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*
Gwen Knapp Center for Lupus and Immunology Research, Department of Medicine,
Ben May Institute for Cancer Research, Department of Pathology, and
Howard Hughes Medical Institute, University of Chicago, Chicago, IL 60637; and
§
Pfizer Pharmaceuticals, Groton, CT 06340
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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CTLA-4-mediated inhibition of T cell activation may be accomplished by
competition with CD28 for binding to B7 ligands. CTLA-4 may also
interfere with TCR and/or CD28-initiated intracellular signaling
pathways. In support of this, ligation of CTLA-4 on the surface of T
cells has been shown to inhibit TCR/CD28-mediated IL-2 production and
to delay cell cycle progression (8, 9), and biochemical studies have
demonstrated that CTLA-4 ligation inhibits TCR- and CD28-mediated ERK
and JNK activation (10). In addition, T cells from CTLA-4-deficient
mice exhibit hyperphosphorylation of the CD3
chain as well as
increased activity of the TCR-associated kinases Fyn, Lck, and ZAP70
(11), suggesting that CTLA-4 may also regulate early events associated
with TCR activation.
The cytoplasmic domain of CTLA-4 contains 36 amino acid residues, with tyrosines present at positions 201 and 218. It has been suggested that phosphorylation of Y201 may regulate interactions between CTLA-4 and potential cytoplasmic signaling molecules. The sequence surrounding Y201 forms a consensus binding motif for the SH2 domains of the p85-regulatory subunit of phosphatidylinositol 3-kinase (PI(3)K) (12). Lipid kinase activity has been reported to coprecipitate with CTLA-4 in T cells (13), and phosphorylated peptides containing the Y201 motif have been demonstrated to bind to PI(3)K with high affinity (13, 14). CTLA-4 has also been shown to coimmunoprecipitate with the SH2 domain-containing tyrosine phosphatase SHP-2, and peptide binding studies have suggested that this association is also mediated by the phosphorylated Y201 motif of CTLA-4 (11).
In addition to PI(3)K and SHP-2, CTLA-4 Y201 has been demonstrated to associate with the medium chain of the clathrin-associated adaptor complex AP-2 (14, 15, 16, 17). This interaction mediates internalization of CTLA-4 and provides a mechanism for regulating CTLA-4 cell surface expression. In contrast to the association with SHP-2 and PI(3)K, the association of CTLA-4 with AP-2 does not require tyrosine phosphorylation. In vitro studies with peptides corresponding to the cytoplasmic domain of CTLA-4 have suggested that tyrosine phosphorylation of Y201 inhibits the association of CTLA-4 with AP-2 (14, 15, 17). Although CTLA-4 tyrosine phosphorylation has been difficult to demonstrate in vivo, a low level of phosphorylation was reported to be detected in primary T cells treated with pervanadate (15). Taken together, these studies suggest that CTLA-4 Y201 may play a critical role in the regulation of CTLA-4-mediated signal transduction by first preventing the internalization of CTLA-4 from the plasma membrane and then creating a binding site for SH2 domain-containing molecules in the CTLA-4 present on the cell surface.
In the present study, we have addressed the role of tyrosine phosphorylation in regulating CTLA-4 intracellular trafficking and signal transduction. We have found that CTLA-4 can be phosphorylated by the tyrosine kinases Lck and Fyn, but not by ZAP70. Tyrosine phosphorylation was found to regulate CTLA-4 cell surface expression. CTLA-4 was found to associate with Lck and Fyn, and Lck coexpression induced the association of CTLA-4 with SHP-2. The formation of a CTLA-4 signaling complex which includes Src kinases and SHP-2 could provide a molecular basis for the regulation of T cell activation by CTLA-4.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The anti-CD3 145-2C11 and anti-CTLA-4 4F10 mAbs have been described previously (18). Phycoerythrin (PE)-conjugated anti-CTLA-4 and hamster IgG anti-trinitrophenol were purchased from PharMingen (San Diego, CA). Mouse monoclonal anti-phosphotyrosine (4G10), rabbit polyclonal anti-PI(3)K, and rabbit polyclonal anti-Lck Abs were purchased from Upstate Biotechnology (Lake Placid, NY). Anti-SHP-2 antiserum (19) was kindly provided by Dr. G.-S. Feng (Indiana University Medical Center, Indianapolis, IN). Goat polyclonal anti-CTLA-4 Ab to the CTLA-4 extracellular domain used for Western blotting and goat polyclonal anti-CD28 Abs to the murine CD28 extracellular and intracellular domains were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit polyclonal anti-Fyn Ab was purchased from Transduction Laboratories (Lexington, KY).
