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The Journal of Immunology, 2000, 165: 6933-6940.
Copyright © 2000 by The American Association of Immunologists

The Physical Association of Protein Kinase C{theta} with a Lipid Raft-Associated Inhibitor of {kappa}B Factor Kinase (IKK) Complex Plays a Role in the Activation of the NF-{kappa}B Cascade by TCR and CD281

Ali Khoshnan, David Bae2, Charles A. Tindell2 and Andre E. Nel3

Division of Clinical Immunology and Allergy, Department of Medicine, Center for Health Sciences, University of California, Los Angeles, CA 90095


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We investigated the role of protein kinase C {theta} (PKC{theta}) in the activation of the NF-{kappa}B cascade in primary human CD4+ lymphocytes. Among six or so PKC isoforms expressed in T cells, only PKC{theta} participates in the assembly of the supramolecular activation clusters at the contact site of the TCR with Ag. Signaling via both the TCR and CD28 is required for optimal activation of the multisubunit I{kappa}B kinase (IKK) complex in primary human T lymphocytes; this activation could be inhibited by a Ca2+-independent PKC isoform inhibitor, rottlerin. Moreover, endogenous PKC{theta} physically associates with activated IKK complexes in CD3/CD28-costimulated primary CD4+ T cells. The same set of stimuli also induced relocation of endogenous PKC{theta} and IKKs to a GM1 ganglioside-enriched, detergent-insoluble membrane compartment in primary T cells. IKKs recruited to these lipid rafts were capable of phosphorylating a recombinant I{kappa}B{alpha} sustrate. Confocal microscopy further demonstrated that exogenously expressed PKC{theta} and IKKß colocalize in the membrane of CD3/CD28-costimulated Jurkat T cells. Constitutively active but not kinase-inactive PKC{theta} activated IKKß in Jurkat T cells. Expression of dominant-active PKC{theta} also had stimulatory effects on the CD28 response element of the IL-2 promoter. Taken together, these data show that the activation of PKC{theta} by the TCR and CD28 plays an important role in the assembly and activation of IKK complexes in the T cell membrane.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CD28 is a key accessory receptor that enhances cellular activation via the TCR. Not only is CD28 an integral component of the supramolecular activation clusters (SMAC)4 that assemble at the TCR contact site with the APC, but it also plays an active role in the recruitment of signaling molecules to the TCR (1, 2, 3, 4, 5). The latter effect is mediated by the coclustering of lipid rafts to the TCR synapse. Lipid rafts are detergent-resistant, cholesterol and glycosphingolipid-enriched membrane domains which contain proteins that contribute to signal transduction (6). These rafts also include signaling molecules belonging to the src-family, LAT (linker for activation of T cells) and Ras (1, 4, 6). Through this action, CD28 enhances signal transduction by the TCR, including engagement of signaling pathways that cannot be activated by TCR alone. One example is activation of the c-Jun N-terminal kinase (JNK) cascade and another engagement of the NF-{kappa}B pathway (7, 8). These cascades appear to be functionally linked through the action of a mitogen-activated protein kinase kinase kinase (MAPKKK), MEKK1, which induces JNK as well as inhibitor of {kappa}B factor (I{kappa}B) kinase (IKK)ß activity during CD28 coligation (7, 8, 9, 10). Both cascades are critical for the activation of the IL-2 gene, including activation of the CD28 response element (RE) in the promoter of that gene (7, 11, 12). This composite RE, which includes c-Rel and AP-1 binding sites, is activated in a synergistic fashion by the NF-{kappa}B and JNK cascades during TCR/CD28 coligation (7, 13). Although the mechanism of NF-{kappa}B activation by the TCR/CD28 is unclear, we have recently shown that CD28 synergizes with TCR in the activation of homo- and heterodimeric IKK complexes in primary human T cells (8). Activated IKKs phosphorylate the inhibitory proteins, I{kappa}B{alpha} and I{kappa}Bß, leading to their degradation and release of NF-{kappa}B transcription factors into the nucleus (8, 14, 15).

IKK{alpha}, IKKß, and IKK{gamma} are the core components of a 700- to 900-kDa multimolecular kinase complex, which is responsible for stimulus-induced phosphorylation of I{kappa}Bs (16, 17, 18, 19, 20, 21, 22). Although proteins like MEKK1 and the NF-{kappa}B-inducing kinase have been shown to associate with the IKKs, there is considerable debate as to whether these are functionally involved in the regulation of IKK activity (18, 23, 24). In addition, two other serine-threonine kinases, namely Cot kinase and protein kinase C {theta} (PKC{theta}), have now been implicated in IKK activation by CD3/CD28 (25, 26, 27, 28). Whether these kinases are involved in individual signaling pathways or act in hierarchical fashion is still uncertain. In this communication, we will focus on PKC{theta} because this is the only PKC isoform that is recruited to the TCR synapse and the SMAC (1, 5, 29, 30). Moreover, PKC{theta} has been shown to be involved in NF-{kappa}B activation in T lymphocytes, in addition to its ability to induce the JNK/AP-1 pathway (6, 26, 27, 28, 31, 32, 33). This suggests that PKC{theta} plays a key role in the regulation of signaling cascades influenced by CD28 receptor.

Because activated PKC{theta} is recruited to the T cell membrane, it is possible that IKK activation may commence in a signalsome that assembles at the TCR synapse. We demonstrate that CD3/CD28 coligation in primary human T lymphocytes induces relocation of endogenous PKC{theta} as well as IKKs to a GM1 ganglioside-enriched, detergent-insoluble membrane compartment that overlaps with lipid rafts. IKKs recruited to lipid rafts were actively capable of phosphorylating the recombinant I{kappa}B{alpha} in vitro. Although exogenously expressed PKC{theta} induced IKKß activity in Jurkat cells, it was also possible to show that endogenous PKC{theta} associated with activated IKK complexes in primary human CD4+ T cells. Expression of dominant-active (DA) and dominant-negative (DN) PKC{theta} had stimulatory and inhibitory effects, respectively, on the CD28RE. Taken together, these data suggest that activation of PKC{theta} by TCR and CD28 plays an important role in the assembly and activation of IKK complexes in the T cell membrane.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents

OKT3 (anti-CD3) was obtained from Ortho Pharmaceuticals (Raritan, NJ), and the 9.3 (anti-CD28) and 187.1 mAb were generously provided by Bristol-Meyer Squibb (Princeton, NJ). Polyclonal anti-PKC{theta} and anti-Lck were purchased from Santa Cruz Biotechnology (Santa Cruz, CA), and anti-IKK{alpha}, anti-IKKß, and anti-IKK{gamma} were purchased from PharMingen (San Diego, CA). Anti-Flag (M2) and FITC-conjugated anti-Flag Abs were obtained from Sigma (St. Louis, MO). Tosyl-activated magnetic beads and M-450 anti-CD4 beads were purchased from Dynal (Great Neck, NY). The PKC inhibitors Gö6976 and rottlerin were purchased from Calbiochem (San Diego, CA). Recombinant IL-2 was from Chiron (Emeryville, CA).

