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C-CBL PROMOTES TCR-INDUCED THYMOCYTE APOPTOSIS BY ACTIVATINGTHE PI3K/AKT PATHWAY

Christine B F Thien1, Samantha A Dagger1, James H. Steer2, Frank Koentgen3, Elisa S.Jansen4, Clare L. Scott4 and Wallace Y Langdon1*

From 1School of Pathology and Laboratory Medicine, University of Western Australia, Crawley,Western Australia, 2School of Medicine and Pharmacology, University of Western Australia, 3Ozgene

Pty Ltd, Bentley, Western Australia, 4The Walter and Eliza Hall Institute of Medical Research,Melbourne, Australia.

Address correspondence to: W Y Langdon, School of Pathology and Laboratory Medicine, Universityof Western Australia, Crawley, WA 6009, Australia.Tel : 61 8 9346 2939; Fax : 61 8 9346 2891; Email : wally.langdon@uwa.edu.au

The ability of thymocytes to assess T cellreceptor (TCR) signaling strength and toinitiate the appropriate downstreamresponse is crucial for determining theirfate. We have previously shown that a c-Cbl RING finger mutant knock-in mouse,in which the E3 ubiquitin ligase activity ofc-Cbl is inactivated, is highly sensitive toTCR-induced death signals that causethymic deletion. This high intensity signalinvolves the enhanced tyrosinephosphorylation of the mutant c-Cblprotein promoting a marked increase in theactivation of Akt. Here we show that thishigh-intensity signal in c-Cbl RING fingermutant thymocytes also promotes theenhanced induction of two mediators ofTCR-directed thymocyte apoptosis, Nur77and the pro-apoptotic Bcl-2 family member,Bim. In contrast, a knock-in mouseharbouring a mutation at Y737, the site inc-Cbl that activates phosphatidylinositol 3-kinase (PI3K), shows reduced TCR-mediated responses including suppressionof Akt activation, a reduced induction ofNur77 and Bim and greater resistance tothymocyte death. These findings identifytyrosine-phosphorylated c-Cbl as a criticalsensor of TCR signal strength that regulatesthe engagement of death-promoting signals.

The development of T cells in the thymusinvolves stringent selection processes toensure the generation of T cells that are notstrongly self-reactive (1-3). Thus thymocytesexpressing T cell receptors (TCRs) thatrecognise self-peptide-MHC ligands with highaffinity trigger strong signaling responses thatresult in cell death. Intriguingly, similar TCRsignaling pathways are also employed for thesurvival and differentiation of thymocytes thatinteract with self-peptide-MHC complexes

with lower affinity. The TCR is therefore anexceptional example of a receptor whoseengagement directs signaling responses that canlead to opposing cell fates of survival or death.For example, in mature T cells a strong signalstimulates proliferation and cytokine productionwhereas the same signal received by immaturethymocytes induces an apoptotic response. Incontrast, engagement of a weaker TCR signal inimmature thymocytes is ineffective in producing adeath signal but promotes survival. As aconsequence there has been much interest inidentifying the signaling molecule or moleculesthat can sense TCR signal strength and act as aswitch to direct signaling pathways towards eithera survival or a death pathway.

It has been proposed that since both low-and high-intensity TCR signals in thymocytesactivate the Ras and calcium pathways, yet onlythe high intensity signal activates a death program,the TCR signal may be re-directed or “split” toactivate an additional pathway by a molecule thatsenses the intensity of the signal (4). Recently weshowed that while either the complete loss of c-Cbl or inactivation of its RING finger domainmarkedly enhances thymocyte signaling from theTCR, only the RING finger mutant mouse exhibitsthymic deletion and increased sensitivity tothymocyte death (5). These differences between c-Cbl knockout (KO) and RING finger knockin micehave provided a unique opportunity to furthercharacterize signaling pathways that direct TCR-mediated deletion of thymocytes.

c-Cbl is a multi-adaptor proto-oncogenethat possesses E3 ubiquitin ligase activity by virtueof its RING finger domain which recruits ubiquitinconjugating enzymes (E2s). It is abundantlyexpressed in the thymus where it functions as anegative regulator of TCR signaling (6,7). c-CblKO mice have elevated levels of TCR and CD3 onthe surface of CD4+CD8+ double positive (DP)thymocytes, increased levels of Lck and Fyn, and

http://www.jbc.org/cgi/doi/10.1074/jbc.M109.094920The latest version is at JBC Papers in Press. Published on February 4, 2010 as Manuscript M109.094920

Copyright 2010 by The American Society for Biochemistry and Molecular Biology, Inc.

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enhanced activity of the ZAP-70 tyrosinekinase (8-11). However despite the resultantincrease in the intensity of TCR signals, c-CblKO thymi appear to develop normally,although experiments with MHC class II-restricted TCR transgenic mice do show that,in the absence of c-Cbl, the selection of CD4+

thymocytes is enhanced on a weakly-selectingbackground (9).

To better understand the mechanismsinvolved in c-Cbl’s regulation of thymocytesignaling and fate we have been studying aknock-in mouse with a loss-of-functionmutation in the c-Cbl RING finger domain (5).This mutation of the N-terminal cysteine in theRING finger at position 379 (i.e. C379A)disrupts c-Cbl’s interactions with E2 enzymesand abolishes its E3 ligase activity (12-15).Mice with this mutation have manycharacteristics identical to the c-Cbl KO suchas increased expression of TCR, CD3, Lck andFyn in DP thymocytes (5). Remarkablyhowever, unlike the c-Cbl knockout, the RINGfinger mutation results in a progressive loss ofthe thymus even though DP thymocytes fromboth mutants show equivalent increases inTCR-directed activation of ZAP-70, the Rasand Rac pathways, JNK and p38 MAPkinases, and calcium mobilisation (5). Weshowed that the thymic loss is not caused by adevelopmental block, a lack of thymicprogenitors or peripheral T cell activation (5).Rather the phenotype correlates with a markedincrease in expression of the CD5 and CD69activation markers on DP thymocytes andincreased sensitivity to cell death whencultured with plate-bound anti-CD3, a signalthat in wt thymocytes is insufficient to inducea death response. In addition, expression of aBcl-2 transgene rescues thymic loss andblocks anti-CD3-mediated thymocyteapoptosis (5). These findings support thehypothesis that thymic deletion in the c-CblRING finger mutant mouse is caused by anabnormally intense TCR-directed apoptoticsignal.

