The JI PBL Intereron Source
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Related articles in The JI
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by von Essen, M.
Right arrow Articles by Geisler, C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by von Essen, M.
Right arrow Articles by Geisler, C.
The Journal of Immunology, 2006, 176: 7502-7510.
Copyright © 2006 by The American Association of Immunologists

Protein Kinase C (PKC){alpha} and PKC{theta} Are the Major PKC Isotypes Involved in TCR Down-Regulation1

Marina von Essen*, Martin W. Nielsen*, Charlotte M. Bonefeld*, Lasse Boding*, Jeppe M. Larsen*, Michael Leitges{dagger}, Gottfried Baier{ddagger}, Niels Ødum* and Carsten Geisler2,*

* Institute of Medical Microbiology and Immunology, University of Copenhagen, Copenhagen, Denmark; {dagger} Medical University, Hanover, Germany; and {ddagger} Department for Medical Genetics, Molecular and Clinical Pharmacology, Innsbruck Medical University, Innsbruck, Austria


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
It is well known that protein kinase C (PKC) plays an important role in regulation of TCR cell surface expression levels. However, eight different PKC isotypes are present in T cells, and to date the particular isotype(s) involved in TCR down-regulation remains to be identified. The aim of this study was to identify the PKC isotype(s) involved in TCR down-regulation and to elucidate the mechanism by which they induce TCR down-regulation. To accomplish this, we studied TCR down-regulation in the human T cell line Jurkat, in primary human T cells, or in the mouse T cell line DO11.10 in which we either overexpressed constitutive active or dominant-negative forms of various PKC isotypes. In addition, we studied TCR down-regulation in PKC knockout mice and by using small interfering RNA-mediated knockdown of specific PKC isotypes. We found that PKC{alpha} and PKC{theta} were the only PKC isotypes able to induce significant TCR down-regulation. Both isotypes mediated TCR down-regulation via the TCR recycling pathway that strictly depends on Ser126 and the di-leucine-based receptor-sorting motif of the CD3{gamma} chain. Finally, we found that PKC{theta} was mainly implicated in down-regulation of directly engaged TCR, whereas PKC{alpha} was involved in down-regulation of nonengaged TCR.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The TCR is a multisubunit receptor complex composed of the Ag recognizing TCR{alpha}beta dimer (1, 2), and the CD3{gamma}{epsilon}, CD3{delta}{epsilon}, and TCR{zeta}{zeta} dimers responsible for signal delivery to the interior of the cell (3, 4). Cell surface expression of TCR is a dynamic process. During steady state, the amount of surface-expressed TCR is dependent on protein turnover (5) as well as constitutive cycling of the receptor (6, 7). At least two different, but interrelated pathways of TCR down-regulation are induced when the TCR is engaged by specific MHC/peptide complexes, anti-TCR Abs, or other ligands (8). One pathway depends on tyrosine kinase activity, Cbl, and ubiquitin, and leads to TCR degradation in the lysozymes (9, 10, 11, 12, 13, 14). The other pathway is dependent on protein kinase C (PKC)3-mediated activation of the CD3{gamma} dileucine-based (diL) receptor-sorting motif and leads to TCR recycling (7, 15). In addition to down-regulation of directly engaged TCR, a second mechanism of TCR down-regulation exists. This process involves down-regulation of nonengaged TCR and is called TCR comodulation (16, 17, 18, 19, 20). It has been shown recently that TCR comodulation is dependent on PKC (21).

To date, nine distinct PKC isotypes have been described. Of these, six have a binding site for phorbol ester/1,2-diacylglycerol (DAG) and are present in T cells (PKC{alpha}, -beta, -{delta}, -{epsilon}, -{eta}, and -{theta}) (22). In the majority of studies describing the role of PKC in TCR down-regulation phorbol esters, like phorbol 12,13-dibutyrate (PDB) and PMA, have been used to activate PKC. With the present knowledge on phorbol ester substrates, it is most likely that the six different PKC isotypes as well as other proteins, for instance, chimaerin (23) and Ras exchange factor RasGRP3 (24), became activated by the phorbol esters applied in these studies. In several PKC studies, commercial PKC inhibitors (e.g., rottlerin and members of the Ro, Bis, and Gö series of inhibitors) were also applied. However, these inhibitors have been found to inhibit a broad spectrum of kinases other than PKC (25, 26), and hence, they are not reliable as tools for determining the involvement of specific PKC isotypes in cellular processes. In addition to the six PKC isotypes with binding sites for phorbol ester/DAG, T cells express two PKC isotypes (PKC{zeta} and -{iota}) without this binding site. Thus, the specific role in TCR down-regulation of each of the eight PKC isotypes expressed in T cells remains to be determined.

The aim of this study was to identify the PKC isotypes involved in TCR down-regulation and to elucidate the mechanism by which they induce TCR down-regulation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Cells and Abs

The human Jurkat T cell line JTag, the B cell line Raji (American Type Culture Collection), and a stable transfectant of the mouse T cell hybridoma DO11.10 expressing two different TCR comprising either Vbeta2 or Vbeta8 (21) were cultured in complete medium (C medium): RPMI 1640 medium (Sigma-Aldrich) supplemented with 10% FBS (Invitrogen Life Technologies), 2 x 105 U/L penicillin (Leo Pharmaceuticals Products), and 50 mg/ml streptomycin (Merck) at 37°C in 5% CO2. Primary human Vbeta8+ T cells were generated from freshly isolated PBL and expanded in culture, as previously described (27). Mouse lymph node cells from 8-wk-old wild-type (WT), PKC{alpha} (–/–), and PKC{theta} (–/–) knockout (KO) mice were expanded in culture for 3 days at 1 x 106 cells/ml in C medium supplemented with 3.2 ng/ml murine rIL-2 (R&D Systems), 0.5 µM 2-ME, and 4 x 105 mouse CD3-CD28 expander beads/ml (Dynal Biotech). Production and genotyping of the PKC{theta} (28) and PKC{alpha} (29) KO are described elsewhere. Abs against the TCR included mAb F101.01 (anti-TCR) (30), mAb UCHT1 (anti-CD3{epsilon}; DakoCytomation), pAb A452 (anti-CD3{epsilon}; DakoCytomation), mAb BV8 (anti-Vbeta8; BD Biosciences), mAb BV3 (anti-Vbeta3; BD Biosciences), mAb Vbeta2 and Vbeta8 (BD Biosciences), mAb 6B10.2 (anti-TCR{zeta}; Santa Cruz Biotechnology), and H57-597 (anti-TCRbeta; BD Biosciences). Other Abs used included mAb against PKC{alpha}, PKC{theta}, PKCbeta, PKC{delta}, PKC{epsilon}, PKC{zeta}, and PKC{iota} (BD Biosciences); W6beta2 (anti-panMHC-I, a gift from S. Buus, University of Copenhagen, Copenhagen, Denmark); mAb GFP (BD Clontech); mAb CD28 (BD Biosciences); mAb fyn (Santa Cruz Biotechnology); PE-conjugated goat anti-mouse IgG, Cy5-conjugated donkey anti-mouse IgG, and FITC-conjugated goat anti-Armenian hamster IgG (Jackson ImmunoResearch Laboratories); and HRP-conjugated swine anti-rabbit Ig and HRP-conjugated rabbit anti-mouse Ig (DakoCytomation). Beads coated with Vbeta-specific mAb in combination with CD28 were prepared from M-450 Epoxy Dynabeads (Dynal Biotech). A total of 20 µg of Vbeta-specific mAb and 10 µg of anti-CD28 was bound to 6 x 107 M-450 Epoxy Dynabeads, according to the protocol of the manufacturer.

