From the Research Institute for Biological Sciences,
Science University of Tokyo, 2669 Yamazaki, Noda, Chiba 278-0022, Japan, the § Department of Applied Biological Science,
Faculty of Science and Technology, Science University of Tokyo, Chiba
278-0022, Japan, the ¶ Naval Medical Research Center, Bethesda,
Maryland 20889-5607, the
Faculty of Pharmaceutical Sciences,
Hokkaido University, Sapporo 060-0812, Japan, the ** Department of
Applied Biological Chemistry, Faculty of Agricultural and Life Science,
University of Tokyo, Tokyo 113-8657, Japan, and the
Abramson Family Cancer Research Institute, University
of Pennsylvania, Philadelphia, Pennsylvania 19104
Received for publication, June 12, 2000, and in revised form, December 1, 2000
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ABSTRACT |
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Ligation of the CD28 surface receptor provides a
major costimulatory signal for full scale T cell activation. Despite
extensive studies, the intracellular signaling pathways delivered by
CD28 ligation are not fully understood. A particularly controversial matter is the role of phosphatidylinositol 3-kinase (PI3K) in CD28-mediated costimulation. It is known that the binding site for PI3K
and Grb-2 lies nested within the YMNM motif of the CD28 cytoplasmic
domain. To elucidate the role of PI3K during CD28-mediated interleukin-2 (IL-2) production, CD28 YMNM point and deletion mutants
were expressed in Jurkat cells. We then measured IL-2 promoter
activation after CD28 ligation. Our results showed that the Y189F
mutant, which disrupts binding by PI3K, and the YMNM deletion mutant
both demonstrated reduced but significant activity for IL-2 promoter
activation. In contrast, the N191A mutant, which retains PI3K binding
ability, resulted in a complete abrogation of activity, suggesting that
PI3K mediates a negative effect upon transcriptional activation of the
IL-2 gene. Consistent with this idea, we found that the
addition of a PI3K pharmacological inhibitor augmented IL-2 promoter
activity, whereas coexpression of a constitutively active form of PI3K
reduced this activity. Taken together, these data indicate that PI3K,
when associated with the YMNM motif, may act as a negative mediator in
CD28-mediated IL-2 gene transcription.
In addition to the signaling provided by recognition of
antigen-major histocompatibility complex by the T cell receptor,
other receptor-ligand interactions play critical roles for full
activation of T cells by providing costimulatory signals to T cells.
Among them, the CD28-mediated signal is considered to be one of the most important costimulatory signals. Costimulation delivered by CD28
is involved in T cell activation and subsequent expression of T cell
functions such as cytokine production (1-3). Although the importance
of CD28 in T cell activation has been well documented, the
intracellular signaling pathways required for CD28-mediated costimulation of T cells have yet to be clearly defined.
A number of signaling molecules such as phosphatidylinositol 3-kinase
(PI3K)1 (4-7), Grb-2 (8),
ITK (9), and Tec (10) have been shown to be involved in CD28-mediated
costimulatory signals. PI3K is a heterodimer, composed of a p85 adaptor
subunit linked to a p110 catalytic subunit that phosphorylates the D-3
position of the inositol ring of phosphatidylinositol,
phosphatidylinositol 4-phosphate, and phosphatidylinositol
4,5-bisphosphate, generating phosphatidylinositol 3-phosphate,
phosphatidylinositol 3,4-bisphosphate, and phosphatidylinositol 3,4,5-triphosphate, respectively (11). Ligation of CD28, either by its
natural ligands (B7 molecules on antigen-presenting cell) or by
monoclonal antibodies, triggers phosphorylation of tyrosine residues in
CD28 cytoplasmic domain. Phosphorylation of CD28 on Tyr189
within the YMNM motif has been shown to provide a binding site for the
SH2 domain of the p85 regulatory subunit of PI3K (4-7). Through its
lipid products, PI3K is involved many cellular responses including
proliferation, cell survival, adhesion, and actin rearrangement (12).
