PKCθ/β and CYLD Are Antagonistic Partners in the NFκB and NFAT Transactivation Pathways in Primary Mouse CD3+ T Lymphocytes

In T cells PKCθ mediates the activation of critical signals downstream of TCR/CD28 stimulation. We investigated the molecular mechanisms by which PKCθ regulates NFκB transactivation by examining PKCθ/β single and double knockout mice and observed a redundant involvement of PKCθ and PKCβ in this signaling pathway. Mechanistically, we define a PKCθ-CYLD protein complex and an interaction between the positive PKCθ/β and the negative CYLD signaling pathways that both converge at the level of TAK1/IKK/I-κBα/NFκB and NFAT transactivation. In Jurkat leukemic T cells, CYLD is endoproteolytically processed in the initial minutes of stimulation by the paracaspase MALT1 in a PKC-dependent fashion, which is required for robust IL-2 transcription. However, in primary T cells, CYLD processing occurs with different kinetics and an altered dependence on PKC. The formation of a direct PKCθ/CYLD complex appears to regulate the short-term spatial distribution of CYLD, subsequently affecting NFκB and NFAT repressional activity of CYLD prior to its MALT1-dependent inactivation. Taken together, our study establishes CYLD as a new and critical PKCθ interactor in T cells and reveals that antagonistic PKCθ/β-CYLD crosstalk is crucial for the adjustment of immune thresholds in primary mouse CD3+ T cells.


Introduction
The central role of PKCh in signal transduction pathways during an adaptive immune response has extensively focused on the exact biochemical mechanisms of PKCh function (reviewed in [1][2][3]). A recent study by Kong et al. identified the structural requirement in PKCh for its localization to the immunological synapse as a prerequisite for activation of downstream signaling [4]. Several transcription factors essential for the T cell activation response (i.e. NFkB, AP1, and NFAT) are regulated by PKCh [5,6]. In vivo analysis of PKCh 2/2 mice revealed the importance of PKCh for Th2- [7] and Th17-mediated immune responses [8,9] but not for host-protective antiviral responses [10]. Nevertheless, despite a profound understanding of the cellular role of PKCh, little is known about its molecular function, specifically the effector proteins downstream of PKCh during T cell activation.
Ubiquitylation and deubiquitylation are established posttranslational mechanisms for regulating immune responses, as well as the development and activation of immune cells. The tumor suppressor gene CYLD encodes an evolutionary conserved and ubiquitously expressed protein of approximately 120 kDa and was originally discovered as gene mutated in familial cylindromatosis, an autosomal dominant inherited disease characterized by the development of multiple benign skin tumors, principally on the head and neck [11]. Functionally it is a deubiquitylating enzyme (DUB) which removes mainly K63-linked polyubiquitin chains from several specific substrates, influencing in a negative way the activation status and/or spatial distribution of these target proteins in different signaling pathways. Numerous studies both in vitro and in vivo provided us with new insights in its established function as an important negative regulator of inflammatory responses, by counteracting the aberrant activation of NFkB signaling: Cyld 2/2 animals spontaneously develop intestinal inflammation and autoimmune symptoms due to the constitutive activation of the TAK1/IKK/IkBa axis [12,13]; the study of Lim et al. described a CYLD dependent negative NFkB regulation during bacteria induced lung inflammation in mice via deubiquitylation of TRAF6 and TRAF7 [14]; moreover, the same scientific group showed that Cyld knockout mice are protected from Streptococcus pneumonia infection and lethality via a negative crosstalk with p38 MAPK [15]; a synergistic crosstalk between the E3 ligase Itch and CYLD for TAK1 inactivation and termination of tumor necrose factor dependent inflammatory signaling was recently described [16]. The PKC isoform expression profile in whole cell extracts of naive thymocytes (Thy) and peripheral CD3 + T cells derived from wild-type and PKCh/b 2/2 mice. PKCh/b inhibition leads to an CYLD plays also an essential role in regulating T cell development and activation. Cyld-deficient mice show a delayed thymocyte development due to a constitutively K48-ubiquitylated and degraded LCK protein [17]. In addition, Cyld-deficient T cells are hyperresponsive to TCR/CD28 stimulation and CYLD has been firmly established as negative regulator of NFkB and JNK activation in response to antigen receptor activation in T cells [12,13,18].
In the current study, we defined physiologically redundant roles for the PKCh and PKCb isotypes in TCR/CD28-dependent NFkB and NFAT transactivation by examining PKCh/b single and double knockout mouse lines. Additionally, we provide experimental evidence that a constitutive interaction of PKCh with CYLD apparently leads to CYLD sequestration that affects the transactivation of the critical transcription factors NFkB and NFAT. Therefore, the results described here elucidate some aspects of PKCh and PKCb function during TCR activation and the processes that modulate CYLD function upstream of NFkB and NFAT activation in primary CD3 + T lymphocytes.

