Protein Kinase C
Cooperates with Vav1 to Induce JNK
Activity in T-cells*
Andreas
Möller,
Oliver
Dienz,
Steffen P.
Hehner,
Wulf
Dröge, and
M. Lienhard
Schmitz
From the German Cancer Research Center, Division of Immunochemistry
(G0200), Im Neuenheimer Feld 280, 69120 Heidelberg, Germany
Received for publication, December 11, 2000, and in revised form, February 13, 2001
 |
ABSTRACT |
Here we show that in human T-cell leukemia cells
Vav1 and protein kinase C
(PKC
) synergize for the activation of
c-Jun N-terminal kinase (JNK) but not p38 MAP kinase. Vav1 and PKC
also cooperated to induce transcription of reporter genes controlled either by AP-1 binding sites or the CD28RE/AP composite element contained in the IL-2 promoter by stimulating the binding of
transcription factors to these two elements. Dominant negative
versions of Vav1 and PKC
inhibited CD3/CD28-induced activation of
JNK, revealing their relative importance for this activation pathway.
Gel filtration experiments revealed the existence of constitutively
associated Vav1/PKC
heterodimers in extracts from unstimulated
T-cells, whereas T-cell costimulation induced the recruitment of Vav1
into high molecular weight complexes. Several experimental approaches showed that Vav1 is located upstream from PKC
in the control of the
pathway leading to synergistic JNK activation. Vav1-derived signals
lead to the activation of JNK by at least two different pathways. The major contribution of Vav1 for the activation of JNK relies on the PKC
-mediated Ca2+-independent
synergistic activation pathway, whereas JNK is also activated by a
separate Ca2+-dependent signaling route.
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INTRODUCTION |
Activation of the T-cell antigen receptor complex is not
sufficient for T-lymphocyte activation and requires additional signals provided by the occupancy of costimulatory receptors such as CD28 (1).
Full activation of T-cells by T-cell antigen receptor/CD28 costimulation initiates a series of intracellular signaling events. A
hallmark of costimulated T-cells is the synergistic activation of
JNK,1 NF-
B, and IL-2
expression (2). Receptor clustering leads to the activation of protein
tyrosine kinases of the Src and Syk families, which phosphorylate
numerous substrate proteins, thus leading to dynamic formation (3) or
disruption (4) of multi-protein signaling complexes. These complexes
and the T-cell antigen receptor itself associate with lipid rafts,
which form a structural scaffold and promote further signaling events
(5).
Activated protein tyrosine kinases phosphorylate multiple target
proteins including phospholipase C
, which controls the
phosphatidyl inositol lipid metabolism, thereby producing inositol
triphosphate and diacylglycerols (6). Whereas inositol triphosphate
results in a rapid and sustained calcium increase, diacylglycerol
mediates activation of PKC family members (6, 7). Among those, the novel Ca2+-independent PKC isoform PKC
is of special
importance for T-cells, because it is rapidly recruited to the site of
contact between T-cells and antigen-presenting cells (8). Another
protein tyrosine kinase-induced signaling route is mediated by the Vav1
protein family member Vav1, which is exclusively expressed in
hematopoietic cells (9). T-cell costimulation induces the membrane
recruitment of Vav1 via indirect, adaptor protein SLP-76-mediated
binding to the membrane protein LAT (linker for activation of T-cells) (10). Protein tyrosine kinase-induced phosphorylation and
phosphatidylinositol-3,4,5-triphosphate binding of Vav1 activate its
GDP/GTP exchange factor activity for the Rho family of GTPases such as
Rac and Cdc42 (11, 12) and results in the stimulation of signaling
pathways and alterations in cell shape and motility.
