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The Journal of Immunology, 2000, 165: 2927-2931.
Copyright © 00 by The American Association of Immunologists

CUTTING EDGE

Cutting Edge: T Cell-Specific Adapter Protein Inhibits T Cell Activation by Modulating Lck Activity

Vibeke Sundvold*, Knut Martin Torgersen{dagger}, Nicholas H. Post§, Francesc Marti§, Philip D. King§,¶, John Arne Røttingen{ddagger}, Anne Spurkland* and Tor Lea1,*

* Institute of Immunology, The National Hospital, Oslo, Norway; Departments of {dagger} Anatomy and {ddagger} Physiology, University of Oslo, Oslo, Norway; § Immunology and Inflammation, Hospital for Special Surgery, Weill Medical College of Cornell University, and Weill Graduate School of Medical Sciences of Cornell University, New York, NY 10021


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
We previously reported the isolation of a cDNA encoding a T cell-specific adapter protein (TSAd). Its amino acid sequence contains an SH2 domain, tyrosines in protein binding motifs, and proline-rich regions. In this report we show that expression of TSAd is induced in normal peripheral blood T cells stimulated with anti-CD3 mAbs or anti-CD3 plus anti-CD28 mAbs. Overexpression of TSAd in Jurkat T cells interfered with TCR-mediated signaling by down-modulating anti-CD3/PMA-induced IL-2 promoter activity and anti-CD3 induced Ca2+ mobilization. The TCR-induced tyrosine phosphorylation of phospholipase C-{gamma}1, SH2-domain-containing leukocyte-specific phosphoprotein of 76kDa, and linker for activation of T cells was also reduced. Furthermore, TSAd inhibited Zap-70 recruitment to the CD3{zeta}-chains in a dose-dependent manner. Consistent with this, Lck kinase activity was reduced 3- to 4-fold in COS-7 cells transfected with both TSAd and Lck, indicating a regulatory effect of TSAd on Lck. In conclusion, our data strongly suggest an inhibitory role for TSAd in proximal T cell activation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
Engagement of the TCR/CD3 complex initiates intracellular signaling processes leading to transcription of genes regulating T cell proliferation and differentiation (1, 2). An early event is the phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs)2 within the TCR/CD3 complex. Phosphorylation of ITAMs by the Src family protein tyrosine kinases (PTKs) Lck and Fyn recruits and activates the Syk family PTK, Zap-70 (3). Zap-70 mediates phosphorylation of the linker for activation of T cells (LAT), which subsequently recruits a variety of signaling molecules to the plasma membrane, including phospholipase C-{gamma}1 (PLC-{gamma}1), Grb2-Sos, Grap, p85, c-Cbl, Vav, and SH2-domain-containing leukocyte-specific phosphoprotein of 76 kDa (SLP-76) (4, 5, 6, 7, 8, 9). This results in activation of PLC-{gamma}1 and subsequent Ca2+ mobilization as well as activation of the Ras signaling pathway, leading to cytokine production and T cell proliferation (10).

Adapter proteins are intracellular molecules with no enzymatic activity or DNA binding motifs, but with domains or motifs mediating protein-protein interactions (11). Thus, they are well suited to couple proximal activation events initiated by receptor ligation with more distal signaling processes. In T cells, adapter proteins may promote signaling through the TCR/CD3 complex by recruiting catalytically active signaling molecules to their substrates. The adapter protein LAT, for instance, assembles signaling proteins at the plasma membrane following TCR triggering (7). Its functional importance is evident from cell lines deficient in LAT, which fail to activate PLC-{gamma}1 and Ras (10, 12), and LAT-deficient mice, which do not develop mature T cells (13). However, adapter proteins may also have a negative influence on TCR signaling. Cbl is an adapter protein involved in down-regulation of the TCR/CD3 complex (14) and degradation of Fyn (15), in addition to having a negative regulatory effect on Syk and Zap-70 (16, 17, 18). By sequestering Grb2, Cbl may also inhibit activation of Ras by preventing recruitment of Sos to the plasma membrane (19).

