1 Department of Molecular Biology and Biochemistry, Center for Immunology, University of CaliforniaIrvine, Irvine, CA 92697-3900, USA
2 Department of Biology, University of California, San Diego, La Jolla, CA 92093-0687, USA
3 University of California, San Diego Cancer Center
4 Present address: Genomics Institute of the Novartis Research Foundation, La Jolla, CA 92121, USA
5 Present address: Department of Pathology and Committee on Immunology, University of Chicago, Chicago, IL 60637, USA
Correspondence to: C. M. Walsh; E-mail: cwalsh{at}uci.edu
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Abstract |
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Keywords: DAP kinase, thymocytes, positive selection, T cells, signal transduction, central tolerance
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Introduction |
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There are five known mammalian members of the DAPK family; additional homologs have also been found in Caenorhabditis elegans and Drosophila melanogaster. All family members share a characteristic serine/threonine kinase domain at the extreme amino-terminus with high primary amino acid sequence homology to myosin light chain kinases, and all are known to possess intrinsic kinase activity (2). However, these proteins share little homology outside of this kinase domain, suggesting that these factors regulate, or are regulated by, disparate processes in cells. In addition to this conserved kinase domain, the prototype DAP kinase (DAPK1) contains a calmodulin-binding domain, an ankyrin repeat region, a cytoskeletal-binding domain and a carboxy-terminal death domain (3). The latter is thought to physically link DAPK1 to death receptor-mediated apoptosis, though the means by which DAPK1 potentiates this form of apoptosis is unclear (46). DRAK2 was originally cloned as a result of its sequence homology with DAPK1, and was shown to promote apoptosis upon expression in murine NIH3T3 cells (7, 8). While DAP kinase family members are thought to be involved in both extrinsic and intrinsic pathways of apoptosis, a physiological role for DRAK2-dependent apoptosis has not been firmly established. DRAK2-deficient mice have no defects in apoptosis, and as described here, retroviral expression of DRAK2 in NIH3T3 cells did not induce apoptosis. Rather, we have recently described a role for DRAK2 in negative regulation of T cell activation (1); T cells from mice deficient in DRAK2 are hypersensitive to TCR stimulation, proliferate in response to sub-optimal stimulation and produce higher levels of cytokines (especially IL-2 and IL-4) when compared with wild-type T cells.
With an interest in characterizing a potential role for DRAK2 in thymocyte development, we investigated the expression of this gene in distinct thymocyte subsets. We found that DRAK2 expression was regulated differentially during lymphoid development, with increasing expression corresponding to lymphocyte maturation. In the thymus, DRAK2 was expressed at low levels in immature double-negative(DN) cells, whereas CD4/CD8 double-positive (DP) cells lacked appreciable expression of DRAK2 mRNA. In contrast, DRAK2 mRNA was found to exist at the highest levels in CD4 and CD8 single-positive (SP) thymocytes. In DP thymocytes, treatment with phorbol myristate acetate (PMA) strongly induced DRAK2 mRNA expression, and this occurred in a protein synthesis-independent fashion, suggesting that DRAK2 is a primary response gene in this thymocyte subset. Stimulation of purified DP cells with TCR cross-linking antibodies, especially with cross-linking of CD4 or CD8 co-receptors, similarly enhanced DRAK2 expression. Pharmacologic inhibition with agents that block non-classical protein kinase C (PKC) isoforms abrogated the PMA-dependent up-regulation of DRAK2, suggesting that non-classical PKCs are involved in DRAK2 transcription in activated DP thymocytes. Similar to previous reports using ectopically expressed DRAK2, endogenous DRAK2 was primarily localized in the nucleus. This nuclear localization required an 80-residue domain carboxy-terminal to the kinase domain. However, in Jurkat T cells and thymocytes, TCR stimulation led to export of a large fraction of DRAK2 out of the nucleus.