T-cell costimulation also leads to the activation of mitogen-activated
protein kinase pathways. However, the synergistic activation appears to
be unique for the mitogen-activated protein kinases JNK and p38 (13),
because neither extracellular signal-regulated kinase nor transcription
factor nuclear factor of activated T-cells require
coreceptor-derived signals. JNK phosphorylates various transcription
factors including ATF2, ELK-1, and components of the AP-1 heterodimer,
namely JunB, JunD, and c-Jun (14). Because AP-1 contributes to the
induced expression of numerous target genes including IL-2
and IL-4, the JNK pathway has been implicated in
various functions including cell proliferation, effector T-cell function (15), T-cell activation (16), and the regulation of apoptosis
(14). However, these functions are dependent on the inducing signal and
the cell type (15, 16). JNK is activated by the dual specificity JNK
kinases (JNKKs) MKK4/JNKK1/SEK1 and JNKK2/MKK7 (17). A variety of
different kinases can activate the JNKKs, but the cell type and the
nature of the JNK-inducing stimulus determines which of these kinases
is operational (14).
Vav1 and PKC
are constitutively associated in unstimulated T-cells
(18), and both proteins synergistically activate transcription factor
NF-
B, JNK activity, and the expression of IL-2, CD69, and IL-4 (4,
19, 20). In this study we have addressed the question whether Vav1 and
PKC
synergize for the activation of JNK for two reasons. 1)
Gain-of-function approaches have revealed that both proteins contribute
to the activation of JNK (10, 21-26) and cooperate with constitutively
active calcineurin or Ca2+ signals to activate JNK. 2) We
have previously seen that the Vav1/PKC
module synergistically
triggers binding of transcription factors to the P1 and PRE-I elements
contained within the IL-4 promoter (19). Because both DNA elements are
bound by AP-1 family members, we tested the effects of Vav1 and PKC
on the activation of JNK. A variety of experimental approaches revealed
cooperative activation of JNK and AP-1-dependent gene
expression by Vav1 and PKC
.
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EXPERIMENTAL PROCEDURES |
Cell Culture, Transfections, and Stimulations--
Jurkat T
leukemia cells expressing the large T antigen were grown in RPMI 1640 medium at 37 °C containing 10% (v/v) heat-inactivated fetal calf
serum, 10 mM HEPES, 1% (v/v) penicillin/streptomycin, 2 mg/ml G418, and 2 mM glutamine (all from Life Technologies, Inc.). Cells were electroporated using a gene pulser (Bio-Rad) at 250 V/950 microfarads. In all transfections, the amount of total DNA (20 µg) was kept constant by the addition of empty expression vector.
Stimulations were performed in a volume of 400 µl by adding agonistic
CD3 (final concentration of 10 µg/ml) and/or
CD28 (final
concentration of 10 µg/ml) antibodies.
Antisera, Plasmids, and Reagents--
The following antibodies
were obtained from the indicated suppliers:
Phospho-p38, New England
Biolabs;
Flag (M2), Sigma;
Myc (9E10), Santa Cruz
Biotechnology, Inc.;
Vav1, Upstate Biotechnology;
PKC
,
Transduction Laboratories;
HA antibody (12CA5), Roche Molecular
Biochemicals. The
CD3 (OKT3),
CD28 (9.3), and isotype-matched control antibodies were kindly provided by Dr. R. Breitkreutz. The
luciferase constructs (AP-1)3-Luc (10), RE/AP-Luc, and
mutant derivatives thereof (27) and expression vectors for myc-tagged Vav1 wild type (27), Flag-tagged Vav1 wild type and DN Vav1 variants
(10), Flag-tagged p38 (28), PKC
A/E, PKC
K/R (19), MKK7 K/L (10),
MEKK1
K/M (29), and MKK4 K/R (30) have been described.
Electrophoretic Mobility Shift Assays (EMSAs) and Luciferase
Determination--
EMSAs were performed using nuclear extracts
essentially as described (19). Equal amounts of nuclear protein were
tested for protein binding to oligonucleotides containing either an
AP-1 binding site or a CD28RE/AP element. The coding strands of the oligonucleotides used were as follows: AP-1,
5'-CGCTTGATGACTCAGCCGGAA-3'; CD28RE/AP,
5'-TCTGGTTTAAAGAAATTCCAAAGAGTCATCAG-3'. The free and the
oligonucleotide-bound proteins were separated by electrophoresis on a
native 4% polyacrylamide gel. Following electrophoresis the gel was
dried and exposed to an x-ray film (Amersham Hyperfilm).
