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INTRODUCTION |
CD28 is a 44-kDa homodimer surface glycoprotein expressed by most
mature T cells (1-3). This molecule is critical for T cell activation.
Thus, mice lacking CD28 because of targeted gene disruption show
significant immune system defects consistent with disrupted T cell
function (4, 5). Furthermore, CD28-initiated signals regulate T cell
receptor (TCR)1-mediated
activation, as TCR-induced T cell proliferation and cytokine release
are markedly reduced in the absence of CD28 ligation (2, 3). The
molecular mechanisms by which CD28 augments TCR function are not clear.
Recent studies, however, have shown that CD28 and the TCR complex
initiate distinct intracellular signals (2, 3). For example,
TCR-induced IL-2 production is almost completely inhibited by
cyclosporin A, yet this drug has no effect on the production of IL-2 by
the ligation of CD28 in conjunction with phorbol 12-myristate
13-acetate (PMA) (6-8). These data, together with the fact that CD28
but not TCR induces the tyrosine phosphorylation of the adaptor protein
Dok (9-13), support the notion that TCR and CD28 induce biochemically
distinct intracellular signals.
Protein tyrosine kinases (PTKs) are activated after the ligation of
various receptors, including TCR and CD28 (14-19). Ligating TCR
rapidly activates the Src PTKs Fyn and Lck, leading to the phosphorylation of the immunoreceptor tyrosine-based activation motifs
in the CD3 complex (16, 18-20). Phosphorylated immunoreceptor tyrosine-based activation motifs interact with the Src homology (SH) 2 domains of the Syk/Zap family PTKs, leading to the activation of these
PTKs and in turn to the phosphorylation of an array of cellular
proteins (16, 18-21). Similarly, CD28 signaling pathways involve the
phosphorylation of several proteins on tyrosine residues (11-13,
22-28). Indeed, such phosphorylations are critical for CD28 function
(12, 23). The cytoplasmic region of CD28 becomes rapidly
tyrosine-phosphorylated after CD28 ligation, presumably by Fyn and Lck
(24, 29). Tyrosine-phosphorylated CD28 associates with Src homology
(SH)2-containing proteins including Itk (a member of the Tec family),
phosphatidylinositol 3-kinase (PI 3-kinase), and growth factor
receptor-bound protein 2 (Grb2) (22, 27-29, 30-35). CD28-Grb2
association may link CD28 signaling pathways to Ras and in turn to
downstream events such as mitogen-activated protein (MAP) kinase (36,
37). Recent studies suggest that CD28 signaling pathways also involve
the activation of Rho family GTPases. For example, CD28 ligation
induces tyrosine phosphorylation and activation of Vav, a guanine
nucleotide exchange factor for Rho family GTPases (13, 22, 35, 36), and
promotes the formation of focal adhesion-like sites where these GTPases
accumulate (38). Rho family GTPases are implicated in regulating the
activation of the MAP kinase c-Jun NH2-terminal kinase
(JNK) (37, 39, 40). Accordingly, JNK activation has been reported to be
regulated by signals initiated after CD28 aggregation (41).
Recently, a new family of PTKs has been identified as the focal
adhesion PTK family. This family consists of the non-receptor, proline-rich PTKs Fak (Focal Adhesion
Kinase) and Pyk2 (Proline-rich tyrosine kinase 2, also designated CAK
,
RAFTK, FAK2, or CADTK) (42-48). These kinases have a molecular mass of
110-125 kDa and are closely related in their overall structures. Fak
is expressed in almost all tissues, whereas Pyk2 is expressed mainly in
the central nervous system and in cells and tissues derived from
hematopoietic lineages. Although in adherent cells Fak is localized to
focal adhesion sites, Pyk2 is mainly diffused throughout the cytoplasm (49, 50). Fak and Pyk2 become tyrosine-phosphorylated and activated
after the stimulation of various receptors including TCR (44, 45, 47,
51-64), and both kinases have been linked to the signaling pathways
that regulate MAP kinases (45, 48, 58, 61, 59).
In the present study, we examined the involvement of Fak/Pyk2 in the
CD28 signaling pathways. We found that Pyk2, but not Fak or its
substrate paxillin, is rapidly tyrosine-phosphorylated and activated
after CD28 ligation. We also found that simultaneously ligating CD28
and TCR had an additive effect on tyrosine phosphorylation of Pyk2.
