Homology Domain Interface Independently of Its Pleckstrin Tec

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

This information is current as Fabien Garçon, Georges Bismuth, Daniel Isnardon, Daniel of September 28, 2021. Olive and Jacques A. Nunès J Immunol 2004; 173:770-775; ; doi: 10.4049/jimmunol.173.2.770 http://www.jimmunol.org/content/173/2/770 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2004 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

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

Fabien Garc¸on,* Georges Bismuth,† Daniel Isnardon,‡ Daniel Olive,* and Jacques A. Nune`s2*

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 Downloaded from 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. The Journal of Immunology, 2004, 173: 770–775.

n efﬁcient T cell activation requires the appropriate in- of function, the Tec kinases play an important role in signaling

teraction between T cells and APCs. Upon Ag recogni- through Ag receptors, and during lymphocyte development, dif- http://www.jimmunol.org/ A tion, a complex compartmentalization of molecules in- ferentiation, and apoptosis (10). However, despite numerous data volved in T cell stimulation occurs in the T cell-APC interface, about their regulation, the presence of Tec kinases in the immu- termed the immunological synapse (IS)3 (1, 2). Furthermore, this nological synapse has not been investigated yet. compartmentalization is accompanied by and depends on a rapid The PH domain of the Tec kinases recognizes the phosphati- cytoskeletal polarization (3, 4). Altogether, these events lead to the dylinositol 3,4,5 trisphosphate (PI-3,4,5-P3) (11Ð13). This PH do- polarized secretion by T cells of cytokines directed toward target main is involved for the translocation of the Tec kinases at the cells (5). plasma membrane (14). During T cell-APC conjugate formation,

Many molecules participate in the synapse formation. For in- PI-3,4,5-P3 accumulates in T cells at the contact area (15, 16). In stance, early events of T cell signaling, such as the activation of addition to phosphorylation events and membrane recruitment, by guest on September 28, 2021 protein tyrosine kinase (PTKs), occur even before the formation of these kinases are strongly regulated by intra- and intermolecular a mature synapse (6). Among the PTKs, Src kinase family mem- interactions involving their protein binding domains: Src homol- bers are recruited at the IS (7, 8). ogy 2 (SH2) and 3 (SH3) domains (10). Three members of Tec Another PTK family, the Tec kinases are structurally similar to kinases are expressed in T cells: Tec, Itk/Emt, and Rlk/Txk, but Src kinases, but they are distinguished by a unique amino-terminal only Tec and Itk contain a PH domain. This PH domain is involved region containing a pleckstrin homology (PH) domain, followed in the Tec kinase colocalization with the TCR/CD3 complex upon by a Tec homology (TH) domain. This family consists of five TCR cross-linking with Abs (17, 18). Among these Tec kinases, members: Tec, Btk, Itk/Emt, Rlk/Txk, and Bmx/Etk (9). In terms 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. *Institut National de la Sante«et de la Recherche Me«dicale Unite«Mixte de Recherche 599, Institut du Cancer de Marseille, Equipe labellise«e 2001 par la Ligue Nationale contre le In this study, we use transient transfections in human resting T Cancer, Institut Fe«de«ratif de Recherche 137, Universite«delaMe«diterrane«e, Marseille, cells to demonstrate that Tec can be translocated at the IS formed France; †De«partement de Biologie Cellulaire, Institut Cochin, Institut National de la Sante« et de la Recherche Me«dicale Unite« 537, Centre National de la Recherche Scientifique between a T cell and a dendritic cell (DC). Furthermore, we show Unite«Mixte de Recherche 7627, Universite«Rene«Descartes, Paris, France; and ‡Atelier de that this Tec accumulation occurs in a PH-independent manner, Bio-Imagerie, Institut Paoli-Calmettes, Marseille, France whereas its SH3 domain is necessary to this recruitment. In dif- Received for publication February 19, 2003. Accepted for publication May 6, 2004. ferent types of T cell-APC contacts using primary T cells or Jurkat The costs of publication of this article were defrayed in part by the payment of page cells, Tec is located at the plasma membrane in a PH-independent charges. This article must therefore be hereby marked advertisement in accordance manner. However, the presence of PH domain remains important with 18 U.S.C. Section 1734 solely to indicate this fact. for Tec kinase to induce functional events. 1 This work was supported by grants from Institut National de la Sante«etdela Recherche Me«dicale and the Ligue Nationale Contre le Cancer (Equipe labellise«e Finally, these data implicate Tec in the early events of the syn- 2001). F.G. is supported by fellowship from the Ligue Nationale Contre le Cancer. apse formation and emphasize the role of the PH and SH3 domains 2 Address correspondence and reprint requests to Dr. Jacques A. Nune`s, Institut Na- in these events. tional de la Sante«et de la Recherche Me«dicale Unite«Mixte de Recherche 599, Institut du Cancer de Marseille, 27 bd Leõ¬Roure, 13009 Marseille, France. E-mail address: [email protected] Materials and Methods 3 Abbreviations used in this paper: IS, immune synapse; DC, dendritic cell; DIC, Plasmid constructs differential interference contrast; GFP, green fluorescence protein; KO, knockout; PH, Expression plasmid pCDNA3-Flag-Tec was previously described (19). Ex- 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, supe- pression plasmid pCDNA3-⌬PHTec-Flag, containing the murine Tec rantigen; SEE, staphylococcal enterotoxin E; SH, Src homology; TH, Tec homology. cDNA lacking the PH domain (deletion of aa 1Ð113), was generated by