Luciferase activity in cell extracts was measured in a luminometer (Duo
Lumat LB 9507, Berthold) by automatically injecting 50 µl of assay
buffer and measuring light emission for 10 s after injection
according to the instructions of the manufacturer (Promega Inc.). To
ensure comparable transfection efficiencies, results were normalized to
-galactosidase produced by a cotransfected Rous sarcoma
virus-
-galactosidase expression vector.
Cell Extracts and Western Blotting--
Cells were washed with
phosphate-buffered saline, and the pellets were resuspended on ice for
15 min in Nonidet P-40 lysis buffer (20 mM Tris/HCl, pH
7.5, 150 mM NaCl, 1 mM phenylmethylsulfonyl fluoride, 10 mM NaF, 0.5 mM sodium vanadate,
leupeptin (10 µg/ml), aprotinin (10 µg/ml), 1% (v/v)
Nonidet P-40, and 10% (v/v) glycerol). Cell debris was pelleted upon
centrifugation, and the supernatant was either directly analyzed by
Western blotting or used for the determination of JNK activity as
described below. After separation of cell extracts on reducing
SDS-polyacrylamide gels, the proteins were transferred onto a
polyvinylidene difluoride membrane (Millipore) using a semi-dry
blotting apparatus (Bio-Rad). The membrane was then incubated in a
small volume of TBST buffer (25 mM Tris/HCl, pH 7.4, 137 nM NaCl, 5 mM KCl, 0.7 mM
CaCl2, 0.1 mM MgCl2, 0.1% (v/v)
Tween 20) containing various dilutions of the primary antibodies. After
extensively washing the membrane, the immunoreactive bands were
visualized by enhanced chemiluminescence according to the instructions
of the manufacturer (PerkinElmer Life Sciences). Western blots
were quantitated using the Lumi-ImagerTM from Roche
Molecular Biochemicals.
JNK Assays--
Two days posttransfection, cell lysates were
prepared and precleared with protein A/G-Sepharose. The HA-tagged JNK
proteins contained in the cell lysate were precipitated by the addition of 1 µg of
HA antibody and 25 µl of protein A/G-Sepharose. The precipitate was washed three times in lysis buffer and two times in
kinase buffer (20 mM Hepes/KOH, pH 7.4, 25 mM
-glycerophosphate, 2 mM dithiothreitol, 20 mM MgCl2). The kinase assay was performed in a
final volume of 20 µl of kinase buffer containing 2 µg of glutathione S-transferase (GST)-c-Jun-(5-89), 20 µM ATP, and 5 µCi of [
-32P]ATP for 20 min at 30 °C. The reaction was stopped by the addition of 5× SDS
loading buffer, followed by reducing SDS-PAGE, gel fixation, and
quantification of the results in a PhosphorImager.
Gel Filtration--
The analysis of cellular multi-protein
complexes was performed on a Superose 6 column (Amersham Pharmacia
Biotech). Total cell extracts from 3 × 108
Jurkat cells contained in 75 µl of octylglycoside lysis buffer (20 mM Tris/HCl, pH 7.5, 150 mM NaCl, 1 mM phenylmethylsulfonyl fluoride, 5 mM
dithiothreitol, leupeptin (10 µg/ml), aprotinin (10 µg/ml),
1% (v/v) octylglycoside, and 10% (v/v) glycerol) were analyzed at a
flow rate of 0.5 ml/min, and fractions of 500 µl were collected.
Aliquots from the respective fractions were analyzed by Western
blotting for the occurrence of Vav1 and PKC
. The washed columns were
calibrated with pre-made molecular weight standards (Amersham Pharmacia Biotech).
