(Received for publication, December 1, 1995; and in revised form, February 1, 1996)
From the
Through the interaction with its ligands, CD80/B7-1 and
CD86/B7-2 or B70, the human CD28 molecule plays a major functional role
as a costimulator of T cells along with the CD3TcR complex. We
and others have previously reported that phosphatidylinositol 3-kinase
inducibly associates with CD28. This association is mediated by the SH2
domains of the p85 adaptor subunit interacting with a cytoplasmic YMNM
consensus motif present in CD28 at position 173-176. Disruption
of this binding site by site-directed mutagenesis abolishes
CD28-induced activation events in a murine T-cell hybridoma transfected
with human CD28 gene.
Here we show that the last 10 residues of the intracytoplasmic domain of CD28 (residues 193-202) are required for its costimulatory function. These residues are involved in interleukin-2 secretion, p85 binding, and CD28-associated phosphatidylinositol 3-kinase activity. In contrast, the CD28/CD80 interaction is unaffected by this deletion, as is the induction of other second messengers such as the rise in intracellular calcium and tyrosine phosphorylation of CD28-specific substrates. Furthermore, we also demonstrate that, within these residues, the tyrosine at position 200 is involved in p85 binding, probably together with the short proline-rich motif present between residues 190 and 194 (PYAPP).
In the absence of a costimulatory signal, activation of the
CD3TcR (
)complex is not sufficient to induce the
complete activation of T lymphocytes. The interaction between CD28 on T
lymphocytes and its counter-receptors CD80 (B7-1) and CD86 (B70 or
B7-2) on antigen-presenting cells provides a costimulatory signal
required for IL-2 production, T-cell proliferation, and effector
functions such as T-cell-mediated cytotoxicity and differentiation of
Th cells into Th1 or Th2 subsets (for recent reviews, see (1, 2, 3) ). This CD28/CD80 interaction has
also been shown to prevent anergy and to boost anti-tumor
immunity(4, 5, 6) .
Sequence comparisons
between human, rat, mouse, and chicken CD28 cytoplasmic domains (7, 8, 9, 10) demonstrates high
interspecies conservation, suggesting a crucial role for this domain in
coupling to signal transduction pathways. In the absence of catalytic
motifs in this sequence, an indirect coupling via adaptor molecules was
the most likely mechanism of action. Indeed, we and others have
demonstrated previously that ligand stimulation of the human CD28
molecule induces its association with PI 3-kinase activity (11, 12, 13, 14, 15) by
means of a cytoplasmic YMNM motif at position 173-176 which, when
phosphorylated, interacts with the SH2 domains of the p85 adaptor
subunit. Similarly, the SH2 domain of the adaptor protein Grb-2 has
been shown to interact with this motif although with a lower
affinity(16) , and the CD28-associated Grb-2Sos complexes
are likely to link the activated CD28 receptor to the activation of
p21
and downstream events such as Raf-1
hyperphosphorylation and ERK2 stimulation(17) , as well as Jun
kinase activation(18) .
The primary events leading to CD28
phosphorylation are becoming better understood. The T-cell-specific
protein-tyrosine kinase ITK has been shown to associate with CD28 and
to be phosphorylated on tyrosine residues after CD28
stimulation(19) , and the Src-related tyrosine kinases
p56 and p59
have been
found in CD28 immune complexes from stimulated T cells(20) .
Recently, it has been shown that CD28 is phosphorylated by
p56
and p59
in vitro leading to the recruitment of ITK, Grb-2, and p85 (21) .
Interestingly, the pattern of tyrosine phosphoproteins induced by a
CD28 stimulation is similar but not superimposable to that induced by a
CD3
TcR stimulation (22, 23) and, among the
identified products, are p36-38, p95
, and
PLC-
1 as well as a CD28-specific 64-kDa protein which has yet to
be formally identified (reviewed in (24) ).