Constructs and transfection

The pPKC constructs included the constitutive active (A/E) and the dominant-negative (K/R) forms of the various PKC isotypes cloned into the pEF vector. Using the pEGFP-N1 vector (BD Clontech), various CD3{gamma}-GFP variants were constructed. CD3{gamma}WT, -S126A, -S123A, and -LLAA inserts were obtained by PCR using CD3{gamma} sequences from already existing and published constructs (pTbeta-CD3{gamma}-Fneo) as templates (31). The PCR products were cut with EcoRI and BamHI, cloned into the EcoRI-BamHI fragment of the expression vector pEGFP-N1, and confirmed by complete DNA sequencing. For transient transfection of JTag cells, 3 µg of pPKC and 1 µg of pEGFP (or pEGFP-CD3{gamma}) were used with the lipid DMRIE-C reagent (Invitrogen Life Technologies), according to the manufacturer. For transient transfection of activated primary human Vbeta8+ T cells, Nucleofector technology (Amaxa Biosystems) was used. A total of 1 x 106 Vbeta8+ T cells was mixed with Nucleofector solution from the Human T Cell Nucleofector kit (Amaxa Biosystems) and 3 µg of pPKC and 1 µg of pEGFP. The program T-14 was used for transfection. The transfected cells were transferred to C medium and incubated for 24 h at 37°C, 5% CO2.

Cell stimulation and TCR down-regulation

Jurkat and mouse lymph node T cells were washed twice in C medium and adjusted to 0.5 x 106 cells/ml. A total of 20 nM PDB (Sigma-Aldrich) or CD3-CD28 expander beads (Dynal Biotech) at a cell:bead ratio of 1:2 for Jurkat cells and 1:1 for mouse T cells was added to the cell suspensions and incubated for the time indicated at 37°C, 5% CO2. The beads were removed, and the cells were stained with PE-UCHT1 in case of Jurkat cells and with H57-597 plus FITC-conjugated goat anti-Armenian hamster IgG in the case of mouse T cells. For Staphylococcus aureus enterotoxin E (SEE) superantigen (Sigma-Aldrich) stimulation of human primary Vbeta8+ T cells, Raji cells were either unpulsed or pulsed with 2 ng/ml SEE and mixed with the Jurkat cells at a ratio of 2:1. The cells were incubated for 20 min at 37°C, 5% CO2, and stained with PE-BV8. The mean fluorescence intensity (MFI) of the stained TCR was determined by flow cytometry and used to calculate the percentage of TCR down-regulation as (1 – (MFI2 ng/ml SEE/MFI0 ng/ml SEE))x 100%. As control, cells were stained for surface expression of MHC class I (MHC-I).

TCR recycling and confocal microscopy analyses

JTag cells were cotransfected with pEGFP and either pEF, pPKC{alpha}A/E, pPKC{theta}A/E, or pPKC{theta}myr. Forty-eight hours posttransfection, 1 x 106 transfected cells were washed twice and resuspended in C medium. The cell suspension was incubated for 2 h at 12°C with PE-UCHT1, and the temperature was then switched to 37°C. At the time points indicated, cell samples were transferred to a precooled solution of FACS medium (PBS with 2% FCS, 0.1% NaN3). The cells were washed in FACS medium and subjected to flow cytometric analysis. Confocal microscopy analyses were performed on a Zeiss LSM510 connected to a Zeiss Axiovert 100 M microscope (Carl Zeiss). GFP and Cy5 fluorescence were detected using band pass filter BP 505–550 and long pass filter LP 650, respectively.

Biotinylation and TCR degradation

Cells were washed twice in PBS and resuspended in 0.5 mg/ml sulfosuccinimidyl-6-(biotinamido)hexanoate/PBS (Pierce). The cells were incubated for 30 min on ice, washed, and resuspended in C medium to a concentration of 1 x 106 cells/ml. The biotinylated cells (4 x 106 cells/sample) were stimulated for 6 h with anti-TCR mAb or PDB and lysed in lysis buffer (50 mM Tris-base (pH 7.5), 150 mM NaCl, and 1 mM MgCl2) supplemented with 1 mg/ml Pefabloc SC, 10 µg/ml leupeptin, 10 µg/ml pepstatin, 5 mM EDTA, 10 mM NaF, 1 mM Na3VO4, and 1% Triton X-100. Cell lysates were precleared with protein A-agarose (Kem-En-Tec), and biotinylated proteins were precipitated with streptavidin-agarose beads (Pierce), followed by Western blotting using A452 (anti-CD3{epsilon}) or 6B10.2 (anti-TCR{zeta}) Abs.

Small interfering RNA (siRNA) and comodulation

A total of 1 x 106 Jurkat cells was cotransfected with 5 µM nonsense-siRNA (silencer negative control 1 siRNA; Ambion), 5 µM PKC{alpha}-siRNA (ID301; Ambion), or 2 µM PKC{theta}-siRNA (ID782 and ID783; Ambion) together with 1 µg of pEGFP and 4 µg of pcDNA6-HA1.7 TCRbeta-5 (construct expressing the TCRbeta variant Vbeta3) (21). The amount of siRNA used was selected to ensure proper knockdown of target mRNA without targeting other PKC isotype mRNAs. For transfection, Nucleofector technology was used with the Cell Line Nucleofector kit V and program G-10 (Amaxa Biosystems). The transfected cells were transferred to C medium at 37°C, 5% CO2. Twenty-four hours posttransfection, the cells were retransfected with siRNA and pcDNA6-HA1.7 TCRbeta-5, and incubated for additional 24 h. The cells were washed, adjusted to 0.5 x 106 cells/ml in C medium, and incubated in flat-bottom Nunclon Microwell plates (Invitrogen Life Technologies) for 20 min at 37°C with anti-Vbeta3/anti-CD28 or anti-Vbeta8/anti-CD28 Ab-conjugated beads at a cell:bead ratio of 1:5 and 1:10, respectively. The beads were removed from the cell samples, and the cells were stained with PE-BV3 and analyzed by flow cytometry. TCR double-positive DO11.10 cells were transfected with pEGFP and dominant-negative pPKC{alpha}K/R, pPKC{theta}K/R, or pEF; stimulated with anti-Vbeta2/anti-CD28 or anti-Vbeta8/anti-CD28 Ab-conjugated beads at a cell:bead ratio of 1:10; and subsequently analyzed, as described above.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Identifying PKC{alpha} and PKC{theta} as the PKC isotypes involved in TCR down-regulation

As a first approach to identify the PKC isotypes involved in TCR down-regulation, we transfected the human T cell line JTag with constructs expressing the constitutive active (A/E) forms of all PKC isotypes identified in T cells (PKC{alpha}, -beta, -{delta}, -{epsilon}, -{zeta}, -{eta}, -{theta}, and -{iota}) (22). The pPKCA/E constructs were cotransfected with pEGFP to identify cells with high plasmid expression levels. TCR cell surface expression levels were subsequently determined in this cell population by flow cytometry. Of the constitutive active PKC isotypes, only PKC{alpha} and PKC{theta} induced significant TCR down-regulation (Fig. 1A). Furthermore, a myristylated form of WT PKC{theta} (PKC{theta}myr) also induced TCR down-regulation, illustrating the importance of membrane recruitment for activation of this kinase. In addition, a minor effect of the constitutive active PKCbetaI on TCR down-regulation was observed, and hence, we cannot completely rule out a role of this particular PKC isotype in TCR down-regulation. Cell surface expression of MHC-I was not affected by the various constitutive active PKC isotypes, indicating that the effect of constitutive active PKC{alpha} and PKC{theta} on TCR expression was specific and not due to a general influence on receptor expression (Fig. 1A). The expression levels of GFP and the various PKCA/E isotypes were comparable (Fig. 1, B and C). Furthermore, the constitutive activity of the various PKCA/E isotypes has previously been confirmed (32, 33, 34), excluding the possibility of artifacts caused by deficient kinase activity.


