Phosphorylation of STAT2 on Serine-734 negatively regulates the IFNα-induced antiviral response

28 Pages·2016·2.79 MB·English

by  Håkan Steen

©2016.PublishedbyTheCompanyofBiologistsLtd|JournalofCellScience(2016)129,4190-4199doi:10.1242/jcs.185421
RESEARCHARTICLE
Phosphorylation of STAT2 on serine-734 negatively regulates the
α
IFN- -induced antiviral response
HåkanC.Steen1,*,KevinP.Kotredes1,*,ShokoNogusa2,MicheleY.Harris3,SiddharthBalachandran2and
AnaM.Gamero1,‡
ABSTRACT dimerizationofthetwoSTATs,thattogetherwiththeDNA-binding
proteinIRF9formtheheterotrimericIFN-stimulatedgenefactor3
Serine phosphorylation of STAT proteins is an important post-
(ISGF3) complex (Fu et al., 1990, 1992; Improta et al., 1994).
translational modification event that, in addition to tyrosine
ISGF3 translocates to the nucleus and binds to the promoters of
phosphorylation, is required for strong transcriptional activity.
multipleIFN-stimulatedgenes(ISGs)toactivatetheirtranscription
However, we recently showed that phosphorylation of STAT2 on
S287 induced by type I interferons (IFN-α and IFN-β), evoked the (Fuetal.,1990;Lietal.,1997a).Theresultinggeneproductsdrive
thecellularresponsestoIFNsthatincludeantiviral,antiproliferative
oppositeeffect.S287-STAT2phosphorylationinhibitedthebiological
effectsofIFN-α.Wenowreporttheidentificationandcharacterization and, in certain instances, pro-apoptotic responses (Chawla-Sarkar
etal.,2001;deVeeretal.,2001).
ofS734ontheC-terminaltransactivationdomainofSTAT2asanew
phosphorylation site that can be induced by type I IFNs. IFN-α- ThespecificroleofSTAT2intheISGF3complexistoprovideits
transactivation domain (TAD) for full transcriptional activity (Li
inducedS734-STAT2phosphorylationdisplayeddifferentkineticsto
etal.,1996).TheSTAT2TADissituatedattheC-terminalendand
that of tyrosine phosphorylation. S734-STAT2 phosphorylation was
spans ∼100 amino acid residues (amino acids 747–851) (Kraus
dependent on STAT2 tyrosine phosphorylation and JAK1 kinase
et al., 2003) and binds p300 and CBP (hereafter p300/CBP, also
activity.MutationofS734-STAT2toalanine(S734A)enhancedIFN-
α-driven antiviral responses compared to those driven by wild-type known as EP300 and CREBBP, respectively) and GCN5 (also
known as KAT2A), both of which are involved in facilitating
STAT2.Furthermore,DNAmicroarrayanalysisdemonstratedthata
transcription through their histone acetylation activities
smallsubsetoftypeIIFNstimulatedgenes(ISGs)wasinducedmore
byIFNαincellsexpressingS734A-STAT2whencomparedtowild- (Bhattacharya et al., 1996; Paulson et al., 2002). Earlier studies
have suggested that the most crucial region for transactivation
typeSTAT2.Takentogether,thesestudiesidentifyphosphorylation
resides between residues 800 and 832 because deletion of this
of S734-STAT2 as a new regulatory mechanism that negatively
region abolished the transcriptional activity of ISGF3 (Qureshi
controlsthetypeIIFN-antiviralresponsebylimitingtheexpressionof
et al., 1996). Later studies have suggested T800 and Y833 as
aselectsubsetofantiviralISGs.
additionalphosphorylationsites(Farraretal.,2000;Hornbecketal.,
KEYWORDS:STAT2,Serinephosphorylation,JAK1,Interferon, 2012; Shiromizu et al., 2013). Although mutation of Y833 to a
Virusinfection,Vesicularstomatitis phenylalanine residue impaired dimerization of STAT2–STAT4
inducedbytypeIIFNs,evidenceislackingtosupportthebiological
INTRODUCTION significance of T800 and Y833 in their phosphorylated state
Signaltransducerandactivatoroftranscription(STAT)2isapivotal (Hornbecketal,2012;Shiromizuetal2013).
componentofthetypeIinterferon(IFN)signalingpathway(Leung Aside from tyrosine phosphorylation, serine phosphorylation
etal.,1995;SteenandGamero,2013).TypeIIFNs,whichinclude modulates the transcriptional activity of the STATs. STATs are
IFN-α, -β, -ε, -κ and -ω, activate the tyrosine phosphorylation of phosphorylated on serine residues located predominantly in the
bothSTAT2andSTAT1throughengagementoftheheterodimeric N-terminalregionoftheTAD,usually20–30aminoacidresidues
type I IFN receptorcomposed of IFNΑR1 and IFNΑR2 (Darnell, C-terminaloftheconservedtyrosine(DeckerandKovarik,2000).
