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Journal of Virology, January 2006, p. 108-118, Vol. 80, No. 1
0022-538X/06/$08.00+0     doi:10.1128/JVI.80.1.108-118.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Association of Herpesvirus Saimiri Tip with Lipid Raft Is Essential for Downregulation of T-Cell Receptor and CD4 Coreceptor

Nam-Hyuk Cho,^1,2* Dior Kingston,¹ Heesoon Chang,¹ Eun-Kyung Kwon,² Jo-Min Kim,² Jung Hee Lee,² Hyuk Chu,² Myung-Sik Choi,² Ik-Sang Kim,² and Jae Ung Jung^1*

Department of Microbiology and Molecular Genetics and Tumor Virology Division, New England Primate Research Center, Harvard Medical School, Southborough, Massachusetts 01772-9102,¹ Department of Microbiology and Immunology, Seoul National University College of Medicine, Seoul 110-799, South Korea²

Received 13 June 2005/ Accepted 4 October 2005

Top Abstract Introduction Materials and Methods Results Discussion References

 
Lipid rafts are membrane microdomains that are proposed to function as platforms for both receptor signaling and trafficking. Our previous studies have demonstrated that Tip of herpesvirus saimiri (HVS), which is a T-lymphotropic tumor virus, is constitutively targeted to lipid rafts and interacts with cellular Lck tyrosine kinase and p80 WD repeat-containing endosomal protein. Through the interactions with Lck and p80, HVS Tip modulates diverse T-cell functions, which leads to the downregulation of T-cell receptor (TCR) and CD4 coreceptor surface expression, the inhibition of TCR signal transduction, and the activation of STAT3 transcription factor. In this study, we investigated the functional significance of Tip association with lipid rafts. We found that Tip expression remarkably increased lipid raft fractions in human T cells by enhancing the recruitment of lipid raft-resident proteins. Genetic analysis showed that the carboxyl-terminal transmembrane, but not p80 and Lck interaction, of Tip was required for the lipid raft localization and that lipid raft localization of Tip was necessary for the efficient downregulation of TCR and CD4 surface expression. Correlated with this, treatment with Filipin III, a lipid raft-disrupting agent, effectively reversed the downregulation of CD3 and CD4 surface expression induced by Tip. On the other hand, Tip mutants that were no longer present in lipid rafts were still capable of inhibiting TCR signaling and activating STAT3 transcription factor activity as efficiently as wild-type (wt) Tip. These results indicate that the association of Tip with lipid rafts is essential for the downregulation of TCR and CD4 surface expression but not for the inhibition of TCR signal transduction and the activation of STAT3 transcription factor. These results also suggest that the signaling and targeting activities of HVS Tip rely on functionally and genetically separable mechanisms, which may independently modulate T-cell function for viral persistence or pathogenesis.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Lipid rafts are cholesterol- and sphingolipid-rich plasma membrane microdomains (13) that may take part in coordinating the signaling functions of the T-cell receptor (TCR). Following interaction with major histocompatibility complex class II (MHC II) molecules containing antigenic peptide, the TCR has been shown to rapidly translocate into Lck-enriched, CD45-deficient lipid rafts, where both TCR and Lck become phosphorylated (13, 31). Upon translocation to the lipid rafts, the TCR transmits signals through several signaling pathways that result in the assembly of lipid rafts through anchoring of the lipid rafts to the underlying actin cytoskeleton and the expression of a variety of genes associated with T-cell activation. Efficient assembly of rafts represents a necessary threshold that must be surpassed to avoid abbreviated signaling and T-cell anergy and is required for a full response by the stimulated T cell (21, 31). The formation of macromolecular structure, referred to as an immunological synapse, through the merging of lipid rafts enhances the accumulation of signaling proteins and amplifies the initial signals from the surface receptors. Subsequently, the clustered lipid raft, including TCR/CD3 complexes, is subjected to endocytosis, which delivers the TCR/CD3 complexes to lysosomes for degradation (8, 22). This targeting process appears to be initiated from the lipid raft, as a portion of the raft constituents, sphingolipid and GM1 ganglioside, is internalized along with the TCR and trafficked to the lysosomal compartments. Thus, current evidence indicates that lipid rafts function as platforms for both the signaling and the endocytosis-targeting functions of the TCR.

Herpesvirus persists in its host through an ability to establish a latent infection and periodically reactivates to produce infectious virus. Herpesvirus saimiri (HVS), an oncogenic 2 herpesvirus, persists in the T lymphocytes of the natural host (squirrel monkey) without any apparent disease, but infection of other species of New World and Old World primates results in fulminant T-cell lymphomas (19). In addition, when HVS infects primary T lymphocytes of humans, Old World primates, New World primates, and rabbits, these cells become immortalized and cytokine independent for growth (4).