Since DRAK2 was expressed at low levels in DP cells, we hypothesized that its increased expression in SP cells might serve as a mechanism to interfere with activation as cells transit through this developmental stage. With evidence indicating that DRAK2 raises the threshold for T cell activation in naive peripheral T cells, we used Drak2-deficient thymocytes to examine the role of DRAK2 in thymocyte activation. An increase in the concentration of free Ca2+ is an essential signal during T cell development and activation, leading to the activation of downstream transcriptional pathways (9). Our experiments using Drak2-deficient thymocytes demonstrate that this DAP kinase sets a threshold during selection for TCR signal strength, as measured by Ca2+ release following TCR cross-linking. Thus, in addition to negatively regulating the activation of mature T cells, DRAK2 also acts as a rheostat to alter the outcome of TCR signaling in developing thymocytes.
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Methods |
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Northern analysis of tissue-specific DRAK2 expression
To study the tissue-specific expression of DRAK2, we generated a multiple tissue northern blot. Indicated organs were isolated from adult C57BL/6 mice and total cellular RNA was produced using Trizol (Invitrogen, San Diego, CA, USA). For northern blotting, 20 µg of total RNA for each sample was loaded onto a 1.0% agarose6% formaldehyde gel, separated electrophoretically and transferred to a GeneScreen (NEN/Perkin Elmer, Boston, MA, USA) nylon membrane. The membrane was hybridized with radiolabeled cDNA probes for DRAK2 (the entire 3.2-kb cDNA) or GAPDH, and analyzed by autoradiography.
Northern analyses of T and B cell developmental subsets and activated DP thymocytes
To characterize DRAK2 expression in thymocyte subsets, pooled C57BL/6 thymocytes were incubated with anti-CD4FITC and anti-CD8PE antibodies, washed in staining buffer (PBS with 2% FCS) and separated on a cell sorter (MoFlow). Four populations were recovered consisting of DP (CD4+/CD8+), CD4 SP, CD8 SP and CD4/CD8 DN cells. Cellular RNA was extracted from these thymocytes using Trizol and 15 µg per condition was used for northern analyses with DRAK2 or GAPDH probes. B cell developmental subsets were assayed for DRAK2 expression following sequential separations using magnetic cell sorting (Miltenyi Biotech, Auburn, CA, USA). Briefly, total bone marrow (BM) cells were isolated from C57BL/6 mice and pooled prior to incubation with antibodies to mouse IgM and purification on a MACS column following binding of paramagnetic beads specific for the primary antibody. These cells were then separated on a MACS column (Miltenyi Biotech); cells retained on this column were labeled IgM+. The flow-through from this column was then incubated with antibodies to BP-1, followed by incubation with isotype-specific antibody-conjugated paramagnetic beads, and these cells were isolated again on a second MACS column. These cells were labeled BP-1+. Finally, the flow-through from this purification step was incubated with antibodies to murine B220, followed by anti-isotype binding to beads and column separation. These cells were labeled B220+. Following isolation, total cellular RNA was purified as described above and subjected to electrophoresis, followed by northern blotting and hybridization with radiolabeled DRAK2 cDNA or radiolabeled GAPDH probes.
To characterize DRAK2 mRNA expression patterns in activated DP thymocytes, we isolated cells from AND.Rag2/;H-2d/d TCR transgenic mice, which lack positive selection (see below) and treated these cells under a variety of conditions, as described previously (11). Although these thymocytes are contaminated with some DN cells, most of the cells (>90%) are DP. A total of 2.5 x 106 thymocytes were used for each sample, and following the activation period, total cellular RNA was isolated. In some cases, cells were treated with PMA at 1 ng ml1 for the indicated times, with or without PD98059 (90-min pre-treatment). A total of 10 µg ml1 cycloheximide (CHX) was added to some samples 90 min prior to addition of PMA. For TCR-induced expression, 5 x 107 cells were incubated for 48 h with plate-bound anti-CD3 (145-2C11, 1 µg ml1) and/or anti-CD4 (GK1.5, 1 µg ml1) or anti-CD8 (3.155, 1 µg ml1) antibodies prior to northern blotting.