The rabbit polyclonal Abs recognizing phospho-Y201 CTLA-4 or phospho-Y218 CTLA-4 were generated using as immunogens either KLH-PLTTGVpYVKMPPT or KLH-EKQFQPpYFIPIN.
Cell lines, cell culture, and transfections
The human T cell leukemia cell line J32 Jurkat (20) was kindly provided by Dr. C. June (Naval Medical Center, Bethesda, MD). Plasmids were introduced by electroporation of 20 µg of plasmid DNA using the Bio-Rad (Hercules, CA) Gene Pulser set at 250 V and 960 µF capacitance. Transfectants were selected by growth in 0.3 mg/ml G418. Single-cell subclones were obtained by the limiting dilution method, and CTLA-4 surface and intracellular expression was determined by flow cytometry. Transient transfections of the human embryonic kidney cell line HEK293 were performed according to standard calcium phosphate methods using 2.5–5 µg of CTLA-4 plasmid DNA and 2.5 µg of Lck plasmid DNA unless otherwise indicated, and cells used for experiments were harvested at 36–40 h after transfection. Cell lines were maintained in either RPMI (Jurkat) or DMEM (HEK 293) media supplemented with 10% FCS, 2–4 mM glutamine, 50 mM HEPES (pH 7.5), 100 U/ml penicillin, and 100 µg/ml streptomycin. Pervanadate treatment of cells was performed by combining 10 mM or 1 mM Na3VO4 with 10 mM H2O2 in PBS for 20 min. Cells were treated for 15 min at 37°C. The OVA-reactive T cell clone used in this experiment has been described previously (21) and was kindly provided by Dr. T. Gajewski (University of Chicago, Chicago, IL). Cells (5 x 106) were stimulated with irradiated splenocytes, 2 µM OVA, and rIL-2 (10 U/ml) for 2 days. Cells were passed over a Ficoll density gradient before lysis for immunoprecipitations.
Plasmids
The mammalian expression plasmids encoding murine CTLA-4 and
murine CD28 were constructed as previously described (16). Point
mutations were generated using the Chameleon mutagenesis system
(Stratagene, La Jolla, CA), and mutations were confirmed by DNA
sequencing. The CTLA-4
tail plasmid was constructed using mutagenesis
to create an XhoI site distal to the transmembrane region
and subcloning into the pcDNA3 plasmid (Invitrogen, San Diego, CA). The
CTLA-4tail plasmid encodes the CTLA-4 amino acids 1–189. The
expression plasmids encoding ZAP70 and Fyn (22) were generously
provided by Dr. L. Samelson (National Institute of Child Health and
Human Development, Bethesda, MD). The pEF plasmid encoding wild-type
(WT) Lck was constructed by cloning the murine Lck cDNA into a
derivative of the pEF-BOS expression vector (23) and was kindly
provided by Dr. D. Straus (University of Chicago).