Using a murine
T-cell hybridoma transfected with the human CD28 gene, we have shown
previously that a point mutation of the Tyr residue into
phenylalanine abolished CD28-induced IL-2 secretion, suggesting that
the PI 3-kinase pathway plays a major role in the CD28
function(12) . Here we report the generation and functional
characterization of a set of intracytoplasmic variants of the human
CD28 molecule. We have generated mutants of CD28 containing progressive
truncations of its intracytoplasmic tail (10, 21, 30, and 41 residues),
as well as a point mutation of the tyrosine residue at position 200.
These variants were expressed in a murine T-cell hybridoma. By
analyzing stable transfectants, we investigated whether these molecules
were able to mediate cell adhesion to human CD80-transfected L-cells,
to be phosphorylated, bind and activate PI 3-kinase, and to costimulate
IL-2 production together with CD3
TcR.
Figure 1:
Intracytoplasmic truncations of the
human CD28 molecule. A, deletion mutants were produced by
replacing original codons by stop codons (arrows) using
oligonucleotide-directed mutagenesis. Sequencing of mutated molecules
before transfection was performed according to the classical dideoxy
method. B, one clone representative of each transfection (wild
type or deleted CD28 molecules) was analyzed by flow cytometry after
staining with the CD28.2 mAb. These fluorescence histograms were
compared with staining of the untransfected murine T-cell hybridoma,
DC27.1. C, adhesion assay was performed using untransfected (LTK-) or CD80-L cells in the absence (LB7+), or presence of the CD28.2 mAb (LB7+/CD28.2).
CD28 is an adhesion molecule since CD28/CD80
interaction allows cell adhesion(31) . Using L cells
transfected with human CD80, we show that wild type CD28-expressing
cells bound to huCD80 cells (LB7
,
34.5% of binding) but not to untransfected cells
(LTK
). In addition, this binding was inhibited by the
addition of the human mAb CD28.2 (Fig. 1C). The del 10
and del 30 transfected mutants were still able to bind
huCD80
-L cells with almost similar efficacy to wild
type CD28. We previously reported the involvement of the tyrosine
residue at position 173 in the activation of the PI 3-kinase
pathway(12) . Fig. 1C shows that this mutation
did not affect CD28/CD80 interaction. Altogether, these data indicate
that all deleted CD28 molecules still bind CD28 mAbs and B7-Ig and, in
addition, are equally able to mediate the CD28/CD80 interaction showing
that their extracellular structure was not modified.
Figure 2:
Function of wild type and deleted CD28
molecules. Transfected cells were stimulated by cross-linked CD5 (closed circles) or CD3 (open circles) mAbs as
negative and positive controls, respectively. CD28 stimulations were
performed with CD80-L cells (triangles), or
soluble CD28 mAb in combination with soluble CD3 (closed
squares). Supernatants were collected after 24 h of stimulation
and titrated by serial dilutions for their ability to support
proliferation of the IL-2-dependent cell line, CTLL-2. Results are
expressed as A
obtained for each dilution of
the supernatants and correspond to the proliferation of CTLL-2 as
assessed by the cell growth determination
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reagent.
Stimulation by soluble CD28 or CD3 mAbs on their own did not induce a
significant IL-2 production.
Figure 3: Association of CD28 mutants with phosphatidylinositol 3-kinase. Each transfected clone, left unstimulated (lanes 1, 3, 5, 7, and 9) or stimulated with the CD28.2 mAb (lanes 2, 4, 6, 8, and 10), was tested for its ability to associate with PI 3-kinase. Immunoprecipitation of CD28 molecules was performed using protein G, and measure of PI 3-kinase activity associated with the various CD28 mutants was performed as described under ``Experimental Procedures.''
Since the total immunoprecipitable PI 3-kinase activity was equivalent in all these cells (data not shown), the observed defect in PI 3-kinase activity could be explained either by the inability of truncated molecules to activate the enzyme or by their failure to associate with its p85 adaptor subunit. p85 Western blotting of CD28 immunoprecipitates revealed that deletion of 10 C-terminal residues decreased the CD28/p85 association by more than 90% while a deletion of 30 C-terminal amino acids including residues 173-176 completely abolished it ( (12) and data not shown).