293T and DP thymocyte DRAK2 western blots
To determine the specificity of the anti-DRAK2 antibody, we transfected 293T cells with DRAK2 expression constructs encoding full-length and a C-terminal deletion mutant lacking the amino acids 351365 epitope. The 293T cells were transfected as above with the indicated constructs. Following a 24-h recovery, the cells were lysed in complete lysis buffer (150 mM NaCl, 50 mM sodium fluoride, 10 mM ß glycerophosphate, 20 mM HEPES pH 7.4, 1% Triton X100 with 1 mM sodium vanadate, 1 mM phenylmethylsulphonylfluoride, 1 mg ml1 aprotinin, 1 mg ml1 leupeptin), and lysates were resolved on a 12% SDS-PAGE gel. The proteins were transferred to PVDF membranes (Immobilon-P, Millipore, Billerica, MA, USA), pre-washed in Tris buffered saline/tween 20 (TBST), then blocked in TBSTM (TBST supplemented with 5% milk) and incubated with anti-DRAK2 polyclonal antibody (Imgenex, San Diego, CA, USA) at a 1:1000 dilution or with anti-HA mouse mAb (Babco/Covance Research Products, Berkeley, CA, USA) at a 1:1000 dilution. In some cases, blots were stripped and reprobed with anti-phospho-Erk1/2 (Cell Signaling, Beverly, MA, USA) at a 1:1000 dilution or anti-ß-actin (Abcam, Cambridge, UK) at a 1:50 000 dilution. Following an overnight incubation with the primary antibody, the blots were washed three times in TBST, and incubated with peroxidase-labeled anti-rabbit or anti-mouse secondary antibodies (Vector Labs, Burlingame, CA, USA) for a 1-hour incubation at room temperature. To visualize these westerns, the blots were washed three times in TBST, then incubated in ECL reagent (Amersham Biotech) and exposed to X-ray film. For DP thymocyte westerns, 10 million thymocytes from MHC0 (MHC class I/class II deficient) mice were stimulated for 4 h with 10 ng ml1 PMA for each condition. Inhibitors were co-cultured at the following concentrations: all PKC inhibitors at 1 µM, PD98059 at 30 µM, SB203580 at 10 µM and UO126 at 30 µM (Calbiochem, San Diego, CA, USA).
In vitro translation and subcellular localization studies
To characterize subcellular localization, DRAK2 was fused to EGFP. GFPDRAK2 was transfected into 293T cells using Lipofectamine (Invitrogen) and assayed by western blotting using an antibody to GFP (Clontech, Palo Alto, CA, USA). For microscopy, COS7 and Jurkat cells were transfected with pcDNA3-GFP-DRAK2 or pcDNA3-GFP. Additionally, COS7 cells were transfected with a C-terminal truncation mutant (pcDNA3-GFP-DRAK2[1290]). Following overnight incubation, the cells were counterstained with Hoechst 33342 (Molecular Probes, Eugene, OR, USA) and analyzed using an epifluorescent microscope equipped with digital image capture capabilities (for COS7 cells) or a confocal microscope (for Jurkat cells). Separate images of each field were captured using filters for UV and green fluorescence, and these images were digitally composited.
Freshly isolated thymocytes from a C57BL/6 mouse were stimulated with plate-bound CD3 and soluble anti-CD4 (eBioscience, San Diego, CA, USA) (low: 25 ng ml1
CD3 and 2 µg ml1
CD4; high: 2 µg ml1
CD3 and 10 µg ml1
CD4; PMA 40 ng ml1, ionomycin 2 µM) for 1 h. Cells were fractionated into cytoplasmic and nuclear fractions by homogenization of cells in hypotonic buffer (10 mM MgCl2, 10 mM KCl, 10 mM HEPES pH 7.0) followed by low-speed centrifugation (800 x g).