Mice
PKCh/b knockout mice are viable, fertile and were generated by crossing PKCh [5] and PKCb [19] single knockout mice. The generation of Cyld-deficient mice was described previously [20]. All mice were on a C57Bl/6 background and housed (under SPF conditions) at the mouse facility of the Medical University of Innsbruck. All animal experiments were performed in accordance with the Austria ''Tierversuchsgesetz'' (BGBI. Nr. 501/1988 i.d.g.F.) and have been granted by the Bundesministerium für Bildung, Wissenschaft und Kultur (bm:bwk).
The pan-PKC low molecular weight inhibitor LMWI [22] was provided by NYCOMED GmbH, and the tetrapeptide inhibitor z-VRPR-fmk (MALT1 LMWI) was a gift from Dr. Margot Thome.

Cell Culture and Transfections
Jurkat-TAg cells [23] (a kind gift from G.R. Crabtree, Stanford University, CA) were maintained in RPMI medium supplemented with 10% FCS (Life Technologies, Inc.) and antibiotics. Transient transfection of cells with 20 mg of plasmids encoding GFP, wildtype Cyld or a cleavage-resistant R324A Cyld mutant was performed by electroporation with a BTX-T820 Electro Square Porator (ITC, Biotech, Heidelberg, Germany) apparatus under predetermined optimal conditions: 2610 7 cells at 450 V/cm and five pulses of 99 ms.
HEK293T cells were cultured in Dulbecco's Modified Eagle's Medium supplemented with 10% FCS, 2 mM L-glutamine, and 100 mg/ml penicillin-streptomycin. HEK293T cells were transfected using MetafecteneTM transfection reagent according to the manufacturer instructions.
Primary human T cells were purified from PBMCs (isolated by standard Hypaque-Ficoll separation from whole blood samples) with the Pan T Cell Isolation Kit (Miltenyi Biotec) according to the manufacturer instructions.
Primary mouse CD3 + T cells were purified from pooled spleens and lymph nodes with mouse T cell enrichment columns (R&D Systems). T cell populations were typically 95% CD3 + as determined by staining and flow cytometry.
increased NF-kB and NFAT transactivation defect in T cells. (C) The nuclear extracts of resting and stimulated (overnight) wild-type, PKCh 2/2 , PKCb 2/2 and PKCh/b 2/2 CD3 + T cells were probed for DNA binding to radio-labeled probes containing NFkB and NFAT binding site sequences, as indicated. One representative experiment of three is shown. (D) Impaired nuclear import of p50, p65 and NFAT in activated PKCh/b2-deficient T cells. Nuclear extracts of resting and stimulated (overnight) wild-type, PKCh 2/2 , PKCb 2/2 and PKCh/b 2/2 CD3 + T cells were probed for p65, p50 and NFAT using immunoblot assays. DNA polymerase served as the loading control. One representative experiment of three is shown. (E) Effect of PKCh/b inhibition on proximal phosphorylation events after a brief stimulation. Western blot analysis was performed with cytosolic extracts from wild-type, PKCh 2/2 , PKCb 2/2 and PKCh/b 2/2 CD3 + T cells. CD3 + T cells were stimulated with anti-CD3/anti-CD28 and probed at different time points for the phosphorylation status of (p)S-32 IkBa, (p)S-473 AKT and (p)ERK1/2, as indicated. Fyn served as loading control. One representative experiment of three is shown. Protein phosphorylation levels were relatively quantitated by densitometric analysis. Numbers beneath bands indicate fold change compared to wt control after normalization to FYN. doi:10.1371/journal.pone.0053709.g001 Table 1. Flow cytometric analyses of the cellularity of the thymus, spleen and lymph nodes from wild-type and PKCh/ b2/2 mice.

Analysis of Proliferative Response and IL-2 Cytokine Production
For in vitro proliferation, 5610 5 T cells in 200 ml proliferation medium (RPMI supplemented with 10% FCS, 2 mM L-glutamine and 50 units/ml penicillin/streptomycin) were added in duplicate to 96-well plates precoated with anti-CD3 antibody (clone 2C11, 5 mg/ml) and soluble anti-CD28 (1 mg/ml; BD Bioscience) was added. For TCR-independent T cell stimulation, 10 ng/ml Phorbol 12,13-dibutyrate (PDBu) and 125 ng/ml of the calcium ionophore ionomycin were added to the media. Cells were harvested on filters after a 64 h stimulation period, pulsed with  IL-2 production in mouse CD3 + T cells after antibody stimulation was determined by BioPlex technology (BioRad Laboratories) from the supernatant.

