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J. Biol. Chem., Vol. 276, Issue 31, 29313-29318, August 3, 2001
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§¶,¶
,,,
í**,,
From the Departments of Medical Biochemistry and
§ Anatomy, Institute of Basic Medical Sciences, University
of Oslo, Box 1112, Blindern, N-0317 Oslo, Norway, the ** Institute of
Molecular Genetics, Academy of Sciences of the Czech Republic, 14220 Prague, Czech Republic, the
Immunomodulation Laboratory of the
Institute for Immunology, Ruprecht-Karls University Heidelberg, 69120 Heidelberg, and the Institute of Immunology,
Otto-von-Guericke-University, 39120 Magdeburg, Germany, and the
§§ La Jolla Cancer Research Center, The Burnham
Institute, La Jolla, California 92037
Received for publication, January 12, 2001, and in revised form, May 21, 2001
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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In resting peripheral T cells, Csk is
constitutively present in lipid rafts through an interaction with the
Csk SH2-binding protein, PAG, also known as Cbp. Upon triggering of the
T cell antigen receptor (TCR), PAG/Cbp is rapidly dephosphorylated
leading to dissociation of Csk from lipid rafts. However, tyrosine
phosphorylation of PAG/Cbp resumes after 3-5 min, at which time Csk
reassociates with the rafts. Cells overexpressing a mutant Csk that
lacks the catalytic domain, but displaces endogenous Csk from lipid
rafts, have elevated basal levels of TCR- Activation of the Src family kinases Lck and Fyn after engagement
of the T cell antigen receptor is an initiating event in T cell
activation and leads to phosphorylation of immunoreceptor tyrosine-based activation motifs
(ITAMs)1 within the TCR
complex (1). The subsequent recruitment of the tandem SH2 domain
containing tyrosine kinase ZAP-70 to phosphorylated ITAMs generates an
activated immune receptor signaling complex that is able to initiate
downstream events leading to a functional T cell response (2, 3). The
control and fine-tuning of the proximal signaling is not only essential
for an effective T cell response to antigen, but also for avoiding
exaggerated T cell activation and autoimmunity. Thus a balance must be
maintained to avoid hypo- as well as hyper-reactivity and
immunopathology. The TCR signaling machinery appears to be controlled
by setting a threshold for activation to avoid too easy triggering.
Suppression of the catalytic activity of Lck and Fyn by phosphorylation
of a C-terminal residue (Tyr505 in Lck,
Tyr528 in FynT) by the C-terminal Src kinase,
Csk, appears to be an important means of negative regulation of TCR
signaling (4-6). Complexed with Csk via binding to its SH3 domain is
also a protein tyrosine phosphatase, PEP, that dephosphorylates the
activating phosphorylation site (Tyr394 in Lck,
Tyr416 in FynT) (7, 8). Recent discoveries
indicate that the assembly of TCR signaling complexes occurs in
specific membrane subdomains with high cholesterol and glycolipid
contents, called glycosphingolipid-enriched microdomains or
lipid rafts (9-11). Key components, including Lck and LAT, are
targeted to rafts by virtue of their lipid modifications, whereas other
proteins such as the The ubiquitously expressed, cytosolic Csk tyrosine kinase is critical
for suppression of Src-like kinases and necessary for normal thymic
development of T cells (17-19). Disruption of the Csk gene leads to
unregulated Src kinase activity and early embryonic lethality (17, 18).
Moreover, loss of Csk in thymocytes uncouples thymocyte development
from control by the TCR (19). Csk consists of an SH3 domain, an SH2
domain and a kinase domain similar to the Src kinases, but lacks the
C-terminal regulatory tyrosine as well as the N-terminal lipid
modification sites that targets Src kinases to lipid rafts (20). Thus,
although Csk has been shown to move to sites of Src activity in
fibroblasts (21), it has for some time been unclear how the cytoplasmic
Csk can effectively regulate the lipid raft-associated Src kinases and whether this inhibition by Csk is temporarily released upon T cell
activation. The recent identification of a lipid raft-associated transmembrane adaptor protein,
phosphoprotein-associated with glycosphingolipid-enriched microdomains (PAG) or
Csk binding protein (Cbp) with the
capacity to bind Csk provides a means for localizing Csk to the
proximity of its substrates (22, 23). It was shown that Csk is
associated with lipid rafts in normal T cells via PAG/Cbp and that Csk
dissociates from the rafts concomitantly with TCR-mediated
dephosphorylation of PAG/Cbp (22). Here, we extend these findings and
show that the raft-associated Csk present in resting T cells is
targeted via interaction of the Csk SH2 domain with PAG/Cbp.