Retroviral expression of DRAK2 and survival/apoptosis studies of 3T3 cells and primary mouse T cells
The open reading frame of full-length DRAK2 was subcloned into MSCV-IRES-Thy1.1 (MiT) (12, 13). This construct (or empty MiT) was co-transfected with the Eco helper virus construct into 293T cells. Following transfection, supernatants were isolated at various times and subsequently titered by infection of murine NIH3T3 cells. To transduce T cells, spleens and lymph nodes were harvested from 6- to 8-week-old mice, and single-cell suspensions were incubated with plate-bound anti-CD3, soluble anti-CD28 and IL-2 to initiate cell cycle. After 24 h, retroviral supernatants were added, along with polybrene, and the cells were spun for 1 h to enhance infection. At various times following transduction, cells were isolated and stained for FACS. Cells were stained with anti-CD8PerCP (BD Biosciences, San Diego, CA, USA), anti-CD4 allophycoerythrin (APC) (eBioscience), anti-Thy1.1PE (eBioscience) and Annexin-V (AnnV)FITC (Caltag, Burlingame, CA, USA). In each case, mock-transduced cells served as negative controls for Thy1.1. For 3T3 retroviral transduction, cells were incubated overnight with polybrene and retroviral supernatant at the indicated dilutions. Following infection, cells were maintained for >40 days in culture. To demonstrate expression, some cells were harvested at 5-day post-infection, lysed and analyzed by western blotting with anti-DRAK2. For investigation of apoptotic potential, cells were harvested at 5-day post-infection, left unstimulated or treated with tumor necrosis factor (TNF)
(20 ng ml1) and CHX (10 µg ml1) for 18 h, and then harvested for FACS. Cells were stained with anti-Thy1.1PE and AnnVFITC, followed by data acquisition using a FACSCalibur (BD Biosciences, Franklin Lakes, NJ, USA). All FACS data were analyzed using FlowJo software (Treestar, Inc., Ashland, OR, USA).
Mice
DRAK2/ mice have been described previously (1). All mice used were back-crossed onto a C57BL/6 background a minimum of six generations, and were used between 6 and 12 weeks of age. Mice were housed in pathogen-free conditions in accordance with regulations of the Institutional Animal Care and Use Committee at University of California, Irvine.
Calcium mobilization assays
Following harvest, thymocytes were re-suspended at 10 x 106 ml1 and were labeled in RPMI + 2% FCS with 4.6 µM Fura red (Molecular Probes), 3.4 µM Fluo-3 (Molecular Probes) and 0.02% Pluronic (Molecular Probes) for 1 h at 37°C. Cells were washed with cold (unsupplemented) RPMI, then incubated on ice for 15 min with biotinylated CD3 and
CD4 (PharMingen and eBioscience) as well as
CD4APC and
CD8PE (eBioscience and Caltag), and then washed and re-suspended in cold RPMI. Samples were pre-warmed for 15 min prior to analysis, then stimulated by cross-linking with 20 µg ml1 streptavidin (Sigma). Calcium mobilization was plotted as a ratio of Fluo-3 : Fura red using the kinetics suite of FlowJo Software (Treestar, Inc.).
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Results |
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DRAK2 is a primary response gene activated by TCR ligation in DP thymocytes
As described above, DRAK2 mRNA was expressed at low levels in the DP population, whereas it was found at high levels in positively selected, mature CD4+ and CD8+ thymocytes. It is known that many signals derived through the TCR complex are necessary for the maturation of DP cells to become CD4+ and CD8+ thymocytes. Thus, we sought to determine if signaling through the TCR complex might increase the levels of DRAK2 mRNA in isolated DP thymocytes. Treatment of isolated DP thymocytes with PMA resulted in the accumulation of DRAK2 mRNA (Fig. 2a). This induction of DRAK2 message accumulation occurred rapidly (within 30 min) and in a PMA dose-dependent fashion, with maximal levels achieved between 0.1 and 1.0 ng ml1. This induction occurred in the presence of the MEK inhibitor PD98059, demonstrating that the activation of the Erk MAP kinase (MAPK) pathway (downstream of Ras-GRP) was not involved in the induction of DRAK2 mRNA accumulation in DP thymocytes (Fig. 2b). Furthermore, the ability of PMA to up-regulate DRAK2 mRNA was found to be independent of protein synthesis since the elongation factor inhibitor CHX failed to inhibit this (Fig. 2c).