We previously reported the cloning of a cDNA encoding a novel SH2-containing, T cell-specific adapter protein, TSAd3 (20). Here we demonstrate an inhibitory function of TSAd on both distal and proximal anti-CD3-mediated signaling. TSAd expression is rapidly induced after TCR triggering. Overexpression of TSAd in Jurkat T cells inhibits T cell activation, probably by directly regulating Lck activity. Together our data support a model by which TSAd is induced after TCR ligation to down-modulate proximal PTK activity.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 References
 
Plasmids

TSAd cDNA was cloned into the EcoRI site of the mammalian expression vector pEF/hemagglutinin (pEF/HA). The IL-2 luciferase reporter plasmid was a gift from Dr. Tomas Mustelin (The Burnham Institute, La Jolla, CA). Lck cDNA was cloned into the HindIII/EcoRI sites of pMH-Neo (21). The TSAd cDNA used in the Lck kinase studies was subcloned into the BamHI/XbaI sites of pEF-FLAG (22).

Antibodies

The mAbs used were anti-CD3{zeta} and -Lck (Santa Cruz Biotechnology, Santa Cruz, CA), anti-Zap-70 (Transduction Laboratories, Lexington, KY), anti-HA (Babco, Richmond, CA), anti-human CD3{epsilon} (OKT3, American Type Culture Collection, Manassas, VA), anti-phosphotyrosine (4G10, Upstate Biotechnology, Lake Placid, NY), and anti-CD28 (PharMingen, San Diego, CA). The polyclonal Abs used were anti-PLC-{gamma}1 and -SLP-76 (Santa Cruz Biotechnology), and anti-LAT and -SLP-76 (Upstate Biotechnology). Antiserum raised against a synthetic peptide of 370–389 aa of TSAd was made as previously described (20). Sheep anti-mouse Ig-coated Dynabeads were obtained from Dynal (Oslo, Norway).

Cell cultures and transfections

Jurkat T cells (clone E6.1), COS-7 cells (both from American Type Culture Collection), and Jurkat TAg cells (a gift from Dr. Tomas Mustelin, The Burnham Institute) were used. PBMC were obtained from healthy blood donors by standard gradient centrifugation and depleted of non-T cells using anti-CD14- and anti-CD19-coated Dynabeads (Dynal). Cells were cultured in RPMI 1640/10% FCS (c-RPMI). Transfections of 20 x 106 Jurkat or Jurkat TAg cells in RPMI with 5–40 µg of DNA were performed using a Gene Pulser (Bio-Rad, Richmond, CA) at 250 V and 960 µF. Transient transfectants were cultured in c-RPMI for 16–48 h. Stable transfectants were selected and cloned by limiting dilution in c-RPMI with 7.5 mg/ml G418 (Duchefa, Haarlem, The Netherlands). Stable clones expressing TSAd (clones 1B2, 2D6, 3A3, 4B1, and 5B5) or empty vector (clone B2) were established, and similar CD3 expression verified by flow cytometry. In Lck assays, 5 x 106 COS-7 cells were transiently transfected as described above with 10 µg of each construct.

Cell stimulation, lysis, immunoprecipitation, Western blot, and luciferase assay

T cells were stimulated with 5 µg/ml OKT3 and 1 µg/ml anti-CD28 mAbs and were cross-linked with goat anti-mouse Ig-coated Dynabeads. In immunoprecipitation (IP) experiments, Jurkat TAg cells (10–20 x 106 cells/IP) were washed in RPMI 1640, resuspended to 5 x 107 cells/ml, and stimulated with 5 µg/ml OKT3. Cells were lysed in 2x lysis buffer (2% Nonidet P-40, 50 mM Tris (pH 7.5), 200 mM NaCl, 40 mM NaF, 2 mM Na3VO4, 20 µg/ml leupeptin, and antipain). Lysates were precleared for 1–2 h with protein A/G-agarose (Santa Cruz Biotechnology) and incubated with the relevant Abs overnight followed by protein A/G agarose for 2 h. IPs were separated by SDS-PAGE and blotted onto polyvinylidene difluoride membranes (Immobilon-P, Millipore, Bedford, MA). Blots were probed with the indicated Abs in Tris-buffered saline/Tween 20/5% skim milk (or 2.5% BSA/2.5% skim milk for phosphotyrosine blots). Signals were detected by HRP-labeled secondary Abs (Jackson ImmunoResearch Laboratories, West Grove, PA) and Super Signal (Pierce, Rockford, IL). Activation of the IL-2 promoter requires combined stimulation with anti-CD3 and PMA (23, 24) or PMA and ionomycin. Thus, in the luciferase assay, 5 x 105 Jurkat T cells were stimulated with 5 µg/ml OKT3/25 ng/ml PMA or with PMA/5 µM ionomycin for 5–6 h and assayed for luciferase activity according to the manufacturer’s instructions (Luciferase Assay System kit; Promega, Madison, WI).