We also
examined the ability of other transducing pathways to associate with
CD28 deletion mutants. A rise in Ca reflecting
PLC
1 activation was detected in cells expressing either wild type
CD28 or del 10 mutant upon stimulation by CD3, as well as by CD28 mAbs
(data not shown). CD28 and CD3 stimulations induce the tyrosine
phosphorylation of specific
substrates(17, 22, 23) . A 2-min stimulation
of both WT and del 10 transfected cells by CD3 mAbs induced the
tyrosine phosphorylation of several substrates, the two most prominent
bands corresponding to molecular masses of 100 and 36 kDa (Fig. 4, lanes 2 and 5(32, 33) ). CD28 stimulation led to a strong
phosphorylation of two proteins of 95 and 64 kDa, the former being vav. (
)As shown in Fig. 4, deletion of the
10 C-terminal amino acids did not prevent phosphorylation of these
substrates.
Figure 4: CD28-induced tyrosine phosphorylations in wild type and del 10 CD28 transfectants. Wild type and del 10 transfected cells were stimulated with CD3 (3, lanes 2 and 5) and CD28 (28, lanes 3 and 6) mAbs and goat anti-mouse Ig antiserum for 2 min, or left unstimulated (NS, lanes 1 and 4). Whole cell lysates were separated on SDS-PAGE, transferred onto polyvinylidene difluoride, and probed with anti-phosphotyrosine monoclonal antibody (4G10) as described under ``Experimental Procedures.''
Figure 5:
Binding of CD28 peptides to purified p85
SH2 and SH3 domains. Peptides corresponding to CD28 residues
166-180 (lanes 1 and 2) including a
phosphorylated Tyr (lanes 3 and 4) and
residues 186-202 (lanes 5-7) with phosphorylated
Tyr
(lanes 8-10) were coupled on beads and
used to precipitate recombinant GST (lanes 1, 3, 5, and 8), GST-C-SH2 (lanes 2, 4, 6, and 9), and GST-SH3 (lanes 7 and 10) p85 fusion proteins. Precipitates were run on 10% SDS-PAGE
and revealed by silver staining.
Figure 6:
Point mutation of the Tyr
Phe
. A, wild type and mutated
transfected cells were left unstimulated (lanes 1 and 3) or stimulated with the CD28.2 mAb (lanes 2 and 4) before immunoprecipitation with protein G. PI 3-kinase
activity associated with CD28 was analyzed as in Fig. 3. B, PtdIns(3,4,5)P
generation. Wild type (open
circles) or mutated (Tyr
, open squares;
Tyr
, closed circles) CD28 transfectants were
labeled with [
P]orthophosphate as described
under ``Experimental Procedures.'' Cells were then stimulated
by the addition of CD80
-L cells. Phospholipids were
extracted and analyzed by HPLC as described in (28) .
It has never been
proven that PI 3-kinase association with CD28 was necessary for
activation of the enzyme. We have therefore tested CD28-induced
accumulation of D-3 phosphoinositides in the transfectants expressing
wild type or mutated (Tyr, Tyr
) CD28
molecules. In wild type transfectants, B7 ligation induces a transient
accumulation of PtdIns(3,4,5)P
(Fig. 6B).
However, point mutation of the Tyr
residue completely
abolished CD28-induced PI 3-kinase activation as assessed by
PtdIns(3,4,5)P
accumulation. In contrast, a point mutation
of the Tyr
residue, however, only delayed and attenuated
its activation. Thus, the defect in the PI 3-kinase activation observed
in the DYF200 transfectant occurs at the level of p85 association.
