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International Immunology Advance Access originally published online on February 27, 2007
International Immunology 2007 19(4):487-495; doi:10.1093/intimm/dxm015
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Dok-1 and Dok-2 are negative regulators of T cell receptor signaling

Tomoharu Yasuda1, Kenji Bundo1, Ayako Hino2, Kazuho Honda3, Akane Inoue1, Masaki Shirakata1, Mitsujiro Osawa4, Toshiki Tamura5, Hideo Nariuchi6, Hideaki Oda3, Tadashi Yamamoto7 and Yuji Yamanashi1

1 Department of Cell Regulation, Medical Research Institute, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan
2 Division of Mucosal Immunology, Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan
3 Department of Pathology, Tokyo Women's Medical University, Tokyo 162-8666, Japan
4 Laboratory of Stem Cell Therapy
5 Division of Immunology, Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan
6 Ogata Institute for Medical and Chemical Research, Tokyo 102-8230, Japan
7 Division of Oncology, Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan

Correspondence to: Y. Yamanashi; E-mail: yamanashi.creg{at}mri.tmd.ac.jp


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
Interaction of the TCR complex with self- or foreign peptides is a central event in the immune responses. Upon TCR stimulation, a protein–tyrosine kinase (PTK), ZAP-70, is recruited to signaling units of the TCR complex, such as TCR{zeta}, to play an essential role in T cell activation. Here, we find that mice lacking adaptor proteins Dok-1 and Dok-2 show augmented responses to thymus-dependent, but not thymus-independent, antigens, and that their T cells show elevated responses to TCR stimulation, including the activation of ZAP-70 and subsequent proliferation and cytokine production. Furthermore, the forced expression of Dok-1 or Dok-2 in a CD3+CD4+ T cell clone inhibited the activation of ZAP-70 upon TCR stimulation. Interestingly, the Dok-1 and Dok-2 COOH-terminal moieties bearing the src homology 2 target motifs were dispensable for this negative regulation, even though they are crucial for the known adaptor function of Dok-family proteins. Thus, by an as yet unidentified mechanism, Dok-1 and Dok-2 play an essential role in the negative regulation of TCR signaling. Consistently, all mice lacking these proteins exhibited elevated titers of antibodies to double-stranded DNA and developed lupus-like renal disease.

Keywords: antigen receptor, protein–tyrosine kinase, signal transduction


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
T cells are responsible for the recognition of both self- and foreign peptides, and thus play crucial roles in the immune responses. Because inappropriate T cell responses cause immune disorders, the activation of the cells through TCRs requires tight regulation (1, 2). For example, either enhanced or attenuated activation of TCR signaling leads to lupus-like autoimmune disorders (3, 4). TCR on naive T cells is a functional complex of antigen-binding units, {alpha} and ß chains, and signaling units, CD3{epsilon}/{gamma}/{delta} and TCR{zeta} (5). Engagement of the TCRs initiates signaling cascades beginning with the tyrosine phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs) in the TCR complex by Src-family protein–tyrosine kinases (PTKs), leading to activation of a cytoplasmic PTK, ZAP-70. The activation of ZAP-70 leads to tyrosine phosphorylation of downstream targets, including LAT, SLP-76 and PLC-{gamma}1, and is an essential event in the propagation of TCR-mediated signaling.

Dok-1 was originally identified as a major substrate of many PTKs (6, 7), and the Dok family has since been expanded to seven members, Dok-1 to Dok-7 (8–10). These proteins share structural similarities characterized by the NH2-terminal pleckstrin homology (PH) and phosphotyrosine-binding (PTB) domains, followed by the src homology 2 (SH2) target motifs in the COOH-terminal moiety, suggesting an adaptor function; upon tyrosine phosphorylation, Dok proteins might recruit or, in a few cases, sequester other signaling molecules bearing SH2 domains. The PH and PTB domains may act as lipid/protein-interacting modules. Indeed, upon tyrosine phosphorylation various Dok-family members have been shown to recruit SH2-containing molecules such as p120 rasGAP and Nck via the COOH-terminal SH2 target motifs (6, 7, 11). The recruitment of rasGAP appears to be essential for negative regulation of the Ras–Erk pathway by Dok-1 and Dok-2 (12, 13). However, it should be noted that the distantly related Dok-7 can activate MuSK, a muscle-specific receptor PTK, in myotubes (9), indicating that Dok-family proteins are not necessarily limited to playing an adaptor function downstream of PTKs.