Gel Mobility Shift Assays
Nuclear extracts were harvested from 1610 7 cells according to standard protocols. Briefly, purified CD3 + T cells were washed in PBS and resuspended in 10 mM HEPES (pH 7.9) 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT and protease inhibitors. Cells were incubated on ice for 15 min. NP-40 was added to a final concentration of 0.6%, cells were vortexed vigorously, and the mixture was centrifuged for 5 min. The nuclear pellets were washed twice and resuspended in 20 mM HEPES (pH7.9), 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, and 1 mM DTT and protease inhibitors, and the tube was rocked for 30 min at 4uC. After centrifugation for 10 min, the supernatant was collected. Extracted proteins (2 mg) were incubated in binding buffer with [32P]-labeled, double-stranded oligonucleotide probes (NFkB: 59-GCC ATG GGG GGA TCC CCG AAG TCC-39; NFAT: 59-GCC CAA AGA GGA AAA TTT GTT TCA TAC AG-39) (Nushift; Active Motif). In each reaction, 3610 5 c.p.m. of labeled probe was used, and the band shifts were resolved on 5% polyacrylamide gels. All experiments were performed at least three times with similar outcomes.

Flow Cytometry
Single-cell suspensions from the spleen, lymph node and thymus were prepared and incubated for 30 min on ice in staining buffer (PBS containing 2% fetal calf serum and 0.2% NaN 3 ) with FITC, PE or APC antibody conjugates. Surface marker expression was analyzed using a FACScan TM cytometer (Becton Dickinson & Co., Mountain View, CA) and CellQuestPro TM software according to standard protocols. Antibodies against murine CD3, CD4, and CD8 were obtained from Caltag Laboratories; CD19, CD69, CD44, and CD25 were obtained from BD PharMingen.

Retroviral Transduction of Primary Mouse T cells
The packaging cell line platE was transfected with a pMX retroviral vector encoding an EGFP-Cyld fusion cDNA. Approximately 36 h later, supernatants were collected and used directly to infect 24 h to 48 h preactivated CD3 + cells using spin inoculation (1 h, 20006g, 32uC), followed by a 5-6 h incubation period at 37uC. Infected cells were washed, resuspended in full supplemented medium and incubated for an additional 48 h to 72 h. From these cultures, GFP-expressing cells were analyzed using confocal microscopy to track the subcellular distribution of the protein of interest.
Monitoring CYLD Localization Using Confocal Microscopy CD3 + cells from wild type and PKCh/b knockout mice that were transduced with a retrovirus expressing an EGFP-CYLD were not stimulated or PDBu-and ionomycin-stimulated, transferred to a polylysine-coated slide and fixed with 2% paraformaldehyde. After permeabilization (0.1% TritonX-100 in PBS) and a blocking step (5% goat serum in PBS), the cells were stained with Alexa595-CTB (for lipid raft staining) and TOPRO 3 (Nucleus) (Molecular Probes). Immunofluorescence was analyzed with a Zeiss LSM 510 confocal laser scanning microscope and Zeiss LSM software v3.2.

Statistical Analysis
Differences between genotypes were analyzed using the unpaired Student's t test.

