Introduction

T cell activation requires T cell receptor (TcR) signalling upon binding to the peptide–MHC complex on the antigen-presenting cell (APC), and a second antigen-independent signal called co-stimulation.¹ Signal transduction events delivered into T cells after APC contacts, are dependent on the formation of intracytoplasmic multiprotein networks. Adapter molecules play a key role in the organization of these complexes.² These molecules can be major substrates for cytoplasmic protein tyrosine kinases (PTKs), linking membrane receptors to signaling pathways involved in many functions in T cells such as cytokine gene expression, cell adhesion or proliferation. Identification of these adapter molecules sets up the first step on dissecting the implication of signalling networks in T cell functions.

Dok-1 protein meets these criteria since this intracellular molecule is phosphorylated by PTKs upon receptor stimulation in hematopoietic cells. Dok-1 is constitutively phosphorylated on tyrosine residues in chronic myelogenous leukemia cells expressing the oncoprotein Bcr-Abl.³ The amino-terminal part of Dok-1 contains a pleckstrin homology (PH) domain and a phospho-tyrosine-binding (PTB) domain, which are thought to mediate the association with the plasma membrane and with phosphotyrosine-containing Y/MxxNxLpY motifs, respectively.^4,5 The carboxy-terminal part of Dok-1 likely functions as a molecular platform for signal complex assembly induced by activated PTKs. On the one hand, Dok-1 contains some proline-rich regions that may constitute docking sites for various Src homology 3 (SH3) domain-containing PTKs such as Src family kinases or the Abl kinase. On the other hand, several tyrosine phosphorylation sites are present creating docking sites for Src homology 2 (SH2) domain-containing adapter molecules such as Nck, the Ras GTPase-activating protein (RasGAP) or the product of the X-lymphoproliferative syndrome gene, SH2D1A/SAP.^3,6,7,8 The structure of Dok-1 presents some similarities with other adapter molecules such as Gab proteins or insulin receptor substrate (IRS) proteins.^9,10 Two proteins sharing extensive sequence homology with the amino-terminal part (PH and PTB domains) of Dok-1 have been cloned: Dok-2 (also known as FRIP or Dok-R)^11,12,13,14 and Dok-3 (also known as Dok-L).^15,16 These proteins are essentially expressed in hematopoietic tissues.¹⁵ Among the Dok family members, only Dok-1 and Dok-2 are expressed in T cells. These adapters can be phosphorylated by an antigen-independent signal involving costimulatory molecules such as CD2 or CD28.^17,18,19,20

Cloning of three members (Dok1, Dok2 and Dok3) raises the possibility to perform database searches to identify new putative Dok protein members. A first screening has been performed with a sequence containing a high homology between Dok-1, Dok-2 and also Dok-3.^11,14 Among putative proteins displaying a Dok homology (DKH) sequence motif, a search has been performed for proteins containing a PH domain followed by a PTB domain.

Following these criteria, two new proteins have been recently identified in mice: Dok-4 and Dok-5.²¹ These proteins are expressed mostly in non-hematopoeitic cells and act downstream of PTKs. In the present study, we report the characterization of the human DOK4 and DOK5 genes. We show that these genes are expressed in peripheral blood T cells, which raises the possibility of the role of Dok-4 and Dok-5 proteins in the regulation of the immune response induced by T cells.

