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The Journal of Immunology, 2004, 173: 770-775.
Copyright © 2004 by The American Association of Immunologists

Tec Kinase Migrates to the T Cell-APC Interface Independently of Its Pleckstrin Homology Domain1

Fabien Garçon*, Georges Bismuth{dagger}, Daniel Isnardon{ddagger}, Daniel Olive* and Jacques A. Nunès2,*

* Institut National de la Santé et de la Recherche Médicale Unité Mixte de Recherche 599, Institut du Cancer de Marseille, Equipe labellisée 2001 par la Ligue Nationale contre le Cancer, Institut Fédératif de Recherche 137, Université de la Méditerranée, Marseille, France; {dagger} Département de Biologie Cellulaire, Institut Cochin, Institut National de la Santé et de la Recherche Médicale Unité 537, Centre National de la Recherche Scientifique Unité Mixte de Recherche 7627, Université René Descartes, Paris, France; and {ddagger} Atelier de Bio-Imagerie, Institut Paoli-Calmettes, Marseille, France


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Note added in proof.
 References
 
Tec is the prototypical member of the Tec tyrosine kinases family, which plays an important role in T cell signaling. We show in this study that Tec translocates to the immunological synapse when a T cell contacts a dendritic cell. Surprisingly, the presence of the pleckstrin homology (PH) domain of Tec is not required for this accumulation, and despite a strong activation of 3'-phosphorylated phosphoinositide lipids synthesis during the synapse formation, the Tec PH domain is not redistributed to the T cell plasma membrane. In contrast, we demonstrate that an active Src homology 3 domain is absolutely required, underlining the essential role played by this part of the molecule in the recruitment and/or stabilization of Tec at the immunological synapse. Our results nevertheless suggest that the PH domain controls the kinase activity of the molecule in vivo. We finally demonstrate that the two domains are necessary to trigger transcriptional events following Ag presentation. These data support a model in which the plasma membrane recruitment of the PH-containing protein Tec is not dependent on the production of 3'-phosphorylated phosphoinositide lipids by the PI3K, but rather on an intact Src homology 3 domain.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Note added in proof.
 References
 
An efficient T cell activation requires the appropriate interaction between T cells and APCs. Upon Ag recognition, a complex compartmentalization of molecules involved in T cell stimulation occurs in the T cell-APC interface, termed the immunological synapse (IS)3 (1, 2). Furthermore, this compartmentalization is accompanied by and depends on a rapid cytoskeletal polarization (3, 4). Altogether, these events lead to the polarized secretion by T cells of cytokines directed toward target cells (5).

Many molecules participate in the synapse formation. For instance, early events of T cell signaling, such as the activation of protein tyrosine kinase (PTKs), occur even before the formation of a mature synapse (6). Among the PTKs, Src kinase family members are recruited at the IS (7, 8).

Another PTK family, the Tec kinases are structurally similar to Src kinases, but they are distinguished by a unique amino-terminal region containing a pleckstrin homology (PH) domain, followed by a Tec homology (TH) domain. This family consists of five members: Tec, Btk, Itk/Emt, Rlk/Txk, and Bmx/Etk (9). In terms of function, the Tec kinases play an important role in signaling through Ag receptors, and during lymphocyte development, differentiation, and apoptosis (10). However, despite numerous data about their regulation, the presence of Tec kinases in the immunological synapse has not been investigated yet.

The PH domain of the Tec kinases recognizes the phosphatidylinositol 3,4,5 trisphosphate (PI-3,4,5-P3) (11, 12, 13). This PH domain is involved for the translocation of the Tec kinases at the plasma membrane (14). During T cell-APC conjugate formation, PI-3,4,5-P3 accumulates in T cells at the contact area (15, 16). In addition to phosphorylation events and membrane recruitment, these kinases are strongly regulated by intra- and intermolecular interactions involving their protein binding domains: Src homology 2 (SH2) and 3 (SH3) domains (10). Three members of Tec kinases are expressed in T cells: Tec, Itk/Emt, and Rlk/Txk, but only Tec and Itk contain a PH domain. This PH domain is involved in the Tec kinase colocalization with the TCR/CD3 complex upon TCR cross-linking with Abs (17, 18). Among these Tec kinases, Tec is particularly active to induce cytokine gene expression (19). So, Tec can be a useful model to analyze both the translocation and function of Tec kinases in T cells.