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RESULTS |
A possible synergism between Vav1 and PKC
for the activation of
JNK was tested by transfecting T-cell leukemia Jurkat cells with
expression vectors for HA-tagged JNK together with vectors encoding
constitutively active PKC
(PKC
A/E) and/or wild type Vav1.
The tagged JNK protein was immunoprecipitated, and its kinase activity
was determined by immune complex kinase assays (Fig. 1A). JNK activation triggered
by expression of Vav1 and PKC
alone was synergistically
stimulated upon coexpression of both proteins. Costimulation with
agonistic
CD3/
CD28 antibodies further triggered JNK activity
elicited by Vav1, PKC
A/E, or both. To determine whether the synergy
between Vav1 and PKC
also occurs for the activation of p38, Jurkat
T-cells were cotransfected with expression vectors for Flag-tagged p38
together with different combinations of vectors encoding Vav1 and
PKC
A/E. The tagged p38 protein was immunoprecipitated and analyzed
for its activation (as seen by Thr-180/Tyr-182 phosphorylation) in
Western blot experiments (Fig. 1B). These experiments showed
that Vav1 and PKC
activated p38 only in an additive,
non-synergistic manner, thereby revealing that the cooperation only
occurs for the activation of JNK. To test whether this synergism is
also apparent at the level of induced AP-1 binding to its cognate DNA,
different combinations of expression vectors for Vav1, PKC
A/E, or
the empty expression vector as a control were expressed in Jurkat
cells. Analysis of AP-1 DNA binding activity by EMSAs showed that
either Vav1 or PKC
A/E alone induced DNA binding of AP-1, albeit to
different extents. Coexpression of both proteins synergistically
stimulated the DNA binding activity of AP-1 (Fig.
2B). The impact of Vav1/PKC
expression on the activation of AP-1-dependent
transcription was tested in reporter gene assays. An
AP-1-dependent luciferase gene was transfected into Jurkat
cells together with increasing amounts of Vav1 and/or PKC
A/E
expression vectors (Fig. 2B). The slight induction of AP-1-dependent transcription mediated by Vav1 was strongly
enhanced even by moderate amounts of coexpressed PKC
A/E. These
experiments revealed that the synergism also occurs at the level of DNA
binding and gene expression.

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Fig. 1.
Synergistic activation of JNK by Vav1 and
PKC . A, Jurkat cells were
transiently transfected with HA-tagged JNK and the indicated
combinations of expression vectors for Vav1 (5 µg) and PKC A/E (5 µg). Two days later, cells were either left untreated or stimulated
for 30 min with agonistic CD3/ CD28 antibodies as indicated. JNK
was immunoprecipitated from cell lysates, and its activity was
determined by immune complex kinase assays (KA) using
recombinant GST-c-Jun-(5-89) as substrate. An autoradiogram from a
reducing SDS-gel shows phosphorylation of the recombinant substrate
protein and a quantitative evaluation obtained by phosphorimaging. A
sample of each lysate was analyzed by Western blotting (WB)
for protein expression of PKC (upper panel), Vav1
(middle panel), and JNK (lower panel).
B, an expression vector for Flag-tagged p38 (1 µg) and
Vav1 and/or PKC A/E (5 µg, respectively) were transfected in
Jurkat cells, which were either left untreated or costimulated for 30 min as shown. The tagged p38 protein was immunoprecipitated and
analyzed by Western blotting for p38 expression and phosphorylation.
Samples of whole cell lysates were immunoblotted for the expression
levels of the various transfected proteins (lower
panels).
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Fig. 2.
Synergistic activation of AP-1 by Vav1 and
PKC . A, Jurkat cells were
transfected either with empty expression vector or with plasmids
encoding Vav1 (10 µg) and/or PKC A/E (10 µg) at the indicated
combinations. The next day, nuclear cell extracts were prepared, and
the DNA binding activity of AP-1 was determined by EMSAs. An
autoradiogram is displayed; the arrow indicates the location
of the DNA-AP-1 complex, and the circle indicates the
position of the unbound oligonucleotide. B, Jurkat cells
were transiently transfected with 5 µg of an AP-1 luciferase reporter
construct together with increasing amounts of PKC A/E and/or Vav1 at
the indicated combinations. Luciferase activity was determined 30 h posttransfection. Gene expression is displayed as the average fold
activation relative to vector-transfected cells. Mean values from three
independent experiments are shown.
