Cutting Edge: T Cell-Specific Adapter
Protein Inhibits T Cell Activation by
Modulating Lck Activity
This information is current as
of May 24, 2020.
Vibeke Sundvold, Knut Martin Torgersen, Nicholas H. Post,
Francesc Marti, Philip D. King, John Arne Røttingen, Anne
Spurkland and Tor Lea
J Immunol 2000; 165:2927-2931; ;
doi: 10.4049/jimmunol.165.6.2927
http://www.jimmunol.org/content/165/6/2927
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http://www.jimmunol.org/content/165/6/2927.full#ref-list-1
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References
●
Cutting Edge: T Cell-Specific Adapter
Protein Inhibits T Cell Activation by
Modulating Lck Activity
Vibeke Sundvold,* Knut Martin Torgersen,†
Nicholas H. Post,§ Francesc Marti,§ Philip D. King,§¶
John Arne Røttingen,‡ Anne Spurkland,* and
Tor Lea1*
E
ngagement 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
*Institute of Immunology, The National Hospital, Oslo, Norway; Departments of
†
Anatomy and ‡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
Received for publication April 14, 2000. Accepted for publication July 10, 2000.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
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-g1 (PLC-g1), Grb2-Sos, Grap, p85, c-Cbl, Vav,
and SH2-domain-containing leukocyte-specific phosphoprotein of 76
kDa (SLP-76) (4 –9). This results in activation of PLC-g1 and subsequent Ca21 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-g1 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 –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
Plasmids
1
Address correspondence and reprint requests to Dr. Tor Lea, Institute of Immunology,
The National Hospital, 0027 Oslo, Norway. E-mail address: tor.lea@labmed.uio.no
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.
Copyright © 2000 by The American Association of Immunologists
●
TSAd cDNA was cloned into the EcoRI site of the mammalian expression
vector pEF/hemagglutinin (pEF/HA). The IL-2 luciferase reporter plasmid
3
The TSAd protein is encoded by the SH2D2A gene (http://www.gene.ucl.ac.uk/
cgi-bin/nomenclature/searchgenes.pl).
0022-1767/00/$02.00
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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
Ca21 mobilization. The TCR-induced tyrosine phosphorylation of phospholipase C-g1, 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 CD3z-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. The Journal of Immunology, 2000,
165: 2927–2931.
2928
CUTTING EDGE
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-CD3z and -Lck (Santa Cruz Biotechnology,
Santa Cruz, CA), anti-Zap-70 (Transduction Laboratories, Lexington, KY),
anti-HA (Babco, Richmond, CA), anti-human CD3e (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-g1 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 3 106
Jurkat or Jurkat TAg cells in RPMI with 5– 40 mg of DNA were performed
using a Gene Pulser (Bio-Rad, Richmond, CA) at 250 V and 960 mF.
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 3 106 COS-7 cells were transiently transfected as
described above with 10 mg of each construct.
Cell stimulation, lysis, immunoprecipitation, Western blot, and
luciferase assay
T cells were stimulated with 5 mg/ml OKT3 and 1 mg/ml anti-CD28 mAbs
and were cross-linked with goat anti-mouse Ig-coated Dynabeads. In im-
munoprecipitation (IP) experiments, Jurkat TAg cells (10 –20 3 106 cells/
IP) were washed in RPMI 1640, resuspended to 5 3 107 cells/ml, and
stimulated with 5 mg/ml OKT3. Cells were lysed in 23 lysis buffer (2%
Nonidet P-40, 50 mM Tris (pH 7.5), 200 mM NaCl, 40 mM NaF, 2 mM
Na3VO4, 20 mg/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 antiCD3 and PMA (23, 24) or PMA and ionomycin. Thus, in the luciferase
assay, 5 3 105 Jurkat T cells were stimulated with 5 mg/ml OKT3/25 ng/ml
PMA or with PMA/5 mM 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 Ca21 concentrations in single cells were measured as previously
described (25). Jurkat TAg cells (3– 4 3 105) were incubated with 5 mM
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 mg/ml OKT3 at 37°C.