Immunoprecipitations and Western blotting
HEK 293 cells (5–10 x 106) or Jurkat T cells (30–40 x 106) were lysed in buffer containing 150 mM NaCl, 50 mM HEPES (pH 7.5), 1 mM EDTA, 1% Nonidet P-40, 10% glycerol, 10 mM NaF, 1 mM Na3VO4, 1 mM PMSF, 8 µg/ml aprotinin, and 2 µg/ml leupeptin. For experiments using the T cell clone, 25 x 106 cells were used for each immunoprecipitation. Lysates were precleared by incubating with protein G-Sepharose for 1 h, and immunoprecipitations were performed by the addition of 5 µg of anti-CTLA-4 4F10 Ab or control hamster Ig and protein G-Sepharose for 3–16 h at 4°C. The precipitates were separated by SDS-PAGE, transferred onto nitrocellulose, and Western blotted with the indicated Abs. Results were visualized using enhanced chemiluminescence (Amersham, Arlington Heights, IL). Where indicated, the blots were stripped with 1% SDS and 100 mM 2-ME in PBS for 30 min at 55°C and reprobed using the indicated Abs.
For the in vitro phosphorylation assay, 1 µg of bacterially expressed glutathione S-transferase (GST) or GST-CTLA-4c, a fusion protein containing the cytoplasmic domain of CTLA-4 fused to GST, was incubated with 0.5 µg of baculovirus-expressed Lck in PBS with 24 mM MgCl2 and 10 µM ATP for 20 min at 37°C. The reaction was electrophoresed using SDS-PAGE and tyrosine phosphorylation detected by Western blotting with an anti-phosphotyrosine Ab.
Flow cytometry
Cells were washed with PBS containing 1% BSA and 0.1% sodium azide and incubated with PE-conjugated anti-CTLA-4 or control hamster Ig for 30 min at 4°C or fixed with 1% paraformaldehyde and permeabilized with 0.3% saponin before incubation with the Abs for intracellular staining. Cells were analyzed on a FACSort flow cytometer (Becton Dickinson Immunocytometry Systems, Mountain View, CA) using Cellquest software.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Phosphorylation of Y201 has been proposed to negatively regulate
the interaction between the CTLA-4 internalization motif and
clathrin-association adaptor complex proteins (14, 15, 17). To analyze
the role of tyrosine-phosphorylated Y201 in CTLA-4 cell surface
expression, Jurkat T cells were stably transfected with full length
(WT) CTLA-4 or a CTLA-4 mutant in which a valine substitution is
present at position 201 (CTLA-4 Y201V). The effect of the tyrosine
phosphatase inhibitor pervanadate on CTLA-4 cell surface expression was
assessed. As shown in Fig. 1
A,
the majority of WT CTLA-4 protein was detected intracellularly, whereas
significant amounts of the CTLA-4 Y201V mutant protein accumulated on
the cell surface. This is consistent with our previous findings that
the CTLA-4 Y201V mutant protein interacts poorly with AP-2 and is
internalized less rapidly than the WT molecules (16). As shown in Fig. 1B, treatment of the cells with pervanadate resulted in a
dose-dependent enhancement of WT CTLA-4, but not CTLA-4 Y201V cell
surface expression. The increase in cell surface expression correlated
with pervanadate-induced tyrosine phosphorylation of WT CTLA-4 (Fig. 1C). In contrast, tyrosine phosphorylation of the CTLA-4
Y201V mutant was significantly reduced, and pervanadate treatment had
little effect on its cell surface expression. These results are
consistent with a role for phosphorylation of Y201 in the regulation of
CTLA-4 intracellular trafficking. In the unphosphorylated state, Y201
of WT CTLA-4 interacts with AP-2, and CTLA-4 is rapidly internalized
from the cell surface. Upon its phosphorylation, the affinity of Y201
for AP-2 is diminished, resulting in the accumulation of CTLA-4 on the
cell surface. In the CTLA-4 Y201V mutant, the AP-2 binding site has
been mutated, and its cell surface expression is not regulated by
clathrin-mediated endocytosis or by tyrosine phosphorylation. As a
result, CTLA-4 Y201V is constitutively expressed at high levels on the
cell surface.