We also examined the ability of p85 C-SH2 domain fusion proteins to
precipitate wild type or mutated CD28 molecules following ligation. Fig. 7A shows that upon ligation by CD80-L
cells, the GST-p85 C-SH2 fusion protein precipitated wild type CD28 (lane 2), while a point mutation of Tyr
strongly
decreased this interaction (lane 8). Mutation of Tyr
did not prevent CD28 interaction with p85 C-SH2 domain (lane
5). Mutation of both Tyr
and Tyr
to
phenylalanine abrogated most of the CD28 ability to be recognized by
the C-SH2 domain of p85 (lane 11).
Figure 7:
Association of CD28 molecules with the
C-SH2 domain of p85 and CD28 phosphorylation. A, cells
transfected by wild type (lanes 1-3), Tyr (lanes 4-6), Tyr
(lanes
7-9), or double-mutated Tyr
(lanes 10-12) CD28 molecules were stimulated by
CD80
-L cells in the absence (lanes 2, 5, 8, and 11) or presence (lanes 3, 6, 9, and 12) of CTLA-4/Ig or left
unstimulated (lanes 1, 4, 7, and 10). Whole cell lysates were precipitated using a p85 C-SH2
fusion protein. Precipitates were run on SDS-PAGE, and transferred onto
polyvinylidene difluoride. Membranes were blotted using CD28.6 mAbs and
revealed by ECL. B, blots were stripped and reprobed using an
anti-phosphotyrosine mAb, and bands were quantified using the BioImage
System (Millipore). Fold induction corresponds to the ratio of CD28
phosphorylation obtained in cells stimulated by CD80
-L
cells versus unstimulated cells.
CD28 is
tyrosine-phosphorylated upon activation (12, 21) and
mainly on the Tyr residue(21) . Mutation of the
Tyr
residue did not prevent CD28 phosphorylation, while
point mutation of Tyr
strongly decreased it. The double
mutant Tyr
lost most of its ability to be
tyrosine-phosphorylated after stimulation (Fig. 7B).
Hence, CD28 phosphorylation is further decreased by a double point
mutation.
The tyrosine residue at position 200 is therefore involved
in PI 3-kinase binding and activation. We have also tested its role in
CD28 function. Fig. 8shows that CD28 mAbs in combination with
CD3 mAbs induced IL-2 secretion although to a lesser extent than wild
type CD28. In contrast, stimulation by CD80-L cells
did not induce IL-2 secretion (Fig. 8) while binding to
CD80
-L cells was retained (not shown).
Figure 8:
IL-2 secretion in DYF200 transfectant.
Cells transfected by mutated CD28 molecule were stimulated by
cross-linked CD5 or CD3 mAbs (closed and open
circles, respectively), by soluble CD28 mAbs in combination with
soluble CD3 (closed squares) or by CD80-L
cells (triangles). Supernatants were collected after 24 h of
stimulation and analyzed as described in Fig. 2.
In this report, we studied the structural requirements of the
cytoplasmic domain of human CD28 for its signaling. For this analysis,
the wild type CD28 molecule and various cytoplasmic mutants (deletion
of 10, 21, 30, and 41 amino acids, or point mutation of Tyr
Phe) were expressed into the murine T-cell hybridoma
DC27.1. We have shown previously that transfection of the full-length
CD28 cDNA in these cells allowed surface expression of functional
molecules which induce either early or late events of T-cell
activation(27) . Flow cytometric analysis showed that deletion
of 10, 21, or 30 residues did not affect the cell surface expression of
CD28. Deletion of the whole intracellular domain, however, impaired the
expression of the construct. This observation has previously been
reported for mutational analysis of the human CD2 molecule and could be
explained by a partial instability of the molecule due to the removal
of positively charged amino acids which are responsible for
transmembrane stabilization(35) .
