of November 21, 2018.This information is current as

T Cells+Distribution of CD40 Ligand mRNA in CD4

Critical for the Turnover and Subcellular Polypyrimidine Tract-Binding Protein Is

Valentina Marcelli and Lori R. CoveyCastro-Faix, Anibal Valentin-Acevedo, Karnail Singh, Rodrigo Matus-Nicodemos, Stefano Vavassori, Moraima

http://www.jimmunol.org/content/186/4/2164doi: 10.4049/jimmunol.1003236January 2011;

2011; 186:2164-2171; Prepublished online 17J Immunol 

Referenceshttp://www.jimmunol.org/content/186/4/2164.full#ref-list-1

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The Journal of Immunology

Polypyrimidine Tract-Binding Protein Is Critical for theTurnover and Subcellular Distribution of CD40 LigandmRNA in CD4+ T Cells

Rodrigo Matus-Nicodemos, Stefano Vavassori,1 Moraima Castro-Faix, Anibal Valentin-

Acevedo, Karnail Singh,2 Valentina Marcelli,3 and Lori R. Covey

CD40L (CD154) is regulated at the posttranscriptional level by an activation-induced process that results in a highly stable tran-

script at extended times of T cell activation. Transcript stability is mediated by polypyrimidine tract-binding protein (PTB)-

containing complexes (complex I and II) that bind to three adjacent CU-rich sequences within the 39 untranslated region. To

assess the role of PTB in the expression and distribution of CD40L mRNA, PTB was targeted using short hairpin RNA in both

primary T cells and a T cell line that recapitulates the stability phase of regulated CD40L mRNA decay. PTB knockdown resulted

in a marked decrease in the mRNA stability that resulted in lowered CD40L surface expression. PTB was also critical for

appropriate distribution of CD40L mRNA between the nucleus and cytoplasm and in the cytoplasm between the cytosol and

the translating polysomes. The activation-induced formation of PTB-specific ribonucleoprotein complexes was observed only with

cytoplasmic and not nuclear PTB indicating functional differences in the protein defined by cellular localization. Finally, we

observed that cytoplasmic and nuclear PTB isoforms were differentially modified relative to each other and that the changes in

cytoplasmic PTB were consistent with activation-induced phosphorylation. Together this work suggests that differentially mod-

ified PTB regulates CD40L expression at multiple steps by 1) retaining CD40L mRNA in the nucleus, 2) directly regulating mRNA

stability at late times of activation, and 3) forming a ribonuclear complex that preferentially associates with translating ribosomes

thus leading to an enhanced level of CD40L protein. The Journal of Immunology, 2011, 186: 2164–2171.

The interaction of CD40L (CD154), expressed primarilyon CD4+ T cells, with CD40 on APCs is a critical eventresulting in the activation of select pathways in both innate

and adaptive immunity (1–3). Early work on CD40–CD40L inter-actions underscored the critical role of this receptor–ligand pair inhumoral immunity through the initiation of signaling pathwaysrequired for B cell proliferation, Ab class switching, somatichypermutation, memory cell development, enhanced expression ofcostimulatory molecules, and survival (4–7). Subsequent workrevealed the importance of CD40L in the priming of CD4+ T cellsand enhanced innate responses through the direct interaction withCD40 expressed on macrophage and dendritic cells. Recent workhas also supported a role for CD40 signaling in augmenting TLR

responses leading to the enhanced secretion of multiple cytokinesand chemokines by a variety of cell types (1, 7).The near constitutive nature of CD40 expression on APCs

necessitates that regulation of signaling pathways occurs prima-rily through modulated CD40L expression. Although CD40Ltranscription is rapidly induced after T cell activation, post-transcriptional processes also have a critical role in regulatingCD40L gene expression throughout T cell activation (reviewed inRef. 8). Because lymphocyte activation is characterized by tran-sitions between different checkpoints, diversification at the level ofmRNA stability provides a unique mechanism to regulate the ex-pression of a number of responding genes (8, 9). The stability of theCD40L message in both human and mouse CD4+ T cells signifi-cantly increases with activation either through prolonged signalingthrough the TCR or via exposure to PMA/ion (10, 11). Transcriptstability is mediated by two complexes (complex I and II) that bindto cis-acting elements within the 39 untranslated region (39UTR)(8, 12, 13). The major binding protein in complex I and II is thepolypyrimidine tract-binding protein (PTB) or hnRNP I, whichbelongs to a family of ribonuclear proteins (RNPs) displaying di-verse roles in multiple aspects of RNA metabolism (14). PTBshuttles between the nucleus and cytoplasm and is important forregulating alternative splicing, IRES-mediated translation initia-tion, and enhanced stability of a number of transcripts (15). Cel-lular localization and/or interactions with specific factors appear tobe critical for PTB specificity (15, 16), which is consistent with itsrole in CD40L stabilization where two additional RNA bindingproteins, nucleolin and hnRNP L, have been identified as addi-tional components of complex I/II (17–19). Although the relation-ship between PTB and CD40L mRNA stability has been firmlyestablished, how PTB-mediated regulated decay influences theexpression of CD40L protein through T cell activation is less clear.