Preparation of CD4+ primary T cells

Human CD4+ T cells were purified from PBLs and were stimulated with anti-CD3 and anti-CD28 mAbs coupled to tosyl-activated magnetic beads as previously described (8, 34). After stimulation for ~14 days, beads were removed and cells returned to their resting state over a 48-h time period (8). These rested cells were restimulated as described below.

Cellular stimulation with mAbs and PMA (P) plus ionomycin (I)

The Jurkat T cell clone BMS2 and resting primary human CD4+ T lymphocytes were stimulated with 2 µg/ml each anti-CD3(OKT3) or OKT3 plus anti-CD28 (9.3) mAb, secondarily cross-linked with 10 µg/ml mAb 187.1 for 30 min. Stimulation with P (100 nM) plus I (1 µg/ml) was used as positive control. Alternatively, Jurkat T cells were stimulated by OKT3/9.3 Abs coupled to magnetic beads for 30 min before conducting confocal microscopy.

Gene constructs and cellular transfection

Flag-IKK{alpha} and IKKß (wild-type and kinase-inactive mutants) were provided by Tularik (San Francisco, CA) (19). PKC{theta}, wild-type (wt), DA, and DN (kinase-inactive) were provided by Dr. Baier (University of Innsbruck, Innsbruck, Austria) (31). The CD28RE/AP-1 luciferase reporter was previously described (8, 13). For cellular transfection, we used the indicated amounts of cDNA for electroporation (240 V, 950 µF) as previously described (8, 35)

Immunoprecipitation and Western blot analysis

A total of 2 x 107 primary CD4+ or Jurkat T cells were stimulated with anti-CD3 (aCD3) plus anti-CD28 (aCD28) mAb (2 µg/ml each), secondarily cross-linked with 187.1 (10 µg/ml), or P + I (100 nM and 1 µg/ml, respectively). Cells were lysed in buffer A (50 mM HEPES (pH 7.6), 250 mM NaCl, 1% Triton X-100, 2 mM MgCl2, 2 mM DTT, 0.1 mM Na3VO4, 20 µM ß-glycerophosphate, and 20 µM p-nitrophenylphosphate) and cleared by centrifugation. Lysate (200 µg) was treated with 2 µg of the indicated Abs, bound to protein A-Sepharose, and rocked for 2 h at 4°C. Immunoprecipitated complexes were washed, separated by SDS-PAGE, and transferred to nitrocellulose membrane (8). Membranes were sequentially overlaid with the indicated concentrations of the primary Abs, followed by a 1:2000 dilution of the secondary HRP-conjugated Ab.

Cellular fractionation into detergent-soluble and detergent-insoluble material

Cellular fractionation was performed as previously described (36). Briefly, 1 x 108 primary CD4+ cells were suspended for 15 min in hypotonic buffer E (10 mM Tris (pH 7.4), 10 mM KCl, 1.5 mM MgCl2, 0.2 mM PMSF, 1 mM sodium vanadate, 10 µg/ml leupeptin, and 0.01 TIU/ml aprotonin), then lysed with 50 mechanical strokes in a Dounce homogenizer. Lysates were spun for 30 min at 100,000 x g, and the supernatant, designated cytosol, was collected. The pellet was rinsed twice in buffer E and resuspended in buffer E, containing 1% Nonidet P-40 for 30 min. Samples were recentrifuged at 100,000 x g, and the supernatant, designated detergent-soluble material (DSM), was collected (33). The remaining pellet was rinsed once in buffer E plus detergent, followed by sonication in RIPA buffer (20 mM Tris (pH 7.5), 250 mM NaCl, 10 mM DTT, 10 mM MgCl2, 1% Nonidet P-40, 0.1% SDS, 0.5% sodium deoxycholate), and cleared by centrifugation as described above. This supernatant was designated detergent insoluble material (DIM) (36).

Sucrose gradient fractionation

Aliquots of 1 x 108 Jurkat or primary human CD4+ lymphocytes stimulated or unstimulated were lysed in 1 ml of ice-cold MBS (25 mM MES pH6.5, 150 mM NaCl, 0.5% Triton X-100, 1 mM Na3VO4, 1 mM PMSF, and 10 µg/ml aprotinin). Lysates were homogenized using 15 strokes of a Dounce homogenizer, mixed with an equal volume of 85% sucrose (w/v) in MBS, and transferred to a SW41 centrifuge tube. The mixture was first overlaid with 6 ml of 35% sucrose, then 3 ml of 5% sucrose in MBS with 1 mM Na3VO4, and centrifuged at 200,000 x g for 16 h at 4°C. Following centrifugation, eleven 1-ml fractions were sequentially collected from the top of the centrifuge tube. The low-density fractions (i.e., fractions 2–4) contained lipid raft material, whereas cytosolic material and soluble membranes were predominantly recovered in fractions 9–11. Primary human CD4+ T cells were similarly processed, except cells were stimulated with anti-CD3 + anti-CD28 mAb coupled to magnetic beads.

GM1 ganglioside staining

To determine the efficiency of the isolation of lipid rafts via sucrose gradient centrifugation, 2 µl of each fraction was dot blotted onto Immobilon-P Nitrocellulose Transfer membrane (Millipore, Bedford, MA). After drying, the membrane was blocked in 6% BSA for 1 h at room temperature and washed once in PBS with 0.1% Tween 20 (PBS-T). HRP-linked cholera toxin ß subunit (Sigma) was diluted in PBS-T to 4.2 µg/µl and was used to overlay the membrane for 1 h. The membrane was washed three times, incubated with SuperSignal Chemiluminescent Substrate (Pierce, Rockford, IL), and subjected to autoradiography.

Immune complex kinase assays

A total of 107 Jurkat or resting CD4+ T cells in 1 ml of RPMI were either unstimulated or stimulated with anti-CD3 + CD28 mAb as described above (8). For the effects of PKC inhibitors on IKK activity, CD4+ T cells were incubated with the indicated concentrations of these drugs for 1 h before stimulation. Two hundred microgram lysate was incubated with 2 µg anti-IKK{gamma} Ab, adsorbed onto protein A-Sepharose for 2 h. In vitro kinase assays using GST-I{kappa}B1–55(1–55) as substrate were performed as previously described (8, 17).