After ligand binding and
dimerization, many growth factor receptors phosphorylate several
substrates on tyrosine residues leading to a cascade of signaling
events. The antigen-binding T-cell receptor does not possess intrinsic
enzymatic activity, and its coupling to the cellular signaling
machinery is mediated by adaptor molecules. Mutagenesis studies of
several molecules involved in T-cell functions (CD3 chain, CD2)
have identified cytoplasmic consensus motifs which couple these
receptors to early events of T-cell activation. The ITAM motif
(Yxx(I/L))
present in several subunits of the CD3
complex (36, 37, 38) couples the T-cell
receptor to tyrosine kinase activation. Sequence comparison of CD28
with these molecules failed to identify any common motifs. Nonetheless,
analysis of cytoplasmic sequences from human, mouse, rat, and chicken
CD28 (7, 8, 9, 10) showed high
interspecies sequence similarity, suggesting a role for this domain in
signal transduction. Functional characterization of clones carrying
mutations of CD28 confirms that the CD28 cytoplasmic domain plays a
major role in signal transduction. We show that deletion of the 10
C-terminal amino acids severely impairs IL-2 secretion induced by a
CD28 stimulation. This impairment was not merely due to a modification
of CD28 extracellular structure, since all epitopes recognized by 6
different CD28 mAbs (25) were retained on the various deleted
molecules, and since these transfectants were equally able to bind to
B7-Ig and CD80-transfected L cells. Furthermore, cells carrying a
deletion of 10 C-terminal residues were able to exhibit wild type
levels of calcium mobilization as well as tyrosine phosphorylation of
cellular substrates in response to CD28 stimulation. This suggests that
the most C-terminal region of CD28 (residues 193-202) is crucial
for the coupling of this receptor to IL-2 secretion. Interestingly,
this region of CD28 is also involved in PI 3-kinase binding and
activation. Together with the previously described loss of CD28
function following mutations of the PI 3-kinase binding site at
residues Tyr
(12) and
Met
(39) , our data argue for the major role of
this enzyme and/or its associated molecules in coupling the CD28
receptor to the cellular events leading to IL-2 secretion.
Upon
ligand interaction, CD28 becomes tyrosine-phosphorylated and associates
with p85 via a YMNM motif present in its cytoplasmic
domain since a point mutation of Tyr
completely abolished
p85 binding to CD28(12, 13, 20) . Recently,
Raab et al.(21) have shown that p56
and
p59
can phosphorylate the Tyr
residue of
CD28 in vitro. Interestingly, mutation of this residue did not
completely abolish CD28 phosphorylation denoting the presence of other
phosphorylation sites(21) . Here we show that a deletion of 10
C-terminal residues greatly diminished the ability of CD28 to bind PI
3-kinase without affecting other signaling pathways such as PLC
1
activation and tyrosine phosphorylations. Within this region, we have
further identified two putative motifs involved in p85 binding. The
first is a short proline-rich region (residues 190-194), and the
second a tyrosine residue at position 200. We mutated this tyrosine
residue (Tyr
) and confirmed its involvement in p85
binding and PI 3-kinase activation in vivo. Interestingly,
deletion of the last 10 amino acids and point mutation of Tyr
only decreased PI 3-kinase binding to CD28. A low, but
detectable, amount of the p85 still associated with mutated CD28.
Furthermore, in vitro binding experiments showed that while
the binding of the p85 SH2 domain to this Tyr
residue was
dependent upon its phosphorylation, it was weak compared to binding to
the
YMNM motif. This observation was not surprising since
Tyr
is not located within a consensus binding site for
SH2 domains of p85 (YxxM, (40) ). Two alternative
non-consensus binding sites, YVXV (41) and
YVNA(42) , have also been described as novel p85 recognition
motifs in the tyrosine kinase receptors HGF-R and Flt-1, respectively.
The results we present here demonstrate that two regions in the
intracytoplasmic domain of CD28 are involved in PI 3-kinase binding,
one corresponding to the consensus p85 binding site YMNM
and another one at the C terminus of the molecule (residues 193 to 202)
including tyrosine 200 within a non-consensus p85 binding site.