Since the T cell responses that are induced by CD28-mediated cosignaling overlap the reported functions of PI3K in lymphocyte activation, it is conceivable that PI3K may be the critical signaling molecule in T cell costimulation. For example, wortmannin, a potent inhibitor of PI3K, is reported to inhibit CD28-dependent
IL-2 production in human peripheral T cells (13-15). Furthermore, CD28 mutants, which are unable to bind to PI3K, demonstrate that PI3K is
required for CD28-mediated IL-2 production in mouse T cell hybridoma
cell lines (4, 16). However, conflicting results concerning the
requirement for PI3K in CD28-mediated costimulation have been reported.
For instance, some investigators reported that wortmannin fails to
block CD28-mediated costimulation of IL-2 production by Jurkat cells
and murine CD4+ splenic T cells (14, 17-20). Moreover,
they showed that mutation of Tyr189 to Phe, which disrupts
PI3K binding, had no effect on the ability of CD28 to deliver a
costimulatory signal to Jurkat cells (17). Therefore, while the role of
PI3K in CD28 costimulation has been extensively investigated, there is
as yet no consensus about the role of PI3K for CD28 function.
CD28-mediated costimulation is involved in regulation of various T cell
functions such as proliferation, cytokine production, T cell survival,
prevention of anergy, and expression of cell surface antigens. We
postulate that binding of multiple intracellular signaling molecules to
the CD28 receptor may selectively regulate multiple CD28-induced
cellular functions. As a part of efforts to define the relationship
between CD28-mediated intracellular signaling pathways and T cell
functions, we constructed a panel of point and deletion mutants of CD28
including the critical PI3K binding motif, YMNM, and tested their
costimulatory ability for activation of IL-2 gene transcription.
Recombinant DNA Constructs--
Murine CD28 cDNA was
generously gifted by K. Lee (University of Miami School of Medicine,
Miami, FL). Murine CD28 cDNA was subcloned into pBluescript
(Stratagene, La Jolla, CA). Mutant CD28 constructs were generated by
oligonucleotide-directed site-specific mutagenesis and verified by DNA
sequencing. CD28 wild-type and mutant constructs were subcloned into
the mammalian expression vector pcDNA3.1/Zeo (Invitrogen, Carlsbad,
CA). cDNA for BD110, an active form of PI3K, was described
previously (25).
Cell Lines and Transfections--
Jurkat cells were maintained
in RPMI 1640 supplemented with 10% fetal calf serum, penicillin,
streptomycin, 10 mM HEPES (pH 7.55), and 50 µM 2-mercaptoethanol. For transient transfections, exponentially growing cells were harvested, washed in
phosphate-buffered saline, and resuspended at 2.8 × 107 cells/ml. 7 × 106 cells (0.25 ml)
were combined 10 µg of effector construct and 5 µg of the
IL-2-luciferase reporter gene in a 4-mm cuvette and electroporated with
a Bio-Rad Gene Pulser at 220 V and 950 microfarads (Bio-Rad).
Stable transfectants were derived by electroporating 7 × 106 Jurkat cells with 20 µg of plasmid DNA at 220 V and
950 microfarads. After 24 h, the cells were subjected to Zeocin
(Invitrogen) selection at 400 µg/ml. Zeocin-resistant cells were
screened for expression of the relevant construct by
fluorescence-activated cell sorting (FACScaliber; Becton Dickinson, San
Jose, CA) analysis with anti-mouse CD28 antibody.
FACS Analysis--
Cell surface expression of stably transfected
cells was determined by incubating 106 cells with
fluorescein isothiocyanate-conjugated anti-mouse CD28 (PV-1 (21)) at
room temperature for 20 min. Data were collected and analyzed with a
FACScaliber and Cell quest software (Becton Dickinson).
IL-2 Promoter Activity Assay--
Jurkat cells were transiently
transfected with effector and reporter constructs. After 24 h,
cells were treated with PMA (5 ng/ml; LC Services Corp, Woburn, MA) and
anti-mouse CD28 mAb PV-1 (5 µg/ml) or PMA and ionomycin (200 ng/ml;
Sigma), with or without anti-CD28 antibody. In the PI3K inhibition
experiments with LY294002, stimulation was performed with PMA and
anti-CD28 mAb, in the presence of the indicated concentration of
LY294002 (Sigma). After 18 h, cell lysates were analyzed for
luciferase activity using a luciferase assay kit (Promega). Briefly,
cells were resuspended in 100 µl of lysis buffer and incubated at
room temperature for 15 min. After a brief centrifugation, 50 µl of
the supernatant was used with 100 µl of luciferase assay reagent.