Furthermore, rapid Csk reassociation with lipid rafts coincides with
termination of proximal TCR signaling. In addition, overexpression of a
catalytically inactive mutant Csk that displaces endogenous Csk from
lipid rafts leads to both constitutive and sustained activation. Thus,
Csk acts as a gatekeeper that must be temporarily sent off duty for
efficient TCR signaling to occur.
Cell Culture, Stimulation, and Transfection--
The human
leukemic T cell line Jurkat TAg, a derivative of the Jurkat cell line
stably transfected with the SV40 large T antigen (24), and the
Lck-deficient JCaM1 cell line (25) were kept in logarithmic growth in
RPMI medium supplemented with 10% fetal calf serum and antibiotics.
Human peripheral blood T cells were purified from normal donors by
negative selection (26). T cells were activated by addition of 5 µg/ml anti-CD3 Lipid Raft Purification--
Isolation of lipid rafts or
glycoprotein-enriched membrane microdomains was performed as described
in detail elsewhere (11). Briefly, cells were homogenized in 1 ml of an
ice-cold lysis buffer (25 mM MES, pH 6.5, 100 mM NaCl, 5 mM EDTA, 1.0% Triton X-100 with 1 mM sodium orthovanadate, 1 mM PMSF, 10 mM sodium pyrophosphate, and 50 mM sodium
fluoride) by 10 pestle strokes in a Dounce homogenizer, loaded at the
bottom of a 40 to 5% sucrose gradient, and centrifuged at 200,000 × g for 20 h. Fractions (0.4 ml) were collected from the top.
Immunoprecipitation and Immunoaffinity Purification--
For
immunoprecipitation, cells (5 × 107) were disrupted
in precipitation buffer (50 mM Tris-HCl, pH 7.4, 100 mM NaCl, 5 mM EDTA, 60 mM
n-octyl- Immunoblot Analysis--
Detection of phosphotyrosine by
anti-Tyr(P) mAb (4G10, Upstate Biotechnology, Lake Placid, NY),
and immunoblotting with anti-Csk, anti-PAG, anti-LAT, anti-PLC- Generation of Csk Constructs and Recombinant Protein--
The
gene encoding human Csk (29) was subcloned into the expression vector
pEF-BOS/HA at NheI-XbaI sites. Vectors encoding HA-Csk-SH3-SH2 and GST-Csk-SH3-SH2 were generated by stop mutations in
amino acid 175 using full-length Csk in the respective vectors as
template and using a site-directed mutagenesis kit (QuikChange, Stratagene, La Jolla, CA). Subsequently, HA-Csk-SH3-SH2(R107K), HA-Csk-SH3-SH2(S109C), and HA-Csk-SH3(W47A)-SH2 were made in a second
round of mutagenesis to eliminate SH2 and SH3 binding capacity, respectively (30-32), whereas a full-length, kinase-dead Csk,
HA-Csk(K222R) (6), was generated by mutagenesis of the wild type
HA-Csk. GST-Csk-SH3-SH2 was purified as described previously (29).
Mutants were verified by sequencing. The FLAG-PAG construct was
described previously (22).
Csk Affinity Chromatography--
For purification of
Csk-associated proteins, purified peripheral T cells (5 × 108) were disrupted in precipitation buffer (as described
above including 60 mM
n- IL-2 Promoter Activity--
To assess activity of the proximal
IL-2 promoter, cells were transfected by electroporation with a
construct consisting of the NFAT-AP-1 elements from the proximal IL-2
promoter inserted in a luciferase reporter construct, incubated with 5 µg/ml OKT-3 or 25 ng/ml PMA and 5 µM ionomycin for
6 h, lysed, and assayed for luciferase activity (Promega, Madison, WI).