Overlapping Roles of PKCh and PKCb in NFkB and NFAT Transactivation Processes in Primary Mouse CD3 + T cells
Studies using targeted gene disruption defined a critical role for PKCh in the activation of the IL-2 promoter in the NFkB and Ca 2+ /NFAT pathways [5,6]. Surprisingly, the phenotypic characterization of PKCh-deficient T cells revealed a strong upregulation of PKCb protein levels in PKCh single knockout T cells (Fig. 1A). To investigate potentially compensatory and overlapping roles of these two PKC family members in T cell activation processes, PKCh/b double knockout mice were generated. These mice were viable, fertile and breed at normal Mendelian ratios. The null mutations for PKCh and PKCb were confirmed by PCR and immunoblotting of whole cell lysates from naive thymocytes and peripheral CD3 + T cells (Fig. 1B).
Flow cytometric analysis of thymocyte populations in PKCh/ b 2/2 double knockout mice revealed a slightly diminished percentage of CD3-, CD4-and CD8-positive cells, comparable to the PKCh single knockout phenotype and in agreement with previous research [24,25], which might indicate an involvement of PKCh in the positive selection process during thymocyte development. Nevertheless, in the periphery, PKCh/b 2/2 mice revealed no gross differences in the distribution of CD3-, CD4-, CD8-positive cells, leading to the conclusion that the concomitant loss of PKCh and PKCb did not additively affect T cell development (Table 1+ Table 2).
Examination of the stimulation-dependent upregulation of CD25, CD69 and CD44 surface markers on CD4 + and CD8 + subsets revealed no gross differences in the total percentage of positive cells between the genotypes, but the total protein amount per cell, monitored by median fluorescence intensity, was strongly reduced in PKCh/b 2/2 and to an intermediate extend in PKC singly-deficient T cells. These data might indicate a possible defect in the upregulation of both the IL-2 receptor chain alpha (CD25) and the activation marker CD69 in PKC-deficient T cells in both CD4 + and CD8 + T cells (Fig. S1).
indicated phospho-specific and pan-antibodies. Protein phosphorylation levels were relatively quantitated by densitometric analysis. Numbers beneath bands indicate fold change compared to wt control after normalization to FYN. doi:10.1371/journal.pone.0053709.g003 In contrast to the relatively normal T-cell development observed, the T-cell response of peripheral T cells after TCR stimulation was affected by the single and simultaneous loss of PKCh and PKCb. The H 3 -thymidine uptake and IL-2 secretion response of PKCh/b-deficient T cells stimulated with anti-CD3 and with or without anti-CD28-activated did not significantly exacerbate the defects already observed in the absence of PKCh alone (Fig. S2A-C). To exclude the proliferative defects being caused by deregulated apoptosis, we analyzed the activationinduced cell death (AICD) of CD4 + and CD8 + T-cell blasts derived from wild-type and double knockout animals using CD3 engagement in vitro; in addition also the Fas ligand induced cell death was monitored, but no enhanced apoptotic responses of PKCh/b 2/2 were detected ( Fig. S3A-B).
However, analysis of the pathways leading to IL-2 transcription revealed additively reduced binding of NFkB and NFAT to DNA in PKCh/b double-deficient CD3 + T cells after CD3/CD28 stimulation (Fig. 1C). Immunoblot analysis of nuclear extracts demonstrated that the weaker DNA binding of NFkB and NFAT was due to the reduced nuclear entry of two NFkB subunits, p50 and p65, and NFAT upon stimulation (Fig. 1D). Activation of NFkB involves the phosphorylation of I-kBa by IKKb and its subsequent proteasomal degradation. Consistent with the additive affect on NFkB translocation, the double knockout showed a weaker I-kBa phosphorylation after stimulation with CD3/CD28. Also the activation of the Map kinase pathway was partially affected by PKCh/b deficiency, visible through a reduced ERK phosphorylation, whereas the activation of Akt/PKB was normal (Fig. 1E).

PKCh and PKCb Synergistically Regulate TAK1 and JNK Activation
In agreement with the defective IKK/I-kBa axis, PKCh/b 2/2 CD3 + cells revealed a drastic activation defect in TGF b activated kinase 1 (TAK1), which is known to be a key regulator of IKKb signaling. The loss of both PKC isotypes appears to be required to abolish the signal, because TAK1 activation levels were similar between the wild-type and PKC single knockout T cells (not shown). Additionally, the JNK signal was attenuated by the targeted disruption of PKCh and PKCb, whereas ERK1/2 activation was only marginally affected ( Fig. 2A). Similar outcomes were observed in PDBu-and ionomycin-stimulated primary mouse wild-type T cells pretreated with 500 nM of a PKCspecific low molecular weight inhibitor (PKC LMWI). The stronger effect of the pharmacological pan-PKC inhibitor on MAP kinase activation can be best explained by its established inhibition of additional PKC family members next to PKCh and PKCb (Fig. 2B).