Within the Dok family, only Dok-1, -2 and -3 are preferentially expressed in hematopoietic cells. Previously, we demonstrated that Dok-1 is a negative regulator of B cell receptor signaling (14); however, mice lacking Dok-1 showed normal responses to thymus-independent (TI) antigens, suggesting functional redundancy among Dok-family proteins. In fact, we and others have recently generated mice lacking Dok-1 and/or Dok-2 and found that these proteins are indispensable negative regulators for maintenance of myeloid homeostasis, suppressing leukemia and innate immune responses to LPS (15–17). Since the dok-3 transcript was barely detectable in the thymus, as well in as many T cell lines (18, 19), we have further studied the roles of Dok-1 and Dok-2 in T cells. Here, we report that these proteins work as key negative regulators of TCR signaling by inhibiting the activation of ZAP-70, and that this inhibitory function requires their PH and PTB domains, but not the COOH-terminal moiety, suggesting a non-adaptor mechanism. Furthermore, the Dok-1/2-mediated negative regulation of TCR signaling appeared to be important for immune tolerance, because mice lacking Dok-1 and Dok-2 developed a lupus-like renal disease.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Mice
The generation of Dok-1–/–, Dok-2–/– and Dok-1/Dok-2 double-deficient (dKO) mice has been described elsewhere (14, 15). Mice were backcrossed to C57BL/6J for at least eight generations, unless otherwise indicated, and maintained under specific pathogen-free conditions. Experiments and animal care were performed according to the institutional guidelines.

Flow cytometry and reverse transcription–PCR
Mononuclear cells from 7- to 9-week old mice were stained with FITC-, PE- or biotin-conjugated mAbs (BD Biosciences, San Jose, CA, USA or eBioscience, San Diego, CA, USA) for fractionation and enumeration with FACSCalibur or FACSVantage (BD Biosciences) of Thy1.2+ T, CD3+CD4+ T (CD4+), CD3+CD8+ T (CD8+), CD25+ T (CD4+CD25+), CD69+ T (CD4+CD69+) or B220+ B cells in the spleen, and CD4–CD8– (DN), CD4+CD8+ (DP), CD4+CD8– or CD4–CD8+ cells in the thymus, pro-B (B220+CD43+IgM–), pre-B (B220+CD43–IgM–), immature and recirculating B (B220+IgM+) or myeloid (Gr-1+Mac-1+) cells in the bone marrow (Figs 1, 2A, 3F and G). Streptavidin–quantum red (Sigma, St Louis, MO, USA) or –texas red (Invitrogen, Carlsbad, CA, USA) conjugate was used to label the biotin-coupled mAbs. Fractionated cells were subjected to normalized reverse transcription–PCR as described (20; Fig. 1) with primers: 5'-TTGGAGATGCTGGAGAATTCGC-3' and 5'-AGTCAGTTCTGAGGATATCCTG-3' (dok-1), 5'-AGTGACTGGATACAGGCCATC-3' and 5'-AGCAATGACCTTTTCTAAGGC-3' (dok-2) and 5'-TACTCCTCCTGGCAGGAAGTG-3' and 5'-ACATGGCCAGCTCTGGAAGAC-3' (dok-3).


Figure 1
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  		Fig. 1. Expression of dok-family genes in hematopoietic cells. Mononuclear cells from the spleen (Sp), thymus (Thy) or bone marrow (BM) of C57BL/6 mice at 8 weeks of age were fractionated with flow cytometry and their RNA was subjected to reverse transcription–PCR for each mRNA. imm + rec B, immature and recirculating B cells.

 

Figure 2
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  		Fig. 2. Dok-1 and Dok-2 negatively regulate T cell-dependent immune responses. (A) The absolute number of each cell type in the thymus or spleen of mice was counted using flow cytometry. Statistical significance was determined with Student's t-test. *P < 0.05 and **P < 0.01. (B) Dilutions of the serum from dKO or WT littermates with mixed backgrounds (129 x 1/SvJ and C57BL/6J) were evaluated for DNP-specific IgA or IgG on day 14 and IgM on day 7 after immunization with DNP-KLH. Data are the mean ± SD from four individual mice.

 

Figure 3
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  		Fig. 3. Augmented activation of T cells from dKO mice upon TCR stimulation. The proliferation (A and C) and cytokine production (B, D and E) of CD4+ splenic T cells purified by magnetic field exclusion from mice were determined following stimulation with PMA (10 ng ml–1) and ionomycin (1 µg ml–1) (P + I), or with mAbs to CD3{epsilon} in 96-well plates pre-treated with various concentrations of mAbs, in the absence or presence of exogenous IL-2. ND, not detectable. Data are the mean ± SD from triplicate experiments. (F and G) Splenocytes from the indicated mice were stained for CD4, and CD4+ T cells were gated and examined for the expression of CD3 (F), CD25 or CD69 (G). Percentages of each population are indicated in (G).