Results

In silico identification of new Dok adapter family members

Analysis of the public databases revealed the presence of putative candidate members of the Dok adapter family.^11,14 These studies have led to the identification of an amino acid sequence in the PTB domain displaying a high similarity between the first three Dok family members: Dok-1 (human DOK1 and mouse Dok1 genes), Dok-2/FRIP/DokR (human DOK2 and mouse Dok2 genes) and also Dok3/DokL (mouse Dokl-pending gene). This sequence has been defined as a Dok homology (DKH) sequence motif (WPxxxLRxxGxDxxxFxFExGR, where x is any amino acid residue).^11,14 To identify potential new members of the Dok adapter family, we performed public database similarity searches using this DKH motif and also the sequence of human DOK1 as probes. Based on the screening with the DKH motif, we identified several candidates containing this signature as it has been shown previously. The DKH motif is present in the functional PTB domains of Dok1/2/3. By using a three-dimensional structure prediction program, we showed that all the new candidates contain a putative PTB domain where the DKH motif is located. The general structure of Dok1/2/3 displays a PH domain followed by a PTB domain at their amino-terminus. Some of the candidates do not present a PH domain upstream the PTB domain. These proteins correspond to another group of adapter molecules which bind to some receptor tyrosine kinases:SNT-1/2 (suc1-associated neurotrophic factor target, also known as FRS2/).²² However, we identified five candidates containing a PH domain followed by a PTB domain. The same results were obtained by using human DOK1 sequence for screening public databases. These molecules from human and their mouse homologues are denoted as Dok-1, Dok-2, Dok-3, Dok-4 and Dok-5. The sequences of DOK1/2 in human and Dok1/2/3 in mice have been reported previously.^{3,7,,12,13,15,16} Recently, Dok4/5 genes have been cloned from mouse tissues.²¹ The amino acid sequences from human DOK3/4/5 genes have been deduced (see Material and Methods).

Phylogenetic analysis

The human or mouse five dok genes encode for two potential functional domains (PH and PTB domains). The PH and PTB domains from the human and mouse Dok family members were analyzed separately. If the phylogeny trees are congruent, this suggests that both domains result from the same evolution story since exon shuffling did not occur during these evolutionary events. The PH and PTB domains were aligned including an outgroup as the PH or PTB domain of IRS-1. Like Dok proteins, the IRS-1 adapter molecule contains a PH domain followed by a PTB domain at the amino-terminus part.^7,11

The two phylogenetic trees are congruent and can discriminate two groups denoted as A and B, since group A contains DOK1, DOK2, DOK3 and group B contains DOK4, DOK5 (Figure 1). The presence of two groups is consistent with previously published studies in mouse.²¹

Figure 1.

Phylogenic trees of human and mouse dok family members. Phylogenics trees were calculated using the sequences of PH and PTB domains of dok family members using IRS-1 as outgroup. The Dok members cluster in two groups noted as A and B. The bootstrap values at the nodes are expressed in percentage.

Full figure and legend (71K)

In group A, the PH domain of Dok-1 and Dok-3 forms a monophylogenetic cluster indicating that these genes could be derived from a common ancestor different from the Dok-2 ancestor. However, this cannot be accessed for the PTB domain of group A due to a lack of resolution. Dok-4 and Dok-5 form a monophylogenetic cluster suggesting that the dok genes from group B could derive from a common ancestor. An important point is that each human dok gene has its orthologue in mouse. Thus, a complex set of five dok genes could predate to the mammalian radiation.

Genomic structure of dok genes

A gene prediction program was combined to a comparison of the matching ESTs, for identifying different human dok genes, named DOK1 to DOK5. Figure 2 shows a deduced exon/intron structure of the different dok genes. The two groups of genes defined by the phylogenetic analysis display a different general exon/intron structure. The dok genes of group A are composed of four or five putative exons and the dok genes of group B are composed of eight putative exons. The putative exons encoded, in order, a PH domain, a PTB domain and a region containing some phosphorylable tyrosine residues and proline-rich sequences. The PH or PTB domain of Dok from group A is encoded by one or two exons. The classification of DOK3 in group A by the phylogenetic analysis suggests that the nature of exon 4 is a result of the fusion of two ancestral exons. However, the PH or PTB domain of Dok from group B is encoded by four exons.

Figure 2.

Schematic diagram showing the comparison of the genomic structure of the human Dok family members. Exons are shown by open boxes, and introns by the connecting lines. Numbers inside boxes indicate exon lengths in base pairs. The 5' UTR and 3' UTR extremities are not represented. The introns are not drawn to scale. Roman numbers indicate intron phases. The positions of the PH and PTB domains are indicated by the dotted lines.

Full figure and legend (36K)

Analysis of phylogenetic trees and exon/intron structure of Dok family members are congruent, since the same groups (A and B) can be defined in both experimental approaches. These results could predict the general exon/intron structure of the two common ancestors of the dok genes.