1997;Pestkaet al.,2004). IFN binding toits receptor triggersthe STAT1, STAT3, STAT4, STAT5a and STAT5b all share a
activation of protein tyrosine Janus kinases (JAK)1 and TYK2 conservedmotifconsistingoftwoprolinesflankingeitherasingle
through reciprocal transphosphorylation in response to receptor serineresidueoraserineresidueprecededbyamethionineresidue,
dimerization(Barbierietal.,1994;Mulleretal.,1993;Velazquez usually referred to as the ‘P(M)SP’ motif, that is targeted for
et al., 1995; Yan et al., 1996). JAK1 and TYK2 phosphorylate phosphorylation.STAT2andSTAT6aretheexceptionastheylack
STAT1 and STAT2 on a conserved tyrosine residue, resulting in the PMSP motif. STAT6, however, is phosphorylated at a serine
e
residue in the TAD, but its function is to inhibit gene activation c
n
(Shirakawaetal.,2011).Mostrecently,wemadethediscoverythat e
1Dept.ofMedicalGeneticsandMolecularBiochemistry,LewisKatzSchoolof S287-STAT2, which is mapped to the coiled-coil domain, is ci
Medicine,TempleUniversity,Philadelphia,PA19140,USA.2ImmuneCell S
DevelopmentandHostDefenseProgram,FoxChaseCancerCenter,Philadelphia, phosphorylated in response to IFN-α stimulation (Steen et al., l
PA19111,USA.3Dept.ofAnatomyandCellBiology,LewisKatzSchoolof 2013). In contrast to other STATs, but similarly to STAT6, el
Medicine,TempleUniversity,Philadelphia,PA19140,USA. C
phosphorylation of S734-STAT2 did not augment, but rather
*Theseauthorscontributedequallytothiswork f
inhibited,genetranscription. o
‡Authorforcorrespondence([email protected]) In this study, we report that S734 is a new STAT2 al
n
phosphorylation site targeted by type I IFN stimulation. This
A.M.G.,0000-0001-7843-1415 r
moleculareventreliesonJAKkinaseactivityandSTAT2tyrosine u
o
Received23December2015;Accepted19September2016 phosphorylation.PhosphorylationofS734-STAT2playsaspecific J
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role in type I IFN signaling by contributing to the inactivation of acids.WeestablishedthespecificityofthepS734-STAT2antibody
the antiviral, but not the anti-proliferative, response to IFN-α and intwoways.First,weshowedtheabsenceofapS734-STAT2signal
IFN-β. Our findings indicate that phosphorylation of STAT2 at inimmunoblotsofproteinlysatesfromparentalU6AcellsandU6A
specific serine residues dictate the specificity and kinetics of an cellsstablyexpressingmutantSTAT2inwhichS734waschanged
IFN-αandIFN-βresponse. toalanine(S734A)(Fig.2A;Fig.S1A).Ofnote,asecondbandwas
often detected migrating immediately below the pS734-STAT2-
RESULTS specifcband.GiventhatthisbandwasalsodetectedinSTAT2-null
IdentificationofanewIFN-α-inducedS734-STAT2 U6AcellsandinS734A-STAT2-expressingU6Acells,weinterpret
phosphorylationbymassspectrometry thislowerproteinbandtobenon-specific.Second,toconfirmthatthe
To gain further insight into the regulation of type I IFN signaling observed protein band recognized by our pS734-STAT2 antibody
by post-translational modifications of STAT2, we reconstituted was indeed a phosphorylated amino acid, immunoprecipitated
STAT2-nullU6AcellswithhumanSTAT2.Wetreatedthesecells wild-type(WT-)STAT2wastreatedwithorwithoutlambdaprotein
withorwithoutIFN-αandimmunoprecipitatedSTAT2fromthese phosphatase (LPP). LPP treatment eliminated the protein band
cells, which was then subjected to mass spectrometry. From this correspondingtopS734-STAT2(Fig.2B).Withthesetwosequential
analysis, we identified phosphorylation of S734 as a new IFN-α- approaches, we convincingly demonstrate the specificity of the
induced post-translational modification of STAT2 (Fig. 1). S734 anti-pS734-STAT2antibody.
maps to the TAD of STAT2 (Fig. 1B) and appears to be
evolutionarily restricted to primates (Fig. 1C). To confirm our S734-STAT2phosphorylationbyIFN-αisobservedinmultiple
mass spectrometry data and show that STAT2 was indeed celllines
phosphorylated on S734 after stimulation with type I IFNs, we We consistently detected constitutive phosphorylation of S734-
generated a polyclonal rabbit antibody against an immunopeptide STAT2 in resting U6A cells reconstituted with WT-STAT2. The
containing phosphorylated (p)S734 and its surrounding amino level of phosphorylated S734-STAT2 was increased with IFN-α
Fig.1.Massspectrometryidentifies
S734asatypeIIFN-inducible
phosphorylatableresidueinhuman
STAT2thatisconservedinprimates.
(A)Tandemmassspectrometry(MS/
MS)spectrumofSTAT2aftertreatment
withIFN-α.+P(lowerpanel)indicates
S734asbeingphosphorylatedandthe
underlinedpeptidemass(lowerpanel)
indicatesthepresenceofthepeptidein
thespectrum(upperpanel).
P,phosphorylgroup(79.97Da).Notall
peaksareannotatedinthemass
spectrum.Matchesweremadewitha
masstoleranceof±2Da.(B)S734is
mappedtothetransactivationdomain
(TAD)ofSTAT2.(C)Cross-species
conservationofthisphosphorylation
siteisseeninprimateSTAT2.
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Fig.2.STAT2isphosphorylatedatS734inresponsetoIFN-αtreatment.(A)U6Acellsstablyexpressingemptyvector,WT-STAT2orS734A-STAT2weretreated i
c
withhumanIFN-α(1000U/ml)fortheindicatedtimes.PhosphorylationofS734-STAT2wasdetectedbywesternblotanalysisusinganaffinitypurifiedpolyclonal S
antibodyraisedagainstthisphosphorylatedepitope.IFN-α-inducedtyrosinephosphorylationofSTAT2(pY-STAT2)andSTAT1(pY-STAT1)togetherwithtotallevels l
l
ofSTAT2andSTAT1werealsoassessedbywesternblotanalysis.(B)ImmunoreactivityofthepS734-STAT2antibodyisconfirmedbyalambdaproteinphosphatase e