Cyld 2/2 T cells Show a Hyperactive Phenotype in NFkB and NFAT Transactivation Responses
Since the deubiquitinating enzyme CYLD has been shown to be a negative regulator of Tak1 [12] and JNK signaling [26], we investigated a possible link between CYLD and the PKCh/b isotypes in NFkb and NFAT driven IL-2 upregulation.
Despite of an observed thymocyte maturation defect, recent work on T cell signaling in Cyld 2/2 mice demonstrated hyperresponsiveness to TCR stimulation by constitutive activation of NFkB [12]. We confirmed these results, as we also observed that Cyld 2/2 T cells showed elevated activation-induced IL-2 responses (Fig. 3A). This hyper-responsive IL-2 secretion correlated with an increase of NFkB DNA binding to the IL-2 promoter in the nuclear fractions of stimulated Cyld-deficient T cells (Fig. 3B) and hyper-phosphorylated I-kBa levels in the cytosol compared to wild-type controls (Fig. 3C). Interestingly, and in accordance with a previous publication [27], we determined that CYLD also acts as a negative modulator of the NFAT pathway. The examination of NFAT transactivation using immunoblot and EMSA technology revealed increased nuclear translocation and subsequent binding of NFAT to DNA in Cyld-deficient cells (Fig. 3B). Our EMSA result was confirmed by the elevated activation status of phospholipase Cc1, which has been identified as a key regulator of Ca2+/Calcineurin/NFAT signaling. However, ERK signaling was not affected by the loss of Cyld (Fig. 3C).

Association of CYLD with PKCh
Considering the reciprocal phenotypes of Cyldand PKCh/bdeficient T cells involving NFAT and NFkB transactivation we investigated a potential direct interaction between this enzymes. Interestingly and indeed, we identified a physical and functional PKCh-CYLD interaction in the cytosol of primary T cells. The coimmunoprecipitation analysis of CYLD and the PKCh isotype from cell extracts of unstimulated and CD3/CD28-activated peripheral CD3 + cells revealed that PKCh and CYLD physically associate in a complex in resting conditions (Fig. 4A). Next, we mapped the PKCh interaction domain in the CYLD protein by co-transfection of HEK293T cells with a vector encoding PKCh and with vectors expressing full-length Flag-tagged wild-type or Nand C-terminally truncated forms of CYLD (encoding residues 1-212, 318-956 and 587-986 of CYLD). The CYLD pull down with a specific Flag antibody revealed increased binding of PKCh to CYLD mutants containing the deubiquitinase domain (Fig. 4B). We observed identical results when the co-immunoprecipitation was performed to precipitate PKCh. A strep-tagged PKCh construct was co-transfected with the Flag-tagged CYLD constructs. A GFP control for each CYLD construct was included to identify unspecific binding to the Streptactin-beads. PKCh precipitation confirmed that the C-terminal part of CYLD is necessary for complex formation between the two interacting protein (Fig. 4C).

