andNMIIA,indicating thattheUBL1domain isnecessarytobindtothese proteins (Figure3D).SinceUBL1interactswithHsp70,weinvestigated whethertheassociationofFAF1withHsp70isaprerequisitefor variousinteractionsofFAF1withIQGAP1,NMIIA,andactinby employingFlag-FAF1His160Ala mutant,whichisunabletobindto Hsp70.First,weexaminedwhetherHis160ofFAF1isimportant forthe interaction with Hsp70. As shown in Figure 3E,FAF1 wild-type(WT)interactedwithHsp70,poly-ubiquitin,andVCP asreportedpreviously(Songetal.,2005),whereasFAF1His160Ala
mutant interacted with poly-ubiquitin and VCP but not with Hsp70.Intriguingly,thismutantcouldnotinteractwithIQGAP1 aswellasHsp70.
SilencingFAF1inducessignificantmorphologicalchanges throughRhoAactivation
To investigate whether FAF1 interacting with protein com- plexesinthecelladherensjunctionplaysaroleinmorphogene- sis,weexaminedmorphologicalchangesinHeLacellssilencing FAF1. Wefound that thesilencing ofFAF1 increased thecell sizeandtransformedthecellstoamoresphericalshape.Since polymerizedcytoskeletonsareresponsibleforcellmorphology, weinvestigatedwhetherFAF1alterstheformationofactinstress fibers, which are contractile actomyosin bundles containing actinfilamentsandactiveNMII.Weexaminedthestressfibersin HeLacellsaftersilencingFAF1byconfocalmicroscopyalongwith immunostainingwithanti-actinandanti-pMLC2.HeLacellswith silencedFAF1(FAF1KD)showedsignificantlyincreasedcentrally located F-actinstressfibersandpMLC2thatconformationally alteredNMIIintobipolarfilaments(Figure4A).Thesefilaments associatewithactinfilamentsinbundles(Vicente-Manzanares et al.,2009), suggesting that FAF1regulates stressfiber for- mation. Inaddition, HeLa cellswerestainedwith antibodies forpaxillinandNMIIA,whichareexpressedinfocaladhesions andaconstituentofactinfilament,respectively.Consistentwith Figure4A,paxillinandNMIIAlevelswerealsoincreasedinHeLa cellsafterFAF1silencing(SupplementaryFigureS4).
FAF1 KD cells clearly showed an increase in actin stress fiber formation,but notlamellipodia orfilopodia, which isa well-knownphenotypeofRhoactivation(Hall,1998;Maekawa et al., 1999). To investigate how FAF1 inhibits stress fiber formation,weexaminedRhoAactivationbecauseactiveRhoA activatesROCKandthenphosphorylatesMLC,whichleadstothe assembly ofcontractileactomyosinbundleswithunbranched polymerized actin (Hall, 1998). We quantified GTP-bound activeRhoA throughapull-downassayusingtheGST-RBDof Rhotekinthat interactswithactiveRhoA and thenperformed western blottinganalysisusing aspecific antibody.Weused two differentsiRNAstosilence endogenousFAF1 expression.
AsshowninFigure4B,silencingofendogenousFAF1increased GTP-bound active RhoA but not Rac1 (Supplementary Figure S5A–C). Then, to confirm the restoration of RhoA activation inFAF1-silencedcellsbyaddingFAF1,wemeasuredtheRhoA activation in HeLa cells with endogenously silenced FAF1 followedbytransfectionswithcontrolorFlag-FAF1.ActiveGTP- boundRhoAwasincreasedinFAF1KDcells,andtheincrease inactiveRhoAlevelwasreducedbyaddingFlag-FAF1compared tocontrol(Figure4C).Theeffectswereverifiedbyquantifying pMLC2asatargetproteinofactiveRhoAviaROCKactivation.
AsshowninFigure4D,thepMLC2levelsignificantlyincreased in cells silencing FAF1, and this increase was abolished by addingFlag-FAF1viatransfection.Theseresultsconfirmedthat FAF1suppressesRhoAactivationanditsdownstreamsignaling, ROCK,andphosphorylationofMLC2.
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Songetal.,J.Mol.CellBiol.(2022),14(6),mjac037
Figure3 FAF1formsacomplexwithIQGAP1andactomyosin.(AandB)GSTandGST-FAF1(A)orGST,GST-FAF1WT,andGST-FAF1(aa362–
650)(B)wereincubatedwithglutathione-agarosebeadsfor3handwashedsixtimeswithwashingbuffer.GST-fusedprotein-boundbeads werefurtherincubatedwithHeLacelllysatesfor3h,andthenthebeadswerewashedsixtimes.Proteincomplexeswereseparatedby SDS–PAGEanddetectedbysilverstaining(A)oranalysedbywesternblotting(B).(C)HeLacellsweretransfectedwithFlagorFlag-FAF1.