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JNK induces IL-2 promoter activity not only by targeting the AP-1 site
but also via the CD28RE/AP composite element, which is bound by various
proteins including members of the NF-
B/Rel and AP-1 families of
transcription factors and so far only partially characterized proteins
(27, 31). Because the CD28RE/AP element is absolutely required for the
transmission of CD28-derived signals on the IL-2 promoter (27), we
asked whether Vav1/PKC
-mediated JNK activation also targets gene
expression directed from this sequence element. A construct controlled
by four repeats of the CD28RE/AP element fused to the luciferase
reporter gene (4xRE/AP-Luc) was cotransfected with increasing amounts
of Vav1 and/or PKC
expression vectors (Fig.
3A). Both proteins
cooperatively boosted CD28RE/AP-dependent transcription. To
test whether the Vav1/PKC
-derived JNK activity targets the CD28RE or
the AP-1 half-site within the composite element, reporter constructs
with mutations in either or both half-sites were tested for their
inducibility. Mutation of either half-site completely prevented
Vav1/PKC
-mediated transcription (Fig. 3B), revealing a
strong interdependence of both half-sites. The effects of Vav1 and
PKC
on protein binding to the CD28RE/AP element were investigated by
EMSAs using the labeled CD28RE/AP element (Fig. 3C). Neither
Vav1 nor PKC
alone were able to induce DNA binding, but coexpression
of both proteins caused DNA binding of transcription factors contained
in the inducible DNA-protein complexes Ia and Ib. Formation of
both complexes, which are known to contain predominantly c-Fos and
c-Jun proteins (31), could be further triggered upon CD3/CD28
stimulation. In contrast to complex I, DNA-protein complexes II and III
were already present in extracts from the untransfected, nonstimulated
cells and showed no inducibility upon Vav1/PKC
A/E coexpression and
T-cell activation. The relatively moderate effects of Vav1/PKC
on
induced binding of proteins to the CD28RE/AP element can be attributed
to the limited transfection efficiency of Jurkat cells.

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Fig. 3.
Cooperative effects of
Vav1/PKC on the CD28RE/AP element.
A, the experiment was done as in Fig. 2B, with
the exception that a construct controlled by four repeats of the
CD28RE/AP element fused to the luciferase reporter gene (4xRE/AP-Luc)
was used. WT, wild type. B, Vav1 and/or
PKC A/E (4 µg, respectively) were expressed in Jurkat cells, and
their effects on cotransfected reporter constructs with intact or
mutated CD28RE/AP elements was measured. Results are expressed as the
average fold activation relative to vector-transfected cells, and error
bars indicate standard deviations from three independent experiments.
C, Jurkat cells were transfected either with empty
expression vector or with plasmids encoding Vav1 (10 µg) and/or
PKC A/E (10 µg) as shown. The next day, cells were stimulated for
4 h with CD3/ CD28 antibodies as indicated, and equal amounts
of protein contained in nuclear extracts were assayed for binding to a
labeled CD28RE/AP element by EMSAs. The positions of the constitutive
complexes II and III and of the inducible complexes Ia and Ib are
indicated. The arrowhead indicates the position of the
unbound oligonucleotide; a representative experiment is shown.
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To investigate the relative importance of Vav1 and PKC
for the
CD3/CD28-mediated activation of JNK, the costimulation-induced activation of this mitogen-activated protein kinase was tested in the
presence of DN versions of both signaling proteins. Coexpression of the
kinase-deficient point mutant PKC
K/R prevented JNK activation induced either by CD3/CD28 ligation or by treatment of cells
with the pleiotropic PKC activator phorbol 12-myristate 13-acetate together with CD28 or the ionophore ionomycin (Fig.