An HVS protein called Tip (for tyrosine kinase-interacting protein) is encoded by an open reading frame at the left end of the viral genome. Tip is not required for viral replication, but it is required for T-cell transformation in culture and for lymphoma induction in primates (9). Tip interacts with the Src homology 3 (SH3) domain of Lck tyrosine kinase, and this interaction interferes with early events of the TCR signal transduction pathway (7, 20). Our recent study demonstrated that, due to the sequestration of Lck by Tip, TCR stimulation fails to activate ZAP-70 tyrosine kinase and to initiate downstream signaling events. Consequently, Tip expression not only markedly inhibits TCR-mediated intracellular signal transduction, but also blocks TCR engagement with MHC II on the antigen-presenting cells and immunological synapse formation (7).

In an effort to delineate the role of Tip in the modulation of the TCR signal transduction pathway, we have identified a novel cellular endosomal protein, p80, that contains an amino-terminal WD repeat domain and a carboxyl-terminal coiled-coil domain and that efficiently interacts with Tip in living cells (29). Interaction of Tip with p80 facilitates endosomal-vesicle formation and subsequent recruitment of Lck and TCR/CD3 complexes into the lysosomes for degradation. Consequently, the interaction of Tip with Lck and p80 results in downregulation of both TCR/CD3 and CD4 surface expression. Remarkably, these actions of Tip are functionally and genetically separable: the interaction of its amino-terminal region with p80 is responsible for TCR downregulation, and the interaction of its carboxyl region with Lck is responsible for CD4 downregulation (29). We have also demonstrated that Tip is constitutively present in lipid rafts and exploits cellular proteins, Lck and p80, to downregulate TCR/CD3 complexes and CD4 (28). Furthermore, Tip and Lck interaction is required for the recruitment of the TCR/CD3 complexes to lipid rafts, and the interaction of Tip and p80 is critical for the aggregation and internalization of lipid rafts. Interestingly, in contrast to the downregulation of TCR/CD3 complexes, the downregulation of CD4 induced by Tip is dependent on the interaction with Lck but independent of Lck kinase activity and p80 interaction (28).

In this report, we describe the molecular details of lipid raft localization of Tip and evaluate the functional significance of Tip targeting to lipid rafts in the modulation of T-cell functions. We demonstrate that the carboxyl transmembrane domain of Tip is critical for lipid raft targeting and is essential for the downregulation of TCR and CD4 coreceptor induced by Tip. On the other hand, the inhibition of TCR signal transduction and the activation of STAT3 transcription factor activity induced by Tip do not rely on its lipid raft localization. These results provide evidence that T-cell functions, such as endocytosis and signal transduction, modulated by Tip are differentially affected by the interactions of Tip with lipid rafts and other cellular signaling molecules.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cell culture and reagents. Jurkat T cells and 293T cells were grown in RPMI and Dulbecco's modified Eagle's media, respectively, supplemented with 10% fetal bovine serum (FBS). Jurkat T cells were electroporated using the Bio-Rad (Hercules, CA) electroporator at 260 V and 975 µF in serum-free RPMI medium. Fugene 6 (Roche, Indianapolis, IN) or calcium phosphate (Clontech, Palo Alto, CA) was used for transient expression of Tip in 293T cells. A stable Jurkat T-cell line expressing the AU1-tagged Tip was selected and maintained by the presence of puromycin (5 µg/ml). Anti-Lck, anti-CD3, anti-phospho-CD3, anti-CD71, and anti-STAT3 antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA); anti-phosphotyrosine (4G10) antibody was purchased from Upstate (Charlottesville, VA); AU1 tag antibody was obtained from BAbCO (Berkeley, CA); cholera toxin B subunit (CTB)-biotin and streptavidin 633 were purchased from Molecular Probes (Eugene, OR); and anti-CD3 antibody for TCR stimulation was purchased from Pharmingen (San Diego, CA). Filipin III was purchased from Sigma-Aldrich (St. Louis, MO). Tip and its mutant DNA fragments were cloned into pFJ, pBabe, or pTracer vector (Invitrogen, San Diego, CA) (29). Green fluorescent protein (GFP) fusions of Tip or its mutants were made using pEGFP-C2 plasmids (Clontech, Palo Alto, CA). PCR-based mutagenesis was performed to create the mutations in the transmembrane domain of Tip, and the sequences changed in each mutant were as follows: Tip TM containing the deletion from amino acid 227-Ile to 256-Ser, TipCD45TM containing the replacement of Tip TM (227-Ile to 254-Met) with CD45 TM (415-Ala to 438-Ile), and TipCD71TM containing the replacement of Tip TM (227-Ile to 254-Met) with CD71 TM (62-Cys to 89-Cys).

Isolation of lipid rafts. Lipid rafts were isolated using the method of flotation on a discontinuous sucrose gradient (28). Briefly, Jurkat T cells (10⁸ cells) were washed with ice-cold phosphate-buffered saline (PBS) and lysed for 30 min on ice in 1% Triton X-100 in TNEV (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA) containing phosphatase inhibitors and protease inhibitor cocktail (Roche, Germany). The lysis solution was further homogenized with 10 strokes in a Wheaton loose-fitting Dounce homogenizer. Nuclei and cellular debris were pelleted by centrifugation at 900 x g for 10 min. For the discontinuous sucrose gradient, 0.5 ml of cleared cell lysates was mixed with 0.5 ml of 85% sucrose in TNEV and transferred to the bottom of a Beckman 14- by 89-mm centrifuge tube. The diluted lysates were overlaid with 4 ml of 35% sucrose in TNEV and, finally, with 1 ml 5% sucrose in TNEV. Samples were then centrifuged in an SW41 rotor at 200,000 x g for 20 h at 4°C, and 0.5-ml fractions were collected from the top of the gradient.