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CTLA-4 phosphorylation by Src kinases
The above findings suggested that tyrosine phosphorylation
of CTLA-4 can be a mechanism for regulating its cell surface
expression. To identify tyrosine kinases that potentially phosphorylate
CTLA-4 in vivo, CTLA-4 was transiently expressed in HEK293 cells
together with candidate tyrosine kinases, and the presence of CTLA-4
tyrosine phosphorylation was determined. As candidate kinases, the Syk
family kinase ZAP70 and the Src family kinases Lck and Fyn were chosen
because of their activity at sites of MHC/TCR interactions. These are
sites to where CTLA-4 may be colocalized through interactions with B7
molecules. As shown in Fig. 2A, coexpression of CTLA-4
with Lck or Fyn in HEK293 cells resulted in the tyrosine
phosphorylation of CTLA-4, whereas coexpression of ZAP70 did not. Since
optimal activation of ZAP70 requires its phosphorylation by Src
kinases, Fyn and ZAP70 were coexpressed together with CTLA-4. However,
this did not lead to phosphorylation of CTLA-4 above the levels
achieved by expression of Fyn alone, despite the presence of ZAP70
activity as assessed by the detection of increased tyrosine
phosphorylation of proteins with molecular masses of 70 to 100
kDa in cells that express both ZAP70 and Fyn (Fig. 2B).
Furthermore, the lack of an additive effect of ZAP70 in Fyn-expressing
cells was not due to saturation of CTLA-4 tyrosine phosphorylation,
since increasing the quantity of transfected DNA encoding Fyn alone
resulted in the detection of increased CTLA-4 phosphorylation (Fig. 2A). A similar increase was seen when the quantity of Lck
cDNA transfected into cells was increased (data not shown). The above
findings indicate that Fyn and Lck, but not ZAP70, induce the tyrosine
phosphorylation of CTLA-4. To determine whether CTLA-4 is a direct
substrate for Src kinases, a fusion protein containing the cytoplasmic
domain of CTLA-4 fused to GST (GST-CTLA-4c) was combined with
recombinant Lck protein in an in vitro phosphorylation assay. As shown
in Fig. 3, incubation of Lck with GST
proteins results in the tyrosine phosphorylation of GST-CTLA-4c, but
not GST. The results demonstrate that Lck can directly phosphorylate
CTLA-4 and that phosphorylation occurs within the cytoplasmic domain of
CTLA-4.
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The cytoplasmic domain of CTLA-4 contains two tyrosines. To
determine which tyrosine was phosphorylated by Src kinases, mutagenesis
of Y201 or Y218 was performed, and the effect of these mutations on
CTLA-4 phosphorylation by Lck was analyzed. As shown in Fig. 4, substitution of phenylalanine for Y201
significantly diminished the amount of phosphorylation detected.
Substitution of phenylalanine for Y218 also diminished the observed
levels of tyrosine phosphorylation. These findings suggest that both
tyrosines are phosphorylated by Lck. To directly confirm the presence
of phosphorylated Y201 or Y218, Abs recognizing these epitopes were
generated. Western blotting using these phosphospecific Abs confirmed
that in the WT CTLA-4 molecule, both Y201 and Y218 were phosphorylated
by Lck. The specificity of these Abs is confirmed by the specific
detection of mutant CTLA-4 with these Abs. These findings demonstrate
that both Y201 and Y218 are substrates for the tyrosine kinase activity
of Lck. Due to the different affinities of the anti-phosphotyrosine
Abs for their epitopes, however, the relative contribution of the
individual phosphorylated tyrosine residues to the total tyrosine
phosphorylation detected in the WT CTLA-4 molecule cannot be
determined.
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The above studies demonstrated that Src kinases can induce the
phosphorylation of CTLA-4. To determine whether CTLA-4 could also
associate with Src kinases, the Western blot shown in Fig. 4
was
reprobed with an anti-Lck Ab (Fig. 5A). Lck was found to
coimmunoprecipitate with CTLA-4. Mutation of either Y201 or Y218 did
not reduce the amount of Lck detected, suggesting that these residues
were not required for binding to Lck. To confirm the association of
CTLA-4 with Lck found in HEK293 cells, CTLA-4 was immunoprecipitated
from Jurkat cells that were stably transfected with either WT CTLA-4 or
the CTLA-4 Y201V mutant. Western blotting using an anti-Lck Ab
indicated that Lck coimmunoprecipitated with CTLA-4 (Fig. 5B), and the amount of Lck precipitated correlated with the
amount of CTLA-4 present (Fig. 5B, top and
bottom left panels). Finally, to determine whether CTLA-4
and Lck associations could be observed in nontransfected T cells, an
activated T cell clone expressing CTLA-4 was investigated for
CTLA-4/Lck interactions. As shown in Fig. 5C, CTLA-4
immunoprecipitates were found to contain Lck.