Further analysis of CD28- and TCR-induced tyrosine phosphorylation of
Pyk2 showed that CD28 appears to utilize signaling pathways to
phosphorylate Pyk2 distinct from those used by TCR.
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EXPERIMENTAL PROCEDURES |
Reagents--
RPMI was purchased from Mediatech Cellgro
(Herndon, VA), and EMEM was purchased from BioWhittaker (Walkersville,
MD). Fetal calf serum was purchased from HyClone Laboratories, Inc.
(Logan, UT). Antibiotic/antimycotic mixture and glutamine were from
Life Technologies, Inc. Aprotinin, piceatannol, phenylmethylsulfonyl fluoride, PMA, protease-free bovine serum albumin (BSA), protein A-agarose beads, and wortmannin were from Sigma. LumiGLO
chemiluminescence substrate kit was purchased from Kirkegaard & Perry
Laboratories (Gaithersburg, MD). Polyvinylidene fluoride membranes were
from Millipore (Bedford, MA). Chelerythrine chloride were obtained from
Biomol (Plymouth Meeting, PA).
Antibodies--
Anti-human CD28 mAb (Leu-28, clone L293) was
purchased from Becton Dickinson (San Jose, CA). This anti-CD28 mAb does
not induce IL-2 production from Jurkat T cells, but it significantly
enhances IL-2 release induced by the combination of PMA and
Ca2+ ionophore.2
Anti-human CD3 mAb was from Ancell (Bayport, MN). Anti-phosphotyrosine mAb PY20, anti-Pyk2 mAb, anti-paxillin mAb, anti-Fak mAb, anti-PKC
mAb, and anti-PKC
mAb were obtained from Transduction Laboratories (Lexington, KY). Anti-PKC
rabbit polyclonal antibody was purchased from Promega (Madison, WI). Normal mouse IgG (NMG), rabbit anti-mouse IgG, and horseradish peroxidase-conjugated goat anti-mouse or rabbit
immunoglobulins were from Jackson ImmunoResearch (Bar Harbor, ME).
Cell Activation and Preparation of Cell Lysates--
Acute human
T cell leukemia (Jurkat) cells, clone E6-1, were obtained from American
Type Culture Collection (Bethesda, MD). Before activation, cells were
washed with RPMI containing 0.01% BSA, resuspended in the same media
in polyethylene tubes (Fisherbrand, Fisher). The cells were
then incubated with NMG, anti-CD28 mAb, or anti-CD3 mAb, and the tubes
were placed in a 37 °C water bath for the indicated time. Following
incubation, the cells were solubilized by adding an equal volume of 2×
ice-cold solubilizing buffer (10 mM Tris-HCl, pH 7.5, 1%
Triton X-100, 0.5% sodium deoxycholate, 50 mM NaCl, 50 mM NaF, 2 mM Na3VO4,
0.5 unit/ml aprotinin, and 1 mM phenylmethylsulfonyl
fluoride; final concentrations) and immediately placed on ice (65). The
cells in the tubes were vortexed vigorously every 10 min. Following
incubation, solubilized cells were moved to chilled 1.5-ml Eppendorf
tubes, and insoluble material was removed by centrifugation at
21,000 × g for 30 min at 4 °C.
For costimulation studies, T cells in 0.01% BSA/RPMI were stimulated
in suspension for 10 min at 37 °C with different concentrations of
anti-human CD3 mAb in the presence or absence of 3 µg/ml of anti-CD28
mAb. Cell lysates were processed as described above. For stimulation in
the absence of extracellular Ca2+, cells in suspension were
divided into 2 aliquots (66). In 1 aliquot, the cells were washed in
EMEM containing 0.01% BSA and then resuspended in the same media and
stimulated with anti-CD28 mAb or anti-CD3 mAb as above. The cells in
the other aliquot were washed with Ca2+-free EMEM
containing 0.01% BSA and 4 mM EDTA, suspended in EMEM containing 0.01% BSA and 50 µM EDTA, and then stimulated
with anti-CD28 mAb or anti-CD3 mAb. Cell activation was as described above. To study the effects of inhibitors, T cells were pretreated in
suspension for 30 min at 37 °C with different concentrations of the
PTK Syk/Zap inhibitor, piceatannol, or with the lipid kinase PI
3-kinase inhibitor wortmannin. After washing, the cells were resuspended in 0.01% BSA/RPMI and stimulated with anti-CD3 mAb or
anti-CD28 mAb as above. To deplete protein kinase C (PKC), cells were
incubated for 16 h at 37 °C with 400 nM PMA. After washing, the cells were resuspended in 0.01% BSA/RPMI and stimulated with anti-CD3 mAb or anti-CD28 mAb as above.
