(Received for publication, June 14, 1995; and in revised form, September 20, 1995)
From the
This study compares the biochemical responses in T cells activated with the CD28 ligands B7-1 and B7-2. The patterns of tyrosine phosphorylation induced in T cells by these two CD28 ligands are identical, but clearly different from the tyrosine phosphorylation induced by the T cell receptor (TCR). The TCR regulates protein complexes mediated by the adapter Grb2 both in vivo and in vitro. In contrast, there is no apparent regulation of in vivo Grb2 complexes in response to B7-1 or B7-2. Rather, B7-1 and B7-2 both induce tyrosine phosphorylation of a different adapter protein, p62. The regulation of p62 is a unique CD28 response that is not shared with the TCR. These data indicate that B7-1 and B7-2 induce identical tyrosine kinase signal transduction pathways. The data show also that the TCR and CD28 couple to different adapter proteins, which could explain the divergence of TCR and CD28 signal transduction pathways during T cell activation.
T lymphocyte activation is controlled by the T cell antigen
receptor (TCR) ()in combination with additional signals
triggered by accessory molecules present on the surface of the
antigen-presenting cells(1, 2) . CD28, a 44-kDa
homodimeric glycoprotein expressed by most mature T lymphocytes, is a
costimulatory signal receptor for this process of T cell
activation(3, 4) . Two physiological ligands for CD28
have been described: B7-1 (CD80) and B7-2/B70 (also called
CD86)(5, 6, 7, 8, 9, 10, 11) .
B7-2/B70 shares 25% sequence identity with the extracellular domains of
B7-1(6, 8, 9) . B7-1 and B7-2/B70 have
similar binding properties for CD28 and can provide apparently
identical costimulatory signals for interleukin-2 production by T
cells(12, 13, 14) . However, these molecules
are differentially expressed on antigen-presenting cells: B7-1
expression is detected only on activated antigen-presenting cells,
whereas B7-2/B70 expression is detected on unactivated quiescent
monocytes(6) . It has also been reported that these molecules
are differentially involved in the differentiation of Th1/Th2
subpopulations of T cells(14, 15) . Thus, one crucial
question in CD28 signal transduction is whether B7-1 and B7-2 use
similar signal transduction pathways to costimulate T cells.
Triggering of the TCRCD3 complex activates intracellular
protein-tyrosine kinases (PTKs) that couple the TCR to phospholipase C,
phospholipase C-
1(16) , and the guanine nucleotide-binding
protein p21
(17) . Triggering of the CD28
receptor with antibodies induces phospholipase C and Ras
activation(18, 19) . Previous studies using CD28
antibodies have suggested that PTKs involved in CD28 signal
transduction include the Src kinases p56
and p59
(20, 21) and the Tec family
kinase ITK/EMT(22) . The TCR regulates these PTKs, but also
activates the T cell-specific kinase ZAP70. Triggering of CD28 with the
natural ligand B7-1 also activates cellular PTKs, but the pattern of
phosphoproteins differs from that seen in CD28 antibody-activated
cells(19, 23) . In addition, CD28 stimulation by the
natural ligand B7-1 does not induce phospholipase C activity or Ras
activation, but is associated with activation of phosphatidylinositol
3`-kinase(19, 24) .
Recently, there has been
considerable analysis of the role of the adapter molecule Grb2/SEM5 in
T cells(25) . The importance of Grb2 for PTK signaling was
first established in the Ras signaling pathway(26) . Many
receptors in a variety of cells regulate p21 by
stimulating the guanine nucleotide exchange protein Sos via a mechanism
involving Grb2. Grb2 is composed of one SH2 domain and two SH3 domains.
The Grb2 SH3 domains bind the carboxyl-terminal proline-rich domain of
Sos, and the Grb2 SH2 domain binds to tyrosine-phosphorylated
molecules. In TCR-stimulated cells, there is rapid formation of a
complex between Sos/Grb2 and a 36-kDa membrane protein that is a
substrate for TCR-induced PTKs(27) . Grb2 and p36 may link the
TCR to multiple signaling pathways. Thus, p36 is an apparent link
between the TCR and both Grb2 and phospholipase C(28) . Grb2
may also be important in coupling the TCR to more than just
Sos/p21
because additional Grb2 effector
molecules have been identified in T cells. The best characterized of
these novel Grb2 effector molecules is a 75-kDa molecule, SLP-76, that
is constitutively associated with Grb2 SH3 domains (29, 30, 31) and is a substrate for
TCR-activated PTKs.