After 24h,celllysateswereimmunoprecipitatedwith anti-Flagantibody,andtheimmunecomplexwasanalysedbywesternblotting.
(D)HeLacellsweretransfectedwith Flag,Flag-FAF1WT,Flag-FAF1(aa1–201),orFlag-FAF1(aa82–650).After24h,celllysateswere immunoprecipitatedwithanti-Flagantibody,andtheimmunecomplexwasanalysedbywesternblotting.(E)HeLacellsweretransfectedwith FAF1WTorFAF1H160Amutantandthenimmunoprecipitatedwithanti-Flagantibodies.Immunoprecipitatedproteinswereimmunoblottedwith theindicatedantibodies.
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Figure4 FAF1silencingpromotesactinstressfiberformationandFAF1negativelyregulatesRhoAactivation.(A)HeLacellstransfectedwith controlsiRNAorFAF1siRNA#2for72hwerefixedandstainedforF-actin(phalloidin)ordoublephosphorylatedMLC2(Thr18/Ser19)to investigatestressfiberformation.ThebargraphindicatesthefluorescenceintensitiesforF-actinordoublephosphorylatedMLC2intheleft panel.Theexperimentswereconductedinquadruplet.Scalebar,10μm.(B)HeLacellsweretransfectedwithtwotypesofFAF1siRNAs,#1 and#2.After72h,cellswerelysed,andthelysateswereincubatedwithGST-RBDofRhotekintomeasurethelevelofGTP-boundRhoA.Cell extractswereusedtoanalysetheamountoftotalRhoAbywesternblotting.TherelativeamountofRhoA-GTPincontrolandFAF1KDcellswas determinedbywesternblottingfollowedbydensitometryanalysis.(C)HeLacellswithsilencedFAF1(bytransfectingwithcontrolsiRNAor FAF1siRNA#2for48h)weretransfectedwithFlagorFlag-FAF1asindicatedforaddingbackFAF1.At24hpost-transfection,cellswerelysed, andthelysateswereincubatedwithGST-RBDofRhotekintomeasurethelevelofGTP-boundRhoA.Cellextractswereanalysedusingthe indicatedantibody.TherelativeamountofRhoA-GTPwasdeterminedbywesternblottingfollowedbydensitometryanalysis.(D)HeLacells weretransfectedwithcontrolsiRNAorFAF1siRNA#2for48handtransfectedwithFlagorFlag-FAF1asindicated.At24hpost-transfection, cellswerelysedwithgelsamplebufferandanalysedusingtheindicatedantibodies.
FAF1isvitalforstabilizingadherensjunction
RhoGTPasesareregulatorsofthedynamicorganizationand contractilityofjunctionalactomyosin.Sincesilencing ofFAF1 activatedRhoA(Figure4),we examinedwhetherFAF1 affects theintegrityofthecelladherensjunction.Theadherensjunc- tionintegrityincontroland FAF1-depleted HeLacellswasvi- sualizedby stainingthe cellswith β-catenin, sinceβ-catenin isa cytosolicadapter protein betweenextracellularcadherin andintracellularβ-catenin/F-actin.Imageanalysisshowedthat β-cateninincontrolcellswithastableadherensjunctionhada thinlinearappearance,whereasβ-cateninintheFAF1-depleted cellsshowedastretchedanddiffusedappearance(Figure5A). Whenthefluorescenceintensitiesofβ-catenin(dashlines)were plotted,thepeakofadherensjunctionwassharpincontrolcells butbroadin FAF1KDcells(Figure 5B).These resultsdemon- stratedthatsilencingofFAF1impairstheintegrityofadherens junction.
Next,toconfirmtheeffectsofFAF1ontheformationofcell–
celljunctions,weconductedacalciumswitchassay.Alowcal- ciumleveldisruptsadherensjunctionbyremovingcalciumfrom thebindingsitesonE-cadherinextracellulardomains,causing a conformational changeand interruptingcell–cell adhesion.