Figure 1
View larger version (28K):
[in this window]
[in a new window]
  		FIGURE 1. The active forms of PKC{alpha} and PKC{theta} induce TCR down-regulation. A, JTag cells were cotransfected with pEGFP and the constitutive active (A/E) forms of the PKC isotypes or the empty vector (pEF). A myristylated (myr) form of WT PKC{theta} was also included. Two days after transfection, PKC-induced TCR down-regulation was determined by measuring the levels of cell surface TCR expression. The ordinate gives the percentage of surface-expressed TCR (bars) and control MHC-I (line) relative to TCR and MHC-I expression in pEF-transfected cells and calculated as (MFIpPKC/MFIpEF) x 100%. The data are given as mean ± SD of four independent experiments. B, Western blot of GFP expression in the transfected cells using anti-GFP. C, Western blots of PKC (upper panels) and CD3{epsilon} (lower panels) expression in the transfected cells using anti-PKC isotype-specific (PKCx) and anti-CD3{epsilon} Abs, respectively. D–F, Examples of FACS dot plots and histograms from cells cotransfected with pEGFP and pPKC/pEF. Dot plots of cells transfected with pEGFP and pEF (D) or pEGFP and PKC{theta}myr (E). F, FACS histograms showing TCR expression on gated cells from D and E.

 
To further investigate whether both PKC{alpha} and PKC{theta} were involved in TCR down-regulation, the dominant-negative (K/R) forms of these kinases (34) were transfected into JTag cells. The transfected cells were stimulated with CD3-CD28 expander beads or with PDB, and the TCR surface expression levels were determined by flow cytometry. Both the dominant-negative PKC{alpha} and PKC{theta} partially inhibited TCR down-regulation in response to the applied stimuli (Fig. 2, A–E).


Figure 2
View larger version (27K):
[in this window]
[in a new window]
  		FIGURE 2. Involvement of PKC{alpha} and PKC{theta} in TCR down-regulation in T cell lines, primary human T cells, and T cells from PKC KO mice. A and B, JTag cells were cotransfected with pEGFP and the dominant-negative (K/R) forms of PKC{alpha} and PKC{theta} or the empty vector (pEF). Two days after transfection, the cells were stimulated with CD3-CD28 expander beads (A) or PDB (B) for the times indicated. Surface expression levels of TCR were determined by flow cytometry and calculated as (1 – (MFIt=n/MFIt=0))x100%. The ordinate gives the percentage of down-regulated surface TCR, and the abscissa the time in minutes. The data are given as mean ± SD of three independent experiments. C, Western blots of PKC, GFP, and TCR{zeta} expression in the transfected cells using anti-PKC{alpha} (lanes 1 and 2, upper panel), anti-PKC{theta} (lanes 3 and 4, upper panel), anti-GFP (middle panel), and anti-TCR{zeta} (lower panel) Abs, respectively. D and E, Examples of FACS profiles of cells cotransfected with pEGFP and pPKC/pEF. Cells tranfected with pEGFP and pEF (gray histograms) or pEGFP and PKC{theta}K/R (open histograms) were either left untreated (D) or treated with 25 nM PDB for 45 min (E). F, Vbeta8+ T cells were isolated from human PBL and expanded in culture. Subsequently, the cells were cotransfected with pEGFP and either pEF, pPKC{alpha}A/E, pPKC{theta}A/E, pPKC{alpha}K/R, or pPKC{theta}K/R. For pEF- and pPKCA/E-transfected cells, the percentage of down-regulated surface TCR was directly determined by flow cytometry and calculated as (1 – (MFIPKCA/E/MFIpEF)) x 100%. The PKCK/R- and pEF-transfected cells were stimulated for 20 min with Raji cells prepulsed with 0 or 2 ng/ml SEE before flow cytometry analysis. The percentage of TCR down-regulation was calculated as (1 – (MFI2 ng/ml SEE/MFI0 ng/ml SEE)) x 100%. The ordinate to the left gives the percentage of TCR down-regulation (bars), and the ordinate to the right gives the expression levels of control MHC-I (line). The data are given as mean ± SD of three independent experiments. G and H, Expanded T cells from WT, PKC{alpha} (PKC{alpha}–/–), and PKC{theta} (PKC{theta}–/–) KO mice were stimulated with mouse CD3-CD28 expander beads (G) or PDB (H) for the times indicated. The surface TCR expression levels were determined by flow cytometry and calculated as (1 – (MFIt=n/MFIt=0)) x 100%. The ordinate gives the percentage of TCR down-regulation, and the abscissa gives the time in hours. The data are given as mean ± SD of three independent experiments. I, Western blots of PKC{alpha} (lanes 1 and 2, upper panel), PKC{theta} (lanes 3 and 4, upper panel), and fyn (lower panel) expression in the WT (lanes 1 and 3), PKC{alpha} (lane 2), and PKC{theta} (lane 4) KO mice. J–L, Examples of FACS profiles of T cells obtained from WT (J), PKC{alpha} (K), and PKC{theta} (L) KO mice. The cells were either left untreated (open histograms) or treated with 56 nM PDB for 30 min (gray histograms).

 
We next extended the study to primary human T cells. For this, we purified human PBL from which the Vbeta8+ T cells were isolated and subsequently expanded in culture (27). The Vbeta8+ T cells were transiently transfected with PKC{alpha}A/E, PKC{theta}A/E, PKC{alpha}K/R, PKC{theta}K/R, or the empty vector pEF. As seen for JTag cells, PKC{alpha}A/E and PKC{theta}A/E induced TCR down-regulation in primary T cells (Fig. 2F). Furthermore, PKC{alpha}K/R and PKC{theta}K/R partially inhibited TCR down-regulation in primary T cells stimulated with SEE-pulsed Raji cells (Fig. 2F).

To study the role of PKC{alpha} and PKC{theta} in T cells from genetically deficient mice, we next performed TCR down-regulation experiments on T cells from PKC KO mice. Lymph node cells from WT, PKC{alpha}, and PKC{theta} KO mice were isolated and expanded in culture. The cells were then stimulated with CD3-CD28 expander beads or with PDB, and the cell surface expression level of the TCR was determined by flow cytometry. In agreement with the results obtained for transfected cells, we found impaired TCR down-regulation in PKC{alpha} and PKC{theta} KO mice in response to both stimuli (Fig. 2, G–L).