In Jurkat Cells, CYLD is Cleaved by MALT1 in a PKCdependent Mechanism
When Jurkat cells overexpressing an N-terminally HA-tagged CYLD construct were stimulated for 30 min with PDBu and ionomycin, a CYLD fragment of approximately 40 kDa was Figure 4. Association of CYLD with PKCh. (A) CYLD directly interacts with PKCh in primary mouse CD3 + cells. The complex is formed constitutively and is not affected by TCR activation. One representative experiment of three is shown. The C-terminal region of CYLD is important for interaction with PKCh. (B) Co-immunoprecipitation of PKCh using CYLD pulldown. Increased binding of PKCh to the C-terminus of CYLD was shown in HEK293T cells transiently co-transfected with vectors encoding PKCh (PEFneo) and a full-length Flag-tagged wild-type CYLD, N-or C-terminally truncated forms of CYLD (residues 1-212, 318-956 and 587-986 of CYLD). Untransfected and GFP-transfected controls were included. One representative experiment of three is shown. A schematic representation depicts the CAP-Gly and peptidase domains in wild-type and truncation mutants of CYLD. (C) Co-immunoprecipitation of CYLD using PKCh pulldown. A strep-tagged full-length PKCh construct was co-transfected with Flagtagged CYLD constructs into HEK293T cells. As previously, the importance of the C-terminal region of CYLD for binding is shown. GFP controls for each CYLD construct were included. One representative experiment of three is shown. doi:10.1371/journal.pone.0053709.g004 detected using the anti-HA antibody. Because the administration of PDBu mimics TCR signaling by activating PKC family members, we wanted to identify a role for PKC in this cleavage event. Therefore, Jurkat cells were pretreated with the specific pan-PKC pharmacological inhibitor, which resulted in the disappearance of this fragment (Fig. 5A). This finding emphasized that the endoproteolytic cleavage of CYLD is PKC dependent. We also isolated primary T cells from human whole blood and analyzed the CYLD processing under endogenous conditions. In addition, human T cells were treated with the pan-PKC inhibitor to investigate the PKC dependency in this process. Comparable to the results with Jurkat cells, CYLD underwent a stimulation dependent processing also in primary human T cells, which could be blocked by PKC inhibition. The generation of a 40 kDa NH 2terminal and a 70 kDa C-terminal cleavage fragment was confirmed via the use of NH 2 -and C-terminus recognizing specific CYLD antibodies (Fig. 5B).
Because Coornaert et al. showed that the paracaspase MALT1 directly cleaved the deubiquitinating protein A20 to generate a fragment with a smaller molecular size in stimulated T cells [28], we asked if MALT1 was also responsible for the cleavage of CYLD by treating Jurkat cells with the tetrapeptide inhibitor z-VRPRfmk, which has been shown to inhibit specifically the MALT1 protease activity [29]. As a result, cells treated with the MALT1 inhibitor showed a reduced CYLD cleavage after activation (Fig. 5A).
Based on the size and the molecular weight of the CYLD Nterminal 40 kDa proteolytic fragment, we identified the cleavable arginine residue at position 324 in the human CYLD protein and generated a Cyld mutant with alanine substituted for arginine at position 324 (Cyld-R324A). Next, we investigated if this mutant was cleavable when overexpressed in Jurkat cells or was resistant to proteolysis. Wild-type CYLD was processed after stimulation, whereas the mutant could no longer be cleaved (Fig. 5C). This led to permanent inhibition of the NFkB pathway by the inactivation resistant CYLD mutant and result in slightly diminished phosphorylation of I-kBa. The MAPK pathway was not affected by the expression of uncleavable CYLD.
Because NFkB is important for IL-2 upregulation in activated T cells, we tested the influence of the protease-resistant Cyld mutant on IL-2 transactivation using an IL-2-promoter-dependent luciferase assay. The diminished NFkB signal observed by immunoblot was correlated with impaired IL-2 transcription (Fig. 5D). This provides experimental evidence that CYLD processing, which leads to an inactivation of its repressor function within a positive feedback loop, is an important prerequisite for robust IL-2 activation. Consistent with our investigation of CYLD cleavage at arginine 324 in the Jurkat tumor cell line, Staal et al. independently published that TCR-induced JNK activation required CYLD proteolysis by MALT1 [18]. Nevertheless, our findings extend the function of CYLD cleavage to NFkB activation. To identify the physiological role of this candidate process, we examined primary CD3 + T cells derived from wildtype and knockout mice.

PKC-dependence, Cleavage Site and Kinetics of CYLD Cleavage Differ in Primary Mouse T cells
Stimulation induced a CYLD fragment not only in human cells but also in primary mouse T cells. Interestingly activation of primary mouse T cells by CD3 with or without CD28 costimulation generated a NH 2 -terminal CYLD fragment of approximately 25 kDa, smaller in size than the human fragment (Fig. 6A), implicating a different cleavage site in the mouse CYLD protein. Although we did not determine the exact cleavage site, arginine 235 was the best candidate for the cleavage site. Importantly, the fragment was first detectable after 4 h of stimulation, indicating different kinetics leading to CYLD inactivation in primary mouse cells. Unexpectedly, PKCh/bdeficient T cells showed normal CYLD processing after stimulation, implicating additional protein kinases in this process (Fig. 6B). As a consequence, the activation defects of the TAK1/IKK axis in PKCh/b-deficient T cells cannot solely be explained by CYLD inactivation.
Alternatively, the existence of a constitutive CYLD/PKCh complex might suggest that PKCh, which shows activationdependent subcellular translocation, is important for removing CYLD from its NFkB-related targets and attenuates the negative regulatory function of CYLD, enabling feedback control of NFkB activation. To address this hypothesis, we analyzed the subcellular distribution of a retrovirally introduced mutant CYLD-GFP fusion protein in unstimulated and stimulated wild-type and PKCh/bdeficient T cells with confocal microscopy. CD3 + T cells from both genotypes were retrovirally infected with a CYLD-GFP fusion construct, and the colocalization of CYLD-GFP with lipid rafts was monitored with Cholera Toxin B. Interestingly, CYLD colocalization with lipid rafts was strongly diminished in PKCh/bdeficient T cells compared to control cells, suggesting PKCdependent CYLD membrane shuttling. A statistically significant decrease in CYLD translocation in double knockout cells was detected in unstimulated cells, whereas after 15 min of PDBu and ionomycin stimulation, CYLD translocation to the membrane was observed in both wild-type and knockout cells (Fig. 6C).