Luminescence was measured immediately with a Lumat LB9501 (Berthold,
Bundoora, Australia).
GST Fusion Proteins--
The cDNA encoding the cytoplasmic
domain of CD28 was amplified by polymerase chain reaction and cloned
into the pGEX 4T-1 vector (Amersham Pharmacia Biotech).
Nonphosphorylated GST-CD28 was expressed in the Escherichia
coli BL21(DE3) pLysS strain (Novagen). Phosphorylated GST-CD28 was
expressed E. coli TKB1 strain (Stratagene), a BL21(DE3)
derivative strain that harbors a plasmid-encoded, inducible tyrosine
kinase gene. Bacterial cultures were grown to log phase, induced by 0.3 mM
isopropyl-1-thio- GST Precipitation, Immunoprecipitations, and Western
Immunoblots--
Jurkat cells were lysed in the lysis buffer (1%
Nonidet P-40, 20 mM Tris (pH 7.5), 150 mM NaCl,
5 mM EDTA, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 1 mM Na3VO4, and 50 mM
NaF). The lysate was centrifuged at 20,000 × g for 10 min, and the supernatant was incubated with immobilized GST fusion
proteins on glutathione beads for 2 h at 4 °C. The beads were
washed three times with lysis buffer and boiled in the presence of SDS
sample buffer. The protein complexes were resolved by SDS-PAGE (12%)
and transferred to polyvinylidene difluoride (PVDF) membranes, and
immunoblotted with antiserum specific for the p85 subunit of PI3K
(Upstate Biotechnology, Lake Placid, NY) or anti-Grb-2 antibody (C-23;
Santa Cruz Biotechnology, Santa Cruz, CA).
The Jurkat cells expressing the mouse CD28 wild-type and mutant
constructs were activated by incubating in the presence of anti-CD28
mAb for 10 min at 37 °C. The cells were then lysed in the lysis
buffer. After centrifugation, the lysates were incubated for 2 h
at 4 °C with protein A-Sepharose (Amersham Pharmacia Biotech) beads
coated with anti-mouse CD28 mAb. The proteins were eluted from the
beads by boiling in SDS sample buffer, separated on 10% polyacrylamide
gels, and transferred to polyvinylidene difluoride membranes.
Immunoblotting was performed with antiserum specific for the p85
subunit of PI3K or anti-CD28 antibody (I-20; Santa Cruz Biotechnology).
Mutational Analysis of CD28-mediated IL-2 Promoter
Activation--
In the CD28 cytoplasmic domain, four tyrosines that
potentially bind to SH2 domains and two potential motifs
(PXXP motif) that bind to SH3 domains exist (Fig.
1). The YMNM motif, which exists in the
CD28 cytoplasmic region, is known to associate with PI3K and Grb-2
(4-8). To determine which amino acid is critical for IL-2
gene transcription, we generated various mutants of the mouse
CD28 gene, and co-transfected them with the
IL-2-luciferase reporter gene into Jurkat cells (Fig. 1). The
transfectants were then activated by treating with phorbol ester, PMA,
and anti-mouse CD28 antibody, and subsequently luciferase activity was
measured to determine the IL-2 promoter activity. It has been shown
that upon CD28 ligation, the tyrosine residue within the YMNM motif is
phosphorylated and that this tyrosine phosphorylation makes YMNM motif
available for the association of PI3K p85 subunit and Grb-2 through
their SH2 domains. These associations have been proposed to be critical
to trigger the CD28 signal transduction pathway for IL-2
production.
As shown in Fig. 2, the Y189F mutant,
which is mutated at Tyr189 to Phe in the YMNM motif, showed
reduced ability to activate the IL-2 promoter. On the other hand,
mutation of Asn191 to Ala (N191A) resulted in complete
abrogation of activity. In contrast, the M192L mutant retained full
activity (Fig. 2). Since it has been shown that PI3K binds to the
YXXM motif and that Grb-2 binds to the
YXNX motif (22), it is predicted that the N191A mutant would associate with only PI3K, and that the M192L mutant would
selectively bind to Grb-2. In fact, anti-p85 immunoblotting of
anti-CD28 immunoprecipitates showed that when cells expressing the
mouse CD28 WT or N191 mutant were stimulated with anti-mouse CD28
antibody, p85 was found to associate with CD28, whereas stimulation of
the Y189F or M192L mutants failed to recruit p85 (Fig.