Surprisingly the only signaling eventin c-Cbl(C379A) thymocytes that we found todiffer from the c-Cbl KO involved themarkedly elevated activation of Akt inresponse to anti-CD3 crosslinking (5).Further, we demonstrated that the mutant c-Cbl protein itself is responsible for theenhanced Akt activation through an increasedinteraction with the p85 regulatory subunit of

PI3K (5). This is caused by high levels of TCR,CD3 and Fyn in the mutant thymocytes whichresults in a greater proportion of c-Cblphosphorylation on tyrosine 737, the siterecognized by the SH2 domains of p85 (16-19).Thus analysis of this mouse has provided evidencethat tyrosine-phosphorylated c-Cbl can play apositive role in directing thymocyte apoptosisthrough its interaction with p85 and the resultantactivation of the PI3K/Akt pathway.

Here we expand the study of this pathwayby examining the regulation of Nur77 and Bim,two key proteins involved in thymocyte negativeselection. Our analysis of the c-Cbl RING fingermutant mouse and a c-Cbl knockin mouse with aphenylalanine substitution of tyrosine 737 hasidentified a novel apoptotic pathway directed by c-Cbl that activates Akt and promotes enhancedlevels of Bim and Nur77.

Experimental ProceduresMice - The generation of c-Cbl-/-, c-

Cbl(C379A) and c-Cbl(Y737F) mice have beenpreviously described (5,8,30). Mice weremaintained on a mixed C57BL/6J x129Sv/Jbackground and experiments were performed incompliance with the Animal Ethics Committee atUWA (approvals 03/100/275 and 07/100/578).

Inhibitors - Pharmacological inhibitorsused and their working concentrations are: Aktinhibitor VIII, Akti-1/2 10µM (Calbiochem,124018), histone deacetylase inhibitor TrichostatinA 33nM or 400nM (Sigma, T 8552), PI3Kinhibitors Wortmannin 100nM and LY 29400225µM (Alomone Labs, W-400 and L-300respectively), PKC inhibitor Ro 31-8220 200nM(Alexis Biochemicals, 270-020-M001), PKC/PKDinhibitor Go 6976 400nM (Alexis Biochemicals,270-021-MC05), and MEK inhibitor PD 9805940µM (Calbiochem, 513000).

Flow cytometry and antibody staining -Antibody-stained thymocyte, spleen and lymphnode cell suspensions were acquired on a BDFACSCanto and the data analyzed using FlowJosoftware (Tree Star Inc). Antibodies used wereagainst: TCRβ (H57-597), CD4 (RM4-5), CD8(53-6.7), CD5 (53-7.3), CD69 (H1.2F3) and B220(RA3-6B2) (BD). To detect intracellular levels ofNur77 thymocytes were stained with anti-CD4 andanti-CD8, fixed and permeabilized inCytofix/Cytoperm (BD) for 20 minutes at roomtemperature and washed in FACS buffer.Thymocytes were then incubated with ice-coldmethanol for 30 min at 4˚C, washed twice inFACS buffer before incubation with mouse anti-

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Nur77 (BD Tranduction Labs) for 45 min at4˚C. Following two washes in FACS bufferthymocytes were incubated with FITC-labelled anti-mouse IgG1 (BD). For thedetection of Bim thymocytes were fixed andpermeabilized in Cytofix/Cytoperm, washedtwice in FACS buffer + 0.03% Saponin andincubated on ice for 45 minutes with rat anti-Bim (3C5/10B12 from Andreas Strasser) inFACS buffer containing 0.3% Saponin. Bimwas detected by sequential incubations withbiotinylated anti-rat IgG2a (BD) followed bystreptavidin PE (Molecular Probes), both inFACS buffer containing 0.3% Saponin and onice for 45 minutes. Between each stepthymocytes were washed twice with FACSbuffer + 0.03% Saponin and the thymocyteswere finally stained with anti-CD4-FITC andanti-CD8-APC. For the detection ofintracellular levels of Lck and Fyn inthymocytes antibodies were purchased fromSanta Cruz Technologies (sc-433 and sc-434respectively). The cells were fixed andpermeabilized in Cytofix/Cytoperm (BD) andall procedures were carried out in FACS buffercontaining 0.1% saponin as previouslydescribed (5). The Lck and Fyn antibodieswere detected with APC-conjugated goat anti-mouse IgG (BD) and the thymocytes finallystained with anti-CD4-APC-Cy7 and anti-CD8-PE (BD).

T h y m o c y t e s t i m u l a t i o n ,immunoprecipitation and immunoblotting -Thymocytes were incubated with biotinylatedantibodies against CD3 (500A2), CD4(GK1.5) or CD28 (37-51) (BD Pharmingen),and stimulated by streptavidin crosslinking at37˚C. Cells were lysed in 0.2%NP-40/60mMn-β-D-glucopyranoside containing buffer andlysates analyzed by immunoprecipitation andimmunoblotting as previously described (10).PLCg-1 and Erk antibodies were purchasedfrom Santa Cruz Technology, anti-ZAP-70antibodies from Transduction Labs, anti-p85and c-Cbl from Upstate Cell SignalingSolutions and anti-actin from Sigma. Anti-phosphotyrosine (4G10) was provided byBrian Druker and anti-LAT by LarrySamelson. Antibodies to Bim, Akt, p-Akt(S473), p-Erk(T202/Y204), p-c-Cbl(Y731and Y774), p-LAT(Y191), p-PKD(S916) andPKD were from Cell Signaling and p-PLCγ-1(Y783) antibodies were from Biosource.Quantitation of protein bands was determinedby densitometric analysis of scanned X-ray

films using Kodak Molecular Imaging SoftwareVersion 4.0 (Eastman Kodak Company, NewHaven CT).

