Biochem.J.(2010)428,429–437(Printed in Great Britain)
doi:10.1042/BJ20091660
429
T-cell receptor early signalling complex activation in respon to interferon-αreceptor stimulation
Claire N.STEVENS*1,Ann-Marie SIMEONE †1,Susan JOHN ‡,Zamal AHMED*†,Orso M.LUCHERINI §,C.Tatiana BALDARI §and John E.LADBURY*†2
*Department of Biochemistry and Molecular Biology,University College London,Gower Street,London WC1E 6BT,U.K.,†Department of Biochemistry and Molecular Biology,
Anderson Cancer Center,University of Texas M.D.,Houston,TX 77030,U.S.A.,‡Peter Gorer Department of Immunobiology,King’s College,London School of Medicine at Guy’s,King’s and St Thomas’Hospitals,Great Maze Pond,London SE19RT,U.K.,and §Department of Evolutionary Biology,University of Siena,via Aldo Moro,2–53100Siena,Italy药物过敏症状
Signalling through the IFN αR (interferon-αreceptor)and TCR (T-cell receptor)in Jurkat T lymphocyte
s results in distinct immune respons.Despite this both receptors elicit ERK (extracellular-signal-regulated kina)/MAPK (mitogen-activated protein kina)phosphorylation.Vav and Slp76are shown to be required for IFN α(interferon-α)-stimulated ERK activity.The form a subt of proteins which behave identically on stimulation of both receptors.TCR deletion abrogates IFN αR-stimulated MAPK activity,whereas the canonical JAK/STAT (Janus kina/signal transducer and activator of transcription)pathway is unaffected.Thus recruitment of the intact TCR ESC
(early signalling complex)is necessary for this downstream MAPK respon.Despite using a common ESC,stimulation of the IFN αR does not produce the transcriptional respon associated with TCR.Up-regulation of the MAPK pathway by IFN αR might be important to ensure that the cell responds to only one stimulant.Key words:interferon-αreceptor (IFN αR),Janus kina/signal transducer and activator of transcription pathway (JAK/STAT pathway),mitogen-activated protein kina pathway (MAPK pathway),Slp76,T-cell receptor early signalling complex (TCR ESC),Vav.
INTRODUCTION
Lymphocytic class I IFNR (interferon receptor)and TCR (T-cell receptor)have distinct respons to extracellular stimulation.The former is a member of the cytokine family of receptors and signals prim
arily through the JAK/STAT (Janus kina/signal transducer and activator of transcription)pathway resulting in antiviral and growth inhibitory effects.In contrast,activation of the TCR through antigen prentation during an adaptive immune respon leads to cell proliferation and cretion of cytokines.The discrete cellular respons are derived from the distinct downstream gene transcriptional activity.Despite the contrasting cellular outcomes arising from their activation,both receptors invoke up-regulation of the ERK (extracellular-signal-regulated kina)/MAPK (mitogen-activated protein kina)pathway [1–3].The different cellular respons to the cytokine-and antigen-stimulated receptors have been shown to involve recruitment of a subt of common proteins.The proteins have all previously been identified as being involved in the ESC (early signalling complex)formed at the TCR within minutes of stimulation [4,5].For example,the proto-oncogene product Vav,which is phosphorylated upon stimulation of the TCR in human peripheral blood lymphocytes and human leukaemic T-cells [6],also interacts with the IFNR-associated tyrosine kina,Tyk2[7,8].Similarly,the tyrosine kinas Zap70and Lck,and the phosphata CD45,which are all fundamental components of the TCR signal respon,are also recruited to the IFN αR (interferon-αreceptor)in Jurkat cells and primary lymphocytes [9].The MAPK respons elicited from both IFN αR-and TCR-stimulated signals are dependent on the prence of both Zap70
and Lck [2,3,10]and the resultant tyrosine phosphorylation of Zap70occurs by the same mechanism upon IFN αR stimulation as when it is activated as part of the TCR signalling pathway [11].Furthermore,both Lck and Zap70were shown to play an integral role in IFN αsignalling as stimulated cells where the proteins were deleted were unable to demonstrate the typical anti-proliferative effects of this cytokine [9].The abnce of Lck and Zap70in the cells did not however affect IFN αR signalling through JAK or offer protection to cells from viral infection [9].The utilization of common proteins in transducing two signals with distinct cellular outcomes suggests a potential cross-talk between the pathways in lymphocytes.In the prent study we show further that IFN αstimulation of cells recruits the TCR ESC protein machinery,and requires a functional TCR to produce the MAPK respon in both lymphoma and ex vivo in healthy human primary CD4+T-cells.This respon has a more limited time cour than the sustained signalling at the TCR and leads to a different gene expression profile.The activation of the MAPK pathway via TCR after IFNR stimulation may be a way of committing the lymphocyte to a single cour of action by occupying/blocking a subquent TCR respon to extracellular antigen prentation.