In this study, we use transient transfections in human resting T cells to demonstrate that Tec can be translocated at the IS formed between a T cell and a dendritic cell (DC). Furthermore, we show that this Tec accumulation occurs in a PH-independent manner, whereas its SH3 domain is necessary to this recruitment. In different types of T cell-APC contacts using primary T cells or Jurkat cells, Tec is located at the plasma membrane in a PH-independent manner. However, the presence of PH domain remains important for Tec kinase to induce functional events.

Finally, these data implicate Tec in the early events of the synapse formation and emphasize the role of the PH and SH3 domains in these events.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Note added in proof.
 References
 
Plasmid constructs

Expression plasmid pCDNA3-Flag-Tec was previously described (19). Expression plasmid pCDNA3-{Delta}PHTec-Flag, containing the murine Tec cDNA lacking the PH domain (deletion of aa 1–113), was generated by PCR amplification of pCDNA3-Flag-Tec using the following primers: sense primer, 5'-ATAAAGGGATCCAATAATATCATGATTAAATAC3'; antisense primer, 5'-ACTTAACTCGAGTCATTTGTCATCATCGTCCTTATAGTCTCTTCCAAAAGTTTCTTCACA-3' (containing Flag epitope). The plasmid pCDNA3-TecSH3* encodes for a Tec protein including substitutions of Leu for Trp at residues 215 and 216 of the SH3 domain (Tec W215/216L or Tec SH3*). These mutations were previously described for Btk (20). Point mutations were introduced by overlap-extension PCR. All PCR products were resolved on a 1% agarose gel, isolated, and purified with the NucleospinExtract kit (Macherey-Nagel, Hoerdt, France).

GFP fusion protein expression vectors, {Delta}PHTec-GFP and TecSH3*-GFP, were obtained by removing stop codon from pCDNA3-{Delta}PHTec-Flag and pCDNA3-Flag-TecSH3*, respectively, and introducing cDNAs tagged with Flag epitope into the XhoI/BamHI sites of the pEGFP-N3 vector (BD Clontech, Palo Alto, CA).

The Flag-tagged PH domain (aa 1–151) of Tec was amplified from pCDNA3-Flag-Tec with the following primers: sense primer, 5'-CAAGCTCGAGCCGAGATGGACTAC-3'; antisense primer, 5'-CTGGCTGGATCCACTCTCAAAAAG-3'. The PCR product was subcloned into XhoI/BamHI sites of pEGFP-N3 expression vector. PHAkt-GFP was provided by T. Meyer (Stanford University School of Medicine, Stanford, CA) and used in previous studies (16). Tec-GFP was generated in the lab by W.-C. Yang. Phospholipase C-{gamma}1 (PLC-{gamma}1) into pRK5 expression vector is a kind gift of B. Margolis (University of Michigan Medical Center, Ann Arbor, MI). Plasmids were transfected into COS-7 cells and analyzed by SDS-PAGE, followed by immunoblotting for the presence of the GFP fusion constructs using mAbs against GFP. All constructs were verified by DNA sequencing.

The promoter assay plasmids pIL-2-Luc composed of IL-2 promoter, fused to firefly luciferase reporter gene, and p{beta}-actin-Rluc composed of {beta}-actin promoter, fused with Renilla luciferase gene, were previously reported (19).