PCR amplification of pCDNA3-Flag-Tec using the following primers: Luciferase assays sense primer, 5Ј-ATAAAGGGATCCAATAATATCATGATTAAATAC ϫ 6 ␮ 3Ј; antisense primer, 5Ј-ACTTAACTCGAGTCATTTGTCATCATCG Jurkat cells (10 10 ) were electroporated at 960 F and 250 V using a ␮ TCCTTATAGTCTCTTCCAAAAGTTTCTTCACA-3Ј (containing Flag Bio-Rad (Hercules, CA) Gene Pulser with 10 g of pIL-2-Luc plasmid, 5 ␮ ␤ ␮ epitope). The plasmid pCDNA3-TecSH3* encodes for a Tec protein including gofp -actin-Rluc, and 10 g of the other expression plasmids. Stimu- substitutions of Leu for Trp at residues 215 and 216 of the SH3 domain (Tec lations with APC were conducted for 8 h with Raji cells (ratio 1:1) pulsed W215/216L or Tec SH3*). These mutations were previously described for Btk for 20 min at 37¡C with SEE at 10 ng/ml. Following cell lysis, proteins ␮ (20). Point mutations were introduced by overlap-extension PCR. All PCR were quantified by Bradford reagent (Bio-Rad), and 30 l of cell lysates products were resolved on a 1% agarose gel, isolated, and purified with the was then subjected to dual luciferase reporter assay, according to the man- NucleospinExtract kit (Macherey-Nagel, Hoerdt, France). ufacturer’s instructions (Promega France SARL, Charbonnie`res, France). GFP fusion protein expression vectors, ⌬PHTec-GFP and TecSH3*- Results were corrected by the activity of firefly luciferase standardized by GFP, were obtained by removing stop codon from pCDNA3-⌬PHTec-Flag that of Renilla luciferase and quantification of proteins in lysates. and pCDNA3-Flag-TecSH3*, respectively, and introducing cDNAs tagged with Flag epitope into the XhoI/BamHI sites of the pEGFP-N3 vector (BD Results Clontech, Palo Alto, CA). Synapse formation induces Tec recruitment at the contact zone The Flag-tagged PH domain (aa 1Ð151) of Tec was amplified from in a PH-independent manner pCDNA3-Flag-Tec with the following primers: sense primer, 5Ј- CAAGCTCGAGCCGAGATGGACTAC-3Ј; antisense primer, 5Ј-CTG The recruitment of Tec family kinases during the IS formation has GCTGGATCCACTCTCAAAAAG-3Ј. The PCR product was subcloned not been studied yet. Using a wild-type Tec molecule fused to GFP into XhoI/BamHI sites of pEGFP-N3 expression vector. PHAkt-GFP was provided by T. Meyer (Stanford University School of Medicine, Stanford, (Tec-GFP), we first analyzed its distribution in T cell/APC conju- CA) and used in previous studies (16). Tec-GFP was generated in the lab gates. Tec-GFP was expressed in human peripheral blood resting by W.-C. Yang. Phospholipase C-␥1 (PLC-␥1) into pRK5 expression vec- T cells, and these cells were incubated with mature DCs pulsed Downloaded from tor is a kind gift of B. Margolis (University of Michigan Medical Center, with a SAg mixture, as previously described (16). Tec-GFP has a Ann Arbor, MI). Plasmids were transfected into COS-7 cells and analyzed diffuse distribution in unstimulated T cells (Fig. 1A, upper panel). by SDS-PAGE, followed by immunoblotting for the presence of the GFP fusion constructs using mAbs against GFP. All constructs were verified by The molecule was significantly expressed both in the nucleus and DNA sequencing. the cytoplasm. This distribution was dramatically modified in T The promoter assay plasmids pIL-2-Luc composed of IL-2 promoter, cells contacting DCs. After 20 min at 37¡C, in a large percentage fused to firefly luciferase reporter gene, and p␤-actin-Rluc composed of of cells (78%, n ϭ 120), most Tec-GFP molecules were recruited http://www.jimmunol.org/ ␤-actin promoter, fused with Renilla luciferase gene, were previously reported (19). to the IS formed between T cells and DCs (Fig. 1A, lower panels). Previous studies in Jurkat T cells have suggested that the PH Cell culture and transfections domain is required to recruit Tec kinase family members such as Human PBL-T and DCs were isolated and prepared, as previously de- Itk or Tec to the plasma membrane upon TCR triggering by Ab scribed (16). Human primary T cells were transfected with the Amaxa cross-linking (17, 18). We therefore used a PH-deleted form of Tec Nucleofector technology (Ko¬ln, Germany), according to the manufactur- (⌬PHTec-GFP) to investigate its contribution in the observed ac- er’s instructions, and were used 18 h after transfection (www.amaxa.com). ⌬ The percentages of transfection in resting PBL-T reach 40Ð50% for most cumulation of Tec at the IS. In unstimulated cells, PHTec-GFP of the GFP constructs. COS-7 cells were maintained in DMEM medium has a diffuse distribution, such as Tec-GFP (Fig. 1B, upper panel). by guest on September 28, 2021 supplemented with 10% FCS, 2 mM L-glutamine, and 1 mM sodium pyru- A strong accumulation of this probe at the IS was still observed in vate. Jurkat JA16 T cell subclone (21) and Raji B cell lymphoma were a large majority of T cells contacting DCs (Fig. 1B, lower panels) grown in RPMI 1640 medium supplemented with 10% FCS, 2 mM L- (74%, n ϭ 150). Both Tec-GFP and ⌬PHTec-GFP showed the glutamine, and 1 mM sodium pyruvate. Jurkat T cells were electroporated, as described before (19), and were used 18 h after transfection. same pattern of accumulation at the IS, with a relatively uniform distribution throughout the contact zone. Reagents and Abs These data show that Tec is efficiently mobilized at the T Superantigen (SAg) mixture (staphylococcal enterotoxin A, B, C3, and E cell-DC interface during Ag recognition. They also indicate that (SEE)) was purchased from Toxin Technology (Sarasota, FL). The 3-amin- the PH domain of Tec is dispensable for this process. opropyltriethoxysilane, poly(L-lysine), and PMA were purchased from Sig- ma-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-␥1 and anti-PLC-␥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 ␮g/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 ϫ60 oil objective equipped with differential interference contrast (DIC). Jurkat T cells can form conjugates with Raji B cells pulsed with 1 ␮g/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 FIGURE 1. Synapse formation induces Tec relocalization to the T cell- onto poly-L-lysine-coated coverslips, let sediment for 3 min, and then cen- trifugated at 300 rpm for 1 min. Finally, these cells were fixed for 30 min APC contact area in a PH-independent manner. A and B, Fluorescence ⌬ at room temperature with 3.7% paraformaldehyde in PBS. Slides were images show the distribution of Tec-GFP (A) and PHTec-GFP (B)in mounted with fluorescent mounting medium. Images were taken and pro- isolated human peripheral blood T cells (upper panel) or T cells contacting cessed using a confocal microscope (Leica TCS NT confocal microscope; DCs pulsed with SAg mixture (lower panels). The right-hand panels are Leica Microsystems, Heidelberg, Germany). DIC panels. Data are representative of three independent experiments. 772 Tec AND IMMUNOLOGICAL SYNAPSE

