Mechanism of Focal Adhesion Kinase Mechanosensing

16 Pages·2015·1.93 MB·English

RESEARCHARTICLE
Mechanism of Focal Adhesion Kinase
Mechanosensing
JingZhou1☯,CamiloAponte-Santamaría1☯,SebastianSturm2,JakobTómasBullerjahn2,
AgnieszkaBronowska1,FraukeGräter1,3*
1HeidelbergInstituteforTheoreticalStudies,Heidelberg,Germany,2LeipzigUniversity,Institutefor
TheoreticalPhysics,Leipzig,Germany,3InterdisciplinaryCenterforScientificComputing(IWR),Heidelberg
University,Heidelberg,Germany
☯Theseauthorscontributedequallytothiswork.
a11111 *[email protected]
Abstract
Mechanosensingatfocaladhesionsregulatesvitalcellularprocesses.Here,wepresent
resultsfrommoleculardynamics(MD)andmechano-biochemicalnetworksimulationsthat
OPENACCESS
suggestadirectroleofFocalAdhesionKinase(FAK)asamechano-sensor.Tensileforces,
Citation:ZhouJ,Aponte-SantamaríaC,SturmS, propagatingfromthemembranethroughthePIP bindingsiteoftheFERMdomainand
2
BullerjahnJT,BronowskaA,GräterF(2015)
fromthecytoskeleton-anchoredFATdomain,activateFAKbyunlockingitscentralphos-
MechanismofFocalAdhesionKinase
phorylationsite(Tyr576/577)fromtheautoinhibitoryFERMdomain.Varyingloadingrates,
Mechanosensing.PLoSComputBiol11(11):
e1004593.doi:10.1371/journal.pcbi.1004593 pullingdirections,andmembranePIP concentrationscorroboratethespecificopeningof
2
theFERM-kinasedomaininterface,duetoitsremarkablylowermechanicalstabilitycom-
Editor:PeterMKasson,UniversityofVirginia,
UNITEDSTATES paredtotheindividualalpha-helicaldomainsandthePIP -FERMlink.Analyzingdown-
2
streamsignalingnetworksprovidesfurtherevidenceforanintrinsicmechano-signalingrole
Received:June5,2015
ofFAKinbroadcastingforcesignalsthroughRastothenucleus.ThisdistinguishesFAK
Accepted:October12,2015
fromhithertoidentifiedfocaladhesionmechano-responsivemolecules,allowinganew
Published:November6,2015
interpretationofcellstretchingexperiments.
Copyright:©2015Zhouetal.Thisisanopen
accessarticledistributedunderthetermsofthe
CreativeCommonsAttributionLicense,whichpermits
unrestricteduse,distribution,andreproductioninany
AuthorSummary
medium,providedtheoriginalauthorandsourceare
credited.
Focaladhesionsintegrateexternalmechanicalsignalsintobiochemicalcircuitsallowing
DataAvailabilityStatement:Allrelevantdataare cellularmechanosensing.Althoughthezooofmechanosensingproteinsatfocaladhesions
withinthepaperanditsSupportingInformationfiles.
issteadilygrowing,force-inducedenzymaticmechanisms,asthoseuncoveredforautoin-
Funding:FundingfromtheKlausTschiraFoundation hibitedkinasesinmuscle,remaintobeidentifiedforfocaladhesiondownstreamsignaling.
(toJZandFG),theBMBFSYSTECprogramme(to Here,weprovideevidencethatfocaladhesionkinase(FAK)canactasadirectmechano-
JZandFG),theDeutscheForschungsgemeinschaft
enzymeatfocaladhesions,usingmoleculardynamicssimulationsandkineticmodelling.
(DFG)researchgroupFOR1543(toCASandFG),
WeshowthatanchorageofFAKtothemembraneviaPIP-2iscriticalforthismechanical
andtheBIOMSprogrammeatHeidelbergUniversity
activation.Ourresultssuggestsimilarmechanismstobeatplayforothermembrane-
(toAB)isgratefullyacknowledged.JTBandSS
acknowledgefinancialsupportfromtheEuropean boundautoinhibitedkinases.
UnionandtheFreeStateofSaxony,andtheDFG
throughFOR877andtheLeipzigSchoolofNatural
Sciences-BuildingwithMoleculesandNano-objects
(BuildMoNa).Thefundershadnoroleinstudy
PLOSComputationalBiology|DOI:10.1371/journal.pcbi.1004593 November6,2015 1/16
FAKMechanosensing
design,datacollectionandanalysis,decisionto Introduction
publish,orpreparationofthemanuscript.
Focaladhesions(FAs)actaskeycellularlocationsformechanosensingbyintegratingmechani-
CompetingInterests:Theauthorshavedeclared
calandbiochemicalsignalsbetweentheoutsideandinsideofthecell,therebyregulatingpro-
thatnocompetinginterestsexist. cessessuchascellproliferation,motility,differentiation,andapoptosis[1–3].Theycontain
numerousadapteroranchorproteins,whichestablishthemechanicallinkofthecytoskeleton
withtheextracellularmatrix[4].Someoftheseproteinshavebeenidentifiedasmechano-
responsiveelements[5–7].