Cell culture and transfections

Human PBL-T and DCs were isolated and prepared, as previously described (16). Human primary T cells were transfected with the Amaxa Nucleofector technology (Köln, Germany), according to the manufacturer’s instructions, and were used 18 h after transfection (www.amaxa.com). The percentages of transfection in resting PBL-T reach 40–50% for most of the GFP constructs. COS-7 cells were maintained in DMEM medium supplemented with 10% FCS, 2 mM L-glutamine, and 1 mM sodium pyruvate. Jurkat JA16 T cell subclone (21) and Raji B cell lymphoma were grown in RPMI 1640 medium supplemented with 10% FCS, 2 mM L-glutamine, and 1 mM sodium pyruvate. Jurkat T cells were electroporated, as described before (19), and were used 18 h after transfection.

Reagents and Abs

Superantigen (SAg) mixture (staphylococcal enterotoxin A, B, C3, and E (SEE)) was purchased from Toxin Technology (Sarasota, FL). The 3-aminopropyltriethoxysilane, poly(L-lysine), and PMA were purchased from Sigma-Aldrich (St. Louis, MO). Fluorescent mounting medium FluorSave was purchased from Calbiochem (VWR International SAS, Fontenay-sous-Bois, France). The anti-Tec rabbit polyclonal antiserum was previously described (19). Anti-Flag mAb (M2) was purchased from Sigma-Aldrich. Anti-phospho-Y783-PLC-{gamma}1 and anti-PLC-{gamma}1 polyclonal Abs were purchased from Cell Signaling Technology (Beverly, MA). Cell lysates, immunoprecipitations, and immunoblots were performed, as described previously (19).

Fluorescence analysis

For T cell-DC conjugate analysis, DCs were plated on glass coverslips coated with 2 µg/ml poly(L-lysine) and mounted on petri dishes, and were incubated with 10 ng/ml SAg mixture at 37°C for 20 min. Transfected T cells were then added to DCs and incubated for 20 min. Conjugates were then fixed in PBS plus 4% paraformaldehyde and stored in PBS at 4°C before analysis. T cell-DC conjugates were analyzed on an inverted microscope (Nikon, Melville, NY) with a x60 oil objective equipped with differential interference contrast (DIC). Jurkat T cells can form conjugates with Raji B cells pulsed with 1 µg/ml SEE for 15 min (22). Transfected Jurkat cells were mixed at a 2:1 ratio with SEE-pulsed APC, and then incubated at 37°C for 20 min. After stimulation, the cells were deposed onto poly-L-lysine-coated coverslips, let sediment for 3 min, and then centrifugated at 300 rpm for 1 min. Finally, these cells were fixed for 30 min at room temperature with 3.7% paraformaldehyde in PBS. Slides were mounted with fluorescent mounting medium. Images were taken and processed using a confocal microscope (Leica TCS NT confocal microscope; Leica Microsystems, Heidelberg, Germany).

Luciferase assays

Jurkat cells (10 x 106) were electroporated at 960 µF and 250 V using a Bio-Rad (Hercules, CA) Gene Pulser with 10 µg of pIL-2-Luc plasmid, 5 µg of p{beta}-actin-Rluc, and 10 µg of the other expression plasmids. Stimulations with APC were conducted for 8 h with Raji cells (ratio 1:1) pulsed for 20 min at 37°C with SEE at 10 ng/ml. Following cell lysis, proteins were quantified by Bradford reagent (Bio-Rad), and 30 µl of cell lysates was then subjected to dual luciferase reporter assay, according to the manufacturer’s instructions (Promega France SARL, Charbonnières, France). Results were corrected by the activity of firefly luciferase standardized by that of Renilla luciferase and quantification of proteins in lysates.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Note added in proof.
 References
 
Synapse formation induces Tec recruitment at the contact zone in a PH-independent manner

The recruitment of Tec family kinases during the IS formation has not been studied yet. Using a wild-type Tec molecule fused to GFP (Tec-GFP), we first analyzed its distribution in T cell/APC conjugates. Tec-GFP was expressed in human peripheral blood resting T cells, and these cells were incubated with mature DCs pulsed with a SAg mixture, as previously described (16). Tec-GFP has a diffuse distribution in unstimulated T cells (Fig. 1A, upper panel). The molecule was significantly expressed both in the nucleus and the cytoplasm. This distribution was dramatically modified in T cells contacting DCs. After 20 min at 37°C, in a large percentage of cells (78%, n = 120), most Tec-GFP molecules were recruited to the IS formed between T cells and DCs (Fig. 1A, lower panels).