ConfluentHeLacellswithendogenoussilencing ofFAF1were culturedinalowCa2 + medium(∼2μMCa2 + )for30h,andthe assemblyofcell–celljunctionswasinitiatedbyswitchingthe mediumtoanormalCa2 + level(∼2mMCa2 + ).Thedegreeof junctionformationand maturationwasevaluatedbystaining for β-catenin with an anti-β-catenin antibody. When calcium wasdepletedfromthemediumfor30h,β-cateninstainingin bothcontrolandFAF1-depletedcellswasdispersedasshown indisruptedadherensjunctions(0h).β-cateninincontrolcells appearedstretchedat3hafterre-incubationwiththemedium containing a normalconcentration ofcalcium (2mM),but it hadathinlinearappearanceafter6hincubation.However,in
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Songetal.,J.Mol.CellBiol.(2022),14(6),mjac037
Figure5 FAF1alterstheintegrityofadherensjunction.(A)HeLacellsweretransfectedwithcontrolsiRNAorFAF1siRNA#2andreplatedon coverslipsfor72h.Cellswerefixedandstainedwithβ-catenintoinvestigatetheintegrityofadherensjunction.Scalebar,10μm.(B)The fluorescenceintensitiesofβ-cateninarerepresentedalongthedashedlineinA.Normalizationwasperformedwithrespecttobothendpoints ofthedashedlineinthreedifferentexperiments.(C)HeLacellsweretransfectedwithcontrolsiRNAorFAF1siRNAfor48handreplatedon coverslipsinalow-calciummedium.After30h,cellswerereturnedtothenormal-calciummediumtoinitiatejunctionassembly.Thecells werethenfixedandstainedwithβ-cateninattheindicatedtimepoints.Scalebar,10μm.(D)HeLacellsweretransfectedwithcontrolsiRNA orFAF1siRNAfor72handlysedwithgelsamplebuffer,andthelysateswereanalysedbywesternblotting.(E)HeLacellsweretransfected withcontrolsiRNAorFAF1siRNAandreplatedoncoverslipsfor72h.CellsweretreatedwithvehicleorY-27632(10μM)for6handthen stainedwithF-actin(phalloidin)andβ-catenin.Scalebar,10μm.
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FAF1-depleted cells, β-catenin remained discontinuous and stretchedeven after 6h incubation in the medium contain- inganormalcalciumconcentration(Figure5C).Toinvestigate whether thesechanges weredue totheproteinlevelsin ad- herensjunction,weexamined theexpressionlevelsofactin, N-cadherin, β-catenin, and γ-catenin in control and FAF1- depletedcells.SilencingofFAF1didnotaffecttheproteinex- pressionlevelsinadherensjunction(Figure5D).Thesefindings confirmedthatFAF1activelyregulatesthestabilityanddynam- icsofadherensjunctions.
TreatmentwithaROCKinhibitorabolishestheimpairmentof adherensjunctionduetothesilencingofFAF1
Impairmentofadherensjunctioninthe FAF1-silencedcells occursviaRhoAactivation,whichactivatesROCK,upregulates pMLC,andtriggersstressfiberformation.Toconfirmthis,weex- aminedβ-cateninandpMLClevelsincontrolandFAF1-depleted HeLacellsbytreatingthecellswiththeROCKinhibitorY-27632.
As shown in Figure 5E, silencing ofFAF1 impaired adherens junctiondetected bytheelevatedβ-catenin andpMLClevels, whereasY-27632treatment inhibitedthe disassemblyofad- herensjunctionbysuppressingpMLCinduction.TheROCKin- hibitorabolishedtheimpairmentofadherensjunctioninduced byFAF1knockdown.TheseresultssuggestedthatFAF1deple- tion impairs adherensjunction by activating RhoA and then increasesstressfiberformationbyactivatingROCK.
TheFAF1–Hsp70complexisrequiredforsuppressingRhoA activationorsuppressingstressfiberformation
ElevatedHsp70expressionishighlycorrelatedwithincreased cancercellproliferationaswellasmetastasisandthusissug- gestedasa therapeutictargetforcancer (Hall,1998;Juhasz etal.,2013;Yietal.,2017).Theregulationofcelladherence by Hsp70 is also proposed as one of the mechanisms con- tributingtometastasis(Juhaszet al.,2013;Boudesco etal., 2018).FAF1interacts withIQGAP1,NMIIA,and actinthrough theUBL1domain,whichinteractswithHsp70.Thesefindings encouragedustoinvestigatewhetherthebindingofHsp70to FAF1isrequiredforitsinteractionwithIQGAP1,NMIIA,andactin andformaintainingtheintegrityofadherensjunction.Asshown in Figure 3E,FAF1 WT interacted with Hsp70, poly-ubiquitin, andVCPasreportedpreviously (Songet al.,2005),whereas FAF1His160Ala mutantinteractedwithpoly-ubiquitinandVCPbut notwithHsp70.Intriguingly,thismutantcouldnotinteractwith IQGAP1 as well as Hsp70. Weexamined RhoA activation by addingbackFAF1WTandFAF1His160Ala mutantinFAF1-depleted cells,asrecentstudiesdemonstratedthatIQGAP1inhibitsRhoA activation.Asshown earlierinFigure 4,silencingofFAF1 in- ducedRhoAactivation,whichwasabolishedbyoverexpressing FAF1WTbutnotFAF1His160Ala mutant(Figure6A).Thequantifi- cationofpMLC,adownstreamtargetofRhoAactivation,further confirmedtheseeffects(Figure6B).FAF1activatesRhoAbyinter- actingwithIQGAP1viatheassociationofHsp70withtheUBL1 domainofFAF1.StressfiberformationinFAF1-silencedcellswas
diminishedbyaddingbackFAF1WTbutnotFAF1His160Ala mutant (Figure6C).