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 larg- est 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, Downloaded from FIGURE 3. The SH3 domain of Tec is required for the recruitment of these data strongly suggest that another interacting domain of Tec, Tec at the immunological synapse. Fluorescence images show the distri- distinct from the PH domain, is important for the translocation of bution of TecSH3*-GFP in isolated T cell (upper panel) or T cells con- Tec to the IS. tacting DCs pulsed with SAg mixture (lower panels). The right-hand panels are DIC panels. Data are representative of three independent The SH3 domain of Tec is necessary for the recruitment of Tec experiments. at the immunological synapse http://www.jimmunol.org/ The role of SH3 domains in targeting signaling molecules at the present in the nucleus through the binding of its SH3 domain to plasma membrane has been repeatedly emphasized (23), including importin ␣ (24). Mainly, we observed that in most T cells con- for Tec kinase family members in which the SH3 domain is sup- tacting DCs (n ϭ 140), the SH3 mutant did not clearly concentrate posed to bind various proteins in the signalosome complex ex- to the IS contrary to Tec and ⌬PH Tec (Fig. 3, lower panels). In pressed at the IS (10). That is why to investigate its role in the our cell system, in which T cells and DCs were from different recruitment of Tec at the IS, we generated a mutated molecule (Tec donors, a few stable conjugates were formed in the absence of Ag SH3*) by a two-residue mutation (W215/216L), reported to inac- (Fig. 4). They most likely correspond to an allogenic T cell re-