Department of Cell Biology and Neuroscience, Rutgers University, Piscataway, NJ08854

1Current address: Experimental Immunology, Department of Biomedicine, Univer-sity Hospital Basel, Basel, Switzerland.

2Current address: Department of Surgery, Emory Transplant Center, Emory Univer-sity School of Medicine, Atlanta, GA.

3Current address: Novartis Pharma Schweizerhalle AG, Schweizerhalle, Switzerland.

Received for publication September 29, 2010. Accepted for publication December 8,2010.

This work was supported by a National Institutes of Health grant (PO1 AI-57596)and an American Heart grant-in-aid (to L.R.C.).

Address correspondence and reprint requests to Dr. Lori R. Covey, Department ofCell Biology and Neuroscience, Rutgers University, 604 Allison Road, Piscataway,NJ 08854. E-mail address: covey@biology.rutgers.edu

Abbreviations used in this article: DRB, 5,6-dichlorobenzimidizole 1-b-D-ribofura-noside; IEF, isoelectric focusing; MFI, mean fluorescence intensity; PTB, polypyr-imidine tract-binding protein; RNP, ribonucleoprotein; shRNA, short hairpin RNA;39UTR, 39 untranslated region.

Copyright� 2011 by TheAmericanAssociation of Immunologists, Inc. 0022-1767/11/$16.00

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In this study, the effect of downregulating PTB on CD40LmRNA expression and distribution was analyzed both in an acti-vated T cell line (Jurkat/D1.1) and in primary CD4+ T cells. Wefound that PTB-mediated regulation of CD40L mRNA stabilityhad a considerable effect on levels of surface CD40L in bothprimary CD4+ T cells and cell lines. In addition, PTB influencedthe cellular distribution of CD40L transcript between the nucleusand cytoplasm as well as within the polysomal cytoplasmic frac-tion. Finally, PTB was differentially modified as a function of itsdistribution within the nucleus and cytoplasm and in responseto stimulation, suggesting a link between T cell activation, PTBmodification, and CD40L expression.

Materials and MethodsAbs and cell lines

The anti–human-PTB mAb (ATCC CRL-2501) was purified as previouslydescribed (20). Anti–b-actin (C-11), anti-poly(ADP-ribose) polymerase,and anti-nucleolin (C23 Ab 4E2) mAbs were purchased from Santa CruzBiotechnology. Anti-S6 ribosomal protein (54D2) mAb was purchasedfrom Cell Signaling. Anti-DDK (Flag Ab 4C5) mAb was purchased fromOrigene. Anti–human-CD154 (21–28) was purchased from Biolegend. TheJurkat/D1.1 cell line was obtained from Dr. S. Lederman (ColumbiaUniversity, New York, NY) and cultured as previously described (12). TheFlag-PTB-Jurkat/D1.1 line was generated by the stable expression of thevector pCDNA.3 expressing PTB-1 downstream of the Flag epitope.

Short hairpin RNA knockdown and analysis of PTB

The pLV-CTRL and pLV-PTB together with virus production techniqueshave been previously described (29). Briefly, the following pairs of primerswere cloned into the HindIII and EcoRI sites of the pSilencer2.1-U6 hygro(Ambion): U6-shPTB: 59-GAT CCA ACT TCC ATC ATT CCA GAGAAC TTG CTT CTT CTC TGG AAT GAT GGA AGT TTT TTT TGGAAA-39 (forward) and 59-AGC TTT TCC AAA AAA AAC TTC CATCAT TCC AGA GAA GAA GCA AGT TCT CTG GAA TGATGG AAGTTG-39 (reverse); and U6-shCTRL: 59-GAT CCA ATC AGA CGT GGACCA GAA GAG AGA TCT TCT GGT CCA CGT CTG ATT TTT TTTGGA AA-39 (forward) and 59-AGC TT TTC CAA AAA AAATCA GACGTG GAC CAG AAG ATC TCT CTT CTG GTC CAC GTC TGATTG-39(reverse). The sequence containing the U6 promoter and short hairpinRNA (shRNA) was PCR amplified using the primer set pLVTHM: 59-CCATCG ATG GAG CTT TTC CAA AAA AAA CTT-39 (forward) and 59-CAG AAA GGT GAC CCC TTA AGC TTC TAG AAG-39 (reverse).These PCR products were cloned into the pLVTHM vector (Addgeneplasmid 12247).

Viral particles were packaged by transfection of 293T cells withpLVTHM-U6-shPTB and pLVTHM-U6-CTRL together with the viruspackaging plasmids psPAX2 (Addgene plasmid 12260) and pCI-VSVG(Addgene plasmid 1733), both obtained from D. Trono (Ecole Poly-technique Federale de Lausanne, Lausanne, Switzerland), using FuGene. Atotal of 5 3 106 Jurkat/D1.1 cells were incubated with lentivirus for 24 hfollowed by replacement of the conditioned medium with RPMI-complete(RPMI 1640 medium supplemented with 10% heat-inactivated FBS, 50 U/ml penicillin, 50 mg/ml streptomycin, and 1 mM L-glutamine at 37˚C), and48 h postinfection, cells were checked for GFP expression by flow cy-tometry.