Luciferase assays

A total of 107 Jurkat cells were transfected with 10 µg of the consensus NF-{kappa}B or CD28RE luciferase (Luc) reporters in the absence or presence of one of the following constructs: wt-PKC{theta}, DA-PKC{theta}, or DN-PKC{theta}. In separate experiments, CD28RE Luc reporter was coexpressed with DA-PKC{theta} and either DN-IKKß or DN-IKK{alpha}. Cells were rested for 24 h and then stimulated for 6 h with anti-CD3 + CD28 mAb or P + I. Luciferase assays were performed as previously described (8). Transfection efficiency was monitored by cotransfection of a ß-galactosidase plasmid (CMV-ß-gal).

Confocal microscopy

Jurkat T cells were transfected with 10 µg of Flag-IKKß plus 10 µg of wt PKC{theta} by electroporation as described. (35). Cells were rested for 24 h and stimulated for 1 h with anti-CD3 + CD28 mAb coupled to magnetic beads, at a ratio of 4 beads/cell (34). Cells were vigorously resuspended and the magnetic beads were removed. In a separate experiment, Jurkat T cells transfected with Flag-IKKß and DA-PKC{theta} were used without any stimulations. Cells spun onto a glass slide were fixed in 4% glutaraldehyde for 30 min and permeabilized in 70% methanol for 1 h. Slides were overlaid with 5 µg/ml goat anti-PKC{theta} for 2 h. After washing in PBS (three times), slides were treated with 5 µg/ml of PE-conjugated anti-goat plus 5 µg/ml FITC-conjugated anti-Flag for 2 h. Appropriate controls consisted of omitting the primary Abs to rule out nonspecific binding of the secondary reagent. Samples were washed extensively and examined under a LEICA inverted TCS-SP confocal microscope, using a x100 objective lens.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
PKC{theta} plays a role in CD3/CD28-induced activation of IKKs in primary human CD4+ T lymphocytes as well as Jurkat cells

We have previously shown that CD28 costimulation is essential for the activation of heterogeneous IKK complexes, as well as activation of the IL-2 promoter (8). We focused on the role of PKC{theta}, because this is the only PKC isoform that is recruited to the TCR (29, 30). Moreover, it was recently demonstrated by the cellular transfection approach that PKC{theta} plays a role in the activation of the IKKs in Jurkat cells (26, 27). To demonstrate the relevance of these findings in NF-{kappa}B activation in primary human CD4+ T cells, we examined the effect of PKC inhibitors on IKK activation during CD28 costimulation (Fig. 1Go). As previously shown, CD3/CD28 coligation but not anti-CD3 alone, induced IKK activation (Fig. 1Go, lanes 1–3). Rottlerin, a Ca2+-independent PKC isoform inhibitor, interfered in IKK activation (lanes 4–6), whereas Gö6976, a Ca2+-dependent PKC isoform inhibitor, had no effect (lanes 7–9). Similar findings were made in Jurkat cells (data not shown). These data suggest that Ca2+-independent PKCs participate in the activation of IKK complexes by the TCR. These data do not rule out the role of other PKC isoforms in NF-{kappa}B pathway activation, e.g., stimulation by cytokines.



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  		FIGURE 1. Rottlerin blocks anti-CD3/anti-CD28-mediated activation of IKKs. CD4+ T cells were treated with DMSO, 10 µM rottlerin, or 50 nM Gö6976 for 1 h before stimulation with aCD3 or aCD3/aCD28 mAb as described (7 ). After cellular lysis, IKK immunoprecipitates were washed, equilibrated in kinase buffer, and incubated with 2 µg of GST-I{kappa}B{alpha}(1–55) and 10 µCi [{gamma}-32P]ATP. Proteins were separated by SDS-PAGE, followed by autoradiography.

 
Because PKC{theta} has been implicated in CD28 costimulatory events (29, 32), we examined whether this PKC isoform physically associates with the IKK signalsome. PKC{theta} immune precipitates were analyzed for the presence of IKK subunits by Western blotting. Compared with resting CD4+ T cells, which did not show any association between these proteins, a small amount of IKKß coprecipitated with PKC{theta} in anti-CD3-treated cells (Fig. 2GoA). The amount of IKKß coprecipitaiton was dramatically increased by CD28 costimulation (lane 4), which is in agreement with the key role of this receptor in IKK activation (Fig. 1Go). To show the specificity of PKC{theta} in this costimulatory event, we also performed an analysis in which we compared PKC{theta} with PKC{alpha} immune complexes. As shown in Fig. 2GoB, both IKK{alpha} and IKKß coprecipitated with PKC{theta} in anti-CD3 + CD28 or P + I-stimulated CD4+ T cells. In contrast, PKC{alpha} immune complexes did not contain any IKKs under resting or stimulated conditions (Fig. 2GoA), showing a specific role for PKC{theta} in CD3/CD28-induced NF-{kappa}B activation (26, 27). In a reverse protocol, anti-IKK{alpha} immune complexes could be seen to coprecipitate with PKC{theta} in CD3 + CD28 or P + I-treated cells (Fig. 2GoC). Parallel in vitro kinase assays showed that PKC{theta} was associated with active IKK complexes capable of phosphorylating the substrate, I{kappa}B{alpha} (Fig. 2GoD). In contrast, anti-PKC{alpha} precipitates were not associated with active IKK complexes (Fig. 2GoD). Taken together, these data show that PKC{theta} associates with a complex that also includes activated IKKs in CD3/CD28-stimulated T cells. Although we do not know whether this is a direct interaction or an indirect association via intermediary components in a macromolecular complex, other workers have failed to show a direct interaction of PKC{theta} with IKKs (27).



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  		FIGURE 2. PKC{theta} interacts with activated IKK complexes in primary CD4+ T cells. A, Coimmunoprecipitation of PKC{theta} and IKKß. Two hundred micrograms of cleared lysate from resting and stimulated T cells were incubated with 2 µg of anti-PKC{theta} mAb bound to protein A beads for 3 h. Immune complexes were resolved by SDS-PAGE and transferred to nitrocellulose membranes. The membrane was overlaid with anti-IKKß (1:250) and anti-PKC{theta} (1:1000), followed by HRP-conjugated goat-anti-mouse (1;2000 antiserum. B, Same as in A, except that parallel immunoprecipitation was performed with 2 µg anti-PKC{alpha} Ab. The membranes were overlaid with anti-PKC{theta} (1:1000), anti-PKC{alpha} (1:1000), anti-IKK{alpha} (1:1000), and anti-IKKß (1:250) as indicated. The reduced amount of PKC{theta} in lane 2 was not due to a decreased amount of cellular protein, but is the result of steric hindrance by an unknown mechanism in the lysates from unstimulated cells (also see lane 2 in A). This effect was negated by inclusion of a small amount of SDS in the lysis buffer, but could not be used for co-precipitation analysis. C, Same as in B, except IKK complexes were immune precipitated with anti-IKK{alpha} before immunoblotting with a goat-anti PKC{theta} Ab (1:1000). D, In vitro kinase assay on PKC{alpha} and PKC{theta} immunoprecipitates. The same immune complexes as in B were incubated with 2 µg of GST-I{kappa}B{alpha}(1–55) and 10 µCi [{gamma}-32P]ATP. Kinase assays were conducted as described in Fig. 1Go.