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  		FIGURE 1. Synapse formation induces Tec relocalization to the T cell-APC contact area in a PH-independent manner. A and B, Fluorescence images show the distribution of Tec-GFP (A) and {Delta}PHTec-GFP (B) in isolated human peripheral blood T cells (upper panel) or T cells contacting DCs pulsed with SAg mixture (lower panels). The right-hand panels are DIC panels. Data are representative of three independent experiments.

 
Previous studies in Jurkat T cells have suggested that the PH domain is required to recruit Tec kinase family members such as Itk or Tec to the plasma membrane upon TCR triggering by Ab cross-linking (17, 18). We therefore used a PH-deleted form of Tec ({Delta}PHTec-GFP) to investigate its contribution in the observed accumulation of Tec at the IS. In unstimulated cells, {Delta}PHTec-GFP has a diffuse distribution, such as Tec-GFP (Fig. 1B, upper panel). A strong accumulation of this probe at the IS was still observed in a large majority of T cells contacting DCs (Fig. 1B, lower panels) (74%, n = 150). Both Tec-GFP and {Delta}PHTec-GFP showed the same pattern of accumulation at the IS, with a relatively uniform distribution throughout the contact zone.

These data show that Tec is efficiently mobilized at the T cell-DC interface during Ag recognition. They also indicate that the PH domain of Tec is dispensable for this process.

The PH domain of Tec does not translocate to the immunological synapse

The Tec PH domain has been shown to have affinity for PI-3,4,5-P3 produced at the plasma membrane after PI3K activation (18). However, our data (Fig. 1) suggest that it is not necessary for the recruitment of Tec at the contact zone between a T cell and a DC. We therefore directly analyzed its recruitment to the plasma membrane of T cells contacting DCs using a molecule containing the Tec PH domain fused to GFP. As a control, we used in parallel the PH domain of Akt/PKB also fused to GFP (AktPH-GFP). This probe has been shown to translocate to the plasma membrane of T cell during IS formation due to the PI-3,4,5-P3 increase induced by T cell activation (15, 16). Both PH domains show the same diffuse distribution into the cytoplasm and the nucleus of unstimulated T cells (Fig. 2, upper panels). As previously described (16), the largest part of AktPH-GFP accumulated at the plasma membrane of T cells contacting Ag-pulsed DCs (Fig. 2A, lower panels). In contrast, TecPH-GFP remained mostly nuclear and cytoplasmic (Fig. 2B, lower panels). Taken together with the results shown in Fig. 1, these data strongly suggest that another interacting domain of Tec, distinct from the PH domain, is important for the translocation of Tec to the IS.



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  		FIGURE 2. Distribution of the Tec PH domain in human T cell-DC conjugates. A, Images of PHAkt-GFP in T cells interacting with DCs pulsed with SAg mixture (0.1 µg/ml). B, TecPH-GFP distributions in T cells forming synapses with DCs. Results are from one of two replicate experiments. The right-hand panels are DIC panels. Representative conjugates are shown of at least 70 analyzed per experiment.