Infection of primary human CD4+ T cells

PBMCs were isolated from freshly drawn blood and CD4+ T cells isolatedusing Miltenyi beads. Concentrated control (pLV-CTRL) and PTB (pLV-PTB) lentiviruses were cocultured with 1 3 106 CD4+ T cells with 0.1 mg/ml PHA (Calbiochem) and 100 U/ml rIL-2 in RPMI-complete. Cells wereexpanded by adding 100 U/ml rIL-2 at 3 d and 50 U/ml rIL-2 every otherday following. At 10 d postactivation, the cells were placed into RPMI-complete medium minus rIL-2, and 3 to 4 d later, cells were activated for 2h with 1 ng/ml PMA and 1 mg/ml ionomycin. Cells were stained with anti-CD40L mAb and analyzed by flow cytometry. Intracellular surface mo-bilization assay was performed as previously described (30). Briefly, CD4+

T cells were infected and prepared as above. Thirty minutes prior to ac-tivation, cells were treated with 10 mg/ml cycloheximide. Activation waswith 1 mg/ml ionomycin, 10 ng/ml PMAwith 10 mg/ml cycloheximide for30 min.

Steady-state expression and decay analyses of the CD40LmRNA

RNA stability was measured by incubating 5 3 106 Jurkat/D1.1 cells with50 mg/ml 5,6-dichlorobenzimidizole 1-b-D-ribofuranoside (DRB) andaliquots collected every 30 min for 2 h. Approximately 1 mg of RNA wasreverse transcribed and real-time PCR performed on an ABI HT7000 PCRCycler. For quantification of CD40L and the b-actin transcripts, the fol-lowing primer sets were used: CD40L: 59-TTC CAC CCT GTC CCTATCTC-39 (forward) and 59-CTG GAA ACA ATG GAG ACT GC-39 (reverse);and b-actin: 59-GCA TCC TCA CCC TGA AGT A-39 (forward) and 59-TGT GGT GCC AGA TTT TGT CC-39 (reverse).

Cell fractionation and protein immunoblots

Total, nuclear, and cytoplasmic extracts were prepared as previously de-scribed (29) and analyzed using immunoblotting with anti-PTB Abs ata dilution of 1:3000 and anti-Flag (DDK) Abs, anti-S6 ribosomal proteinAbs, anti-actin Abs, and anti-nucleolin Abs at a dilution of 1:1000. HRP-conjugated secondary Abs were used for detection by ECL. To obtain thenon-polysomal and polysomal proteins and RNA, cell fractionation wasperformed essentially as previously described (31).

RNA electromobility shift assay and RNA immunoprecipitation

Isolation of CD4+ T cells, CD3 activation of T cells, and preparation ofcytoplasmic fractions and RNA electromobility shift assays were carriedout as previously described (18). To analyze the CD40L mRNA bound byPTB, 5 3 106 Jurkat/D1.1 cell equivalents of cytoplasmic and nuclearextracts were incubated overnight at 4˚C with agarose-A/G beads bound toanti-PTB Abs in NT2 buffer (50 mM Tris [pH 7.4], 150 mM NaCl, 1 mMMgCl2, and 0.05% Nonidet P-40). The beads were washed six times withNT2 buffer and resuspended in 100 ml NT2 buffer with 0.1% SDS and 30mg proteinase K, followed by incubation at 55˚C for 3 h. RNA wasextracted from the supernatant, reverse transcribed, and the cDNA am-plified by PCR.

Two-dimensional gel electrophoresis and phosphatase assay

Cytoplasmic (20 mg) and nuclear (5 mg) extracts from differentially acti-vated CD4+ T cells were dialyzed against DeStreak Rehydration Solution(GE Healthcare) for 2–4 h. Isoelectric focusing (IEF) was carried out onsamples using 7 cm Immobiline DryStrip Gels with immobilized pHgradient range 6–11. IEF was performed in an IPGphor according to theprotocols provided. After IEF, strips were equilibrated in 50 mM Tris-HCl,pH 8.8, 6 M urea, 2% SDS, 30% glycerol with 1% DTT for 15 min,followed by 15 min in the same buffer without DTT but with 4% iodoa-cetamide. Equilibrated strips were run through 4–20% linear gradientpolyacrylamide gels, transferred to PVDF membranes, and immunoblottedwith anti-PTB Abs. Twenty micrograms from Jurkat/D1.1 cytoplasmicextracts were mock-treated or treated with 800 U l phosphatase in 20 mlfinal volume of 50 mM HEPES (7.5), 100 mM NaCl, 2 mM DTT, 0.01%Brij 35, 2 mMMnCl2, and 13 PIC for 4 h. The samples were subsequentlydialyzed and focused as described.