 
Immunization and assays for antibodies
Mice of 7–9 weeks old were immunized i.p. with 100 µg of DNP-conjugated keyhole limpet hemocyanin (DNP-KLH) in incomplete Freund's adjuvant. DNP-specific antibodies in the serum from immunized mice (Fig. 2B) or double-stranded DNA (dsDNA)-specific antibodies in the serum from non-immunized mice (Fig. 6B) were evaluated as previously described (21).


Figure 6
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  		Fig. 6. Lupus-like renal diseases developed in dKO mice. (A) Kidney sections from 12-month-old mice were stained with periodic acid–Schiff (PAS) or antibodies to each class of Ig or C3. Arrows indicate positions of the glomeruli. (B) IgG to dsDNA in the sera from littermates were evaluated. Mixed background mice (129 x 1/SvJ and C57BL/6J) were studied.

 
Preparation of CD4+ splenic T cells by magnetic field exclusion
Splenocytes from 7- to 9-week old mice were incubated with mAbs to CD8, B220 and CD11b (BD Biosciences) and anti-rat IgG magnetic beads (MACS, Miltenyi Biotech, Bergisch Gladbach, Germany) followed by magnetic field exclusion to purify CD4+ splenic T cells (>85%) (Figs 3A–E and 4A).

T cell proliferation and cytokine production assay
CD4+ splenic T cells (5 x 104 cells 100 µl–1) purified by magnetic field exclusion were treated with soluble or immobilized mAbs to CD3{epsilon} (145-2C11), in the presence or absence of exogenous IL-2, or with phorbol myristate acetate (PMA) and ionomycin in culture medium (RPMI-1640 supplemented with 10% FCS and 50 µM ß-mercaptoethanol). For the proliferation assay, [3H]thymidine (0.2 µCi per well) was added 48 h after stimulation, and its incorporation was measured 12 h later. For cytokine assay, culture supernatants at 48 h after stimulation were removed into 96-well plates coated with mAbs to mouse IL-2, IFN{gamma} or IL-4 (BD Biosciences), and subjected to ELISA using biotinylated mAbs to each cytokine (BD Biosciences) and HRP-conjugated goat antibodies to biotin (Vector Laboratories, Burlingame, CA, USA).

Dok cDNAs and transfection
Mouse cDNAs encoding Dok-1 mutants lacking the PH domain ({Delta}PH, 121–482) or COOH-terminal region ({Delta}C, 1–276) or having a mutation in the PTB domain (PTB-AA, R207A and R222A) and those encoding Dok-2 mutants ({Delta}PH, 120–412; {Delta}C, 1–270; PTB-AA, R202A and R217A) were generated by PCR. These Dok cDNAs and those encoding mouse TCR{zeta} with or without the Y72F substitution were inserted into the expression vector pAPIG-Flag-bearing COOH-terminal FLAG-tag and IRES-GFP sequences. All plasmids were confirmed by sequencing and transfected into 25-14 T cells, which express exogenous TCR{alpha}ß and CD4 (22). Comparable expression levels of the exogenous genes in transfected clones were confirmed by flow cytometry. Data were representative among at least three independent clones.

Immunoblotting and immunoprecipitation
CD4+ splenic T cells (2 x 107 per 0.2 ml) purified by magnetic field exclusion or 25-14 T cells (1 x 107 per 0.2 ml) were treated with biotinylated anti-CD3{epsilon} mAbs (10 µg ml–1) followed by cross-linking with streptavidin (25 µg ml–1) at 37°C. These cells were solubilized with the lysis buffer (1% NP-40, 150 mM NaCl, 10 mM Tris at pH 7.4, 0.5 mM EDTA, 10 mM NaF, 1 mM sodium orthovanadate, 10 µM sodium molybdate, 2 µg ml–1 aprotinin, 1 mM benzamidine, 5 µg ml–1 chymostatin, 5 µg ml–1 leupeptin, 1 µg ml–1 pepstatin A and 0.2 mM phenylmethylsulphonylfluoride), and cleared lysates (whole cell lysates) were subjected to immunoblotting with antibodies to phospho-ZAP-70, phospho-LAT, phospho-Erk (Cell Signaling, Danvers, MA, USA), Erk2 (Santa Cruz, Santa Cruz, CA, USA) or FLAG-tag (Sigma). Otherwise, they were sequentially incubated with antibodies to TCR{zeta} (Santa Cruz), Dok-1 or Dok-2 (Santa Cruz) and protein G-Sepharose (Amersham Biosciences, Buckinghamshire, UK), and immunoprecipitates were subjected to immunoblotting with antibodies to TCR{zeta}, phosphotyrosine (4G10, Millipore, Billerica, MA, USA), Dok-1, Dok-2 or FLAG-tag. In Fig. 5E, 293T cells were transfected with the indicated combinations of expression plasmids for myc-tagged TCR{zeta}{Delta}C (1–110 amino acids of mouse TCR{zeta}) or CD3{epsilon}, FLAG-tagged Dok-1 or Dok-2, hemagglutinin (HA)-tagged ZAP-70 and LckYF, an active form of PTK known to phosphorylate TCR{zeta} and CD3{epsilon}. These cells were solubilized with the lysis buffer as described above, and whole cell lysates or anti-myc-tag immunoprecipitates were subjected to immunoblotting with antibodies to myc-tag (Cell Signaling), FLAG-tag (Sigma) or HA-tag (Cell Signaling).