From these experiments, we concluded that both PKC{alpha} and PKC{theta} are involved in down-regulation of the TCR. At first sight, this seemed surprising as PKC{alpha} and PKC{theta} belong to two different PKC subfamilies, and hence depend on partially different cofactors and lipid metabolites for their activation. Possible explanations could be that PKC{alpha} and PKC{theta} use different mechanisms to induce TCR down-regulation or that they are involved in different types of TCR down-regulation.

PKC{alpha} and PKC{theta} both induce TCR down-regulation via the CD3{gamma} diL motif-dependent recycling pathway

Previous studies have demonstrated that CD3{gamma} of the TCR contains a phosphoserine-dependent diL-based receptor-sorting motif involved in TCR down-regulation (31). Following TCR triggering, PKC becomes activated. Activated PKC phosphorylates Ser126 of CD3{gamma}, which makes the diL motif accessible to binding by AP-2. Binding of AP-2 to CD3{gamma} subsequently leads to TCR endocytosis (31, 35, 36). To examine whether both PKC{alpha}- and PKC{theta}-induced TCR down-regulation were dependent on the CD3{gamma} diL motif, we studied Jurkat cells expressing TCR with mutations in the CD3{gamma} chain. We cotransfected JTag cells with constitutive active PKC and various mutated forms of CD3{gamma}-GFP. The TCR surface expression levels of the individual transfected cells were analyzed by flow cytometry (Fig. 3, A, C, and D). Cotransfection of the constitutive active PKC{alpha} or PKC{theta} with CD3{gamma}WT-GFP or CD3{gamma}S123A-GFP (Fig. 3, A, {blacksquare} and dark gray, and C) led to the same degree of TCR down-regulation as seen in cells with native TCR (Fig. 1A). In contrast, cotransfection of the constitutive active PKC{alpha} or PKC{theta} with either CD3{gamma}LLAA-GFP or CD3{gamma}S126A-GFP (Fig. 3, A, {square} and light gray, and D) led to an almost complete block in TCR down-regulation. The expression levels of the various PKC isotypes and CD3{gamma}-GFP molecules were similar (Fig. 3B). Furthermore, all CD3{gamma}-GFP variants were expressed at similar levels on the cell surface (Fig. 3E). These results indicated that both PKC{alpha}- and PKC{theta}-induced TCR down-regulation depend on the phosphoacceptor Ser126 and the diL-based receptor-sorting motif of the CD3{gamma} chain.


Figure 3
View larger version (45K):
[in this window]
[in a new window]
  		FIGURE 3. PKC{alpha}- and PKC{theta}-induced TCR down-regulation is dependent on the diL-based receptor-sorting motif of CD3{gamma}. A, JTag cells were cotransfected with WT or mutated forms CD3{gamma}-GFP together with pEF, pPKCA/E, or pPKCmyr. TCR cell surface expression levels were determined by flow cytometry using GFP to identify the cell populations with high plasmid expression and calculated as (MFIpEF/pPKC/MFIpEF(WT)) x 100%. The ordinate gives the percentage of TCR cell surface expression levels relative to JTag cells cotransfected with CD3{gamma}WT-GFP and pEF. The data are given as mean ± SD of three independent experiments. B, Western blots of PKC, CD3{gamma}-GFP, and CD3{epsilon} expression in cells cotransfected with pEF (lanes 1–4), PKC{alpha} (lanes 5–8), or PKC{theta} (lanes 9–16) using anti-PKC{alpha} (lanes 1–8, upper panel), anti-PKC{theta} (lanes 9–16, upper panel), anti-GFP (middle panel), and anti-CD3{epsilon} (lower panel) Abs. C and D, Examples of FACS dot plots and profiles from cells cotransfected with CD3{gamma}WT-GFP (C) or CD3{gamma}S126A-GFP (D) together with pEF (gray histograms) or pPKC{theta}A/E (open histograms). The FACS histograms show TCR expression on cells gated as shown in the dot plots. E, Transfected cells were surface biotinylated and lysed. The biotinylated proteins were precipitated and subjected to Western blotting using anti-GFP Abs to determine the expression of CD3{gamma}-GFP chimeric molecules.

 
Earlier studies have indicated that TCR down-regulation induced by PKC leads to increased recycling of the TCR (7, 21, 37). Therefore, we investigated whether both PKC{alpha} and PKC{theta} induced TCR down-regulation through the recycling pathway. JTag cells were transfected with constitutive active PKC, and surface-expressed TCR were stained with PE-UCHT1 at 12°C for 2 h. By using this condition, TCR trafficking was blocked and complete labeling of the cell surface-expressed TCR was obtained. The temperature was then shifted to 37°C in the continuous presence of PE-UCHT1. After ~20 min, the entire pool of cycling TCR was labeled, and at this time point we found no significant difference in the level of labeled TCR in PKCA/E and mock-transfected cells, indicating that both PKC{alpha} and PKC{theta} induced TCR down-regulation by increased internalization and localization of the TCR in the endocytotic recycling compartments (Fig. 4, A–C). Confocal microscopy analyses confirmed that a higher fraction of TCR was found in vesicles in cells transfected with constitutive active PKC compared with mock-transfected cells (Fig. 4, D and E). This supported that PKC activation shifts the ratio between cell surface-expressed TCR and TCR located in endocytotic recycling compartments in favor of the recycling compartments (7). Although these results indicated that TCR degradation did not take place in cells transfected with constitutive active PKC, we further examined whether PKC activation induces TCR degradation. For this, Jurkat cells were surface biotinylated and stimulated for 6 h with either PDB or by TCR triggering that is known to induce dose-dependent TCR degradation (9, 11, 27, 38). Following stimulation, the biotinylated proteins were precipitated and subjected to SDS-PAGE, followed by Western blotting using anti-CD3{epsilon} (data not shown) or anti-TCR{zeta} Abs (Fig. 4F). TCR triggering led to a dose-dependent TCR degradation, as expected, whereas PDB stimulation and hence PKC activation did not induce TCR degradation, confirming the results described above.


Figure 4
View larger version (30K):
[in this window]
[in a new window]
  		FIGURE 4. PKC{alpha} and PKC{theta} both induce TCR down-regulation via the recycling pathway. A–C, TCR recycling was examined on JTag cells transfected with pPKC{alpha}A/E (A), pPKC{theta}A/E (B), or pPKCmyr (C) and compared with JTag cells transfected with pEF. Transfected cells were stained for 2 h at 12°C with PE-UCHT1, and the temperature was then shifted to 37°C. At the time points indicated, cell samples were transferred to ice and subsequently analyzed by flow cytometry. TCR staining levels were compared with TCR staining of pEF-transfected cells at time zero and calculated as (MFIt=n/MFI(pEF)t=0) x 100%. The ordinate gives the MFI of stained TCR relative to MFI of stained TCR of pEF-transfected cells at time zero, and the abscissa gives the time in minutes. The data are given as mean ± SD of three independent experiments. D and E, Confocal microscopy analyses of cells cotransfected with pEGFP and pEF (D) or pEGFP and pPKCA/E (E). The cells were stained with anti-TCR mAb F101.01, followed by F(ab')2 of Cy5-conjugated donkey anti-mouse Ig. Cy5 (frames labeled 1) and GFP (frames labeled 2) fluorescence were detected using long pass filter LP 650 and band pass filter BP 505–550, respectively. F, Jurkat cells were biotinylated and stimulated with the indicated concentrations of the anti-TCR mAb F101.01 or PDB for 6 h. The cells were subsequently lysed, and the biotinylated proteins were precipitated by streptavidin-agarose beads. Western blotting was performed by using anti-TCR{zeta} mAb.