Plate-bound antibody-mediated assays todetermine thymocyte apoptosis and the inductionof Nur77 and Bim - To determine the extent ofantibody-mediated thymocyte apoptosis cultureplates (96 well) were coated overnight at 4˚C with10µg/ml of anti-CD3 (2C11) with or withoutvarying amounts anti-CD28 (37.51) antibodies(BD Pharmingen). Thymocytes at 2 x106/ml werecultured in triplicate in IDM/10% FCS for 16 hrsat 37˚C then harvested and stained with propidiumiodide (PI) at 0.2µg/ml for analysis by flowcytometry. For the analysis of Nur77 and Bim thethymocytes were cultured as above but in 48 wellantibody-coated dishes and harvested after 2 and 4hours respectively.

Real-time PCR quantitation of Bim mRNA- Thymocytes (2x107 cells per timepoint) werecultured in IDM/10% FCS for 2 or 4 hours at37˚C in 96 well culture plates that had been coatedovernight at 4˚C with 10µg/ml anti-CD3 (2C11)with or without 10ug/ml anti CD28 (37.51)antibodies. At the end of each timepoint, cellswere harvested and RNA isolated using Trizolreagent (Invitrogen) according to themanufacturer’s instructions. First strand cDNAwas prepared from 2µg RNA using TaqManReverse Transcription Reagent (Roche - AppliedBiosystems). Real-time PCR was performed usingthe ABI Prism 7900 (Applied Biosystems) and thePower SYBR Green PCR Master Mix (AppliedBiosystems) in 15 µ l reaction volumes. Dataanalyses were performed with the CT methodusing b-actin as an internal control.

Quantitative reverse-transcription PCR(qRTPCR) was performed using the followingforward and reverse primers (a kind gift from DrD C S H u a n g ) : B i m, 5 ’ -GAGTTGTGACAAGTCAACACAAACC-3’( s e n s e ) a n d 5 ’ -GAAGATAAAGCGTAACAGTTGTAAGATAAC C - 3 ’ (a n t i s e n s e ) ; N u r 7 7 , 5’-CCTGTTGCTAGAGTCTGCCTTC-3’ (sense)and 5’-CAATCCAATCACCAAAGCCACG-3’( a n t i s e n s e ) ; a n d a c t i n , 5 ’ -TATTGGCAACGAGCGGTTC-3’ (sense) and 5’-CCATACCCAAGAAGGAAGGCT-3’(antisense).

RESULTSEnhanced Nur77 and Bim induction in c-CblRING finger mutant thymocytes. As previouslydescribed c-Cbl RING finger mutant mice were

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generated through matings betweenheterozygous c-Cbl C379A mice (termed +/Amice) and c-Cbl -/- mice which produce c-CblA/- and c-Cbl +/- littermates (5). The c-CblA/- offspring carry only a single copy of themutated allele and have improved survivalrates compared to homozygous c-Cbl A/Amutant mice (5).

Since Nur77 and Bim are keymediators of negative selection in DPthymocytes (20-22) we tested whether thegreater susceptibility of c-Cbl A/- thymocytesto TCR-mediated death may be linked to anenhanced induction of these proteins. Time-course analyses found that the optimalexpression of Nur77 and Bim proteinsoccurred after 2 and 4 hours of culturerespectively ((23) and supplemental Fig. S1).Levels of Nur77 and Bim were thereforeexamined after culturing thymocytes withplate-bound anti-CD3 or anti-CD3 + anti-CD28 antibodies for 2 and 4 hoursrespectively. Flow cytometry of fixed,permeabilized and antibody stained DPthymocytes revealed that Nur77 protein levelsare induced to markedly higher levels in c-CblA/- thymocytes compared to c-Cbl +/-thymocytes, and to slightly higher levels thanin c-Cbl -/- thymocytes (Figure 1A). The highlevel of Nur77 induction in both mutant miceby anti-CD3 stimulation was surprising sincewe had previously shown that this activationalone is unable to mediate c-Cbl -/- thymocytedeath, while producing a potent death signal inc-Cbl A/- thymocytes (5). This findingsuggests that additional signaling events, otherthan a high level of Nur77 induction, are likelycontributing to thymocyte death.

Analysis of Bim induction in DPthymocytes in response to anti-CD3 and anti-CD3 + anti-CD28 stimulation showed thatBim levels were also induced to much higherlevels in c-Cbl A/- thymocytes (Figure 1B).Interestingly however, in contrast to Nur77, nosignificant difference was seen in the extent ofBim induction between c-Cbl +/- and c-Cbl -/-DP thymocytes. Both genotypes showedsimilar modest but consistent increases of 10-20% in the levels of Bim in response toculturing with the plate-bound antibodies(Figure 1B, bar graph). These findingssuggest that the enhanced induction of Bim inc-Cbl A/- thymocytes may be an importantfactor in mediating thymic deletion. Theenhanced levels of Bim protein in c-Cbl A/-

thymocytes was further confirmed byimmunoblotting lysates from anti-CD3 stimulatedthymocytes with an anti-Bim antibody (Figure1C). These results showed that both the BimEL andBimL isoforms were induced to higher levels in c-Cbl A/- thymocytes compared to wt and c-Cbl -/-thymocytes.

Enhanced levels of protein Bim in c-Cbl RINGfinger mutant mice are caused by post-transcriptional events. Since enhanced sensitivityto a stimulatory death signal correlated withincreased Bim protein levels in c-Cbl A/-compared to c-Cbl -/- or c-Cbl +/- thymocytes(Figure 1B and C), we performed quantitative real-time PCR to determine if this effect was due toincreased transcription of Bim in c-Cbl A/-thymocytes. Thymocytes from c-Cbl A/-, c-Cbl+/- and c-Cbl -/- mice were cultured for 2 or 4hours in media alone or in the presence of plate-bound anti-CD3 or anti-CD3 + anti-CD28antibodies. At each time point cells wereharvested and total RNA extracted for analysis.PCR quantitation showed that while stimulationwith either anti-CD3 or anti-CD3+CD28antibodies induced an increase in the levels of BimmRNA, there was no significant difference in theextent of this increase amongst the three genotypes(Figure 1D). Thus the higher level of Bim proteinin c-Cbl A/- thymocytes appears to be due to post-transcriptional events.