FocalAdhesionKinase(FAK)centrallyregulatesFAsbyestablishingadhesiveinteractions
atthecellperiphery[8].Actingasasignalinghubbetweenintegrinandmultipleproteinspar-
ticipatingindownstreamsignalingpathways,itcarriesoutdiversefunctionsinembryonic
development,cellmigration,andsurvival,anditsmalfunctionisassociatedwithcancerpro-
gressionandcardiovasculardiseases[9,10].FAKcomprisesacentraltyrosinekinasedomain
flankedbytwolargenon-catalyticdomains:FERMandFAT(Fig1A).TheN-terminalthree-
lobed4.1ezrinradixinmoesin(FERM)homologydomainisconnectedtothekinaseN-lobe
Fig1.MechanicalactivationofFK-FAK.A)DomainorganizationofFAKanditsrelativepositionatthecell
peripheryinthecytosol.Thekinasedomain(orange)containsthelobesNandCandtheFERMdomain
(blue)consistsoflobesF1–3,withF2bindingtoPIP lipids(red)concentratedattheinnerleaflet(IL)ofthe
2
membrane(grey).ThemajorphosphorylationsiteTyr576/577(magentastar),locatedattheactivationloop
(green)inthekinasedomain,isautoinhibitedbytheFERMdomain.TheautophosphorylationsiteTyr397
(greystar)ispositionedintheloopconnectingthekinaseandFERMdomains(grey).TheFATdomain(violet)
isnotconsideredinourstudy.B)StretchingforceappliedtothebasicpatchinFERMandthekinaseC-
terminalresidue(red),informofvirtualsprings,inducesFERM-kinasedissociation.Representative
structuresofFAKinitsinitialautoinhibitedconformation(1),afterdissociationofthekinaseC-lobefromthe
FERMF2lobe(2),andafterTyr576/577releaseandpartialC-terminalunfolding(3)areshown.Color-code
andorientationoftheproteinasinA.C)Cumulativenumberofdissociationeventsasafunctionofthe
distancebetweenthepulledelementsatthemomentofdissociation(De−e).Thisindicatestheextentof
unfoldingpriordissociation.Twoeventsweremonitored:dissociationofthetheFERMF2lobefromthe
kinaseC-lobe,F2-C(transitionfrom(1)to(2)inB),andseparationofFERMdomainfromtheTyr576–577
phosphorylationsite,F-YY(transitionfrom(2)to(3)inB).Totalnumberofsimulations(83)isindicatedwith
thedashedline.
doi:10.1371/journal.pcbi.1004593.g001
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FAKMechanosensing
througha50-residuelinker.TheC-terminalFAT(focaladhesiontargeting)domainfollowsa
220-residuelongproline-richanddisorderedlinker,throughwhichitisconnectedtothe
kinaseC-lobe.TheactivationofFAKfirstrequiresautophosphorylationofTyr397,which
offersaSrchomology2(SH2)bindingsite.SrcbindingtoFAKincreasesSrckinaseactivity,
inducingthephosphorylationofTyr576/577withinthekinasedomainactivationloop[11].
ThisisneededformaximalFAK-associatedactivityandleadstotheformationofaSrc-FAK
complex,whichtriggerssubsequentphosphorylationsintheFATdomainandbindingof
downstreamsignalingproteins[12].TheFERMdomainauto-inhibitsthekinasedomainby
blockingtheTyr576/577phosphorylationsite[13,14].Exposureofthissiteisanessentialstep
topermititsphosphorylation,andtherebyrendermaximumFAKactivity.
FAKlocatesatsitesofintegrinclusteringthroughprotein-proteininteractionsofitsFAT
domain,whichcontainsbindingsitesforintegrin-andactin-associatedproteins[4,15].Integ-
rinsignalingandinteractionswithgrowthfactorreceptorsweredeterminedasFAKactivators
[16,17].Recentstudiesprovidedevidencethatphosphoinositidephosphatidylinsositol-
4,5-bis-phosphate(PIP )iscriticalforefficientFAKactivationandautophosphorylation[18,
2
19].PIP ,aubiquitoussecondmessengerenrichedintheinnerleafletoftheplasmamembrane
2
andconcentratedatFAs,regulatestheinteractionofcytoskeletalproteinswiththemembrane
[20,21].PIP interactsdirectlywiththebasicpatch(216KAKTLR221)intheFERMdomain[18,
2
19],whichinducesconformationalchangesinFAK.TheglobalconcentrationofPIP inthe
2
cellmembraneisonlyapproximately1%[22].However,PIP -proteininteractions[22,23]or
2
divalentions,suchasCa2+,[24,25]canleadtolocalPIP2accumulation.Localizedincrements
ofCa2+werealsosuggestedtoincreasetheresidencyofFAKatFAs[26].