Immunoprecipitation and Immunoblotting--
Cell lysates from
8 × 106 cells were precleared by incubating for
1 h with 10 µg of rabbit anti-mouse immunoglobulin that had been
preincubated for 2 h with 50 µl of protein A-agarose. Meanwhile, the primary antibody was added to 10 µg of rabbit anti-mouse
immunoglobulin that had been preincubated for 2 h at 4 °C with
50 µl of protein A-agarose beads. Precleared lysates were then added
to the protein A-antibodies mixture and incubated for 2 h at
4 °C. After incubation, the beads were pelleted by centrifugation
and washed 5 times with ice-cold solubilization buffer. After the final
centrifugation, the beads were resuspended in 2× SDS-polyacrylamide
gel electrophoresis sample buffer (final concentration, 75 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol) and boiled for
5 min. For immunoblotting, aliquots from whole cell lysates (WCL) or
immunoprecipitates were separated by SDS-polyacrylamide gel
electrophoresis (10%) and electrotransferred onto polyvinylidene
fluoride membranes. Immunoblotting with the horseradish
peroxidase-conjugated anti-phosphotyrosine antibody PY-20 was as
described previously (62, 66, 67). To confirm similar loading of
samples, antibodies were stripped from the membranes as previously
reported (66), and the proteins were reprobed with specific antibodies
followed by horseradish peroxidase-coupled rabbit anti-mouse
immunoglobulin (1:50,000 dilution). The signals were visualized using
the LumiGLO kit according to the manufacturer's recommendations.
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RESULTS |
Induction of Pyk2 Tyrosine Phosphorylation by Aggregating
CD28--
To examine whether the PTKs Pyk2 and Fak are involved in
CD28 signaling, Jurkat T cells were treated with anti-CD28 mAb, and the
immunoprecipitates of Pyk2 and Fak were transferred to membranes and
probed with anti-phosphotyrosine mAb. Immunoblotting of WCL with
anti-phosphotyrosine mAb showed that aggregating CD28 on Jurkat T cells
induces tyrosine phosphorylation of several proteins, the most
prominent of which were 42-46-, 65-, 78-, and 95-kDa proteins (Fig.
1A). In contrast, NMG did not
induce detectable protein tyrosine phosphorylation. CD28-induced
tyrosine phosphorylation was apparent within 2 min, peaked at 5 min,
and decreased slowly after that (Fig. 1B). As shown in Fig.
2A, treating the cells with 1 or 3 µg/ml anti-CD28 mAb increased tyrosine phosphorylation of Pyk2.
No detectable increase in the tyrosine phosphorylation of the kinase
was seen in cells treated with NMG (Fig. 2A). Time course
studies showed that the phosphorylation of Pyk2 by CD28 cross-linking
was first apparent within 2 min, reached a peak by 10 min, and
decreased at 20 min (Fig. 2B). In contrast, incubating the
cells with anti-CD28 mAb did not induce any detectable tyrosine phosphorylation of Fak (Fig. 2C). Fak tyrosine
phosphorylation was not detected after CD28 ligation even when more
cells were used in immunoprecipitation, when cells were stimulated with
plate-bound anti-CD28 mAb, or when the probed membranes were exposed
for extended periods to films (data not shown). The cytoskeletal
protein, paxillin, becomes strongly tyrosine-phosphorylated in response
to the ligation of various receptors including TCR (66, 68, 69).
However, CD28 ligation failed to induce the tyrosine phosphorylation of paxillin (Fig. 2D). These data show that Pyk2 is involved in
CD28 signaling and suggest that neither Fak nor paxillin is involved in
the signaling pathways of CD28.

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Fig. 1.
Induction of protein tyrosine phosphorylation
by aggregating CD28. A, Jurkat T cells were treated
with 3 µg/ml NMG or anti-CD28 mAb for 5 min at 37 °C. Proteins in
cell lysates were separated by SDS-polyacrylamide gel electrophoresis,
transferred to membranes, and immunoblotted with anti-phosphotyrosine
mAb ( -PY). Stim., stimulus. B,
Jurkat T cells were stimulated with 3 µg/ml anti-CD28 mAb for the
indicated time. Tyrosine-phosphorylated proteins were detected as in
A. Arrows indicate proteins that become
tyrosine-phosphorylated after CD28 ligation.