SilencingofFAF1increasescollectiveinvasionintranswelland 3Dspheroidcellculture
To investigate whether morphological changes induced by FAF1 depletion affect cancer metastasis, we utilized in vitro transwellinvasion and a3D spheroidmodelofMDA-MB-231 breastcancercells.FAF1depletionleadstoincreasedtranswell invasion(SupplementaryFigureS6).A3Dspheroidmodelre- flectstheinvivomicroenvironmentmorepreciselythan anin vitromonolayercellculturemodelandis,hence,calledan‘in vivo-likemodel’.The3Darrangementofcellsallowsustostudy thecell–cellinteractionsandotherinvivoprocesses.Asshown inFigure7AandB,FAF1silencinginthismodelpromotedthe invasionofcancercells.Theresultsverifiedourhypothesisthat theexpansionofcellsizeandweakeningofadherensjunction affecttheinvasivepotentialofcancercells.
Discussion
Inthisstudy,wedeterminedthecrystalstructureofacomplex of FAF1 UBL1 and Hsp70 NBD and found that this complex playsakeyroleinstabilizingadherensjunctionbysuppressing RhoA activation through its interaction with IQGAP1 via the UBL1domain. SilencingofFAF1 in HeLacellsinducesstress fiberformationbypromotingRhoAactivation,ROCKactivation, andMLCphosphorylationasdownstreamsignalingcomponents (Figure7C).
Thecrystalstructureofthecomplexrevealedthatboththe 10 or so residues preceding the UBL1 domain as well as residues146–149and158–166ofFAF1areimportantinthe binding to Hsp70 NBD. Furthermore, ITCanalysis and struc- tural data suggested that His160 of FAF1 is critical for the bindingtoHsp70. Residueslining theHsp70 surfaceforthe β-structuredlinkerbindingarehighlyconserved.Surprisingly, theinterdomainlinkerofHsp70,forminga β-structure,binds tothe samebindingcleft asthe FAF1linker,resulting inthe β-domainofSBDpositionedneartheUBL1ofFAFintheATP- boundDnaK(SupplementaryFigureS2A).Therefore,onceFAF1 bindstoHsp70,theinterdomainlinkerofHsp70cannotbindto thecleft,i.e. thetwowillbecompetingforthesame binding surface. Theinterdomain linkermodulatesvariousstructure–
function featuresofHsp70, suchas its global conformation, theaffinityforpeptidesubstrates,andtheinteractionwithco- chaperones(Kampingaand Craig,2010; Clericoetal.,2019; MayerandGierasch,2019;Rosenzweigetal.,2019).Inother words,thisinterdomainlinkerfacilitatesinterdomaincommuni- cationbetweentheNBDandSBDofHsp70.Unlikethehighly conservedinterdomainlinkerofHsp70,thesequenceidentity onthelinkerregionofFAF1 islow,andthereisnosequence homologytothe inter-domainlinkerofHsp70.However,this isnotcritical,sincebackbone-to-backboneinteractionoccurs at‘interfaceI’betweenHsp70andFAF1(Figure1E).However, Gln97 andHis160ofFAF1are highlyconserved amongstthe
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Songetal.,J.Mol.CellBiol.(2022),14(6),mjac037
Figure6 FAF1–Hsp70interactioniscrucialforFAF1regulationofRhoAactivation.HeLacellswithFAF1knockdownweretransfectedwithFAF1 WTandFAF1H160Amutant.Then,RhoAactivation(A)andMLC2phosphorylation(B)levelsweremeasured,andactinstressfiberformation wasdetectedbyconfocalmicroscopy(C).Scalebar,10μm.
species.AlthoughFAF1andHsp70inthecrystalstructureare notintheirfullforms,FAF1bindingcertainlycaninhibitHsp70 function.Withtherecentimprovementinomicstechnologies, avastnumberofpost-translationalmodifications(PTMs)have beenuncoveredontheHsp70familyofproteins(Nitikaetal., 2020and the referencestherein).Althoughtherespectiveroles are not yet fully understood, the PTMs that lie close to the interactionsiteofHsp70andFAF1mayaltertheinteraction.The interactionbetweenthetwoproteinsisexothermicwithaKD of 3–16μMinthepresenceofnucleotides.Also,theinterfacearea of∼2400Å2 suggeststhattheinteractionbetweenthetwoisnot verystrongbutenoughtofunctionasaregulator.