 
In an extension of these studies in Jurkat cells, we asked whether coexpression of wt, DA-, or DN-PKC{theta} with epitope-tagged IKKs could induce IKK activity. Although DA-PKC{theta} induced IKKß activity, no activation was obtained with wt or DN-PKC{theta} (Fig. 3Go, top). HA-IKKß was equally expressed in these samples (Fig. 3Go, middle). Blotting for PKC{theta} confirmed overexpression of wt, DA-, and DN-PKC{theta} (Fig. 3Go, bottom). In contrast, a parallel experiment using Flag-IKK{alpha} cotransfection failed to show an effect of PKC{theta} on IKK{alpha} activity (data not shown). This is in agreement with the findings of Lin et al. (27) who showed that DA-PKC{theta} induced the phosphorylation of coexpressed IKKß but not IKK{alpha}. Taken together, the above data show that PKC{theta} plays a key role in IKK activation in primary human CD4+ lymphocytes as well as in overexpressing Jurkat cells.



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  		FIGURE 3. IKKß activation by PKC{theta}. Jurkat cells were cotransfected with 20 µg of wt, DA-, or DN-PKC{theta} plus 2 µg HA-IKKß. Total cDNA was corrected to 22 µg, using an empty vector. After resting for 24 h, cells were lysed, and 200 µg cleared lysate was immunoprecipitated with 2 µg anti-HA mAb bound to protein A-Sepharose beads. The immunoprecipitates were washed, equilibrated in kinase buffer, and incubated with 3 µg GST-I{kappa}B{alpha}(1–55) and 10 µCi [{gamma}-32P]ATP. Proteins were separated by SDS-PAGE, followed by autoradiography (upper panel). Aliquots of the lysates were also transferred to immunoblotting membranes, which were overlaid with 1:1000 anti-HA mAb (middle panel) or anti-PKC{theta} Ab (bottom panel). Blots were developed as described in Fig. 2Go. PKC{theta} overexpression can be seen as increased staining intensity in lanes 2–4 compared with the endogenous protein in lane 1.

 
Evidence that PKC{theta} is involved in the activation of the CD28RE

A biologically relevant target for the CD28 accessory receptor is the IL-2 gene, in particular the CD28RE in the promoter of that gene (7, 13). We have recently shown that IKKß, but not IKK{alpha}, contributes to the activation of that RE during CD3/CD28 costimulation (7, 8). The importance of PKC{theta} in the activation of that RE was confirmed by the use of wt, DA-, or DN-PKC{theta}, which were equally expressed in Jurkat cells together with a CD28RE-Luc reporter (Fig. 4GoA). Although the response of this reporter was minimally affected by wt-PKC{theta}, DA-PKC{theta} itself was sufficient in CD28RE activation (Fig. 4GoB). This activity was further enhanced by CD3 + CD28 or P + I costimulation (Fig. 4GoB). In contrast, DN-PKC{theta} interfered in CD28RE activation (Fig. 4GoB). Similar inhibitory effects on CD28RE activation were obtained with rottlerin, but not Gö6976 (Fig. 4GoC). To demonstrate that the IKKs function downstream of PKC{theta} in the activation of that RE, we used DN-IKK{alpha} and DN IKKß cotransfection with DA-PKC{theta}. Although DN-IKKß interfered in the DA-PKC{theta}-induced response, DN-IKK{alpha} had minimal effects on CD28RE-Luc activity (Fig. 4GoD). These findings are in agreement with the ability of DA-PKC{theta} to induce IKKß, but not IKK{alpha}, activation under cotransfection conditions (Fig. 3Go).



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  		FIGURE 4. Luciferase assay showing the effect of PKC{theta} on the activation of the CD28RE. A total of 107 Jurkat cells were transfected with 15 µg empty vector (pEF), wt-PKC{theta}, DA-PKC{theta}, or DN-PKC{theta} together with 6 µg CD28RE-Luc and 6 µg CMV-ß-gal (12 ). Alternatively, cells were cotransfected with 10 µg DA-PKC{theta} and 10 µg empty vector (pRK), DN-IKK{alpha}, or DN-IKKß plus 6 µg CD28RE-Luc and 6 µg CMV-ß-gal. After resting for 24 h, an aliquot of each transfection was examined for PKC{theta} expression. The rest of the cells was washed, divided into aliquots, and stimulated with anti-CD3 + CD28 mAb or P + I, as indicated, for 6 h. Cells were washed, lysed, and 25 µg of each lysate was used for luciferase assays as described in Materials and Methods. Luciferase activity was standardized by using the ß-gal assay. Similar results were observed in two additional experiments. A, PKC{theta} immunoblotting performed by sequential overlay with 1:1000 anti-PKC{theta} and 1:2000 HRP-conjugated goat-anti-mouse antiserum. Overexpression can be seen as increased staining intensity in lanes 2–4 compared with the endogenous protein in lane 1. B, Luciferase assay showing effect of wt- and mutant PKC{theta} constructs. C, Luciferase assay showing the effect of 10 µM rottlerin or 50 nM Gö6976 incubated for 1 h before the addition of anti-CD3 + CD28 mAb for 6 h. D, Luciferase assay showing the effect of DN-IKK{alpha} or DN-IKKß on CD28RE-Luc activity.

 
PKC{theta} relocates to a detergent-resistant membrane compartment together with talin and IKKs

Because PKC{theta} is recruited to the signal assembly complex at the TCR (29, 30), it raised the question whether IKKs and their associated components are also recruited to that membrane site. We have previously shown that TCR ligation leads to the translocation of several signaling components, including the {zeta}-chain of the TCR, cytoskeletal components, p56lck, p36(LAT), PLC-{gamma}1, and ZAP-70 to DSM and DIM (36). Interestingly, the same signaling molecules are recruited to the SMAC, where PKC{theta} localizes in the center cluster, whereas talin appears in the peripheral zone (29, 30). Talin is a member of the cytoskeletal complex that assembles at the TCR synapse (37). To determine whether PKC{theta} and talin are corecruited to the DIM, we fractionated primary human CD4+ T cells into cytosolic DSM and DIM components. Western blot analysis showed talin relocation from the cytosol to the DIM during CD3 + CD28 or P + I, and, to a lesser extent, anti-CD3 stimulation (Fig. 5GoA). Although endogenous PKC{theta} underwent similar redistribution to the DIM during CD3 + CD28 or P + I stimulation (Fig. 3Go), this kinase also relocated to the DSM under these stimulatory conditions (Fig. 5GoA). In a related set of experiments, we also asked whether IKK components relocate to these cellular fractions. IKK{alpha}, IKKß, and IKK{gamma} were recruited to the DIM, and to a variable degree the DSM, during cellular stimulation with anti-CD3 + CD28 or P + I (Fig. 5GoB).