Pharmacologic inhibitors reveal differingrequirements for MEK and Akt in the induction ofNur77 and Bim. We have previously shown thatthe PI3K/Akt pathway is markedly enhanced inthymocytes from the RING finger knockin mousebut not the c-Cbl -/- mouse. In contrast all otherpathways examined, including the Erk pathway,are equivalently enhanced in both mutantscompared to thymocytes from wt mice (5). Inaddition we have shown that culturing c-Cbl A/-thymocytes in the presence of the PI3K inhibitorLY294002 suppresses anti-CD3-induced death (5).In this study we examined the roles of thePI3K/Akt and Erk/MEK pathways in the inductionof Nur77 and Bim through the use ofpharmacological inhibitors. The inhibitorsincluded Wortmannin, a potent inhibitor of thePI3K superfamily that shows excellent specificityexcept for its inhibition against the smooth muscleisoform of Myosin light chain kinase (SmMLCK,IC50 of 260 nM) (24), and Akti, a specific inhibitoragainst the Akt1 and Akt2 isoforms of Akt thatexhibits no inhibitory effects against the closely

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related AGC family kinases, PKA, PKC andSGK (25). We found that Nur77 and Bimprotein induction in thymocytes from c-CblA/- mice were inhibited by both Wortmanninand the Akti treatment (Figures 2A-D),although of the two compounds Akticonsistently showed a more inhibitory effecton Bim than Nur77. We found similar levelsof inhibition of Nur77 and Bim when weexamined the effects of LY 294002, anothercommonly used inhibitor of PI3K(supplemental Fig. S2). LY 294002 does notshow activity against SmMLCK but is activeagainst casein kinase II whereas Wortmanninis not (24).

In contrast to inhibitors of thePI3K/Akt pathway, the MEK inhibitor PD98059 only minimally affected the inductionof Bim whereas Nur77 was markedly inhibited(Figure 2A- D). PD 98059 is a selectiveinhibitor of MEK and to date no other proteinkinases have been identified that are inhibitedby PD 98059 (24). These findings indicatethat Bim and Nur77 are both regulated by aTCR-activated PI3K/Akt pathway, but theenhanced induction of Bim in c-Cbl RINGfinger thymocytes is largely independent ofactivated MEK. This finding supports ourearlier findings that the PI3K/Akt pathway, butnot the Erk pathway, is a key component indirecting the strong death signal in c-Cbl A/-thymocytes (5). Consistent with these findingswe found that Nur77 and Bim induction in wtthymocytes showed a similar pattern ofdependence for MEK and Akt respectively(Figure 2E). We found a small inhibitoryeffect of Akti on Nur 77 induction whereasBim was markedly suppressed. In contrastinhibition of MEK by PD98059 was veryeffective in blocking Nur77 whereas Bimlevels remained unaffected.

Trichostatin A inhibits rather than inducesNur77 in DP thymocytes. Since histonedeacetylase 7 (HDAC7) is highly expressed inDP thymocytes and has been found to inhibitthe activity of the Nur77 promoter in the T cellhybridoma DO11.10 (26), we tested the effectof the HDAC inhibitor trichostatin A (TSA) onNur77 induction in c-Cbl A/- thymocytes.TSA is a potent inhibitor of HDACs that ishighly active at nanomolar concentrations. Inmarked contrast to findings in DO11.10 Tcells, where TSA was found to induce Nur77expression, we found that TSA markedly

inhibited the induction of Nur77 in c-Cbl A/-thymocytes (Figure 2A and C). In addition wealso found that TSA had a similar inhibitory effecton the induction of Bim (Figure 2B and D). Thereason for the conflicting results of the effect ofTSA on Nur77 is not clear, however Dequiedt andcolleagues only examined the effects of TSA onDO11.10 T cells, not primary thymocytes, andused TSA at a higher concentration of 400nM(26), compared to 33nM in our experiments.However, even when we treated wt thymocyteswith 400nM TSA for up to 6 hours we found noevidence of Nur77 induction (Figure 3A). Inaddition 400nM TSA was found to inhibit Nur77induction in anti-CD3 and anti-CD3 + anti-CD28stimulated wt thymocytes to a level equivalent tothat observed with 33nM (Figure 3B). Thesefindings indicate that DO11.10 hybridoma cellsdiffer in their regulation of Nur77 compared toprimary thymocytes and as such may not be anappropriate model for examining thymocytesignaling.

An additional observation in DO11.10 Tcells relating to the regulation of Nur77 that isrelevant to this study is the finding that theinduction of Nur77 by phorbol 12-myristate 13-acetate (PMA) is suppressed by Gö6976, aninhibitor that targets both calcium-dependentisoforms of protein kinase C (PKC) and proteinkinase D (PKD) (27-29). Consistent with theirfindings with PMA we found that Gö6976 also hadan inhibitory effect on the induction of Nur77 byanti-CD3, whereas no inhibition was seen with Ro31-8220, a selective inhibitor of PKC which doesnot affect PKD (27,28) (Figure 4A and B). It hasbeen found in DO11.10 T cells that PKDparticipates in the induction Nur77 byphosphorylating HDAC7 which results in itsnuclear export, thus removing its inhibitorypresence (29). However since our findings withTSA indicate that HDACs are not inhibitory inthymocytes, but appear to be required forpromoting the induction of Nur77, this suggeststhat another mechanism for the activity of PKD isinvolved. An analysis of this mechanism isbeyond the scope of this study however since PKDclearly has a role in regulating Nur77 weinvestigated whether the induction of phospho-PKD differed between c-Cbl A/- and c-Cbl -/-thymocytes. As shown in Figure 4C, althoughanti-CD3 + anti-CD4-stimulated thymocytes fromboth mutants show a greater induction of phospho-PKD than wt, each were activated to equivalentlevels and with similar kinetics. This finding issimilar to that observed for phospho-PLCγ1 and

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phospho-Erk, but in marked contrast to themarked activation of Akt seen only in theRING finger mutant (Figure 4C and (5)).Thus, as with PLCγ1 and Erk, a perturbation toPKD signaling activity does not provide anexplanation for the marked differences insensitivity to thymocyte death that existsbetween the two c-Cbl mutants.