EXPERIMENTAL
DNA constructs and mutagenesis
The SLP76construct ud in this paper was cloned,in frame,into the pcDNAhygro3.1-mRFP fusion vector [3],between the NheI and XhoI sites of the multiple cloning site so that Slp76was C-terminally tagged with monomeric RFP (red fluorescent
Abbreviations ud:ERK,extracellular-signal-regulated kina;ESC,early signalling complex;FBS,fetal bovine rum;IFN α,interferon-α;IFN αR,IFN αreceptor;IFNR,interferon receptor;IL,interleukin;JAK,Janus kina;LAT,linker for activation of T -cells;MAPK,mitogen-activated protein kina;MEK,MAPK/ERK kina;NFAT,nuclear factor of activated T -cells;NF-κB,nuclear factor κB;NP-40,Nonidet P40;PBMC,peripheral blood mononuclear cell;RFP ,red fluorescent protein;RLU,relative light unit;STAT,signal transducer and activator of transcription;TCR,T -cell receptor.1
The authors contributed equally to this work.2
To whom correspondence should be addresd (email jeladbury@mdanderson).
www.biochemj
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430 C.N.Stevens and others
protein).The Slp76-Y3F–RFP mutant was generated through
single A→T ba pair mutations using the Stratagene mutagenesis
kit according to the manufacturer’s instructions.The mutations
resulted in replacement of tyrosine residues with phenylalanine
residues at positions112,128and145of Slp76.The integrity of
all three point mutations was confirmed by gene quencing. Cell culture
Jurkat E6.1cells were purchad from the ECACC(European
Collection of Animal Cell Cultures),the J14(Slp76-deficient)
and PF2.4(TCRβ-reconstituted)cell lines were a gift from A.
Weiss(Dept.of Medicine and HHMI,University of California
San Franciso,San Franciso,CA,U.S.A.),the J.Vav1cell line
and clone15-11reconstituted J.Vav1cell line were kindly
provided by R.Abraham(Program in Signal Transduction
Rearch,The Burham Insitute,La Jolla,CA,U.S.A.),the JRT3-
T3.5(TCRβ-deficient)cell line was purchad from A.T.C.C.
The cell lines above and primary human peripheral CD4+
自编操T-cells(purification of the is described below)were all
cultured in RPMI-1640containing L-glutamine(Cambrex).This
medium was additionally supplemented with10%(v/v)heat-
inactivated,γ-radiated FBS(fetal bovine rum)(Biora)and
antimycotic/antibiotic(BioWhittaker).Cells were maintained in
a humidified incubator at5%(v/v)CO2and37◦C.J.Vav1cells
and reconstituted J.Vav1cells were cultured in the above medium,
supplemented with500μg/ml G418.
Purification of primary CD4+T-cells
PBMCs(peripheral blood mononuclear cells)were isolated from
buffy coats purchad from the Blood Bank(St.Georges Hospital,
Tooting,London U.K.).The PBMCs were isolated through
Ficoll density-gradient centrifugation paration according to the
supplier’s instructions(Amersham Biosciences).PBMCs were
collected and washed and subjected to negative lection in
order to isolate CD4+T-cells.This was achieved using MACS
(magnetic cell sorting)in accordance with the protocol described
by the manufacturer(Miltenyi Biotec).FACS analysis showed
that purity of the resultant CD4+T-cells was above97%in
all experiments.Following purification,cells were incubated妇联活动
overnight in RPMI1640medium supplemented with10%
(v/v)FBS and antimycotic/antibiotic.The following day5×106
cells per time point were placed into2-cm-diameter dishes
and stimulated with1mg/ml UCHT1monoclonal antibody or
6000units/ml IFNα(Roferon-α;Roche).Cells were incubated
山东省考试院
for the time period required at37◦C.Where an inhibitor was
ud,cells were pre-incubated for2h with20nm Lck II inhibitor
(Calbiochem)or15nM JAK inhibitor I(Calbiochem)before
being stimulated with1mg/ml UCHT1or6000units/ml IFNα.