Discussion
Numerous studies emphasize PKChs key role as a regulator of NFkB and Ca 2+ /NFAT signaling in T cells downstream of the TCR [5,6,[30][31][32]. The PKCb isotype is also expressed in T cells. However, PKCb-deficient primary mouse T cells have a fairly normal activation response [33], although Volkov et al. established a major role for PKCb in LFA1-dependent T-cell locomotion [34,35].
Our recent work defines a redundant role for both the novel PKCh variant and the classical isotype PKCb in the NFkB and NFAT signaling pathways. T cells isolated from PKCh/b-deficient mice had a stronger impairment in NFkB and NFAT nuclear entry and DNA binding compared to CD3 + T cells from control and single knockout mice. Impaired TAK1 activation in doubledeficient T lymphocytes is the best candidate for restricted IKK/ IkBa signaling. Functional redundancy of PKCh with other members of the PKC family in NFkB and/or NFAT activation has been shown in previous studies. For instance, stimulation-PKC dependency of this process was verified by pan-PKC LMWI treatment. The generation of a 40 kDa NH 2 -terminal and a 70 kDa C-terminal cleavage fragment was confirmed via the use of NH 2 -and C-terminus recognizing specific CYLD antibodies. (C) Mapping of the R234 cleavage site in CYLD using site-directed mutagenesis. The cleavage-resistant CYLD mutant R324A, when overexpressed in Jurkat cells, is not cleaved and leads to an increased suppression of NFkB-driven signals (as detected by the decreased phosphorylation of IkBa. (D) PKC-and MALT1-induced proteolysis at R234 appears critical for complete IL-2 promoter transactivation, shown by an IL-2 promoter luciferase assay. doi:10.1371/journal.pone.0053709.g005 The activation of primary mouse T cells by CD3 with or without CD28 costimulation leads to the formation of an NH 2 -terminal CYLD fragment of dependent colocalization of atypical PKCf/i with PKCh in the lipid raft fraction of T lymphocytes leads to cooperation of these isotypes in modulating the NFkB signaling pathway [36]. The collaborative activity of PKCh and PKCa in the NFAT pathway was examined in a PKCa/h double knockout mouse strain. Compared to PKCa and PKCh single-deficient T cells, doubledeficient CD3 + cells showed additively reduced IL-2 secretion levels correlated with strongly impaired nuclear translocation and DNA binding of NFAT after stimulation. Of note, the PKCa/h double knockout mice showed an impaired alloimmune response, leading to significantly prolonged allograft survival in heart transplantation experiments [37].
Similar to phosphorylation, K63 ubiquitination is a reversible process that influences protein activity, trafficking and signaling complex assembly. The removal of ubiquitin chains is mediated by a family of deubiquitinases, of which the cylindromatous gene product CYLD and the Tumor necrosis factor a-induced protein 3, also called A20, is currently receiving broad scientific attention. Both CYLD and A20 have been implicated as a modulator of the activity of NFkB-related molecules, such as NEMO (IKKc), TRAF2 and TRAF6 [38][39][40]. Both enzymes overlap functionally by targeting a similar set of substrates, which was explained by the different expression pattern of A20 and CYLD. A20 function depends on its transcriptional upregulation, whereas CYLD is constitutively expressed, influencing the different time windows of NFkB activation differently. However, a constitutive expression and activity pattern requires a posttranslational regulatory mechanism to inactivate the repressor during signal-induced NFkB signaling.
The cleavage-dependent inactivation of a deubiquitinase as a posttranslational regulatory mechanism in activated T cells was first described by Coornaert et al. [28], in which A20 was defined as a MALT1 substrate, which upon antigen receptor engagement undergoes cleavage for functional NFkB signaling. In our study, we showed that PKCh and CYLD are constitutively bound in a physical complex in the cytosol of primary mouse CD3 + cells. Direct crosstalk between CYLD and a PKC family member has not been described to date; therefore, we aimed to elucidate the biological relevance of this protein-protein interaction in T-cell signaling by examining genetic knockout mouse models in combination with selective pharmacological inhibitors. The reciprocal phenotypes of T-cell signaling pathways in Cyld 2/2 and PKCh/b 2/2 mice prompted us to analyze the activity of key molecules linked to NFkB and NFAT transactivation to uncover a regulatory mechanism to address the modulation of TAK1 activity In agreement with Koga et al. [27], who demonstrated negative regulation of NFAT activity by CYLD via the TAK1/MKK3/6/p38a/b axis, our experimental data clearly attest to CYLD involvement in NFAT activity modulation downstream of TCR signaling.
Our results show that PKC, particularly the PKCh/b isotypes, can influence CYLD repressive activity in different ways. In the human Jurkat leukemic T-cell line, PKC enzymatic activity was important for rapid MALT1-dependent CYLD processing, which is required for TCR-linked NFkB transactivation and leads to functional IL-2 induction. The requirement for MALT1-mediated CYLD cleavage for intact JNK signaling downstream of the TCR has been previously described [18]. Thus, proteolytic inactivation of CYLD affected IL-2 transactivation via the JNK/AP1 pathway; here, we provide experimental evidence that NFkB activity is also specifically dependent on CYLD cleavage, subsequently modulating IL-2 signals. Of note, we independently confirmed arginine 324 as the CYLD cleavage site. Recently, caspase 8 has been shown to cleave CYLD at aspartate 215 in Jurkat cells following TNFa stimulation, generating a pro-survival signal to save the cells from necrotic cell death [41]. Additionally, phosphorylation of CYLD was found to downregulate CYLD activity: transient phosphorylation by IKK in a serine cluster just upstream of the TRAF2 binding site, attenuates DUB function [42]. However, NFkB itself can regulate CYLD expression in a negative feedback loop [43].
In primary T cells isolated from PKCh/b deficient mice, CYLD was processed to the same extent as in wild-type control cells. Additionally, the kinetics of CYLD cleavage was different in mouse T cells compared to Jurkat cells, starting approximately 4 hours after stimulation, later then the rapid response through TAK1 activation. The different requirement for PKC and the altered kinetics in the mouse system led to the analysis of the stimulation-dependent spatial and temporal organization of the PKCh/CYLD complex using immunofluorescence microscopy. Interestingly, we found decreased CYLD lipid raft localization in PKCh/b-deficient T cells under resting conditions, likely affecting activation-induced signaling.