Kinetics of Csk Dissociation from Lipid Rafts following T Cell
Activation--
The presence of Csk in rafts, together with the
observation that Csk association with these membrane microdomains is
regulated upon T cell activation via dephosphorylation of PAG/Cbp (22), suggest that Csk maintains a certain level of tonic inhibition of Src
kinases, which is released during perturbation of the TCR. To address
in more detail the level of inhibition by Csk, we first examined the
kinetics of Csk dissociation and re-association with rafts. Purified T
cells were stimulated by cross-ligation of the TCR/CD3, and samples
were taken out at different time points, lysed, and subjected to lipid
raft purification. In lipid rafts prepared from resting T cells (0 min), we observed tyrosine-phosphorylated PAG/Cbp and Lck (Fig.
1A) as well as Csk (Fig.
1B). The identity of PAG/Cbp was established based on
mobility, localization, immunoreactivity, and Csk SH2-association (Fig.
1, B-D). Upon T cell activation, we observed
dephosphorylation of PAG/Cbp and phosphorylation of LAT at 2 min,
whereas tyrosine-phosphorylated PAG/Cbp reappeared at 5 min (Fig.
1A). The kinetics of Csk association was assessed by
analysis of fractions containing lipid rafts (numbers 3 and 4 pooled)
at the different time points. Fig. 1B shows that whereas the
amounts of PAG/Cbp remained constant, the majority of Csk was
dissociated at 2 min following cross-ligation of the TCR/CD3 at the
same time as dephosphorylation of PAG/Cbp occurred. However, concurrently with PAG/Cbp rephosphorylation, Csk rapidly reassociated with lipid rafts and was present with increasing levels at 5 and 10 min
following activation. As expected, PLC Overexpression of the Csk SH2 Domain Displaces Endogenous Csk from
Lipid Rafts--
Expression of Csk mutants lacking the kinase domain
or with a kinase-dead mutation (K222R) (30), and with functional or deficient SH2 (wt, R107K or S109C (30, 31)) and SH3 domains (wt, W47A
(32)) (Fig. 2A) show that a
minor, but clearly detectable, fraction of Csk proteins with an intact
SH2 domain (either full-length Csk-wt, Csk-K222R, or truncated
Csk-SH3-SH2) are targeted to lipid rafts (Fig. 2, B and
D). In contrast, mutation of critical residues for SH2
function (Arg107, Ser109) reduces raft
localization. Furthermore, in far-Western analysis, PAG expressed in
COS cells only interacted with wild type Csk or recombinant Csk SH2
domain when cotransfected with Fyn, indicating that the Csk SH2 domain
requires a phosphotyrosine in PAG/Cbp for interaction (Fig.
2C, see also Fig. 1D). These observations support
the notion that targeting of Csk to rafts is mediated only via
interaction of the Csk SH2 domain with phospho-PAG/Cbp. Therefore, we
next expressed the truncated Csk-SH3-SH2 or Csk-SH3(W47A)-SH2 at higher
levels, which showed that these mutant proteins could compete out
endogenous Csk from the lipid rafts (Fig. 2D). Presumably, displacement of wild type Csk occurs by saturating the SH2-binding phospho-Tyr317 in human PAG/Cbp, since the majority of the
mutant Csk was in the soluble fractions numbers 7-12 (not shown). The
soluble pool of wild type Csk (fractions numbers 7-12, data not shown)
was only slightly affected by overexpression of mutant Csk, since the
displaced, raft-associated pool is less than 5% of the total Csk (Ref.
34 and Fig. 2B).
Displacement of Csk from Lipid Rafts Leads to Constitutive and
Sustained T Cell Activation--
To assess the significance of the
lipid raft-associated pool of Csk, we next examined the effect of
overexpression of the truncated Csk (Csk-SH3(W47A)-SH2) with intact SH2
domain on proximal T cell activation in Jurkat T cells (Fig.