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Fig. 2.
CD28 ligation induces tyrosine
phosphorylation of Pyk2 but not of Fak or paxillin. A,
Jurkat T cells were treated with 1 or 3 µg/ml NMG or anti-CD28 mAb
for 5 min at 37 °C. Pyk2 was immunoprecipitated from 8 × 106 cells and analyzed by immunoblotting with
anti-phosphotyrosine mAb ( -PY, upper panel) or with
anti-Pyk2 mAb (lower panel). Stim., stimulus.
B-D, Jurkat T cells were stimulated with 3 µg/ml
anti-CD28 mAb for the indicted time at 37 °C. Cell lysates were
immunoprecipitated with anti-Pyk2 mAb (B), anti-Fak mAb
(C), or anti-paxillin mAb (D). Immunoprecipitates
were analyzed by immunoblotting with anti-phosphotyrosine mAb
( -PY, upper panel) or with the specific mAb (lower
panel).
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Concurrent Ligation of CD28 and TCR Leads to Additive Tyrosine
Phosphorylation of Pyk2--
It is now well established that signals
from TCR and CD28 cooperate in the activation of T cells. Previous
studies have shown that cross-linking TCR increases the tyrosine
phosphorylation of Pyk2 (52, 53). Thus, we examined the effect of
simultaneously aggregating CD28 and TCR on tyrosine phosphorylation of
Pyk2. Immunoblotting WCL with anti-phosphotyrosine mAb showed that
CD28- and CD3-induced protein tyrosine phosphorylation was different in
pattern and intensity (Fig.
3A). Thus, some tyrosine
phosphoproteins were common to TCR- and CD28-activated cells, but in
general, the level of phosphorylation was stronger in the TCR-activated cells (Fig. 3A). These results are similar to those
previously reported (11-13, 24). No enhanced tyrosine phosphorylation
could be detected in WCL as a result of concurrently ligating CD28 and TCR (Fig. 3A). To examine the effect of simultaneously
ligating CD28 and TCR on Pyk2 tyrosine phosphorylation, Jurkat T cells were stimulated for 10 min at 37 °C with different concentrations of
anti-human CD3 mAb in the presence or absence of 3 µg/ml anti-CD28 mAb. As shown in Fig. 3B, concurrent ligation of CD28 and
TCR increased tyrosine phosphorylation of Pyk2. Interestingly, when low
concentrations of anti-CD3 mAb were used, the extent of phosphorylation by both receptors was equivalent to the sum of that induced by each
receptor alone, whereas at high concentrations the phosphorylation by
ligating both receptors was less than the sum of that induced by each
receptor alone (Fig. 3, B and C). Similar results
were obtained when the cells were stimulated with 0.5 instead of 3 µg/ml anti-CD28 mAb (data not shown).

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Fig. 3.
Concurrent ligation of CD28 and TCR leads to
additive tyrosine phosphorylation of Pyk2. Jurkat T cells were
stimulated for 10 min at 37 °C with different concentrations of
anti-human CD3 mAb in the presence or absence of 3 µg/ml anti-CD28
mAb. A, WCL immunoblotted with anti-phosphotyrosine mAb
( -PY). B, Pyk2 immunoprecipitates prepared
from 8 × 106 cells were immunoblotted with
anti-phosphotyrosine mAb ( -PY, upper panel) or with
anti-Pyk2 mAb (lower panel). C, densitometry of
the data in the upper panel of B.
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Optimal CD28-induced Pyk2 Tyrosine Phosphorylation Requires
Ca2+--
The engagement of CD28 has been shown to
increase the levels of intracellular Ca2+ (8); thus, we
examined the role of Ca2+ in CD28-induced tyrosine
phosphorylation of Pyk2 by stimulating the cells with anti-CD28 in the
presence or absence of external Ca2+. As shown in Fig.
4A, tyrosine phosphorylation
of several proteins induced with anti-CD28 mAb or anti-CD3 mAb was
profoundly reduced or completely abolished in the absence of
Ca2+. As previously reported, anti-CD3 mAb-induced tyrosine
phosphorylation of Pyk2 was reduced by Ca2+ depletion (Fig.