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It has been proposed that tyrosine phosphorylation of CTLA-4 may
create a binding site for SH2 domain-containing proteins. Peptide-based
binding studies have suggested that the association of CTLA-4 with
PI(3)K and SHP-2 occurs via the SH2 domains of these molecules with the
phosphorylated Y (201)VKM motif contained within the cytoplasmic domain
of CTLA-4. To determine whether the tyrosine-phosphorylated full length
CTLA-4 molecule could associate with PI(3)K or SHP-2, the
coprecipitation of endogenous PI(3)K or SHP-2 with CTLA-4 was analyzed.
Phosphorylation of CTLA-4 by Lck did not increase binding of PI(3)K
over the basal level (Fig. 6A,
top). However, coexpression of Lck with CTLA-4 induced
binding of SHP-2 to CTLA-4 (Fig. 6A, bottom). In
contrast, Lck-induced phosphorylation of CD28 recruited significant
amounts of PI(3)K but did not recruit SHP-2. Interestingly, a
60–69-kDa tyrosine-phosphorylated protein was found to
coimmunoprecipitate with CTLA-4 under conditions of Lck coexpression
(Fig. 6B, left). Stripping and reprobing of this
blot with an Ab to SHP-2 demonstrated that SHP-2 comigrates with this
tyrosine-phosphorylated protein (Fig. 6B, right).
These results confirm that Lck enhances the formation of a complex
between CTLA-4 and SHP-2. Furthermore, under these conditions a subset
of the SHP-2 which is coassociated with CTLA-4 is tyrosine
phosphorylated.
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Both Lck and Fyn can induce the phosphorylation of CTLA-4. To
determine whether, like Lck, Fyn could also associate with CTLA-4, Fyn
and CTLA-4 were transiently expressed in HEK293 cells, and the presence
of Fyn in CTLA-4 immunoprecipitates was determined. In some samples,
ZAP70 was coexpressed to assess the effects of ZAP70 expression and
kinase activity on potential CTLA-4/Fyn associations. As shown in Fig. 7, Fyn coimmunoprecipitates with CTLA-4,
and this association was unaffected by ZAP70 coexpression.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Both ZAP70 and Lck are critical elements for TCR-initiated signal transduction (24, 25, 26). Whereas ZAP70 appears to function primarily within the TCR signaling pathway, the Src kinases have been implicated in the regulation of multiple T cell activation-signaling pathways, including those initiated by costimulatory molecules, adhesion molecules, and cytokine receptors (27). Our findings that Lck both associates with and phosphorylates CTLA-4 suggest that Lck is a strong candidate to phosphorylate CTLA-4 in vivo. However, Lck may not be the only kinase that can perform this function, as Fyn appears to share an ability to associate with and phosphorylate CTLA-4.
Using mutagenesis as well as Abs that specifically detect phosphorylated Y201 or Y218, we have shown that phosphorylation of CTLA-4 by Lck occurs on both tyrosines. These findings are also consistent with a recent report in which both tyrosines of CTLA-4 were required for optimal phosphorylation by Lck in transfected COS cells (17). Furthermore, treatment of CTLA-4-expressing Jurkat T cells with pervanadate resulted in tyrosine phosphorylation of both WT CTLA-4 and a CTLA-4 Y201 mutant, suggesting that phosphorylation of Y218 also occurs in cells that have not been transfected with tyrosine kinases. Although the role of Y201 in regulating CTLA-4 internalization and signal transduction is becoming better understood, the role of Y218 in CTLA-4 biology is not known at the present; additional studies will be directed at determining the function of Y218 in CTLA-4 signal transduction.