2D gel electrophoresis and protein identification by MALDI-TOF MS. For two-dimensional (2D) gel analysis, lipid raft-enriched samples (the second and third fractions of sucrose gradients) were resuspended in 8 M urea, 4% (wt/vol) CHAPS {3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate}, and 1% (wt/vol) dithioerythritol after the concentration step. 2D electrophoresis was performed using an Immobiline polyacrylamide system as described previously (5). Isoelectric focusing was carried out on a nonlinear wide-range immobilized pH gradient (pH 3 to 10) using the IPGphor system (Amersham Biosciences, Uppsala, Sweden). The second dimension was carried out on 4 to 12% polyacrylamide linear-gradient gels at 40-mA/gel constant current. After visualization of proteins by silver nitrate staining, each protein of 2D-separated spots was identified by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) peptide mass fingerprinting in the Taplin biological mass spectrometry (MS) facility of Harvard Medical School (Boston, MA).

Flow cytometry. Cells (5 x 10⁵) were washed with RPMI medium containing 10% fetal calf serum and incubated with fluorescein isothiocyanate-conjugated or phycoerythrin-conjugated monoclonal antibodies for 30 min at 4°C. After being washed, each sample was fixed with 4% paraformaldehyde solution, and flow cytometry analysis was performed with a FACS Scan (Becton Dickinson Co., Mountainview, CA). Antibodies for CD3 (SK7), CD4 (Leu-3a), and CD45 (HI30) were purchased from Becton Dickinson, and antibody for TCRß (BW242/412) was purchase from T Cell Diagnostics (Cambridge, MA).

Confocal immunofluorescence. Cells were fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.2% Triton X-100 for 15 min, blocked with 5% bovine serum albumin in PBS for 30 min, and reacted with 1:100 to 1:2,000 dilutions of primary antibody in PBS for 30 min at room temperature. After incubation, the cells were washed extensively with PBS, incubated with 1:2,000-diluted Alexa 488- or Alexa 568-conjugated anti-rabbit or anti-mouse antibody (Molecular Probes, Eugene, OR) in PBS for 30 min at room temperature, and washed three times with PBS. In some experiments, Topro-3 (Molecular Probe) was used to stain the nucleus. Confocal microscopy was performed using a Leica TCS SP laser-scanning microscope (Leica Microsystems, Exton, PA) fitted with a 40x Leica objective (PL APO; 1.4 numerical aperture). Images were collected at 512- by 512-pixel resolution using Leica imaging software. The stained cells were optically sectioned in the z axis, and the images in the different channels (photomultiplier tubes) were collected simultaneously. The step size in the z axis varied from 0.2 to 0.5 µm to obtain 8 to 16 slices/imaged file. The images were transferred to a Macintosh G4 computer (Apple Computer, Cupertino, CA), and NIH Image v1.61 software was used to render the images.

Calcium mobilization analysis. Cells (2 x 10⁶) were loaded with 1 µM indo-1 in 100 µl of RPMI medium containing 10% FBS at 37°C for 30 min, washed once with the medium, resuspended in 1 ml of cold RPMI medium containing 10% FBS, and then put on ice until analysis. Baseline calcium levels were established for 1 min prior to the addition of the antibody. The cells were stimulated with 2 µg of anti-human CD3 antibody. Data were collected and analyzed on a FACS Vantage (Becton Dickinson).

Immunoprecipitation and immunoblotting. For immunoprecipitation, cells were harvested and resuspended in lysis buffer (150 mM NaCl, 0.5% Nonidet P-40, and 50 mM HEPES buffer [pH 8.0]) containing protease and phosphatase inhibitors. Immunoprecipitated proteins from precleared cell lysates were used for immunoblotting. For immunoblotting, polypeptides from 2 x 10⁶ cells were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. Immunoblot detection was performed with a 1:1,000 or 1:3,000 dilution of primary antibody with the enhanced chemiluminescence system (Amersham, Chicago, IL).