3A). Whereas activation of
vector-transfected cells by anti-CD3 antibody leads to a rapid and
transient tyrosine phosphorylation of the TCR
To assess downstream T cell activation events with relevance to T cell
function, Jurkat T cells were transfected with a luciferase reporter
directed by the proximal IL-2 promoter containing the NFAT and AP1
response elements together with constructs directing expression of
various Csk mutants (Fig. 3D). Cells transfected with empty
vector displayed low basal levels of NFAT-AP1 reporter activity
and a TCR-induced increase in activity, while overexpression of
wild-type Csk, as expected, completely abolished reporter activity. In
contrast, cells transfected with constructs directing expression of Csk
mutants with an intact SH2 domain (Csk-K222R, Csk-SH3-SH2, or
Csk-SH3(W47A)-SH2), and with the capacity to displace endogenous Csk
from lipid rafts, exhibited severalfold elevated levels of NFAT-AP1
reporter activity in the absence of TCR stimulation. In cells
transfected with Csk-SH3-SH2 or Csk-SH3(W47A)-SH2, stimulation through
TCR activated the NFAT-AP1 reporter to higher total levels, although
the elevated basal levels reduced the relative effect of TCR
stimulation. Surprisingly, cells transfected with Csk-K222R in three
independent experiments displayed no induction in reporter activity
upon TCR stimulation, although basal levels were substantially increased. It may be speculated that mutated full-length Csk has a
better conformation of the SH3 domain, which interacts more strongly
with other proteins (e.g. PEP). In contrast, the
Csk-SH3(W47A)-SH2 that does not recruit tyrosine phosphatases gave
strong TCR-mediated activation (W47A > SH3-SH2 > K222R).
Alternatively, the increased basal level but lack of further
stimulation through TCR observed with the Csk-K222R mutation in the ATP
binding site may be due to substrate trapping (i.e. Lck is
sequestered) or due to other Csk kinase domain interaction partners.
Transfection of constructs with mutated SH2 domains (Csk-SH3-SH2(R107K)
and Csk-SH3-SH2(S109C)) gave NFAT-AP1 reporter activities that were
significantly lower than those of the Csk-SH3-SH2 construct but higher
than basal levels. This is probably due to the presence of residual
phosphotyrosine binding capacity related to these mutants (Fig.
2D and Refs. 30 and 31).
Unlike Src family PTKs, Csk does not have a C-terminal negative
regulatory tyrosine phosphorylation site and therefore cannot be
suppressed by the intramolecular "tail-bite" mechanism that is
characteristic for Src-like kinases (35). Indeed, it appears that Csk
is constitutively active and exerts a tonic suppression of the Src
family kinases Lck and Fyn in the lipid rafts of resting T cells (Fig.
4, left panel). Such basal
levels of inhibition appear to set the threshold for T cell activation
to prevent an aberrant immune response (20). In the presence of this
tonic negative regulation, T cell activation requires two independent early steps: 1) initiation of activating pathways and 2) release from
inhibition. A molecular mechanism for the latter is apparently provided
by the TCR-induced, specific, and rapid dephosphorylation of PAG/Cbp,
resulting in dissociation of Csk from the lipid rafts where Lck and Fyn
reside (22) (Fig. 4, middle panel). This allows a stronger
and prolonged activation of Lck and Fyn and an improved TCR signaling.
However, the relief is only temporary in that PAG/Cbp is again
phosphorylated after several minutes and recruits Csk back to the lipid
raft environment, where it can suppress the Src family PTKs (Fig. 4,
right panel). If PAG/Cbp is phosphorylated by Lck and/or Fyn
as proposed (22), this mechanism is a classical negative feedback loop
that would limit the time course of tyrosine phosphorylation of
substrates for Lck and Fyn.
-chain phosphorylation and spontaneous activation of an NFAT-AP1 reporter from the proximal interleukin-2 promoter as well as stronger and more sustained responses
to TCR triggering than controls. We suggest that a transient release
from Csk-mediated inhibition by displacement of Csk from lipid rafts is
important for normal T cell activation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-chain can localize via interaction with raft
components only after TCR engagement (11-13). Lipid rafts serve to
concentrate and promote specific protein-protein interactions and the
tyrosine phosphorylation of signaling intermediates by the Src family
kinases during the proximal phases of immunoreceptor signaling via the
TCR as well as via the B cell and Fc receptors (14-16).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
mAb OKT-3 or by pervanadate treatment
(0.01% H202 in 100 µM
Na3VO4). For transfections, cells (2 × 107) in 0.4 ml of Opti-MEM were mixed with 2-80 µg of
each DNA construct in electroporation cuvettes with 0.4-cm electrode
gap (Bio-Rad) and subjected to an electric field of 250 V/cm with
960-microfarad capacitance. The cells were expanded in complete medium
and harvested after 20 h.