4A). Similarly, two Vav1
variants with a mutation or a deletion in the Dbl homology domain (Vav1
LLL/QIF and Vav1
319-356), which is responsible for the activation
of Rac, efficiently prevented CD3/CD28-triggered 32P
incorporation into GST-c-Jun by JNK (Fig. 4B).

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Fig. 4.
Effects of DN forms of Vav1 and
PKC on CD3/CD28-induced JNK activation.
A, Jurkat cells were stimulated as shown in the absence or
presence of coexpressed PKC K/R (10 µg). JNK activity was
determined using the GST-c-Jun direct phosphorylation assay. A fraction
of the lysate was analyzed by immunoblotting as shown
(lower panels). B, the experiment was
performed as in A, with the exception that two different DN
forms of Vav1 (10 µg) were used. PMA, phorbol 12-myristate
13-acetate; KA, kinase assay; WB, Western
blot.
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Vav1 and PKC
are constitutively associated in unstimulated T-cells
(18) but can also interact with a battery of further proteins, as
revealed by various experimental approaches (9, 32). Whereas T-cell
costimulation leads to the recruitment of Vav1 into an inducible
multi-protein complex (10), the association partners of Vav1 in
unstimulated cells are not well characterized. To directly address this
question, unstimulated Jurkat cells were lysed, and proteins contained
in the lysates were separated according to size by gel filtration. The
analysis of fractions for the distribution of Vav1 and PKC
revealed
an identical elution profile for both proteins, as revealed by Western
blotting (Fig. 5A). The two
fractions (22 and 23) containing most of Vav1 and PKC
correspond to
a molecular mass of ~170 kDa, suggesting that both proteins exist
predominantly as heterodimers. Because previous studies showed that
T-cell costimulation leads to the disruption of the Vav1/PKC
heterodimer (4), we investigated the distribution of Vav1 and PKC
in
extracts from CD3/CD28-stimulated cells. These experiments revealed the
majority of Vav1 in high molecular weight complexes of various sizes up to very large aggregates of more than 1 MDa (Fig. 5B). In
contrast, most of the PKC
protein coeluted with the 158-kDa marker
protein, and only a minor fraction was found in larger complexes (Fig. 5B).

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Fig. 5.
Gel filtration analysis of endogenous Vav1
and PKC . A, total cell lysates
prepared from unstimulated Jurkat cells were separated on a Superose 6 column. Aliquots of the fractions were analyzed by Western blotting for
the occurrence of Vav1 and PKC . Molecular masses of a protein marker
are given at the left, and the fractions containing the
maximum of the marker proteins bovine serum albumin (BSA)
and catalase are indicated. B, the experiment was performed
as above, with the exception that extracts from cells stimulated for 15 min with agonistic CD3/ CD28 antibodies were used.
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The signaling pathways were characterized by monitoring the effect of
coexpressed DN forms of protein kinases from the JNK activation pathway
on the activation signals derived from Vav1, PKC
, or both (Fig.
6A). A kinase-dead form of
MEKK1 affected Vav1-mediated AP-1 activity only moderately but
significantly inhibited PKC
- and PKC
/Vav1-generated signals. A
comparison between DN forms of the JNKKs MKK4 and MKK7 revealed that
Vav1-derived activation signals were only moderately blocked by
expression of DN forms of each of these kinases. In contrast,
simultaneous expression of kinase-inactive forms of MKK4 and MKK7
completely prevented Vav1-mediated AP-1 activation, indicating that
both kinases can mutually compensate the functions of each other.
PKC
- and Vav1/PKC
-derived signals were only partially impaired in the presence of DN MKK4 but efficiently blocked by MKK7 K/L, indicating the special importance of MKK7 for this pathway.

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Fig. 6.