Expression analysis

The dok family members from group A are mainly expressed in hematopoeitic tissues.¹⁵ For group B in mouse, Dok4 is broadly expressed in many tissues and Dok5 is essentially expressed in brain.²¹ To establish the expression pattern of DOK4 and DOK5 in humans, Northern blotting analysis was performed (Figure 3). Among human tissues, DOK4 and DOK5 are highly expressed in heart and skeletal muscles. An expression of DOK4 can be clearly detected also in liver, small intestine and lung. An expression of DOK5 can be clearly detected in brain and kidney. Using this approach, little or no DOK4 and DOK5 were detectable in other tissues as lymphoid organs. To detect a low expression level of DOK4 and DOK5 transcripts, PCR analysis was used. We prepared cDNA from heart where dok genes of group B are expressed (Figure 3) or from spleen where dok genes of group A are expressed¹⁵ and also from resting or activated T cells. We used them for PCR with specific primers for the different dok genes. To verify the RT-PCR specificity, the PCR products were cloned and sequenced. As shown in Figure 4 dok genes of group B are expressed in heart and dok genes of group A are expressed in spleen. In group A, only DOK1 and DOK2 are expressed in human T cells. These data are consistent with previously published studies showing that Dok3 gene is not expressed in mouse T cells.¹⁵ In the new dok group B, DOK4 can be detected in T cells. However, DOK5 is only detectable in T cells upon stimulated conditions. DOK4, but not DOK5, is detectable in spleen. Our results indicate that four dok genes (DOK1, 2, 4 and 5) can be expressed in human T cells and the expression of DOK5 is regulated after T cell activation.

Figure 3.

Gene expression patterns of DOK genes from group B. Northern blot analysis of DOK4 and DOK5 genes in various tissues. A filter containing poly(A)+ selected RNA prepared from multiple human tissues was hybridized with a radiolabeled DOK4 and DOK5 probe (A and B) or -actin probe (C). The positions of migration of RNA molecular weight markers are indicated at left.

Full figure and legend (194K)

Figure 4.

DOK genes expression in human T cells as determined by RT-PCR. 2-microglobulin was used as a control gene.

Full figure and legend (85K)

Discussion

Over the past 5 years, a new family of adapter proteins: Dok molecules appears to play a role in diverse physiological processes in hematopoeitic organs, especially in lymphoid tissues.^{12,15,16,23,24} Recently, two new Dok family members have been identified in mouse tissues, but the expression of these molecules has not been reported in T cells.²¹ In search for new genes that may be involved in T cell activation, we investigated the possible existence of new Dok family members expressed in human T cells. By using computer programs for gene prediction and available public databases, we were able to identify two new human genes, named DOK4 and DOK5. Based on analysis of phylogenetic trees (Figure 1) and exon/intron structure (Figure 2) of Dok family members, we identified two groups: on the one hand, DOK1, DOK2 and DOK3 (group A), and on the other DOK4 and DOK5 (group B). Structural studies have shown that PH and PTB domains adopt the same fold²⁵ It is noteworthy that a PH domain or PTB domain is encoded by an equal number of exons into the same group of dok genes (Figure 2). So, PH and PTB domains encoded by dok genes may be derived from a duplication of a common ancestral domain.

By finding new human dok genes, we investigated the possibility that these genes are expressed in hematopoietic organs and especially in T cells. Northern blot analysis did not permit detection of DOK4 or DOK5 gene expression in lymphoid tissues like thymus, spleen or peripheral blood leukocytes (Figure 3). However, these genes are expressed in other human tissues consistent with previous data in mouse.²¹ Using RT-PCR technique that is more sensitive than Northern blot analysis, the expression of the dok genes from groups A and B has been studied in human peripheral blood T cells (Figure 4). In group A, only DOK1 and DOK2 genes are expressed in T cells, consistent with data published in mouse tissues.¹⁵ In group B, expression of DOK4 or DOK5 gene can be detected in human peripheral blood T cells. An interesting point is that DOK5 gene is expressed in activated but not resting T cells. This suggests that the expression of an adapter molecule as Dok-5 is regulated upon T cell activation. Taken together, these data point to a possible role of two new dok family members in human T cells.