Calcium mobilization assay

Cytosolic Ca2+ concentrations in single cells were measured as previously described (25). Jurkat TAg cells (3–4 x 105) were incubated with 5 µM fura-2 (Teflabs, Austin, TX) for 1 h, washed twice with RPMI 1640, seeded in a 9-mm well for 10 min, and stimulated with 1 µg/ml OKT3 at 37°C. Fluorescence data were treated as previously reported (25, 26). The Ca2+ concentration was calculated as previously described (27).

Lck kinase assay

Lck activity was assayed as previously described (28). Briefly, three quarters of Lck-IP beads were incubated at 30°C for 15 min with 20 µCi of [{gamma}-32P]ATP (DuPont-NEN, Boston, MA) and 10 µg Src peptide (Sigma, St. Louis, MO) in 150 µl of 25 mM HEPES (pH 7.5), 7.5 mM MgCl2, 1.5 mM MnCl2, and 1 mM Na3VO4. Beads were then pelleted and 25 µl of supernatant was spotted in triplicate onto p81 phosphocellulose paper disks and washed in 1% phosphoric acid, and associated radioactivity was determined by scintillation counting. Results are expressed as mean counts per minute ± 1 SE. Lck autokinase activity was assessed by SDS-PAGE and autoradiography of Lck-IP. The remaining one-quarter of Lck-IP beads was used to ascertain equivalent precipitation of Lck by SDS-PAGE and immunoblotting as described above.


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
TSAd is induced by TCR triggering and inhibits IL-2 promoter activity and intracellular Ca2+ mobilization

Expression of TSAd mRNA is induced upon triggering of the CD3, CD4, or CD8 molecules (20). To study the kinetics of TSAd expression, normal peripheral blood T cells were purified and stimulated with Abs to CD3 and CD28. Anti-CD3 mAbs induced TSAd expression after 4 h, with maximal expression after 30 h. Anti-CD3 plus anti-CD28 stimulation resulted in a stronger and more sustained TSAd expression, reaching maximum levels after 48 h (Fig. 1GoA). This expression pattern indicates that the SH2D2A gene is regulated by signals through the TCR/CD3 complex and that TSAd could be involved in regulating TCR-mediated signaling.



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  		FIGURE 1. TSAd is induced by TCR triggering and inhibits activation of T cells. A, Normal peripheral blood T cells (1 x 107) were stimulated with either anti-CD3 mAbs or anti-CD3- plus anti-CD28 mAbs, cross-linked with Dynabeads for the indicated time, and lysed. Western blot of cell lysates was probed with anti-TSAd Abs (upper panel), or anti-Zap-70 mAbs (lower panel) as a control. B, Jurkat TAg cells stably transfected with either empty vector (clone B2) or TSAd (clones 3A3, 4B1, and 5B5) were transiently transfected with an IL-2 luciferase reporter plasmid. Cells were left untreated ({square}) or were stimulated with anti-CD3/PMA () or PMA/ionomycin ({blacksquare}). One representative of three experiments is shown. Luciferase activity is given as arbitrary units (AU). C, Jurkat TAg cells transiently transfected with empty vector or TSAd were loaded with the indicator dye fura-2 and stimulated with anti-CD3 mAbs. The average Ca2+ responses in 36 (empty vector) and 72 (TSAd) cells are shown. The arrow indicates the addition of anti-CD3 mAbs.