EvidenceforadecisiveroleofFAKinmechanotransductionissteadilygrowing[27].FAK
isrecruitedtotheleadingedgeandphosphorylatedinmigratingcellsundershearstress[28,
29].Ithasalsobeenshowntomediateforce-guidedcellmigration[30,31]aswellasstrain-
inducedproliferation[32].Recently,themechano-sensitivityofFAKhasbeenascribedtothe
force-sensingfibronectin-integrinlink[33].However,untilnow,theavailabledataon
mechano-sensingthroughFAKisindirect.ItremainsunknownifFAKonlyliesdownstream
ofmechano-sensingprocessessuchasthoseinvolvingintegrins,orifFAKisalsoperse
exposedtoandactivatedbymechanicalforce.
WeherehypothesizethatmechanicalforceactsasadirectstimulusofFAKactivity,indica-
tionsforwhicharetwo-fold.First,FAKistetheredbetweenthePIP -enrichedmembraneand
2
thecytoskeleton,likelyactingasaforce-carryinglinkinFAs.Second,theFERM-kinasestruc-
turesuggestsitselfasamechano-responsivescaffold,inwhichforcecouldspecificallydetach
theautoinhibitoryFERMdomainfromtheactivesite.FAKwouldbethefirstmechanoenzyme
ofFAs,allowingadirecttransductionofamechanicalsignalintoanenzymaticreactionand
downstreameventsintothenucleus,whichwouldyieldamechanisticexplanationofFAK’s
mechano-sensingrole[10].Indeed,twoanalogouscasesofmechanicallyactivatedenzymes
havebeenpreviouslyidentified,bothofwhicharekinasesandfeatureforce-inducedactivation
byremovalofanautoinhibitorydomain,namelytitinandtwitchinkinaseinmuscle[34–36].
IncontrasttotheFATdomain[37],theforceresponseoftheautoinhibitedFERM-kinasefrag-
mentiscurrentlyunknown.TotestthehypothesisofFAKasaforce-sensor,weperformed
extensiveequilibriummoleculardynamics(MD)andforce-probemoleculardynamics
(FPMD)simulationsoftheFERM-kinasefragmentofFAKundervariousconditions.Force
propagatingontoFAKfromaPIP -enrichedmembraneandthecytoskeletonspecifically
2
opensthehydrophobicFERM-kinaseinterface,preparingFAKforactivationviaphosphoryla-
tionpriortotheunfoldingofthekinasedomain.Giventhelowstabilityofthelargelyα-helical
kinaseandFERMdomains,thisisremarkable.Theenforcedactivationisrobustwithregardto
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FAKMechanosensing
alargerangeofpullingvelocities,butsensitivetothesiteofforceapplication.Ourforce-
inducedactivationpathwaysuggestsadirectmechanoenzymaticfunctionofFAKinFAs.
MaterialsandMethods
EquilibriumMDsimulationsoftheFERM-kinasefragment(FK-FAK)fragment[14](PDB
code:2J0J)intheapostate,bothintheabsenceandinthepresenceofamembranecontaining
PIP andPOPElipids,aswellasofonlythemembrane,wereperformedusingtheGROMACS
2
package[38].FPMDsimulations[39,40]ofFK-FAKwithoutamembranewereperformedby
subjectingtheC-terminalC-alphaatomandthecenter-of-massoftheC-alphaatomsofthe
basicpatch216KAKTLR221toharmonic-springpotentialswhichweremovedawayfromeach
otherwithconstantvelocity.FAThasbeensuggestedtointeractwithFERM,bindingtothe
samesiteasPIP2does[41].However,PIP2bindingisrequiredforFAKactivation[18,19],
thusexcludingthepossibilityofFAT-FERMstableinteractionsforPIP2-mediatedFAKactiva-
tion.OtherinteractionsbetweenFATandtheFERM/Kinasecomplexarenotknownoratleast
suggestedtobeverydynamicandweak[42],andtherebyeasiertobreakunderforcecondi-
tions.Thissuggeststhatundertensileforce,FATismaintainedsufficientlyfarfromthecom-
plex,andthattheforceistransducedtowardsthecomplexthroughthefully-stretched200
amino-acidproline-richdisorderedlinker.Inconsequence,inourFPMDsimulationsneither
FATnorthelinkerwereconsidered.
Theforceresponseofmembrane-boundFK-FAKwasinvestigatedbysubjectingitsC-ter-
minustoaharmonicpotentialthatwasthenmovedawayfromthemembraneeithervertically
ordiagonally,whilekeepingthemembranepositionatitsoriginalposition.PLS-FMA[43]was
usedtodetectcollectivemotionsmaximallycorrelatedwiththeopeningoftheFERM-kinase
interface.Theunderlyingfreeenergylandscapewascharacterizedbyanalyzingtherupture
forcesasafunctionofloadingrate,usingboththeHSmodelbyHummer&Szabo[44]andthe
BSKmodelbyBullerjahnetal.[45].Kineticmodelswerebasedonpreviousbiochemicalnet-
works[46–49]andsimulatedusingCOPASI[50].DetailsofthemethodsaregiveninS1Text.