Analysis of the signaling pathways mediated
by Vav1 and/or PKC . A, Jurkat
cells were transfected with an AP-1-dependent reporter gene
along with Vav1 and/or PKC A/E (5 µg, respectively) and 10 µg of
Flag-tagged DN forms of kinases as shown. The next day, cell extracts
were prepared and analyzed for luciferase activity (upper
panel) and protein expression (lower panels). To
facilitate comparison, full activation by Vav1 and/or PKC was
arbitrarily defined as 100%. Mean values from three to five
independent experiments are shown; bars indicate standard deviations.
Western blots (WB) showing expression of the Flag-tagged DN
forms of kinases are shown, and the relative levels of Vav and
PKC were similar (data not shown). B, Jurkat cells were
transfected with the AP-1 reporter gene and expression vectors for Vav1
and/or PKC A/E (5 µg, respectively). Cells were grown for 24 h in the presence or absence of cyclosporin A (1 µM) or
the PKC inhibitor bisindolylmaleimide (2 µM) as
indicated. Luciferase activity in the absence of inhibitory compounds
was arbitrarily set as 100%; error bars display standard deviations
from two independent experiments performed in duplicate.
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We also tested the impact of pathway-specific inhibitory compounds on
Vav1- and/or PKC
-induced AP-1-dependent transcription (Fig. 6B). Vav1-derived signaling, but not PKC
- and
Vav1/PKC
-mediated AP-1 activation, was preferentially inhibited by
cyclosporin A, a compound that blocks the
Ca2+-dependent activation of the phosphatase
calcineurin. The PKC inhibitor bisindolylmaleimide blocked Vav1- and
PKC
-mediated AP-1 activity, raising the possibility that Vav1 acts
upstream from PKC
. To address this question directly, Jurkat cells
were transfected with various combinations of active and inactive
variants of Vav1 and PKC
prior to stimulation with
CD3/
CD28
antibodies and subsequent analysis of JNK activity. Vav1-induced JNK
activation was efficiently prevented by kinase-dead PKC
K/R, but
PKC
A/E-mediated JNK activation was not affected by DN Vav1 LLL/QIF
(Fig. 7A). To investigate
whether directional signaling also occurs at the level of
AP-1-dependent transcription, an analogous experimental approach was taken by monitoring AP-1-dependent luciferase
activity. Similarly, Vav1-induced transcription of the
AP-1-dependent luciferase gene was inhibited upon
coexpression of PKC
K/R (Fig. 7B) or the PKC inhibitor
bisindolylmaleimide (data not shown). In contrast, gene activation
induced by PKC
A/E was not affected by the DN Vav1 variant
Vav1
319-356. In summary, these data clearly indicate that PKC
acts downstream from Vav1 in a pathway leading to the activation of
JNK.

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Fig. 7.
Analysis of the directional signaling from
Vav1 to PKC . A, JNK activation
triggered by CD3/ CD28 stimulation or expression of Vav1 (5 µg)
or PKC A/E (5 µg) was analyzed in the absence or presence of DN
forms of Vav1 (10 µg) or PKC (10 µg) as shown. JNK activity was
determined by immune complex kinase assays (KA) (upper
panel). A sample of each lysate was analyzed by immunoblotting for
protein expression of JNK, Vav1, and PKC (lower panels).
WB, Western blot. B, Jurkat cells were
transfected with an AP-1-dependent reporter gene and
various combinations of expression vectors encoding active and DN forms
of Vav1 and PKC (10 µg, respectively). AP-1-dependent
gene expression is displayed as the average fold activation relative to
vector-transfected cells. Mean values from three independent
experiments are shown; bars display standard deviations.
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 |
DISCUSSION |
Here we show that Vav1 and PKC
synergize for the induction of
JNK. Because these two proteins also cooperate for the up-regulation of
NF-
B, the simultaneous activation of several pathways may be
important for the efficient transcription of target genes, because many
promoters (e.g. the IL-2 and IL-4 promoter) depend on the
coordinated activation of several transcription factors. Interestingly,
PKC
does not only synergize with Vav1 (this study) but also with
calcineurin for the activation of JNK (22). The Vav1 protein cooperates
with SLP-76 and Syk family kinases for the activation of IL-2
transcription and nuclear factor of activated T-cells
activation, respectively (33, 34). One could speculate that the
synergistic behavior of signaling proteins for the initiation of T-cell
signaling pathways may be an important regulatory principle, especially
early in infection when only low doses of antigen are present, but an
efficient cellular response is required. In that respect it will be
interesting to analyze signaling pathways and the immune response in
Vav1/PKC
double knockout mice in future experiments.