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Regulation of DRAK2 in DP thymocytes by non-classical PKC pathways
To characterize the regulation of DRAK2 protein levels in DP thymocytes, we utilized a polyclonal antibody produced to a peptide corresponding to human DRAK2 amino acids 351365. In this region, murine and human DRAK2 differ by only a single amino acid. To test the specificity of this antibody against murine DRAK2, we transfected 293T cells with 3x HA-tagged versions of full-length and carboxy-terminal-deleted DRAK2 and probed these by western blot using antibodies to HA or the polyclonal DRAK2 antibody (Fig. 3a). Anti-DRAK2 detected a 43-kDa band in wild-type DRAK2 transfected cells, but failed to identify the lower molecular weight mutant form since this mutant lacks the epitope recognized by the polyclonal antibody. The anti-DRAK2 antibody detected an artifactual high-molecular weight band, as this band, but not the 43-kDa band, was present in DRAK2-deficient thymocytes (data not shown). Thus, this antibody is highly specific for DRAK2.
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Given that PMA induced the accumulation of DRAK2 in DP thymocytes and that this process did not explicitly involve MEK (at least at the mRNA level), we wanted to know if PKC activity in thymocytes was involved in its up-regulation, since PKC function is thought to be crucial for T cell activation and thymocyte selection (2022). To test this notion, we utilized cell-permeable inhibitors of classical and non-classical PKC isoforms (Fig. 3d). PKC inhibitors Ro31-8425 and Ro31-8220 blocked the PMA-dependent accumulation of DRAK2 protein in DP thymocytes; these inhibitors are only moderately isoform selective and are known to inhibit the activities of both classical and non-classical PKC isoforms (23). However, addition of Go-6976, a potent and selective inhibitor of classical isoforms alone (PKC IC50 = 2.3 nM, PKCß IC50 = 6.2 nM), did not block the up-regulation of DRAK2 protein (24). Reprobing this blot with anti-phospho-Erk1/2 demonstrated that the blockade in DRAK2 up-regulation correlated with the phosphorylation status of the upstream MAPKs. These results suggest that the PMA-dependent up-regulation of DRAK2 in DP thymocytes requires the activation of a signaling pathway downstream of a non-classical PKC isoform(s). Taken together, we conclude that this pathway is comprised, at least partly, by a Raf
MEK
Erk signaling cascade.
DRAK2 expression alone is not sufficient to induce apoptosis in 3T3 cells or primary T cells
To determine if murine DRAK2 could promote apoptosis upon ectopic expression, we employed retroviral transduction of 3T3 cells and primary T cells. The DRAK2 open reading frame was subcloned into MiT, a murine stem cell retroviral construct useful for ectopic expression in primary lymphocytes (12, 13). In this retroviral system, DRAK2 expression is driven from a bicistronic mRNA also encoding the open reading frame of murine Thy1.1. Thus, cells transduced with DRAK2-expressing retrovirus can be conveniently assessed on the basis of surface Thy1.1 expression. First, retroviral supernatants from 293T cells transfected with MiT-DRAK2 or MiT (empty vector) were used to infect 3T3 cells at different dilutions. After 5 days of infection, the cells were lysed and analyzed by western blotting with an anti-DRAK2 antibody. Mock- and MiT-infected cells lacked any detectable DRAK2 expression, whereas MiT-DRAK2-infected cells expressed DRAK2 in a dose-dependent fashion (Fig. 4a). To determine if such ectopic expression of DRAK2 induced apoptosis in 3T3 cells, cells were infected with a 1:1 dilution of MiT or MiT-DRAK2, and then stained with anti-Thy1.1 and Annexin-V to detect apoptosis in uninfected and infected cells (Fig. 4b). Treatment of uninfected and MiT- and MiT-DRAK2-infected cells with TNF and CHX (TNF + CHX) induced appreciable apoptosis, as assessed by AnnV+ events. However, no difference in the proportion of apoptotic cells was observed with MiT-DRAK2 infection alone. Concerned that DRAK2 might manifest apoptotic effects on a longer time-scale, we maintained MiT-DRAK2-infected 3T3 cells over 40 days in culture. Our rationale was that if DRAK2 was capable of inducing apoptotic events slowly, the proportion of cells expressing the retrovirally maintained transgene would decrease over time. However, even at the highest concentration of MiT-DRAK2 supernatant, no appreciable difference in the proportion of cells expressing the transgene was observed (Fig. 4c).