4B) (52). CD28-induced Pyk2 tyrosine phosphorylation was
also reduced in the absence of Ca2+ (Fig. 4B).
Whether the incomplete inhibition of receptor-mediated Pyk2 tyrosine
phosphorylation is due to the release of Ca2+ from
intracellular stores or due to the tyrosine phosphorylation of Pyk2 by
mechanisms that do not require Ca2+ awaits further
investigation. Nonetheless, these results show that optimal tyrosine
phosphorylation of Pyk2 by CD28 signaling requires
Ca2+.

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Fig. 4.
Optimal CD28-induced Pyk2 tyrosine
phosphorylation requires external Ca2+. Jurkat T cells
were divided into two aliquots. In one aliquot
(Ca2+, lane +), the cells were washed
in EMEM containing 0.01% BSA and then resuspended in the same media
and stimulated with 3 µg/ml anti-CD28 mAb or anti-CD3 mAb for 5 min
at 37 °C. The cells in the other aliquot
(Ca2+, lane ) were washed with
Ca2+-free EMEM containing 0.01% BSA and 4 mM
EDTA, suspended in EMEM containing 0.01% BSA and 50 µM
EDTA, and then stimulated with 3 µg/ml anti-CD28 mAb or anti-CD3 mAb
for 5 min at 37 °C. A, WCL immunoblotted with
anti-phosphotyrosine mAb ( -PY). Stim.,
stimulus. B, Pyk2 immunoprecipitates prepared from 8 × 106 cells were immunoblotted with anti-phosphotyrosine mAb
( -PY, upper panel) or with anti-Pyk2 mAb (lower
panel).
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The Syk/Zap Inhibitor Piceatannol Blocks Both TCR and CD28-induced
Tyrosine Phosphorylation of Pyk2--
Syk and the related PTK Zap are
critical for TCR function; however, their role in CD28 signaling is not
clear. Syk has been shown to be critical for Fc
RI-mediated tyrosine
phosphorylation of Pyk2 in mast cells (where Fc
RI is high affinity
IgE receptor) (51). Thus, we used the specific Syk/Zap inhibitor
piceatannol (70) to examine the role of these PTKs in CD28- and
TCR-induced Pyk2 tyrosine phosphorylation. As shown in Fig.
5A, pretreating the cells with
piceatannol reduced tyrosine phosphorylation of several proteins
induced by CD28 or TCR. This is not surprising, as Syk/Zap become
activated early in signaling cascades. Interestingly, piceatannol
inhibited both CD28- and TCR-induced tyrosine phosphorylation of Pyk2
(Fig. 5B). Such inhibition was evident at concentrations that have been reported previously to selectively influence Syk/Zap activity (70). Furthermore, piceatannol appears to be more effective in
inhibiting CD28-induced tyrosine phosphorylation of Pyk2 than inhibiting CD3-induced Pyk2 phosphorylation. Although these data suggest that Syk/Zap are involved in TCR- and CD28-induced tyrosine phosphorylation of Pyk2; the possibility that piceatannol is also inhibiting PTKs other than Syk/Zap (or directly inhibiting Pyk2) cannot
be ruled out. Thus, the precise role of Syk/Zap in CD28- and
TCR-induced tyrosine phosphorylation of Pyk2 requires further investigation.

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Fig. 5.
The Syk/Zap inhibitor piceatannol blocks TCR
and CD28-induced tyrosine phosphorylation of Pyk2. Jurkat T cells
were pretreated for 30 min at 37 °C with the indicated
concentrations of piceatannol (PIC). After washing, the cells were
resuspended in 0.01% BSA/RPMI and stimulated with 3 µg/ml anti-CD28
mAb or anti-CD3 mAb for 5 min at 37 °C. A, WCL
immunoblotted with anti-phosphotyrosine mAb ( -PY).
Stim., stimulus. B, Pyk2 immunoprecipitates
prepared from 8 × 106 cells were immunoblotted with
anti-phosphotyrosine mAb ( -PY, upper panel) or with
anti-Pyk2 mAb (lower panel).