Figure 5
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  		Fig. 5. Interaction of Dok-1 and Dok-2 with signaling units of the TCR complex. (A) Structures of TCR{zeta} and CD3{epsilon} and amino acid sequences of their ITAMs in various mammals. Core residues of the ITAMs are in bold and PTB target motifs are indicated by asterisks, including Tyr-72 of mouse TCR{zeta}. TM, transmembrane and IC, intracellular. (B) 25-14 T cells expressing FLAG-tagged TCR{zeta} or Y72F mutant ({zeta}YF-FLAG), which has a Tyr/Phe substitution at Tyr-72 in ITAM1, were treated with or without mAbs to CD3{epsilon} for 3 min (CD3 stim.). Anti-Dok-1 or Dok-2 immunoprecipitates (IP) or whole cell lysates (WCL) were subjected to IB. (C) 293T cells transfected with expression plasmids for myc-tagged TCR{zeta} ({zeta}-myc-WT) or its mutant bearing the Y72F substitution ({zeta}-myc-YF) alone or together with LckYF were subjected to GST pull-down analysis with the indicated fusion proteins. Pulled-down proteins (GST-PD) or WCL were subjected to IB. The band of about 20 kD weakly detected with antibodies to myc-tag in every GST-PD in the upper panel is non-specific. (D) 293T cells transfected with expression plasmids for myc-tagged CD3{gamma} or CD3{epsilon} were subjected to GST pull-down analysis, as in (C). (E) 293T cells were transfected with expression plasmids for myc-tagged TCR{zeta}{Delta}C ({zeta}{Delta}C-myc) or CD3{epsilon} (CD3{epsilon}-myc), HA-tagged ZAP-70, FLAG-tagged Dok-1 or Dok-2, and LckYF. Anti-myc IP or WCL were subjected to IB. Arrowheads indicate positions of proteins detected by each IB.

 
GST pull-down analysis
cDNAs encoding the intact PTB domain of mouse Dok-1 or Dok-2 (153–269 or 150–254) and their mutants bearing the R207A/R222A or R202A/R217A substitutions, respectively, were amplified by PCR and inserted into the pGEX4T-1 (Amersham Biosciences) to generate the GST fusion proteins. 293T cells were transfected with expression plasmids for myc-tagged CD3{gamma} (CD3{gamma}-myc), CD3{epsilon} (CD3{epsilon}-myc), TCR{zeta} ({zeta}-myc-WT) or its mutant bearing the Y72F substitution ({zeta}-myc-YF) alone or together with LckYF. These cells were solubilized with the lysis buffer as described above, and whole cell lysates were incubated with 25 µg of purified GST fusion proteins bound to glutathione sepharose for 2 h at 4°C. Pulled-down proteins were washed and subjected to immunoblotting with antibodies to myc-tag (Cell Signaling) or GST (Santa Cruz). Whole cell lysates were also subjected to immunoblotting with antibodies to myc-tag.

Immunohistochemistry
Kidneys were fixed with 10% phosphate-buffered formalin (pH 7.2), embedded in paraffin, cut into 4-µm sections and stained with periodic acid–Schiff for light microscopy. For immunohistochemistry, kidneys were embedded in OCT compound and frozen. Cryostat sections (5-µm) were fixed in 95% acetone, incubated with FITC-conjugated goat IgG to mouse IgG, IgA, IgM or C3 (MP Biomedicals, Solon, OH, USA), washed with PBS, mounted and viewed on an AX80 fluorescence microscope (Olympus, Tokyo, Japan).