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  		FIGURE 5. Relocation of PKC{theta}, talin, and IKKs to the DSM and DIM from stimulated human CD4+ T lymphocytes. A total of 1 x 108 primary human CD4+ T lymphocytes were stimulated with anti-CD3, anti-CD3 + CD28, or P + I for 30 min before fractionation into cytosolic, DSM, and DIM as described in Materials and Methods. Seventy-five micrograms from each cytosolic fraction and a representative aliquot (same number of cells) from DSM and DIM were resolved by SDS-PAGE and processed for Western blotting. A, Western blot for PKC{theta} and talin using 1:1000 dilution of the primary Abs. These results were reproduced once in primary lymphocytes and once in Jurkat cells. B, Western blot for IKK{alpha}, IKKß, and IKK{gamma} using 1:1000 dilution of the primary Abs on the same fractions analyzed in A. Above results were reproduced once.

 
Recently, it has been shown that lipid rafts are important in controlling the assembly of signaling molecules at the TCR synapse. Moreover, co-cross-linking of the CD28 receptor enhances the clustering of lipid rafts at the TCR synapse, thereby enhancing TCR signaling (1, 5, 38). Lipid rafts are defined by their insolubility in cold, nonionic detergents, unlike the bulk of the cell membrane. Because this suggests that DIM may overlap with lipid rafts, we used sucrose density fractionation to prepare raft fractions from unstimulated and anti-CD3 + CD28-treated Jurkat cells. Using the cholera toxin ß-subunit to identify the fractions containing GM1 ganglioside, we showed that lipid rafts were mostly present in the hypodense layers (fractions 3–5) from resting as well as stimulated T cells (Fig. 6GoA). Immunoblotting showed PKC{theta} and talin recruitment to the hypodense fractions during cellular stimulation (Fig. 6GoB). Although PKC{theta} and talin did not appear in the low density fractions from unstimulated cells, these fractions did show the constitutive presence of Lck, which was further increased by CD3 + CD28 costimulation (Fig. 6GoB). Moreover, Western blotting for IKKß and IKK{alpha} showed their relocation to lipid rafts in CD3 + CD28-treated cells (Fig. 6GoB). Taken together, these data indicate that IKK components are recruited to lipid rafts in parallel with PKC{theta} and talin.



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  		FIGURE 6. Recruitment of PKC{theta}, talin, and IKK{alpha} to lipid rafts. A, 108 Jurkat T cells were used for control and anti-CD3 + CD28-stimulated samples. Each sample was separated into 11 fractions after being subjected to sucrose gradient centrifugation as described in the Materials and Methods. Fractions 2–4 contain low density lipid rafts, whereas fractions 9–11 contain cytosolic and soluble membrane material. Two microliters of each fraction were dot-blotted onto Immobilon-P membrane and treated with HRP-linked cholera toxin ß subunit to detect the ganglioside GM1 as described in the Materials and Methods. B, Equal cellular amounts (60 µl) of fractions 1, 3, 4, 6, 10, and 11 were loaded onto SDS-PAGE gels and processed for Western blotting for PKC{theta}, talin IKK{alpha}, IKKß, and Lck using 1:1000 dilution of the primary Abs. C, Primary human CD4+ T cells were expanded by magnetic beads coupled to anti-CD3/CD28 Abs. A total of 1 x 108 proliferating cells were processed for lipid raft separation as described in A. Top row shows Western blotting of the sucrose gradient fractions for IKKß. Lower panel demonstrates the activity of IKKs recruited to the lipid rafts. Kinase activity was examined on anti-IKK{gamma} immunoprecipitates as described in the legend of Fig. 1Go.

 
Presence of active IKK complexes in lipid rafts from primary human CD4+ T cells

Although for logistic reasons the entire experiment in Fig. 6GoB could not be repeated in primary CD4+ T cells, we did perform sucrose gradient fractionation on CD3 + CD28-stimulated primary T cells. As for Jurkat cells, the lipid rafts were identified by cholera toxin staining and shown to reside in fractions 3–5. Immunoblotting showed the presence of IKKs in these low density fractions, in addition to their presence in the DSM (fractions 10 and 11; Fig. 6GoC). Moreover, immune precipitation with anti-IKK{gamma} and performance of in vitro kinase assays showed the presence of activated IKK complexes in fractions 2–4 as well as 10 and 11 (Fig. 6GoC, lower panel). These findings indicate that lipid rafts from CD3/CD28 costimulated T cells contain activated IKK complexes. Similar results were obtained in Jurkat T cells.

Exogenously expressed PKC{theta} and IKKß relocate and colocalize in the membranes of Jurkat T cells

To confirm the recruitment of PKC{theta} and IKKs to the membranes, we used confocal microscopy. Exogenously transfected wt-PKC{theta} and Flag-IKKß were expressed in a diffuse manner in unstimulated cells (Fig. 7Go). However, upon stimulation with anti-CD3 + CD28-conjugated beads, both PKC{theta} and IKKß were redistributed to membrane cap sites (Fig. 7Go). Overlay of the green (IKKß) and red (PKC{theta}) images yielded a composite yellow fluorescent pattern, which indicates colocalization of these proteins in the membrane cap sites of stimulated cells (Fig. 7Go). When cotransfected with DA-PKC{theta}, Flag-IKKß colocalized in the membrane in a diffuse distribution without the need for cellular stimulation (Fig. 7Go, right panel). These data suggest that PKC{theta} activity is involved in the membrane recruitment of the IKK signalsome, which is also consistent with coimmunoprecipitation studies shown in Fig. 2Go. In accordance with this notion, DN-PKC{theta} resides in the cytosol of unstimulated cells, similar to wt-PKC{theta} (data not shown). Although endogenous PKC{theta} undergoes a similar redistribution as exogenously expressed PKC{theta} during CD28 costimulation (data not shown), it was not possible with the available anti-IKK Abs to obtain a strong enough signal to study endogenous IKK redistribution. This was, however, achieved by the biochemical and coimmunoprecipitation studies shown in Figs. 2Go and 6Go.