 
Taken together, these results demonstrated that both PKC{alpha} and PKC{theta} induced TCR down-regulation via the CD3{gamma} diL motif-dependent recycling pathway.

Additive effect of PKC{alpha} and PKC{theta} in TCR down-regulation

Up to this point, our study did not explain why T cells make use of two different PKC isotypes in TCR down-regulation. One possibility was that the two PKC isotypes operate in an additive way to induce TCR down-regulation. To investigate this hypothesis, JTag cells were transfected with either PKC{alpha}K/R, PKC{theta}K/R, or a combination of both PKC{alpha}K/R and PKC{theta}K/R. The transfected cells were stimulated for 20 min with PDB, and the TCR surface expression levels were subsequently determined. Expression of either PKC{alpha}K/R or PKC{theta}K/R led to a ~50% reduction in TCR down-regulation as compared with the control cells transfected with pEF. Simultaneous expression of both PKC{alpha}K/R and PKC{theta}K/R caused an additional 50% reduction in TCR down-regulation as compared with cells transfected with only one of the dominant-negative isotypes (Fig. 5A). The observed additive effect of PKC{alpha}K/R and PKC{theta}K/R was not caused by a higher total expression level of PKCK/R in the double-transfected cells (Fig. 5B). These experiments could suggest that PKC{alpha} and PKC{theta} acted on TCR located in different compartments of the plasma membrane.


Figure 5
View larger version (22K):
[in this window]
[in a new window]
  		FIGURE 5. Additive effect of PKC{alpha} and PKC{theta} on TCR down-regulation. A, JTag cells were cotransfected with pEGFP and pPKC{alpha}K/R, pPKC{theta}K/R, or a combination of pPKC{alpha}K/R and pPKC{theta}K/R. The transfected cells were left untreated or were stimulated for 20 min with 20 nM PDB. The percentage of TCR down-regulation was determined by flow cytometry and calculated as (1 – (MFIPDB treated/MFIuntreated)) x 100%. The data are given as mean ± SD of three independent experiments. B, Western blots of PKC{alpha} (lanes 1–3, upper panel), PKC{theta} (lanes 4–6, upper panel), GFP (middle panel), and TCR{zeta} (lower panel) expression in the transfected cells.

 
PKC{theta} down-regulates engaged TCR, whereas PKC{alpha} down-regulates nonengaged TCR

Triggering of the TCR leads to internalization not only of engaged, but also of nonengaged receptors (16, 17, 18). We have demonstrated recently that down-regulation of nonengaged TCR (TCR comodulation) is dependent on PKC (21). We therefore wanted to investigate the role of PKC{alpha} and PKC{theta} in down-regulation of engaged vs nonengaged TCR. The Jurkat cell line expresses the TCRbeta chain Vbeta8. To obtain cell lines with two different Vbeta chains and reduced PKC isotype-specific activity, we transfected Jurkat cells with constructs expressing Vbeta3, GFP, and the dominant-negative forms of PKC. However, this approach did not result in significant expression of Vbeta3 and PKC, and we therefore shifted to an experimental design using siRNA. The PKC{alpha}- and PKC{theta}-siRNA sequences used (PRKCA 301 + PRKCQ 783) almost exclusively target the RNA of interest according to the manufacturer. In agreement, we found that PRKCA 301 + PRKCQ 783 specifically targeted PKC{alpha} and PKC{theta}, respectively (Fig. 6A). A siRNA experiment was also performed using a different PKC{theta}-siRNA (PRKCQ 782) from which we obtained similar results (data not shown). Cells transfected with Vbeta3, GFP, and siRNA were stimulated with anti-Vbeta3/anti-CD28 or anti-Vbeta8/anti-CD28 Ab-conjugated beads. Following stimulation, the beads were removed and the level of Vbeta3 down-regulation was measured in the Vbeta3/Vbeta8 double-positive cell population by flow cytometry. Stimulation with either Vbeta3 beads or Vbeta8 beads of cells transfected with nonsense-siRNA led to down-regulation of the Vbeta3-TCR complex (Fig. 6B). This demonstrated that comodulation of the nontriggered Vbeta3-TCR took place following stimulation with anti-Vbeta8. In contrast, in cells transfected with PKC{alpha}-siRNA, only stimulation with Vbeta3 beads led to optimal Vbeta3-TCR down-regulation, and anti-Vbeta8-induced comodulation of Vbeta3-TCR was severely inhibited. Furthermore, in cells transfected with PKC{theta}-siRNA, only stimulation with Vbeta8 beads led to Vbeta3-TCR down-regulation, whereas anti-Vbeta3-induced down-regulation of Vbeta3-TCR was inhibited. Collectively, these experiments indicated that PKC{theta} is involved in down-regulation of engaged TCR, whereas PKC{alpha} is implicated in down-regulation of nonengaged TCR.


Figure 6
View larger version (39K):
[in this window]
[in a new window]
  		FIGURE 6. PKC{theta} induces down-regulation of engaged TCR, and PKC{alpha} induces down-regulation of nonengaged TCR. A and B, JTag cells were transfected with nonsense-, PKC{alpha}-, or PKC{theta}-siRNA together with a construct expressing the TCR-Vbeta3 chain. A, Western blots of PKC{alpha} (lanes 1–3, upper panel), PKC{theta} (lanes 4–6, upper panel), and TCR{zeta} (lower panel) expression in the transfected cells. B, The cells were stimulated with anti-Vbeta3/anti-CD28 ({blacksquare}) or anti-Vbeta8/anti-CD28 ({cjs2108}) Ab-conjugated beads for 20 min. Cell surface expression levels of TCR-Vbeta3 were subsequently measured by flow cytometry of the Vbeta3-Vbeta8 double-positive cell population. TCR-Vbeta3 down-regulation was calculated as (1 – (MFIstimulated cells/MFIunstimulated cells)) x 100%. The ordinate gives the percentage of TCR-Vbeta3 down-regulation. The data are given as mean ± SD of three independent experiments. C and D, Double-positive TCR-Vbeta2/Vbeta8 DO11.10 cells were transfected with pEF, pPKC{alpha}K/R, or pPKC{theta}K/R. C, Western blots of PKC{alpha} (lanes 1–3, upper panel), PKC{theta} (lanes 4–6, upper panel), and TCR{zeta} (lower panel) expression in the transfected cells. D, The cells were stimulated with anti-Vbeta2/anti-CD28 ({blacksquare}) or anti-Vbeta8/anti-CD28 ({cjs2108}) Ab-conjugated beads for 20 min. Cell surface expression levels of TCR-Vbeta2 were subsequently measured by flow cytometry. TCR-Vbeta2 down-regulation was calculated as (1 – (MFIstimulated cells/MFIunstimulated cells)) x 100%. The ordinate gives the percentage of TCR-Vbeta2 down-regulation. The data are given as mean ± SD of three independent experiments.