There has been no analysis of CD28 links to adapter molecules analogous to the TCR studies, although such experiments will almost certainly afford insight into the mechanisms that transduce the signals generated by CD28-activated PTKs. In this context, it has been reported that CD28 cross-linking with antibodies can induce an association between tyrosine-phosphorylated CD28 and Grb2(32) . Also, ligation of CD28 with antibodies has been shown to induce tyrosine phosphorylation of the Grb2-associated p36 molecule, whereas triggering of CD28 with the natural ligand B7-1, which also activates cellular PTKs, does not(19) . It thus seems that there is the potential for CD28 to regulate Grb2, but this potential is not always realized in response to ligation with the natural ligand for CD28, B7-1. Nevertheless, it is possible that the effects of the CD28 antibodies on p36 phosphorylation mimic the effects of the ligand B7-2/B70 triggering of CD28.
Accordingly, the object of this study was to compare the effects of CD28 triggering with antibodies or B7-1 or B7-2 on the regulation of Grb2 protein complexes. The data show that the CD28 ligands B7-1 and B7-2 induce tyrosine phosphorylation of apparently identical cellular substrates. There are recent data suggesting that B7-1 and B7-2 activate differentially Th1 and Th2 cytokines(14, 15) . In this context, it is worth considering if B7-1 and B7-2 differ in their ability to regulate signal transduction pathways. These studies have identified proteins that are tyrosine-phosphorylated in response to either TCR or CD28 signaling. Thus, the p36 Grb2 SH2 domain-binding protein and SLP-76, the 75-kDa Grb2 SH3 domain-binding protein, are selective substrates for TCR (but not CD28) signaling pathways. Recent studies have identified a tyrosine-phosphorylated 62-kDa molecule, p62, as a multifunctional adapter molecule in many different cells and receptor systems(33) . Herein, we show that this adapter molecule, p62, is a substrate for CD28-activated (but not TCR-regulated) PTKs, which implies that p62 may have a selective function in CD28 (but not TCR) signal transduction. The present data thus indicate that the regulation of adapter molecules is a point of divergence of CD28 and TCR signal transduction pathways.
The monoclonal anti-phosphotyrosine antibody 4G10 and the monoclonal
anti-human GTPase-activating protein (GAP) antibody were purchased from
Upstate Biotechnology, Inc. (Lake Placid, NY). The monoclonal anti-Grb2
antibody was purchased from Affiniti (Nottingham, United Kingdom). The
anti-peptide antiserum Vav-1 was used to immunoprecipitate and detect
in immunoblotting the p95 protein as described previously (36) . The monoclonal anti-phosphotyrosine antibody Fb2 was
used to immunoprecipitate tyrosine-phosphorylated proteins as
described(37) . The polyclonal Cbl antibody was purchased from
Santa Cruz Biotechnology Inc. (Santa Cruz, CA).
The EGFR-pY1068 phosphotyrosine peptide has the sequence PVPEYINQS and was used to precipitate endogenous Grb2 via its SH2 domain as described (29) .
The human CTLA4-Ig fusion protein (CTLA4-Ig) was a kind gift of Dr. P. S. Linsley (Oncogene, Seattle, WA). CTLA4 binds to B7 molecules with high affinity(12) ; a preincubation of L cells expressing B7-1 or B7-2/B70 with CTLA4-Ig at 5 µg/ml prevents the interaction of B7 with the CD28 receptor. Glutathione S-transferase fusion proteins encoding full-length Grb2 (GST-Grb2) and double SH3 mutant GST-Grb2 49L/203R (named GST-Grb2µSH3) as well as carboxyl-terminal GST-mSos1 (where ``m'' is murine; residues 1135-1336) (GST-C-Sos) have been described previously(38) . The isolated amino-terminal GST-huGrb2 (where ``hu'' is human) SH3 domain (residues 1-58) (N-SH3) and the carboxyl-terminal GST-huGrb2 SH3 domain (residues 159-217) (C-SH3) have been described(39) .