Results
Force-inducedreleaseofFAKautoinhibition
Invitro,phosphorylationoftheactivationloopofFAKisenhancedbyrelievingtheautoinhibi-
tionthroughY180/M183mutation[14]orPIP -binding[18].Catalyticturnoverofwild-type
2
FAK,however,requiresanadditionalbiochemicalstimulus.Here,weaskifmechanicalforce
couldpromotefulldomaindissociationofFAKasrequiredforauto-andSrc-phosphorylation–
analogoustotheeffectoftheY180/M183mutation.Weexaminedtheeffectoftensileforceon
theautoinhibitedFK-FAKusingFPMDsimulations.TetheringFAKbetweenthemembrane
andthecytoskeletonresultsinforcetransmissionfromthemembraneontothebasicpatchof
theFERMdomainandfromthepaxillin-interactingFATdomainthroughtheproline-rich
linkerontothekinaseC-terminus(Fig1A).Accordingly,inoursimulations,apullingforcewas
appliedtothebasicpatchofFERMandtheC-terminusofthekinasedomaininoppositedirec-
tionswith13differentpullingvelocitiesfrom6×10−3nm/nsto1nm/ns(1inFig1B).Foreach
pullingvelocity,multiplerunswerecarriedout(83runsintotal),yieldingaconcatenatedsimu-
latedtimeofabout7μs,withtheslowestpullingsimulationcovering1μs.Weobservedthe
autoinhibitoryFERMdomaintodissociatefromthekinasedomainin76outof83FPMDsimu-
lations(morethan90%ofthecases).ConformationaldamageofeithertheFERMF2-lobeor
kinaseC-lobeoccurredintheremaining7simulations.ReleaseofTyr576/577fromFERM
occurredalwayslater,i.e.atlargerend-to-enddistances,thandissociationoftheF2-lobefrom
theC-lobe(Fig1Band1C),suggestingtheF2-Cdetachmenttobearequirementformechanical
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FAKMechanosensing
FAKactivation.WeobservedpartialunfoldingatthekinaseC-terminuspriortoexposureof
Tyr576/577toanonlyminorextentandmostlyathigherloadingrates,comprisingatmosta15
nmincreaseinend-to-endlength(Fig1Band1C),ornomorethan30residuesoftheC-termi-
nalα-helix(S1Fig).Asthesecondhalfofthishelix(ormore)istypicallydisorderedinother
kinases(e.g.inproteinkinaseAorSrc),itspartialunfoldingunderforceislikelynottoimpair
FAKenzymaticfunction.Hence,ourdatasuggestdomain-domaindissociationtolargelythwart
theunfoldingofthemoderatelystableα-helicaldomainstructures.However,moresubstantial
unfoldingfromtheC-terminusofthekinasedomainwasthedominantpathwaywhenpulling
FK-FAKfromitsNandC-terminus(S2Fig).Thus,weconcludethatforceactingspecifically
betweentheFERMbasicpatchandthekinaseC-terminusremovestheinhibitoryFERMdomain
andtherebyfacilitateskinaseactivation,insteadofdomainunfoldingandkinaseinactivation.
FAK-membraneinteractionsunderforce
AtFAs,thespecificinteractionoftheFERMbasicpatchwithPIP2isrequiredfortheanchor-
ingofFK-FAKtothemembrane.Otherphospholipidsonlydisplaybackgroundlevelsofbind-
ing[18].Inourpreviousstudy,weobservedanallostericchangeattheFERM-kinaseinterface
uponPIP bindingtoFK-FAK,butnofullopening[18].Thisraisedthequestioniffulldomain
2
openingunderforce,asobservedforisolatedFK-FAKinsolution,alsooccurswhenFK-FAKis
anchoredtoamembraneviaPIP .ThiswouldrequireboththePIP -containingmembraneas
2 2
wellasthePIP -FERMlinktobemechanicallymorerobustagainstrupturethantheFERM-
2
kinaseinteraction.Totestthis,wesetupapalmitoyloleoylphosphatidylethanolamine(POPE)
membranecontaining15%(mol/mol)ofPIP intheinnerleafletofthemembrane,whichwas
2
surroundedbywaterandneutralizedbyCaCl .Within100nsofMDsimulationsstarting
2
fromindividualPIP moleculesinthemembrane,weobservedtheformationofsmallPIP
2 2
clustersinvolvingtwoormorelipidsandCa2+(S3Fig),accompaniedbyadecreaseofareaper
lipidby*1Å2(S1Table),inagreementwithdivalent-cation-mediatedPIP -enrichmentin
2
membranes[18,24,51].FK-FAKwasanchoredtothemembraneandthedynamicsofthe
resultingcomplexwasmonitoredover150nsofMD.Anchoragefurtherincreasedclustering.
TheproteinremainedstablyboundtothemembranethroughtheFERM-PIP andadditional
2
interactionsbetweenthekinaseC-lobeandthemembrane,independentfromtheinitialorien-
tationoftheproteinrelativetothemembraneplane(Fig2AleftandS3Fig).Thesamewas
observedforamembranewith1%PIP ,which,however,showedlessclusteringandprovided
2
onlyasinglePIP lipidforanchorageofFK-FAK.