Characterization of mice with a c-Cbl Y737Fmutation. Previously we showed that a greaterproportion of c-Cbl protein in RING fingermutant thymocytes is phosphorylated ontyrosine 737, which promotes a greaterassociation with the p85 regulatory subunit ofPI3K resulting in enhanced Akt activation (5).Y737, together with Y706 and Y780, representthe three major tyrosine phosphorylation sitesin mouse c-Cbl (equivalent to Y731, Y700 andY774 of human c-Cbl respectively). Tofurther investigate the involvement ofphosphotyrosine 737 in the activation of thePI3K/Akt pathway we generated a c-Cblknockin mouse where tyrosine 737 has beenmutated to a phenylalanine (i.e. c-Cbl Y737F)(30). We find homozygous c-Cbl Y737F mice(termed F/F) are viable and born at close toexpected frequencies from heterozygousmatings (i.e. of 250 mice 23% were genotypedas F/F, 48% +/F and 29% +/+). Thymocytesfrom homozygous F/F mutant mice werefound to be present in normal numbers(18.35±1.77 x 106 for +/+, n=27, vs22.03±2.11 x 106 for F/F, n=25; Mean±SE),showed no developmental perturbations andthe cell surface expression of TCR, CD3, CD4and CD8 were unaltered compared to wtlittermates (Figures 5A and B). In additionlevels of the CD5 and CD69 activationmarkers were unchanged compared to normallittermates, as were levels of protein tyrosinekinases, Lck, Fyn and ZAP-70 (Figure 5B andsupplemental Figure S3). Further, no changesin T or B cell numbers were evident in lymphnodes or spleen (Figure 5C) nor were spleensizes affected (87.9±5.0mg for +/+, n=17, vs84.5±4.2mg for F/F, n=18; Mean±SE).

c-Cbl Y737F mutation abolishes binding to thep85 regulatory subunit of PI3K. The lack ofphosphorylation at Y737 in c-Cbl F/Fthymocytes following anti-CD3 + anti-CD4stimulation was shown by immunoblottingwith a phospho-specific antibody directedagainst this residue (Figure 6A). In addition,

the mutation also reduced the level ofphosphorylation of Y780, one of the other majortyrosine phosphorylation sites in c-Cbl (18,31).Thus Y737 plays an important role in determiningthe level of c-Cbl tyrosine phosphorylation.However the tyrosine phosphorylation of othersubstrates in thymocytes did not appear affectedby the Y737F mutation when total lysates wereexamined by immunoblotting with anti-phosphotyrosine antibodies (Figure 6B).Significantly the immunoprecipitation of lysateswith anti-p85 antibodies showed that the Y737Fmutation completely abolishes the inducibleassociation between c-Cbl and the p85 (Figure6C). This finding demonstrates that thephosphorylation of Y737 is an absoluterequirement for this interaction.

The activation of Akt is dependent on thephosphorylation of Y737. To further characterizethe effects of the Y737F mutation on thymocytesignaling we immunoblotted lysates with a rangeof phospho-specific antibodies following theirstimulation by cross-linking with anti-CD3 + anti-CD4 or anti-CD3 + anti-CD28 antibodies. Anti-phospho-Akt immunoblotting showed thatphosphorylation of S473 was markedly reduced inc-Cbl F/F thymocytes compared to thymocytesfrom normal littermates, most notably at the latertime points of 10 and 15 minutes (top panel,Figure 7A and B). This finding is consistent withour observations that hyperphosphorylation ofY737 is responsible for the marked enhancementin Akt activation seen in c-Cbl C379A RINGfinger mutant thymocytes (5). Additional analysisshowed tha t the ac t iva t ion- inducedphosphorylation of PLCγ1, Erk and LAT were alsosuppressed in c-Cbl F/F thymocytes, with themarked effects being evident at later time points(Figure 7). Interestingly we always observed anincrease in pErk induction in F/F thymocytescompared to wt thymocytes at 5 minutes, but thisactivation rapidly diminished by 10 and 15minutes. This is consistent with Erk activationbeing regulated by multiple signaling moleculesbut the data clearly demonstrates that the sustainedactivation of Erk is largely reliant on c-Cbltyrosine phosphorylation.

Mutation of the p85 binding site in c-Cbl inhibitsTCR-mediated induction of Nur77 and Bim. Tofurther characterize the downstream effects ofblocking the phosphorylation of Y737 in c-Cbl weexamined the induction of Nur77 and Bimfollowing incubation of thymocytes with plate-

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bound anti-CD3 and anti-CD3 + CD28antibodies. Analysis by flow cytometry of DPthymocytes from wt and c-Cbl Y737F mutantmice revealed a reduced level of induction ofboth Nur77 and Bim in thymocytes from c-CblF/F mice (Figure 8A and B). This findingfirmly establishes a link between the extent ofc-Cbl tyrosine phosphorylation and the degreeof induction of two key mediators ofthymocyte apoptosis. The wide-rangingeffects of this mutation on signaling pathwaysother than the PI3K/Akt pathway, includingErk, may explain the reduced induction ofNur77, which from our inhibitor studies is lessdependent on Akt signaling but highlydependent on the activity of MEK. Althoughwe were aiming for a mutation that wouldspecifically perturb the PI3K/Akt pathway thefindings support the hypothesis that c-Cbltyrosine phosphorylation has a profoundimpact on the signaling events in thymocytesthat regulate Nur77 and Bim.