For immunoprecipitation, lysates were clarified and incubated with purified anti-Vav antibody coupled to protein A-Sepharose, anti-phosphotyrosine or anti-GAP antibody coupled to protein G-Sepharose, glutathione-agarose beads preloaded with 5 µg of glutathione S-transferase fusion proteins, or synthetic peptides precoupled to Affi-Gel 10-activated ester-agarose beads (Bio-Rad) for 2 h at 4 °C. Immunoprecipitates or precipitates were washed four times in 1 ml of lysis buffer and then boiled in reducing SDS gel sample buffer for 5 min. Samples were resolved by standard 8% SDS-polyacrylamide gels.
For immunoblotting, membranes were blocked and probed with specific antibodies. Blots were then incubated with the appropriate second antibodies, anti-rabbit IgG or anti-mouse IgG (Amersham Corp.), both conjugated with horseradish peroxidase. Immunoreactive bands were visualized by enhanced chemiluminescence (Amersham Corp.).
Figure 1:
Interaction of CD28 with B7-1 or
B7-2/B70 L cells induces tyrosine phosphorylation of cellular proteins.
Jurkat cells (4 10
/point) were unstimulated
(control) or stimulated with UCHT1 (10 µg/ml); CD28.2 (10
µg/ml); untransfected L cells; or B7-1, B7-2, or B70 L cells (at a
ratio of 1:2) for the indicated times. The data show Western blot
analyses of whole cell lysates with monoclonal anti-phosphotyrosine
antibody 4G10. The experiment shown is representative of three separate
experiments. The positions of molecular mass markers (in kilodaltons)
are shown on the left. Arrowheads indicate the positions of
the 62- and 95-kDa phosphoproteins.
The data in Fig. 1show
that the patterns and intensities of tyrosine phosphorylation in TCR-
and CD28-activated T cells are different. Some tyrosine phosphoproteins
were common to TCR- and CD28-induced cells, but, in general, the level
of phosphorylation was stronger in the TCR-activated cells. A 95-kDa
tyrosine phosphoprotein was detected in Jurkat cells within 1 min of
contact of the cells with B7-1, B7-2, or B70 L cells. The 95-kDa
tyrosine phosphoprotein was also detected after stimulation of T cells
with UCHT1 or CD28.2. One candidate for the 95-kDa tyrosine
phosphoprotein in the TCR- and CD28-activated cells is
p95, which is known to be tyrosine-phosphorylated in
response to TCR, CD28 mAb, or B7-1
triggering(19, 22, 36, 40) . Fig. 2A shows anti-phosphotyrosine Western blot analyses of
p95
immunoprecipitates. Weak basal tyrosine
phosphorylation of p95
was detectable in unstimulated
Jurkat cells. B7-2 or B70 L cells, like mAbs UCHT1 and CD28.2 or B7-1 L
cells, induced an increase in p95
tyrosine
phosphorylation. The Vav tyrosine phosphorylation induced by B7-1 or
B7-2/B70 was inhibited by preincubation of the B7-expressing L cells
with CTLA4-Ig.
Figure 2:
B7-1 and B7-2/B70 ligation induces
tyrosine phosphorylation of the vav and c-cbl proto-oncogene products in Jurkat cells. A, Jurkat cells
(4 10
/point) were stimulated with UCHT1 (10
µg/ml); CD28.2 (10 µg/ml); B7-1, B7-2, or B70 L cells; or
untransfected L cells (at a ratio of 1:2) for 5 min. The plus signs mean that the L cells were preincubated with CTLA4-Ig at 10
µg/ml for 5 min, and the minus signs mean that the L cells
were used without CTLA4-Ig preincubation. The experiment shown is
representative of two separate experiments. Vav immunoprecipitations
were analyzed by Western blot analyses with anti-phosphotyrosine
antibody 4G10 (upper panel) or anti-Vav antibodies (lower
panel). B, Jurkat cells (4
10
/point)
were stimulated with UCHT1 (10 µg/ml), CD28.2 (10 µg/ml), or
B7-1 or B7-2 L cells (at a ratio of 1:2) for 5 min. The plus signs mean that the L cells were preincubated with CTLA4-Ig at 10
µg/ml for 5 min, and the minus signs mean that L cells
were used without CTLA4-Ig preincubation. The experiment shown is
representative of two separate experiments. Anti-phosphotyrosine
immunoprecipitations were analyzed by Western blot analyses with
anti-p120
antibody.