Fluorescence data were treated as previously reported (25, 26). The Ca21
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 mCi of
[g-32P]ATP (DuPont-NEN, Boston, MA) and 10 mg Src peptide (Sigma,
St. Louis, MO) in 150 ml 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 ml 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 6 1 SE. Lck autokinase activity was assessed by SDS-PAGE
and autoradiography of Lck-IP. The remaining one-quarter of Lck-IP beads
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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 3 107) 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 (M) or were
stimulated with anti-CD3/PMA (o) or PMA/ionomycin (f). 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 Ca21 responses in 36 (empty vector) and 72 (TSAd) cells are shown. The arrow indicates the addition of anti-CD3 mAbs.
The Journal of Immunology
was used to ascertain equivalent precipitation of Lck by SDS-PAGE and
immunoblotting as described above.
Results and Discussion
TSAd is induced by TCR triggering and inhibits IL-2 promoter
activity and intracellular Ca21 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. 1A). 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.
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 antiCD3/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. 1B).
To investigate possible effects on anti-CD3-induced Ca21 mobilization, Jurkat TAg cells were transiently transfected with TSAd or
empty vector and loaded with the indicator dye fura-2. The TCRinduced Ca21 mobilization in TSAd transfectants was delayed and
reduced compared with that in empty vector transfectants (Fig. 1C).
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-g1, SLP-76, and LAT
The adapter proteins LAT and SLP-76 are necessary for T cell
development and activation of the PLC-g1 and Ras pathways (10,
13, 29, 30). Importantly, recruitment of SLP-76 to phosphorylated
LAT seems essential for signaling downstream of PLC-g1 (29,
31). The observation that TSAd has no effect on PMA/ionomycin-
FIGURE 3. TSAd inhibits recruitment of Zap-70 to the CD3z-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-CD3z mAbs (lower panel). B, The CD3z-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 CD3z-chains.
induced IL-2 promoter activity indicates that TSAd inhibits T cell
activation upstream of Ca21 mobilization. Indeed, Jurkat TAg
cells transfected with TSAd demonstrated abolished tyrosine phosphorylation of PLC-g1 after CD3 triggering (Fig. 2A), indicating
that reduced Ca21 mobilization in TSAd transfectants is due to
improper activation of PLC-g1. Moreover, TSAd expression inhibited anti-CD3-induced tyrosine phosphorylation of both
SLP-76 and LAT (Fig. 2, B and C), indicating that TSAd prevents
the generation of a multimeric protein complex necessary for activation of PLC-g1 and Ras. Together, these data point to a negative function of TSAd on T cell signaling by modulating proteinprotein interactions close to TCR.
Alteration of Zap-70 and z-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 CD3zchain is believed to be essential for T cell activation, as peptides
blocking the association of Zap-70 with the CD3z-chain inhibit
TCR-mediated signaling (32). To address the ability of TSAd to
regulate this proximal event, we precipitated Zap-70 from antiCD3-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. 3A, upper panel). Furthermore,
coprecipitation of CD3z-chains with Zap-70 was dramatically reduced (Fig. 3A, lower panel). This finding could be due to reduced
tyrosine phosphorylation of the CD3z-chains, leading to reduced
recruitment and activation of Zap-70. Indeed, the tyrosine phosphorylation of CD3z-chains precipitated from the same lysates was
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FIGURE 2. TSAd interferes with tyrosine phosphorylation of PLC-g1,
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-g1 (A) or anti-SLP-76 (B) Abs, respectively. The immunoprecipitates were resolved by SDS-PAGE and immunoblotted with antiphosphotyrosine 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.
2929
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CUTTING EDGE
scriptional 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.
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.
References
reduced in TSAd transfectants (Fig. 3B). Furthermore, Jurkat TAg
cells transiently transfected with increasing amounts of TSAd
cDNA displayed reduced levels of tyrosine-phosphorylated CD3zchains coprecipitating with Zap-70 in a dose-dependent manner
(Fig. 3C). These results suggest an ability of TSAd, either directly
or indirectly, to regulate Lck activity.
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. 4A). 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 (33–35). 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. 4B, 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 –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 tran4
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.
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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).
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