Stat3 luciferase reporter assay. Jurkat T cells were electroporated with wild-type Tip or its mutant expression vectors, together with the Stat3-dependent reporter vector pLucTKS3. To normalize transfection efficiency, pGK-ßgal vector, which expresses ß-galactosidase from a phosphoglucokinase promoter, was included in the transfection mixture. At 48 h posttransfection, the cells were washed with cold PBS and lysed in lysis solution (25 mM Tris, pH 7.8, 2 mM EDTA, 2 mM dithiothreitol, 10% glycerol, and 1% Triton X-100). Luciferase activity was measured with a luminometer using a luciferase assay kit (Promega, Madison, WI) and normalized with ß-galactosidase activity.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Tip induces the dramatic increase of lipid rafts in T cells. Previously, we reported that Tip not only localizes to lipid rafts, but also induces the aggregation of lipid rafts through interaction with p80 (28, 29). To gain insight into the dynamics of lipid rafts induced by Tip, a global analysis of lipid raft-associated proteins was performed by 2D gel electrophoresis and mass spectrometry analysis. Lipid rafts were purified by sucrose gradient centrifugation from Jurkat T cells transfected with Babe, Babe-Tip wt, or Babe-Tip 2 mutant vector. The Tip 2 mutation has been shown to abolish p80 interaction (28, 29). The position and integrity of lipid rafts from sucrose gradient centrifugation were determined by the presence of the raft-associated GM1 ganglioside and Lck (data not shown). Proteins recovered from lipid raft-enriched fractions were then resolved by 2D gel electrophoresis and visualized by silver staining. The intensity of each protein spot was also determined by densitometry. The protein-staining pattern of Jurkat-Tip cells showed dramatic differences from those of Jurkat-Babe cells and Jurkat-Tip 2 cells (Fig. 1). Not only the number of detected protein spots, but also the amount of protein from each spot, was dramatically increased in Jurkat-Tip cells compared to those in Jurkat-Babe and Jurkat-Tip 2 cells (Fig. 1). Approximately 100 different spots of lipid raft proteins were reproducibly detected in Jurkat-Babe, Jurkat-Tip, and Jurkat-Tip 2 cells, whereas approximately 50 spots appeared to be unique to Jurkat-Tip cells (Fig. 1). This indicated that increases in the protein number and amount were likely correlated with the aggregation of lipid rafts induced by Tip and p80 interaction. Over 40 silver-stained lipid raft proteins from Jurkat-Tip cells were cut and subjected to mass spectrometry analysis for identification (Table 1). This showed that these proteins were primarily components of cytoskeletons, heat shock proteins, and signaling proteins (Table 1). In fact, the proteins have been reported to also be part of the lipid raft resident proteins (5, 11). These results indicate that Tip expression induces a marked increase of lipid raft residential proteins in T cells and that this activity of Tip requires p80 interaction.

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FIG. 1. Tip expression increases the quantity of lipid raft proteins in T cells. Lipid raft fractions were purified from Jurkat T cells transfected with vector alone, wt Tip, or Tip 2 vector; resolved by 2D gel electrophoresis; and visualized by silver staining. The migration of molecular weight markers (y axis) and the pI gradient (x axis) are indicated. The yellow numbers indicate the proteins that are present only in Jurkat-Tip cells, and the white numbers indicate the proteins that are present in Jurkat-vector, Jurkat-Tip, and Jurkat-Tip 2 cells. The identities of these proteins were determined by MALDI-TOF MS and are listed in Table 1.

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TABLE 1. Identification of lipid raft-associated proteins by MS

Lipid raft association of Tip requires the carboxyl-terminal transmembrane domain. The association of a protein with lipid rafts is primarily mediated by three known mechanisms: a hydrophobic membrane-spanning sequence or transmembrane domain, a hydrophobic tail with a glycosylphosphatidylinositol anchor or acylation, or protein-protein and protein-lipid interaction (23). To identify the sequence motif required for the association of Tip with lipid rafts, we tested several Tip mutants for their lipid raft localization. They were a Tip mLBD mutant containing the loss of Lck interaction, a Tip 2 mutant containing the loss of p80 interaction, and a Tip TM mutant containing the deletion of the carboxyl-terminal transmembrane domain (TM) (Fig. 2A). Jurkat T cells expressing Tip or its mutants were lysed in 1% Triton X-100 and subjected to discontinuous sucrose density gradient centrifugation. The positions and integrity of the lipid rafts in the sucrose gradient were determined by the presence of the raft-associated GM1 ganglioside and Lck. CD71 transferrin receptor was also included as a marker of the soluble fractions (1). Immunoblot assays showed that wt Tip, Tip mLBD, and Tip 2 were efficiently associated with lipid rafts in T cells, whereas Tip TM mutants were excluded from the lipid raft fractions and primarily present in the soluble fractions (Fig. 2B). To further elucidate the role of Tip TM for lipid raft localization, we constructed the chimeric Tip proteins that contained the replacement of their own TM with CD45 TM, called TipCD45TM, or with CD71 transferrin receptor TM, called TipCD71TM. Both CD45 and CD71 proteins have been shown to be primarily present in the soluble fractions (3, 18). Purification of lipid raft fractions showed that TipCD45TM and TipCD71TM chimeras did not partition into lipid rafts (Fig. 2B). These results indicate that the carboxyl-terminal TM sequence, not Lck and p80 interaction, of Tip is necessary for lipid raft localization.

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FIG. 2. Construction of Tip mutants and their association with lipid rafts. (A) Schematic representation of Tip and its mutant constructs. Each mutant construct was generated as described in Materials and Methods. E-R, glutamate-rich region; S-R, serine-rich region; CSKH, c-Src kinase homology domain; SH3B, SH3-binding domain; TM, transmembrane domain; CD71, CD71 TM; CD45, CD45 TM. "X" indicates the mutation at the CSKH and SH3B motifs that has been previously described (15). (B) Lipid raft association of wt Tip or its mutants. Jurkat T cells were transfected with plasmids encoding Tip or its mutants and applied for lipid raft purification. Polypeptides from each fraction of the sucrose gradient were subjected to immunoblotting with an anti-AU1 antibody to detect Tip or its mutants. Anti-Lck and anti-CD71 antibodies and CTB-horseradish peroxidase conjugate were also used to confirm the locations and integrity of lipid rafts.