ResultsPTB is critical for maintaining steady-state levels of CD40LmRNA

To determine the effect of attenuated PTB expression on surfaceCD40L levels, Jurkat/D1.1 cells expressing a highly stable CD40Ltranscript were infected with a modified pLVTHM-U6 lentiviralvector expressing GFP and either an shRNA against PTB (pLV-PTB) or a scrambled-sequence control (pLV-CTRL). Infectionefficiency was consistently between 70 and 100% (GFP+) usingboth viruses (data not shown). To control for off-target effects,a Jurkat/D1.1 subclone was generated that stably expressedFLAG-tagged PTB-1 from a cDNA lacking the small interferingRNA target sequence within the 39UTR. This subclone was sim-ilarly infected with pLV-PTB and pLV-CTRL viruses and analyzedin parallel with the infected, noncomplemented populations. Twodays after infection, Jurkat/D1.1 and Flag-PTB-Jurkat/D1.1 cy-toplasmic and nuclear fractions were prepared and analyzed forPTB expression using immunoblotting. Cells expressing shPTBhad decreased PTB in both fractions with a particularly sharp drop

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in the cytoplasmic level (20% of shCTRL), which may reflecta preferential distribution of PTB in the nucleus prior to its re-distribution into the cytoplasm. In contrast, PTB expression inFlag-PTB-shPTB Jurkat cells was 70 and 60% of that observedfor the Flag-PTB-shCTRL cytoplasmic and nuclear fractions,respectively (Fig. 1A). Overall, the steady-state levels of CD40LmRNA were reduced more than 50% in cells expressing shPTB,whereas cells complemented with Flag-tagged PTB had higher(Flag-PTB-shCTRL) or equal (Flag-PTB-shPTB) levels of tran-script compared with those of the parent cell line (Fig. 1B).To confirm that the change in PTB levels affected the decay

rate of CD40L mRNA, infected Jurkat/D1.1 cells were treated withthe transcriptional inhibitor DRB and decay measured over a 2-h

FIGURE 1. PTB directly regulates the stability of the CD40L transcript.A,

Extracts were prepared from23 104 cell equivalents of Jurkat/D1.1 (lanes 1–

4) or Flag-PTB-Jurkat/D1.1 (lanes 5–8) cells infected with pLV-CTRL (lanes

1, 2, 5, and 6) or pLV-PTB (lanes 3, 4, 7, and 8) and assayed for PTB ex-

pression by immunoblotting with anti-PTB mAb. Membranes were stripped

and rehybridized with anti-Flag and anti-nucleolin Abs. Numbers represent

the fraction of PTB in the indicated lanes relative to the appropriate CTRL

lane. B, Steady-state levels of CD40L mRNAwere determined by quantita-

tive PCR for Jurkat/D1.1 and Flag-PTB-Jurkat/D1.1 cells infected with either

pLV-CTRL or pLV-PTB. Results were normalized to b-actin levels, and the

average plus the SEM of three independent experiments are presented. C, To

determine the effect of PTB on the decay rate of CD40L mRNA, quantitative

PCR was performed on mRNA isolated from infected Jurkat/D1.1 or Flag-

PTB-Jurkat/D1.1 cells exposed to the transcriptional inhibitor DRB (50 mg/

ml) for 0, 30, 60, and 120 min. Results were normalized to b-actin levels

determined at the same time period. Findings represent the average plus the

SEM of three independent experiments.

FIGURE 2. PTB regulates CD40L surface expression in both Jurkat and

primary T cells. A, CD40L expression (x-axis) of Jurkat/D1.1 (left panel) or

Flag-PTB- Jurkat/D1.1 (right panel) cells infected with either pLV-CTRL

(gray line) or pLV-PTB (black line). The MFIs are given for the individual

populations, and results are representative of five independent experiments. B,

Expression of GFP (x-axis) and CD40L (y-axis) was monitored in primary

CD4+ T cells infected with pLV-CTRL (top graphs) or pLV-PTB (bottom

graphs). Cells were either nonactivated (left graphs) or activated (right

graphs) with PMA/ion for 2 h prior to analysis. Within a specific graph, un-

infected cells are present in the left quadrants and infected cells are in the right

quadrants. Numbers in each quadrant represent MFI for representative pop-

ulations.C, CD4+ T cells infectedwith either pLV-CTRL (top graphs) or pLV-

PTB (bottom graphs) were treated with 10 mg/ml cycloheximide for 30 min

prior to analysis for CD40L expression without activation (left graphs) or after

activation with PMA/ion for 30 min (right graphs). Cells in left quadrants are

uninfected, and those in the right quadrants are infected. Numbers in each

quadrant represent MFI for representative populations.

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time course (Fig. 1C). In cells infected with the pLV-PTB virus,the CD40L mRNA decayed with a t1/2 of ∼45 min, which repre-sented a 50% decrease in stability compared with that of cellsinfected with control virus. Assessment of Flag-tagged PTB-expressing cells harboring either shPTB or shCTRL revealeddegradation profiles very similar to that seen with cells expressingthe control shRNA confirming that the Flag-tagged PTB was com-plementing the decreased stability seen with shPTB.