3B). To examine the effect of
mutation within the CD28 YMNM motif upon Grb-2 association, we
generated a GST fusion protein of WT and each mutant of CD28, and
tested their binding capability to Grb-2 in Jurkat lysates by Western
blot analysis. As shown in Fig. 3D, strong associations of
Grb-2 with WT-GST and M192L-GST protein were present in a
phosphorylation-dependent manner, whereas only minimal
associations with Y189F-GST and N191A-GST were detected with or without
phosphorylation. This weak phosphorylation-independent association may
be due to the binding of the PXXP motif, which is present
downstream of YMNM motif, to the SH3 domain of Grb-2 (23, 24).
The above results suggested that the loss of function phenotype present
in the N191A mutant could be due to the impaired Grb-2 binding
capability. Consistent with the results shown in Fig. 3B,
the N191A GST fusion protein, but neither the Y189F or M192L fusion
proteins were co-precipitated with PI3K. Together with the association
patterns of the mutant CD28 constructs with PI3K and Grb-2, our data
suggested that Grb-2 binding to the YMNM motif might play an essential
role for IL-2 gene transcription, whereas PI3K may have a
different role. Most interestingly, the YMNM deletion mutant, which
loses association to both PI3K and Grb-2, showed comparable activity to
that of Y189F mutant (Fig. 2). The simplest interpretation of these
results is that Grb-2 and PI3K have opposing functions on
IL-2 gene transcription. In this scenario, Grb-2 has a
stimulatory role and PI3K an inhibitory role. Furthermore, our results
are compatible with the possibility that there may be other adaptor or
signaling molecules that associate outside of the YMNM motif in CD28
cytoplasmic domain.
To test whether the tyrosine residues outside of the YMNM motif or the
SH3 binding motif of CD28 are involved in the IL-2 promoter activation,
we generated multiple CD28 mutants (Fig. 1). Constructs were tested
that contained mutated Tyr at positions 204, 207, and 216 to Phe, or
mutated Pro at positions 194, 197, 206, and 209 to Ala, respectively.
These mutant constructs were co-transfected with the luciferase
reporter gene into Jurkat cells. The transfectants were stimulated with
PMA and anti-CD28 antibody, and then luciferase activity was measured.
As shown in Fig. 2, all mutants showed activity comparable to that of
the CD28WT construct. Thus, we were unable to demonstrate that these
regions were necessary for IL-2 expression.
LY294002, Inhibitor of PI3K, Augments CD28-mediated IL-2 Promoter
Activation--
To further analyze the PI3K function in CD28-mediated
signaling, we tested the effect of LY294002, a potent inhibitor of
PI3K, on CD28-mediated IL-2 promoter activation. In the CD28 WT and all
mutants except for the TM mutant, LY294002 enhanced the response to
anti-CD28 antibody cross-linking in a dose-dependent
manner. However, the CD28 WT and the N191A mutant, which retain the
capability to associate with PI3K (Fig. 3), showed greater enhancement
by LY294002 treatment than the other mutants (Y189F, M192L, and YMNMdel mutant), which lost PI3K binding ability (Fig.
4).
These results are consistent with the idea that PI3K is a negative
regulator that suppresses IL-2 promoter activation. Some enhancement of
IL-2 promoter activity by LY294002 treatment observed in CD28 mutants
(Y189F, M192L, and YMNMdel mutant), which do not associate with PI3K,
may be due to the inhibition of constitutive activity of PI3K in Jurkat
cells. However, there remains the possibility of a weak association
between PI3K and these mutants that is undetectable by our assay. This
possible association may work negatively on IL-2 promoter activation.
Alternatively, it is possible that PI3K activation occurs downstream of
CD28 signaling transmitted through site(s) outside of the YMNM region,
which negatively regulates the IL-2 promoter activation. Only the IL-2
promoter activity of the TM mutant decreased with LY294002. Our
interpretation of this result is that since cross-linking of the CD28
TM mutant does not induce a positive signal for IL-2 promoter
activation, overall inhibition of PI3K may hinder the maintenance of
cellular homeostasis, resulting in the decrease of IL-2 promoter
activity. To confirm these results, we tested another inhibitor of
PI3K, wortmannin. In the nanomolar range, wortmannin also had a similar enhancing effect, as did LY294002 (data not shown).