 
The SH3 domain of Tec is necessary for the recruitment of Tec at the immunological synapse

The role of SH3 domains in targeting signaling molecules at the plasma membrane has been repeatedly emphasized (23), including for Tec kinase family members in which the SH3 domain is supposed to bind various proteins in the signalosome complex expressed at the IS (10). That is why to investigate its role in the recruitment of Tec at the IS, we generated a mutated molecule (Tec SH3*) by a two-residue mutation (W215/216L), reported to inactivate the SH3 domain of the Tec kinase family member, Btk (20). This molecule fused to GFP (TecSH3*-GFP) was unable to interact with known partners of the Tec SH3 domain, such as the CD28 costimulatory molecule (F. Garçon, unpublished data). We next transfected this construct into T cells. In contrast to wild-type Tec, this mutant was localized in the cytoplasm and poorly expressed in the nucleus of unstimulated T cells (Fig. 3, upper panel, compare with Fig. 1A). These data fit well with earlier observations that have shown that a fraction of the Tec family member, Itk, is present in the nucleus through the binding of its SH3 domain to importin {alpha} (24). Mainly, we observed that in most T cells contacting DCs (n = 140), the SH3 mutant did not clearly concentrate to the IS contrary to Tec and {Delta}PH Tec (Fig. 3, lower panels). In our cell system, in which T cells and DCs were from different donors, a few stable conjugates were formed in the absence of Ag (Fig. 4). They most likely correspond to an allogenic T cell response. In these conjugates, we also observed a clear accumulation at the IS of Tec and {Delta}PHTec, but not of the Tec SH3 mutant. We conclude that a functional SH3 domain is necessary for the recruitment of Tec to the IS.



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  		FIGURE 3. The SH3 domain of Tec is required for the recruitment of Tec at the immunological synapse. Fluorescence images show the distribution of TecSH3*-GFP in isolated T cell (upper panel) or T cells contacting DCs pulsed with SAg mixture (lower panels). The right-hand panels are DIC panels. Data are representative of three independent experiments.

 


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  		FIGURE 4. Tec recruitment requires its SH3 domain, but not the PH domain in absence of SAg. A–C, DIC images (right) show T cells-DC conjugates after 20 min of interaction without SAg stimulation. Fluorescence images (left) show the subcellular localization of the Tec (A), {Delta}PHTec (B), and TecSH3* (C) molecules. Data are representative of two experiments.

 
Functional analysis of Tec and its mutants

To analyze Tec activation during the IS formation, we next used a gene reporter assay (19) to explore the influence of Tec and its mutants on the IL-2 promoter activity induced by Ag presentation. As this kind of assay requires multiple transfections, we used Jurkat T cells activated by SEE-pulsed Raji B cells as APCs. We first checked the intracellular distribution of the different Tec-GFP molecules in Jurkat after transfection. The same striking differences found in normal resting T cells between Tec and {Delta}PHTec on the one hand and Tec-SH3* on the other were observed. The SH3 mutant was largely excluded from the nucleus, contrasting with the broad distribution of Tec and {Delta}PHTec in the whole cell (Fig. 5A). After Ag presentation, both Tec and {Delta}PHTec, but not Tec-SH3*, strongly translocated to the IS of most conjugates, demonstrating again the key role played by the SH3 domain of the molecule for its redistribution to the IS (Fig. 5B). We next investigated how the expression of the different Tec molecules could alter IL-2 promoter activity in unstimulated T cells or after Ag recognition. In the absence of Ag, Tec, but not {Delta}PHTec or Tec-SH3*, induced a low, but significant IL-2 promoter activity above the control values obtained with an empty vector. Ag presentation induced a strong increase in IL-2 promoter activity in Jurkat cells. This activity was further potentiated by Tec wild type, but not by {Delta}PHTec or Tec-SH3*, which were unable to trigger IL-2 activity above the control (Fig. 5C). Similar results were obtained after stimulation of Jurkat cells with Abs against the CD3/TCR complex (data not shown). Tec activity was also explored using a heterologous COS-7 cell system in which the different Tec mutants were cotransfected with PLC-{gamma}1, a well-known Tec family substrate (25). PLC-{gamma}1 phosphorylation was analyzed after immunoprecipitation and blotting with a PLC-{gamma}1 pY783-specific Ab. The expression of the different Tec mutants was measured in parallel. The results show that both Tec and to a lesser extent Tec-SH3* phosphorylate PLC-{gamma}1. On the contrary, {Delta}PHTec was totally inefficient (Fig. 5D). Taken together, these data suggest that the PH domain, although not required for the membrane localization of Tec, apparently affects its activity, while the SH3 domain mainly controls the localization of the kinase.