The enzymatic and physical association of Lck with CTLA-4 could have important functional consequences for T cell activation since Lck can participate in the signal transduction pathways of the TCR and CD28, the two receptors that CTLA-4 has been proposed to regulate. In TCR signaling, phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs) of the CD3 complex by Lck results in the recruitment and activation of ZAP70 (24). Within the CD28 signaling pathway, Lck has been shown to be required for activation-induced tyrosine phosphorylation of CD28 (28). Phosphorylation of CD28 by Lck has been shown to recruit PI(3)K and growth factor receptor-bound protein Grb-2 to CD28 (29) and may also regulate some of the tyrosine phosphorylation events seen in response to CD28 ligation such as phosphorylation of ITK (30), Vav, and the rasGAP-associated protein p62dok (31, 32). We have found that CTLA-4 associates with Lck when CTLA-4 is transiently expressed in HEK293 cells, when CTLA-4 is stably expressed in Jurkat T cells, and when CTLA-4 is induced in a T cell clone. This association does not appear to be mediated by the Lck SH2 domain, as mutation of individual CTLA-4 tyrosines does not reduce binding. CTLA-4 contains proline-rich sequences in its cytoplasmic domain which could interact with the SH3 domain of Lck. Association of CTLA-4 with the Lck SH3 domain could facilitate phosphorylation of CTLA-4, resulting in the creation of a binding site for SHP-2 and/or other signaling molecules.
We have found that CTLA-4 is associated at low levels with endogenous SHP-2 and that this association was enhanced by the coexpression of Lck. Unlike the tyrosine phosphatase SHP-1, which is expressed predominantly in cells of hemopoietic origin and can negatively regulate activation of the B cell receptor (33), TCR (34, 35), and NK inhibitory receptor (36, 37), the function of the ubiquitously expressed SHP-2 in cells of the immune system has been less well characterized. In fibroblasts, activation of the PDGF receptor induces the recruitment and phosphorylation of SHP-2. Phosphorylation of SHP-2 creates a Grb-2-SOS docking site, leading to activation of the Ras pathway and enhanced cell proliferation (38, 39, 40). In T cells, both positive and negative roles for SHP-2 have been suggested. SHP-2 has been proposed to negatively regulate TCR-mediated activation in primary T cells (11). In contrast, expression of a dominant negative SHP-2 inhibited TCR activation in Jurkat cells (41), suggesting that SHP-2 may have a positive regulatory role in T cell activation. Previous reports addressing the molecular basis of CTLA-4 activation have suggested that the inhibition of TCR signal transduction by CTLA-4 may occur at the level of Ras activation (10, 11). Although SHP-2 has been closely linked to the Ras pathway, under most circumstances it is thought to activate, not inhibit this pathway. Until the role of SHP-2 in T cell signaling is better clarified, it is difficult to speculate how association of CTLA-4 with SHP-2 results in the inhibition of T cell proliferation.
Both CTLA-4 and CD28 contain consensus sequences for binding to the SH2 domains of the p85 subunit of PI(3)K, and it has previously been demonstrated that phosphorylated peptides based on either the CD28 YMNM motif or the CTLA-4 YVKM motif have similar affinities for PI(3)K (13). We have found that although phosphorylation of the CD28 molecule by Lck resulted in recruitment of significant amounts of PI(3)K, phosphorylation of the CTLA-4 molecule by Lck did not. The inability of CTLA-4 to recruit PI(3)K may be due to several factors. Although we have shown that phosphorylation of CTLA-4 by Lck occurs on both Y201 and Y218, it is possible that the level of phosphorylation on Y201 is not sufficient for significant recruitment of PI(3)K. Another possibility is that within the context of the full length CTLA-4 molecule, the phosphorylated Y201 motif may be structurally inaccessible to PI(3)K. Other mechanisms may be related to the overexpression of Lck and include the sequestration of PI(3)K by Lck (42), or a competition between PI(3)K and Lck for CTLA-4 binding. Presumably, the same mechanism is not operative when CD28 is phosphorylated by Lck. These results demonstrate a distinct difference in the recruitment of PI(3)K following the phosphorylation of CTLA-4 and CD28 by Lck and suggest that the regulation of CTLA-4 and CD28 by Lck can result in distinct intracellular effects.
