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The Journal of Immunology, 2005, 175: 4483-4489.
Copyright © 2005 by The American Association of Immunologists

Phosphotyrosine Binding-Mediated Oligomerization of Downstream of Tyrosine Kinase (Dok)-1 and Dok-2 Is Involved in CD2-Induced Dok Phosphorylation1

Iohann Boulay, Jean-Guy Némorin and Pascale Duplay2

Institut National de la Recherche Scientifique-Institut Armand-Frappier, Université du Québec, Laval, Canada


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
To date, five members of the downstream of tyrosine kinase (Dok) family have been characterized. In T cells, two members, Dok-1 and Dok-2, are expressed. CD2 or CD28 stimulation, but not CD3/TCR stimulation, induces Dok phosphorylation. Recent evidence suggests that they act as negative regulators of the CD2 and CD28 signaling pathways. To identify the molecular mechanisms involved in Dok-mediated inhibition, we have identified proteins that bind to the phosphotyrosine-binding (PTB) domain of Dok-1 and Dok-2. We showed that the Dok PTB domain mediates phosphotyrosine-dependent homotypic and heterotypic interactions of Dok-1 and Dok-2. Moreover, in CD2-stimulated Jurkat cells, Dok-1 coimmunoprecipitates with tyrosine-phosphorylated Dok-2. To study the involvement of PTB-mediated oligomerization in Dok function, we have generated Jurkat clones overexpressing Dok-1 or Dok-2 with a mutation that prevents oligomerization (in either the PTB domain or Tyr146 of Dok-1 and Tyr139 of Dok-2). These mutations abrogate CD2-induced phosphorylation and the ability of Dok-1 or Dok-2 to inhibit CD2-induced ERK1/2 and NFAT activation. Moreover, overexpression of Dok-1Y146F or Dok-2Y139F interferes with CD2-induced phosphorylation of endogenous Dok, whereas overexpression of PTB mutant or wild-type Dok does not. Taken together, these data indicate that PTB-mediated oligomerization of Dok-1 and Dok-2 represents an essential step for Dok phosphorylation and function.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Downstream of tyrosine kinase (Dok)3 proteins belong to a newly identified family of adaptor molecules. They contain an N-terminal pleckstrin homology (PH) domain, a central phosphotyrosine-binding (PTB) domain, and a C-terminal domain rich in proline and tyrosine residues. The first member identified, Dok-1 (also known as p62dok), was originally shown to be a target of activated protein tyrosine kinases and to associate with Ras GTPase-activating protein (RasGAP) when phosphorylated (1, 2, 3). Five other members, Dok-2, also known as Dok-R, FRIP, or p56dok (4, 5, 6); Dok-3, also known as Dok-L (7, 8); Dok-4 and Dok-5 (9, 10); and Dok-6 (11) have been characterized. Dok-4 and Dok-5 define a subfamily within Dok proteins that is more related to the insulin receptor substrate family than the other Dok members (12). Dok proteins are implicated in the regulation of multiple biological processes, including cell proliferation, transformation and tumorigenesis, cell motility, and differentiation. In hemopoietic cells, Dok proteins are implicated in the negative regulation of signaling through growth factor, cytokine, and immunoreceptors (5, 8, 13, 14, 15, 16, 17, 18, 19, 20, 21).

In T cells, both Dok-1 and Dok-2 are expressed. Recently, Dok-4 and Dok-5 mRNA have been shown to be present in humans, but not in mice (9, 10). Dok-1 and Dok-2 seem to play a specific role in signal transduction pathways initiated by CD2 and CD28 receptors. The phosphorylation of Dok-1 and Dok-2 occurs after CD2 or CD28 stimulation, whereas CD3/TCR stimulation does not induce Dok phosphorylation (22, 23, 24). Overexpression of Dok-1 in Jurkat cells has a negative effect on CD2-mediated activation of NFAT and ERK1/2 (15). When introduced into lethally irradiated mice, retroviral-mediated expression of Dok-2 in bone marrow cells inhibits their capacity to repopulate thymus by reducing the number of thymic precursors and by inhibiting the transition of CD4–CD8– to CD4+CD8+ thymocytes (25). Moreover, in transient assays, overexpression of Dok-1 or Dok-2 inhibits the CD28/TCR-induced IL-2 promoter activity (26).