 
To investigate the effects of TSAd on TCR-induced signaling events, cDNA encoding TSAd was subcloned into the expression vector pEF/HA. This construct encodes TSAd with an N-terminal HA tag. Jurkat TAg cells stably transfected either with empty vector or TSAd were subsequently transfected with the IL-2 luciferase reporter plasmid. TSAd-transfected cells showed a profound inhibition of anti-CD3/PMA-induced IL-2 promoter activity compared with empty vector transfectants, whereas PMA/ionomycin-induced IL-2 promoter activity was similar in TSAd and empty vector transfectants (Fig. 1GoB). To investigate possible effects on anti-CD3-induced Ca2+ mobilization, Jurkat TAg cells were transiently transfected with TSAd or empty vector and loaded with the indicator dye fura-2. The TCR-induced Ca2+ mobilization in TSAd transfectants was delayed and reduced compared with that in empty vector transfectants (Fig. 1GoC). Together with the inhibition of IL-2 promoter activity, these data indicate an inhibitory role of TSAd in T cells.

TSAd results in impaired TCR-induced tyrosine phosphorylation of PLC-{gamma}1, SLP-76, and LAT

The adapter proteins LAT and SLP-76 are necessary for T cell development and activation of the PLC-{gamma}1 and Ras pathways (10, 13, 29, 30). Importantly, recruitment of SLP-76 to phosphorylated LAT seems essential for signaling downstream of PLC-{gamma}1 (29, 31). The observation that TSAd has no effect on PMA/ionomycin-induced IL-2 promoter activity indicates that TSAd inhibits T cell activation upstream of Ca2+ mobilization. Indeed, Jurkat TAg cells transfected with TSAd demonstrated abolished tyrosine phosphorylation of PLC-{gamma}1 after CD3 triggering (Fig. 2GoA), indicating that reduced Ca2+ mobilization in TSAd transfectants is due to improper activation of PLC-{gamma}1. Moreover, TSAd expression inhibited anti-CD3-induced tyrosine phosphorylation of both SLP-76 and LAT (Fig. 2Go, B and C), indicating that TSAd prevents the generation of a multimeric protein complex necessary for activation of PLC-{gamma}1 and Ras. Together, these data point to a negative function of TSAd on T cell signaling by modulating protein-protein interactions close to TCR.



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  		FIGURE 2. TSAd interferes with tyrosine phosphorylation of PLC-{gamma}1, SLP-76, and LAT. Jurkat TAg cells stably transfected with empty vector (clone B2) or TSAd (clone 3A3) were kept unstimulated or were stimulated with anti-CD3 mAbs for 3 min, lysed, and subjected to immunoprecipitation with anti-PLC-{gamma}1 (A) or anti-SLP-76 (B) Abs, respectively. The immunoprecipitates were resolved by SDS-PAGE and immunoblotted with anti-phosphotyrosine mAbs (upper panels) or specific Abs (lower panels). C, Jurkat TAg cells transiently transfected with empty vector or TSAd were treated as described above, but anti-LAT Abs were used for immunoprecipitation.

 
Alteration of Zap-70 and {zeta}-chain association in TSAd transfectants

The reduced LAT phosphorylation in TSAd transfectants indicates that TSAd influences either the ZAP-70/Syk protein kinases or the Src kinases, Lck or Fyn. Zap-70 is recruited to phosphorylated ITAMs in the TCR/CD3 complex and is activated by phosphorylation on Tyr493 by Lck (3). The binding of Zap-70 to the CD3{zeta}-chain is believed to be essential for T cell activation, as peptides blocking the association of Zap-70 with the CD3{zeta}-chain inhibit TCR-mediated signaling (32). To address the ability of TSAd to regulate this proximal event, we precipitated Zap-70 from anti-CD3-stimulated Jurkat TAg cells stably transfected with either empty vector or TSAd. Phosphorylation of Zap-70 after anti-CD3 stimulation was reduced in TSAd transfectants compared with empty vector transfectants (Fig. 3GoA, upper panel). Furthermore, coprecipitation of CD3{zeta}-chains with Zap-70 was dramatically reduced (Fig. 3GoA, lower panel). This finding could be due to reduced tyrosine phosphorylation of the CD3{zeta}-chains, leading to reduced recruitment and activation of Zap-70. Indeed, the tyrosine phosphorylation of CD3{zeta}-chains precipitated from the same lysates was reduced in TSAd transfectants (Fig. 3GoB). Furthermore, Jurkat TAg cells transiently transfected with increasing amounts of TSAd cDNA displayed reduced levels of tyrosine-phosphorylated CD3{zeta}-chains coprecipitating with Zap-70 in a dose-dependent manner (Fig. 3GoC). These results suggest an ability of TSAd, either directly or indirectly, to regulate Lck activity.