2
Next,wemonitoredthemechanicalresponseofmembrane-anchoredFK-FAK.InFPMD
simulations,wesubjectedtheproteintoforcebymovingaharmonicspringattachedtothe
kinaseC-terminuswithconstantvelocityalongadirectionverticalordiagonaltothemem-
brane,whilepositionrestrainingthecenter-of-massofthemembranebilayer(Fig2A).At15%
PIP concentration,independentofthepullingdirection,weobservedalossofcontactsofthe
2
kinasedomainwiththemembraneandwiththeFERMdomain,whiletheFERM-membrane
interactionremainedintact(Fig2B).Whilediagonalpullingledtoaconcurrentdissociationof
thekinasefromthemembraneandtheFERMdomain,verticalpullingresultedinkinase-mem-
branedissociationpriortokinase-FERMdissociation.Innoneofthesesimulations,we
observedkinaseunfoldingpriortodissociation.Also,forbothpullingdirections,themem-
braneandthePIP -FERMinteractionweremechanicallymorerobustthanthoseatthe
2
FERM-kinaseinterface.Thus,themembranesimulationsreproducedtheprocesspredomi-
nantlyobservedforisolatedFK-FAKinsolution(compareFig2BwithFig1Band1C).Namely,
theyallshowedforce-inducedremovaloftheautoinhibitoryFERMdomainandexposureof
theactivationloopcarryingtheTyr576/577phosphorylationsite.
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FAKMechanosensing
Fig2.MechanicalactivationofFAKboundtothemembrane.A)ForcewasappliedtotheC-terminusof
FK-FAK,inverticalordiagonaldirectionwithrespecttothemembrane,withacounter-forceactingonthe
membrane,leadingtothereleaseofautoinhibition(lefttorighttransition).FK-FAKisshownasinFig1B,PIP
2
lipidsinthemembrane(hereat15%)incyan/redandPOPElipidsingrey.B-C)NumberofcontactsN
betweentheFERMF2-lobe(F2)andthekinaseC-lobe(C)comparedtothenumberofcontactsbetween
bothlobesandthemembrane(mem),at15%(B)and1%(C)PIP concentration.Numberofcontacts
2
betweenlobeswasdefinedasthenumberofatomsinoneofthelobescloserthan0.6nmtoatleastone
atomoftheotherlobe.Upperpanelsshowresultsfordiagonalpullingwhilelowerpanelsforverticalpulling.
DensitiesofN(forapullingvelocityof0.03nm/ns)areshownasagreygradient,withapolynomialfittothe
datashownasasolidblackline.Thelabelsi,a,b,anducorrespondtotheinactive,active,boundand
unboundstatesofFK-FAK,respectively,sketchedattherightside.
doi:10.1371/journal.pcbi.1004593.g002
WhenweappliedforcetoFK-FAKanchoredtoamembranecontainingonly1%PIP ,i.e.
2
toasinglePIP molecule,detachmentofthekinasedomainfromthemembranewasfollowed
2
bythedetachmentofalsotheFERMdomain(Fig2C).Fulllossofmembraneanchoringnatu-
rallystopsforcetransmissionandimpedesactivation.Thus,aninteractionoftheFERMbasic
patchwithmultiplePIP ,whichislikelyinPIP -enrichedmembranes,isrequiredformechani-
2 2
calFK-FAKactivation.ThisisinlinewiththefactthatPIP5Koverexpressionincreasesand
PIP5KknockdowndecreasestheopenFAKconformation[19].Thepullingdirection,instead,
appearstobelessrelevant.
Mechanismofforce-inducedFK-FAKopening
Wenextanalyzedinfurtherdetailthedynamicsunderlyingtheforce-triggeredFK-FAK
domain-domainrupture.Applyingpartialleastsquaresfunctionalmodeanalysis(PLS-FMA)
[43]tothesimulationsofisolatedFK-FAKinsolution,weobtainedacollectiveopening
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FAKMechanosensing
motionthatmaximallycorrelateswiththeincreaseinminimaldistancebetweentheF2andC
lobes(S4Fig).ThisopeningmotionalsostronglycorrelatedwiththeF2-Clobedistances
obtainedforthetrajectoriesofmembrane-boundFK-FAK,suggestingthatitcapturesthe
essentialopeningdynamicsofFK-FAKbothisolatedandboundtothemembrane.Thisimplies
thesimplifiedsystemofisolatedFK-FAKinsolutiontofollowaFERM-kinasedissociation
mechanism,whichishighlysimilartotheoneofthemorerealisticsystemincludingthemem-
brane,eventhoughitlackseffectsfromFERM/kinase-membraneinteractions.