The absence of Dok-1 expression does not lead to an obvious defect in T cell development and function (13, 27). The remaining expression of Dok-2 in Dok-1–/– T cells might compensate for the absence of Dok-1. However, it should be noted that specific contributions of Dok-1 and Dok-2 exist because Con A-induced proliferation of Dok-1–/– thymocytes is reduced (27). Recently, Dok-1/Dok-2 double-knockout mice have been generated, but characterization of the T cell development and T cell responses of these mice has not been yet reported (27, 28). Therefore, it remains to be established whether in T cells, Dok-1 and Dok-2 are redundant adaptors or play a unique function in T cell signaling and form complexes with a different (probably overlapping) set of molecules.

The Dok PTB domain is involved in Dok recruitment to membrane receptors such as epidermal growth factor receptor and IL-4R (5, 29). The PTB domain of Dok-1 has also been shown to bind negative regulators such as SHIP-1 (30). In addition, the Dok-1 PTB domain mediates phosphotyrosine-dependent homotypic interactions through residue Tyr146, and this homotypic interaction is necessary for Dok-1-mediated inhibition of v-Src-induced transformation (31). In this study we have addressed the role of the PTB domain of Dok-1 and Dok-2 by identifying proteins that bind to their PTB domain and studying the functional effects of PTB mutants on Dok-mediated signaling. We show that Dok-1 and Dok-2 can form homo- and heterodimers in a phosphorylation-dependent manner through interaction of the PTB domain with Tyr146 of Dok-1 and Tyr139 of Dok-2. This oligomerization is required for full phosphorylation of Dok proteins and Dok-mediated signaling.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Cell lines and Abs

The Jurkat cells, clone 77-6, were grown in RPMI 1640 supplemented with 10% FCS and 2 mM L-glutamine. Puromycin was added at 1 µg/ml to the medium when required. The mAbs used included anti-CD2 (anti-T11-2 and T11-3; provided by E. Reinherz, Harvard Medical School, Boston, MA), anti-hemagglutinin (anti-HA; 12CA5; provided by M. Tremblay, McGill University, Montreal, Canada), and anti-phosphotyrosine (4G10; Upstate Biotechnology). Polyclonal Abs used included anti-Dok-1 directed against Dok-1 C-terminal domain (produced by immunizing rabbits with a GST fusion protein bearing residues 260–482); anti-Dok-1 (used for Western blotting; Fig. 2) directed against aa 425–439 (provided by B. Stillman, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY); anti-Dok-2 directed against the Dok-2 C-terminal domain (produced by immunizing rabbits with a GST fusion protein bearing residues 253–412); anti-Dok-2 (Tyr351) and anti-phospho-Dok-2 (pTyr351) directed against a peptide and phospho-peptide, respectively, corresponding to residues surrounding Tyr351 of human-Dok-2 (Cell Signaling Technology); anti-phospho- p44/42 MAPK; and anti-p44/42 MAPK (Cell Signaling Technology). Abs were biotinylated with EZ-Link Sulfo-NHS-LC-Biotin (Pierce) according to the manufacturer’s instructions.



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  		FIGURE 2. Dok-1 and Dok-2 association after CD2 stimulation. Jurkat cells overexpressing HA-Dok-2 WT cells were left unstimulated (–) or were stimulated (CD2) with an anti-CD2 mAb pair for 10 min. A, Postnuclear lysates were immunoblotted in parallel with anti-phosphotyrosine mAb and anti-phospho-Dok-2 (Y351) Abs. B and C, Postnuclear lysates were immunoprecipitated with anti-phosphoDok-2 (Y351) or anti-Dok-2 Abs (Y351). Precipitates were immunoblotted with biotinylated anti-phosphotyrosine or anti-Dok-1 Abs as indicated. Blots were stripped and reprobed with biotinylated anti-Dok-2. These experiments were repeated at least twice. IP, Immunoprecipitation; WB, Western blot.