In addition, the colocalization of Tip or its mutants with lipid rafts was investigated in Jurkat T cells using a confocal immunofluorescence microscope. Cells were electroporated with plasmids encoding Tip or its mutants. At 24 h after electroporation, the cells were fixed, permeabilized, and reacted with anti-AU1 antibody or a biotin-streptavidin 633 conjugate of CTB that detected the raft-associated GM1 ganglioside. This showed that Tip- and Tip mLBD-GFP fusions were primarily in patches at the plasma membrane, with considerable colocalization with CTB (Fig. 3A). However, Tip TM- and TipCD71TM-GFP fusions were not robustly colocalized with lipid rafts (Fig. 3A). Specifically, the Tip TM-GFP fusion was localized throughout the cytoplasm but only weakly associated with the plasma membrane (Fig. 3A). These results further support the notion that the ability of Tip to associate with lipid rafts is primarily dependent on the carboxyl-terminal transmembrane domain but independent of Lck interaction.

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FIG. 3. Localization of Tip or its mutants. The localization of Tip or its mutants with lipid rafts was analyzed under a confocal microscope. (A) Jurkat T cells were electroporated with plasmids encoding wt Tip or its mutants and stained with rabbit anti-AU1 antibody, followed by anti-rabbit Alexa 488 conjugate (green). Lipid rafts were stained with CTB-biotin, followed by streptavidin 633 conjugate (blue). (B) Jurkat T cells were transfected with plasmids encoding the GFP fusion of Tip or its mutant (green) and stained with an antibody specific to EEA1 (red), an early endosomal marker, or an antibody specific to LAMP2 (red), a lysosomal marker.

We have previously shown that Tip targeting activity directs its own complex containing Lck and p80 to the lysosomal compartments (29). To correlate the lipid raft-targeting activity of Tip with the lysosomal localization, Jurkat T cells expressing Tip-GFP or its mutant-GFP fusions were reacted with antibodies specific to EEA1, an early endosomal marker, or LAMP2, a late endosomal/lysosomal marker, and then examined under a confocal microscope (Fig. 3B). Tip-GFP was weakly colocalized with EEA1 but extensively colocalized with LAMP2 (Fig. 3B). Tip mLBD-GFP displayed a localization pattern similar to that of Tip-GFP, even though its localization in the intracellular compartments was detectably lower than that of Tip-GFP (Fig. 3B). In contrast, Tip TM- and TipCD71TM-GFP fusions were detectably colocalized with EEA1 but hardly with LAMP2 (Fig. 3B). These results suggest that the association with lipid rafts is required for the efficient targeting of Tip to the lysosomal compartments.

Association of Tip with lipid rafts is required for the efficient downregulation of CD3 and CD4. We next examined whether the lipid raft-targeting activity of Tip played a role in the downregulation of TCR/CD3 and CD4 surface expression. Jurkat T cells stably expressing wt Tip or the Tip TM or TipCD71TM mutant were compared for their levels of CD3, CD4, and CD45 surface expression by flow cytometry. As shown previously (29), wt Tip expression effectively downregulated the surface expression of CD3 and CD4 (Fig. 4). By striking contrast, the downregulation of CD3 and CD4 surface expression was severely impaired on Jurkat T cells expressing Tip TM or TipCD71TM (Fig. 4). In addition, wt Tip and its mutants did not affect the surface expression of CD45, showing the specificity of CD3 and CD4 downregulation induced by Tip (Fig. 4). These results indicate that the association of Tip with lipid rafts is required for the efficient downregulation of CD3 and CD4 surface expression.

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FIG. 4. Downregulation of CD3 and CD4 by Tip or its mutants. Jurkat T cells were transfected with pBabe, pBabe/Tip, pBabe/Tip TM, or pBabe/TipCD71TM as indicated and selected with puromycin (5 µg/ml). Jurkat T cells stably expressing Tip or its mutants were analyzed by flow cytometry to detect the level of surface expression of CD3, CD4, and CD45.

To further delineate the functional significance of Tip association with lipid rafts in the downregulation of TCR and CD4 surface expression, we used Filipin III, a sterol-binding agent, which disrupts the structure of lipid rafts (36). Jurkat T cells were treated with Filipin III (5 µg/ml) for up to 6 h and analyzed for their surface expression of CD3 and CD4 by flow cytometry. The surface expression of CD3 and CD4 on Jurkat-Tip cells was gradually recovered upon Filipin III treatment and reached up to 60% and 90% of the CD3 and CD4 expression of Jurkat-vector cells after 6 h of treatment with Filipin III (Fig. 5). In addition, the slight reduction of CD45 surface expression was reproducibly detected on Jurkat-Tip cells (Fig. 5). By contrast, no detectable change in CD3, CD4, and CD45 surface expression was detected on Jurkat-Babe cells under the same conditions (Fig. 5). These results indicate that the disruption of lipid raft structure induced by Filipin III readily blocks the downregulation of CD3 and CD4 surface expression induced by Tip.