An Activated Form of PI3K Suppresses CD28-mediated IL-2 Promoter
Activation--
To further examine whether activation of PI3K
suppresses IL-2 promoter activation, we tested the effect of a
constitutively active form of PI3K, BD110 (25), on CD28-mediated
signaling. The BD110 protein has an inter-SH2 domain of p85, which
binds to the p110 amino terminus, but does not have the two SH2 domains of p85. Thus, this active form of PI3K does not have a binding site for
the CD28 cytoplasmic domain. Jurkat cells were transfected with empty
vector or the plasmid encoding BD110 together with the mouse CD28
construct and IL-2 reporter plasmid, and their CD28-induced IL-2
promoter activity was measured. As shown in Fig.
5, expression of the BD110 strongly
inhibited promoter activation by PMA plus anti-CD28 antibody
stimulation. On the other hand, this suppressive effect of BD110 was
weaker, when transfectants were stimulated with anti-CD28 antibody in
the presence of both PMA and ionomycin and, it was not seen, when cells
were treated with PMA/ionomycin alone (Fig. 5). These results are
consistent with the hypothesis that the activation of PI3K negatively
regulates the CD28-mediated IL-2 promoter activation, and that this
negative regulation primarily works in CD28-mediated costimulation.
Alternatively, it is possible that PMA and ionomycin activate signal
transduction that is downstream of CD28.
The role of PI3K in CD28 costimulation remains controversial. In
this study, we found that the YMNM motif deletion mutant and the Y189F
mutant had reduced, but yet retained significant activity for IL-2
promoter activation, whereas the M192L mutant was not altered after
CD28 stimulation. Because each of these three mutations eliminated the
PI3K association to CD28, we concluded that PI3K is not absolutely
required for CD28-mediated IL-2 gene transcription. We also
found that the mutation of Asn191 completely abolished
CD28-mediated signaling. Since the mutation of Asn191 to
Ala reduces Grb-2 binding but does not affect PI3K binding, Grb-2 may
have a critical role in CD28-mediated IL-2 gene
transcription. Together, these results lead us to propose the following
hypothesis. 1) Two molecules that bind to the YMNM motif may control
CD28-mediated activation of the IL-2 promoter positively and
negatively. 2) Since the N191A mutant retains its ability to bind PI3K
and reduces binding to Grb-2, PI3K could be considered to be a negative
regulator, while Grb-2 may be a positive regulator, and 3) yet
undefined positive regulators may exist, which bind to the CD28
cytoplasmic domain outside of the YMNM motif.
Several groups demonstrated that PI3K has a crucial role in
CD28-mediated IL-2 production. For example, using a similar approach, Cai et al. (16) showed that murine T cell hybridomas
expressing point mutations of Tyr189 or Met192
within the YMNM motif eliminated IL-2 production after CD28 ligation. In contrast, Truitt et al. (17) reported that transfection
of the mouse CD28 Y189F mutant into Jurkat cells, which showed partial reduction of IL-2-transcriptional activation, did not inhibit CD28-dependent IL-2 production. Furthermore, in Jurkat
cells, it was reported that wortmannin treatment did not decrease or, in some cases, even increased CD28-dependent costimulation
of IL-2 production (14, 17-20). Truitt et al. (17) also
found that wortmannin did not inhibit CD28-dependent IL-2
production from freshly isolated murine CD4+ T cells. On
the contrary, wortmannin was reported to inhibit CD28-mediated
costimulation of IL-2 in human primary T cells (13-15). One of the
possible explanations for the apparent discrepancies among these
reports may be the nature of the cell used in the respective studies.
We have recently found that T cells from transgenic mice expressing the
CD28 Y189F mutation are able to produce a significant amount of IL-2
following CD28
cross-linking.2 This result
suggests that PI3K is dispensable for CD28-mediated IL-2 production in
primary T cells.