WethenidentifiedthefirststepsalongtheopeningmotionofFK-FAKgivingrisetorup-
tureforces.Fig3AshowstypicalforceprofilesandF2/C-lobeinteractionareasasafunctionof
thespringlocationsrecoveredfromtheFPMDsimulations.ForbothFK-FAKinisolationand
boundtothemembrane,andindependentoftheloadingrate,weobservedthattheinterface
areabetweenthetwolobeswasreducedintwosteps,bothofwhichcoincidedwithnoticeable
forcepeaks.Themaximalforcewasreachedwhenthefirstdecreaseininter-lobeareaoccurred
Fig3.MechanismofFK-FAKmechanicalactivation.A)InterfacialareabetweentheF2-andC-lobe(grey)
andaverageforceexertedbythetwosprings(blue)asafunctionofthedistancebetweensprings,D .
spring
ResultsfromsixindependentFPMDsimulationsareshown:(1–3)withoutthemembranepullingatV=0.006,
0.006and0.014nm/ns,respectively,and(4and5)pullingdiagonallyawayfromthemembraneatV=0.03and
0.05nm/ns,respectively.Theinterfacialareadropsfrominitialvaluesof3–4.5nm2tointermediatevaluesof
1.5–2.8nm2.Afterwardsitdecreasestozero.Ruptureforce(highestforcepeak)alwayscorrespondedtothe
firstdropintheinterfacialarea(redline).Thepeakforceassociatedtotheseconddropintheareais
highlightedwiththegreenline.B)DistributionofinterfaceareasreflectingthetwostatesofFK-FAKduringits
force-inducedopening(highlightedwitharrows).AllFPMDsimulationswereconsideredtocomputethe
distribution.C)Residuesinvolvedintherupturestepsarehighlightedassticks.FERMF2-andKinaseC-lobe
areshowninsurfacerepresentation.Rupturestepsareassociatedtothedisruptionofhydrophobic
interactions(green);saltbridges(blue)andotherelectrostaticinteractions(magenta),andinteractionswith
otherpartners(cyan).ResidueswereidentifiedbyTRFDA(S5Fig).TheyarelistedinS2Table.
doi:10.1371/journal.pcbi.1004593.g003
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FAKMechanosensing
(from3–4.5nm2to1.5–2.8nm2).Thisledtoashort-livedintermediate,asreflectedbyasec-
ondpeakinthedistributionoftheF2/C-lobeinterfacearea(Fig3B),beforethetwolobesfully
dissociated.Wenotethattheintermediatebecomeslessevidentforfasterpullingvelocities.
Topinpointtheload-carryingresidue-residueinteractionsacrosstheinterface,wecalcu-
latedthepunctualstressofeachresidue,usingtimeresolvedforcedistributionanalysis
(TRFDA)[52],andtherebydetectedthelossofinter-lobeinteractionsduringpulling(seeS5
Fig,S2Table,andS1Text).Inter-lobeinteractionswhichrupturedreproduciblyatoneofthe
twodissociationstepsarehighlightedinFig3C.Thefirstmajorrupturesteprequiredthe
breakupofahydrophobicclustercomposedofresiduesY180,M183,N193,V196,andF596,
andofanadditionalsaltbridge(D200-R598).Ruptureoftheseinteractionsgaverisetothe
maximalforce,thusstressingtheircriticalstabilizingrole.Ourresultsareinagreementwith
theobservationthatmutationsY180A,M183A,andF596DresultinconstitutivelyactiveFAK
withanopenFERM-kinaseinterface[14,18].Residuepairsrupturingatthesecondstep
includedresiduesofmostlyelectrostaticnature(E182,R184,K190andN595,N628,N629,
E636)andarelocatedfurtherawayfromthemembraneanchor.Thissecondrupturestepis
immediatelyfollowedbytheopeningoftheremainingFERM-kinaseinterfaceestablished
betweentheF1andtheN-lobe,includingtheexposureofTyr576/577.Thus,therupturepro-
cessresemblesazipper-likemechanism,duringwhichtheFERMandkinaseinterfaceis
sequentiallyopened.Herein,themembrane-proximalhydrophobicpatcharoundF596repre-
sentsthemostrobustmechanicalclamptobeopenedfirst.OurPLS-FMAcalculationsfurther
supportthissequentialmodeofopening(S4CFig).
ForcerequiredforFAKactivation
AretheforcespredictedbythesimulationstorelieveFAKautoinhibitionrelevanttoFAKat
FAs?Atthirteendifferentloadingrates,coveringtwoordersofmagnitude,weobtainedmaxi-
malruptureforcesforFK-FAKactivationbetween150and450pN(Fig4A).Thisforceregime
issimilartotheoneobservedfortitinkinase(400pNat0.2pN/ps),akinaseknowntobe
mechanicallyactivatedbyforcespresentinmuscle[35,36,53].Ourruptureforcesarealsosim-
ilartoorslightlyhigherthanthosepredictedbyMDsimulationsofthefocaladhesionproteins
talinandvinculin(250–400pNfornanosecondscaleactivationoftalin[54,55]and100pNfor
sub-nanosecondactivationofvinculin[56],respectively).WethenusedboththeHSmodel
[44]andtheBSKmodel[45]tofittheobservedruptureforcesasafunctionoftheloadingrate.