 
DNA constructs and transfection

The plasmid construct pSR{alpha}-HADok-1 has been described previously (15). This construct yielded expression of Dok-1 with an N-terminal HA tag. The HA tag does not interfere with Dok function and localization (15). To generate the plasmid pSR{alpha}-HA-Dok-2, an EcoRI-BamHI fragment from pcMV5.I/HAp56Dok (provided by M. Resh, Memorial Sloan-Kettering Cancer Center, New York, NY) was subcloned into the plasmid pSR{alpha}-puromycin. The PTB domain mutants (Dok-1R207-208A and Dok-2R200-201A) and the PH-PTB interdomain Tyr mutants (Dok-1Y146F and Dok-2Y139F) were generated by site-directed mutagenesis using PCR and subcloned into the pSR{alpha}-puromycin vector. Stable transfectant Jurkat cells overexpressing wild-type (WT) or mutant versions of Dok-1 or Dok-2 were selected as previously described (15). The expression levels of HA-Dok-1 or HA-Dok-2 mutants were evaluated by immunoblotting of cell extracts with anti-Dok-1 or anti-Dok-2 Abs. The expression levels of CD2 and CD3 were evaluated by flow cytometric analysis with an EPICS XL (Coulter Electronics). Clones expressing similar levels of CD3 and CD2 compared with the parental Jurkat cells were kept for additional studies.

GST fusion protein constructs and expression

To generate GST fusion proteins constructs, the DNA fragment encompassing residues 151–259 for Dok-1 and 144–252 for Dok-2 was PCR amplified and cloned into the BamHI-EcoRI sites of the pGEX-2-thymidine kinase (TK) vector (Amersham Biosciences) using HA-Dok-1, HA-Dok-2, HA-Dok-1R207-208A, or HA-Dok-2R200-201A as templates. GST fusion proteins were purified using glutathione-Sepharose beads (Amersham Biosciences).

Binding assays

Postnuclear lysates were incubated for 1 h at 4°C with glutathione-Sepharose beads coupled to GST (Amersham Biosciences). Supernatants were incubated for 2 h at 4°C with the GST-PTB fusion protein preabsorbed to glutathione-Sepharose beads. The complexes were washed under the same conditions as the immune complexes and eluted by boiling in the presence of SDS sample buffer or by incubating twice, for 10 min each time, at 4°C in the presence of 50 mM phenyl phosphate (a structural analog of phosphotyrosine) in 1% detergent lysis buffer under constant agitation.

Cell stimulation, immunoprecipitation, and immunoblotting

Cells were washed in RPMI 1640 and resuspended at 4 x 107 cells/ml in RPMI 1640. Cells were left unstimulated or were stimulated with anti-CD3 (UCHT1 at a 1/500 dilution of ascites) or anti-CD2 (a combination of T11-2 and T11-3 at a 1/500 dilution of ascites) at 37°C for the time indicated. Cells were harvested and lysed in 1% Nonidet P-40 containing 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM MgCl2, and 1 mM EGTA in the presence of protease and phosphatase inhibitors. Immunoprecipitations and immunoblotting were performed as previously described (15). In several experiments, to avoid IgH detection we used biotinylated Abs and streptavidin-biotinylated HRP complex to detect immunoreactive products (Amersham International).

Transient transfection and luciferase assays

Jurkat cells (106 cells) were transfected with 2.5 µg of NF-AT-firefly luciferase (provided by O. Acuto, Institut Pasteur, Paris, France) and 0.5 µg of TK-Renilla luciferase constructs (Promega) using FuGene transfection assays (Roche). After 24 h, 3 x 105 cells were stimulated with plate-bound anti-CD3 or soluble anti-CD2 Abs for 6 h in a 24-well plate. Maximal stimulation was obtained by a combination of PMA (10 ng/ml) and ionomycin (1 µM). Cells were then lysed and assayed for luciferase activity using the dual luciferase reporter assay system (Promega) and a luminometer (Berthold; LUMAT LB 9507). The NF-AT-firefly luciferase values were normalized based on the constitutive Renilla luciferase activity.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Homotypic and heterotypic interactions of Dok proteins through their PTB domain

To gain better insight into the function of Dok proteins in CD2 signaling, we performed in vitro binding assays to identify binding partners of Dok-1 and Dok-2 PTB domains (Fig. 1A). Although many tyrosine-phosphorylated proteins were present in lysates isolated from CD2-stimulated cells, only two proteins of 54–56 and 62 kDa were associated with the Dok-2 PTB domain (Fig. 1B). In unstimulated cells, smaller amounts of these two phosphorylated proteins bound to the PTB domain, indicating that their phosphorylation was increased by CD2 stimulation. We previously showed that Dok-1 and Dok-2 are phosphorylated upon CD2 stimulation (22). Moreover, the Dok-1 PTB domain has been shown to mediate phosphotyrosine-dependent homotypic interactions (31). Therefore, to investigate whether these two proteins corresponded to Dok-1 and Dok-2, we performed immunoprecipitations with anti-Dok-1 or Dok-2 Abs after elution of PTB domain-bound proteins. As expected, the 54- to 56- and 62-kDa proteins that bound in vitro to the PTB domain of Dok-2 corresponded to Dok-2 and Dok-1 proteins, respectively (Fig. 1B). Similarly to Dok-2, the Dok-1 PTB domain is involved in homo- and heterodimerization (Fig. 1C). To verify the specificity of this PTB domain-mediated interaction, we mutated two Arg residues to alanine within the PTB domain of Dok-1 and Dok-2. One of these Arg residues (Arg207 of Dok-1 and, on the basis of sequence homology, Arg200 of Dok-2) coordinates phosphotyrosine binding. As expected, mutation of Arg207 (Dok-1R207–208A) and Arg200 (Dok-2R200–201A) dramatically reduced PTB domain ligand binding (Fig. 1C). Taken together, these results suggest that in T cells, homotypic and heterotypic interactions may take place between two Dok family members, Dok-1 and Dok-2.