Mechanicalstressinducesheatshockresponse,increasing the expression of heat shock proteins, Hsp70, Hsp90, and Hsp27.Inhibitorstargetingheatshockproteinshavebeendevel- opedasanticancerdrugs,sinceupregulatedheatshockproteins areresponsibleforcancerprogressionandmetastasis.Forex- ample,geldanamycin,anHsp90inhibitor,increasesstressfiber formationbyregulatingRhoA-dependentcytoskeletonremodel-
ing,thusinhibitinginvasionandmetastasis(Amirietal.,2007). Hsp70 increasesmetastatic growththrough theupregulation ofcytoskeleton-dependentsignalingaswellaschaperoneand anti-apoptoticfunctions(Juhaszetal.,2013).Hsp70regulates manycell adhesionmolecules,among which upregulation of RhoAexpressionbyextracellularHsp70toincreasecellmigra- tion and invasion in hepatocarcinoma hasbeen reported (Yi et al.,2017).Inaddition, theexpressionofFAF1and Hsp70 isassociatedwithcancerdevelopment.Mostpreviousstudies have been conducted on either FAF1 or Hsp70 individually.
However,Kangetal.(2014)reportedacorrelationbetweenFAF1 andHsp70expressioninovariancancer.TheyshowedthatFAF1 expression decreases in advanced stages of ovarian cancer, while theexpressionofHsp70increases; therefore,FAF1 ex- pressioninverselycorrelateswithHsp70expression.Thisreport supportedourfindingsthatFAF1structurallyinhibitstheHsp70 cycle.WefurtherinvestigatedwhetherinhibitionofHsp70activ- ityisnecessaryfortheregulationofRhoAbyFAF1viainteraction withIQGAP1.
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Figure7 FAF1inhibitscancercellinvasioninMDA-MB-231cellsbyregulatingtheintegrityofadherensjunction.(AandB)MDA-MB-231cells weretransfectedwithcontrolsiRNAorFAF1siRNAfor48handthenspheroidformationwasinitiatedin2.5%Matrigel.Then,sizekinetics weremeasuredbycalculatingvolumechangesbasedontheradiusofeachspheroid.Representativeimages(A)andrelativevolumeofthe 3Dspheroidcellinvasion(B)areshown.Errorbarsrepresentstandarddeviation(n=3).Scalebar,200μm.(C)Schematicdiagramofthe effectofFAF1onstabilizingadherensjunctionbysuppressingRhoAactivation.
Dysregulationofcelladhesionplaysacriticalroleinmalignant transformationandmetastasis.Despite thefunctionalsignifi- canceofRhoAactivation,intactadherensjunctions,andreorga- nizationoftheactincytoskeletonmachinery,detailsregarding howtheassociatedproteinsparticipateintheprocessarenot yet known.RhoA hasbeen widely studied to understandits roleintheinvasionandmetastasisofmalignanttumorsandis consideredapromising therapeutictarget.Yoonet al.(2016) showedthatRhoAactivityishigheringastricadenocarcinoma, andRhoAinhibitioncanreversechemotherapyresistance.Both theexpressionlevelandactivityofRhoAareimportantforthe progressionofcancers,e.g.breastcancer(Zhengetal.,2020). UsinganactiveRhoApull-downassay,weshowedthatFAF1de- pletionpromotesRhoAactivitywithoutalteringtheexpression
ofRhoA. Additionally,FAF1depletionincreasedbreastcancer cellinvasioninthetranswellassayand3Dspheroidmodel.FAF1 is knowntoregulatethe proteasomaldegradationofcancer- relatedproteinsrecruitingtheVCPandE3ligase(Mengesetal., 2009). Forinstance, FAF1 destabilizes TβRII on the cell sur- face by recruitingthe VCP/E3 ligase complex, thereby limit- ingpro-oncogenicresponsestoTGF-β(Xieetal.,2017).TβRII regulatesRhoAexpressionandactivity(Ozdamaretal.,2005; HuangandChen,2012),implyingthatFAF1regulatecancercell proliferationandinvasionthroughdirectorindirectregulation ofRhoA.
RhoA-mediatedregulationofinvasionandmetastasisoccurs through the stimulation of contractility by activating down- streammolecules,ROCKandMLC,whichleadstotheformation
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Songetal.,J.Mol.CellBiol.(2022),14(6),mjac037
ofstressfibersandfocaladhesions.TreatmentwithaROCKin- hibitorabolishedtheimpairmentofadherensjunctioninduced bysilencingofFAF1(Figure5E),suggestingthatFAF1regulates theintegrityofadherensjunctionviatheRhoA–ROCKpathway.