 
The differential use of PKC isotypes in down-regulation of engaged and nonengaged TCR would allow T cells to selectively regulate trafficking of these TCR subpopulations during an immune response. To substantiate the results obtained by using siRNA, we next studied a stable transfectant of the mouse T cell line DO11.10 that expresses two distinct TCR comprising either Vbeta2 or Vbeta8 (21). The double-TCR-positive DO11.10 cells were transfected with pEGFP, and the dominant-negative PKC{alpha}K/R, PKC{theta}K/R, or pEF and expression of the transfected PKC isotypes were analyzed by Western blotting (Fig. 6C). The cells were subsequently stimulated with anti-Vbeta2/anti-CD28 or anti-Vbeta8/anti-CD28 Ab-conjugated beads. Following stimulation, the beads were removed and the level of Vbeta2 down-regulation was determined. Stimulation with either Vbeta2 or Vbeta8 beads of mock-transfected cells resulted in down-regulation of the Vbeta2-TCR complex (Fig. 6D). This demonstrated that comodulation of the nontriggered Vbeta2-TCR took place following stimulation with anti-Vbeta8. In contrast, in cells transfected with PKC{alpha}K/R, only stimulation with Vbeta2 beads led to optimal Vbeta2-TCR down-regulation, and anti-Vbeta8-induced comodulation of Vbeta2-TCR was inhibited. Furthermore, in cells transfected with PKC{theta}K/R, only stimulation with Vbeta8 beads led to Vbeta2-TCR down-regulation, whereas anti-Vbeta2-induced down-regulation of Vbeta2-TCR was inhibited. Thus, this experiment supported that PKC{theta} is involved in down-regulation of engaged TCR, whereas PKC{alpha} is implicated in down-regulation of nonengaged TCR.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Despite the large amount of information on the critical role of PKC in TCR down-regulation (6, 7, 35, 39, 40, 41), the identification of the specific PKC isotypes involved in this process still remained to be determined. In the present study, we systematically addressed the function of T cell-expressed PKC isotypes in TCR down-regulation, using in vitro studies with human and mouse T cell lines as well as human and mouse primary T cells. Of the PKC isotypes studied, only the constitutive active forms of PKC{alpha} and PKC{theta} induced significant TCR down-regulation. In line with this, overexpression of dominant-negative PKC{alpha} and PKC{theta}, as well as PKC{alpha} and PKC{theta} gene ablation by siRNA and PKC KO, reduced TCR down-regulation significantly. Thus, our study defined PKC{alpha} and PKC{theta} as the two major PKC isotypes involved in TCR down-regulation. This finding is in agreement with an earlier study showing that PKC{alpha} and PKC{theta} are the main PKC isotypes recruited to the plasma membrane in Jurkat cells within the first 15 min of TCR triggering (42). The kinetics of PKC recruitment described by Szamel et al. (42) correlates with the kinetics of PKC-induced TCR down-regulation in Jurkat cells.

The observation that PKC{theta} is involved in TCR down-regulation is in accordance with the established role of PKC{theta} as a regulator of T cell activation. PKC{theta} is known to translocate to the center of the immunological synapse, where it colocalizes with the TCR. Once activated, PKC{theta} plays a critical role in TCR signaling (43, 44). In mice lacking PKC{theta}, T cells are partially unresponsive to TCR triggering and deficient in IL-2 production due to impaired NF-{kappa}B, AP-1, and NF-AT activation (28, 45). In addition, PKC{theta} is required for the development of a robust immune response controlled by Th2 cells (46), and lack of PKC{theta} leads to induction of T cell anergy (47). PKC{alpha} also has critical roles in T cell activation and T cell immunity. Thus, PKC{alpha} plays a key role in TCR-induced IFN-{gamma} production and Th1-dependent immune responses (G. Baier, unpublished observations).

We were surprised to find that PKC isotypes belonging to two different PKC subfamilies were involved in TCR down-regulation. PKC{alpha} is a member of the conventional PKC subfamily, and PKC{theta} is a member of the novel PKC subfamily. Both subfamilies contain a binding site for phorbol ester/DAG, but only conventional PKC contain a functional binding site for Ca2+ (48, 49). The activity of PKC{alpha} can be modified by the intracellular Ca2+ level of the cell, whereas the activity of PKC{theta} is not dependent on the Ca2+ level (50). Early tyrosine phosphorylation events induced by TCR triggering recruit PLC{gamma} and PI3K to the membrane. Activated PLC{gamma} generates DAG and inositol 1,4,5-triphosphate that leads to increased intracellular Ca2+ concentrations. Activated PI3K generates second messengers that are capable of activating several PKC isotypes via 3'-phosphoinositide-dependent kinase-1 or related enzymes (51, 52). Thus, the activity of PKC{alpha} and PKC{theta} is differently regulated following TCR triggering. We investigated whether PKC{alpha} and PKC{theta} used different mechanisms to induce TCR down-regulation. We found that both PKC{alpha} and PKC{theta} induced TCR down-regulation by the TCR recycling pathway in which the phosphoacceptor group Ser126 and the diL-based receptor-sorting motif of the CD3{gamma} chain were indispensable. These results were in agreement with previous studies on substrate requirements for PKC during TCR down-regulation in general (31, 35).

Interestingly, we observed an additive effect of PKC{alpha} and PKC{theta} in TCR down-regulation. Previous studies have shown that PKC{theta} is recruited to the contact zone between the T cell and the APC following TCR triggering, whereas PKC{alpha} is recruited to the entire plasma membrane (53, 54). Giving the different membrane localizations of PKC{alpha} and PKC{theta} in response to T cell activation, it was reasonable to consider whether these kinases cooperated in TCR down-regulation of engaged and nonengaged TCR. The theory of TCR comodulation states that down-regulation of nonengaged TCR is initiated through intracellular signaling involving tyrosine kinases and PKC rather than by direct physical interaction between the TCR and MHC/peptide complexes (16, 17, 18, 20). In accordance, we have demonstrated recently that TCR comodulation is dependent on PKC (21). In the present study, we observed that Vbeta3-TCR in Vbeta3/Vbeta8 double-positive T cells was down-regulated following both Vbeta3- and Vbeta8-TCR triggering, confirming the occurrence of TCR comodulation. By using siRNA to knockdown specific PKC isotypes, we found that PKC{alpha} was mainly involved in down-regulation of nonengaged TCR, whereas PKC{theta} was mainly involved in down-regulation of engaged TCR. To validate this intriguing observation, we examined the effect on TCR down-regulation of dominant-negative forms of PKC{alpha} and PKC{theta} in stable double-TCR-positive DO11.10 cells. By using this model, we confirmed that PKC{alpha} was mainly involved in down-regulation of nonengaged TCR, whereas PKC{theta} was mainly involved in down-regulation of engaged TCR. These results are in agreement with our results obtained with T cells from PKC KO mice. Thus, the reduced TCR down-regulation observed in PKC{alpha} KO T cells following TCR triggering most likely represented impaired comodulation of nonengaged TCR, whereas down-regulation of directly triggered TCR was intact. Likewise, the reduced TCR down-regulation observed in PKC{theta} KO T cells following TCR triggering most likely represented impaired down-regulation of directly triggered TCR, whereas down-regulation of nonengaged TCR was intact in these mice. If directly triggered TCR are down-regulated by PKC{theta} and nontriggered TCR are down-regulated/comodulated by PKC{alpha}, this would also explain why TCR down-regulation was not completely blocked in neither PKC{alpha} nor PKC{theta} KO mice.