The medium was then aspirated and cells were washed with
1ml of1×Dulbecco’s PBS(Biowhittaker)and centrifuged at
900g for5min.Finally cells were lyd in50–100μl ice-cold
NP-40(Nonidet P40)-containing lysis buffer and centrifuged at
22000g for20min.Protein concentration was then determined
and Western blotting was performed as described below. Western blot analysis车载电热杯
Cells were grown to approx.50%confluency and5×106
cells were then ud per time point.Cells were rum-starved
for2h before being stimulated with either1mg/ml OKT3
monoclonal antibody(eBioscience)or6000units/ml of Roferon-αand incubated at37◦C for the time periods indicated. Cells were then lyd in NP-40-containing lysis buffer[50mM
Hepes,pH7.5,1%NP-40,1mM sodium pervanadate,10mM
sodiumfluoride,10%(v/v)glycerol,50mM NaCl and1mM EDTA]supplemented with1%(v/v)protea inhibitor cocktail
III(Calbiochem).The protein concentration of the whole-cell
extracts obtained was determined through u of Bradford assay
reagents(BioRad Laboratories).For Western blot analysis,50μg
of cell lysates were parated by SDS/PAGE(12%gels).Proteins
were then transferred on to nitrocellulo membranes(Millipore).
Membranes were then incubated with specific antibodies in3%
(w/v)BSA or5%(w/v)dried low-fat milk.β-Actin was ud
as a loading control.Protein bands were visualized by enhanced
chemiluminescent detection(Cell Signaling Technology).Images
were scanned using an Alpha Innotech Densitometer.
Polyclonal antibodies specific for phospho(Tyr1054/Tyr1055)-
Tyk2,phospho(Tyr701)-STAT1,phospho(Tyr705)-STAT3,
phospho(Tyr694)-STAT3,STAT5,phospho(Ser217/Ser221)-MEK
(MAPK/ERK kina)1/2,and phospho(Thr202/Tyr204)-ERK1/2
and ERK1/2were all purchad from Cell Signaling Technology.
Polyclonal antibodies against phospho(Tyr1022/Tyr1023)-JAK1,
phospho(Tyr174)-Vav,Vav(H-211),Slp76(H-300)and pTyr(phos-
photyrosine)were purchad from Santa Cruz Biotechnology. Immunoprecipitation of protein complexes
Cells were grown at a concentration of10×103cells per ml prior
to stimulation,10×106cells were then ud for each time point.
Cells were stimulated either with1mg/ml OKT3or6000units/ml
Roferon-αand incubated at37◦C for the amount of time
indicated before being transferred on to ice and lyd immediately
with1ml of ice-cold lysis buffer as above.Nuclei and
unbroken cells were pelleted through centrifugation at22000g
for20min and the supernatant was decanted into a fresh tube.
The lysate concentration was determined through u of Bradford
Assay reagents(BioRad Laboratories).Antibody(2μg)was then
added to2mg of lysate and placed in continuous rotation at
4◦C overnight.After18h,80μl of hydrated Protein A–agaro
beads(in a1:1slurry in1×phosphate-buffered solution)were
added to each sample and returned to continuous rotation for
a further4h.Cells were then spun for1min at22000g and
washed with ice-cold lysis buffer.This wash step was repeated
four times.The beads were then dried and protein was eluted from
the beads through addition of an equal amount of2×Laemmli
buffer followed by boiling at95◦C for5min.The supernatant was
then resolved via SDS/PAGE(10%gels).The proteins were then
transferred on to a nitrocellulo membrane for2h at250mA,
blocked with3%(w/v)BSA and probed with specific antibodies. Transfection of cells and generation of stable cell lines
Cells(20×106–30×106)were washed with unsupplemented
RPMI1640medium,resuspended in350μl RPMI1640medium
and transferred into an electroporation chamber.Plasmid DNA
(50μg)was added to the cells and mixed gently.The chamber
containing the cells plus DNA was then puld,using a BioRad
gene pulr,at a charging pul of0.27V and a capacitance of960μF.Cells were resuspended in RPMI1640medium supplemented with10%(v/v)FBS.After48h,400μg/ml hygromycin was
added and the cells were maintained,with regular medium
changes,in the prence of antibiotic for2–3weeks in order
to lect for transfected cells.Antibiotic-resistant cells were then
dilution-cloned in96-well plates.Individual clones were lected
for expression of the construct through Western blot analysis
彰化肉圆and on the basis of visualization offluorescence intensity using
fluorescence microscopy.