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FIG. 5. Recovery of CD3 and CD4 surface expression on Jurkat-Tip cells by Filipin III treatment. Jurkat-vector and Jurkat-Tip cells were treated with Filipin III (5 µg/ml) for the indicated times, and the surface expression of CD3, CD4, and CD45 was analyzed by flow cytometry. The mean fluorescent intensity (MFI) of each plot is shown in each box: the black numbers indicate MFI on Jurkat-Tip cells, and the red numbers indicate MFI on Jurkat-vector cells. Dashed line, isotype control; red line, Jurkat-vector cells; filled line, Jurkat-Tip cells.

Inhibition of TCR signal transduction and activation of STAT3 by Tip are independent of the association with lipid rafts. We have previously demonstrated that Tip expression efficiently blocks the activation of TCR signal transduction upon stimulation and that this activity of Tip requires Lck interaction (7). We first tested whether the loss of lipid raft association of Tip affected its interaction with Lck. 293T cells were cotransfected with Lck expression vector, together with wt Tip or the Tip mLBD or Tip CD71TM mutant, followed by a coimmunoprecipitation assay. This showed that TipCD71TM interacted with Lck as efficiently as wt Tip, whereas Tip mLBD did not, indicating that the ability of Tip to associate with lipid rafts is independent of its ability to interact with Lck (Fig. 6A). To examine whether the association of Tip with lipid rafts contributed to the inhibition of TCR signal transduction, Jurkat T cells were electroporated with pTracer plasmids encoding wt Tip or its mutants and then measured for their levels of intracellular free-calcium mobilization. At 24 h postelectroporation, these cells were gated for GFP expression and then stimulated with an anti-CD3 antibody to measure intracellular calcium mobilization. Jurkat T cells expressing GFP or the Tip mLBD mutant exhibited a rapid increase in intracellular calcium concentration immediately after anti-CD3 antibody stimulation, whereas Jurkat T cells expressing wt Tip were not capable of increasing the intracellular calcium concentration under the same conditions (Fig. 6A). Surprisingly, a Tip CD71TM mutant that was no longer present in lipid rafts was still capable of blocking the modulation of the intracellular calcium concentration induced by anti-CD3 antibody as efficiently as wt Tip (Fig. 6A). These results indicate that the inhibitory effect of Tip on TCR signal transduction is dependent on the interaction with Lck, but not on the association with lipid rafts.

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FIG. 6. Association of Tip with lipid rafts is not required for inhibition of TCR signal transduction. (A) Interaction of Tip or its mutants with Lck. Lck and Tip or its mutants were expressed in 293T cells, and Lck interaction was analyzed after immunoprecipitation (IP) using an anti-Lck antibody, followed by immunoblotting (IB) with anti-Lck and anti-Tip antibodies. Whole-cell lysates (WCL) were included as controls. Lanes 1, Tip; lanes 2, Lck plus Tip; lanes 3, Lck plus Tip mLBD; lanes 4, Lck plus TipCD71TM. (B) Intracellular calcium mobilization upon TCR stimulation. At 24 h after electroporation with pTracer, pTracer/Tip, pTracer/Tip mLBD, or pTracer/TipCD71TM mutant, GFP-positive Jurkat T cells were gated and stimulated with anti-CD3 antibody, followed by flow cytometry analysis to measure intracellular calcium mobilization. Calcium mobilization was monitored over time by changes in the ratio of violet to blue (405 nm to 485 nM) fluorescence of cells loaded with indo-1 and analyzed by flow cytometry. The data are presented as histograms of the number of cells with a particular fluorescence ratio (y axis) versus time (x axis). Ionomycin was added as a control for intracellular calcium mobilization. The breaks in the graphs represent the time intervals during which the anti-CD3 antibody was added. The data were similar in three independent experiments.

The previous report also showed that the phosphorylated tyrosine residue 114 of Tip induced by Lck binds to the SH2 domain of STAT3, which recruits STAT3 into the vicinity of Lck associated with Tip. Subsequently, Lck phosphorylates STAT3, resulting in robust activation of STAT3 transcription factor activity (25). We also tested whether the association of Tip with lipid rafts affected the activation of STAT3 factor activity. Jurkat T cells were electroporated with wt Tip, Tip Y₁₁₄F, Tip mLBD, or TipCD71TM, together with the STAT3-dependent reporter vector pLucTKS3. The Tip Y₁₁₄F mutant that was incapable of binding to STAT3 and the Tip mLBD mutant that was incapable of binding to Lck were included as controls in this assay. At 48 h posttransfection, cells were harvested to measure STAT3 transcription factor activity. Wt Tip strongly induced STAT3 transcriptional activity, whereas both Tip Y₁₁₄F and Tip mLBD mutants showed no activation of STAT3 activity (Fig. 7A). Interestingly, the Tip CD71TM mutant induced STAT3 activation as proficiently as wt Tip (Fig. 7A). Upon stimulation with cytokine, an activated STAT3 forms a homodimer through reciprocal interaction between its own phosphorylated tyrosines and SH2 domains. The dimer translocates into the nucleus, where it recognizes specific DNA elements and activates transcription. We used confocal immunofluorescence microscopy to examine the effect of Tip on STAT3 localization. Jurkat T cells were transfected with Tip or its mutant, together with STAT3 expression vector. STAT3 was primarily present in the cytoplasm, whereas it efficiently translocated into the nucleus upon coexpression of wt Tip or TipCD71TM (Fig. 7B). In contrast, coexpression of the Tip Y₁₁₄F or Tip mLBD mutant did not induce the nuclear localization of STAT3 under the same conditions (Fig. 7B). These results indicate that Tip efficiently induces the nuclear localization of STAT3, but its lipid raft association is not required for this activity.