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Dok-1 and Dok-2 negatively regulate T cell-dependent immune responses
To gain insight into the roles of Dok-1 and Dok-2 in the T cell lineage, we examined their expression and found that peripheral T cells (Thy1.2+) and most thymocytes express dok-1 and dok-2, but not dok-3, transcripts (Fig. 1). Interestingly, the amounts of both dok-1 and dok-2 mRNA increased upon thymocyte maturation. In addition, the thymic cellularity of dKO mice was typically double that of the wild type (WT) due to an increase in the number of thymocytes, except for the DN population, whereas such changes were moderate in Dok-1–/– or Dok-2–/– mice (Fig. 2A). A similar increase was also seen in the number of CD4+ or CD8+ T cells in the spleens of these mutant mice. However, the numbers of DN thymocytes and B-lineage cells in the spleen were not significantly changed (Fig. 2A). These results suggest that Dok-1 and Dok-2 act co-operatively as negative regulators in T cells that have functional TCRs. Therefore, we tested the primary antibody response of dKO mice to the thymus-dependent antigen DNP-KLH. Upon immunization, dKO mice showed elevated levels of DNP-specific IgA and IgG in the serum (Fig. 2B), an abnormality not found in either Dok-1–/– or Dok-2–/– single knockout mice (data not shown). However, IgM production, which is independent of Th (23), was intact in dKO mice (Fig. 2B). These dKO mice also showed normal responses to the TI antigen DNP-Ficoll (data not shown). Together, these data indicate that Dok-1 and Dok-2 are key negative regulators of T cell-dependent immune responses.

Dok-1 and Dok-2 are negative regulators of TCR signaling
Because TCR plays a pivotal role in T cell-dependent immune responses, we next examined the roles of Dok-1 and Dok-2 in the TCR-mediated activation of peripheral T cells. Upon TCR cross-linking with mAbs to CD3{epsilon}, CD4+ T cells from mice lacking Dok-1 or Dok-2 displayed increased proliferation, as well as IL-2 production (Fig. 3A and B). Furthermore, CD4+ T cells from dKO mice responded even more vigorously to TCR cross-linking. However, these mutant T cells showed normal proliferative and IL-2 responses to a combination of PMA and ionomycin, a treatment known to bypass TCR signaling (Fig. 3A and data not shown). The augmented proliferation of dKO T cells was likely due to elevated IL-2 production, because exogenous IL-2 neutralized the negative effects of Dok-1 and Dok-2 in WT T cells upon TCR cross-linking (Fig. 3C). We also examined IFN{gamma} or IL-4 response to TCR stimulation and confirmed the augmented production of these cytokines by CD4+ T cells from dKO mice (Fig. 3D and E). Although we confirmed that CD4+ T cells from dKO mice expressed normal levels of TCRs (Fig. 3F), augmented activation of dKO T cells upon TCR stimulation could be due to an activated phenotype of the mutant T cells. However, the proportions of splenic CD4+ T cells positive for each of the activation markers, CD25 and CD69, were apparently normal in dKO mice (Fig. 3G). Consistently, the proportions of naive (CD4+CD44lowCD45RBhigh) and memory T cells (CD4+CD44highCD45RBlow) were also intact in the spleens of dKO mice (data not shown), indicating that hyperactivation of dKO T cells upon TCR stimulation was not due to accumulation of non-naive CD4+ T cells. Although there are reports that Dok-1 becomes tyrosine phosphorylated upon the cross-linking of CD28, a co-receptor of T cells that can enhance TCR signaling (24), CD4+ T cells from dKO mice showed normal increases in their proliferative responses to the cross-linking of CD28 in the presence of basal TCR stimulation (data not shown). Together, these findings demonstrate that Dok-1 and Dok-2 work co-operatively as negative regulators of TCR signaling.

Dok-1 and Dok-2 negatively regulate ZAP-70 upon TCR signaling
Engagement of the TCR activates Lck and Fyn to phosphorylate ITAMs of the receptor complex, which then recruit ZAP-70 by binding to the tandem SH2 domains of the PTK. Consequently, ZAP-70 is activated and phosphorylates essential downstream targets, including an adaptor protein, LAT, to facilitate calcium mobilization and activation of Erk mitogen-activated protein kinase, which is a crucial event in the activation of T cells (1, 25, 26). To delineate where Dok-1 and Dok-2 interfere with signaling downstream of TCR, we examined the phosphorylation, and thereby activation, of ZAP-70, LAT and Erk upon TCR stimulation of CD4+ T cells from dKO mice. The loss of Dok-1 and Dok-2 resulted in enhanced phosphorylation of ZAP-70, LAT and Erk in CD4+ T cells, indicating that Dok-1 and Dok-2 play an essential role in the negative regulation of ZAP-70 and its downstream signaling (Fig. 4A).