The data in Fig. 1show that TCR or CD28
engagement leads to an increase in tyrosine phosphorylation of a
120-kDa band. A 120-kDa protein, p120, has been described
to be tyrosine-phosphorylated upon TCR
stimulation(41, 42) . Fig. 2B shows
anti-p120
Western blot analyses of anti-phosphotyrosine
immunoprecipitates. Basal tyrosine phosphorylation of p120
was detectable in unstimulated Jurkat cells. Like mAb UCHT1, mAb
CD28.2 and B7-1 and B7-2 L cells induced an increase in p120
tyrosine phosphorylation. The Cbl tyrosine phosphorylation
induced by B7-1 or B7-2 was inhibited by preincubation of the
B7-expressing L cells with CTLA4-Ig. There was one tyrosine
phosphoprotein of
62 kDa that was detected in the B7-1- or
B7-2/B70-stimulated T cells, but not in the TCR-stimulated Jurkat cells (Fig. 1).
Triggering of CD28 with the ligand B7-1 induced tyrosine phosphorylation of multiple cellular proteins. In initial experiments to determine whether these tyrosine phosphoproteins include Grb2-binding proteins, a series of binding experiments with GST-Grb2 fusion proteins were performed. Western blot analysis with anti-phosphotyrosine antibodies of GST-Grb2-binding proteins isolated from CD28-activated cells (Fig. 3) showed that CD28 induced a major tyrosine phosphorylation of Grb2-binding proteins of 62 and 120 kDa. The pattern of CD28-induced Grb2-binding proteins had some similarities, but also some differences from that observed in TCR-activated cells, where tyrosine phosphoproteins of 36, 75, 95, and 120 kDa were induced in response to ligation of the TCR complex.
Figure 3:
Comparison of the TCR- and CD28-induced
Grb2 complexes. A, Jurkat cells (4
10
/point) were stimulated with UCHT1 (10 µg/ml), B7-1 L
cells (B7-L cells), or control L cells (at a ratio of 1:2) for
5 min. Proteins were precipitated from postnuclear cell lysates with
GST-Grb2, double SH3 mutant GST-Grb2 49L/203R (GST-Grb2µSH3), or
carboxyl-terminal GST-mSos1 (residues 1135-1336) (GST-C-Sos)
fusion protein immobilized on glutathione beads. Bound proteins were
analyzed with anti-phosphotyrosine antibody 4G10. The experiment shown
is representative of three separate experiments. The positions of p36
and p62 are indicated by arrows. B, Jurkat cells were
stimulated (4
10
/point) with UCHT1 (10 µg/ml)
or with L cells expressing B7-1 (B7-L cells) or not expressing
B7-1 (L cells) (at a ratio of 1:2) for the indicated times.
Proteins were precipitated from the postnuclear cell lysates with the
EGFR-pY1068 peptide coupled to Affi-Gel 10 beads. The experiment shown
is representative of three separate experiments. Bound proteins were
analyzed with anti-phosphotyrosine antibody 4G10 (upper panel)
or anti-Grb2 antibody (lower panel). p75 is indicated by an arrow. C, Jurkat cells (4
10
/point) were stimulated with UCHT1 (10 µg/ml), L
cells expressing B7-1 or B7-2, or untransfected L cells (at a ratio of
1:2) for 5 min. The plus signs mean that the L cells were
preincubated with CTLA4-Ig at 10 µg/ml for 5 min, and the minus
signs mean that the L cells were used without CTLA4-Ig
preincubation. Proteins were precipitated from the postnuclear cell
lysates with double SH3 mutant GST-Grb2 49L/203R (GST-Grb2µSH3)
fusion protein immobilized on glutathione beads. Bound proteins were
analyzed with anti-phosphotyrosine antibody 4G10. The experiment shown
is representative of three separate experiments. The positions of p36
and p62 are indicated by arrows.