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  		FIGURE 5. PH and SH3 domains are required for Tec function. A, Fluorescence images show Jurkat T cells electroporated with Tec-GFP, {Delta}PHTec-GFP, or TecSH3*-GFP. B, Fluorescence images show Jurkat T cells, electroporated with Tec-GFP, {Delta}PHTec-GFP, or TecSH3*-GFP, and incubated for 20 min with Raji cells prepulsed with SEE (1 µg/ml). C, Jurkat T cells, electroporated with vector alone; pCDNA3; or indicated Tec constructs, pCDNA3-Tec (TecWT), pCDNA3-{Delta}PHTec ({Delta}PHTec), and pCDNA3-TecSH3* (TecSH3*), were cotransfected with pIL-2-Luc and {beta}actinR-Luc. After 24-h recovery, cells were left unstimulated (NS) or stimulated for 8 h with an equivalent number of Raji cells or Raji cells pulsed with SEE (10 ng/ml). Inset, Comparable Flag epitope expression of kinases after anti-Flag immunoprecipitations, followed by anti-Tec immnunoblots. Data shown are representative of three experiments ± SD. D, COS-7 cells, transfected with the different versions of Tec, were cotransfected with PLC-{gamma}1. PLC-{gamma}1 was immunoprecipitated and analyzed for phosphotyrosine content. Following stripping, membrane was reblotted with anti-PLC-{gamma}1 Abs. Expression level of the different forms of Tec, in the whole cell lysates, is controlled by an anti-Tec Western blot (lower panel). Data are representative of two experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Note added in proof.
 References
 
Tec kinase plays an important role in T cell activation leading to IL-2 production (18, 26). Plasma membrane association is involved in the activation of many cytosolic kinases. The current model of Tec kinase activation involves their PH domain. The PH domain of Tec kinases is required for the translocation to the plasma membrane during a cell surface receptor cross-linking via soluble ligands (14, 17, 18, 27). However, the localization of Tec has not been studied under physiological conditions in which T cells are in contact with APCs. In this study, we evaluate the contribution of the PH domain of Tec for its accumulation at the IS and we demonstrate that another interaction domain of Tec is important for this recruitment at the plasma membrane.

To evaluate the recruitment of Tec to the plasma membrane following Ag presentation, we used an experimental approach in which resting primary human T cells can be transfected with different Tec-GFP constructs and cell conjugates between T cells and DCs can be visualized (16). Following Ag presentation, we demonstrate that Tec is recruited to the IS formed between T cells and DCs. However, our results also reveal that the presence of the PH domain is not required for this recruitment because a Tec molecule lacking the PH domain also translocates very efficiently to the IS. This translocation is not dependent on the PH domain of Tec, but rather on an intact SH3 domain. This result is particularly important, as the necessity of the PH domain in the translocation of Tec kinases has been an accepted dogma.