Figure 4
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  		Fig. 4. Dok-1 and Dok-2 negatively regulate ZAP-70 upon TCR signaling. CD4+ splenic T cells purified by magnetic field exclusion from mice (A) or 25-14 T cells expressing exogenous Dok proteins (B and C) were stimulated with mAbs to CD3{epsilon} (CD3 stim.) and subjected to immunoprecipitation (IP) and/or immunoblotting (IB). pTCR{zeta}, tyrosine-phosphorylated TCR{zeta}. Arrowheads indicate positions of proteins detected by each IB. Unlike primary T cells, TCR{zeta} is apparently unphosphorylated in the absence of TCR stimulation in 25-14 T cells (C).

 
It is generally accepted that Dok-family adaptors recruit downstream effectors, including p120 rasGAP, via the SH2 target motifs in their COOH-terminal regions in a manner dependent on tyrosine phosphorylation. However, tyrosine phosphorylation of Dok-1 and Dok-2 upon TCR stimulation is still controversial, and there might be an as yet unidentified mechanism downstream of the TCR (24, 27, 28). Therefore, we established CD4+ T cell clones exogenously expressing WT or mutant Dok-1 or Dok-2 to investigate the molecular mechanism underlying their function against ZAP-70. The forced expression of Dok-1 or Dok-2 in 25-14 T cells inhibited the phosphorylation of ZAP-70, LAT and Erk upon TCR stimulation, confirming their negative role (Fig. 4B). However, forced expression of the Dok-1 mutant lacking the PH domain ({Delta}PH), or bearing a mutation in the PTB domain (PTB-AA), failed to suppress TCR signaling, indicating essential roles for these membrane/lipid and protein-interacting domains. To our surprise, the Dok-1 mutant lacking the COOH-terminal moiety ({Delta}C) inhibited ZAP-70 normally along with downstream signaling. The corresponding mutants of Dok-2 showed similar results (data not shown). Taken together, these data suggest that Dok-1 and Dok-2 inhibit ZAP-70 via the PH and PTB domains independent of the COOH-terminal moiety bearing the SH2 target motifs.

To further understand how these Dok-family proteins interfere with the activation of ZAP-70, we next examined whether they inhibit the tyrosine phosphorylation of TCR{zeta} upon TCR stimulation. The forced expression of Dok-1 in 25-14 T cells did not significantly affect tyrosine phosphorylation of the TCR subunit, a result independent of whether the PTB domain was intact or mutated (Fig. 4C). Likewise, the forced expression of Dok-2 resulted in the normal phosphorylation of TCR{zeta} (data not shown), suggesting that these Dok-family proteins inhibit the recruitment of ZAP-70 to the phosphorylated ITAMs in the receptor complex and/or a downstream event required for the activation of ZAP-70. Notably, the Dok-1 and Dok-2 PTB domains bind to the phosphotyrosine (pY)-containing peptide sequences NXLpY and NPXpY (12, 29), which are present on the CD3{epsilon} ITAM and the most juxtamembrane ITAM (ITAM1) of TCR{zeta} (Fig. 5A). These ITAMs are essential for tyrosine phosphorylation-dependent recruitment and activation of ZAP-70 (5, 30). As ITAM1 and the CD3{epsilon} ITAM are binding targets of the ZAP-70 SH2 domains upon tyrosine phosphorylation, interference between these Dok-family proteins and ZAP-70 might occur through their binding to the ITAMs upon TCR stimulation. Consistently, TCR stimulation induced binding of Dok-1 and Dok-2 with TCR{zeta}, a response that was largely inhibited by Tyr/Phe substitution (Y72F) in the PTB target motif (NQLY72) of TCR{zeta} ITAM1 (Fig. 5B). In addition, GST pull-down experiments with 293T cells over-expressing each TCR chain demonstrated that the Dok-1 or Dok-2 PTB domains can bind TCR{zeta} and CD3{epsilon}, but not CD3{gamma}, which has no PTB target motif, and this binding requires LckYF, which is known to phosphorylate these signaling units of the TCR complex (Fig. 5C and D). As expected, such interactions were abolished by a mutation in the PTB domain (PTB-AA) or a Y72F substitution in ITAM1 (Fig. 5C), suggesting an interaction of the Dok PTB domain with ITAM1 upon TCR stimulation. As mentioned earlier, these findings imply that Dok-1 and Dok-2 may compete with ZAP-70 for binding to the ITAMs of TCR{zeta} and CD3{epsilon} upon TCR signaling. Therefore, the effects of forced expression of Dok-1 or Dok-2 on the association of ZAP-70 with TCR{zeta} or CD3{epsilon} in the presence of LckYF were examined in 293T cells (Fig. 5E). It should be noted that we used a truncation form of TCR{zeta} ({zeta}{Delta}C) lacking ITAM2 and ITAM3, which can bind to ZAP-70, because TCR{zeta} ITAM1 is fully responsible for the activation of ZAP-70 upon TCR stimulation (30, 31). Although LckYF efficiently induced the binding of ZAP-70 to {zeta}{Delta}C or CD3{epsilon}, the forced expression of Dok-1 or Dok-2 significantly suppressed these interactions. Therefore, our findings suggest that Dok-1 and Dok-2 can compete with ZAP-70 for binding to ITAM1 of TCR{zeta} and the ITAM of CD3{epsilon} upon tyrosine phosphorylation.