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(LPP)assay.(C–F)ImmortalizedhTERTcells(C)andhumancolorectalcancercellsF6-8(D),T29(E)and(F)Stat2−/−MEFsreconstitutedwithhumanSTAT2were
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stimulatedwitheitherhumanIFN-α(1000U/ml)ormurineIFN-β(1000U/ml),accordingly,andexaminedasinA.GAPDHwasusedasaninternalloadingcontrol. o
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a
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after 1h of treatment that then peaked at 4h (Fig. 2A). We next endogenous STAT2. No basal levels of phosphorylated S734-
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evaluated the basal phosphorylation and the kinetics of IFN-α- STAT2wereobservedinimmortalizedhumanmammaryepithelial u
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inducedphosphorylationofS734-STAT2incelllinesthatexpress hTERT-HME1cells.InresponsetoIFN-αtreatment,inductionof J
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serine phosphorylation was first detected at 8h of cytokine Moreover,immunoprecipitationofSTAT1fromU6AWT-STAT2
exposure that peaked around 12h and decreased thereafter cells revealed that, in the absence of IFN-α treatment, STAT2
(Fig.2C).Asexpected,given thatSTAT2isan ISG,totalSTAT2 associated with STAT1 was serine phosphorylated at a basal level
levels also increased over time in hTERT-HME1 cells. The (Fig. 3D). Treatment with IFN-α increased pS734-STAT2, but did
observed decrease in pS734-STAT2 detected at 24h did not not alter pS734-STAT2 binding to STAT1, indicating that pS734-
correlate with elevated STAT2 levels, indicating at least a partial STAT2 is neither enriched in nor excluded from IFN-α-induced
independence between STAT2 levels and S734-STAT2 STAT2–STAT1 dimers. Moreover, analysis of cytoplasmic and
phosphorylation in hTERT-HME1 cells. Similarly, IFN-α nuclearextractsfromIFN-α-treated2fTGHcells(theparentofU6A
treatment also induced S734-STAT2 phosphorylation in two cells) showed a time-dependent accumulation of nuclear pS734-
human colon carcinoma cell lines (Fig. 2D,E). Additionally, we STAT2.After1hofIFN-αstimulation,theamountofcytoplasmic
show that human STAT2 was phosphorylated on S734 when pS734-STAT2 was decreased, which coincides with an initial
reconstituted in Stat2−/− mouse embryonic fibroblasts (MEFs) accumulation of nuclear pS734-STAT2. Between 4 h and 8h of
following treatment with murine IFN-β (Fig. 2F; Fig. S1B). It is IFN-αtreatment,pS734-STAT2peaks,andahigherlevelisseenin
important to remark that in all the cell lines we studied, with the thenucleuswithsomedetectedinthecytoplasm,althoughatreduced
exceptionofT29cells,IFN-α-stimulatedphosphorylationofS734- levels(Fig.4A).WealsodetectedpS734-STAT2accumulationinthe
STAT2 peaked later when compared against Y690-STAT2 nucleus of IFN-α-treated hTERT cells (Fig. 4B). As expected,
phosphorylation. Furthermore, we found no differences in the phosphorylation of S734-STAT2 in hTERT cells required a much
kineticsoftyrosinephosphorylationofSTAT2andSTAT1between longerincubationtimewithIFN-α,aswepreviouslydeterminedin
U6Acellsexpressingwild-typeandmutantS734A-STAT2atupto Fig. 2C. Our data, therefore, show that JAK1-dependent tyrosine
8hofIFN-αtreatment(Fig.2A).However,whenexaminedat24h, phosphorylation of STAT2 is a prerequisite for enhancing STAT2
we found STAT2 tyrosine phosphorylation to be prolonged in serine phosphorylation with optimal S734-STAT2 phosphorylation
S734A-STAT2U6AcellsandbarelydetectableinWT-STAT2U6A peaking when STAT2 is tyrosine phosphorylated. This finding
cells (Fig. 7D), indicating that S734-STAT2 plays a role in correlatedwithasteadyincreaseinthelevelofphosphorylatedS734-
regulatingSTAT2activation. STAT2 in the nucleus. Further, U6A cells expressing the RKAA-
STAT2 mutant showed intact IFN-α-induced pS734-STAT2
IFN-αcaninducephosphorylationofSTAT2atS734only (Fig. 3A). Thus, these results reveal that phosphorylation of S734-
inthepresenceofJAKkinasesandifitsY690is STAT2 by type I IFNs must be occurring in the cytoplasm before
phosphorylated STAT2translocatestothenucleus.
To define the signaling requirements for S734-STAT2
phosphorylation, we first examined the role of STAT2 tyrosine S734-STAT2phosphorylationdoesnotaffecttheIFN-α-
phosphorylation as a potential pre-requisite event, as previously inducedantiproliferativeactivity,butdiminishestheIFN-α-
demonstrated with STAT1 serine phosphorylation (Sadzak et al., inducedantiviralstate
2008). We chose two well-characterized STAT2 mutants for this ToelucidatethebiologicalrelevanceofphosphorylatedS734-STAT2
analysis:Y690F-STAT2andR409A/K415A-STAT2(RKAA).The intypeIIFNsignaling,wecomparedtheanti-proliferativeresponses
Y690F-STAT2mutantcannotbephosphorylatedbytypeIIFNsand to IFN-α in U6A cells expressing WT-STAT2 or S734A-STAT2.
is thus unable to translocate to the nucleus (Li et al., 1997b). The First, no significant differences in growth rates were observed
RKAA-STAT2 mutant is retained in the cytosol due to a non- between U6A cells expressing WT-STAT2 and those expressing
functioning nuclear localization signal, but preserves its capacity S734A-STAT2 (Fig. 5A). Second, the anti-proliferative effects of
to become phosphorylated on tyrosine (Melen et al., 2001). IFN-αwerealsounaffectedbyS734A-STAT2.Similarresultswere
Accordingly, we reconstituted STAT2-null U6A cells with WT-, obtainedwiththephosphomimeticmutantSTAT2-S734D(Fig.5B).