Mutation of the c-Cbl Y737 site reducesthymocyte sensitivity to CD3+CD28 inducedcell death. c-Cbl A/- thymocytes are moresensitive to induction of cell death (5) and inthis study we have shown that this correlateswith increased induction of the pro-apoptoticproteins Nur77 and Bim (Figure 1). Sincemutation of the c-Cbl Y737 site has a markedeffect in inhibiting TCR-mediated Nur77 andBim expression (Figure 8), we examinedwhether this mutation also reversed thesensitivity of thymocytes to in vitro cell death.

Thymocytes from wt or c-Cbl Y737Fmutant mice were cultured in media alone orin the presence of plate-bound anti-CD3antibodies (10µg/ml) and varying amounts ofanti-CD28 antibodies. The extent of cell deathinduced by each treatment was measured asthe percentage of propidium iodide (PI)positive cells at the end of a 16hr cultureperiod (Figure 9). Efficient thymocyteapoptosis in vitro requires triggering of bothCD3 and CD28 receptors and, consistent withthis, we observed negligible death when cellswere cultured with anti-CD3 alone (Figure 9Aii). However when cultured with anti-CD3and anti-CD28 a lower proportion of c-CblY737F thymocytes became PI positivecompared to wt thymocytes (Figure 9A iii-iv,and Figure 9B). Compared to cells stimulatedwith anti-CD3 alone, the addition of 2.5µg/mlof anti-CD28 antibody induced a 23% increase

in PI positive wt thymocytes but only a 14%enhancement of PI positive thymocytes from c-CblY737F mice (Figure 9A iii and Figure 9B).Similarly, addition of 5µg/ml anti-CD28 led to a27% increase in PI positive wt thymocytes overCD3 stimulation alone, compared to a 10%increase in c-Cbl Y737F thymocytes (Figure 9A ivand Figure 9B). These results demonstrate that thephosphorylation of c-Cbl Y737 plays a role indetermining the potency of signaling responsesthat direct thymocyte death. We also observedthat the Y737F mutation was not as effective insuppressing thymocyte death when cultured in thepresence of 10µg/ml of both anti-CD3 and anti-CD28 (Figure 9A v), suggesting the c-Cbl-directeddeath pathway can be bypassed or overwhelmedby a very high intensity signal.

DISCUSSIONThe T cell receptor is a rare example of a receptorwhose engagement directs signaling responses thatcan lead to opposing fates of cell survival or death.In mature T cells a strong signal, such as thatdelivered by co-activation of the CD3 and CD28receptors, stimulates proliferation and cytokineproduction. In contrast, the same signal receivedby DP thymocytes will induce an apoptoticresponse. However engagement of the CD3receptor alone, a weaker signal, is ineffective inpromoting a death signal in DP thymocytes. As aconsequence there has been a great deal ofinvestigation to identify the signaling molecule ormolecules that can act as a switch to activatesignaling pathways towards either survival ordeath. Neilson and colleagues (4) proposed amodel for thymocyte fate determination whereby akey molecule acts as a “signal splitter” that sensesthe strength of a signal initiated at the TCR andhence whether a cell death program should beengaged. Our findings here, and in a previousstudy (5), suggest that c-Cbl may be a point atwhich this “signal splitting” can occur.

We previously showed that thymocyteslacking a functional c-Cbl RING finger domainwere induced to die when stimulated by anti-CD3cross-linking, and that this signal correlated withgreatly increased c-Cbl tyrosine phosphorylationand the activation of Akt (5). This enhancedsusceptibility to TCR-directed death demonstratedthe importance of the c-Cbl RING finger domainin regulating thymocyte signaling, however otherdomains of c-Cbl were clearly required as thecomplete loss of c-Cbl did not cause thymicdeletion or hyperactivation of Akt (5).

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To further examine the role of c-Cbl indetermining the fate of thymocytes we firstsought to identify proteins that might beresponsible for thymic loss in c-Cbl RINGfinger mutant thymocytes. Nur77 and Bimwere identified as two such proteins whoseinduction was found to be increased in c-CblRING finger mutant thymocytes compared towt and c-Cbl-/- thymocytes under conditionsthat promote apoptosis (Figure 1). This wasan important finding as the involvement ofNur77 and Bim in thymic deletion has beendemonstrated in numerous studies(20,21,32,33). Bim best fits the criteria as akey causative molecule that distinguishes thec-Cbl A/- and c-Cbl KO phenotypes becausec-Cbl KO and wt thymocytes show anequivalent minimal induction of Bimcompared to the large Bim induction found inc-Cbl A/- thymocytes (Figure 1). In contrast,although Nurr77 levels are enhanced in c-CblA/- thymocytes, they are also elevated in c-CblKO compared to wt thymocytes. Thepossibility that Bim may be a major player inthe proposed c-Cbl/Akt death pathway wasfurther supported by a greater dependence ofBim protein induction on the activity of Akt,while showing little dependence on MEK(Figure 2). In contrast Nur77 was found to behighly sensitive to MEK inhibition but lessdependent on the activity of Akt (Figure 2).These findings, as well as data shown inFigure 4B, are consistent with our previousfindings that Erk activation is enhanced in c-Cbl A/- and KO thymocytes equivalently, andis therefore unlikely to be causing thymocytedeath, whereas it is only c-Cbl A/- thymocytesthat show a marked activation of Akt (5)

Following TCR stimulation, themutant c-Cbl protein in A/- thymocytes ismore highly phosphorylated on Y737 resultingin increased c-Cbl/p85 association andPI3K/Akt activation (5). These results suggestthat thymic loss in the c-Cbl RING fingermutant mouse is at least in part due toenhanced activation of the PI3K/Akt pathwayby phosphorylated Y737. To more closelyinvestigate the roles of Y737 and p85 inthymocyte signaling we examined a mousewith a Y737F knockin mutation (i.e. the c-CblF/F mouse). We found that mutation of Y737effectively abolished the TCR-inducedassociation between p85 and c-Cbl, and as aconsequence markedly suppressed theactivation of Akt (Figures 6 and 7). The

extent of this suppression indicates that c-Cbl is animportant, if not the most important, activator ofthe PI3K/Akt pathway in TCR-stimulatedthymocytes. c-Cbl has been well characterized asa prominent substrate of protein tyrosine kinases inthymocytes and this study has definitivelyidentified a functional role for thisphosphorylation. Mutation of Y737 also causedother inhibitory effects, notably the reducedphosphorylation of Y780, which associates withthe SH2 domain CrkL (34) (Figure 6A). Thereduced phosphorylation of Y780 may be a resultof reduced interactions with Fyn and Syk, twokinases which primarily associate with c-Cblthrough Y737 (18,19). This may affect other sitesof c-Cbl phosphorylation and explain why we alsoobserve reduced activation of Erk, PLCg1 andLAT in Y737F thymocytes compared to wtthymocytes (Figure 7).