Lucifera assays
Jurkat cells stably transfected with a construct containing the
gene encodingfirefly lucifera under the control of a trimer of
Interferon-αactivates the TCR complex
431
夏色蜜汗
Figure1Abnce of Vav or Slp76abolishes downstream ERK1/2phosphorylation in respon to IFNαin Jurkat cells
Protein lysates were obtained from(a)Jurkat cells,Vav-deficient J.Vav1cells and J.Vav1cells reconstituted with a Vav-expressing construct,(b)Jurkat cells,Slp76-deficient J14cells and J14cells reconstituted with Slp76–RFP or(c)Slp76-deficient J14cells and J14cells reconstituted with the Slp76-Y3F–RFP mutant.Cells were stimulated with IFNαover the time cour indicated.Western blotting was ud to determine the expression levels of phospho(Thr202/Tyr204)-ERK1/2,ERK1/2and to determine Vav and Slp76expression levels,to ensure the prence or abnce of each protein in the respective cell lines.β-Actin was ud as a loading control.
the NFAT(nuclear factor of activated T-cells)-binding site from the IL(interleukin)-2gene promoter were activated by immobilized anti-CD3monoclonal antibody alone,Roferon-αalone(6000units/ml)or a combination of Roferon-αand anti-CD3antibody as described previously[12].Cells were collected 2–24h after activation and procesd for lucifera assays as described previously[13].Light emission was quantified in a luminometer(Berthold technologies Junior LB9509)and expresd in RLUs(relative light units).
RESULTS
Vav1is required for MAPK activation upon IFNαR stimulation
The stimulation of Jurkat T lymphocytes by IFNαresults in incread activity of the MAPK pathway shown by elevated levels of ERK1/2phosphorylation for up to approx.5min[3].This is abrogated in the abnce of Lck or Zap70.We considered whether two other known proteins of the TCR ESC,namely Vav1and Slp76,are also implicated in an IFNαR-stimulated respon.J.Vav1cells,in which Vav expression is completely suppresd(through somatic cell gene targeting),show multiple signalling defects including the virtual abolition of ERK1/2 phosphorylation in respon to TCR stimulation[14].The cells also exhibited a significant reduction in ERK1/2phosphorylation upon stimulation with IFNα(Figure1a).This loss of ERK phosphorylation is reverd in reconstituted J.Vav1cells,in which the Vav1gene is restored(Figure1a).The elevation of MAPK activity in Jurkat cells and reconstituted J.Vav1cells was maximal at5min,mirroring the time cour obrved previously in IFNα-stimulated cells[3].In resting T-cells Vav1 adopts an inactive conformation whereby the N-terminus forms an inhibitory loop that occludes the catalytic GEF(guanine-nucleotide-exchange factor)domain from accessing its substrates. TCR-stimulated phosphorylation of the key tyrosine residue,
432 C.N.Stevens and others
Tyr174,by upstream kinas,such as Zap70,releas this loop allowing full catalytic activation[15,16].Western blots using an antibody against phospho(Tyr174)-Vav1showed increasing levels of phosphorylation over a30min time cour post-stimulation by IFNα(Figures2a and2b).To demonstrate that this effect was bad upon Zap70kina activity we showed that Vav1was not phosphorylated upon stimulation by IFNαin Zap70-deficient P116cells(Figure2c).Thus our results suggest that the MAPK respon,elicited by IFNα,requires Vav1function in an identical manner to that reported for TCR activation.