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  		FIGURE 1. In vitro binding of Dok to Dok-1 and Dok-2 PTB domains. A, Schematic representation of the primary structures of Dok-1 and Dok-2. The locations of the tyrosine residues (Y) within the C-terminal and PH-PTB interdomain are indicated by amino acid numbers. The positions of the arginine residues in the PTB domain involved in phosphotyrosine binding are also indicated. The asterisk indicates the positions of the mutations used in this study. B, Jurkat cells overexpressing HA-Dok-2 WT cells were left unstimulated (–) or were stimulated (CD2) with an anti-CD2 mAb pair for 10 min. Postnuclear lysates were incubated with GST-Dok-2 PTB domain and eluted with SDS sample buffer (GST-Dok-2 PTB) or 50 mM phenyl-phosphate (GST-Dok-2 PTB*) and subjected to immunoprecipitation (IP) with anti-Dok-1 or anti-Dok-2 Abs, as indicated. Postnuclear lysates or precipitates were immunoblotted with anti-phosphotyrosine mAb. It should be noted that Dok-2 migrates as a triplet or a doublet when revealed with anti-phosphotyrosine Abs and as a doublet when revealed with anti-Dok-2 Abs. Dok-1 is often seen as a doublet in anti-phosphotyrosine, but as a single band in anti-Dok-1 Western blots. WB, Western blot. C, Jurkat cells were left unstimulated (–) or were stimulated with an anti-CD2 mAb pair for the time indicated. Upper panel, Postnuclear lysates of cells overexpressing HA-Dok-1 WT were incubated with GST-Dok-1-PTB WT and GST-Dok-1-PTB R207–208A. Lower panel, Postnuclear lysates of cells overexpressing HA-Dok-2 WT were incubated with GST-Dok-2-PTB WT and R200–201A. Proteins bound to the PTB domains were eluted with SDS sample buffer and were revealed by immunoblotting with mAb anti-phosphotyrosine. These experiments were repeated at least twice. WB, Western blot.

 
Dok-1 and Dok-2 interact in vivo in a phospho-dependent manner

To determine whether the Dok-1/Dok-2 interaction occurs in vivo, we performed immunoprecipitations with Dok-2 Abs. Because this interaction is probably phosphotyrosine dependent, we used phosphotyrosine-specific Abs directed against the Tyr351 of Dok-2. Lysates from unstimulated and CD2-stimulated parental Jurkat cells and clones overexpressing Dok-2 were probed by immunoblotting with anti-phospho-Dok-2 Abs. A 54- to 56-kDa doublet corresponding to Dok-2 was detected, and its intensity was increased with Dok expression and CD2 stimulation (Fig. 2A and data not shown). This result indicates that the anti-phosho-Dok-2 Abs are highly specific for the phosphorylated form of Dok-2 and do not cross-react with Dok-1 or other phosphorylated proteins. Two phosphoproteins of 62 and 120 kDa coimmunoprecipitated with phospho-Dok-2 in unstimulated and CD2-stimulated cells (Fig. 2B). By contrast, only a small amount of the phosphorylated 62-kDa protein in CD2-stimulated cells was detected in immunoprecipitates performed with Abs directed against total Dok-2 (Fig. 2B). It is important to note that a much greater amount of total Dok-2 was immunoprecipitated with anti-Dok-2 Abs compared with phospho-specific Dok-2 Abs. This indicates that association of the 62- and 120-kDa proteins occurred only with the phosphorylated form of Dok-2. Because phosphorylated Dok-2 associates with RasGAP (6), the 120-kDa protein associated with phospho-Dok-2 might correspond to RasGAP. However, we were unable to detect RasGAP by Western blotting with commercially available Abs, probably because the amount of RasGAP present in phospho-Dok-2 immunoprecipitates is too low. To determine whether the 62-kDa protein corresponded to Dok-1, phospho-Dok-2 immunoprecipitates were probed with anti-Dok-1 Abs. As shown in Fig. 2C, there was a correlation between the amount of Dok-1 and the amount of phosphorylated Dok-2 present in the immunoprecipitates. Taken together, these data show that there is a phospho-dependent association of Dok-1 with Dok-2.