RKAA-orY690F-STAT2.TheSTAT2-reconstitutedU6Acellswere Next,weaskedwhetherphosphorylationonS734affectedIFN-
treated with or without IFN-α and assayed for pS734-STAT2 by α-mediatedantiviralresponses.Tothisend,weleftuntreatedorpre-
immunoblot analysis. We detected a basal level of pS734-STAT2 treated U6A cells expressing WT-STAT2 or S734A-STAT2 with
with all three versions of STAT2 (Fig. 3A). In response to IFN-α 1000U/ml of IFN-α, before infection with a prototypic IFN-
treatment, RKAA-STAT2 showed an increase in S734 sensitivevirusvesicularstomatitisvirus(VSV)thatalsoencodesfor
phosphorylation (Fig. 3A). In contrast, Y690F-STAT2 did not GFPexpression(VSV-GFP)(Fig.6A).Quantifyingprogenyvirus
undergo S734-STAT2 phosphorylation, supporting the notion that yield from these cells demonstrated that, although there was no
tyrosine phosphorylation of STAT2 is necessary for the inducible detectabledifferenceat24hbetweencellsexpressingWT-STAT2,
phosphorylationofS734-STAT2bytypeIIFNs.Totestthepotential and phospho-null or phosphomimetic STAT2 mutants (data not
contributionofJAKkinasesinthephosphorylationofS734-STAT2, shown), virion production at36hfrom S734A-STAT2-containing
U6A cells expressing WT-STAT2 were pre-treated with pan-JAK cells was significantly reduced compared to cells expressing
e
inhibitor followed by treatment with IFN-α. Western blot analysis WT-STAT2 (∼3×105 pfu ml−1 versus ∼4×106 pfu ml−1) or c
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demonstratedthatJAKactivitywasnecessaryforIFN-α-dependent phosphomimetic S734D-STAT2 mutant (Fig. 6A). Note that e
i
induction of pS734-STAT2 (Fig. 3B). To further validate these expression of S734A-STAT2 or WT-STAT2 in the absence of c
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results, we evaluated pS734-STAT2 in JAK1-deficient U4A cells. IFN-α treatment rendered U6A cells similarlysusceptible to VSV l
LackofJAK1notonlyimpairedpS734-STAT2byIFN-α,butalso infection.ItisalsoimportanttomentionthatS734A-STAT2ledto el
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basal STAT2 serine phosphorylation (Fig. 3C). Collectively, these increased STAT2 tyrosine phosphorylation compared to WT-
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results identify tyrosine phosphorylation on Y690 as a prerequisite STAT2in U6Acells pre-treatedwithIFN-αat3hbutnot at36h o
forenhancingbasalS734phosphorylationofSTAT2inresponseto post-VSVinfection(Fig.S2).Nodifferenceswerefoundinthelevel al
IFN-α treatment. Furthermore, IFN-α-induced S734-STAT2 of tyrosine-phosphorylated STAT1 under these conditions. n
r
phosphorylation appears to take place in the cytosol as evidenced Expression of S734D-STAT2 had no significant effect on VSV u
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bythebehavioroftheRKAA-STAT2mutant. yieldateither24or36hpostinfection,comparedtoWT-STAT2- J
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Fig.3.PhosphorylationofS734-STAT2isdependentonSTAT2tyrosinephosphorylationandcatalyticallyactiveJAKkinases.(A)U6Acellsstably
expressingWT-,R409A/K415A(RKAA)-,orY690F-STAT2wereleftuntreatedortreatedfor1and4hwithIFN-α(1000U/ml).Tyrosineandserine
phosphorylationofSTAT2,andGAPDHwereassessedbywesternblotanalysis.DatawerequantifiedandshownastheratioofpS734-STAT2toSTAT2
andpY690-STAT2toSTAT2.Thearrowindicatesanon-specificproteinband(NS).(B)U6AcellsexpressingWT-STAT2werepre-treatedornotwithpan-JAK
inhibitorovernight(o/n)and(C)U4AcellsandU6cellsexpressingWT-STAT2stimulatedwithIFN-α(1000U/ml)forthespecifiedtimeswereevaluatedforpS734-
e
STAT2,pY-STAT2,pY-STAT1,STAT1,STAT2andGAPDHasindicated.(D)STAT1immunecomplexesobtainedfromU6AcellsexpressingWT-STAT2that c
wereleftuntreatedortreatedwithIFN-α(1000U/ml)wereevaluatedforinteractionswithpS734-STAT2andtotalSTAT2bywesternblotanalysis. n
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expressingcells.Wespeculatethattheeffectofthephosphomimetic expression of VSV-GFP 36h later by flow cytometry to l
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mutant is masked by phosphorylation of S287-STAT2 because, determine the degree of viral infection. We found that compared e
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as reported by us previously (Steen et al. 2013), it negatively toIFN-β-pre-treatedStat2−/−MEFsexpressinghumanWT-STAT2,
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regulates type I IFN and because pS287-STAT2 led to a much IFN-β-pre-treatedStat2−/−MEFsexpressinghumanS734A-STAT2 o
stronger phenotype than pS734-STAT2. To confirm our findings showedenhancedviralprotectionat36hpostinfection(Fig.6B). al
using a different cell line, we infected Stat2−/− MEFs expressing Taken together, these results indicate that phosphorylation of n
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either human WT-STAT2 or S734A-STAT2 that had been pre- STAT2onS734servestonegativelyregulatethetypeIIFNantiviral u
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treated with or without IFN-β with VSV-GFP. We quantified response. J
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Fig.4.SubcellularlocalizationofphosphorylatedS734-STAT2.Nuclearand
cytoplasmicfractionsofhuman(A)2fTGHand(B)hTERTcellstreatedwithIFN-
α(1000U/ml)forthegiventimeswereevaluatedforpS734-STAT2,pY-STAT2,
pY-STAT1,STAT1andSTAT2bywesternblotanalysisasindicated.Presence
ofGAPDHinthecytoplasmicextractandpresenceofLaminB1inthenuclear
extractconfirmedpurityoffractions.Thearrowindicatestheupperband
correspondingtopS734-STAT2.Actinwasusedasaninternalloadingcontrol.
Fig.5.PhosphorylationofS734-STAT2doesnotaffectthe
S734-STAT2phosphorylationenhancestheexpressionofa antiproliferativeeffectsofIFN-α.(A)GrowthrateofU6Acellsstably
subsetofIFN-stimulatedantiviralgenes expressingWT-,S734A-orS734D-STAT2wasmeasuredat48hand72hby
We next conducted whole-genome DNA microarray analysis to anMTSassay.TheinsetshowsawesternblotanalysisofSTAT2expressionin
identifypotentialdifferencesinthetranscriptionalresponsestoIFN- U6Acellsexpressingemptyvector,WT-,S734AorS734D-STAT2.(B)The
αbetweenWT-STAT2andS734A-STAT2thatmightexplainhow samepanelofcellswastreatedwith100U/mlor1000U/mlofIFN-αfor72h
phosphorylation of STAT2 on S734 regulated the type I IFN andcellviabilitydeterminedbyMTSassay.Resultsarepresentedasthe
percentageofviablecellsinthepresenceofIFN-αcomparedagainstuntreated
antiviral state. We used total RNA extracted from U6A cells
cells.Datashownarefromthreeorfourindependentexperimentsand
expressingWT-STAT2orS734A-STAT2thathadbeenstimulated
presentedasmean±s.e.m.