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FIG. 7. Activation and nuclear translocation of STAT3 by Tip or its mutants. (A) Luciferase assay. Jurkat T cells were electroporated with wild-type Tip or its mutant expression vectors, together with the STAT3-dependent reporter vector pLucTKS3. To normalize transfection efficiency, pGK-ßgal vector was included in the transfection mixture. At 48 h posttransfection, luciferase activity was determined and normalized with ß-galactosidase activity. (B) Nuclear translocation of STAT3. Jurkat T cells were electroporated with pVR/STAT3 vector, together with the pFJ-Tip or pFJ/Tip mutant. At 24 h after electroporation, cells were fixed and stained with anti-AU1 antibody to detect Tip (green) and anti-STAT3 antibody to detect STAT3 (red), respectively. Topro-3 staining was used to visualize the nucleus.

Top Abstract Introduction Materials and Methods Results Discussion References

 
In this study, we demonstrated that Tip efficiently associated with lipid rafts and that this association induced a marked increase of lipid raft components in human T cells. The association of Tip with lipid rafts was dependent on the carboxyl-terminal transmembrane domain. Biochemical and genetic analyses indicate that the association of Tip with lipid rafts is required for the efficient targeting to the lysosomal compartments and the downregulation of CD3 and CD4 induced by Tip. However, the inhibition of TCR signal transduction and the activation of STAT3 induced by Tip are not regulated by the raft-targeting property of Tip. This suggests that the targeting and signaling activities of Tip are functionally and genetically separable.

A growing body of evidence shows that detergent-resistant rafts comprise anatomically distinct subsets of the plasma membrane and that these subsets are enriched for a variety of important signaling proteins and trafficking machineries (2, 13, 30, 33). Recent studies have also demonstrated that lipid rafts are not static entities but instead are dynamic microdomains on the cell surface for which proteins and lipids have various affinities (14, 23, 35). Upon TCR engagement with antigenic peptides associated with the MHC molecule, lipid raft domains undergo redistribution to induce patched aggregation at the plasma membrane, and TCR-associated signaling molecules and coreceptors then acquire increased affinities for lipid raft domains (13, 31, 34). Thus, the integrity of these domains has been shown to be necessary for efficient signal transduction by the TCR. We have previously demonstrated that Tip expression induces a massive aggregation of lipid raft domains through interactions with Lck and p80 and redistribution of TCR/CD3 complexes into lipid raft domains (28, 29). Here, we further demonstrated that Tip expression dramatically increased the quantity of lipid raft microdomains by enhancing the recruitment of lipid raft-resident components. This phenotypic resemblance between Tip expression and TCR signal transduction suggests that HVS Tip may pirate cellular signaling molecules to emulate TCR stimulation, which powerfully activates virus-infected T cells and provides the virus an opportunity for efficient replication, dissemination, and oncogenesis.

Association of a protein with lipid rafts is mediated by several distinct mechanisms (23). Membrane-spanning domains of many cellular and viral proteins have been implicated in targeting to lipid rafts (32, 37). The length and specific amino acid sequence of the transmembrane domain may contribute to the level and strength of lipid raft association (26, 32). In addition to the transmembrane domain, protein acylation has been also shown to play a role in the efficient association with lipid rafts (27, 37). We found that the carboxyl-terminal transmembrane domain, not Lck and p80 interaction, of Tip was required for its association with lipid rafts. While TipCD45TM and TipCD71TM chimeras were efficiently integrated into the plasma membrane, they were mainly localized in the soluble fractions of the plasma membrane without detectable colocalization with GM1 ganglioside, a marker for lipid rafts. We also found that while the cysteine residue 232 in the transmembrane region of Tip was readily palmitoylated in living cells, this palmitoylation was not required for lipid raft association (data not shown). These results indicate that the carboxyl-terminal transmembrane domain of Tip principally governs its association with lipid rafts. In addition, the specific amino acid sequences, rather than modification, of the Tip transmembrane domain may confer an intrinsic property for lipid raft targeting. It is also notable that lipid raft localization of Tip2 is less efficient in 293T epithelial cells than in Jurkat T cells (Fig. 2) (28), indicating that T cells may provide a better environmental milieu for Tip lipid raft localization.