Grb2 has a single SH2 domain and two SH3 domains. To explore the SH
domain specificity of the CD28-induced tyrosine phosphoproteins,
binding experiments were carried out with GST-Grb2µSH3, which has
an intact SH2 domain, but a single mutation in both the COOH- and
NH-terminal SH3 domains. The data in Fig. 3A show that the TCR-induced p36 tyrosine phosphoprotein binds to
wild-type GST-Grb2 and GST-Grb2µSH3. The remainder of the
TCR-induced Grb2-binding proteins do not bind to GST-Grb2µSH3.
Thus, as described previously(29) , their interaction with Grb2
is SH3 domain-mediated. The 62-kDa B7-1-induced tyrosine phosphoprotein
can bind to the intact GST-Grb2 fusion protein and to GST-Grb2µSH3,
suggesting that p62 binds to Grb2 SH2 domains. In contrast, mutation of
Grb2 SH3 domains abrogated binding of the 120-kDa tyrosine
phosphoprotein that is induced in response to both TCR and B7-1
stimulation.
Experiments with GST fusion proteins are valuable for studying protein/protein interactions, but it is important to establish whether the interactions between proteins that are identified in such in vitro studies actually occur in vivo. To study the proteins interacting with the SH2 domain of endogenous Grb2, we adopted a previously described technique that uses a GST fusion protein of the COOH-terminal proline-rich regions of mSos1(27) . This fusion protein binds effectively to Grb2 SH3 domains. It competes for any interactions between endogenous proteins and Grb2 SH3 domains, thereby allowing purification of endogenous Grb2 and associated SH2 domain-binding proteins. Fig. 3A shows the tyrosine-phosphorylated proteins that can bind to the SH2 domains of GST-Grb2 and GST-Grb2µSH3 or to the SH2 domains of endogenous Grb2 complexes isolated with GST-C-Sos. The TCR-induced tyrosine phosphoprotein of 36 kDa could bind to GST-Grb2 and GST-Grb2µSH3 and, more important, was copurified with endogenous Grb2. In contrast, the B7-1-induced 62-kDa tyrosine phosphoprotein could bind to the GST-Grb2 fusion proteins, but did not copurify with endogenous Grb2.
The GST-Grb2 binding experiments showed that tyrosine phosphorylation of the 120-kDa Grb2 SH3 domain-binding protein was induced by B7-1. This protein, which could correspond to the product of the c-cbl proto-oncogene(41) , was also tyrosine-phosphorylated in the TCR-activated cells, as was an additional TCR unique molecule, p75. To determine whether the B7-1-induced p120 protein binds to the SH3 domains of endogenous Grb2, cellular Grb2 complexes were affinity-purified using a tyrosine-phosphorylated peptide from the cytoplasmic domain of the epidermal growth factor receptor, EGFR-pY1068, as an affinity matrix. This peptide binds with nanomolar affinity to the SH2 domains of Grb2 and allows the purification of Grb2 and associated SH3 domain-binding proteins. The data in Fig. 3B show that the TCR induces tyrosine phosphorylation of a 75-kDa tyrosine phosphoprotein that copurifies with the Grb2 complexes isolated with EGFR-pY1068. Anti-Grb2 Western blotting (Fig. 3B, lower panel) indicates that the amount of immunoprecipitated protein is similar between the different lanes of the gel. These data show also that B7-1 does not induce tyrosine phosphorylation of this p75 molecule. There was no 120-kDa tyrosine phosphoprotein isolated with endogenous Grb2 complexes and thus no evidence that these proteins form a complex with the SH3 domains of endogenous Grb2 (Fig. 3B).