withorwithoutIFN-αfor4h.Thisgenomicapproachidentified10
ISGs that were induced more by IFN-α in U6A cells expressing 60h, however, ISG15 protein levels were reduced in WT-STAT2
S734A-STAT2 than they were in cells expressing WT STAT2 cells, but remained elevated in S734A-STAT2 cells. In contrast,
(Fig. 7A; full microarray data is available in the Gene Expression expressionofOAS1wasfoundtobeslightlyelevatedonlyat24hin
Omnibus under accession number GSE57017). We then selected S734A-STAT2 cells when compared to WT-STAT2 cells. This
representative genes from this list to validate by quantitative real- observation also correlated with increased STAT2 tyrosine
time PCR (qPCR). At 6h, we consistently detected an enhanced phosphorylation in cells expressing S734A-STAT2 at 24h,
induction of OAS1 and IFIT2 by IFN-α in U6A cells containing indicating that S734 regulates STAT2 activity. Thus, these results
S734A-STAT2 compared to that in cells expressing WT-STAT2 supporttheideathatphosphorylationofS734-STAT2contributesto
(Fig. 7B). Similarly, we found that Stat2−/− MEFs reconstituted thetranscriptionalregulationofasmallsubsetofISGswithantiviral
e
with human S734A-STAT2 and stimulated with murine IFN-β activity. c
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showedenhancedtranscriptionofOas1bandIsg15whencompared e
to IFN-β-treated MEFs expressing human WT-STAT2 (Fig. 7C). DISCUSSION ci
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AlthoughdifferencesinOas1bmRNAlevelswerenotevidentuntil Regulation of type I IFN signaling through STAT2 has remained l
24h after IFN-β stimulation, this could be attributed to the understudiedchieflybecausethediversityofSTATTADsandthe el
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activation of human STAT2 in mouse cells. To confirm these sequence divergence of the STAT2 TAD among different species
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resultsattheproteinlevel,U6AcellsreconstitutedwitheitherWT- have made it challenging to predict amino acids in STAT2 that o
STAT2orS734A-STAT2werestimulatedwith1000U/mlofIFN-α might be important for proper transcriptional activity (Park et al., al
n
for24and60handanalyzedbywesternblotanalysisforISG15and 1999; Paulson et al., 1999). For example, serine phosphorylation
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OAS1 expression (Fig.7D). Wefound ISG15proteinlevelsto be within the P(M)SP motifs found in several STAT transactivation u
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similar in WT-STAT2 and S734A-STAT2 U6A cells at 24h. At domainshasbeenshowntoberequiredforSTAT-dependentgene J
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Fig.6.S734A-STAT2enhancesIFN-α-inducedprotectionagainstVSVinfection.(A)TheU6Acellpanelwasleftuntreatedorpre-treatedwithIFN-α(1000U/ml)for
16handtheninfectedwithGFP-taggedvesicularstomatitisvirus(VSV)for36h.Viraltitersafter36hpost-viralinfection(h.p.i.)weredeterminedbyplaqueassay.Viral
titersareshownonalogscale.(B)WesternblotanalysisofSTAT2expressioninStat2−/−MEFsreconstitutedwitheitherhumanWT-orS734A-STAT2.(C)Stat2−/−MEFs
expressinghumanWT-orS734A-STAT2werepre-treatedornotwithmurineIFN-β(1000U/ml)for16handVSVinfectionwasquantifiedbyflowcytometryanalysisof
GFP-positivecells.Dataareshownasthemean±s.e.m.fromthreeorfourindependentexperiments.**P<0.01(Student’st-test).NS,notstatisticallysignificant.
regulation.Incontrast,STAT2lacksthisconservedmotif,andthis Our study provides new evidence that serine phosphorylation of
opens the door to investigate the biological role of serine- STAT2 plays a pivotal role in selectively regulating the biological
phosphorylatedSTAT2. actions of typeI IFNs. Wehaveidentifiedaserinephosphorylation
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Fig.7.S734-STAT2regulatestheexpressionofasubsetofISGs.(A)MicroarrayanalysisidentifiedasmallsubsetofISGsforwhichmRNAlevelswerefound
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tobeenhancedinS734A-STAT2U6AcellswhencomparedagainstWT-STAT2U6Acellsafter4hofIFN-α(1000U/ml)treatment.GenesmarkedinAwithan o
asteriskweresubsequentlyvalidatedbyqPCRusing(B)U6Aor(C)Stat2−/−MEFsreconstitutedwitheitherhumanWT-orS734-STAT2treatedwithorwithout l
a
IFN-αorIFN-β(1000U/ml)for6,18or24h.ThemRNAlevelswerenormalizedtoactinexpressionandcalculatedbyusingΔΔCtmethod.Dataarepresentedas n
themean±s.e.m.foldchangefromuntreatedcellsfromthreeorfourindependentexperiments.*P<0.05,**P<0.01(Student’st-test).(D)WT-STAT2andS734A- r
u
STAT2U6AcellswerestimulatedornotwithIFN-α(1000U/ml)for24or60handlevelsofISG15andOAS1aswellaspY-STAT2,pY-STAT1,STAT1,STAT2and o
GAPDHasindicated,wereassessedbywesternblotanalysis. J
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site(S734)onSTAT2thatresidesintheTAD,butthatisawayfrom
thecrucialregionpreviouslymappedfortransactivation.Thisnewsite
appearsto specifically regulate the antiviral activityof type I IFNs,
withoutdisturbingtheanti-proliferativeactivity.Weconsistentlyshow
thatS734-STAT2isphosphorylatedinresponsetoIFN-αstimulation
inseveralcelllines,withkineticsthataredistinctlyslowerthatthose
observed for STAT2 tyrosine phosphorylation. We found JAK1
activity and STAT2 tyrosine phosphorylation to be necessary for
pS734-STAT2induction.Furthermore,wepresentevidencethatIFN-
α-induced pS734-STAT2 translocates to the nucleus. Although the
serinekinaseresponsibleforphosphorylatingS734isyetunknown,
with the use of prediction tools, we have identified two candidate
serinekinasesthatwearecurrentlyevaluatingfortheirroleinpS734-
STAT2phosphorylation.
Thefunctionofthisnewlydiscoveredpost-translationalmodification
appearstodifferfromtheS287sitewerecentlyidentifiedonSTAT2.In
response to IFN-α stimulation, disabling phosphorylation of S287-
STAT2hastwoconsequences.First,itaugmentsthegrowthinhibitory
effectsoftypeIIFNand,second,itprotectscellsfromviralinfection
resulting in prolonged cell survival due to reduced virus replication
(Steenetal.,2013),twofeaturesnotentirelysharedwithS734-STAT2.