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Subcellular localization of DRAK2 in T cells and thymocytes
Since DRAK2 lacks any apparent homology with other proteins outside of its kinase domain, we endeavored to characterize its cellular function. One important clue to the function of a protein of interest can be obtained by the investigation of its subcellular localization. Analysis of the primary amino acid sequence using the Swiss Prot PSORT II server predicts that DRAK2 is either a nuclear (score = 43.5%) or mitochondrial (score = 43.5%) protein (25). This algorithm detects a putative nuclear localization signal (NLS) near position 65 (KKRRR) within the kinase domain. To assay the localization of DRAK2, we produced an amino-terminal fusion between GFP and the DRAK2 open reading frame. This protein fusion migrated with an apparent molecular mass of 70 kDa on western blots probed with anti-GFP polyclonal antibodies (data not shown). COS7 cells were transiently transfected with expression vectors encoding GFPDRAK2 or GFP alone and cells were analyzed by fluorescent microscopy (Fig. 5a). To identify nuclear morphology, the cells were counterstained with the nucleic acid-intercalating Hoechst 33342 dye, and composite images were produced. Transfection with a GFP-only vector yielded green fluorescence throughout the cell, whereas the GFPDRAK2 fusion was found to be highly concentrated in the nucleus; similar results have been reported for human and rat DRAK2 (7, 8). Closer inspection of GFPDRAK2 localization revealed that the fusion was excluded from nuclear areas characteristic of nucleoli. Although mutation of residues in the putative NLS sequence failed to reliably alter the nuclear localization of DRAK2 (data not shown), deletion of the 80-residue carboxyl-terminus of DRAK2 (1290) completely inhibited nuclear accumulation in COS7. However, in this case, DRAK2 GFP fluorescence appeared punctate, presumably due to accumulation in perinuclear organelles.
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DRAK2 raises the threshold for calcium mobilization in SP thymocytes
We sought to determine a functional role for DRAK2 in developing thymocytes. It has been previously shown that peripheral naive T cells from Drak2-deficient mice receive a TCR signal of greater intensity and have an enhanced calcium flux (1). To determine if DRAK2 modulates TCR signaling within distinct stages in the thymus, we examined calcium flux in developing thymocytes. Freshly isolated thymocytes from C57BL/6 wild-type and Drak2-deficient mice were labeled with the calcium-binding indicators Fluo-3 and Fura red, stimulated with biotinylated antibodies to CD3 and CD4 and cross-linked with streptavidin. To discern the effect of a Drak2 deletion in specific developmental subsets, these thymocytes were first labeled with fluorescently tagged anti-CD4 and anti-CD8. CD4+, DP and transitional cells were electronically gated for subsequent analyses (Fig. 6a). In the DP population, the extent of calcium mobilization following TCR/CD4 cross-linking in Drak2/ thymocytes was similar to that seen in wild-type cells (Fig. 6b). At various sub-optimal doses of anti-CD3 tested, no obvious difference between Drak2/ and wild-type DP cells was observed (data not shown). In contrast, CD4+ thymocytes from Drak2/ mice generated a maximal calcium response following cross-linking with sub-optimal levels of anti-CD3 and -CD4. These results confirm that, similar to the situation in naive peripheral T cells, the presence of DRAK2 is required for negatively regulating TCR signaling in positively selected thymocytes. We also wished to determine how DRAK2 might affect the TCR activation threshold of transitional thymocytes (CD4lowCD8low DP cells), a subset thought to have undergone recent selection events (26). At sub-optimal levels of anti-CD3, there was a modest but reproducible release of Ca2+ in this population in Drak2/ cells, but not in wild-type thymocytes. These results are consistent with the pattern of expression of DRAK2 within distinct thymocyte subsets described above, and suggest that DRAK2 participates in setting the threshold for TCR signaling during thymocyte selection.