-D-glucoside, with 1 mM
sodium orthovanadate, 1 mM PMSF, 10 mM sodium
pyrophosphate, and 50 mM sodium fluoride). When stimulated
with OKT-3, cell lysates were precleared by incubation with protein
A/G-agarose beads (Santa Cruz Biotechnology Inc., Santa Cruz,
CA) for 1 h at 4 °C and subjected to immunoprecipitation with
anti-HA mAb (Babco, Richmond, CA), anti-Csk antibody (Santa Cruz,
catalog number SC-286) or anti-PAG mAb (MEM-250) (22). After overnight
incubation at 4 °C, protein A/G-Sepharose was added and the
incubation continued for 1 h. Immune complexes were washed three
times in lysis buffer and subjected to Western blot analysis.
1,
anti-Grb2, anti-HA, and anti- antibodies was as before (27, 28)
except that anti-Csk antibody and anti- antibody from Santa
Cruz Biotechnology Inc. (SC-286 and SC-1239, respectively) were
used. The anti-PAG antibody (and MEM-255) and the far-Western protocol
were described previously (22).
-octyl-D-glucoside to disrupt rafts) and
passed over a column where GST-Csk-SH3-SH2 was bound to
glutathione-agarose, followed by washing with 50 bead volumes and
elution with 50 mM phosphotyrosine in the same buffer.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 was recruited to
rafts upon activation, whereas the amount of LAT in rafts did not
appear to be regulated. Apparent minor differences in immunoreactive LAT levels at 2 min can be explained by the reduced ability of the
anti-LAT antibody to recognize tyrosine-phosphorylated LAT (confer
phospho-LAT blot in Fig. 1B), as previously observed with this antibody (33).
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Fig. 1.
Csk is detached from lipid rafts upon T cell
activation, but rapidly reassociates. A, purified human
T cells were incubated with anti-CD3 antibodies (OKT-3) on ice for 30 min, washed twice, and then the TCR was cross-ligated by
F(ab')2 fragments for the indicated time periods (0-10
min) at 37 °C. Thereafter, standard lipid raft fractionation of T
cell lysates from all time points was performed. Of the 12 fractions
obtained from each separation, anti-phosphotyrosine immunoblots of the
upper six fractions containing the rafts are shown. Longer exposures of
the same blots also showed Vav, Slp-76, Zap-70, and other proteins
phosphorylated on tyrosine. B, peak fractions (numbers 3 and
4) from A containing lipid rafts were mixed and run on
SDS-PAGE, then immunoblots with the indicated antibodies were
performed. Observations are representative of three experiments.
C, Csk was immunoprecipitated from the lipid raft
(R) and soluble (S) fractions after
solubilization with 60 mM
n-octyl- -D-glucoside, precipitates were
subjected to SDS-PAGE separation and anti-phosphotyrosine (upper
panel), and anti-Csk (lower panel) immunoblots were
performed. D, pervanadate-treated human T cells (5 × 108) were lysed in the presence of 60 mM
n-octyl--D-glucoside to allow solubilization
of lipid rafts, and the lysate was subjected to affinity chromatography
with purified GST-Csk-SH3-SH2 fusion protein bound to
glutathione-agarose beads; the column was washed extensively and eluted
with 50 mM phosphotyrosine. Anti-phosphotyrosine and
anti-PAG blots of the crude lysate and the phosphotyrosine eluate are
shown and together with the data in B and C
establish the identity of eluted protein as PAG/Cbp. Note the
difference in mobility of the eluted band versus the band in
lysate in the anti-PAG blot, since only tyrosine phosphorylated PAG/Cbp
with lower mobility elutes from the column.
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Fig. 2.