Examination of the effect of the Y737Fmutation on Nur77 and Bim expression revealedthat weaker TCR-directed signals resulted inreduced induction in the levels of both proteins(Figure 8). This was not surprising given thewidespread impact of the mutation on thymocytesignaling. This finding additionally demonstratedthe central role that c-Cbl tyrosine phosphorylationplays in regulating Bim and Nur77 protein levels.

In this study we found that the enhancedinduction of Bim protein in the c-Cbl RING fingermutant thymocytes did not involve an increase inBim transcription (Figure 1), indicating thatperturbations of post-transcriptional events areresponsible for the increase in protein levels.These events may involve a greater stability ofBim through an inability of the c-Cbl RING fingermutant protein to ubiquitylate Bim, a functionidentified in osteoclasts (35,36). Whether c-Cbldirects the ubiquitylation of Bim in thymocytesfollowing TCR stimulation is not known howeverthe fact that c-Cbl KO thymocytes do not havealtered Bim protein levels suggests other causesare likely to be involved. It has also beenproposed that the thymic phenotype in the c-CblA/- mouse may involve a dominant negative effecton Cbl-b however this is unlikely given the verylow level of Cbl-b protein in thymocytes (37) andthe fact that the Cbl-b KO (38,39) and Cbl-b RINGfinger knock-in mice (WL and CT unpublished)show no thymic phenotype. Furthermore theenhanced signaling events observed in the c-CblRING finger mutant thymus are not evident in thethymus of the c-Cbl/Cbl-b double mutant mouseindicating that these effects of the c-Cbl RINGfinger mutation cannot be explained or

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recapitulated by the additional loss of Cbl-bactivity (40).

In summary the work presented heresupports and expands the two mainobservations from our original study of the c-Cbl RING finger mutant mouse, (i) that the c-Cbl RING finger mutant protein has roles bothas a gain-of-function and as a loss-of-functionprotein and (ii) that the enhanced activation ofthe PI3K/Akt pathway by tyrosinephosphorylated c-Cbl promotes thymocytedeath. Interestingly this gain-of-function rolefor c-Cbl RING finger mutants has recentlybeen appreciated as a key element in the

activation of Akt associated with a range of humanmyeloproliferative neoplasms that carry c-Cblmutations (41). However it remains a conundrumthat Akt signaling can promote thymocyteapoptosis when the universally accepted role ofAkt is to promote survival (42-44). Howevergiven that a major task of the thymus is to carryout an unparalleled program of cell death it maynot be unreasonable to envisage that a uniquemechanism need to be employed. Future studiesof c-Cbl RING finger and p85-binding mutantmice on TCR transgenic and Akt deficientbackgrounds should help to resolve this point.

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FOOTNOTESWe thank Helen Moulder for animal care, Brian Druker for anti-phosphotyrosine and AndreasStrasser for anti-Bim antibodies. This work was supported by project grant 458539 from NHMRC

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(Canberra), an infrastructure grant from MHRIF (Department of Health, Western Australia) andNHMRC Fellowships to CLS (#406675) and WYL (#303112).

FIGURE LEGENDSFigure 1. Nur77 and Bim protein induction is enhanced in DP thymocytes from c-Cbl RINGfinger mutant mice. Thymocytes from wt (+/-), c-Cbl RING finger mutant mice (A/-) and c-CblKO mice (-/-) mice were cultured with plate-bound anti-CD3 or anti-CD3 + anti-CD28 antibodies(at 10µg/ml) for 2 hours or 4 hours before fixation, permeabilization and intracellular staining with(A) anti-Nur77 antibody (12.14), or (B) anti-Bim antibody (3C5/10B12). Representative flowcytometry profiles are from the CD4+ CD8+ gated population. The bar graphs with standarderrors show the mean level of induction relative to unstimulated wt thymocytes. The quantitationwas determined using geometric mean fluorescence (GMF) from four independent experiments.(C) Cell lysates of thymocytes from c-Cbl +/-, A/- and -/- mice were left unstimulated or culturedwith plate-bound anti-CD3 for 2 or 4 hours and immunoblotted with anti-Bim antibody. Thealternatively spliced forms of Bim, i.e BimEL and BimL are indicated. The middle panel shows alonger exposure to more clearly reveal the relative levels of BimL between the three genotypes.The anti-actin blot was used as a loading control. (D) The c-Cbl RING finger mutation does notenhance Bim mRNA induction following anti-CD3 or anti-CD3 + anti-CD3 + anti-CD28stimulation. mRNA prepared from thymocytes cultured for 2 and 4 hours was measured by realtime quantitative PCR. The bar graphs represent Bim mRNA levels in stimulated cells relative tounstimulated controls for each genotype from three independent experiments.