The B7-1-induced 62-kDa tyrosine phosphoprotein did not bind to endogenous Grb2. Nevertheless, unlike p120, it was a specific substrate for CD28-induced (but not TCR-regulated) PTKs. The data in Fig. 3C compare the ability of B7-1 and B7-2 to induce p62 tyrosine phosphorylation. To study the ability of tyrosine-phosphorylated proteins to interact with the Grb2 SH2 domain after B7-1 or B7-2 stimulation, Jurkat cells were incubated with B7-1 or B7-2 L cells or with untransfected L cells for 5 min (Fig. 3C). TCR-activated cells were included in these experiments as a comparison. The data show that B7-2, like B7-1, can induce tyrosine phosphorylation of a 62-kDa protein that is able to bind GST-Grb2µSH3 and interact with the Grb2 SH2 domain in vitro. p62 tyrosine phosphorylation induced by B7-1 or B7-2 was inhibited by CTLA4-Ig preincubation of B7 L cells.
In many cells, Grb2 via its SH2 domain can form a complex with receptors(26) . To probe for the presence of CD28 in Grb2 complexes, Western blot analysis with CD28 antibodies was performed. The data in Fig. 4A indicate that CD28 molecules isolated from CD28-activated (but not quiescent or TCR-activated) T cell lysates could bind to the wild-type GST-Grb2 fusion protein and GST-Grb2µSH3, but not to GST-Grb2 N-SH3 or GST-Grb2 C-SH3. These fusion protein binding experiments suggest that the Grb2 SH2 domain is important for CD28/GST-Grb2 interactions.
Figure 4:
CD28 can bind to the Grb2 SH2 domain in vitro, but not in vivo in CD28-activated T cells
via its ligand B7-1. In A, Jurkat cells (4
10
/point) were stimulated with UCHT1 (10 µg/ml), L
cells expressing B7-1 (B7-L cells), or control L cells (at a
ratio of 1:2) for 5 min. Proteins were precipitated from postnuclear
cell lysates with GST-Grb2, double SH3 mutant GST-Grb2 49L/203R
(GST-Grb2µSH3), GST-Grb2 N-SH3, or GST-Grb2 C-SH3 fusion protein
immobilized on glutathione beads (A) or with GST-Grb2, double
SH3 mutant GST-Grb2 49L/203R (GST-Grb2µSH3), or carboxyl-terminal
GST-mSos1 (residues 1135-1336) (GST-C-Sos) fusion protein
immobilized on glutathione beads (B). The presence of CD28 in
the precipitates was analyzed by immunoblotting with anti-CD28 antibody
CD28.6. The experiments shown are representative of three separate
experiments.
The previous data show that CD28 induced tyrosine phosphorylation of a 62-kDa protein that could bind to the GST-Grb2 fusion proteins, but could not copurify with endogenous Grb2. A similar analysis was performed to see if CD28 could form a complex with endogenous Grb2. The data in Fig. 4B show that CD28 molecules from CD28-activated (but not quiescent or TCR-activated) cells could bind to GST-Grb2 and GST-Grb2µSH3, but not to the SH2 domain of endogenous Grb2. These data ( Fig. 3and Fig. 4) suggest that, after B7 ligation, at least two proteins (p62 and CD28 receptor) are able to interact with the Grb2 SH2 domain in vitro, but not in vivo.
Figure 5:
A tyrosine-phosphorylated 62-kDa protein
coimmunoprecipitates with GAP specifically after B7-1 and B7-2/B70
ligation. A, GAP immunoprecipitates were prepared from Nonidet
P-40-soluble cellular proteins. Jurkat cells were stimulated with UCHT1
(10 µg/ml), CD28.2 (10 µg/ml), L cells expressing CD80 or CD86
(B7-1, B7-2, and B70 L cells), or untransfected L cells (at a ratio of
1:2) for 5 min. The plus signs mean that the L cells were
preincubated with CTLA4-Ig at 10 µg/ml for 5 min, and the minus
signs mean that the L cells were used without CTLA4-Ig
preincubation. The experiment shown is representative of two separate
experiments. p62-associated protein was detected with
anti-phosphotyrosine antibody 4G10 (upper panel), and GAP was
detected with monoclonal anti-human GAP antibody (lower
panel).