Furthermore, we observed that siRNA knockdown of PKC{theta} or overexpression of the dominant-negative form of PKC{theta} did not interfere with PKC{alpha}-induced down-regulation of nonengaged TCR, suggesting that PKC{alpha}-induced TCR down-regulation was independent of PKC{theta} activity. Likewise, siRNA knockdown of PKC{alpha} or overexpression of the dominant-negative form of PKC{alpha} did not interfere with PKC{theta}-induced down-regulation of engaged TCR, suggesting that PKC{theta}-induced TCR down-regulation was independent of PKC{alpha} activity. In a recent study, Alarcón and coworkers (19) showed that two different pathways of internalization are involved in down-regulation of engaged and nonengaged TCR, and that they take place from different compartments of the plasma membrane. Thus, down-regulation of engaged TCR depended upon lipid rafts, whereas down-regulation of nonengaged TCR was independent of rafts and instead mediated by clathrin-coated pits. This might explain why two different PKC isotypes are involved in down-regulation of engaged and nonengaged TCR, and it could be suggested that the key to understand how T cells distinguish between the two pathways of TCR down-regulation might be found in different cellular localizations and requirements for activation of the two PKC isotypes. From the present study and a recent study demonstrating that recycling TCR are transported to the contact site between the T cell and the APC (20), we suggest that the primary role of PKC{alpha} in TCR trafficking is to induce endocytosis of nonengaged TCR that subsequently recycle to the contact zone between the T cell and the APC, whereas the primary role of PKC{theta} is to induce endocytosis of directly triggered TCR at the contact zone (Fig. 7).


Figure 7
View larger version (24K):
[in this window]
[in a new window]
  		FIGURE 7. Proposed model for PKC{alpha}- and PKC{theta}-induced TCR down-regulation and trafficking. Triggering of the TCR activates PKC{alpha} and PKC{theta} that subsequently are recruited to the entire plasma membrane and to the contact zone between the T cell and the APC, respectively. At their respective intracellular sites, the PKC molecules induce TCR down-regulation through the phosphoserine-dependent diL-based receptor-sorting motif of the CD3{gamma} chain. The primary role of PKC{alpha} in TCR down-regulation is internalization of nonengaged TCR that probably recycle to the contact zone between the T cell and the APC, whereas PKC{theta} are responsible for internalization of directly engaged TCR at the contact zone.

 
We cannot exclude that PKC{alpha}- and PKC{theta}-mediated TCR down-regulation represents a negative feedback mechanism to terminate TCR signaling as described in TCR down-regulation-deficient Cbl KO mice (12). However, the observed activation deficiencies in T cells from PKC{alpha} and PKC{theta} KO mice indicate that PKC{alpha}- and PKC{theta}-mediated TCR down-regulation might be required for optimal TCR signaling. Along this line, mutation in the PKC-dependent diL-based receptor-sorting motif of the CD3{gamma} chain leads to T cell activation deficiency in a mouse model (C. M. Bonefeld, M. C. Haks, B. L. Nielsen, M. W. Nielsen, L. Boding, J. P. Christensen, A. R. Thomsen, P. Krimpenfort, M. von Essen, A. M. Kruisebeek, and C. Geisler, unpublished observations). Collectively, these observations may indicate that deficient PKC/CD3{gamma}-dependent TCR down-regulation results in T cell activation deficiency and subsequent immunosuppression, whereas deficient Cbl-dependent TCR down-regulation results in increased T cell responsiveness and subsequent autoimmunity. One possible explanation is that a lower number of activated TCR signaling complexes is generated in T cells with deficient PKC/CD3{gamma}-dependent TCR down-regulation due to deficient endocytosis and subsequent recruitment of nonengaged TCR to the contact zone between the T cell and the APC. As a consequence, the sustained signaling threshold required for induction of proliferation of naive T cells may not be obtained in such cells. Thus, a role of PKC-mediated TCR down-regulation could be to establish and maintain the critical signaling threshold required for optimal TCR-mediated T cell expansion and differentiation.


   Acknowledgments
 
The technical help of Bodil Nielsen and Christina Lutz is gratefully acknowledged.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported in part by grants from the Danish Medical Research Council, the Novo Nordisk Foundation, the Austrian Science Foundation FWF-P16229-B07, the A.P. Møller Foundation for Advancement of Medical Sciences, the Agnes and Poul Friis Foundation, the Danish Medical Association Research Fund, and the Astrid Thaysen Foundation for Basic Medical Sciences. M.v.E. was supported by a Ph.D. scholarship from Faculty of Health Sciences, University of Copenhagen. L.B. was supported by a scholarship from the Novo Nordisk Foundation. Back

2 Address correspondence and reprint requests to Dr. Carsten Geisler, Institute of Medical Microbiology and Immunology, Panum Institute, Building 22.5, University of Copenhagen, Blegdamsvej 3C, DK-2200 Copenhagen, Denmark. E-mail address: c.geisler{at}immi.ku.dk Back

3 Abbreviations used in this paper: PKC, protein kinase C; DAG, 1,2-diacylglycerol; diL, dileucine; KO, knockout; MFI, mean fluorescence intensity; MHC-I, MHC class I; PDB, phorbol 12,13-dibutyrate; SEE, Staphylococcus aureus enterotoxin E; siRNA, small interfering RNA; WT, wild type. Back