Conclusion
We observed a direct functional connection between the positive PKCh/b and the negative CYLD signaling pathways that fine-tune TCR/CD28-induced signaling responses. Our findings suggest the following scenario: PKCh/b are the essential kinases in a physiological signaling cascade that is necessary to counteract CYLD-mediated repression of NFkB and NFAT transactivation. This direct and physical antagonistic crosstalk between the PKCderived signals and the CYLD-derived signals might represent one mechanism of how antigen-receptor-dependent fine-tuning of the amplitude of T lymphocyte activation is processed. Figure S1 Effect of PKCh/b deficiency on CD25, CD44, and CD69 surface expression. T cells were stimulated for 16 h by CD3/CD28 ligation and the surface expression of CD25, CD44, and CD69 for CD4 + and CD8 + subsets were measured by flow cytometry. The relative fluorescence intensities are indicated as the median fluorescence intensity. The results shown are the mean6SE of three independent experiments. (TIF) Figure S2 Proliferative and cytokine secretion responses of PKCh/b CD3 + T cells. (A, B) Proliferative responses of PKCh/b and PKCh-deficient CD3 + T cells were analyzed in comparison to wild-type littermate controls. After incubation using different stimulatory conditions (antibodies or BALB/C splenocytes), cells were analyzed using standard procedures for thymidine incorporation. (C) IL-2 cytokine secretion by knockout CD3 + T cells was analyzed in comparison to wild-type littermate controls. After stimulation with anti-CD3 with or without soluble approximately 25 kDa. In stark contrast to the rapid kinetics in Jurkat cells, the fragment was first detected after 4 h of stimulation. (B) Primary mouse T cells from PKCh/b 2/2 mice showed normal stimulation-dependent CYLD cleavage, comparable to wild-type control mice. (C) Analysis of the subcellular distribution of a retrovirally introduced CYLD-GFP fusion mutant in unstimulated and stimulated wild-type and PKCh/b-deficient T cells. (green): the co-localization with Cholera Toxin B stained lipid rafts (red) was monitored using confocal microscopy. Nuclei are stained in blue. Quantification of CYLD-lipid rafts co-localization is shown in the bars in the right panel and reveals a statistically significant decrease in CYLD translocation in unstimulated double knockout cells in comparison to wt control cells. *p,0.05; **p,0.01; ***p,0.001. doi:10.1371/journal.pone.0053709.g006

Supporting Information
anti-CD28, supernatants were analyzed for IL-2 concentration using Bioplex suspension array technology. One representative experiment of three is shown. (TIF) Figure S3 Activation-induced cell death (AICD) of CD4 + and CD8 + T cell blasts derived from double knockout animals was not increased compared to cells from single knockout littermates. (A, B) AICD was induced by different concentrations of anti-CD3 for 8 hours. The results shown are the means of three independent experiments. (TIF)