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Discussion |
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We have found DRAK2 to be expressed primarily in the nucleus of COS7 and Jurkat cells (Fig. 5), a similar site of localization reported for the human and rat isoforms. Furthermore, we found that DRAK2 was excluded from regions of high Hoechst staining; we believe these data indicate that DRAK2 is prevented access to nucleoli, and thus, it is unlikely that it regulates rDNA transcription. DRAK1 and DRAK2 both contain strong consensus sequences for NLSs present near the kinase active site. DAPK3 and DAPK1 also contain such NLS motifs in this region, though DAPK1 is not normally localized in the nucleus as a result of retention by cytoskeletal proteins outside of this organelle (3, 28). However, this DAPK1 NLS is likely a functional one, since deletion mutants of DAPK1 retaining only the kinase domain do accumulate in the nucleus. DAPK3/ZIP/Dlk is also known to traffic to the nucleus, but appears to be concentrated in pro-myelocytic leukemia nuclear bodies, multiprotein compartments thought to be involved in the suppression of oncogenic transformation (2830). Other DAPKs exist elsewhere in cells, with DAPK1 associating with the cytoskeleton and DAPK2 expressed primarily as a soluble protein in the cytoplasm (31). The nuclear localization of DRAK2 suggests it may play a role in transcriptional regulation in resting cells, whereas it appears to function in the cytoplasm of activated T cells. Although the potential involvement of the putative canonical NLS has not yet been tested, we demonstrated that the 80-residue C-terminal domain was required for DRAK2 nuclear localization. Thus, DRAK2 may utilize an alternative nuclear localization mechanism via the action of this domain, and this process may be subject to regulation by TCR signaling.
Ectopic expression of DRAK2 in murine 3T3 fibroblasts promoted an apoptotic nuclear morphology and decreased the recovery of colonies stably expressing the transgene (7). However, using transduction with a bicistronic retroviral system to track cells expressing the transgenic mRNA, we failed to detect any evidence of apoptosis or decreased survival in primary T cells or murine 3T3 cells ectopically expressing DRAK2 (Fig. 4). This discrepancy may be due to the reduced levels of expression of the transgene driven by retroviral long terminal repeats when compared with CMV promoter-driven expression reported. However, we and others have found that cell lines such as 293T, HeLa, COS7 and SV40 large T antigen-transformed Jurkat T cells are resistant to death following high-level DRAK2 expression (Fig. 5, also data not shown) (7). It has been speculated that this discrepancy in apoptotic sensitivity is dependent upon the presence of the large T antigen in certain transformed cell lines, though it should be noted that DRAK1/2-insensitive HeLa cells are not transformed with large T antigen (2). Given the high expression of DRAK2 in lymphoid tissues, particularly in non-apoptotic differentiated subsets, we believe that such cells would be, of necessity, similarly resistant to any pro-apoptotic function of DRAK2 due to a requisite induction of inhibitory pathways or via the down-modulation of pro-apoptotic mediators. Differential apoptotic sensitivity during development, or following activation, is certainly a common mechanism for regulating cell fate. However, our attempts to define an apoptotic role for DRAK2 have not revealed any clear requirement for this DAP kinase in promoting physiological apoptosis (Fig. 4 and ref. 1).
DRAK2 expression is developmentally regulated during thymocyte development. Additionally, as demonstrated here, DRAK2 exists as a primary response gene induced by TCR stimulation of DP thymocytes. This pattern of expression is intriguing since it suggests that DP cells are subject to counter-regulation by DRAK2 as a consequence of the TCR-dependent signals received during the process of selection. We found that the TCR-dependent increase in DRAK2 expression was greatly enhanced by co-receptor cross-linking (Fig. 2d). DRAK2 mRNA accumulation did not depend on protein synthesis, suggesting that it is a direct target of transcription factors induced by TCR cross-linking (Fig. 2c). Although we found little effect of blocking calcineurin or MEK/Erk signaling on the induction of DRAK2 mRNA (Fig. 2b and e), inhibition of MEK using PD98059 or U0126 blocked the synthesis of DRAK2, as detected by western blotting (Fig. 3c). Although we are presently unclear about the significance of these findings, these results suggest that both DRAK2 transcription and translation are subject to control by signals emanating from the TCR complex. Additionally, we have found that a non-classical PKC is essential for PMA-induced up-regulation of DRAK2 protein in T cells (Fig. 3d). We favor a model in which DRAK2 transcription depends on the activity of a PKC-dependent transcription factor, perhaps via the action of NF-B. DRAK2 mRNA possesses very large 5' and 3' untranslated regions. It may be that these sequences subject DRAK2 mRNA to translational regulatory mechanisms that depend on the action of the Raf
MEK
Erk cascade. Although additional regulatory factors may be involved in the maintenance of DRAK2 expression in quiescent, naive T cells, our results demonstrate that DRAK2 serves as a negative feedback loop for TCR signaling during DP thymocyte selection.