ThisprovideduswithasetofcompatiblemodelparametersΔG,Dandx ,whereΔGdenotes
b
theactivationenergy,Dtheeffectivediffusivityandx theseparationbetweentheinactivestate
b
andthetransitionstate.Focusingonthoseparametercombinationsthatcorrespondtoaphysi-
ologicallyplausiblespontaneousactivationrateofnomorethank =10−3Hz,weobtaineda
0
numberofbest-fitestimatesfromwhichwederivedtheforce-dependentactivationratek(F)
usedinourkineticmodel(Fig4AandS6Fig).Wenotethatmodelparameterscorresponding
toanunphysiologicallyhighspontaneousdissociationratecanimproveourfittotheobserved
forcefluctuations(S7andS8Figs).Onthisbasisitmightbespeculatedthatthereexistsasec-
ondenergybarrieratalargervalueofx thatguaranteesthermalstabilityatlowforces,but
b
vanishesunderthehighforcesusedinourMDsimulations.Nevertheless,thisdoesnotinvali-
dateourqualitativefindingsonFK-FAKactivationasforcesensitivityincreasesexponentially
withthebarrierlocationx (seetheS1Textforamoredetailedanalysis).
b
Discussion
Wehereprovidecomputationalevidenceforaforce-inducedactivationmechanismofFAK,in
whichtensileforcerelievestheblockageofitsactivesite,aswellasitscentralTyr576/577
PLOSComputationalBiology|DOI:10.1371/journal.pcbi.1004593 November6,2015 8/16
FAKMechanosensing
Fig4.FAKmechano-signaling.A)RuptureforceFasafunctionoftheloadingrateF_,whereFisdefinedasthemaximalforceobservedduringFK-FAK
activation(arrowintheinset).LightgreydotsrepresentindividualruptureforcesFobservedinourmembrane-freeFPMDsimulations.Darkgreydots
representtheiraverages(foreachloadingrateF_).ThesolidlineshowsthemeanruptureforcehFipredictedbytheBSKmodel[45]forΔG=28.5k T,x =
B b
0.86nm,andD=6.6×106nm2/s.AfitwiththeHS[44]modelyieldssimilarmodelparameters(notshown).Dashedlinesshowthevariationoftherupture
forcespredictedbytheBSKmodel(2standarddeviations,seeS1Textforadetailedanalysis).Pullingmembrane-boundFK-FAKdiagonallyyieldedsimilarly
largeruptureforces(greendots).Verticalpullingresultedinsignificantlylowerruptureforces(pinkdots),asthisdirectionpromotesthelessresistantzipper-
likedissociationmechanismdescribedinS4BandS4CFig)Timeatwhich50%ofinactiveFAK(B)andGDP-boundRasprotein(C)areconsumed,under
varyingexternalforce.Timesobtainedforthreesetsofparameters(1to3)correspondingtothethreefitspresentedinS6Fig.
doi:10.1371/journal.pcbi.1004593.g004
phosphorylationsite,imposedbytheautoinhibitoryFERMdomain.Thereleaseofautoinhibi-
tionislikelytomakethekinaseactivesiteaccessibleforitssubstrate,Tyr397ofthesameor
anotherFAKmolecule[42,57],and/ortorenderTyr576/577accessibletoSrc.Itisnon-trivial
thattheexertionofapullingforceatoppositesitesoftheFERMandkinasedomainsleadsto
theirdissociation.OtherlikelyscenariosareproteinunfoldingandPIP -proteindissociation,
2
bothofwhichwouldinactivateFAK,becauseanintactkinasestructureandalsoPIP binding
2
[18]arerequiredforFAKactivity.Infact,α-helicalproteinsareknowntounfoldatforcestypi-
callylowerthanβ-sheetproteins[58],andboththekinaseC-lobeandtheFERMF2domain
featuremainlyα-helicalsecondarystructure.Inthisregard,force-inducedFAKunfolding
wouldbeanexpectedresultandwasindeedpreferredoverFERM-kinasedissociationwhen
pullingtheFERMF1-orF3-lobeawayfromthekinaseC-terminus.Instead,intheparticular–
andphysiologicallyrelevant–casethatforceisappliedtothePIP bindingsiteandthekinase
2
C-terminus,wefounddomain-domaindissociationtobestronglypreferredoverunfoldingor
membranedetachment,overalargerangeofpullingvelocities,androbustwithregardtothe
pullingdirectionandpresenceofmembraneinteractions.Thus,theα-helicalregionssubjected
tothepullingforcemostlyrefrainfromunfolding,andtheyinsteadtransducetheloadtothe
F2/C-lobeinterface,whichreadilyopenspriortosubstantialkinaseunfolding.Wesuggestthat
itisthezipper-liketopology,withtheforceapplicationsitesbothlocatedatthemembrane-
proximalbasisofthetwodomains,thatmechanicallyweakensthedomaininterface,resulting
inefficientFAKopeningandactivation.Forcetransductionthroughthetwotermini,incon-
trast,resultsinshearingthetwodomainsrelativetoeachother,makingthemlesspronetodis-
sociate.Thelowermechanicalresistanceofzipperversusshear-typetopologieshasbeen
describedearlier(e.g.[53]),andFAKforce-induceddomain-domainruptureandactivation
appearstobeanothervariationofthistheme.