Slp76is recruited to a TCR-like ESC on IFNαR stimulation
On stimulation of the TCR,Vav1and Slp76physically associate and co-operate to induce sustained ERK1/2signalling[17–19]. J14cells,derived from parental Jurkat cells that completely lack Slp76expression,elicit diminished ERK1/2phosphorylation in respon to TCR activation[20].Similarly,the IFNαstimulation of J14cells,as with the Vav1-deficient cells above,showed no ERK1/2respon over a30min time cour(Figure1b). Wild-type and J14cells reconstituted with RFP-tagged Slp76 show recovery of MAPK activity(Figure1b).Slp76is implicated in the recruitment o
f Vav1to the TCR ESC prior to phosphorylation[21,22].Figure2(c)shows that in J14cells lacking Slp76,no Vav1phosphorylation was apparent on IFNαstimulation suggesting that Slp76is behaving in a similar way on cytokine stimulation.Furthermore,it is known that as part of the TCR ESC,Slp76is phosphorylated at three key tyrosine residues located at positions112,128and145.All three of the residues are required for optimal downstream ERK activation and NFAT activity[21,22].J14cells reconstituted with Slp76–RFP in which the three tyrosine sites are replaced with phenylalanine (Slp76-Y3F–RFP)failed to rescue ERK1/2activation(Figure1c). The results are consistent with the notion that the function of Slp76in IFNαR signalling is similar to that obrved on TCR stimulation[23–25].
Slp76is phosphorylated in primary CD4+T-cells stimulated
by IFNα
Although Jurkat cells provide an excellent model for studying signal transduction,as they are a leukaemic cell line,they bear veral gene mutations and may not completely reprent signalling in healthy T-cells in vivo.For example,Jurkat cells are known not to express the phosphatas PTEN(phosphata and tensin homologue deleted on chromosome10)and SHIP [SH2(Src homology
2)-domain-containing inositol phosphata], which are involved in phospholipid metabolism(reviewed in [26]).To corroborate our Jurkat cell results peripheral primary CD4+T-cells were isolated from fresh blood donors and cells were stimulated in a similar manner to Jurkat cells. Unstimulated cells,or cells stimulated with IFNαfor5min, were lyd and immunoprecipitated with Slp76.The lysates were then subjected to Western blotting and probed with anti-phosphotyrosine antibodies.Figure2(d)illustrates that Slp76was tyrosine-phosphorylated following5min of IFNαstimulation. As a control,cells were also stimulated through the TCR with the UCHT1antibody for the same period of time.This blot demonstrates for thefirst time that Slp76undergoes IFNα-stimulated phosphorylation both in Jurkat cells and primary CD4+primary T-cells in respon to IFNαstimulation.
An intact TCR is required for IFNα-stimulated MAPK activity
It has been demonstrated previously that Lck,Zap70and CD45 all play a role in IFNαsignalling[3,9,27].In the prent study
we Figure2Activation of both Vav and Slp76by IFNαmirrors the TCR ESC respon in Jurkat and primary T-cells
(a)A Western blot was performed to determine the expression levels of phospho(Tyr174)-Vav and Vav in IFNα-stimulated Jurkat cells over the indicated time cour.β-Actin was ud as a loading control.(b)Phospho(Tyr174)-Vav levels normalized to Vav expression by densitometric analysis.
(c)Western blotting was performed to determine the expression levels of phospho(Tyr174)-Vav and Vav in IFNα-stimulated Jurkat,Zap70-deficient P116cells and Slp76-deficient J14cells over the time periods indicated.β-actin was ud as a loading control.(d)Immunoprecipitation(IP) of Vav and Slp76from primary T-cell lysate.Probing with anti-phosphotyrosine antibody(pY99) reveals that Slp76and Vav are tyrosine-phosphorylated following5min of IFNαstimulation. As a control,the TCR was also stimulated for the same time period and the UCHT1antibody was ud to show the expected protein phosphorylation.