Although additional studies are underway to determine the Fig.8.ProposedmodelofS734-STAT2phosphorylationinregulatingthe
combinatory effect of STAT2 dually phosphorylated on serine antiviralactivityoftypeIIFNs.UponbindingofIFN-αandIFN-βtoits
residues287and734intypeIIFNsignaling,weproposethatS734- receptor,JAK1andTYK2becomeactivatedandtyrosinephosphorylate
STAT1(pY701)andSTAT2(pY690).ActivatedSTAT1andSTAT2,together
STAT2phosphorylationhasaspecificcontributoryroleinnegatively
withIRF9,astheISGF3complex,triggertheinductionofantiviralISGs.
regulatingtheantiviraleffectsoftypeIIFN.
Subsequently,tyrosine-phosphorylatedSTAT2(pY690)isphosphorylatedat
HowmightphosphorylationofS734-STAT2negativelyregulate S734(pS734)inaJAK-dependentmanner,andthisinturn,restrictsthe
type I IFN signaling? Early experiments using U6A cells inductionofISGswithantiviralactivity.
reconstituted with C-terminally truncated versions of STAT2
showed that deletions up to amino acid 831 did not affect type I interaction with other proteins. In such a scenario, the
IFNsignalingandISGinduction(Qureshietal.,1996).However, phosphorylated serine residue would increase the negative charge
removing residues to amino acid 812 led to a partial reduction in on the acidic side, speculatively disrupting relevant STAT2–co-
STAT2function,andtruncatingSTAT2toresidue800completely factorinteractionsatcertainISGlociandresultinginadampened
disruptedgeneinduction.TheinteractionbetweentheSTAT2-TAD transcriptional response. Because this particular post-translational
andp300/CBPhasbeenrecentlymappedbyNMRandconfinedto modificationseemstoonlyaffectthelevelofinductionofaselect
amino acids 788–816 of STAT2, with some stabilizing, but non- subset of ISGs, we hypothesize that the proteins interacting with
crucial interactionsbetweenresidues817and833(Wojciaketal., STAT2atthelocioftheseISGsarewhatisprovidingselectivitythat
2009). Owing to the deleterious effects of C-terminal truncations isnotseenwithotherISGs.
after removing the first 50 residues, the importance of the Based on these observations together with our recent report on
N-terminal part of the TAD (residues ∼700–800) has not been S287phosphorylationofSTAT2,weproposeapreliminarymodel
carefully studied. Compared to other STATs, STAT2 has a very (Fig. 8) in which dynamic S734 phosphorylation regulates the
largeTADthatincludestheregionC-terminalofY690.Addingto antiviralactivitiesoftypeIIFNs.Overall,thisdiscoveryhighlights
that, STAT2 is different from all other STATs in that it does not striking differences in the role of serine phosphorylation of
stablyinteractwithDNA(BluyssenandLevy,1997).IntypeIIFN STAT2 compared to other STATs, including STAT1, wherein
signaling,thistaskisperformedbySTAT1andIRF9,whichbind phosphorylation on serine enhances, rather than antagonizes, the
DNAdirectlywhileSTAT2contributesitstransactivationfunction transcriptionalresponsetotypeIIFNs.
totheISGF3complex.Therefore,wecanpostulatethatthefirstpart
MATERIALSANDMETHODS
oftheSTAT2-TAD(fromresidues∼700–760)isrequiredtobring
Cellculture
theremaining90residues(761–851)intoaproperconformationfor
STAT2-deficient human U6A fibrosarcoma cells (obtained from Dr Ana
binding to co-activators such as p300/CBP, GCN5 and, most Costa-Pereira,ImperialCollegeLondon,London,UK),STAT2-nullmouse
recently,RVB1andRVB2proteins(Gnatovskiyetal.,2013). embryonic fibroblasts (provided by Dr Chris Schindler, Columbia e
The S734-STAT2 location, ‘PELSLDLEP’, consists of several University, New York, NY), human colorectal carcinoma cells F6-8 and c
n
leucineresidues(hydrophobic)andacidicresiduesflankedbytwo T29 (obtained from Dr Bert Vogelstein, Johns Hopkins University, e
proline residues. Interestingly, S734 is found within the mapped Baltimore, MD), and 293FT cells were cultured in Dulbecco’s modified ci
nuclear export signal of STAT2 (Frahm et al., 2006). As a serine Eagle’s medium (DMEM; Cellgro Mediatech, Inc., Manassas, VA) lS
phosphorylation motif, this was unknown, but remarkably very supplemented with 10% fetal calf serum (Gemini Bio-Products), el
100units/mlpenicillin(Cellgro),100μgml−1streptomycin(Cellgro),and C
similartoonepresentjustafewresiduesupstreaminastretchthat
1×GlutaMAX(Invitrogen,Waltham, MA).Humanfibrosarcoma2fTGH f
startsataminoacid716(PELESLELELG).Itisworthmentioning o
cellsandepithelialmammarycellshTERT-HME1(kindlyprovidedbyDr
that WT-STAT2 and S734A-STAT2 showed identical kinetics in George Stark, Cleveland Clinic Foundation, Cleveland, OH) and 293FT al
theirsubcellularlocalizationbeforeandafterIFN-βtreatment(data cellspurchasedfromLifeTechnologies(GrandIsland,NY)weregrownin rn
notshown).ThenatureoftheSTAT2TAD,whichcontainsbothan complete DMEM (Cellgro Mediatech). hTERT-HME1 cells required ou
acidic side and an opposite hydrophobic side, might facilitate its DMEMfurthersupplementedwithmammaryepitheliumgrowthmedium J
4197
RESEARCHARTICLE JournalofCellScience(2016)129,4190-4199doi:10.1242/jcs.185421
containing bovine pituitary extract, hydrocortisone, insulin, epithelial SDS-PAGE.AproteinbandcorrespondingtothesizeofSTAT2(113kDa)
growth factor, and gentamicin and amphotericin-B (Clonetics, Basel, wasvisualizedwithCoomassieBluestainandexcisedforfurtheranalysis.