CD40L surface expression is regulated by PTB

We next analyzed the effect of PTB on CD40L protein expressionin the different Jurkat/D1.1 populations by comparing the meanfluorescence intensity (MFI) 2 d after infection with virus. Jurkat/D1.1 cells expressing shPTB were found to have ∼65% lessCD40L on the surface than that of control cells (MFI = 314.5versus 104.5; Fig. 2A, left panel). Analysis of the Flag-PTB-Jurkatpopulations showed that the clonal Flag-PTB-Jurkat/D1.1 cellsexpressed an overall lower level of surface CD40L compared withthat of the parent Jurkat/D1.1 cells (Fig. 2A, right panel). How-ever, the difference in MFI between the shCTRL and shPTB wasstill evident but greatly reduced, indicating that PTB was influ-encing the surface expression of CD40L (Fig. 2A, right panel).Because Jurkat/D1.1 cells have an activated phenotype and

constitutively express PTB-containing stability complexes, wesought to confirm our findings using primary CD4+ T cells in whichthe effect of PTB downregulation could be assessed in both rest-ing and activated cells. To this end, primary CD4+ T cells wereinfected with either virus and surface expression monitored afterPMA/ion activation, conditions that promote a highly stableCD40L transcript (10, 21). Examination of CD40L expressionwithin the whole population allowed for PTB function to be an-alyzed in infected (GFP+) and uninfected (GFP2) cells underidentical conditions of activation. Specifically, the nonactivatedand activated T cells infected with pLV-CTRL (Fig. 2B, uppergraphs) showed similar levels of CD40L expression betweeninfected and noninfected cells (Fig. 2B, right versus left quad-rants). In contrast, cells infected with pLV-PTB (Fig. 2B, lowergraphs) expressed reduced amounts of CD40L in both the non-activated and activated populations (Fig. 2B, upper right quad-rants) compared with those of the noninfected populations (Fig.2B, upper left quadrants). Over several experiments, we observed

an approximate 9% decrease in expression in nonactivated/shPTB-infected cells and a 25% decrease in expression in activated/shPTB-infected cells compared with that of uninfected orshCTRL-infected cells (Table I).Finally, we asked whether PTB influenced the expression of

preformed CD40L in CD4+ T cells by pretreating cells with cyclo-heximide and measuring the upregulation of CD40L on the surfaceafter activation. Again, a large decrease in the MFI of CD40L ex-pression was observed in T cells infected with pLV-PTB both in theresting and activated states (Fig. 2C, Table I). Whereas these datasupport a role for PTB stabilization of CD40L mRNA during T cellactivation, they also clearly indicate that PTB also influencesCD40L expression through an activation-independent mechanism.

The distribution of CD40L mRNA between the nuclear andcytoplasmic fractions and within the polysome non-polysomefraction is PTB dependent

To identify an additional role for PTB in CD40L expression, weused our shRNA expression system to analyze the compartmenta-lization of CD40L mRNA under targeted PTB expression.Nuclear and cytoplasmic extracts were prepared from virus-infectedJurkat/D1.1 cells or Flag-PTB-Jurkat/D1.1 cells and cytoplasmicextracts further fractionated into non-polysomal (S130 and freeribosomes) and polysomal fractions. As shown in Fig. 3A, in cellsexpressing shCTRL, ∼60% of the CD40L transcript was foundin the nucleus and 40% associated with the cytoplasm. Incontrast, downregulating PTB resulted in a reversal in CD40LmRNA distribution with 45 and 55% of the transcript found inthe nuclear and cytoplasmic fractions, respectively. Evaluation ofthe Flag-tagged Jurkat cells revealed a pattern of CD40L mRNAclose to that seen with shCTRL confirming that PTB plays a rolein appropriately partitioning the CD40L transcript between thenucleus and cytoplasm.Further analysis of the distribution of CD40L transcripts in the

cytoplasm revealed that the vast majority associated with trans-lating polysomes; however, under conditions of reduced PTB, therewas a 20% increase in association with the non-polysomal fraction(Fig. 3B). This skewing was observed in the presence or absenceof cycloheximide but was significantly more pronounced withcycloheximide treatment (data not shown). Infection of Flag-PTB-Jurkat/D1.1 cells revealed no difference in the distribution of

Table I. PTB is critical for CD40L expression in both resting and activated CD4+ T cells

Virus

MFI (CD40L) Percentage Change inMFI (Uninfected

to Infected in SamePopulation)aUninfected Infected

UntreatedshCTRL

Nonactivated 33.9 6 0.10 35.32 6 1.2 4.2Activated 245.7 6 40.3 260.6 6 29.1 6.1

shPTBNonactivated 43.1 6 4.3 39.33 6 3.06 28.7Activated 271.3 6 24.9 204.5 6 17.1 224.6

Treated with CycloheximideshCTRL

Nonactivated 38.95 6 0.15 40.0 6 0.22 +2.7Activated 46.4 6 0.4 52.1 6 0.7 +12.3

shPTBNonactivated 38.3 6 0.15 36.1 6 1.3 25.7Activated 46.9 6 1.65 38.4 6 1.3 218.1

aPrimary CD4+ T cells were infected with either pLVTHM-shCTRL or pLVTHM-shPTB virus in the presence of IL-2 andPHA. Ten days later, cells were either untreated (top rows) or treated with cycloheximide for 30 min (bottom rows). Cells werethen left unactivated for 2 h or activated with PMA/ion for 2 h (top rows) or 30 min (bottom rows) and analyzed for GFP(infected) and CD40L (activated) expression. The MFIs represent the average of three (top rows) and two (bottom rows)independent experiments, and the numbers in the right column represent the percentage change in the average MFI betweenthe uninfected and infected subpopulations in the same culture.