Tyr146 (Dok-1) or Tyr139 (Dok-2) and the PTB domain are required for Dok tyrosine phosphorylation and function

Dok-1 homodimerization occurs through binding of phospho-Tyr146 with the PTB domain of Dok-1 (31). Using a combinatorial peptide library approach, the consensus binding motif for the PTB domain of Dok-1 was defined as Y/MXXNXLpY (31). The sequence surrounding residue Tyr139 (MEENELY) of Dok-2 represents a potential binding site for the Dok PTB domain. To test the importance of PTB-mediated oligomerization in Dok function, we mutated the Tyr residue potentially involved in Dok oligomerization (Tyr146 of Dok-1 and Tyr139 of Dok-2) and the PTB domain (Fig. 1A). Jurkat clones that expressed similar amounts of the mutant Dok (average of 5 times more than the parental Jurkat cells) and similar levels of CD2 and CD3 at their cell surface were selected (data not shown). Mutation of Tyr146 (Dok-1) or Tyr139 (Dok-2) dramatically affected both basal and CD2-induced phosphorylation of Dok-1 and Dok-2, respectively (Fig. 3). A functional PTB domain was also required for Dok-1 or Dok-2 phosphorylation, because Dok-1R207–208A and Dok-2R200–201A were not phosphorylated even after CD2 stimulation. Phosphorylation on tyrosine residues is essential for Dok-1 and Dok-2 functions. We therefore examined whether these mutations affected the ability of Dok protein to inhibit CD2 signaling. As we previously reported for Dok-1 (15), Dok-2 overexpression inhibited CD2-induced ERK1/2 activation, although to a lesser extent (Fig. 4A). By contrast, overexpression of Dok-1R207–208A, Dok-2R200–201A, Dok-1Y146F, and Dok-2Y139F did not interfere with CD2-induced ERK1/2 activation (Fig. 4A). The influence of Dok-1 and Dok-2 overexpression on CD2-induced NFAT activation was also evaluated (Fig. 4B). As reported previously (29), the expression of WT Dok-1 inhibited NFAT activation in response to CD2 stimulation. By contrast, CD2-induced NFAT activation was unaffected by the expression of Dok-1R207-208A and was slightly increased by the expression of Dok-1Y146F (Fig. 4B). CD3-induced NFAT activation was not significantly affected by overexpression of Dok-1WT, Dok-1R207-208A, or Dok-1Y146F. Similarly, Dok-2 overexpression inhibited CD2-induced NFAT activation, whereas Dok-2R200-201A and Dok-2Y139F did not (Fig. 4B). Taken together, these results indicate that phosphorylation of Tyr146 (Dok-1) or Tyr139 (Dok-2) and a functional PTB domain are required for Dok function.



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  		FIGURE 3. Mutations that prevent Dok oligomerization inhibit CD2-induced Dok phosphorylation. Jurkat cells overexpressing HA-Dok-1 WT, Y146F, and R207-208A or HA-Dok-2 WT, Y139F, and R200-201A were left unstimulated (–) or were stimulated (CD2) with an anti-CD2 mAb pair for 10 min. Postnuclear lysates were immunoprecipitated with anti-HA mAb. Immunoprecipitates were immunoblotted as indicated with mAb anti-phosphotyrosine for Dok-1-overexpressing clones and with biotinylated anti-phosphotyrosine for Dok-2-overexpressing clones. Immunoprecipitates were also immunoblotted in parallel with anti-Dok-1 or biotinylated anti-Dok-2 Abs to verify the levels of Dok proteins. This experiment was performed on at least two independent transfectants for each of the Dok constructs. All clones selected for our experiments expressed similar amounts of transfected Dok. IP, Immunoprecipitation; WB, Western blot.