RhoAactivityisprimarilyregulatedbyGEFs,GAPs,andguanine nucleotidedissociationinhibitors.IQGAP1isanotheressential factorregulatingtheRhoAactivity.Itisascaffoldproteinthat providesaplatformforsmallGTPaseproteinslikeRhoA,RhoC, andRac1andtheirregulators,suchasp190RhoGAP,Cdc42,and Arf6(Hedmanetal.,2015).Therefore,thefunctionofIQGAP1 asascaffoldmayfacilitatetherapidregulationofRhoAactivity.
In addition,IQGAP1 localizestositesofcell–cellcontactand colocalizeswithp190RhoGAPandRhoA,inactivatingRhoAand suppressing airwaysmoothmusclecontraction(Bhattacharya et al., 2014). IQGAP1 is associated with diverse oncogenic functions likecellproliferation,extravasation,andmetastasis (Hebertetal.,2020).IQGAP1interactswith>100proteinsand exhibitsoncogenicfunctionseitheraloneorincombinationwith bindingproteins.Here,wefoundthatFAF1interactswithIQGAP1 and p190RhoGAP. The interaction ofFAF1 with p190RhoGAP issignificantlyweakcomparedtothatwithIQGAP1.Therefore, IQGAP1 could mediate the interaction of p190RhoGAP with FAF1.TheFAF1–Hsp70complexwasfoundtobindwithIQGAP1, whichmeansthatthiscomplexregulatesRhoAactivitybyrecruit- ingothermodulatorssuchasp190RhoGAP.Complexformation ofFAF1withHsp70mayprovideaninteractionsurfaceneeded forIQGAP1.However,itisalsopossiblethatIQGAP1interacts with onlytheFAF1 UBL1domaininthe Hsp70-boundconfor- mation.Thecrystalstructureofthecomplexshowedthatonlya handfulofresidues(146–149and158–166)ofFAF1UBL1were engagedinitsinteractionwithHsp70.Therefore,alargesurface ofUBL1isavailableforotherpartnerproteins(Figure1C).TheJ domainbindsontothesurfaceabovetheinterdomainlinkerand contactstheβ-domainoftheSBD(Kityketal.,2018).TheUBL1 surfaceissomewhatnegativelycharged(SupplementaryFigure S1C).
Here, we showed another novel function of FAF1 of using Hsp70 asanadapter proteinand interactingwithIQGAP1 to regulatethe assemblyand disassemblyofadherensjunction throughRhoAsignaling.However,furtherstudiesarerequired toinvestigatewhichismoreimportantfortheregulationofcell invasionbytheFAF1–Hsp70complex.
Materialsandmethods
ExpressionandpurificationofFAF1andHsp70
Several constructs, including full-length protein of human FAF1, werecloned intopET-28a with a His-tag added at the N-terminus(Figure1A)andexpressedinE.coliRosetta-gami2 (DE3) cellsorRosetta2(DE3)cells(Novagen).Afterinduction using0.5mMisopropylβ-D-1-thiogalactopyranoside,thecells were allowed togrow for 12 hat 18°C and then harvested.
Proteins werepurifiedemployingthe Ni-NTAaffinity,MonoQ anionexchangechromatographyfollowedbytheSuperdex-200S 26/60orSuperdex-75S26/60gelfiltrationandconcentratedto 20–25mg/ml.Thetagwasremovedaftertheaffinitypurifica-
tion.AllmutantsofFAF1weregeneratedandpurifiedfollowing thesameprocedureasfortheWT.Toobtainselenomethionine (SeMet)-substitutedprotein,FAF1UBL1wasoverexpressedus- ingthesameproceduredescribedaboveexceptwithE.coliB834 (DE3)cellsandM9cellculturemediumasdescribedpreviously (Hendricksonetal.,1990).Thehexapeptidecorrespondingto
91 RQIVER96 ofFAF1wassynthesized.TheNBDofhumanHsp70 (aa1–382)wasclonedintothepET-28avector(Novagen)with theTEVcleavagesiteandoverexpressedinE.coliRosetta2(DE3) cells(Novagen).PurificationwascarriedoutemployingaNi-NTA columnfollowed byremovalofthe tagand filtrationthrough aSuperdex-200S26/60gelfiltrationcolumn(GEHealthcare). Thefinalproteinin50mMHEPES,pH7.5,150mMNaCl,2mM MgCl2 ,and1mMDTTwasconcentratedto20mg/ml.