To underline this point, an isolated Tec PH domain did not significantly accumulate at the plasma membrane of T cells contacting mature DCs. However, we know from a recent report using the PH domain of Akt that there is a rapid and sustained production of 3'-phosphorylated phosphoinositide lipids at the plasma membrane during Ag presentation by DCs (16). Moreover, the intracellular localization of both PH domains of Akt or Tec is different in the 3'-phosphatase and tensin homologue deleted on chromosome 10 (PTEN) mutated Jurkat T cell line. As described previously, an isolated Akt PH domain localizes predominantly and constitutively at the plasma membrane in Jurkat T cells (28). However, the Tec PH domain is just partially localized at the plasma membrane (data not shown). These observations support the fact that the PH domain of Akt and those of Tec kinases can differ on their binding properties to the PI3K products such as phosphatidylinositol 3,4-bisphosphate (PI-3,4-P2) and PI-3,4,5-P3. Among the members of the Tec kinase family, the PH domain of Btk is well characterized and has been compared with the PH domain of Akt. Comparisons of the two structures of Akt or Btk PH domain in complex with inositol (1, 3, 4, 5)-tetrakisphophate show key differences in their mode of interaction (29, 30, 31). Indeed, the D5 phosphate shows no significant interactions with any residue on the PH domain of Akt, contrary to the recognition of the PH domain of Btk that is based on this phosphate. This explains why Akt interacts with similar affinity with both PI-3,4,5-P3 and PI-3,4-P2, but not the PH domain of Tec kinases, which is not able to interact with PI-3,4-P2. However, the major PI3K product generated at the T cell plasma membrane following Ag presentation is PI-3,4,5-P3 (16). Altogether, these observations suggest that the in vivo PI-3,4,5-P3-binding properties of Akt PH domain seem to be higher than those of the Tec PH domain. In addition, the PI3K (p85{alpha}) knockout (KO) B cells showed an impaired activation of Akt, but not of the Tec kinase member, Btk (32). In conclusion, our experiments indicate that the Tec PH domain is neither necessary nor sufficient for recruitment of the full-length protein to the plasma membrane following Ag presentation.

Recent studies in B cells have demonstrated that the recruitment of activated Btk to the plasma membrane is not affected by PI3K inhibitors or in PI3K (p85{alpha}) KO B cells (32). For Tec in T cells, our data suggest also that an interaction domain different from the PH domain is required for attaching this kinase to the membrane. Among the different interaction domains of Tec, we focused our attention on the SH3 domain. By interacting with positive or negative regulators of the lymphocyte activation, the presence of this SH3 domain can play a critical role in the regulation of Tec kinase activation (9). An SH3 point mutant of Itk, which disrupts its ligand-binding capacity to the SH3 domain, is unable to reconstitute functional events in T cells derived from Itk-null mice (33). Using the same kind of SH3 point mutants on Tec, we showed that the SH3 domain is at least required in early steps of Tec activation such as the plasma membrane targeting in both primary T cells and Jurkat T cells. The mechanism of this recruitment remains unknown. Among the numerous partners of the Tec SH3 domain that can be localized at the IS, we can suggest different candidates for attaching Tec kinase via its SH3 domain to the plasma membrane: cell surface receptors such as the CD28 costimulatory molecule (19), molecules involved in the signalosome formation as the adaptor molecule src homology 2 domain-containing leukocyte phosphoprotein of 76 kDa (34), or molecules controlling the actin polymerization as the Wiskott-Aldrich Syndrome protein (35). Recently, it has been demonstrated that another Tec kinase, Itk, is involved in the regulation of the actin polymerization at the vicinity of the IS (36). Although the importance of the SH3 domain of Itk has been assessed in actin-dependent cytoskeletal events upon TCR cross-linking via Abs (37), it will be interesting to revisit this point in the context of the synapse formation. In our study, we showed that the SH3 domain can participate in the recruitment of Tec to the membrane; however, we could not exclude that other domains such as the SH2 domain are involved in the recruitment of Tec to the plasma membrane (18). The analysis of the role of Tec SH2 domain will need further investigation.