Mice lacking Dok-1 and Dok-2 develop lupus-like disorders
In general, the loss of key regulators in the immune system often disrupts its homeostasis. For instance, the T cell-specific loss of both c-Cbl and Cbl-b in mice resulted in an autoimmune disease and the marked activation of peripheral T cells (3). Consistently, all dKO mice developed a lupus-like renal disease with the enlargement of glomeruli at 6–13 months after birth (Fig. 6A). Kidneys from these mice exhibited diffuse proliferative glomerulonephritis and the dKO, but not WT, glomeruli contained large amounts of immune complexes, including IgM, IgG, IgA and complement C3. In addition, at 6 months of age, all dKO mice, but not the controls expressing Dok-1 and Dok-2, exhibited significantly higher titers of antibodies to dsDNA (Fig. 6B), suggesting that the pathology associated with dKO mice is a consequence of circulating autoreactive antibodies, which are seen in patients with systemic lupus erythematosus.


   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
We have demonstrated that Dok-1 and Dok-2 are key negative regulators of TCR-mediated signaling, which controls T cell proliferation and cytokine secretion by the way of ZAP-70 activation and subsequent phosphorylation of LAT (32). Although abrogation of either Dok-1 or Dok-2 alone did not compromise the safety margin for T cell-dependent Ig response in mice, T cells from mice lacking either protein alone showed increased proliferation and cytokine secretion upon TCR cross-linking (Fig. 3A and B). Similarly Dok-1+/–/Dok-2–/– or Dok-1–/–/Dok-2+/– mice exhibited increased titers of antibodies to dsDNA, but to a lesser extent than dKO mice in general (Fig. 6B). Conversely, the forced expression of Dok-1 or Dok-2 alone in 25-14 T cells suppressed TCR-mediated activation of ZAP-70 as well as subsequent phosphorylation of LAT and Erk (Fig. 4B). Therefore, these Dok-family proteins work co-operatively in the negative regulation of TCR-mediated signaling. Similarly, Dong et al. (28) recently reported that knockdown of Dok-1 and Dok-2 in transformed T cells synergistically enhanced TCR-induced phosphorylation of ZAP-70 and LAT as well as secretion of IL-2. However, the molecular mechanisms underlying this negative regulation remain unclear.

Dok-family proteins are generally thought to act as adaptor/docking proteins' downstream of PTKs, where they recruit SH2-containing molecules such as p120 rasGAP and Nck via the SH2 target sites in their COOH-terminal moieties. However, we recently found that Dok-7 binds and activates the muscle-specific receptor PTK MuSK, and a Dok-7 mutant lacking the COOH-terminal moiety was able to activate MuSK in 293T cells (9). Here, we have demonstrated that Dok-1 or Dok-2 mutants lacking the COOH-terminal moiety can inhibit activation of ZAP-70 upon TCR stimulation (Fig. 4B). Since recruitment of signaling molecules via the COOH-terminal moiety is essential for the known adaptor functions of Dok-family proteins, these data strongly suggest that Dok-1 and Dok-2 can also function in a non-adaptor capacity.