Dok-1 and Dok-2 expressed in T cells seem to play a negative role in T cell function.^12,26 These inhibitory effects can be mediated by phosphotyrosyl motifs interacting by recruitment of RasGAP, a negative regulator of Ras/MAP kinase signaling.^3,7,11,12 Dok proteins from group A can also attenuate signal transduction pathways by binding other negative regulators such as a lipid phosphatase SHIP or a PTK Csk.^15,16,27 The inhibitory effects could also be explained by a competition between Dok-1/2/3 and other PTB domain proteins known as positive regulators.^12,28 Dok-4 and Dok-5 proteins have been described in mouse to be substrates of the c-Ret receptor tyrosine kinase.²¹ Dok-4 and Dok-5 do not recruit the RasGAP protein and can enhance MAP kinase activation.²¹ In contrast to Dok-2, Dok-4 and Dok-5 can promote the neurite outgrowth induced by c-Ret tyrosine kinase receptor. Dok proteins from the two groups could play an opposite role in neuronal cell system.

Here, we have shown that Dok-4 and Dok-5 are expressed in T cells and Dok-5 expression is regulated upon T cell activation. We are now exploring the potential role of Dok-4 and Dok-5 in T cells with which we should be able to address the relevance of the two groups of Dok proteins in T cell signaling.

Materials and methods

Database searching

A sequence containing a high homology between the first three members of the Dok family (Dok-1, Dok-2 and also Dok-3) has been described. This sequence has been defined as a Dok homology (DKH) sequence motif. DKH sequence (WPxxxLRxxGxDxxxFxFExGR, where x is any amino acid residue) or the sequence of human full-length Dok1 (GenBank^TM accession number NP_001372) was subjected to similarity search using the tBLASTN algorithm on the National Center for Biotechnology Information web server (http://www.ncbi.nlm.nih.gov/blast/) and on the Institute for Genomic Research web server (http://www.tigr.org/) against the EST database (dbEST) and Non-redundant database (nr). Selected clones were obtained from the I.M.A.G.E. consortium through Resource Center/Primary Database (http://www.rzpd.de). The clones were propagated, purified and sequenced from both directions with an automated sequencer by Genome Express^TM (Grenoble, France) using insert-flanking vector primers.

DOK3 sequence was predicted from three ESTs clones from NCBI database (accession number NM_024872, AK026223, BC004564). DOK4 sequence was predicted from four ESTs clones and one gene product from NCBI database (accession number BC001540, BC003541, NM_018110, AK001350, HUAC004382). DOK5 sequence was predicted from two ESTs clones and three gene products from NCBI database (accession number AF132732, HSM800394, XM_030597, NM_018431, HS805C22). The human and mouse gene nomenclature belongs to the Human Gene Nomenclature Database (http://www.gene.ucl.ac.uk/nomenclature) or the Mouse Genome Database (http://www.informatics.jax.org).

Structure analysis

The sequences of the various Doks molecules were used for prediction domain studies on the Imperial Cancer Research Fund web server using the 3DPSSM program (http://www.bmm.icnet.uk/~3dpssm/).

Phylogenic analysis

Multiple alignment and phylogeny was performed independently on each domain using the Clustal X software package available from the National Center for Biotechnology Information web server (http://www.ncbi. nlm.nih.gov) using the sequences of the PH and PTB domain of the Doks family members. Phylogenetic studies were performed using the Neighbour Joining method. The reliability was visualized by the TreeView software package (http://taxonomy.zoology.gla.ac.uk/rod/rod.html).

Genomic organization

The nucleotide sequences of the Doks family members were subjected to similarity search using the BLASTN algorithm on the National Center for Biotechnology Information web server against the human Unfinished High Throughput Genomic Sequences database (htgs). GENSCAN prediction gene program was used to identify dok genes from the genomic sequences. These sequences were submitted to analysis with a splicing prediction program NetGene2 from the Center for Biological Sequence Analysis web server (http://www.cbs.dtu.dk/) to determine the position of introns and exons and intron phase. The intron phase refers to the location of the intron within the codon; I denotes that the intron occurs after the first nucleotide of the codon, II denotes the intron occurs after the second nucleotide, 0 denotes the intron occurs between codons.