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The PI 3-Kinase Inhibitor Wortmannin Inhibits TCR- but Not
CD28-induced Tyrosine Phosphorylation of Pyk2--
As the PI 3-kinase
has been implicated in the signaling pathways of TCR and CD28, we
examined the effect of the PI 3-kinase inhibitor wortmannin (71) on
CD28- and TCR-induced tyrosine phosphorylation of Pyk2. Notably,
wortmannin enhanced CD28- and TCR-induced tyrosine phosphorylation of
several proteins (Fig. 6A).
Similar enhancement by wortmannin of TCR-induced protein tyrosine
phosphorylation has been previously reported (72). Treating the cells
with wortmannin invariably decreased tyrosine phosphorylation of Pyk2
induced by aggregating TCR in a dose-dependent manner (Fig.
6B). In contrast, wortmannin had no apparent effect on
CD28-induced tyrosine phosphorylation of Pyk2 (Fig. 6B).
These data suggest that TCR and CD28 induce Pyk2 tyrosine
phosphorylation by distinct pathways.

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Fig. 6.
The PI 3-kinase inhibitor wortmannin inhibits
CD3- but not CD28-induced tyrosine phosphorylation of Pyk2. Jurkat
T cells were pretreated for 30 min at 37 °C with the indicated
concentrations of wortmannin (WT). After washing, the cells
were resuspended in 0.01% BSA/RPMI and stimulated with 3 µg/ml
anti-CD3 mAb or anti-CD28 mAb for 5 min at 37 °C. A, WCL
immunoblotted with anti-phosphotyrosine mAb ( -PY).
Stim., stimulus. B, Pyk2 immunoprecipitates
prepared from 8 × 106 cells were immunoblotted with
anti-phosphotyrosine mAb ( -PY, upper panel) or with
anti-Pyk2 mAb (lower panel).
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Depleting PMA-sensitive PKCs Does Not Block CD28- and CD3-induced
Pyk2 Tyrosine Phosphorylation--
PKC is activated after CD28 and
CD3 ligation (2, 3, 15, 16); thus, we examined whether
receptor-mediated tyrosine phosphorylation of Pyk2 requires PKC. Jurkat
cells were treated for 16 h with PMA (400 nM) to
deplete Ca2+-dependent and
Ca2+-independent PKC (66, 73-75). As shown in Fig.
7A, PMA treatment of Jurkat T
cells markedly reduced PKCs, as determined by blotting WCL with
antibody to Ca2+-dependent PKC
and
Ca2+-independent PKC
. Atypical PKC
was not decreased,
as atypical PKCs are insensitive to PMA (73-75). Interestingly, the
marked decrease in PMA-sensitive PKCs did not block CD28- and
CD3-induced tyrosine phosphorylation of Pyk2 (Fig. 7B).
Similarly, pretreating Jurkat T cells for 72 h with
10
6 M of the PKC inhibitor chelerythrine
chloride did not block receptor-mediated tyrosine phosphorylation (not
shown). Together, the data strongly suggest that CD28 and CD3 induce
tyrosine phosphorylation of Pyk2 by mechanisms that are independent of
PMA-sensitive PKCs.

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Fig. 7.
Depleting PMA-sensitive PKCs does not block
CD28- and CD3-induced Pyk2 tyrosine phosphorylation. A,
Jurkat T cells were incubated for 16 h at 37 °C with 400 nM PMA. To confirm PKC depletion, WCL were immunoblotted
with antibody to Ca2+-dependent PKC ,
Ca2+-independent PKC , and atypical PKC . B,
cells treated as in A were resuspended in 0.01% BSA/RPMI
and stimulated with 3 µg/ml anti-CD3 mAb or anti-CD28 mAb for 5 min
at 37 °C. Pyk2 immunoprecipitates were then blotted with
anti-phosphotyrosine mAb ( -PY, upper panel) or with
anti-Pyk2 mAb (lower panel). Stim.,
stimulus.
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DISCUSSION |
PTKs have been shown to be critical for CD28-mediated T cell
activation (12, 23). Here we identify the PTK Pyk2 as a potential player in CD28 signaling, as this kinase became tyrosine-phosphorylated after CD28 ligation. In contrast, the related PTK Fak was not tyrosine-phosphorylated in response to receptor aggregation. Similar tyrosine phosphorylation of Pyk2 but not of Fak has been previously reported for other receptors (47, 56). Although Fak and Pyk2 are
structurally related, they localize to different sites within the cell.