Recently, two groups generated PI3K p85 The physiological significance of the inhibition of IL-2 promoter
activity by PI3K is unclear at present. The serine/threonine kinase
PKB/Akt is a downstream effector for PI3K. Recently, the PI3K-Akt
pathway was shown to inhibit the Raf-Mek-ERK pathway depending on the
differentiation stage of muscle cell (28). Akt activation inhibited the
Raf-Mek-ERK pathway in differentiated myotubes, but not in their
myoblast precursors. In Jurkat cells, PI3K may inhibit IL-2 promoter
activation using the same pathway. We are presently studying whether
this inhibitory pathway exists in Jurkat cells. Given that the PI3K-Akt
pathway inhibits the Raf-Mek-ERK pathway depending on the
differentiation stage of muscle cells, it is possible that PI3K might
play a differential role depending on the activation state of the T
cell. In fact, distinct functions of CD28-mediated signals during T
cell antigen recognition and activation have been documented (29). Our
recent study using CD28 null mice that have been reconstituted with
CD28 mutants supports this hypothesis. Namely, splenic T cells
expressing the CD28 Y189F mutant are deeply impaired in the
proliferative response and IL-2 production induced by CD28-ligation and
CD3 stimulation at 24 h after stimulation, whereas these responses dramatically improved at 48 h after stimulation.2 From
these results, it is conceivable that PI3K activation by CD28
engagement may suppress IL-2 production in late but not in early
activation stages and therefore, may lead T cell responses to
terminate. In this scenario, the phenomenon observed in the present
study using Jurkat cells may reflect IL-2 production in the late
activation state of T cells.
CTLA-4, a member of the CD28 family of receptors, appears to send a
negative signal to T cells (30-33). CTLA-4 also has a YXXM motif in the cytoplasmic domain and Schneider et al. (34)
reported that anti-CTLA-4 antibody ligation resulted in detectable
levels of PI3K activity in human T lymphoblastoid cell line and in
PHA-stimulated peripheral T cells. This result suggests the possibility
that CTLA-4-induced suppression of IL-2 production may be mediated by
the negative function of PI3K. Recently, a third CD28 family receptor,
ICOS, has been identified (35-37). This molecule also possesses a PI3K
binding motif, YMFM. We have generated and expressed chimeric molecules
composed of the CD28 extracellular domain and ICOS intracellular
domain, and found that cross-linking of these molecules by anti-murine
CD28 antibody resulted in a specific association with PI3K, whereas
only slight IL-2 promoter activity was detected. This result is
consistent with the notion of a non-obligatory role of PI3K for IL-2 production.
The costimulatory effects of CD28 are not limited to IL-2 production,
and PI3K is also involved in many cellular responses. Thus, PI3K may
have a critical role in T cell responses augmented by CD28
costimulation other than IL-2 production. For example, it has been
shown that CD28 costimulation enhances T cell survival following T cell
receptor stimulation by increasing the expression of Bcl-XL
(38). PI3K was also shown to be involved in cellular survival through
activation of Akt. Akt is known to block apoptosis through
phosphorylation and inactivation of the Bcl-2 family protein Bad,
caspase-9, and Forkhead family members (39-42). By the activation of
PI3K, CD28 may block apoptosis through the PI3K-Akt pathway. Furthermore, CD28 costimulation can also affect adhesion between T
cells and antigen-presenting cells, which is important for efficient T
cell antigen recognition. Indeed, PI3K has been implicated in CD28-dependent increases in
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-D-galactopyranoside, and incubated
3 h at 37 °C. The bacteria were lysed, and purified on
glutathione-Sepharose beads (Amersham Pharmacia Biotech).
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Amino acid sequence for the CD28 cytoplasmic
domain. The wild-type sequence of mouse CD28 cytoplasmic domain is
shown at the top. Point mutations and deletion mutants are
indicated below the wild-type sequence.
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Fig. 2.
The effect of mutation in the CD28
cytoplasmic domain on the activation of IL-2 promoter. Mouse CD28
mutants were transiently co-transfected with the IL-2-luciferase
reporter gene in Jurkat cells. Twenty-four hours after transfection,
these cells were treated with PMA (5 ng/ml) and anti-CD28 antibody (5 µg/ml). Eighteen hours later, cells were lysed and luciferase
activity in the cell lysates was measured. Bars show the
mean and S.D. of three representative experiments as a percentage of
luciferase activity of mouse CD28 WT transfectants.