Upon TCR cross-linking, tyrosine residues within ITAMs of the receptor complex are phosphorylated by Src-family PTKs, such as Lck and Fyn, and the phosphorylated ITAMs become specific binding sites for the tandem SH2 domains of ZAP-70. This selective recruitment to the cross-linked receptor complex induces activation of ZAP-70 by a combination of autophosphorylation and transphosphorylation mediated by the Src-family kinases (1, 25, 26). The following results in our studies imply that inhibition of ZAP-70 by Dok-1 and Dok-2 upon TCR cross-linking might be a consequence of the competitive binding of Dok-1 and Dok-2 to phosphorylated ITAMs of the TCR complex: (i) the forced expression of Dok-1 or Dok-2 in 25-14 T cells inhibited the phosphorylation of ZAP-70 upon TCR stimulation without reduction of TCR{zeta} phosphorylation (Fig. 4B and C), (ii) a mutation in the PTB domain of Dok-1 or Dok-2 impaired this negative regulation of ZAP-70 (Fig. 4B), (iii) target motifs of the Dok-1 and Dok-2 PTB domains are conserved among species by the ITAM of CD3{epsilon} as well as the membrane-proximal ITAM1 of TCR{zeta} (Fig. 5A), which are essential for the recruitment and activation of ZAP-70, (iv) TCR cross-linking induced binding of Dok-1 and Dok-2 with TCR{zeta} in a manner largely dependent upon the Dok-1/2 PTB target motif within ITAM1 of the TCR chain (Fig. 5B), (v) the PTB domain of Dok-1 or Dok-2 can bind with ITAM1 of TCR{zeta} and the ITAM of CD3{epsilon}, but not with the CD3{gamma} ITAM, which lacks the Dok-1/2 PTB target motif (Fig. 5C and D) and (vi) forced expression of Dok-1 or Dok-2 impaired the binding of ZAP-70 to {zeta}{Delta}C, which retains ITAM1, as well as CD3{epsilon} in 293T cells (Fig. 5E). Consistently, the NH2-terminal YxxL segment of TCR{zeta} ITAM1, which overlaps the Dok-1/2 PTB target motif, is important for the activation of ZAP-70 and Erk (30, 31). Although constitutive binding of ZAP-70 with ITAM2 and ITAM3 of TCR{zeta} may create difficulties (5, 31), a definitive conclusion addressing molecular mechanisms underlying the negative function of Dok-1/2 upon TCR-mediated signaling awaits further studies.

dKO mice showed increased numbers of DP, CD4+ and CD8+ T cells in the thymus and CD4+ and CD8+ T cells in the periphery (Fig. 2A). Consistent with this observation, Gugasyan et al. (33) previously reported that mice reconstituted with bone marrow cells that had been infected with retroviruses expressing Dok-2 showed impaired transition of DN to DP thymocytes, leading to a marked deficit of mature CD4 or CD8 single-positive T cells in the thymus and, consequently, in the spleen. It was also reported that the four tyrosine residues of LAT, which are required for calcium mobilization, NF-AT activation and Erk activation upon TCR stimulation, are essential for transition of DN3 (CD4–CD8–CD44–CD25+) to DN4 (CD4–CD8–CD44–CD25–) thymocytes and subsequent positive selection in the thymus (34). Given that Erk plays an important roles in T cells for TCRß selection of DN3 thymocytes and positive selection in the thymus (35), it is tempting to speculate that augmented activation of LAT and Erk in dKO thymocytes upon TCR-mediated signaling may be a cause of the accumulation of DP, CD4+ and CD8+ T cell populations by facilitating TCRß selection as well as positive selection.

Consistent with the augmented activation of TCR signaling in dKO T cells, all such mutant mice developed a spontaneous autoimmune disorder characterized by autoantibody production and glomerulonephritis as a result of Ig-complement immune complex deposition in the glomeruli. We previously reported that 7 of 13 dKO mice, but not the others, developed myeloproliferative disease at ~1 year of age (15); however, since all dKO mice developed the lupus-like nephritis by 1 year of age, the myeloproliferative disease is not a prerequisite for the lupus-like disease. Therefore, our findings demonstrate that Dok-1 and Dok-2 in T cells co-operatively play an indispensable role in the homeostasis of adaptive immunity, providing a unique model to understand the molecular and cellular mechanisms underlying T cell-mediated development of autoimmune disease.


   Acknowledgements
 
We thank T. Saito for cDNA and K. Nakamura and K. Matsumoto for animal care. This work was supported by Grants-in-Aid for scientific research from the Ministry of Education, Culture, Sports, Science and Technology and by grants from the Astellas and the Naito foundations.


   Abbreviations
 
CD4+, CD3+CD4+
CD8+, CD3+CD8+
dKO, Dok-1/Dok-2 double deficient
DN, CD4–CD8–
DNP-KLH, DNP-conjugated keyhole limpet hemocyanin
DP, CD4+CD8+
dsDNA, double-stranded DNA
HA, hemagglutinin
ITAM, immunoreceptor tyrosine-based activation motif
PH, pleckstrin homology
PMA, phorbol myristate acetate
PTB, phosphotyrosine binding
PTK, protein–tyrosine kinase
SH2, src homology 2
TI, thymus independent
WT, wild type

   Notes
 
Transmitting editor: T. Kurosaki

Received 9 November 2006, accepted 14 January 2007.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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