We have found that DRAK2 raises the threshold for TCR signaling in developing thymocytes. Deficiency of certain negative regulators of TCR signaling, including c-Cbl, SLAP and Csk, results in enhanced selection (32); similarly, we have recently shown that in Drak2/ mice positive selection of CD4+ T cells was enhanced, as demonstrated by increased numbers of CD4+ thymocytes and increased levels of CD5 and CD69 expression (1). We hypothesize that DRAK2 is involved in regulating the signal strength that controls the fate of developing thymocytes. As its expression dramatically increases in DP thymocytes following TCR cross-linking (Figs 2 and 3), DRAK2 negatively regulates calcium mobilization, a critical signaling pathway for the activation of T cells. Once the TCR is activated, phospholipase C hydrolyzes phospholipids at the cell membrane to produce two crucial second messengers, diacylglycerol and inositol 1,4,5-triphosphate. Elevated intracellular Ca2+ results in the activation of the calcineurin pathway, which targets the NFAT family of transcription factors. In the context of the DP population in the thymus, such signals do not promote proliferation, but elicit the selection of these cells (33). Specifically, Ca2+ oscillations trigger thymocytes to become immotile in order to prolong interactions with stromal cells, presumably to help to organize molecular interactions at the immunological synapse (34). Additionally, mature T cells require sustained periods of Ca2+ signaling to modulate gene expression (35). In unselected DP thymocytes, which do not yet express DRAK2, calcium mobilization in wild-type cells was similar to that seen in Drak2/ cells. However, in the selected CD4+ population, which possesses DRAK2 expression by virtue of signals received via the TCR and CD4 itself, cells from Drak2/ mice had a reduced activation threshold for Ca2+ mobilization. These results confirm a specific role for DRAK2 in the negative regulation of thymocyte activation.
The finding that rat DRAK2 binds to a calcineurin-B-like protein named CHP suggests that DRAK2 and CHP may counter-regulate calcium-dependent signaling pathways in thymocytes following TCR stimulation (8, 36). Recent evidence demonstrates an inhibitory function of CHP in calcineurin signaling following TCR activation of Jurkat T cells (37). DRAK2 may modulate the ability of CHP to block calcineurin signaling, thereby setting the threshold for calcium signaling downstream of TCR signaling. Calcineurin has been implicated in thymocyte positive selection since calcineurin-deficient mice have defective positive selection, whereas mice expressing a transgenic active form of calcineurin possess enhanced positive selection; neither of these genetic manipulations of calcineurin affected negative selection (38, 39). These results are coherent with the slight enhancement in positive selection, but normal negative selection, observed in Drak2-deficient mice (1). An alternative hypothesis is that DRAK2 modulates T cell activation at a level more proximal to the TCR itself, a view that is more consistent with the enhanced Ca2+ flux observed after sub-optimal TCR cross-linking in Drak2/ thymocytes. In this manner, DRAK2 might participate in thymocyte selection by regulating the activation thresholds of T cells as they mature in response to antigen receptor-driven maturation. Additional work regarding the pathways governed by DRAK2 within cells of the lymphoid system will likely yield new insight into the processes of lymphopoiesis and immune regulation.
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Acknowledgements |
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Abbreviations |
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AnnV | Annexin-V |
BM | bone marrow |
CHX | cycloheximide |
DAPK | DAP-like family of serine/threonine kinase |
DN | double negative |
DP | double positive |
DRAK2 | DAP-related apoptotic kinase-2 |
MAPK | MAP kinase |
NLS | nuclear localization signal |
NIH | National Institutes of Health |
PMA | phorbol myristate acetate |
PKC | protein kinase C |
SP | single positive |
TNF | tumor necrosis factor |
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Notes |
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Received 17 November 2004, accepted 2 August 2005.
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References |
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