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Fig. 3.
Independent association of mutant CD28
molecules with PI3K and Grb-2. A, Jurkat cells were
stably transfected with the mouse CD28 WT, Y189F-, N191A-, M192L-, and
YMNMdel-mutant constructs and these transfectants were analyzed by flow
cytometry with control antibody (dotted line) or anti-mouse
CD28 antibody (solid line). B, PI3K immunoblot
was performed on a series of mouse CD28 immunoprecipitations from
Jurkat cells expressing the mouse CD28 WT, Y189F-, N91A-, M192L-, and
YMNMdel-mutant. Cells were incubated with (+) or without ( ) anti-CD28
antibody for 10 min before immunoprecipitation. Precipitates were
subjected to gel electrophoresis and immunoblotted with anti-p85
antiserum (upper panel). The membrane was stripped and
reprobed with anti-CD28 antibody (lower panel). Each lane
corresponds to the lysate from 2 × 107 cells.
C, unphosphorylated (
) or phosphorylated (+) GST-CD28
cytoplasmic domains were precipitated with glutathione-Sepharose beads,
separated by SDS-PAGE (12%), and stained with Coomassie Blue. Equal
amounts of each were used in D. D, Jurkat cell
lysates were incubated with immobilized GST or GST-CD28. Precipitates
were subjected to SDS-PAGE (12%) and immunoblotted with anti-p85
antiserum (upper panel) or with anti-Grb-2 antibody
(lower panel).
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Fig. 4.
The effect of LY294002 treatment on
CD28-mediated IL-2 transcriptional activation. Mouse CD28 mutant
genes were transiently co-transfected with the IL-2-luciferase reporter
gene in Jurkat cells as indicated. Twenty-four hours after
transfection, these cells were treated with PMA (5 ng/ml), anti-CD28
antibody (5 µg/ml) in the presence of the indicated concentration of
LY294002. Eighteen hours later, luciferase activity in the cell lysates
was measured. Data were shown as a percentage of luciferase activity by
PMA and anti-CD28 antibody stimulation in the absence of
LY294002.
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Fig. 5.
The effect of constitutively active form of
PI3K on IL-2 promoter activation by CD28-mediated costimulation.
Jurkat cells were transiently transfected with the mouse CD28 WT gene
and IL-2-luciferase reporter gene together with an empty vector or
plasmid encoding active form of PI3K, BD110. Twenty-four hours after
transfection, these cells were treated with PMA (5 ng/ml) and anti-CD28
antibody (5 µg/ml), or PMA and ionomycin (200 ng/ml), or PMA,
ionomycin, and anti-CD28 antibody. Eighteen hours later, luciferase
activity in cell lysates was measured.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
null mutant mice and
reported that these mice had impaired B cell development and functions,
whereas T cell development and proliferation were normal (26, 27).
These reports are consistent with our conclusion that PI3K
(p85
)-CD28 association is not obligatory for CD28-mediated T cell activation.
1-integrin-mediated adhesion to fibronectin (43). In
summary, it is likely that the binding of multiple signaling molecules
to the CD28 cytosolic domain may selectively regulate diverse cellular functions.
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FOOTNOTES |
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* This work supported by a grant-in-aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture, Japan.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.
§§ To whom correspondence should be addressed: Research Institute for Biological Sciences, Science University of Tokyo, 2669 Yamazaki, Noda, Chiba 278-0022, Japan. Tel.: 81-471-23-9756; Fax: 81-471-24-1955; E-mail: rabe@rs.noda.sut.ac.jp.
Published, JBC Papers in Press, December 11, 2000, DOI 10.1074/jbc.M005051200
2 Harada, Y., Tokushima, M., Matsumoto, Y., Ogawa, S., Otsuka, M., Hayashi, K., Weiss, B. D., June, C. H., and Abe, R. (2001) J. Immunol., in press.
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ABBREVIATIONS |
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The abbreviations used are: PI3K, phosphatidylinositol 3-kinase; SH2, Src homology domain 2; PMA, phorbol myristate acetate; IL-2, interleukin 2; PAGE, polyacrylamide gel electrophoresis; GST, glutathione S-transferase; FACS, fluorescence-activated cell sorting.
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