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CD40L between the non-polysomal and polysomal fractions incells infected with either pLV-CTRL or pLV-PTB (Fig. 3B).To extend these findings, we analyzed the expression of PTB

protein in the nuclear, cytoplasmic, and polysomal fractions of

cells expressing shPTB and found decreased levels in all three frac-tions (Fig. 3C, lanes 4–6). Because nucleolin is a second com-ponent of complex I, we also examined its distribution underconditions where PTB levels were reduced. Surprisingly, muchless nucleolin was associated with the polysomes under conditionsof targeted PTB (Fig. 3C, compare lanes 3 and 6), and this de-crease was not observed when PTB was complemented by Flag-PTB (Fig. 3C, compare lanes 9 and 12). This finding strongly sug-gests that the association of nucleolin with the translating poly-somes is dependent, in part, on PTB expression.

Complex I binding is distinct in cytoplasmic and nuclearfractions from resting and activated CD4+ T cells

The presence of substantial levels of PTB in both the nucleus andcytoplasm compelled us to ask whether PTB from both fractionscomplexed to CD40L mRNA. Binding experiments were carriedout with a probe spanning the CD40L stability element (Xba-Hae)and hybridized with nuclear and cytoplasmic extracts from Jurkat/D1.1 cells and differentially activated CD4+ T cells. We found thatstrong complex I binding was observed in both fractions fromJurkat/D1.1 cells (Fig. 4A, lanes 3–5). Similarly, a high level ofbinding activity was observed in the nuclear fractions from restingand activated T cells from all time periods (Fig. 4A, lanes 6–9). Incontrast and as previously observed, only primary T cells stimu-lated for 48 h, and not resting or early activated cells, containeda measurable level of complex binding (Fig. 4A, lanes 10–13). Thespecific requirement for PTB in complex I binding was demon-strated by inhibiting complex I formation with the addition of anti-PTB Abs to the binding reaction (lane 15).To further confirm that both nuclear and cytoplasmic PTB binds

CD40L mRNA and to quantify the binding relative to levels ofPTB, RNA immunoprecipitation was carried out with Jurkat/D1.1nuclear and cytoplasmic extracts. After incubation with anti-PTBmAb cross-linked agarose beads, one half of the eluate was used inWestern blotting to analyze PTB binding, and the other half wasused to isolate RNA for RT-PCR. As shown in Fig. 4B, the patternof PTB was similar between the total and immunoprecipitatedfractions. However, the majority of CD40L mRNA binding ac-tivity was associated with the cytoplasmic fraction as evidencedby the amount released from the complex. This was unexpectedbecause the levels of both PTB and CD40L mRNA were signifi-cantly higher on a per cell basis in the nuclear fraction. Togetherthese results confirm that the binding of nuclear PTB to CD40LmRNA occurs in the absence of activation and is present in CD4+

T cells at all times after activation. However, based on our in vivobinding studies, PTB–CD40L binding activity is highest in thecytoplasmic fraction. Thus, the complexing of CD40L mRNAwith PTB in CD4+ T cells is influenced by both the subcellularlocalization of complex components and by signals that mediateT cell activation.

Cytoplasmic PTB is modified in response to activation

The lack of complex I binding activity in the cytoplasmic fractionfrom resting and early activated CD4+ T cells could be explainedby either the lack of PTB in the cytoplasm or the inability ofcytoplasmic PTB to bind transcript due to the absence of specificmodifications. To distinguish between these two possibilities,cytoplasmic and nuclear extracts were prepared from resting andCD3-activated CD4+ T cells and analyzed by immunoblotting forPTB expression. As predicted from our binding studies, PTB waspresent in all nuclear fractions over the activation time course(Fig. 5A, lanes 6–8). Surprisingly, PTB was also readily presentin the cytoplasmic fractions of unstimulated, 2 h-, and 48 h-stimulated cells (Fig. 5A, lanes 2–4). This finding suggested that

FIGURE 3. PTB is actively involved in the distribution of CD40LmRNA

between the nucleus and cytoplasm. A, Levels of CD40L mRNA in the

cytoplasm (gray bars) and nucleus (black bars) of pLV-CTRL and pLV-PTB

infected Jurkat/D1.1 and Flag-PTB-Jurkat/D1.1 cells as determined by

quantitative PCR. Results represent the average and SEM of three in-

dependent experiments. B, Quantitative PCR analysis of CD40L mRNA

from the non-polysomal (white bars) and polysomal (black bars) fractions of

pLV-CTRL and pLV-PTB infected Jurkat/D1.1 and Flag-PTB-Jurkat/D1.1

cells. C, Western blot analysis using cytoplasmic (23 103 cell equivalents),

nuclear (23 103 cell equivalents), and polysomal (13 104 cell equivalents)

extracts of Jurkat/D1.1 (lanes 1–6) and Flag-PTB-Jurkat/D1.1 (lanes 7–12)

cells infected with pVL-PTB or pVL-CTRLwere analyzed for expression of

PTB and nucleolin in the different cellular fractions by Western blotting.