Overexpression of the Csk-SH2 domain
displaces endogenous Csk from lipid rafts. A,
expression (anti-HA blot) of the different HA-tagged Csk constructs in
transfected Jurkat TAg cells. B, anti-HA blot of fractions
from lipid raft separation of Jurkat T cells transfected with HA-Csk,
HA-Csk-K222R, which is a catalytically deficient mutant, or
HA-Csk-R107K with a defective SH2 domain. C, FLAG-tagged
PAG/Cbp was transfected into COS cells alone or together with Fyn as
indicated, and anti-FLAG immunoprecipitations were performed.
Subsequently, blots were overlaid with an Nonidet P-40 lysate of COS
cells transfected with wild type Csk (left) or with
recombinant Csk SH2 domain (right) and thereafter detected
by anti-Csk. D, Jurkat T cells transfected with vector,
HA-Csk-SH3-SH2, HA-Csk-SH3-SH2(S109C), or HA-Csk-SH3(W47A)SH2 were
subjected to lipid raft separation and the positions of HA-tagged,
truncated, and endogenous Csk were assessed by anti-HA and anti-Csk
immunoblots of the fractions.
-chain and LAT, the
level of phosphorylation of both molecules was higher and more
sustained in cells overexpressing mutant Csk. Furthermore, basal levels
of -chain phosphorylation were elevated in resting Csk-SH3(W47A)-SH2-expressing cells (Fig. 3, A and
B). Similar effects on TCR-induced -chain phosphorylation
were observed with other Csk mutants (Csk-K222R and Csk-SH3-SH2) being
able to displace endogenous Csk from lipid rafts (data not shown). To
address the basal level of -chain phosphorylation in more detail
without background from nontransfected cells, JCaM1 Jurkat cells with defective Lck were transfected with increasing amounts of Csk-SH3-SH2 together with wild type Lck or empty vector. Phosphorylation of the
-chain could then be induced by anti-CD3 antibody in the Lck-transfected cells (not shown). In resting cells, -chain
phosphorylation was absent in cells transfected with Lck alone ((Fig.
3C, fourth lane) but strongly induced by
expression of increasing amounts of Csk-SH3-SH2 (fifth and
sixth lanes), whereas no such effect was seen in the absence
of Lck (first, second, and third
lanes).
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Fig. 3.
Overexpression of kinase-deficient Csk
mutants is associated with elevated basal levels of T cell activation
markers and sustained activity following TCR stimulation.
A, Jurkat TAg cells (2 × 107) were
transfected with 60 µg of plasmid (either empty vector or encoding
Csk-SH3(W47A)-SH2), harvested 20 h later, and washed twice. After
5 min of pre-equilibration at 37 °C, cells were stimulated with
anti-CD3 antibodies (OKT-3) for the indicated time periods, lysed, and
subjected to SDS-PAGE and immunoblotting with the indicated antibodies.
B, densitometric scanning of tyrosine-phosphorylated
-chain shown in A as a function of time is presented for
cells transfected with either empty vector (filled circles)
or vector encoding Csk-SH3(W47A)-SH2 (open circles).
Observations are representative of two experiments. C,
Lck-defective JCaM1 Jurkat cells were transfected with increasing
amounts of plasmid encoding Csk-SH3-SH2 (0-40 µg), together with
empty vector or vector encoding wild type Lck, and analyzed as in
A. D, overexpression of Csk mutants displacing
endogenous Csk from lipid rafts is associated with increased
NFAT-AP1-luciferase activity. Jurkat TAg cells (2 × 107) were cotransfected with NFAT-AP1-luciferase reporter
construct (10 µg) and plasmid encoding the indicated Csk construct
(60 µg), harvested 20 h later, and then incubated for 6 h
untreated, treated with OKT-3, or treated with PMA/ionomycin. After
lysis (and freezing) luciferase activity was measured using a
luminometer. The level of NFAT-AP1 activation in cells transfected with
the different constructs is presented as percent luciferase activity of
that obtained by treatment with PMA and ionomycin of the same cells.
Inset, expression of the transfected constructs. One
representative of three or more experiments is presented.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 4.