tivate the SH3 domain of the Tec kinase family member, Btk (20). by guest on September 28, 2021 sponse. In these conjugates, we also observed a clear accumulation This molecule fused to GFP (TecSH3*-GFP) was unable to inter- at the IS of Tec and ⌬PHTec, but not of the Tec SH3 mutant. We act with known partners of the Tec SH3 domain, such as the CD28 conclude that a functional SH3 domain is necessary for the re- costimulatory molecule (F. Garçon, unpublished data). We next cruitment of Tec to the IS. transfected this construct into T cells. In contrast to wild-type Tec, this mutant was localized in the cytoplasm and poorly expressed in Functional analysis of Tec and its mutants the nucleus of unstimulated T cells (Fig. 3, upper panel, compare To analyze Tec activation during the IS formation, we next used a with Fig. 1A). These data fit well with earlier observations that gene reporter assay (19) to explore the influence of Tec and its have shown that a fraction of the Tec family member, Itk, is mutants on the IL-2 promoter activity induced by Ag presentation. As this kind of assay requires multiple transfections, we used Ju- rkat 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 ⌬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 ⌬PHTec in the whole cell (Fig. 5A). After Ag presentation, both Tec and ⌬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 ⌬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 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 increase in IL-2 promoter activity in Jurkat cells. This activity was ⌬ pulsed with SAg mixture (0.1 ␮g/ml). B, TecPH-GFP distributions in T further potentiated by Tec wild type, but not by PHTec or Tec- cells forming synapses with DCs. Results are from one of two replicate SH3*, which were unable to trigger IL-2 activity above the control experiments. The right-hand panels are DIC panels. Representative con- (Fig. 5C). Similar results were obtained after stimulation of Jurkat jugates are shown of at least 70 analyzed per experiment. cells with Abs against the CD3/TCR complex (data not shown). The Journal of Immunology 773

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), ⌬PHTec (B), and TecSH3* (C) molecules. Data are representative of two experiments.

Tec activity was also explored using a heterologous COS-7 cell membrane (data not shown). These observations support the fact system in which the different Tec mutants were cotransfected with that the PH domain of Akt and those of Tec kinases can differ on PLC-␥1, a well-known Tec family substrate (25). PLC-␥1 phos- their binding properties to the PI3K products such as phosphati- phorylation was analyzed after immunoprecipitation and blotting dylinositol 3,4-bisphosphate (PI-3,4-P2) and PI-3,4,5-P3. Among with a PLC-␥1 pY783-speciﬁc Ab. The expression of the different the members of the Tec kinase family, the PH domain of Btk is