This has been attributed to differences in the focal adhesion targeting
sequence at their COOH-terminal domains (49, 50). Hence, in adherent
cells Fak is mainly localized to focal adhesion sites, whereas Pyk2 is
mainly diffused throughout the cytoplasm (49, 50). Thus, the inability
of CD28 ligation to induce Fak tyrosine phosphorylation could be due to
the compartmentalization of Fak to areas not involved in CD28 signaling.
Our data showed that the potent PI 3-kinase inhibitor wortmannin
inhibited CD3- but not CD28-induced Pyk2 tyrosine phosphorylation. These results suggest that PI 3-kinase activation is involved in TCR-
but not in CD28-induced tyrosine phosphorylation of Pyk2. Recent
studies, however, have shown that some of PI 3-kinase effects are
wortmannin-resistant. For example, the p85 subunit of PI 3-kinase has
been reported to function as an adaptor molecule in a manner that is
unrelated to the lipid or serine kinase activity of the p110 subunit
(76). Thus, at this time we cannot conclusively dismiss the role of the
p85 subunit of PI 3-kinase in CD28-induced tyrosine phosphorylation of
Pyk2. Regardless, the wortmannin data strongly suggest that CD28 and
TCR induce tyrosine phosphorylation of Pyk2 by distinct mechanisms.
The intracellular signals generated after CD28 aggregation integrate
with those initiated after TCR ligation to induce optimal T cell
activation (2, 3, 41, 77). In this report, coligating CD28 and TCR
increased the tyrosine phosphorylation of Pyk2 compared with that
induced by aggregating each receptor alone. The extent of tyrosine
phosphorylation of Pyk2 brought about by simultaneous stimulation of
both receptors was equivalent to the sum of that induced by each
receptor alone; thus, the effect of stimulating both receptors is
additive and not synergistic. These data suggest that the signals
generated by both receptors do not integrate at the level of Pyk2 and
that both receptors induce tyrosine phosphorylation of Pyk2 by distinct
pathways, a possibility supported by our data with wortmannin (see
above). If Pyk2 is playing a role in the costimulatory process, then
Pyk2 tyrosine phosphorylated by each receptor must regulate downstream
signals that eventually integrate to enhance T cell activation.
Pyk2 does not contain SH2 and SH3 binding domains; therefore, the
phosphorylation of this kinase on tyrosine is critical for its
interaction with the SH2 domains of other signaling molecules. Accordingly, the tyrosine phosphorylation of Pyk2 has been reported to
result in its association with several SH2-containing signaling molecules, including Src kinases, and Grb2 (48, 52, 53, 60, 78). The
association of Pyk2 with the Grb2·Sos1 complex may link Pyk2 to the
small GTPase Ras signaling pathway. Recent studies also link Pyk2 to
the signaling pathways of the Rho family GTPases CDC42 and Rac1 (57).
These GTPases are regulated by guanine nucleotide exchange factors such
as Vav and by GTPases such as Ras and have been implicated in
controlling the activation of JNK (37, 39, 40). Indeed, there is
evidence implicating Pyk2 in pathways that regulate JNK activation. For
example, overexpression of Pyk2 in human embryonic kidney cells
activated JNK and induced the phosphorylation of the glutathione
S-transferase-c-Jun fusion protein (57). Furthermore, the
expression of a catalytically inactive mutant of Pyk2 inhibited the
activation of JNK induced by both UV light and by sorbitol treatment of
PC-12 cells (57) and by MIP1
in a murine pre-B lymphoma cell line
(55). In T cells, JNK activation appears to be controlled by signals
triggered by CD28 ligation, as the cross-linking of TCR in the absence
of CD28 ligation failed to activate JNK (41). Because Pyk2 upon phosphorylation associates with signaling proteins, and because it
appears to function upstream of GTPases, the tyrosine phosphorylation of Pyk2 after CD28 ligation may be important for coupling
CD28-initiated signals to GTPases and, in turn, to the JNK cascade.
However, tyrosine phosphorylation of Pyk2 by itself may not be
sufficient to activate JNK, as Pyk2 becomes tyrosine-phosphorylated by
TCR ligation in the absence of JNK activation. Thus, additional
signals initiated by CD28 ligation may also be required for JNK
activation. Alternatively, CD28 and TCR ligation may cause tyrosine
phosphorylation at different sites of Pyk2, and only those
associated with CD28 ligation trigger pathways leading to JNK activation.