show that,in addition,both Vav1and Slp76are also intimately involved in cytokine-induced signalling and respond in a way that is similar to their respon to TCR stimulation.Thusfive
Interferon-αactivates the TCR complex433
of the proteins known to be recruited to the ESC at the TCR on
antigen prentation are also associated with producing a MAPK
respon on IFNαR stimulation.This suggests that either the
proteins act as independent entities in the two distinct signalling
pathways,or that IFNαR and TCR signalling are inextricably
he ERK1/2activation via IFNαstimulation requires
the recruitment and asmbly of the TCR ESC.To test this we
asked whether the TCR itlf is involved in the IFNα-stimulated
respon.The JRT3-T3.5cell line does not express theβ-subunit
of the TCR and conquently proper asmbly of a complete
functional receptor on the cell surface cannot occur[28].As
predicted,only basal levels of MAPK activity were exhibited
upon stimulation of the cells with OKT3compared with the
parental Jurkat cell line(Figure3a).The MAPK respon
was rescued in PF2.4cells,which are reconstituted with the β-subunit and hence have a functional TCR[29].Stimulation of JRT3-T3.5cells with IFNαfailed to produce the elevated
ERK1/2phosphorylation over the time cour en in wild-type
Jurkat cells(Figure3b).This activity can be restored in the
PF2.4cells.In addition to reduced phosphorylation of ERK1/2,
IFNαstimulation of cells lacking the intact TCR also failed
to effect a MEK1/2respon(Figure3c).As MEK is the
rine/threonine kina that phosphorylates ERK this suggests
that the loss of ERK1/2activity reflects a defect in the pathway
upstream of the kinas.Therefore our results show that class
I interferon signalling appropriates at least part of the TCR
ESC and stimulates a MAPK respon via the usual mechanism
associated with active TCR signalling.
Although our results reveal that the IFNαR appears to employ
the TCR ESC to elicit MAPK activity,this does not result in the
transcriptional respon derived from anti-CD3antibody TCR
深海浩劫stimulation.A lucifera-bad assay was ud to asss whether
IFNαstimulation of Jurkat cells resulted in the transcription
of NFAT-dependent genes,which are characteristic of anti-CD3
antibody TCR activation.Whereas anti-CD3antibody elicited the
expected,and previously reported,transcription of NFAT,this did
not occur in respon to different concentrations of IFNα,over
time(Figure3d,lower panel).The effect of IFNαR stimulation in
combination with anti-CD3antibody was to moderately enhance
NFAT activation at earlier time points and blunt it after6h
(Figure3d,upper panel).This effect on the transient NFAT
activation might mean that IFNαis able to reduce the time required
for anti-CD3antibody to activate transcription.Thus although
interferon stimulation elicits TCR ESC asmbly and MAPK
activity,the clear difference in transcriptional respon suggests
that the signal is subject to downstream modulation/modification.
IFNαR-induced activation of MAPK is independent of JAK/STAT signalling
Stimulation-dependent phosphorylation of JAK,and subquent phosphorylation of the STAT proteins,is esntial for mediating many of the anti-viral respons brought about by stimulation of the IFNαR.To asss whether the IFNα-stimulated signal,which us the TCR ESC,affects the JAK/STAT pathway,cells deleted for the TCR were again ud.IFNαstimulation of JRT3-T3.5cells had no effect on Tyk2activity(Figure4a).This confirms that TCR ESC formation does not affect the normal JAK/STAT signalling from the IFNαR.To exemplify this further Figures4(b)–4(d) show that STAT1,STAT3and STAT5respectively were tyrosine-phosphorylated in respon to IFNαstimulation in wild-type Jurkat and in the JRT3-T3.5cells.Thus one can conclude that the JAK/STAT and MAPK respons derived from IFNαR are entirely
independent.Figure3IFNαR-induced MAPK requires a functional TCR,but,despite this, does not induce up-regulation of the same genes as TCR-induced MAPK signalling
The Jurkat,JRT3-T3.5(TCRβ−/−)and PF2.4(stably reconstituted with the TCRβchain)cell lines were stimulated with(a)OKT3,which activates the TCR or(b)IFNαfor the indicated time periods.Western blotting was then performed to determine the expression levels of phospho(Thr202/Tyr204)-ERK1/2and ERK1/2.β-actin was ud as a loading control.(c)Western blotting was ud to determine the expression levels of phospho(Ser217/Ser221)-MEK1/2and ERK1/2in Jurkat,JRT3-T3.5and PF2.4cells that were stimulated with IFNαfor the indicated time periods.β-Actin was ud as a loading control.(d)Relative lucifera activity from an NFAT–lucifera reporter in Jurkat cells activated for the indicated times by immobilized anti-CD3 monoclonal antibody,either alone or in combination with IFNα.The results are expresd as RLUs(upper panel)or as relative lucifera activity in treated compared with untreated samples (fold activation;lower panel).