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  		FIGURE 3. TSAd inhibits recruitment of Zap-70 to the CD3{zeta}-chains after TCR-triggering. A, Zap-70 was immunoprecipitated from lysates of Jurkat TAg cells stably transfected with empty vector (clone B2) or TSAd (clone 3A3). Western blots were probed with anti-phosphotyrosine mAbs (upper panel), anti-Zap-70 mAbs (middle panel), or anti-CD3{zeta} mAbs (lower panel). B, The CD3{zeta}-chains were immunoprecipitated from the same lysates as those in A and probed with anti-phosphotyrosine mAbs. C, Jurkat TAg cells transiently transfected with increasing amounts of TSAd cDNA. TSAd expression was verified by immunoblotting of lysates with anti-HA mAbs (top panel). Zap-70 immunoprecipitated from anti-CD3-stimulated (2 min) lysates was immunoblotted with anti-Zap-70 mAbs (middle panel). Bottom panel, Coprecipitated tyrosine-phosphorylated CD3{zeta}-chains.

 
TSAd down-modulates Lck kinase activity

To test the hypothesis that TSAd influences the kinase activity of Lck, COS-7 cells were transfected with Lck together with empty vector or TSAd. The catalytic activity of Lck immunoprecipitated from the transfectants was measured by its capacity to autophosphorylate and to transphosphorylate a Src peptide. Phosphorylation of the Src peptide was reduced 3- to 4-fold when TSAd was coexpressed with Lck (Fig. 4GoA). Autophosphorylation of Y394 in the activation loop of Lck is necessary for its kinase activity, probably by inducing steric changes that allow the catalytic region to fold into an active structure (33, 34, 35). Consistent with reduced Lck activity in COS-7 cells coexpressing Lck and TSAd, the autophosphorylation capacity of Lck was also clearly reduced in these transfectants (Fig. 4GoB, upper panel). Taken together, our results strongly suggest an inhibitory effect of TSAd on Lck kinase activity. The mechanism by which this occurs could be analogous to Cbl’s effect on Zap-70 and Syk (16, 17, 18). The induction of TSAd expression in normal peripheral blood T cells argues that TSAd is not necessary during early activation of naive T cells, but, rather, has a function later in the activation process. Interestingly, we have recently shown that the SH2D2A promoter region is polymorphic, and two alleles were found to be increased in frequency among multiple sclerosis patients.4 These alleles displayed a lower transcriptional activity of the SH2D2A gene, probably resulting in reduced TSAd expression. Thus, it is possible that TSAd contributes to a genetic variability in tuning T cell responses, rendering some individuals more susceptible to the development of multiple sclerosis or other autoimmune diseases.



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  		FIGURE 4. TSAd down-modulates Lck activity. COS-7 cells were transiently transfected either with Lck and empty vector or with Lck and TSAd. Lck was immunoprecipitated from lysates and analyzed for its ability to transphosphorylate a Src-peptide (A) or for its autophosphorylation capacity (B, upper panel). Equivalent immunoprecipitation of Lck was confirmed by Western blotting (B, lower panel).

 
In conclusion, we have shown that the adapter protein TSAd inhibits T cell activation by down-modulating Lck activity. The underlying mechanism is currently under study in our laboratory.


   Acknowledgments
 
We acknowledge the technical assistance of Ellen Solum Karlstrøm, Kristin Larsen Sand, and Ingebjørg Knutsen.