Northern Blotting

Human tissue poly(A)⁺ RNA blots (MTN 7780-1 CLONTECH^TM) were hybridized with radiolabeled probes corresponding to DOK4, DOK5 and Actin according to the manufacturer's instructions. Probe for Dok-4 was performed by digestion of I.M.A.G.E. consortium's clone (GenBank^TM accession number AI969431) by SalI and NotI and therefore DpnI to obtain a length of 400 bp of specific DNA. Probe for DOK5 was performed using PCR against I.M.A.G.E. consortium's clone (GenBank^TM accession number AA480087) with the following primers: sense 5'-agcgggaacagaatgagaga-3' and antisense 5'-tcatgttgctcagctatgcc-3'.

Cells culture

Peripheral blood mononuclear cells (PBMC) from healthy donors were prepared with standard Ficoll-Hypaque density gradient separation techniques. PBMC are loaded into T cells Enrichment Columns (R&D Systems) to purified T cell population. Human peripheral blood lymphocyte-T (PBL-T) were cultured in RPMI 1640 supplemented with 10% FCS and activated for 24 h with coated CD3 mAb (289 at 2 g/ml) and CD28 mAb (248 at a dilution 1:400 in ascitis liquid).

Cellular and tissue expression by PCR analysis

Total RNA from PBL-T was extracted using Trizol reagent (GibcoBRL, Inc.) following the manufacturer's instructions. Total RNA from spleen and heart were purchased from Clontech Laboratories, Inc. total RNA (2 g) was reverse-transcribed. Then, cDNA were used for PCR with specific primers for DOK1: sense 5'-aggactccctatactcagaccccttg-3' and antisense 5'-ccccagtcttgtcagtccctactcta-3'; for DOK2: sense 5'-cctactctcggccgcatgactcact-3' and antisense 5'-ctgccagccagaagcagagaaatcct-3'; for DOK3: sense 5'-ccatggaccctctggagacccctat-3' and antisense 5'-cctgccaggaggagtagatggagtttt-3'; for DOK4: sense 5'-atgaagagcaggaagctcgggatcta-3' and antisense 5'-ggtagatgttctcgtgggtgatctgc-3'; for DOK5: sense 5'-gagcagacgcctcgggatttatcag-3' and antisense 5'-cctccctgcctcaaaagtgaaccac-3'; or for 2microglobulin: sense 5'-ccagcagagaatggaaagtc-3' and antisense 5'-taagttgccagccctcctag-3'. PCR was carried out in a reaction mixture containing 2 l of cDNA, 20 mM Tris–HCl (pH 8.4), 50 mM KCl, 1.5 mM MgCl₂, 200 M dNTP, 0.5 M each primer and 2.5 units of Taq DNA Polymerase (GibcoBRL). The cycling conditions were 94°C for 5 min followed by 35 cycles of 94°C for 1 min, 55°C (or 60°C for DOK2) for 1 min, 72°C for 2 min and a final extension at 72°C for 10 min. Equal amounts of PCR products were electrophoresed on 1.5% agarose gels and visualized by ethidium bromide staining. To avoid the possibility that the bands could correspond to amplified genomic DNA, the sense and antisense primers have been designed in different exons. The amplified fragments (492 bp for DOK1, 383 bp for DOK2, 471 bp for DOK3, 550 bp for DOK4, 450 bp for DOK5 and 498 bp for 2microglobulin) were cloned into pGEM T-Easy vector (Promega) and sequenced from both directions using vector specific primers by Genome Express^TM (Grenoble, France).

cDNA cloning

Total RNA, extracted from human T cells stimulated for 24 h by CD3 plus CD28 mAbs, was used to make random primed cDNA for the RT-PCR.

PCR was carried out in a reaction mixture containing 2 l of the RT-PCR products, 200 M dNTP, 0.5 M each appropriate primer and 2.5 units of Taq Advantage DNA Polymerase (CLONTECH^TM). The cycling conditions were 94°C for 5 min followed by 35 cycles of 94°C for 1 min, 55°C for 1 min, 72°C for 2 min and a final extension at 72°C for 10 min. For human DOK4 tagged HA epitope, PCR was performed with the following primers: sense 5'-atgtacccatacgacgtcccagactacgct atggcgaccaatttcagtgacatcgtc-3' and antisense 5'-tcactgggatggggtcttggcctcac-3'.