 


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  		FIGURE 4. Oligomerization is required for Dok to inhibit CD2-induced ERK1/2 activation. A, Jurkat J77.6 cells or cells overexpressing HA-Dok-1 WT, Y146F, and R207-208A or HA-Dok-2 WT, Y139F, and R200-201A were left unstimulated (–) or were stimulated with an anti-CD2 mAb pair for the indicated amounts of time. Postnuclear lysates were immunoblotted with anti-phospho ERK1/2 Abs, and equivalent levels of ERK1/2 protein in each lane were evaluated by probing the membranes with anti-total ERK1/2 Abs in parallel Western blots. This experiment was performed at least five times on at least two independent transfectants for each of the Dok constructs. All clones selected for our experiments expressed similar amounts of transfected Dok. WB, Western blot. B, Jurkat J77.6 cells or cells overexpressing HA-Dok-1 or HA-Dok-2 were transfected with NFAT luciferase reporter plasmid and TK-Renilla luciferase constructs. After 24 h, cells were left unstimulated (–) or were stimulated with anti-CD2 mAbs (CD2) or anti-CD3 mAbs (CD3). Cells were harvested after 6 h, and luciferase activity was measured. The results were expressed as a percentage of the fold induction relative to maximal luciferase induction induced by treatment with PMA and ionomycin. This experiment was repeated in triplicate in three independent experiments with similar results.

 
Overexpression of Dok-1Y146F or Dok-2Y139F interferes with the phosphorylation of endogenously expressed Dok protein

The contributions of Dok-2 Tyr139 and the PTB domain to homo- and heterodimerization were assessed by in vitro binding assays with the PTB domain of Dok-2 and lysates from Jurkat cells overexpressing WT or mutated versions of Dok-2 (Y139F and R200-201A). As expected, in unstimulated or CD2-stimulated cells, no phospho-Dok-2Y139F or phospho-Dok-2R200-201A could be detected in this assay. Unexpectedly, overexpression of Dok-2Y139F affected binding of endogenous Dok-1 to the PTB domain of Dok-2 (Fig. 5A). Therefore, this result probably indicates that overexpression of Dok-2Y139F interferes specifically with the phosphorylation of endogenously expressed Dok protein. To test this hypothesis, we analyzed the phosphorylation of endogenously expressed Dok-1 and Dok-2 proteins in clones overexpressing various forms of Dok-2 and Dok-1, respectively (Fig. 5B). Overexpression of Dok-2Y139F caused a reduction of CD2-induced phosphorylation of Dok-1, whereas WT Dok-2 and Dok-2R200-201A did not. Similarly, overexpression of Dok-1Y146F inhibited Dok-2 phosphorylation, whereas Dok-1WT and Dok-1R207-208A did not. Importantly, the effect of Dok-2Y139F or Dok-1Y146F on CD2-induced Dok-1 phosphorylation did not reflect a global defect in CD2 activation, because CD2-induced ERK1/2 activation and CD2-induced protein tyrosine phosphorylation of whole cell lysate were not affected (Fig. 4 and data not shown). Together, these results suggest that there is an interplay between Dok-1 and Dok-2 and that PTB-mediated oligomerization of Dok-1 and Dok-2 represent an essential step for Dok phosphorylation.



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  		FIGURE 5. Overexpression of Dok-1Y146F or Dok-2Y139F interferes with the phosphorylation of endogenously expressed Dok protein. A, Jurkat J77.6 cells or cells overexpressing HA-Dok-2Y139F or R200-201A were left unstimulated (–) or were stimulated (CD2) with an anti-CD2 mAb pair for 5 min. Postnuclear lysates were pulled down with GST-Dok-2 PTB WT, and precipitates were immunoblotted with mAb anti-phosphotyrosine. B, J77.6 Jurkat cells or cells overexpressing HA-Dok-2 WT, Y139F, and R200-201A were left unstimulated (–) or were stimulated with an anti-CD2 mAb pair for 10 min. Anti-Dok-1 immunoprecipitates were immunoblotted with mAb anti-phosphotyrosine or anti-Dok-1 Abs (left panel). Cells overexpressing HA-Dok-1 WT, Y146F, or R207-208A were left unstimulated (–) or were stimulated with an anti-CD2 mAb pair for 10 min. Postnuclear lysates were immunoblotted with anti-phospho-Dok-2 and anti-Dok-2 (right panel). These experiments were performed at least five times on at least two independent transfectants for each of the Dok constructs. All clones selected for our experiments expressed similar amounts of transfected Dok. WB, Western blot.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In this report we have shown that two members of the Dok family, Dok-1 and Dok-2, form homo- and hetero-oligomers. These interactions occur through binding of the PTB domain to the phosphotyrosine located in the PH-PTB interdomain (Tyr146, Dok-1; Tyr139, Dok-2). Consistent with a phosphorylation-dependent interaction, we show that Dok-1 associates only with the tyrosine-phosphorylated form of Dok-2 in vivo.