   Footnotes
 
1 Address correspondence and reprint requests to Dr. Tor Lea, Institute of Immunology, The National Hospital, 0027 Oslo, Norway. Back

2 Abbreviations used in this paper: ITAM, immunoreceptor tyrosine-based activation motif; PTK, protein tyrosine kinase; LAT, linker for activation of T cells; PLC, phospholipase C; c-RPMI, RPMI 1640/10% FCS; IP, immunoprecipitation, immunoprecipitated; HA, hemagglutinin; SLP-76, SH2-domain-containing leukocyte-specific phosphoprotein of 76 kDa. Back

3 The TSAd protein is encoded by the SH2D2A gene (http://www.gene.ucl.ac.uk/cgi-bin/nomenclature/searchgenes.pl). Back

4 K. Dai, H. F. Harbo, E. G. Celius, A. Oturai, P. S. Sørensen, L. P. Ryder, A. Sveigaard, J. Hillert, S. Fredriksom, M. Sandburg-Wollheim, et al. 2000. Multiple sclerosis is associated to a functionally active polymorphism in one SH2D2A promoter. Submitted for publication. Back

Received for publication April 14, 2000. Accepted for publication July 10, 2000.


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 Introduction
 Materials and Methods
 Results and Discussion
 References
 

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S. Granum, T. C. B. Andersen, M. Sorlie, M. Jorgensen, L. Koll, T. Berge, T. Lea, B. Fleckenstein, A. Spurkland, and V. Sundvold-Gjerstad
Modulation of Lck Function through Multisite Docking to T Cell-specific Adapter Protein
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C. E. Perchonock, A. G. Pajerowski, C. Nguyen, M. J. Shapiro, and V. S. Shapiro
The Related Adaptors, Adaptor in Lymphocytes of Unknown Function X and Rlk/Itk-Binding Protein, Have Nonredundant Functions in Lymphocytes
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F. Marti, G. G. Garcia, P. E. Lapinski, J. N. MacGregor, and P. D. King
Essential role of the T cell-specific adapter protein in the activation of LCK in peripheral T cells
J. Exp. Med., February 21, 2006; 203(2): 281 - 287.
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M. J. Shapiro, Y.-Y. Chen, and V. S. Shapiro
The Carboxyl-terminal Segment of the Adaptor Protein ALX Directs Its Nuclear Export during T Cell Activation
J. Biol. Chem., November 18, 2005; 280(46): 38242 - 38246.
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L. Xing, L. T. Donlin, R. H. Miller, and K. Alexandropoulos
The Adapter Molecule Sin Regulates T-Cell-Receptor-Mediated Signal Transduction by Modulating Signaling Substrate Availability
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K.-Z. Dai, F.-E. Johansen, K. M. Kolltveit, H.-C. Aasheim, Z. Dembic, F. Vartdal, and A. Spurkland
Transcriptional Activation of the SH2D2A Gene Is Dependent on a Cyclic Adenosine 5'-Monophosphate-Responsive Element in the Proximal SH2D2A Promoter
J. Immunol., May 15, 2004; 172(10): 6144 - 6151.
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T. A. Greene, P. Powell, C. Nzerem, M. J. Shapiro, and V. S. Shapiro
Cloning and Characterization of ALX, an Adaptor Downstream of CD28
J. Biol. Chem., November 14, 2003; 278(46): 45128 - 45134.
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W. Sun, X. Wei, K. Kesavan, T. P. Garrington, R. Fan, J. Mei, S. M. Anderson, E. W. Gelfand, and G. L. Johnson
MEK Kinase 2 and the Adaptor Protein Lad Regulate Extracellular Signal-Regulated Kinase 5 Activation by Epidermal Growth Factor via Src
Mol. Cell. Biol., April 1, 2003; 23(7): 2298 - 2308.
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J. Goebel, K. Forrest, L. Morford, and T. L. Roszman
Differential localization of IL-2- and -15 receptor chains in membrane rafts of human T cells
J. Leukoc. Biol., July 1, 2002; 72(1): 199 - 206.
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F. Marti, N. H. Post, E. Chan, and P. D. King
A Transcription Function for the T Cell-Specific Adapter (Tsad) Protein in T Cells: Critical Role of the Tsad Src Homology 2 Domain
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