We have previously demonstrated that the downregulation of TCR/CD3 complexes and CD4 induced by Tip occurs through different mechanisms (28, 29). Downregulation of TCR/CD3 complexes by Tip is dependent on the interaction with and kinase activity of Lck, as well as the interaction with p80, whereas downregulation of the CD4 molecule by Tip is dependent on Lck interaction only. Here, we showed that the association of Tip with lipid rafts was required for both TCR/CD3 and CD4 downregulation. Both disruption of lipid raft structures by Filipin III and forced targeting of Tip to detergent-soluble fractions severely impaired the downregulation of TCR/CD3 and CD4 induced by Tip. This indicates that the targeting activity of Tip to lipid rafts may be a prerequisite for the downregulation of TCR/CD3 complexes and CD4. Docking of Tip into lipid raft microdomains may be required, not only for the subsequent aggregation of lipid rafts to enhance the recruitment of TCR/CD3 complexes to lipid rafts, but also for stable complex formation with CD4, which is constitutively associated with lipid rafts (1, 12). It is intriguing that the left half of Jurkat-Tip cells was more responsible for the recovery of CD3 surface expression upon Filipin III treatment than the right half of Jurkat-Tip cells (Fig. 5). This differential effect of Filipin III treatment may be due to the level of Tip expression and/or action in two different populations of Jurkat T cells. Additional study is necessary to further define this differential effect of Filipin III on Tip-expressing T cells for CD3 surface expression.

In addition to the downregulation of TCR/CD3 complexes and CD4, Tip exhibits multiple biological functions, including the inhibition of TCR signal transduction and the activation of STAT3 transcription factor (7, 16, 20, 24). We further investigated whether the association of Tip with lipid rafts was required for the inhibition of TCR signal transduction and the activation of STAT3 transcription factor in T cells. It has been shown that inhibition of TCR signal transduction and activation of STAT3 transcription factor by Tip are mainly dependent on Lck interaction (7, 24). The Tip mutant TipCD71TM, which was forced to be located in the non-lipid raft regions of the plasma membrane, was still capable of not only interacting with Lck but also inhibiting TCR signaling as efficiently as wt Tip. In fact, the transmembrane deletion mutant of Tip has also been shown to inhibit TCR signal transduction (7). These results indicate that the inhibition of TCR signaling induced by Tip is dependent exclusively on Lck interaction. On the other hand, Tip becomes phosphorylated by Lck at two tyrosine residues (Y₁₁₄ and Y₁₂₇) (16). The phosphorylated Y₁₁₄ residue lies within a consensus YXPQ binding motif of the SH2 domains of STAT1 and -3 (16). Indeed, the phosphorylated Tip at the Y₁₁₄ residue interacts with and activates the STAT3 transcription factor (16). We also showed that the activation and nuclear translocation of STAT3 by Tip was independent of lipid raft targeting of Tip but dependent on Lck interaction and Y₁₁₄ phosphorylation. While the constitutive activation of STAT3 by Tip is not required for HVS-mediated T-cell transformation (17), it may contribute to the modulation of T-cell functions in virus-infected natural hosts. Nevertheless, these results indicate that the association of Tip with lipid rafts is essential for the downregulation of TCR and CD4 surface expression, but not critical for the inhibition of TCR signal transduction and the activation of STAT3 transcription factor.

Epstein-Barr virus LMP2A, a homologue of HVS Tip, has also been shown to be constitutively present in lipid rafts of virus-immortalized B-cell lines (10). This viral protein interacts with B-cell signaling proteins, such as Lyn and Syk, through its amino-terminal cytoplasmic tail. LMP2A functions in lipid rafts to block translocation of the B-cell receptor into lipid rafts, which leads to inhibition of the subsequent signaling and accelerated internalization of the BCR-cell receptor upon stimulation (10). Another functional homologue of Tip is the Kaposi's sarcoma-associated herpesvirus K15, which is also targeted to lipid rafts (6). Thus, the study of HVS Tip may provide insight into the conserved mechanisms employed by other gammaherpesvirus signal modulators to regulate lymphocyte functions and may have significant implications for the understanding of viral persistence and pathogenesis.

 
This work was supported by grants from the Korean Health 21 R&D Project Ministry of Health and Welfare (grant 01-PJ10-PG6-01GM01-0004) and from the SNUH Research Fund (09-04-002) (N. H. Cho) and U.S. Public Health Service grants CA109697, CA31363, CA106156, and RR00168 (J. Jung). J. Jung is a Leukemia and Lymphoma Society Scholar.

 
* Corresponding author. Mailing address for Nam-Hyuk Cho: Department of Microbiology and Immunology, Seoul National University College of Medicine, Seoul 110-799, South Korea. Phone: 82-2-740-8392. Fax: 82-2-743-0881. E-mail: chonh{at}snu.ac.kr. Mailing address for Jae Jung: Department of Microbiology and Molecular Genetics, Harvard Medical School, Southborough, MA 01772-9102. Phone: (508) 624-8083. Fax: (508) 786-1416. E-mail: jae_jung{at}hms.harvard.edu.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Journal of Virology, January 2006, p. 108-118, Vol. 80, No. 1
0022-538X/06/$08.00+0     doi:10.1128/JVI.80.1.108-118.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

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