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  		FIGURE 7. DA-PKC{theta} induces IKKß relocation to the surface membrane. A, Jurkat T cells were transfected with IKKß/wt-PKC{theta} (15 µg each) or IKKß/DA-PKC{theta} (10 µg each). After resting for 24 h, an aliquot of the former population was stimulated with anti-CD3 + anti-CD28 mAb conjugated to microspheres (4 beads/cell) for 1 h. Cytospin preparations of cells were stained for PKC{theta} using a goat anti-PKC{theta} (5 µg/ml) primary Ab followed by a PE-conjugated rabbit anti-goat serum. IKKß was stained with a FITC-conjugated anti-Flag antiserum (5 µg/ml each). Slides were examined under a LEICA Confocal Microscope. These images are representative of >80% of the DA-PKC{theta}/Flag-IKKß coexpressing cells, whereas > 50% of the cells transfected with wt-PKC{theta}/Flag-IKKß showed relocation upon stimulation.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this paper, we have demonstrated that PKC{theta} participates in CD3/CD28-induced activation of IKKs in primary human CD4+ lymphocytes. DN-PKC{theta} and a Ca2+-independent PKC inhibitor interfered in the activation of the IKK complexes as well as the CD28RE of the IL-2 promoter. Moreover, IKKs and PKC{theta} were recruited from the cytosol to a talin-enriched, detergent-resistant cellular compartment during CD28 costimulation. Similarly, PKC{theta}, talin, and IKKs were recruited to GM1-enriched lipid rafts, which represent a detergent resistant membrane compartment. IKKs recruited to the lipid rafts were active. It was also possible to show that the activated IKK signalsome coimmunoprecipitate with PKC{theta} from CD3/CD28 costimulated primary CD4+ lymphocytes. Moreover, active PKC{theta} and IKKß were recruited to and colocalized in the membrane of stimulated Jurkat T cells as determined by confocal microscopy. It was also demonstrated that exogenously expressed DA-PKC{theta} activated IKKß, but not IKK{alpha}, in Jurkat cells. Similarly, DN-IKKß, but not DN-IKK{alpha}, interfered in the activation of the CD28RE by DA-PKC{theta}. Taken together, these findings demonstrate stimulus-dependent interaction between PKC{theta} and IKK components, and present a novel mechanism to explain synergistic activation of IKKs by TCR and CD28.

Although some components of the IKK signalsome have been identified, its overall composition and regulation have not been fully characterized (17, 18, 19, 20, 21, 22, 39). Indeed, the evolving scenario points to heterogeneous IKK complexes responding to different stimuli (24, 39, 40), including TCR/CD28 costimulation in primary T cells (8). Stimulus-dependent assembly and activation of IKK complexes is an attractive model, considering the multitude of stimuli involved in NF-{kappa}B activation (24, 40). Four different serine-threonine kinases have now been implicated in the activation of the IKKs by CD3/CD28, namely MEKK1, NF-{kappa}B-inducing kinase, Cot, and PKC{theta} (7, 25, 26, 27, 28). Although our overexpression studies in Jurkat cells confirm the recently published data about the role of PKC{theta} in IKK activation (Fig. 3Go) (26, 27), the current communication extends those findings by showing the recruitment and colocalization of PKC{theta} and IKKs in the membrane of T cells ( Figs. 5–7GoGoGo). Moreover, we demonstrate that PKC{theta} and IKKs are physically associated with a macromolecular complex that can be reversibly precipitated with Abs to PKC{theta} or IKKs from lysates prepared from CD3 + CD28-stimulated cells (Fig. 2Go). Coudronniere et al. (26) recently reported that rottlerin, an inhibitor of novel PKC isoforms, interfered in the activation of the NF-{kappa}B pathway and the CD28RE by the TCR. We show here that treatment of primary human CD4+ with rottlerin inhibits the CD3/CD28-mediated activation of endogenous IKK complexes (Fig. 1Go). Gö6976, an inhibitor of classical PKC isoforms, had no effects on IKK activity and transcriptional activation of the CD28RE (Figs. 1Go and 4Go). Although the role of other PKC isoforms in the activation of the NF-{kappa}B pathway has been demonstrated, our findings implicate PKC{theta} in IKK activation and recruitment during CD3/CD28 costimulation T cells. We found that endogenous PKC{theta} physically associated with the IKK signalsome in the cell membrane (Figs. 2Go and 7Go). Although this interaction was minimal affected by of CD3 ligation, the association was markedly augmented by CD28 costimulation (Fig. 2GoA). Although the importance of PKC{theta} in IKK activation was recently reported (26, 27), this is the first evidence for CD3/CD28-induced association of PKC{theta} with the IKK complex in a physiologically relevant system. Although a previous study could not demonstrate that PKC{theta} directly interacts with IKK components (27), it is possible that this interaction involve additional components of a macromolecular complex, e.g., cytoskeletal or scaffolding proteins. This aspect requires further study.

We propose that PKC{theta}/IKK association takes place in the immunological synapse. PKC{theta} along with IKK{alpha}, IKKß, and IKK{gamma} were recruited to the membrane fractions and lipid rafts in primary human CD4+ lymphocytes (Figs. 5Go and 6Go). Lipid rafts, which are cholesterol- and sphingolipid-enriched membrane domains, are docking sites for the attachment of costimulatory receptors and signaling molecules in the cell membrane (1, 29, 30, 38). Formation of the TCR synapse and the assembly of productive signaling complexes in the lipid rafts require reorganization of the cortical cytoskeleton (30, 35), and may explain our finding that the IKKs and PKC{theta} are recruited to a detergent-insoluble cellular compartment that includes talin and spectrin (Figs. 5Go and 6Go). CD28 participates in the organization of lipid rafts and recruitment of the associated signaling molecules to the TCR synapse (1, 5, 38). Recruitment of the IKK components to the lipid rafts represents a novel aspect of CD28 function, which may also be responsible for activation of the IKK complex (Fig. 6GoC) and the NF-{kappa}B pathway. The mechanism of CD28-induced recruitment of signaling molecules to the lipid rafts is poorly understood. However, recent studies have demonstrated that CD28 contributes to the rearrangement of the cytoskeletal complex at the TCR contact site via the activation of Vav (41). Vav has been shown to participate in the recruitment of PKC{theta} to the T cell membrane and the cytoskeleton during TCR engagement (41) We hypothesize that this process is essential for the recruitment of IKKs to the lipid rafts. Although ectopically expressed wt PKC{theta} and IKKß colocalized in the membrane after CD3/CD28 costimulation, DA-PKC{theta} and IKKß spontaneously localized in the membrane in the absence of any stimuli (Fig. 7Go). This suggests that, in addition to its role in IKK activation (26, 27), PKC{theta} may also play a role in the recruitment of IKKs to the T cell membrane. The idea that the IKKs are activated in the vicinity of the TCR is further strengthened by the demonstration that TNF-{alpha} induces the IKK holoenzyme to associate with components of the p55 TNF receptor site in the cytoplasmic membrane (42).