Although the CD28
YMNM motif is sufficient to associate
with p85 since individual N- or C-SH2 fusion proteins can coprecipitate
CD28 after CD28-B7 interaction and a 15-mer CD28 peptide including
phosphorylated Tyr
precipitates PI 3-kinase from cell
lysate (not shown), we propose that the two SH2 domains of p85 act in
concert to associate with two distinct tyrosine residues of CD28 in
vivo. The first is present within the consensus sequence
YMNM and the other at position 200 is a non-consensus
binding site. This additional domain could either increase the affinity
of p85/CD28 interaction or, alternatively, it could bind an adaptor
molecule which interacts with p85. Although Tyr
is the
major phosphorylation site in the CD28 cytoplasmic domain, our data
support the hypothesis that Tyr
is also phosphorylated
upon CD28 stimulation even though we do not directly demonstrate it.
The observation that CD28 phosphorylation is further decreased by a
double point mutation suggests that either the level of Tyr
phosphorylation is too weak to be detected in the presence of
Tyr
phosphorylation, or, alternatively, that Tyr
plays a role in Tyr
phosphorylation, for instance
by recruiting a tyrosine kinase. A third hypothesis is that
Tyr
, although not being a direct target for
phosphorylation, may be involved in the phosphorylation of other
tyrosine residues of CD28 (residues 188 and 191).
Our in vitro binding experiments also showed that a nonphosphorylated peptide
corresponding to the 17 C-terminal amino acids of CD28 also interacts
with a purified GST-SH3 domain, probably through an interaction with
the short CD28 proline-rich segment at residues 190-194
(PxxPP). Unexpectedly, phosphorylation of this peptide at
position Tyr decreased SH3 binding. The functional
significance of this is at present unknown, and the in vivo relevance of this interaction has not been established since CD28
only associates with p85 after stimulation(12) . This
SH3/proline-rich interaction may, however, increase the affinity of p85
SH2 domains binding to CD28.
It is noteworthy that IL-2 secretion
induced by a CD28 mAb costimulation was reduced, but not abolished, by
mutation of Tyr whereas it was completely impaired in the
del 10 mutant. One explanation for the inability of deleted CD28
molecules to couple to the IL-2 secretory pathway is that a deletion of
10 C-terminal residues is sufficient to disrupt the CD28
intracytoplasmic structure. Nonetheless, this hypothesis is unlikely
since other second messengers such as Ca
rise and
tyrosine phosphorylation of cellular proteins still occur upon CD28
activation. An alternative explanation is that, although PI 3-kinase is
crucial for CD28 function, it is not the only transducing pathway
involved in the coupling of CD28 to IL-2 secretion. Indeed, other
enzymes such as sphingomyelinase have been reported to be involved in
the CD28 costimulatory function(43) , and their coupling to
CD28 might also involve the C-terminal domain of the molecule.
Consistent with this hypothesis is the observation that CD28 can
function independently of PI 3-kinase, and that the CD28/PI 3-kinase
association is not sufficient to mediate the full costimulatory
function of CD28(44) .
Several reports have shown that SH3
domains of v-Src(45) , p59(46) , or
p56
(47) could interact directly with the p85
subunit of PI 3-kinase, probably through proline-rich motifs identified
at positions 88-97 and 299-309. This mechanism of PI
3-kinase coupling increases the complexity of possible interactions
between transducing proteins. Yet another mechanism that might account
for PI 3-kinase coupling to CD28 may involve indirect binding of p85
through an interaction with SH3 domains of one of these kinases
previously coupled to CD28 via its C-terminal part. It has recently
been shown that CD28 was phosphorylated by the Src-related
protein-tyrosine kinases p56
and p59
in
vitro, and that this phosphorylation could increase the binding of
p85, Grb-2, and ITK(21) . In absence of a consensus binding
site for SH2 domains of these kinases (Yxx(I/L), (40) ) in the intracytoplasmic domain of CD28, one of the
questions remaining unanswered is how the Src kinases are recruited to
the CD28 receptor after its stimulation.