For human DOK5 tagged HA epitope, PCR was performed with the following primers: sense 5'-atgtacccatacgacgtcccagactacgctatggcttccaattttaatgacatagtg-3' and antisense 5'-tcagtgctcagatctgtaggctgg-3'. The amplifications were cloned into the pGEM-T easy vector (Promega) and sequenced as described above.

The nucleotide sequences of human DOK4 and DOK5 were submitted to GenBank; they have been assigned the accession numbers AF466369 and AF466368, respectively. A similar sequence to DOK5 can be found with the accession number XP_030597. An alignment of predicted amino acid sequences of human Dok-4 and Dok-5 is available in our web server (http://u119.marseille.inserm.fr/do/dok45.html).

References

1. Schwartz RH. Costimulation of T lymphocytes: the role of CD28 CTLA-4 and B7/BB1 in interleukin-2 production and immunotherapy.Cell1992;71:1065–1068. | Article | PubMed | ISI | ChemPort |
2. Leo A,Schraven B. Adapters in lymphocyte signalling.Curr Opin Immunol2001;13:307–316. | Article | PubMed | ISI | ChemPort |
3. Carpino N,Wisniewski D,Strife Aet al.p62dok: a constitutively tyrosine-phosphorylated GAP-associated protein in chronic myelogenous leukemia progenitor cells.Cell1997;88:197–204. | Article | PubMed | ISI | ChemPort |
4. Zhao M,Schmitz AA,Qin Y,Di Cristofano A,Pandolfi PP,Van Aelst L. Phosphoinositide 3-kinase-dependent membrane recruitment of p62(dok) is essential for its negative effect on mitogen-activated protein (MAP) kinase activation.J Exp Med2001;194:265–274. | Article | PubMed |
5. Songyang Z,Yamanashi Y,Liu D,Baltimore D. Domain-dependent function of the rasGAP-binding protein p62Dok in cell signaling.J Biol Chem2001;276:2459–2465. | Article | PubMed | ISI | ChemPort |
6. Tang J,Feng GS,Li W. Induced direct binding of the adapter protein Nck to the GTPase-activating protein-associated protein p62 by epidermal growth factor.Oncogene1997;15:1823–1832. | Article |
7. Yamanashi Y,Baltimore D. Identification of the Abl- and rasGAP-associated 62 kDa protein as a docking protein, Dok.Cell1997;88:205–211. | Article | PubMed | ISI | ChemPort |
8. Sylla BS,Murphy K,Cahir-McFarland E,Lane WS,Mosialos G,Kieff E. The X-linked lymphoproliferative syndrome gene product SH2D1A associates with p62dok (Dok1) and activates NF-kappa B.Proc Natl Acad Sci USA2000;97:7470–7475. | Article | PubMed | ChemPort |
9. Hibi M,Hirano T. Gab-family adapter molecules in signal transduction of cytokine and growth factor receptors, and T and B cell antigen receptors.Leuk Lymphoma2000;37:299–307. | PubMed | ISI | ChemPort |
10. Van Obberghen E,Baron V,Delahaye Let al.Surfing the insulin signaling web.Eur J Clin Invest2001;31:966–977. | Article | PubMed | ChemPort |
11. Di Cristofano A,Carpino N,Dunant Net al.Molecular cloning and characterization of p56dok-2 defines a new family of RasGAP-binding proteins.J Biol Chem1998;273:4827–4830. | Article | PubMed | ISI | ChemPort |
12. Nelms K,Snow AL,Hu-Li J,Paul WE. FRIP, a hematopoietic cell-specific rasGAP-interacting protein phosphorylated in response to cytokine stimulation.Immunity1998;9:13–24. | Article | PubMed | ISI | ChemPort |
13. Jones N,Dumont DJ. The Tek/Tie2 receptor signals through a novel Dok-related docking protein, Dok-R.Oncogene1998;17:1097–1108. | Article | PubMed | ISI | ChemPort |