In T cells, CD2 or CD28 stimulation, but not CD3 stimulation, specifically increases Dok phosphorylation and induces membrane translocation of Dok proteins (22, 24). There are many examples where maximal phosphorylation of Dok-1 or Dok-2 requires the presence of both intact PH and PTB domains. In these systems, activation of PI3K generates inositol phospholipids at the plasma membrane that serve as binding sites for PH domains. The PTB domain contributes to the recruitment of Dok to the membrane by interacting with receptors such as epidermal growth factor receptor, Tie, Ret, and IL-4R (5, 29, 32, 33). Deletion of the PH domain of Dok-1 and Dok-2 abolishes their CD2-induced phosphorylation (data not shown). CD2-induced activation of PI3K (34, 35, 36) is probably involved in the PH-dependent recruitment of Dok. In addition, we showed that the PTB domain is required for Dok phosphorylation and, therefore, probably for Dok membrane recruitment. However, using an in vitro binding assay with GST-PTB domain fusion protein, we were not able to detect other phosphorylated proteins except Dok. This might indicate that the phosphorylation of the PTB domain-binding protein involved in Dok recruitment is too low to be detectable in our assay or that the PTB domain interaction is independent of phosphorylation. Recruitment at the membrane may involve Dok association with the receptor CD2. If any CD2/Dok complex exists, it may be indirect and require the association with known CD2-binding protein (CD2BP), such as CD2BP1, CD2BP2, CD2BP3, CIN85, CD2AP/CMS, Lck, or Fyn (37, 38, 39, 40, 41, 42, 43). However, none of these proteins, including the cytoplasmic domain of CD2, contains the Y/MXXNX LpY consensus motif predicted to bind to the Dok-1 PTB domain. Alternatively, CD2 stimulation may induce the recruitment of Dok to an unknown membrane protein.

Although Dok-1 and Dok-2 contain multiple phosphorylation motifs, mutation of the tyrosine residue within the PH-PTB interdomain dramatically affected CD2-induced tyrosine phosphorylation of Dok-1 or Dok-2. It should be emphasized that mutation of the tyrosine residue within the C-terminal tail of Dok-1 and Dok-2 (26) (data not shown) had only a modest effect on total CD2-induced Dok tyrosine phosphorylation. The absence of phosphorylation observed for Dok-1Y146F and Dok-2Y139F indicates that tyrosine phosphorylation occurs in an ordered and interdependent fashion, with Tyr139 (Dok-2) and Tyr146 (Dok-1) being the first tyrosines to be phosphorylated. Phosphorylation of these specific tyrosine residues may lead to a conformational change in Dok, causing exposure and availability of tyrosine for additional phosphorylation. Alternatively, but not exclusively, this first phosphorylation event may allow oligomerization of Dok proteins. This may represent a requisite step for additional phosphorylation of Dok proteins.

Dok mutants of the tyrosine residue in the PH-PTB interdomain inhibited the phosphorylation of endogenously expressed Dok, whereas Dok PTB mutants did not, although they are both deficient in oligomerization. There are several ways to explain the dominant negative effect on phosphorylation of Dok-1Y146F and Dok-2Y139F. A likely interpretation of our results is that the interfering mutant competes with the WT for a PTB-binding site located at the plasma membrane. This model is corroborated by our finding that Dok PTB mutants deficient for binding do not interfere. Based on this study, we propose the following model for CD2-induced oligomerization and phosphorylation of Dok-1 and Dok-2 proteins in T lymphocytes (Fig. 6). After CD2 engagement, both PH and PTB domains target cytosolic nonphosphorylated Dok-1 and Dok-2 to cell surface receptor (designated X in Fig. 6, steps 1 and 2). Phosphorylation of Tyr146 (Dok-1) or Tyr139 (Dok-2) by a tyrosine kinase close to the receptor (Fig. 6, step 3) allows oligomerization of Dok proteins (Fig. 6, step 4). This oligomerization is essential for additional phosphorylation of the C-terminal tyrosine residues (Fig. 6, step 5). Oligomers might stay associated with the receptor or might move to another location in the plasma membrane (as represented in Fig. 6).