In this study, we have compared the signal transduction pathways regulated by the CD28 ligands B7-1 and B7-2/B70. The data show that B7-1 and B7-2 both induce rapid tyrosine phosphorylation of cellular proteins in T cells. B7-1- and B7-2-induced patterns of tyrosine phosphorylation are different than the TCR stimulation pattern, which supports the hypothesis that CD28 and the TCR regulate different cellular PTKs. However, there were no discernible differences in the patterns of B7-1- and B7-2/B70-induced tyrosine phosphorylation, which indicates that B7-1 and B7-2/B70 activate similar PTKs and/or tyrosine phosphatases. One obvious difference between the TCR and CD28 is with regard to their ability to regulate adapter molecules. In particular, the present data reveal that the regulation of Grb2 can distinguish TCR and CD28 signaling: TCR ligation is associated with tyrosine phosphorylation of a 36-kDa Grb2 SH2 domain-binding protein and a 75-kDa Grb2 SH3-domain binding protein, whereas B7-1 and B7-2 have no discernible regulatory effect on endogenous Grb2 complexes. Accordingly, p36 and p75 seem to be selective substrates for TCR-activated PTKs rather than CD28-activated PTKs. In contrast, B7-1 and B7-2 induce tyrosine phosphorylation of a 62-kDa adapter molecule. p62 appears to have a selective function in CD28 signaling as it is not a substrate for TCR-regulated PTKs.
The Grb2-associated p36 molecule
has also been described to associate with phospholipase C and may thus
link the TCR to both p21 and calcium signaling
pathways(28) . Previous work has established that the
regulation of intracellular calcium and that of p21
are
TCR-regulated (but not CD28-regulated) responses. The failure of B7-1
or B7-2 to induce tyrosine phosphorylation of p36 may thus explain this
divergence of TCR and CD28 signal transduction mechanisms. CD28
ligation with B7-1 or B7-2 induces identical tyrosine phosphorylation
of 62-, 95-, and 120-kDa proteins that can bind to GST-Grb2 fusion
proteins. The 120-kDa protein complex that binds to the SH3 domains of
GST-Grb2 fusion proteins was shown recently to include the
proto-oncogene c-cbl(41) . The data in Fig. 2B indicate that c-cbl is a common
substrate for TCR- and CD28-regulated PTKs.
The present data show
that the proteins that are common targets for both TCR- and
CD28-activated PTKs include p95. Vav function is
essential for T cell development and activation, indicating that Vav
has an important role in TCR
function(44, 45, 46) . However, if tyrosine
phosphorylation of Vav is a marker for its functional regulation, then
the present data suggest that Vav may also have a role in CD28 signal
transduction. Interestingly, one of the many immune defects in
Vav-deficient mice is a defect in cytokine production by T cells, which
could occur as a consequence of the disruption of either the TCR or
CD28 signaling pathway.
The 95-kDa tyrosine phosphoprotein seen in
the GST-Grb2 protein completes was shown by Western blot analyses to be
p95 (data not shown). GST fusion protein binding
experiments are valuable for mapping protein/protein interactions, but
can generate artifacts and show associations between proteins that are
purely in vitro phenomena, i.e. that are not
physiologically relevant because they do not occur in vivo under normal conditions. Vav/Grb2 association appears to fall in
this latter category because Vav could not be detected in association
with endogenous Grb2 complexes, nor could CD28 or the 62- and 120-kDa
tyrosine phosphoproteins. With this GST-Grb2 approach, we are able to
show a specific substrate of CD28-induced PTKs, p62 (Fig. 3).
There are two tyrosine phosphoproteins, p36 and p75, in endogenous Grb2
complexes in TCR-stimulated T cells; however, no tyrosine
phosphoprotein was detected binding to endogenous Grb2 after CD28
ligation by B7 molecules. Thus, tyrosine phosphorylation of proteins
that bind to endogenous Grb2 is a TCR (but not CD28) response, which
indicates that the adapter Grb2 is an important component of TCR signal
transduction mechanisms, but is not similarly involved in the CD28
costimulatory pathways.