In conclusion, we have shown that, during CD28 costimulation, PKC{theta}-induced IKK activation in primary human CD4+ T cells involves their physical interaction with an activation complex that localizes in the T cell membrane. This may represent an early event in the activation of the NF-{kappa}B cascade during CD3/CD28 costimulation. The signaling molecules that attract IKKs to the lipid rafts remain to be identified. However, because DA-PKC{theta} facilitated the relocation of IKKß to the membranes (Fig. 7Go), it is tempting to speculate that PKC{theta} plays an active role in this process. PKC{theta} activity has also been linked to JNK activation, and this pathway synergizes with the NF-{kappa}B cascade in the activation of the IL-2 promoter (7, 31, 32, 33). In this regard, PKC{theta} may act as a master switch responsible for the regulation of key signaling pathways involved in the delivery of signal two.


   Acknowledgments
 
We thank Ivonne Castaneda and Peggy Shih for secretarial assistance and Martin Kasubowski for technical support.


   Footnotes
 
1 This study was supported by the U.S. Public Health Service (AG14992). A.K. was supported by National Institute of Allergy and Infectious Diseases-funded University of California, Los Angeles Immunology Training Grant. Back

2 D.B. and C.A.T. contributed equally to this work. Back

3 Address correspondence and reprint requests to Dr. Andre Nel, Department of Medicine, Division of Clinical Immunology and Allergy, 10833 Le Conte Avenue 52-175 CHS, University of California, Los Angeles, CA 90095. Back

4 Abbreviations used in this paper: SMAC, supramolecular activation cluster; PKC, protein kinase C; JNK, c-Jun N-terminal kinase; I{kappa}B, inhibitor of {kappa}B factor; IKK, I{kappa}B kinase; RE, response element; P, PMA; I, ionomycin; Luc, luciferase; DIM, detergent-insoluble material; DSM, detergent-soluble material; wt, wild type; DN, dominant negative; DA, dominant active; ß-gal, ß-galactosidase. Back