Switzerland). All cells were grown at 37°C in a humidified atmosphere ExtractedproteinsweretreatedwithProteinaseKpriortomassspectrometry
containing5%CO ,authenticatedandcheckedforcontamination. analysisperformedbyITSIBiosciences(Johnstown,PA)usinganLTQXL
2
massspectrometer(ThermoScientific).Resultingmassspectrawassearched
Reagentsandantibodies against a custom STAT2 human database from UniProt using Proteome
Recombinant human IFN-α-2a (specific activity 2×107 units ml−1) was Discoverer (version 1.3, Thermo Scientific) and the SEAQUEST search
purchasedfromPeproTech,Inc.(RockHill,NJ).RecombinantmurineIFN- engine.ProteinswereidentifiedwhentwoormoreuniquepeptideshadX-
βwasakindgiftfromBiogen,Inc.(Cambridge,MA).JAKinhibitorwas correlationscoresgreaterthan1.5,2.0and2.5forrespectivepeptidecharge
obtainedfromEMDMillipore(Bedford,MA).Rabbitanti-STAT2(C-20, states of +1, +2, and +3, together with a delta correlation value of ≥0.1.
cat.no.sc-476,1:1000),rabbitanti-STAT1(E-23,cat.no.sc-346,1:1000) Carbamidomethylwasusedasastaticmodification.Phosphorylatedserine,
andrabbitanti-LaminB1(C-5,cat.no.sc-365962,1:1000)werepurchased threonine, and tyrosine along with acetylated residues were variable
from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit anti-phospho- modifications.Peptidetolerancewassetto±5000ppmandmasstolerance
Y689-STAT2(cat.no.07-224,1:1000)wasobtainedfromMillipore,mouse was±2Da.
anti-pY701-STAT1 (cat. no. 612233, 1:1000) was purchased from BD
Biosciences, mouse anti-GAPDH (cat. no. 60004-1, 1:5000) and mouse MTScellproliferationassay
anti-actin (cat. no. 60008-1, 1:5000) antibodies were purchased from U6Acellsseededinflat-bottomed96-wellplatesatadensityof500cellsper
ProteintechGroup,Inc.(Chicago,IL).Affinitypurifiedrabbitpolyclonal wellweretreatedwithorwithoutIFN-αfor72hat37°C.Cellgrowthwas
anti-STAT2-pS734 was raised against the phospho-specific peptide determined by using CellTiter 96® AQueous One solution reagent
VPEPEL(pS)LDLEPL; amino acids 728–740 in collaboration with (Promega, Madison, WI) according to the manufacturer’s protocol.
RocklandImmunochemicals,Inc.(Gilbertsville,PA). Absorbance was measured at 490nm using a VICTOR™X5 Multilabel
PlateReader(PerkinElmerLifeSciences,Waltham,MA).Dataareshownas
Plasmids,site-directedmutagenesisandtransduction apercentageofthevalueofcontrol(untreatedcells).
C-terminallyFlag-taggedhumanSTAT2cDNAvectorwasobtainedfrom
Dr Curt Horvath (Northwestern University, Evanston, IL). Site-directed Virusinfection
mutagenesiswasperformedusingtheQuikChangeLightningkit (Agilent Cellsseededin12-wellplateswereleftuntreatedortreatedwithincreasing
Technologies, Santa Clara, CA) to construct R409A/K415A-(RKAA), doses of IFN-α for 16h prior to infection with vesicular stomatitis virus
Y690F- and S734A-STAT2 mutants. Mutant and wild-type STAT2 (VSV) with a GFP-expressing gene (Fernandez et al., 2002). Plaque-
constructs were subcloned into the lentiviral expression vectors purifiedVSV(Indianastrain)wasaddedtocellsatamultiplicityofinfection
pCDH-CMV-MCS-EF1-GreenandpCDH-CMV-MCS-EF1-RFP(System of 0.01 for U6A cells and 0.002 for Stat2−/− MEFs under serum-free
Biosciences,MountainView,CA).Lentiviruseswereproducedin293FT medium conditions for 1h at 37°C. Cells were washed twice with PBS
cells using ViraPower Lentiviral Expression System (Life Technologies, followed by re-addition of complete DMEM. Progeny virion titers were
Carlsbad, CA). U6A cells and Stat2−/− MEFs were transduced with quantifiedinculturesupernatantsbystandardplaqueassayonbabyhamster
lentivirusinthepresenceof1μgml−1Polybrene(Sigma,St.Louis,MO) kidney(BHK-21)cellsorbyflowcytometry.
andsubjectedtoantibioticselectionbyadding2μgml−1puromycintothe
growthmediumat72hpostinfection. Quantitativereal-timePCR
TotalRNAwasextractedfromcellsusingRNABeesolution(AmsbioLLC,
Westernblotting,nuclearextractpreparationand LakeForest,CA).ContaminatingDNAwasremoved(DNA-freeKit,Ambion)
immunoprecipitation andcDNAwassynthesizedfromRNAusingtheHighCapacitycDNAReverse
Cells were lysed in Triton X-100 lysis buffer (50mM Tris-HCl pH 7.5, TranscriptionKit (AppliedBiosystems,Carlsbad,CA).cDNAwascombined
150mM NaCl, 2mM EDTA, 0.5% Triton X-100) supplemented with 1× withgene-specificFAMdye-labeledTaqManMGBprobes(LifeTechnologies)
protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN), 1mM and2×TaqManMasterMix.Real-timeanalysiswasperformedusinga7300
sodiumorthovanadate,1mMPMSFand1×phosphataseinhibitormixture2 real-time PCR system (Applied Biosystems). Actin cDNA was used to
and3(Sigma),for20minonice.Aftercentrifugationfor15minat4°Cand normalize cDNA loading. Each sample was analyzed in triplicates and
15,000 g, supernatants were collected and protein concentration was normalizedagainstactin.TheΔΔCtmethodwasusedtodeterminedifferencesin
determined with the Bio-Rad protein assay kit (Bio-Rad, Hercules, CA). genetranscriptionbetweenWT-STAT2andS734A-STAT2.
Nuclearextracts wereprepared asdescribedpreviously (Steen etal.,2013).
Immunoprecipitationassayswereperformedbyfirstpre-clearingproteinlysates Microarraygeneanalysis
byincubationwithprotein-G–Sepharose(GEHealthcare,LittleChalfont,UK). STAT2-expressingU6A cells were seededintriplicateand treated withor
Pre-clearedlysateswerethenincubatedwiththeindicatedantibodyovernightat without1000Uml−1IFN-αfor4h.TotalRNAwasisolatedusingRNABee
4°Candimmunocomplexescollectedbyfurtherincubationwithprotein-G– solutionandfurtherpurifiedbyRNeasykit(Qiagen,Valencia,CA)according
Sepharose for 2h. Dephosphorylation of STAT2 was performed byadding to the manufacturer’s instructions. The qualityof total RNAwas assessed
400Uoflambdaproteinphosphatase(LPP;NewEnglandBiolabs,Ipswich, usinganAgilent2100Bioanalyzer(AgilentTechnologies,SantaClara,CA).