Crystallization,datacollection,andstructuredeterminationof FAF1UBL1andFAF1UBL1–Hsp70NBD
Diffraction quality crystals of both the native and SeMet- substituted FAF1 UBL1 were obtained by mixing equal vol- umes ofprotein in 50 mM HEPES,pH 7.5, 150 mM NaCl at 20mg/mlandreservoirsolutioncontaining50mMBis–Tris,pH 6.5,50mM(NH4 )2 SO4 ,and30%(v/v)pentaerythritolethoxy- late. Crystals were transferred into a cryoprotectant contain- ingareservoirsolutionwith20%ethyleneglycolbeforedata collection.ThecrystalsofFAF1L-UBL1complexedwithHsp70 NBDwithADPandPiwereobtainedusing200mMimidazole malate,pH 8.5, and 12% PEG10000 and stabilizedin 25%
ethyleneglycolbeforedatacollection.Diffractiondatawerecol- lectedusingsynchrotronradiation(beamline4AofPohangLight Source,Korea)andwereprocessed,integrated,andscaledus- ingtheHKL2000programsuite(OtwinowskiandMinor,1997). ThestructureofFAF1UBL1wasdeterminedbyMADusingthe programsSOLVE(TerwilligerandBerendzen,1996)andRESOLVE (Terwilliger,2000),whilethestructureofFAF1L-UBL1–Hsp70 NBDcomplexwasdeterminedusingmolecularreplacementwith CNS(Brüngeretal.,1998).CrystalstructuresofFAF1UBL1from thisstudyandHsp70NBDwithADP(Srirametal.,1997)were determined.ModelbuildingwasdoneusingCOOT(Emsleyand Cowtan,2004)andrefinedwithCNS(Brüngeretal.,1998)and REFMAC5(Murshudovetal.,1997).Theidealityandgeometry ofthemodelwerecheckedusingPROCHECK(Laskowskietal., 1993). Table 1 summarizes statistics on the data collection andrefinement.PyMOL(www.pymol.org/)wasusedtoprepare figures.
ITCanalysis
BindingwastestedusingtheITC200instrument(MicroCal)at 25°C,andthedatawereanalysedusingORIGIN7.0.Allsamples wereplacedin50mMHEPES,pH7.5,150mMNaCl,and1mM MgCl2 ,withorwithoutADP.Theywerecentrifugedanddegassed beforethemeasurementsat25°C.Allinjectionswereaddedat anintervalof150sectothesamplesolutioninthecellwith gentlestirringusingacomputer-controlledmicro-syringe.After making correctionsfor dilution by subtracting thevalues for
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bufferalone,weanalysedthedatawithaone-sitebindingmodel usingORIGIN7.0.
Plasmidsandreagents
The following antibodies were used in this study: mouse monoclonalFlagantibody(Sigma),rabbitanti-FAF1(AbFrontier), rabbit anti-NMIIA antibody (BioLegend), mouse anti-RhoA (Abcam), mouse anti-p190B RhoGAP, mouse anti-β-catenin, mouseanti-γ-catenin,mouse-anti-N-cadherin(BDBioscience), rabbit anti-MLC2, anti-PP-MLC2 (Cell Signaling Technology), rabbitanti-IQGAP1, mouseanti-actin, andanti-tubulin(Santa Cruz Biotechnology). Y-27632 (Y0503) was purchased from Sigma.GST-FAF1WT,GST-FAF1UAS-UBX,pFlag-CMV-2-FAF1WT, pFlag-CMV-2-FAF1 (82–650), and pFlag-CMV-2-FAF1 (1–120) werepreparedaspreviouslydescribed(Murshudovetal.,1997; Songetal.,2005).pFlag-CMV-2-FAF1H160Awasgeneratedby cloning employingmutagenesis. Allplasmid constructs were verifiedbyDNAsequencing.
Cellcultureandtransfection
HeLacellswerepurchasedfromATCCandculturedinEagle’s MinimumEssentialMedium(EMEM) supplementedwith 10%
fetalbovineserum,100units/mlpenicillinGand100μg/ml streptomycinat37°Cina5%CO2 -containinghumidifiedincuba- tor.HEK293TcellswereculturedinDulbecco’smodifiedEagle’s medium(DMEM)supplementedinthesamemannermentioned above.Forthetransientoverexpressionofspecificproteins,cells weretransfectedusingLT-1andanalysed24hpost-transfection.
Forgenesilencing,FAF1siRNAswereobtainedfromDharmacon (ON-TARGETplus SMARTpool siRNA) and Bioneer. FAF1 siRNA
#1 was obtained from Dharmacon (L-009106-00-0005), and FAF1siRNA#2(CatNo.1049605)andcontrolsiRNAwerefrom Bioneer.CellsweretransfectedwithsiRNAsusingDharmaFECT1 followingthemanufacturer’sprotocolatafinalconcentrationof 50nM.