Figure 2. Pharmacological inhibitors reveal differential regulation of Nur77 and Bim in c-CblRING finger mutant DP thymocytes. Thymocytes were cultured with or without plate-bound anti-CD3 in the presence of DMSO, 100nM PI3K inhibitor Wortmannin, 10µM Akt inhibitor Akti-1/2,40µM MEK inhibitor PD98059 or 33nM histone deacetylase inhibitor Trichostatin A. Nur77protein levels were determined after 2 hours of culture in (A) and Bim protein levels weredetermined after 4 hours (B). Flow cytometry profiles are of the CD4+ CD8+ gated population andthe numbers in the panels represent the degree of stimulation in inhibitor-treated culturesnormalized to DMSO treated controls, with the increase in GMF of anti-CD3 stimulated vsunstimulated cultures treated with DMSO taken as 1.00. (C) and (D) Fold change in the levels ofNur77 and Bim protein relative to the anti-CD3 DMSO controls. The quantitation was determinedby GMF and the data represents three independent experiments. (E) Wt DP thymocytes show thesame pattern of inhibition as c-Cbl RING finger thymocytes to treatment with Akt and MEKinhibitors. Thymocytes from wt mice were cultured with plate-bound anti-CD3 in the presence ofDMSO, Akti-1/2 or PD 98059 and the levels of Nur77 and Bim determined by flow cytometry.The fold changes are normalized to the change in GMF of anti-CD3 vs unstimulated DMSO-treated thymocytes.

Figure 3. Trichostatin A (TSA) inhibits rather than induces Nur77 in DP thymocytes. (A) Wtthymocytes were cultured in the presence of DMSO or 400nM TSA for 2, 4 or 6 hours withoutplate-bound antibodies before analysis by flow cytometry to determine the levels of Nur77 protein.CD4+ CD8+ DP thymocytes gated profiles are shown. (B) The inhibition of Nur77 is equivalentin presence of either 33nM or 400nM TSA. Wt thymocytes were cultured for two hours withplate-bound anti-CD3 or anti-CD3 + anti-CD28 in the presence or absence of the indicatedconcentrations of TSA. Unstimulated thymocytes cultured in the absence of plate-boundantibodies are indicated by the shaded green histogram. Nur77 levels in DP thymocytes weredetermined as in (A).

Figure 4. (A) The induction of Nur77 in c-Cbl A/- thymocytes is inhibited by a PKC/PKDinhibitor but not by an inhibitor of PKC. Flow cytometry profiles of Nur77 protein expressioninduced by culturing thymocytes for two hours in the presence of plate-bound anti-CD3 antibodiesplus DMSO, Gö 6976 (a dual PKC/PKD inhibitor) or Ro 31-8220 (a specific PKC inhibitor) isshown in (A). The level of inhibition relative to the anti-CD3 DMSO control is indicated by

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numbers in each flow cytometry profile and in (B) which represents data from two independentexperiments. (C) The activation of PKD is equivalently enhanced in c-Cbl A/- and c-Cbl-/-thymocytes. Thymocytes from wt (+/-), c-Cbl RING finger mutant mice (A/-) and c-Cbl KO mice(-/-) were left unstimulated or stimulated by crosslinking with anti-CD3 + anti-CD4 antibodies for5 or 15 minutes at 37˚C. Cell lysates were immunoblotted with the indicated phospho-specificantibodies or anti-actin as a loading control.

Figure 5. Thymocyte development in c-Cbl(Y737F) mice is normal. Thymocytes from 7 week-old wt (+/+) and homozygous c-Cbl(Y737F) (F/F) littermates were examined by flow cytometryfor (A) the proportions of CD4 and CD8 single positive, DP and double negative cells and (B) forthe expression of TCR, CD5 and CD69 on total and gated DP thymocytes. (C) Analysis of lymphnode and spleen cells using the anti-TCR and B220 antibodies shows that the proportion of T andB cells in the periphery are not affected by the c-Cbl(Y737F) mutation.

Figure 6. (A) The c-Cbl(Y737F) mutation reduces the level of phosphorylation of Y780 in c-Cbl.Thymocytes from 6 week-old littermates were left unstimulated or stimulated by crosslinking withanti-CD3 + anti-CD4 antibodies for the indicated times at 37˚C. Lysates were immunoblottedwith anti-c-Cbl(pY737), anti-c-Cbl(pY780) or anti-c-Cbl antibodies. (B) The c-Cbl(Y737F)mutation abolishes the induced association between c-Cbl and the p85 regulatory subunit of PI3K.Thymocytes were stimulated as in (A) and lysates immunoprecipitated with anti-p85 antibodiesand immunoblotted with either anti-c-Cbl or anti-p85 antibodies. (C) The total phospho-tyrosinesignal is not affected by the c-Cbl(Y737F) mutation. Thymocytes from wt (+/+) and c-Cbl(Y737F) (F/F) mice were stimulated as above and total lysates were immunoblotted with anti-phosphotyrosine (4G10) or anti-actin antibodies.

Figure 7. The activation of Akt in thymocytes is dependent on the phosphorylation of Y737.Thymocytes from c-Cbl +/+ and F/F littermates were stimulated with (A) anti-CD3 + anti-CD4 or(B) anti-CD3 + anti-CD28 antibodies for the indicated times at 37˚C before lysis andimmunoblotting with phospho-specific or protein-specific antibodies. The numbers under eachstimulated track refer to densitometry readings that have been normalized for protein loading andexpressed as values relative to the phospho-antibody signal for +/+ thymocytes after 5 minutes ofstimulation.

Figure 8. Nur77 and Bim induction is suppressed in c-Cbl(Y737F) thymocytes. Thymocytesfrom +/+ and F/F littermates were cultured in the absence of plate-bound antibody (i.e.unstimulated thymocytes) or with plate-bound anti-CD3 or anti-CD3 + anti- CD28 antibodies for(A) 2 hours for Nur77 induction and (B) for 4 hours for Bim induction. The bar graphs representdata from three independent experiments with the numbers calculated from the GMF relative tounstimulated +/+ thymocytes that have been normalized to 1.00.

Figure 9. c-Cbl(Y737) thymocytes are less susceptible to an anti-CD3 + anti-CD28 death signal.(A) Thymocytes from 7 week-old +/+ and F/F littermates were cultured for 16 hours with orwithout plate-bound anti-CD3 (10µg/ml) and 0, 2.5, 5 or 10µg/ml of anti-CD28 antibodies asindicated above (panels i-v). After harvesting the cells were incubated with propidium iodide (PI)at a final concentration of 0.2µg/ml and analysed by flow cytometry. The gates indicate the PI+populations and the findings shown here are representative of three independent experiments. (B)Bar graph showing the % PI positive cells under each stimulation condition shown in (A).

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