MA) followed by incubation at 37°C for 30min with constant agitation. RNAsampleswerelabeledandhybridizedtotheAffymetrixHumanGene
LysatesandimmunoprecipitateswereresolvedonNuPAGE4–12%Bis-Tris 2.0 ST Array (Affymetrix, Santa Clara, CA). For each condition, three
gels (Life Technologies) and transferred to PVDF membranes (Millipore, biologicalreplicatesampleswereusedformicroarrayexperiments.Scanned e
Temecula,CA).MembraneswereblockedwithBlockerCaseinTBS(Thermo microarray images were analyzed using the Affymetrix Gene Expression nc
Scientific,Rockford,IL)andincubatedwiththecorrespondingprimaryand ConsolewithRMA(RobustMulti-arrayAverage)normalizationalgorithm. e
i
horseradish peroxidase (HRP)-conjugated secondary antibodies in Tris- Furtherstatisticalanalyses wereperformed using BRB-ArrayTools(Simon c
S
buffered saline with 0.2% Tween 20 (TBS-T) plus 3% bovine serum et al., 2007). Gene classification into ontology categories (GO) was
l
albumin. Membranes were developed by chemiluminescence using performed using BLAST2GO® version 2.6.4 (Biobam Bioinformatics, el
SuperSignalWestPicoorFemto(ThermoScientific).Imageswerecaptured Valencia, Spain). The complete data files are available at NCBI, GEO C
withtheAlpha-InnotechHD2imagingsystem(ProteinSimple,SanJose,CA). accessionnumberGSE57017. of
l
a
Massspectrometry Statisticalanalysis
n
STAT2-reconstituted U6A cells were left untreated or treated with Prismsoftware(GraphPad,SanDiego,CA)wasusedforstatisticalanalysis. r
1000Uml−1 IFN-α for 30min and proteins were extracted with Triton Student’s t-test was applied to discern significant statistical differences ou
X-100 lysis buffer. STAT2 was immunoprecipitated and separated by betweensamples.P≤0.05wasconsideredsignificant. J
4198
RESEARCHARTICLE JournalofCellScience(2016)129,4190-4199doi:10.1242/jcs.185421
Acknowledgements experimentallydeterminedpost-translationalmodificationsinmanandmouse.
WethankMrMichaelSlifkerfortheanalysisofthemicroarraydata.Analyseswere NucleicAcidsRes.40,D261-D270.
performedusingtheBRB-ArrayToolsdevelopedbyDrRichardSimonandBRB- Improta,T.,Schindler,C.,Horvath,C.M.,Kerr,I.M.,Stark,G.R.andDarnell,
ArrayToolsDevelopmentTeam. J.E.,Jr.(1994).TranscriptionfactorISGF-3formationrequiresphosphorylated
Stat91protein,butStat113proteinisphosphorylatedindependentlyofStat91
Competinginterests protein.Proc.Natl.Acad.Sci.USA91,4776-4780.
Kraus,T.A.,Lau,J.F.,Parisien,J.-P.andHorvath,C.M.(2003).AhybridIRF9-
Theauthorsdeclarenocompetingorfinancialinterests.
STAT2proteinrecapitulatesinterferon-stimulatedgeneexpressionandantiviral
response.J.Biol.Chem.278,13033-13038.
Authorcontributions
Leung,S.,Qureshi,S.A.,Kerr,I.M.,Darnell,J.E.,JrandStark,G.R.(1995).
A.M.G.preparedthemanuscriptanddesignedexperiments;H.C.S.andK.P.K. Role of STAT2 in the alpha interferon signaling pathway. Mol. Cell. Biol. 15,
conductedanddesignedexperiments,andhelpedwritethemanuscript;S.N.and 1312-1317.
M.Y.H.conductedsomeoftheexperiments,andS.B.helpedwithmanuscript Li,X.,Leung,S.,Qureshi,S.,Darnell,J.E.,JrandStark,G.R.(1996).Formation
preparationandexperimentaldesign. of STAT1-STAT2 heterodimers and their role in the activation of IRF-1 gene
transcriptionbyinterferon-α.J.Biol.Chem.271,5790-5794.
Funding Li,X.,Leung,S.,Ker,I.M.andStark,G.R.(1997a).Functionalsubdomainsof
ThisprojectwassupportedbygrantstoA.M.G.fromtheNationalCancerInstitute STAT2 required for preassociation with the alpha interferon receptor and for
(RO1CA140499)andthePennsylvaniaDepartmentofHealth.DepositedinPMCfor signaling.Mol.Cell.Biol.17,2048-2056.
releaseafter12months. Li,X.,Leung,S.,Kerr,I.M.andStark,G.R.(1997b).Functionalsubdomainsof
STAT2 required for preassociation with the alpha interferon receptor and for
Dataavailability signaling.Mol.Cell.Biol.17,2048-2056.
Melen,K.,Kinnunen,L.andJulkunen,I.(2001).Arginine/lysine-richstructural
ThemicroarraydatareportedinthisstudyareavailablefromtheGeneExpression
elementisinvolvedininterferon-inducednuclearimportofSTATs.J.Biol.Chem.
OmnibusdatabaseunderaccessionnumberGSE57017(https://www.ncbi.nlm.nih.
276,16447-16455.
gov/geo/query/acc.cgi?acc=GSE57017).
Müller,M.,Briscoe,J.,Laxton,C.,Guschin,D.,Ziemiecki,A.,Silvennoinen,O.,
Harpur,A.G.,Barbieri,G.,Withuhn,B.A.,Schindler,C.etal.(1993).The
Supplementaryinformation proteintyrosinekinaseJAK1complementsdefectsintheinterferon-α/βand-γ
Supplementaryinformationavailableonlineat signaltransduction.Nature366,129-135.
http://jcs.biologists.org/lookup/doi/10.1242/jcs.185421.supplemental Park, C., Lecomte, M.-J. and Schindler, C. (1999). Murine Stat2 is
uncharacteristicallydivergent.NucleicAcidsRes.27,4191-4199.
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