Membranes were reprobed with Abs against b-actin and the cytoplasmic

ribosomal protein S6 to validate the efficient separation of nuclear, cyto-

plasmic, and polysomal fractions. The membrane was further hybridized

with anti-Flag Abs to confirm expression of the Flag-PTB in the different

fractions. Boxed lanes show the effect of PTB downregulation on the dif-

ferent proteins, in particular PTB and nucleolin.

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the lack of PTB–CD40L mRNA complexing in resting and early-activated T cells was not due to an absence of cytoplasmic PTBbut to a potential modification in PTB that favored complex for-mation.To examine if PTB was differentially modified in response to

activation, nuclear and cytoplasmic extracts were prepared fromCD4+ T cells stimulated for different time periods and separatedusing two-dimensional gel electrophoresis. After Western blottingwith anti-PTB Abs, we found that the pattern of nuclear and cy-toplasmic PTB isoforms was clearly different (Fig. 5B). Specifi-cally, in resting T cells, there were several cytoplasmic isoformsthat were spread over a wide pH range and the major bands shiftedto the negative pole upon activation. In contrast, the PTB isoformsin the nucleus were more closely aligned, were shifted more to-

ward the positive pole, and did not change position over the timecourse of activation.To test whether phosphorylation was contributing to the different

cytoplasmic isoforms, extract from Jurkat/D1.1 cells was untreatedor treated with phosphatase prior to separation and analysis by two-dimensional gel electrophoresis. As shown in Fig. 5C, we observeda distinct shift in the location of the PTB isoforms toward thenegative pole after phosphatase treatment indicating that phos-phorylation accounts for some if not all the isoform modificationsin this specific cytoplasmic fraction. Together these findings pro-vide compelling evidence that phosphorylation of cytoplasmicPTB is linked to cellular activation and that the nuclear and cy-toplasmic populations are differentially modified suggesting theyrepresent distinct functional subsets [see our model for PTB-me-diated regulation of CD40L expression (Fig. 6)].

DiscussionThe synergy of transcriptional and posttranscriptional processes isa hallmark of dynamically regulated immune-specific genes thatensures the rapid adjustment of critical inflammatory moleculesin response to pathogenic challenges (reviewed in Refs. 22–24).

FIGURE 5. PTB from the cytoplasm and nucleus is differentially

modified and mediated by activation. A, Cytoplasmic (lanes 1–4) and

nuclear (lanes 5–8) extracts were prepared from Jurkat/D1.1 cells (lanes

1 and 5) and CD4+ T cells that were unstimulated (lanes 2 and 6) or

stimulated with anti-CD3 mAb for 2 h (lanes 3 and 7) or 48 h (lanes 4

and 8). PTB expression was assessed by SDS-PAGE and immunoblot-

ting with anti-PTB Abs. Membranes were stripped and rehybridized

with Abs against S6 and poly(ADP-ribose) polymerase (lower two

panels) to verify the separation of cytoplasmic and nuclear fractions,

respectively. B, Two-dimensional gel electrophoresis was carried out

across a pH gradient of 6–11 using cytoplasmic (left panels) and nuclear

(right panels) extracts from CD4+ T cells activated for 0, 2, 24, and 48 h

with anti-CD3 mAb. Black arrows indicate the major isoforms in each

fraction. C, Cytoplasmic extract from Jurkat/D1.1 cells was either left

untreated or treated with 800 U l phosphatase for 4 h prior to separation

by two-dimensional gel electrophoresis across a pH gradient of 6–11.

PTB was visualized by immunoblotting.

FIGURE 4. CD40L mRNA stability complexes are constitutively pres-

ent in the nucleus and only form in the cytoplasm with prolonged CD4+

T cell activation. A, Binding reactions were carried out with the full-length

CD40L 39UTR stability element (nucleotides 1300–1609) that was uni-

formly labeled with [32P]rUTP and either total extract (lane 3), nuclear

(lanes 5–9), or cytoplasmic (lanes 4 and 10–13) extracts from Jurkat/D1.1

cells (lanes 3–5), resting CD4+ T cells (lanes 6 and 10), and activated

CD4+ T cells for the indicated times with anti-CD3 plate-bound Abs (lanes

7–9 and 11–13). Lane 1, Probe alone; lane 2, probe with RNase alone.

CD4+ T cell extract stimulated for 48 h with anti-CD3 mAb (lanes 14–16)

was hybridized with probe alone (lane 14) or with probe and anti-PTB

mAb (lane 15) or isotype control Ab (lane 16) added to the reaction prior

to hybridization to determine presence of PTB in complex. Arrows in-

dicate the locations of complexes I and II. B, Jurkat/D1.1 nuclear and

cytoplasmic extracts from 53 106 cell equivalents were separated by SDS-

PAGE (top panel) or immunoprecipitated with anti-PTB Abs and separated

by SDS-PAGE (second panel) and immunoblotted with anti-PTB Abs.