Csk is temporarily sent off duty during T
cell activation. In resting normal T cells (left
panel), Csk is present in lipid rafts through interaction with
Tyr317 on PAG/Cbp. This imposes a tonic inhibition of T
cell activation through Csk-mediated phosphorylation of the Lck
regulatory site (Tyr505). Engagement of the T cell receptor
(middle panel) leads to dephosphorylation of PAG/Cbp by an
unknown PTPase, dissociation of Csk from lipid rafts, and displacement
from its substrate Lck, leading to activation of Lck and initiation of
the TCR-induced tyrosine phosphorylation cascade. However, after 2-5
min of activation (right panel), PAG/Cbp Tyr317
is re-phosphorylated by Lck and/or Fyn, thereby recruiting Csk back
into lipid rafts. This terminates Lck and Fyn activity and turns off
TCR signaling. Reproduced from Ref. 38 with permission from Cellular
Signaling.
Our observations (Ref. 22 and this paper) imply that a protein-tyrosine phosphatase (PTPase) responds to TCR ligation by dephosphorylating PAG/Cbp. This so far unidentified enzyme may also be present in lipid rafts. Dephosphorylation apparently occurs at all major physiological tyrosine phosphorylation sites as shown by anti-phosphotyrosine immunoblotting. One candidate for the PTPase that removes phosphate from PAG/Cbp is CD45, which also directly activates Lck and Fyn (36). However, unpublished data2 show that PAG/Cbp dephosphorylation also occurs in the CD45-deficient J45.01 Jurkat T cells (22). The phosphatase PEP, which can be complexed with Csk through binding to its SH3 domain (37), is another obvious candidate. PEP may also contribute to tonic inhibition of Lck and Fyn by dephosphorylating the positive regulatory phosphotyrosines in Lck (Tyr394) (7) and Fyn-T (Tyr416) (8). Release of Csk from rafts would in addition remove the associated PEP and thereby improve the phosphorylation of Lck and Fyn at their activating sites. Overexpression of Csk-SH3(W47A)-SH2, which should not bind PEP due to mutation of the SH3 domain, affected NFAT-AP-1 activation more strongly than expression of Csk-SH3-SH2, suggesting a role for PEP or other interaction partners in lipid rafts. Nevertheless, the constitutive promoter activation in cells expressing either of these mutants indicates the important role of Csk in repression of the Src kinases in resting T cells.
In conclusion, we show that Csk localized to lipid rafts via its SH2
domain is constitutively inhibiting T cell activation. When T cells are
activated through the TCR, Csk rapidly dissociates from lipid rafts
leading to release from inhibition. Shortly thereafter, Csk
reassociates, which contributes to shutting down the TCR signaling. Csk
thus functions as a regulatory gatekeeper for T cell activation.
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ACKNOWLEDGEMENT |
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We are grateful for the technical assistance of Guri Opsahl.
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FOOTNOTES |
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* This work was supported by the Norwegian Cancer Society, The Norwegian Research Council, Novo Nordic Research Foundation, Anders Jahre's Foundation for the Promotion of Science, and the Odd Fellow Medical Fund.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ Both authors contributed equally.
Fellow of the Norwegian Cancer Society.
¶¶ Supported by National Institutes of Health Grants AI35603, AI40552, AI41481, and AI48032.
To whom correspondence and reprint requests should be
addressed: Dept. of Medical Biochemistry, Institute of Basal Medical Sciences, University of Oslo, P. O. Box 1112, N-0317 Oslo, Norway. E-mail: kjetil.tasken@basalmed.uio.no.
Published, JBC Papers in Press, June 4, 2001, DOI 10.1074/jbc.C100014200
2 B. Schraven, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: ITAMs, immunoreceptor tyrosine-based activation motifs; LAT, linker for activation of T cells; Csk, C-terminal Src kinase; Cbp, Csk-binding protein; PAG, phosphoprotein associated with glycosphingolipid-enriched membrane domains; HA, hemagglutinin epitope; TCR, T cell antigen receptor; mAb, monoclonal antibody; MES, 4-morpholineethanesulfonic acid; PMSF, phenylmethylsulfonyl fluoride; IL, interleukin; PLC, phospholipase C; wt, wild type; PTK, protein-tyrosine kinase; PAGE, polyacrylamide gel electrophoresis.
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ABSTRACT
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EXPERIMENTAL PROCEDURES
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DISCUSSION
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