To evaluate the Tec function in T cells, two complementary approaches are generally used depending on the phenotype of the T cells derived from Tec family-null mice. For instance, T cells from Itk KO and Rlk/Itk double KO mice have defects in signaling and development (38). Thus, the more representative experiments to perform structure/function studies on Itk kinase will be to try to restore Itk function with different mutants in T cells derived from Itk-null mice. T cells from Tec KO apparently do not present a detectable defect in signaling or development (39). In this case, it will be difficult to use the same strategy for Tec. Therefore, as Tec overexpression is able to activate or potentiate the IL-2 promoter activity in Jurkat cells (19, 26), this cellular model has been used to evaluate the importance of the PH domain on Tec-induced IL-2 promoter activity during cellular interactions. As shown in primary human T cells, Tec and its PH-deleted mutant are equally able to move to the immune synapse during a contact between Jurkat T cells and SEE-pulsed Raji B cells. Although the first events of Tec activation (targeting to the IS) are conserved, it appears that the PH domain of Tec is required to increase the IL-2 promoter activity induced by the contact with SAg-pulsed Raji cells. It has been suggested for other signaling molecules such as PLC-{gamma}1 that its PH domain is not required for the initial translocation of PLC-{gamma}1 to the plasma membrane, but it stabilizes in the membrane for a longer time (40). Our dynamic studies revealed that Tec reorientation occurred between 3 and 5 min after the initial interaction of the T cell and the APC, and was sustained until 20 min after the contact (data not shown). Similar membrane association kinetics was detected using a Tec PH-deleted mutant. These experiments cannot completely exclude that the PH domain can be involved in maintaining Tec kinase at the IS for longer time points after cell contact (>20 min). However, we need to consider another possibility. Tec family kinases play an important role in the PLC-{gamma}1 activation in T cells (26, 41). The PH domain deletion abolished completely the ability of Tec to phosphorylate PLC-{gamma}1, which can be in accordance with the fact that a similar deletion mutant is not hyperphosphorylated upon TCR cross-linking (18). Thus, the Tec PH domain may have functions other than plasma membrane targeting. The PH domain may induce conformational changes, which make Tec more accessible to Src family kinases or less accessible to down-regulatory phosphatases.

The functional studies on Tec activation demonstrate that both PH and SH3 domains are involved in the regulation of this PTK. In all of these regards, the evaluation of the role of PH domain on the catalytic activity of Tec and the identification of SH3 partners of Tec at the IS are interesting issues for further investigation.


   Note added in proof.
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Note added in proof.
References
 
During the reviewing process of this manuscript, Tomlinson et al. (42) showed that Tec can be accumulated at the immunological synapse in a T cell clone upon Ag presentation.


   Acknowledgments
 
We thank Ben Margolis for providing the PLC-{gamma}1 expression vector, and Dr. Wen-Chin Yang for generating some initial reagents in the lab. We are grateful to Dr. Yves Collette, Dr. Bernard Payrastre, and Dr. Andrès Alcover for helpful discussions; Anne-Paule Tomasi from Hulkette Design (www.hulkette.com) for the color art design; and Marie-Claire and Peter Gerhards for the correction of the manuscript.


   Footnotes
 
1 This work was supported by grants from Institut National de la Santé et de la Recherche Médicale and the Ligue Nationale Contre le Cancer (Equipe labellisée 2001). F.G. is supported by fellowship from the Ligue Nationale Contre le Cancer. Back

2 Address correspondence and reprint requests to Dr. Jacques A. Nunès, Institut National de la Santé et de la Recherche Médicale Unité Mixte de Recherche 599, Institut du Cancer de Marseille, 27 bd Leï Roure, 13009 Marseille, France. E-mail address: nunes{at}marseille.inserm.fr Back

3 Abbreviations used in this paper: IS, immune synapse; DC, dendritic cell; DIC, differential interference contrast; GFP, green fluorescence protein; KO, knockout; PH, pleckstrin homology; PI-3,4-P2, phosphatidylinositol 3,4-bisphosphate; PI-3,4,5-P3, phosphatidylinositol 3,4,5 trisphosphate; PTK, protein tyrosine kinase; SAg, superantigen; SEE, staphylococcal enterotoxin E; SH, Src homology; TH, Tec homology. Back

Received for publication February 19, 2003. Accepted for publication May 6, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Note added in proof.
 References
 

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