The TCR and CD28 clearly differ in their
ability to induce tyrosine phosphorylation of a 62-kDa
p120-associated molecule. CD28-induced 62-kDa tyrosine
phosphoproteins were also detected in Western blot analyses of total
cell lysates. It is not yet clear whether these CD28-induced 62-kDa
proteins seen in total cell lysates comprise solely the GAP-associated
p62 molecule or whether there are multiple 62-kDa proteins that are
substrates for CD28-induced (but not TCR-induced) PTKs. We show that
B7-1 and B7-2/B70 both regulate tyrosine phosphorylation of p62. p62
has also been described as a Grb2-binding protein, but although p62
could bind to GST-Grb2 in vitro, no p62
Grb2 complexes in vivo could be detected in CD28-activated cells. The
GAP-associated p62 protein is a substrate for CD28-activated (but not
TCR-regulated) PTKs, which implies that p62 may have a selective
function in CD28 (but not TCR) signal transduction. It has been shown
previously that tyrosine phosphorylation of the p62 protein is
triggered by the accessory receptor CD2(47) . These CD2 data,
when coupled with the present results, raise the interesting
possibility that, in T cells, p62 is involved selectively in accessory
receptor signal transduction mechanisms.
The function of p62 is not
known, although its association with p120 has
implicated p62 function in the regulation of p21
. This
conclusion was premature and based entirely on initial observations
that tyrosine phosphorylation of p62 correlates with p21
activation in many cells(43, 48) . However, in T
cells, there is no obvious correlation between p62 tyrosine
phosphorylation and p21
activation. For example, TCR
triggering stimulates p21
, but does not induce p62
tyrosine phosphorylation, whereas B7-mediated activation of CD28 does
not activate p21
or p21
-dependent kinases
such as extracellular signal regulated kinase 2 (19) despite
inducing a strong tyrosine phosphorylation of p62. Moreover, there is
no direct evidence that p62 regulates RasGAPs. Rather, p62, like Grb2,
is now recognized as a multifunctional adapter protein and can probably
link receptor-coupled PTKs to several downstream signal transduction
pathways(33) . In this context, another protein, p190, is
present in the p62
RasGAP complexes. This 190-kDa protein contains
a domain with GTPase activity for the GTP-binding protein Rho and a
second domain that has homology to RhoGAP(49) . Accordingly,
p62 should be considered to have the potential to link
receptor-activated PTKs to the Rho family of GTP-binding proteins. The
role of Rho in T cells is not known, but Rho and a related protein,
Rac, are essential for control of the actin cytoskeleton in
fibroblasts(50) . It has also been reported that Rho can
regulate the activity of phosphatidylinositol 5`-kinase(51) .
CD28 is known to stimulate phosphatidylinositol metabolism via a
mechanism attributed to the regulation of phosphatidylinositol
3`-kinase(24) , and this regulation can occur in response to
both B7-1 and B7-2 ligation of CD28 in human T cells (52) . (
)Future studies should thus explore whether CD28 regulation
of other lipid kinases such as phosphatidylinositol 5`-kinase
contributes to CD28 control of inositol lipid phosphorylation.
B7-1 and B7-2 can both costimulate T cells to produce interleukin-2 (13) , but it was not previously assessed whether the different CD28 ligands use similar intracellular signaling mechanisms to costimulate human T cells. In this context, it has been reported that B7-1 and B7-2 may have different functions in T cell biology in vivo(15, 53) , but this could reflect that the CD28 ligands are differentially expressed by antigen-presenting cells and would thus not be involved in equivalent stages of the T cell activation. It must also be considered that there is a second receptor for B7-1 and B7-2, CTLA4. This study thus provides the first comparison of the intracellular signals generated by B7-1 and B7-2/B70, and although the data reveal differences in TCR- and CD28 (using its ligands)-induced signaling pathways, no differences in B7-1 and B7-2 signal transduction responses could be detected. In particular, B7-1 and B7-2 can both induce tyrosine phosphorylation of similar cellular substrates. There is differential regulation of adapter proteins by the TCR and CD28 that probably explains the ability of these receptors to initiate divergent signal transduction responses during T cell activation.