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  		FIGURE 6. Model for CD2-induced oligomerization and phosphorylation of Dok-1 and Dok-2. 1) Cytosolic nonphosphorylated Dok-1 or Dok-2; 2) PH and PTB-dependent recruitment of Dok to the membrane; 3) phosphorylation of Y146 (Dok-1) or Y139 (Dok-2); 4) Dok-1 and Dok-2 homo- or heterodimerization; 5) phosphorylation of C-terminal tyrosine residues. X, An unidentified protein or protein complex that binds to the Dok PTB domain after CD2 stimulation. For clarity, only two tyrosine residues are represented in the C-terminal domain of Dok, and only formation of dimers is shown. Higher order oligomers are possible if the monomeric form of Dok, once phosphorylated at the tyrosine in the PH-PTB interdomain, interacts with two distinct Dok monomers.

 
Several pieces of evidence suggest that at least two kinases, Lck and Tec kinases, are involved in CD2- or CD28-dependent phosphorylation of Dok-1 and Dok-2. CD2-induced Dok phosphorylation is dependent on the expression of Lck. Two members of the Tec kinase family, Itk and Tec, also represent potential kinases involved in Dok phosphorylation, because they have been shown to be activated by CD2 and/or CD28 (24, 44, 45). However, in COS-7 cells, Dok is phosphorylated by the Tec kinase, but not by Itk (46). Moreover, we have shown that the C-terminal region of Dok interacts with Tec (26). Therefore, Tec, but not Itk, is likely to be involved in Dok signaling. Additional experiments are required to identify the specific sites phosphorylated by Tec and/or Lck. It should be noted that Tyr146 and Tyr139 represent potential tyrosine phosphorylation sites for Lck (47).

In T cells, phosphorylation of Dok-1 and Dok-2 has been shown to generate docking sites for RasGAP (15). Translocation to the membrane of RasGAP probably negatively regulates the Ras/MAPK pathway. It is therefore not surprising that Dok-1Y146F, Dok-2Y139F, and the PTB domain Dok mutant, which are not phosphorylated upon CD2 stimulation, do not inhibit CD2-induced ERK1/2 activation. However, because we showed that overexpression of Dok-1Y146F or Dok-2Y139F inhibited phosphorylation of endogenously expressed Dok proteins, we would have expected a dominant negative effect on ERK1/2 and NFAT activation upon overexpression of these mutants. The residual phosphorylation of Dok-1Y146F or Dok-2Y139F upon CD2 stimulation (see Fig. 3) might be sufficient for Dok-mediated negative effects.

It is not clear whether Dok-1 and Dok-2 play redundant or specific roles in T cell signaling. The Dok proteins contain common and unique phosphorylation motifs, suggesting that they may interact with different Src homology 2 domain-containing proteins. Heterodimerization of Dok-1 and Dok-2 may therefore provide a mechanism by which the spectrum of responses can be enlarged. Whether homo- or heteromeric complexes are formed may depend on the receptor engaged as well as on the level of expression of Dok-1 and Dok-2 proteins. Additional experiments are required to elucidate the complete molecular interactions involved in Dok-1 and Dok-2 phosphorylations. In particular, it would be of great interest to study how TCR signaling inhibits Dok phosphorylation to release T cells from inhibition.


   Acknowledgments
 
We thank Drs. E. Reinherz, M. Resh and J. Wu for generous gifts of reagents. We also thank Ingrid Saba for critical reading of the manuscript, and Joanne Roger for technical assistance.


   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 by grants from the Canadian Institutes of Health Research. P.D. is the recipient of a Canadian Institutes of Health Research New Investigator Award. Back

2 Address correspondence and reprint requests to Dr. Pascale Duplay, Institut National de la Recherche Scientifique-Institut Armand-Frappier, Université du Québec, 531 boulevard des Prairies, Laval, Quebec, Canada H7V 1B7. E-mail address: pascale.duplay{at}iaf.inrs.ca Back

3 Abbreviations used in this paper: Dok, downstream of tyrosine kinase; CD2BP, CD2-binding protein; HA, hemagglutinin; PH, pleckstrin homology; PTB, phosphotyrosine binding; RasGAP, Ras GTPase-activating protein; TK, thymidine kinase; WT, wild type. Back

Received for publication August 30, 2004. Accepted for publication July 13, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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