Increasing biochemical evidence suggests that cell surface receptor signaling involves the formation of multimeric complexes through intermolecular interactions mediated by adaptor modules contained within individual proteins (24, 40). The positioning of the kinases and phosphatases that exist within these complexes relative to their substrates and the regulation of their enzymatic activities are likely to be important in determining the integrated effects of signals generated through activation of multiple cell surface receptors. We have found that CTLA-4 associates with Lck and SHP-2, two molecules that have been identified as important participants of signaling complexes at the TCR and/or the CD28 receptor. Complex formation with these molecules could provide the molecular basis for CTLA-4-mediated regulation of TCR and/or CD28 signaling pathways. Our findings are consistent with a dual role for the Src kinases in the regulation of CTLA-4 activation: prevention of CTLA-4 endocytosis through tyrosine phosphorylation; and induction of the formation of a CTLA-4 signaling complex at the plasma membrane.
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Acknowledgments |
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Footnotes |
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2 These authors contributed equally to this work.
3 Address correspondence and reprint requests to Dr. Craig B. Thompson, University of Chicago, 924 E. 57th Street, R413A, Chicago, IL 60637-5420.
4 Abbreviations used in this paper: CTLA-4, cytotoxic T lymphocyte-associated molecule-4; PI(3)K, phosphatidylinositol 3-kinase; PE, phycoerythrin; GST, glutathione S-transferase; WT, wild type.
Received for publication July 20, 1998. Accepted for publication October 9, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and ZAP70. J. Exp. Med. 186:1645.This article has been cited by other articles:
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E. L. Masteller, E. Chuang, A. C. Mullen, S. L. Reiner, and C. B. Thompson Structural Analysis of CTLA-4 Function In Vivo J. Immunol., May 15, 2000; 164(10): 5319 - 5327. [Abstract] [Full Text] [PDF]
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T. Cinek, A. Sadra, and J. B. Imboden Cutting Edge: Tyrosine-Independent Transmission of Inhibitory Signals by CTLA-4 J. Immunol., January 1, 2000; 164(1): 5 - 8. [Abstract] [Full Text] [PDF]
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M. L. Baroja, D. Luxenberg, T. Chau, V. Ling, C. A. Strathdee, B. M. Carreno, and J. Madrenas The Inhibitory Function of CTLA-4 Does Not Require Its Tyrosine Phosphorylation J. Immunol., January 1, 2000; 164(1): 49 - 55. [Abstract] [Full Text] [PDF]
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H. Schneider, M. Martin, F. A. Agarraberes, L. Yin, I. Rapoport, T. Kirchhausen, and C. E. Rudd Cytolytic T Lymphocyte-Associated Antigen-4 and the TCR{zeta}/CD3 Complex, But Not CD28, Interact with Clathrin Adaptor Complexes AP-1 and AP-2 J. Immunol., August 15, 1999; 163(4): 1868 - 1879. [Abstract] [Full Text] [PDF]
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F. J. Barrat, F. Le Deist, M. Benkerrou, P. Bousso, J. Feldmann, A. Fischer, and G. de Saint Basile Defective CTLA-4 cycling pathway in Chediak-Higashi syndrome: A possible mechanism for deregulation of T lymphocyte activation PNAS, July 20, 1999; 96(15): 8645 - 8650. [Abstract] [Full Text] [PDF]
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 C.A. CHAMBERS and J.P. ALLISON CTLA-4 -- The Costimulatory Molecule That Doesn't: Regulation of T-cell Responses by Inhibition Cold Spring Harb Symp Quant Biol, January 1, 1999; 64(0): 303 - 312. [Abstract] [PDF]
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