14. Lock P,Casagranda F,Dunn AR. Independent SH2-binding sites mediate interaction of Dok-related protein with RasGTPase-activating protein and Nck.J Biol Chem1999;274:22775–22784. | Article | PubMed | ISI | ChemPort |
15. Lemay S,Davidson D,Latour S,Veillette A. Dok-3, a novel adapter molecule involved in the negative regulation of immunoreceptor signaling.Mol Cell Biol2000;20:2743–2754. | Article | PubMed | ISI | ChemPort |
16. Cong F,Yuan B,Goff SP. Characterization of a novel member of the DOK family that binds and modulates Abl signaling.Mol Cell Biol1999;19:8314–8325. | PubMed | ISI | ChemPort |
17. Harriague J,Debre P,Bismuth G,Hubert P. Priming of CD2-induced p62Dok tyrosine phosphorylation by CD3 in Jurkat T cells.Eur J Immunol2000;30:3319–3328.
18. Némorin JG,Duplay P. Evidence that Lck-mediated phosphorylation of p56dok and p62dok may play a role in CD2 signaling.J Biol Chem2000;275:14 590–14 597.
19. Nune(c)s JA,Truneh A,Olive D,Cantrell DA. Signal transduction by CD28 ligands B7-1 and B7-2: CD28 regulation of tyrosine kinase adapter molecules.J Biol Chem1996;271:1591–1598. | Article | PubMed | ISI | ChemPort |
20. Yang WC,Ghiotto M,Barbarat B,Olive D. The role of tec protein-tyrosine kinase in T cell signaling.J Biol Chem1999;274:607–617. | Article | PubMed | ISI | ChemPort |
21. Grimm J,Sachs M,Britsch Set al.Novel p62dok family members, dok-4 and dok-5, are substrates of the c-Ret receptor tyrosine kinase and mediate neuronal differentiation.J Cell Biol2001;154:345–354. | Article | PubMed | ISI | ChemPort |
22. Dhalluin C,Yan K,Plotnikova Oet al.Structural basis of SNT PTB domain interactions with distinct neurotrophic receptors.Mol Cell2000;6:921–929. | Article | PubMed | ISI | ChemPort |
23. Di Cristofano A,Niki M,Zhao Met al.p62(dok) a negative regulator of Ras and mitogen-activated protein kinase (MAPK) activity, opposes leukemogenesis by p210(bcr-abl).J Exp Med2001;194:275–284. | Article | PubMed | ISI | ChemPort |
24. Suzu S,Tanaka-Douzono M,Nomaguchi Ket al.p56(dok-2) as a cytokine-inducible inhibitor of cell proliferation and signal transduction.Embo J2000;19:5114–5122. | Article | PubMed | ISI | ChemPort |
25. Lemmon MA,Ferguson KM,Schlessinger J. PH domains: diverse sequences with a common fold recruit signaling molecules to the cell surface.Cell1996;85:621–624. | Article | PubMed | ISI | ChemPort |
26. Némorin JG,Laporte P,Berube G,Duplay P. p62dok negatively regulates CD2 signaling in Jurkat cells.J Immunol2001;166:4408–4415. | PubMed | ISI | ChemPort |
27. Tamir I,Stolpa JC,Helgason CDet al.The RasGAP-binding protein p62dok is a mediator of inhibitory FcgammaRIIB signals in B cells.Immunity2000;12:347–358. | Article | PubMed | ISI | ChemPort |
28. Jones N,Dumont DJ. Recruitment of Dok-R to the EGF receptor through its PTB domain is required for attenuation of Erk MAP kinase activation.Curr Biol1999;9:1057–1060. | Article | PubMed | ISI | ChemPort |

Acknowledgements

We would like to thank Y Collette for a critical reading of this manuscript. This research was supported by the Institut National de la Santé et de la Recherche Médicale, by a grant "équipe labellisée" from the Ligue Nationale Contre le Cancer and by Grants 4330 and 5740 from the Association pour la Recherche contre le Cancer. C Favre was supported by a fellowship from the Association pour la Recherche contre le Cancer.