Received for publication June 8, 2000. Accepted for publication September 18, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Viola, A., S. Schroeder, Y. Sakakibara, A. Lanzavecchia. 1999. T lymphocyte costimulation mediated by reorganization of membrane microdomains. Science 283:680.[Abstract/Free Full Text]
  2. Kaga, S., S. Ragg, K. A. Rogers, A. Ochi. 1998. Cutting edge: stimulation of CD28 with B7-2 promotes focal adhesion-like cell contacts where Rho family small G proteins accumulate in T cells. J. Immunol. 160:24.[Abstract/Free Full Text]
  3. Grakoui, A., S. K. Bromely, C. Sumen, M. M. Davis, A. S. Shaw, P. M. Allen, M. L. Dustin. 1999. The immunological synapse: a molecular machine controlling T cell activation. Science 285:221.[Abstract/Free Full Text]
  4. Xavier, R., T. Brennan, Q. Li, C. McCormack, B. Seed. 1998. Membrane compartmentation is required for efficient T cell activation. Immunity 8:723.[Medline]
  5. Malissen, B. Dancing the immunological two-step.. 1999. Science 285:207.[Free Full Text]
  6. Janes, P. W., S. C. Ley, A. I. Magee, P. S. Kabouridis. 2000. The role of lipid rafts in T cells antigen receptor (TCR) signaling. Semin. Immunol. 12:23.[Medline]
  7. Kempiak, S. J., T. S. Hiura, A. E. Nel. 1999. The Jun kinase cascade is responsible for activating the CD28 response element of the IL-2 promoter: proof of cross-talk with the I{kappa}B kinase cascade. J. Immunol. 162:3176.[Abstract/Free Full Text]
  8. Khoshnan, A., S. J. Kempiak, B. L. Bennett, D. Bae, W. Xue, A. M. Manning, C. H. June., A. E. Nel. 1999. Primary CD4+ T cells contain heterogeneous I{kappa}B kinase complexes: role in the activation of the IL-2 promoter. J. Immunol. 163:5444.[Abstract/Free Full Text]
  9. Lee, F. S., R. T. Peters, L. C. Dang, T. Maniatis. 1998. MEKK1 activates both I{kappa}B kinase {alpha} and I{kappa}B kinase ß. Proc. Natl. Acad. Sci. USA 95:9319.[Abstract/Free Full Text]
  10. Nakano, H., M. Shindo, S. Sakon, S. Nishinaka, M. Mihara, H. Yagita, K. Okumura. 1998. Differential regulation of I{kappa}B kinase {alpha} and ß by two upstream kinases, NF-{kappa}B-inducing kinase and mitogen-activated protein kinase/ERK kinase kinase-1. Proc. Natl. Acad. Sci. USA 95:3537.[Abstract/Free Full Text]
  11. Rudd, C. E.. 1996. Upstream-downstream: CD28 cosignaling pathways and T cell function. Immunity 4:527.[Medline]
  12. Powell, J. D., J. A. Ragheb, S. Kitagawa-Sakakidas, R. H. Schwartz. 1998. Molecular regulation of interleukin-2 expression by CD28 co-stimulation and anergy. Immunol. Rev. 165:287.[Medline]
  13. Shapiro, V. S., K. E. Truitt, J. B. Imboden, A. Weiss. 1997. CD28 mediates transcriptional upregulation of the interleukin-2 (IL-2) promoter through a composite element containing the CD28RE and NF-IL-2B AP-1 sites. Mol. Cell. Biol. 17:4051.[Abstract]
  14. Traenckner, E. B., H. L. Pahl, T. Henkel, K. N. Schmidt, S. Wilk, P. A. Baeuerle. 1995. Phosphorylation of human I{kappa}B-{alpha} on serines 32 and 36 controls I{kappa}B-{alpha} proteolysis and NF-{kappa}B activation in response to diverse stimuli. EMBO J. 14:2876.[Medline]
  15. Zandi, E., Y. Chen, M. Karin. 1998. Direct phosphorylation of I{kappa}B by IKK{alpha} and IKKß: discrimination between free and NF-{kappa}B-bound substrate. Science 281:1360.[Abstract/Free Full Text]
  16. Didonato, J. A., M. Hayakawa, D. M. Rothwarf, E. Zandi, M. Karin. 1997. A cytokine-responsive I{kappa}B kinase that activates the transcription factor NF-{kappa}B. Nature 388:548.[Medline]
  17. Mercurio, F., H. Zhu, B. W. Murray, A. Shevchenko, B. L. Bennett, J. Li, D. B. Young, M. Barbosa, M. Mann, A. M. Manning. 1997. IKK-1 and IKK-2: cytokine-activated I{kappa}B kinases essential for NF-{kappa}B activation. Science 278:860.[Abstract/Free Full Text]
  18. Regnier, C. H., H. Y. Song, X. Gao, D. Goeddel, Z. Cao, M. Rothe. 1997. Identification and characterization of an I{kappa}B kinase. Cell 90:373.[Medline]
  19. Woronicz, J. D., X. Gao, Z. Cao, M. Rothe, D. Goeddel. 1997. I{kappa}B kinase-B: NF-{kappa}B activation and complex formation with I{kappa}B kinase-{alpha} and NIK. Science 278:866.[Abstract/Free Full Text]
  20. Zandi, E., D. M. Rothwarf, M. Delhase, M. Hayakawa, M. Karin. 1997. The I{kappa}B kinase complex (IKK) contains two kinase subunits, IKK{alpha} and IKKß, necessary for I{kappa}B phosphorylation and NF-{kappa}B activation. Cell 91:243.[Medline]
  21. Rothwarf, D. M., E. Zandi, G. Natoli, M. Karin. 1998. IKK-gamma is an essential regulatory subunit of the I{kappa}B kinase complex. Nature 395:297.[Medline]
  22. Mercurio, F., B. W. Murray, A. Shevchenko, B. L. Bennet, D. B. Young, J. W. Li, G. Pascual, A. Motiwala, H. Zue, M. Mann, A. M. Manning. 1999. I{kappa}B Kinase (IKK)-associated protein 1, a common component of the heterogeneous IKK complex. Mol. Cell. Biol. 19:1526.[Abstract/Free Full Text]
  23. Ling, L., Z. Cao, D. Goeddel. 1998. NF-{kappa}B-inducing kinase activates IKK-{alpha} by phosphorylation of Ser-176. Proc. Natl. Acad. Sci. USA 95:3792.[Abstract/Free Full Text]
  24. Karin, M.. 1999. The beginning of the end: I-{kappa}B kinase (IKK) and NF-{kappa}B activation. J. Biol. Chem. 274:27339.[Free Full Text]
  25. Lin, X., Jr E. T. Cunningham, Y. Mu, R. Geleziunas, W. C. Greene. 1999. The proto-oncogene Cot kinase participates in CD3/CD28 induction of NF-{kappa}B acting through the NF-{kappa}B-inducing kinase and I{kappa}B kinases. Immunity 10:271.[Medline]
  26. Coudronniere, N., M. Villalba, N. Englund, A. Altman. 2000. NF-{kappa}B activation induced by T cell receptor/CD28 costimulation is mediated by protein kinase C-{theta}. Proc. Natl. Acad. Sci. USA 97:3394.[Abstract/Free Full Text]
  27. Lin, X., A. O’Mahony, Y. Mu, R. Geleziunas, W. C. Greene. 2000. Protein kinase C-{theta} participates in NF-{kappa}B activation induced by CD3-CD28 costimulation through selective activation of I{kappa}B kinase ß. Mol. Cell. Biol. 20:2933.[Abstract/Free Full Text]
  28. Sun. Z., C. W., W. Arendt, E. M. Ellmeier, M. J. Schaeffer, L. Sunshine, J. Gandhi, D. Anne, A. Petrzilka, P. L. Kupfer, P. L. Schwartzberg, D. R. Littman. 2000. PKC-{theta} is required for TCR-induced NF-{kappa}B activation in mature but not immature T lymphocytes. Nature 404:402.[Medline]
  29. Monks, C. R. F., H. Kupfer, I. Tamir, A. Barlow, A. Kupfer. 1997. Selective modulation of protein kinase C-{theta} during T cell activation. Nature 385:83.[Medline]
  30. Monks, C. R. F., B. A. Freiberg, H. Kupfer, N. Sciaky, A. Kupfer. 1998. :Three-dimentional segregation of supramolecular activation clusters in T cells. Nature 395:82.[Medline]
  31. Ghaffari-Tabrizi, N., B. Bauer, A. Villunger, G. Baier-Bitterlich, A. Altman, G. Utermann, F. Uberall, G. Baier. 1999. Protein kinase C {theta}, a selective upstream regulator of JNK/SAPK and IL-2 promoter activation in Jurkat T cells. Eur. J. Immunol. 29:132.[Medline]
  32. Werlen, G., E. Jacinto, Y. Xia, M. Karin. 1998. Calcineurin preferentially synergizes with PKC-{theta} to activate JNK and IL-2 promoter in T lymphocytes. EMBO J. 17:3101.[Medline]
  33. Ayelet, A., S. Jung, Y. Samuels, R. Seger, Y. Ben-Neriah. 1998. Co-stimulation-dependent activation of a JNK-kinase in T lymphocytes. Eur. J. Immunol. 28:2320.[Medline]
  34. Levine, B. L., W. D. Bernstein, M. Connors, N. Craighead, T. Lindsten, C. B. Thompson, C. H. June. 1997. Effects CD28 costimulation on long-term proliferation of CD4+ T cells in the absence of exogenous feeder cells. J. Immunol. 159:5921.[Abstract]
  35. Faris, M., N. Kokot, L. Lee, A. E. Nel. 1996. Regulation of interleukin-2 transcription by inducible stable expression of dominant negative and dominant active mitogen-activated protein kinase kinase kinase in Jurkat T cells. Evidence for the importance of Ras in a pathway that is controlled by dual receptor stimulation. J. Biol. Chem. 271:27366.[Abstract/Free Full Text]
  36. Nel, A. E., S. Gupta, L. Lee, J. A. Ledbetter, S. B. Kanner. 1995. Ligation of the T-cell antigen receptor (TCR) induces association of hSos1, ZAP-70, phospholipase C-{gamma}1, and other phosphoproteins with Grb2 and the Z-chain of the TCR. J. Biol. Chem. 270:18428.[Abstract/Free Full Text]
  37. Penninger, J. M., G. R. Crabtree. 1999. The actin cytoskeleton and lymphocyte activation. Cell 96:9.[Medline]
  38. Montixi, C., C. Langlet, A-M. Bernard, J. Thimonier, C. Dubios, M-A. Wurbel, J-P. Chauvin, M. Pierres, H-T. He. 1998. Engagement of T cell receptor triggers its recruitment to low-density detergent-insoluble membrane domains. EMBO J. 17:5334.[Medline]
  39. Zandi, E., M. Karin. 1999. Bridging the gap: composition, regulation, and physiological function of the I{kappa}B kinase complex. Mol. Cell. Biol. 19:4547.[Free Full Text]
  40. Mercurio, F., A. M. Manning. 1999. Multiple signals converging on NF-{kappa}B. Curr. Opin. Cell. Biol. 11:226.[Medline]
  41. Villalba, M., N. Coudronniere M. Deckert E. Teixeiro, P. Mas, A. Altman. 2000. A novel functional interaction between Vav and PKC{theta} is required for TCR-induced T cell activation. Immunity 12:151.[Medline]
  42. Zhang, S. Q., A. Kovalenko, G. Cantarella, D. Wallach. 2000. Recruitment of the IKK signalosome to the p55 TNF receptor: RIP and A20 bind to NEMO (IKK{gamma}) upon receptor stimulation. Immunity 12:301.[Medline]



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