Received for publication November 1, 2005. Accepted for publication March 31, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Bjorkman, P. J., M. A. Saper, B. Samraoui, W. S. Bennett, J. L. Strominger, D. C. Wiley. 1987. The foreign antigen binding site and T cell recognition regions of class I histocompatibility antigens. Nature 329: 512-518. [Medline]
  2. Dembic, Z., W. Haas, S. Weiss, J. McCubrey, H. Kiefer, H. von Boehmer, M. Steinmetz. 1986. Transfer of specificity by murine {alpha} and beta T-cell receptor genes. Nature 320: 232-238. [Medline]
  3. Malissen, B., A. M. Schmitt-Verhulst. 1993. Transmembrane signalling through the T-cell-receptor-CD3 complex. Curr. Opin. Immunol. 5: 324-333. [Medline]
  4. Cantrell, D.. 1996. T cell antigen receptor signal transduction pathways. Annu. Rev. Immunol. 14: 259-274. [Medline]
  5. Clevers, H., B. Alarcon, T. Wileman, C. Terhorst. 1988. The T cell receptor/CD3 complex: a dynamic protein ensemble. Annu. Rev. Immunol. 6: 629-662. [Medline]
  6. Minami, Y., L. E. Samelson, R. D. Klausner. 1987. Internalization and cycling of the T cell antigen receptor: role of protein kinase C. J. Biol. Chem. 262: 13342-13347. [Abstract/Free Full Text]
  7. Menne, C., S. T. Moller, V. Siersma, M. von Essen, N. Odum, C. Geisler. 2002. Endo- and exocytic rate constants for spontaneous and protein kinase C-activated T cell receptor cycling. Eur. J. Immunol. 32: 616-626. [Medline]
  8. Geisler, C.. 2004. TCR trafficking in resting and stimulated T cells. Crit. Rev. Immunol. 24: 67-86. [Medline]
  9. Valitutti, S., S. Muller, M. Salio, A. Lanzavecchia. 1997. Degradation of T cell receptor (TCR)-CD3-{zeta} complexes after antigenic stimulation. J. Exp. Med. 185: 1859-1864. [Abstract/Free Full Text]
  10. D’Oro, U., M. S. Vacchio, A. M. Weissman, J. D. Ashwell. 1997. Activation of the Lck tyrosine kinase targets cell surface T cell antigen receptors for lysosomal degradation. Immunity 7: 619-628. [Medline]
  11. Niedergang, F., E. San Jose, B. Rubin, B. Alarcon, A. Dautry-Varsat, A. Alcover. 1997. Differential cytosolic tail dependence and intracellular fate of T-cell receptors internalized upon activation with superantigen or phorbol ester. Res. Immunol. 148: 231-245. [Medline]
  12. Naramura, M., I. K. Jang, H. Kole, F. Huang, D. Haines, H. Gu. 2002. c-Cbl and Cbl-b regulate T cell responsiveness by promoting ligand-induced TCR down-modulation. Nat. Immunol. 3: 1192-1199. [Medline]
  13. Duan, L., A. L. Reddi, A. Ghosh, M. Dimri, H. Band. 2004. The Cbl family and other ubiquitin ligases: destructive forces in control of antigen receptor signaling. Immunity 21: 7-17. [Medline]
  14. Wang, H. Y., Y. Altman, D. Fang, C. Elly, Y. Dai, Y. Shao, Y. C. Liu. 2001. Cbl promotes ubiquitination of the T cell receptor {zeta} through an adaptor function of Zap-70. J. Biol. Chem. 276: 26004-26011. [Abstract/Free Full Text]
  15. Dietrich, J., T. Backstrom, J. P. Lauritsen, J. Kastrup, M. D. Christensen, F. von Bulow, E. Palmer, C. Geisler. 1998. The phosphorylation state of CD3{gamma} influences T cell responsiveness and controls T cell receptor cycling. J. Biol. Chem. 273: 24232-24238. [Abstract/Free Full Text]
  16. San Jose, E., A. Borroto, F. Niedergang, A. Alcover, B. Alarcon. 2000. Triggering the TCR complex causes the down-regulation of nonengaged receptors by a signal transduction-dependent mechanism. Immunity 12: 161-170. [Medline]
  17. Niedergang, F., A. Dautry-Varsat, A. Alcover. 1997. Peptide antigen or superantigen-induced down-regulation of TCRs involves both stimulated and unstimulated receptors. J. Immunol. 159: 1703-1710. [Abstract]
  18. Niedergang, F., A. Dautry-Varsat, A. Alcover. 1998. Cooperative activation of TCRs by enterotoxin superantigens. J. Immunol. 161: 6054-6058. [Abstract/Free Full Text]
  19. Monjas, A., A. Alcover, B. Alarcon. 2004. Engaged and bystander T cell receptors are down-modulated by different endocytotic pathways. J. Biol. Chem. 279: 55376-55384. [Abstract/Free Full Text]
  20. Das, V., B. Nal, A. Dujeancourt, M. I. Thoulouze, T. Galli, P. Roux, A. Dautry-Varsat, A. Alcover. 2004. Activation-induced polarized recycling targets T cell antigen receptors to the immunological synapse; involvement of SNARE complexes. Immunity 20: 577-588. [Medline]
  21. Bonefeld, C. M., A. B. Rasmussen, J. P. Lauritsen, M. von Essen, N. Odum, P. S. Andersen, C. Geisler. 2003. TCR comodulation of nonengaged TCR takes place by a protein kinase C and CD3{gamma} di-leucine-based motif-dependent mechanism. J. Immunol. 171: 3003-3009. [Abstract/Free Full Text]
  22. Bauer, B., N. Krumbock, N. Ghaffari-Tabrizi, S. Kampfer, A. Villunger, M. Wilda, H. Hameister, G. Utermann, M. Leitges, F. Uberall, G. Baier. 2000. T cell expressed PKC{theta} demonstrates cell-type selective function. Eur. J. Immunol. 30: 3645-3654. [Medline]
  23. Caloca, M. J., N. Fernandez, N. E. Lewin, D. Ching, R. Modali, P. M. Blumberg, M. G. Kazanietz. 1997. beta2-chimaerin is a high affinity receptor for the phorbol ester tumor promoters. J. Biol. Chem. 272: 26488-26496. [Abstract/Free Full Text]
  24. Lorenzo, P. S., J. W. Kung, D. A. Bottorff, S. H. Garfield, J. C. Stone, P. M. Blumberg. 2001. Phorbol esters modulate the Ras exchange factor RasGRP3. Cancer Res. 61: 943-949. [Abstract/Free Full Text]
  25. Davies, S. P., H. Reddy, M. Caivano, P. Cohen. 2000. Specificity and mechanism of action of some commonly used protein kinase inhibitors. Biochem. J. 351: 95-105. [Medline]
  26. Sigma, R. B. I.. 2005. Application note. Cell Transm. 19: 11-14.
  27. Von Essen, M., C. M. Bonefeld, V. Siersma, A. B. Rasmussen, J. P. Lauritsen, B. L. Nielsen, C. Geisler. 2004. Constitutive and ligand-induced TCR degradation. J. Immunol. 173: 384-393. [Abstract/Free Full Text]
  28. Pfeifhofer, C., K. Kofler, T. Gruber, N. G. Tabrizi, C. Lutz, K. Maly, M. Leitges, G. Baier. 2003. Protein kinase C{theta} affects Ca2+ mobilization and NFAT cell activation in primary mouse T cells. J. Exp. Med. 197: 1525-1535. [Abstract/Free Full Text]
  29. Maly, K., K. Strese, S. Kampfer, F. Ueberall, G. Baier, N. Ghaffari-Tabrizi, H. H. Grunicke, M. Leitges. 2002. Critical role of protein kinase C{alpha} and calcium in growth factor induced activation of the Na+/H+ exchanger NHE1. FEBS Lett. 521: 205-210. [Medline]
  30. Geisler, C., T. Plesner, G. Pallesen, K. Skjodt, N. Odum, J. K. Larsen. 1988. Characterization and expression of the human T cell receptor-T3 complex by monoclonal antibody F101.01. Scand. J. Immunol. 27: 685-696. [Medline]
  31. Dietrich, J., X. Hou, A. M. Wegener, C. Geisler. 1994. CD3{gamma} contains a phosphoserine-dependent di-leucine motif involved in down-regulation of the T cell receptor. EMBO J. 13: 2156-2166. [Medline]
  32. Kampfer, S., K. Hellbert, A. Villunger, W. Doppler, G. Baier, H. H. Grunicke, F. Uberall. 1998. Transcriptional activation of c-fos by oncogenic Ha-Ras in mouse mammary epithelial cells requires the combined activities of PKC-{lambda}, {epsilon} and {zeta}. EMBO J. 17: 4046-4055. [Medline]
  33. 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: 132-142. [Medline]