GSTpull-downassayandidentificationofbindingproteins GST-FAF1andGST-FAF1[UAS-UBX] wereexpressedinE.coli BL21(DE3)cellsandboundtoglutathione-agarosebeadsfor 3hat4°Cwithgentle rotation.Thebeadswerethenwashed sixtimeswithphosphate-bufferedsalinecontaining0.1%Triton X-100.HeLacellswerelysedwithhypotonicbuffercontaining protease inhibitors (10 mM HEPES, pH 7.9, 1.5 mM MgCl2 , 10mMKCl,1mMPMSF,5μg/mlaprotinin,10μg/mlleupeptin, and10 μg/mlpepstatin A)and incubatedfor30 minonice, followedbycentrifugationat12000rpmfor15minat4°C.GST- FAF1bound tobeadswereincubatedwith HeLacell extracts for3hat 4°C, and thenthebeadswere washedthrice with lysisbufferandadditionallytwicewithlysisbufferwithoutany detergent.Thebeadswereresuspendedingelsamplebuffer, andbindingproteinswereseparatedbySDS–PAGEand visu- alizedusingasilver stainingkitorwesternblottinganalysis.
Gel bands ofbindingproteins werede-stained and digested withtrypsin,andtheresultingpeptideswereextractedaspre- viouslydescribed(Seoetal.,2008).Peptideswereseparated
usingtrapcolumncartridge,injectedintoC18reversed-phase analyticalcolumn(nanoACQUITYBEH300particlesize:1.7μm, ID:75μmandlength:250mm)withintegratedelectrosprayion- izationPicoTipTM usingnanoAcquityTM UPLC/ESI/q-TOFMS/MS (SYNAPTTM G2Si;WatersCo.)(equippedatEwhaDrugDevelop- mentResearchCoreCenter).
Immunoprecipitation
Cellswerelysedwithhypotonicbuffer(10mMHEPES,1.5mM MgCl2 ,10mMKCl,60mMoctyl-β-D-glucopyranoside,pH7.9) containingproteaseinhibitors(1mMPMSF,5μg/mlaprotinin, 10μg/mlleupeptin,10μg/mlpepstatinA,5mMNa3 VO4 ,5mM NaF,0.1mMEDAT,and0.1mMEGTA)and anHDACinhibitor (10mMsodiumbutyrate)for30minonice.Thecelllysateswere centrifugedat12000rpmfor15minat4°C.Thesupernatantwas incubatedwithanti-Flagantibodyfor2hat4°C,andthelysate–
antibodycomplexeswereincubatedwithprotein-Gsepharose4 FastFlowbeadsforanother1hat4°C.Theprecipitatedbeads werewashedsixtimeswithlysisbuffertoremovenon-specific bindingandadditionallytwicewithlysisbufferwithoutanyde- tergent.Theimmunecomplexwaselutedwithgelsamplebuffer, separatedbySDS–PAGE,andanalysedbywesternblotting.
Confocalmicroscopy
CellsweregrownontheSecureSlipTM coverslip(Sigma)and fixedwith 4% paraformaldehydeinHanks’ balancedsalt so- lution(HBSS)for10min.AfterwashingwithHBSS,cellswere permeabilizedwith0.1%TritonX-100inHBSSfor10min.After HBSS washing, cells were incubated with 3% bovine serum albumininHBSSfor1htoblocknon-specificproteinadsorption and thenincubatedwithprimary antibodiesfor2hat 37°C.
AfterwashingthricewithHBSS,thecellswerestainedfor1h at37°CwithAlexaFluor-conjugatedsecondaryantibodies.After washing thricewith HBSS,the mountingmediumforfluores- cencewithDAPIwasusedforstainingthenuclei.Afterbeing mounted,thecellswereobservedunderaZeissLSM510Meta laserscanningmicroscope.Imageswerephotographedandpro- cessedusingtheLSM510software(CarlZeiss).
RhoAactivityassay
HeLacellswerecultured ina 100-mmdish, and FAF1 was depletedbytransfectingthecellswithcontrolorFAF1siRNAfor 72h.ActiveRhoApull-downassayswereperformedaccording tothemanufacturer’sinstructions(ThermoScientific).Briefly, HeLacelllysateswereobtainedbyincubationwithlysisbuffer followedbycentrifugationat8000rpm.Cell lysates(750μg) wereincubatedwithGST-RBDofRhotekinfor1hat 4°Cwith rocking.Precipitateswerewashedthreetimeswithlysisbuffer, solubilizedwithgelsamplebuffer,separatedbySDS–PAGE,and detectedbyusinganti-RhoAantibody.
3Dspheroidtumorinvasionassay
ThespheroidcellcultureofMDA-MB-231cellswaspreparedin anEMEM-conditionedmedium(CM)containing2.5%Matrigel.
Thecellsweretransfectedwith100nMcontrolsiRNAorFAF1
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