RNA was purified from both total and immunoprecipitated samples and

used in RT-PCR (lower two panels). The relative amount of protein and

level of RNA binding were quantified and are indicated under the corre-

sponding samples.

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Accordingly, appropriate steady-state levels of CD40L are definedby a still unique posttranscriptional control mechanism involvingribonuclear complexing at defined regions within the 39UTR atdiscrete times of T cell activation (8). Previous work identifieda role for PTB in CD40L mRNA stability, and our current workextends these findings by demonstrating that PTB levels directlyimpact the surface expression of the protein. Importantly, exami-nation of how CD40L mRNA is apportioned within the cellrevealed that optimal PTB levels are required for maintaining theappropriate distribution of both the cytoplasmic and the polysomalsubpopulations. Therefore, these findings highlight a role for PTBin CD40L expression beyond its role in regulating transcript sta-bility.A large body of work has focused on understanding ARE-

regulated mRNA decay, a mechanism used by a vast number ofgenes involved in the inflammatory response that results in therapid turnover of ARE-containing transcripts (i.e., TNF-a, GM-CSF, IL-2, and IL-10) (25–28). Conversely, a program of post-transcriptional regulation that further extends the t1/2 of relativelylong-lived transcripts represents an energy-efficient mechanism tosustain expression of a particular protein (32). The PTB-dependentphase of CD40L expression falls into this second type of regula-tion and coincides with processes that occur at more advancedstages of the humoral response, such as germinal center formationand the generation of differentiated plasma cells and memoryB cells (33). Similar to what has been shown with BCR signaling(34–38), duration and quality of CD40 signals appear to directlyaffect differentiation outcomes of Ag-selected B cells as wellas other APCs (34, 39–43). Thus, the level and duration of CD40signaling is critical for fate decisions in responding populations,and PTB is directly linked to these decisions by its ability toregulate CD40L expression at extended times of T cell activation.The fact that PTB-containing complexes are constitutively active

in the nucleus but active only at late times of activation in thecytoplasm is consistent with the different subsets of complexesbeing functionally distinct. It has previously been shown that PTBacts as a chaperone for nuclear-associated viral RNAs transportedto the nuclear pore (44, 45). If PTB functions in a similar way and

is required to bring CD40L mRNA to the nuclear pore for export,one prediction would be that reducing levels of nuclear PTBwould result in an increased accumulation of nuclear CD40LmRNA. However, our findings indicate that decreasing nuclearPTB results in an increase in cytoplasmic CD40L transcripts,suggesting that PTB functions to retain CD40L mRNA in thenucleus and that a drop in PTB nuclear levels results in an influxof CD40L mRNA into the cytoplasm. This RNA may be highlysusceptible to degradation especially under conditions where PTBis limiting. These results complement other findings showing thatshuttling of PTB between the nucleus and cytoplasm is inde-pendent of RNA binding (46).Our finding that decreased levels of PTB also resulted in re-

duced association of CD40L mRNA with the polysomal fractionsuggests that another function of PTB is to enhance the transla-tional efficacy of specific transcripts. This finding is consistentwith the known role of PTB in viral and endogenous IRES trans-lation (15) as well as the fact that binding of hnRNPL, which isa component of complex II that binds to both PTB and site C inthe stability element (18), alters the translation of the CD40Ltranscript (19). The fact that reducing PTB in the cell also resultedin a decreased level of nucleolin associated with the polysomessuggests a requirement for interaction between these two factorsin translation, a finding extending previous reports of PTB andnucleolin interactions during posttranscriptional processes (17, 47).From our data, we hypothesize that levels of PTB regulate

crucial nuclear and cytoplasmic events leading to optimal CD40Lexpression (Fig. 6). Specifically, at early times of T cell activationwhen transcriptional levels of RNA are at their highest, nuclear-specific PTB complexes retain only a portion of the CD40Lmessage in the nucleus, whereas the vast majority is transportedinto the cytoplasm in the absence of complex. Although theturnover of this “naked” transcript is rapid, the high level oftranscription along with the release of preformed protein stores(30, 48) lead to significant levels of CD40L expressed on the cellsurface. Continued activation results in specific modifications ofcytoplasmic PTB that lead to the formation of complex I/IIribonuclear complexes in the cytoplasm. Precedence for functionalmodifications comes from work on both PTB and other RNAbinding proteins. Whereas the nucleocytoplasmic distribution ofPTB has been linked to direct protein kinase A-dependent phos-phorylation of Ser16 (49, 50), the stability of many ARE-regulatedtranscripts appear to be controlled by signaling through p38MAPK-specific phosphorylation events that result in the cellularredistribution of specific RNA binding proteins including triste-traprolin (9). Thus, pathways involved in mRNA transport, lo-calization, turnover, and potentially translation intersect withPTB expression, and ultimately these PTB-mediated events maybe highly dependent on pathways induced by T cell activation.

AcknowledgmentsWe thank Lauren Martin for generating the PTB–Flag construct and Frank

Sinquett for excellent technical assistance.

DisclosuresThe authors have no financial conflicts of interest.

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