Tec mutants was measured in parallel. The results show that both well characterized and has been compared with the PH domain of Downloaded from Tec and to a lesser extent Tec-SH3* phosphorylate PLC-␥1. On Akt. Comparisons of the two structures of Akt or Btk PH domain the contrary, ⌬PHTec was totally inefﬁcient (Fig. 5D). Taken to- in complex with inositol (1, 3, 4, 5)-tetrakisphophate show key gether, these data suggest that the PH domain, although not re- differences in their mode of interaction (29Ð31). Indeed, the D5 quired for the membrane localization of Tec, apparently affects its phosphate shows no signiﬁcant interactions with any residue on activity, while the SH3 domain mainly controls the localization of the PH domain of Akt, contrary to the recognition of the PH do-

the kinase. main of Btk that is based on this phosphate. This explains why Akt http://www.jimmunol.org/

interacts with similar afﬁnity with both PI-3,4,5-P3 and PI-3,4-P2, Discussion but not the PH domain of Tec kinases, which is not able to interact

Tec kinase plays an important role in T cell activation leading to with PI-3,4-P2. However, the major PI3K product generated at the

IL-2 production (18, 26). Plasma membrane association is in- T cell plasma membrane following Ag presentation is PI-3,4,5-P3 volved in the activation of many cytosolic kinases. The current (16). Altogether, these observations suggest that the in vivo PI- model of Tec kinase activation involves their PH domain. The PH 3,4,5-P3-binding properties of Akt PH domain seem to be higher domain of Tec kinases is required for the translocation to the than those of the Tec PH domain. In addition, the PI3K (p85␣) plasma membrane during a cell surface receptor cross-linking via knockout (KO) B cells showed an impaired activation of Akt, but soluble ligands (14, 17, 18, 27). However, the localization of Tec not of the Tec kinase member, Btk (32). In conclusion, our exper- by guest on September 28, 2021 has not been studied under physiological conditions in which T iments indicate that the Tec PH domain is neither necessary nor cells are in contact with APCs. In this study, we evaluate the con- sufficient for recruitment of the full-length protein to the plasma tribution of the PH domain of Tec for its accumulation at the IS membrane following Ag presentation. and we demonstrate that another interaction domain of Tec is im- Recent studies in B cells have demonstrated that the recruitment portant for this recruitment at the plasma membrane. of activated Btk to the plasma membrane is not affected by PI3K To evaluate the recruitment of Tec to the plasma membrane inhibitors or in PI3K (p85␣) KO B cells (32). For Tec in T cells, following Ag presentation, we used an experimental approach in our data suggest also that an interaction domain different from the which resting primary human T cells can be transfected with dif- PH domain is required for attaching this kinase to the membrane. ferent Tec-GFP constructs and cell conjugates between T cells and Among the different interaction domains of Tec, we focused our DCs can be visualized (16). Following Ag presentation, we dem- attention on the SH3 domain. By interacting with positive or neg- onstrate that Tec is recruited to the IS formed between T cells and ative regulators of the lymphocyte activation, the presence of this DCs. However, our results also reveal that the presence of the PH SH3 domain can play a critical role in the regulation of Tec kinase domain is not required for this recruitment because a Tec molecule activation (9). An SH3 point mutant of Itk, which disrupts its li- lacking the PH domain also translocates very efficiently to the IS. gand-binding capacity to the SH3 domain, is unable to reconstitute This translocation is not dependent on the PH domain of Tec, but functional events in T cells derived from Itk-null mice (33). Using rather on an intact SH3 domain. This result is particularly impor- the same kind of SH3 point mutants on Tec, we showed that the tant, as the necessity of the PH domain in the translocation of Tec SH3 domain is at least required in early steps of Tec activation kinases has been an accepted dogma. such as the plasma membrane targeting in both primary T cells and To underline this point, an isolated Tec PH domain did not Jurkat T cells. The mechanism of this recruitment remains un- significantly accumulate at the plasma membrane of T cells con- known. Among the numerous partners of the Tec SH3 domain that tacting mature DCs. However, we know from a recent report using can be localized at the IS, we can suggest different candidates for the PH domain of Akt that there is a rapid and sustained production attaching Tec kinase via its SH3 domain to the plasma membrane: of 3Ј-phosphorylated phosphoinositide lipids at the plasma mem- cell surface receptors such as the CD28 costimulatory molecule brane during Ag presentation by DCs (16). Moreover, the intra- (19), molecules involved in the signalosome formation as the adap- cellular localization of both PH domains of Akt or Tec is different tor molecule src homology 2 domain-containing leukocyte phos- in the 3Ј-phosphatase and tensin homologue deleted on chromo- phoprotein of 76 kDa (34), or molecules controlling the actin po- some 10 (PTEN) mutated Jurkat T cell line. As described previ- lymerization as the Wiskott-Aldrich Syndrome protein (35). ously, an isolated Akt PH domain localizes predominantly and Recently, it has been demonstrated that another Tec kinase, Itk, is constitutively at the plasma membrane in Jurkat T cells (28). How- involved in the regulation of the actin polymerization at the vicin- ever, the Tec PH domain is just partially localized at the plasma ity of the IS (36). Although the importance of the SH3 domain of 774 Tec AND IMMUNOLOGICAL SYNAPSE