PLOSComputationalBiology|DOI:10.1371/journal.pcbi.1004593 November6,2015 9/16
FAKMechanosensing
Ourfindingsnotonlydecidedlyargueforamechano-sensingfunctionofFAK,butalso
emphasizethecrucialroleofthemembraneinmechanotransduction.First,membranebinding
allowsforcetopropagatetotheF2-lobeofFAK.Second,theFAK-membraneinteractionwith-
stoodtheexternalloadonlyinthecaseofPIP -enrichedmembranes(15%PIP ).Incontrast,
2 2
FAKdetachedfromlowPIP -contentmembranes.ThissupportsthenotionofPIP clustering
2 2
asarequirementforFAKactivationatFAs[18,51].WenotethatFAKcanbeactivatedinvitro
bythesoleactionofPIP andSrc,i.e.intheabsenceoftensileforcesactingonmembrane-
2
boundFAKatFAsinstretchedcells.However,ithasbecomeclearthatFAKactivationcan
proceedalongdifferentroutes,dependingonthecellularenvironment,andpotentiallycanalso
involvepHchanges[59],and/orgrowthfactorreceptors[16].Wehereproposeforcetosubsti-
tuteorcomplementsomeoftheseactivatorsshapingthemulti-dimensionallandscapeofFAK
activity.
UsingTRFDA,werecoveredthestabilizingroleofY180,M183,V196andF596,ahydro-
phobiccorepreviouslyshownbymutagenesistostabilizetheautoinhibitedstate[14],validat-
ingoursimulationdata.Inaddition,wefoundD200andR598tocontributetotherupture
force,andpredicttheirmutationtoresultinincreasedFAKactivity.
Thequestionarises,howforcefeedsintoFAK-mediatedRasGDP/GTPexchangeandregu-
latesERK-dependentgeneexpression,analogoustothechemicalstimulationofgrowthfactor
receptor-dependentRassignaling(S9Fig)[27].ToassesshowFAKasamechanosensorcou-
plesmechanicalsignalsintothedownstreambiochemicalnetwork,wedefinedforce-dependent
FAKactivationastheinitialstepofakineticmodelfortheRassignalingpathway(S10Fig)
[46–49].BothFAKopeningandGDP/GTPexchangeinRasareacceleratedbyexternalforces,
asexpected(Fig4Band4CandS11AFig).Intriguingly,whileFAKforce-inducedactivation
showsanearlylineardependencyonforceonthelogarithmicscale(Fig4B),Ras-GTPproduc-
tionshowsahighlynon-lineardependencyandsaturatesbeyondacriticalforce(Fig4C).The
reasonisthatactivatedFAKincomplexwithitspartners,Grb2andSOSviac-SrcandSHC,
actsasanenzymeforRasactivation.Asadirectconsequence,Rasactivationfollowsmechano-
enzymatickineticsreminiscentofaninhibitoryMichaelis-Mentenmechanisms(S11BFig)
[60],inwhichforceregulatestheenzymeconcentration.
Inconclusion,ourcomputationalstudyprovidesdirectevidenceatthemolecularlevelfora
mechano-sensoryroleplayedbyFAKatPIP -enrichedmembranesofFAs.Throughaspecific
2
domainopeningmechanismregulatedbyforce,FAKcanintegratemechanicalandchemical
stimuliintodownstreamsignalingtothenucleus.Wesuggestthemechano-enzymaticsofFAK
andRastoprovideacaponthecell’smechano-response.Ourresults,onFAKactivationand
signaling,aredirectlytestableamongothersbymolecularforcesensors[61,62]andcell
stretchingexperiments.Howotherputativelymechano-activatedkinases,suchastherelated
Srckinase,followsimilarmechanisms,atfocaladhesionsorelsewhere,remainstobeshown.
SupportingInformation
S1Fig.Partialunfoldingofthekinasedomain.A)Distancebetweenthepulledgroupsatthe
momentofdissociation(De−e)asafunctionoftheappliedloadingrate.Dissociationwasmoni-
toredbetweentheFERMF2-lobeandthekinaseC-lobe(F2-C)andbetweentheFERMdomain
andtheTyr576–577phosphorylationsite(F-YY).Eachdotcorrespondstoonesimulationrun
andthelineisapolynomialfittoallthepointsasaguidetotheeye,indicatingaslightaugment
inDe−ewithincreasingloadingrate.B)NumberofunstructuredresiduesNUNasafunctionof
thetime,forasimulationdisplayingalargeDe−evalue(circleinA).Timetraceisshownin
blackandapolynomialfitasaguidetotheeyeinblue.Residualunfoldingofaround30resi-
dueswasobservedattheC-terminus.Thenumberofunstructuredresidueswasobtainedusing
PLOSComputationalBiology|DOI:10.1371/journal.pcbi.1004593 November6,2015 10/16