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 Downloaded from 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-␥1 that its PH domain is not required for the initial translocation of PLC-␥1

to the plasma membrane, but it stabilizes in the membrane for a http://www.jimmunol.org/ longer time (40). Our dynamic studies revealed that Tec reorien- tation 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 (Ͼ20 min). However, we need to consider another possi- bility. Tec family kinases play an important role in the PLC-␥1 by guest on September 28, 2021 activation in T cells (26, 41). The PH domain deletion abolished completely the ability of Tec to phosphorylate PLC-␥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 tar- FIGURE 5. PH and SH3 domains are required for Tec function. A, Flu- geting. The PH domain may induce conformational changes, orescence images show Jurkat T cells electroporated with Tec-GFP, which make Tec more accessible to Src family kinases or less ⌬PHTec-GFP, or TecSH3*-GFP. B, Fluorescence images show Jurkat T accessible to down-regulatory phosphatases. cells, electroporated with Tec-GFP, ⌬PHTec-GFP, or TecSH3*-GFP, and The functional studies on Tec activation demonstrate that both ␮ incubated for 20 min with Raji cells prepulsed with SEE (1 g/ml). C, PH and SH3 domains are involved in the regulation of this PTK. Jurkat T cells, electroporated with vector alone; pCDNA3; or indicated Tec In all of these regards, the evaluation of the role of PH domain on constructs, pCDNA3-Tec (TecWT), pCDNA3-⌬PHTec (⌬PHTec), and the catalytic activity of Tec and the identiﬁcation of SH3 partners pCDNA3-TecSH3* (TecSH3*), were cotransfected with pIL-2-Luc and ␤actinR-Luc. After 24-h recovery, cells were left unstimulated (NS) or of Tec at the IS are interesting issues for further investigation. stimulated for 8 h with an equivalent number of Raji cells or Raji cells Note added in proof. During the reviewing process of this manu- pulsed with SEE (10 ng/ml). Inset, Comparable Flag epitope expression of script, Tomlinson et al. (42) showed that Tec can be accumulated at kinases after anti-Flag immunoprecipitations, followed by anti-Tec the immunological synapse in a T cell clone upon Ag presentation. immnunoblots. Data shown are representative of three experiments Ϯ SD. D, COS-7 cells, transfected with the different versions of Tec, were co- Acknowledgments ␥ ␥ transfected with PLC- 1. PLC- 1 was immunoprecipitated and analyzed We thank Ben Margolis for providing the PLC-␥1 expression vector, and for phosphotyrosine content. Following stripping, membrane was reblotted Dr. Wen-Chin Yang for generating some initial reagents in the lab. We are ␥ with anti-PLC- 1 Abs. Expression level of the different forms of Tec, in grateful to Dr. Yves Collette, Dr. Bernard Payrastre, and Dr. Andre`s Alcover the whole cell lysates, is controlled by an anti-Tec Western blot (lower for helpful discussions; Anne-Paule Tomasi from Hulkette Design panel). Data are representative of two experiments. (www.hulkette.com) for the color art design; and Marie-Claire and Peter Gerhards for the correction of the manuscript. Itk has been assessed in actin-dependent cytoskeletal events upon References TCR cross-linking via Abs (37), it will be interesting to revisit this 1. Monks, C. R., B. A. Freiberg, H. Kupfer, N. Sciaky, and A. Kupfer. 1998. Three- point in the context of the synapse formation. In our study, we dimensional segregation of supramolecular activation clusters in T cells. Nature showed that the SH3 domain can participate in the recruitment of 395:82. 2. Grakoui, A., S. K. Bromley, C. Sumen, M. M. Davis, A. S. Shaw, P. M. Allen, Tec to the membrane; however, we could not exclude that other and M. L. Dustin. 1999. The immunological synapse: a molecular machine con- domains such as the SH2 domain are involved in the recruitment trolling T cell activation. Science 285:221. The Journal of Immunology 775

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