In this study we show that at least the abundant fraction of PKC
and
Vav1 proteins is present as heterodimers in unstimulated cells under
the conditions used here. Given the importance of both proteins for the
initiation of various signaling pathways (10, 32) and the necessity to
keep activation pathways in nonstimulated T-cells silent (35, 36), this
heterodimerization might serve the purpose of keeping both proteins in
an inactive state. T-cell activation leads to the transient
dissociation of Vav1 and PKC
by an unknown mechanism (4) and the
incorporation of Vav1 into temporally regulated and highly dynamic and
semi-stable multi-protein signaling complexes (37). Under the
conditions used here, only a minor fraction of the PKC
protein is
incorporated into high molecular weight complexes in stimulated
T-cells. It will be interesting to investigate the composition,
stability, and spatial and temporal regulation of these multi-protein complexes.
Our results indicate that Vav1 is located upstream from PKC
in the
JNK activation cascade. This finding is in good agreement with a
previous study that demonstrated a Vav1-dependent membrane and cytoskeleton translocation of PKC
(20). The same study shows
that the effects of Vav1 on PKC
are mediated by Vav1-induced actin
polymerization and cytoskeletal reorganization. Biochemical and genetic
evidence suggests that Vav1 exerts its function by at least two
mechanisms. One pathway leads to the release of Ca2+ and
the subsequent activation of the serine phosphatase calcineurin, which
stimulates nuclear entry and transactivation of transcription factor
nuclear factor of activated T-cells. This pathway is independent from the GDP/GTP exchange factor function of Vav1 (38) and cannot be
inhibited by the regulatory protein Cbl-b (39). The second pathway is
Ca2+-independent, can be inhibited by Cbl-b, and relies on
the GDP/GTP exchange factor function of Vav1, thus leading to actin
polymerization, cytoskeletal reorganization, and further processes
(39). We favor a model where Vav1 and PKC
activate JNK via
overlapping and distinct pathways. The PKC
-derived signals are
Ca2+-independent and cannot be inhibited by calcineurin,
because PKC
is located downstream from it. Accordingly, a DN form of
PKC
inhibits JNK activation mediated by ionomycin, which causes the release of intracellular Ca2+ (compare Fig. 4A).
Similarly, the pathway shared by both signaling proteins and mediating
the synergistic activation of JNK is
Ca2+/calcineurin-independent. In contrast, the
Vav1-mediated JNK activation pathway contains a
Ca2+-dependent component that can be inhibited
by cyclosporin A. This model would also explain earlier results that
showed only moderate JNK activation by phorbol 12-myristate 13-acetate
and ionomycin alone, whereas the simultaneous administration of both
compounds strongly activated JNK activity (23).
This study suggests that Vav1-derived signals target MKK4 and MKK7. In
contrast, PKC
- and Vav1/PKC
-derived signals are
transmitted preferentially via MKK7, which seems to be of special
relevance for this pathway. The importance of MKK4 for the
CD3/CD28-induced activation of JNK is still not clear. One group
describes a defective JNK activation in thymocytes obtained from
MKK4-deficient mice but normal JNK activation in peripheral T-cells
(40). Another study demonstrates normal activation of JNK in response
to CD3/CD28 stimulation in lymph node cell suspensions from
MKK4
/
mice (41). Further studies are
required to resolve these differences, which may also depend on the
developmental stage of the lymphocytes and the cell types studied. In
accordance with the predominant role of MKK7 described here, peripheral
T-cells from mice lacking MKK7 show only low levels of JNK activity
after CD3/CD28 stimulation (15).
The PKC
-mediated signaling pathways are only incompletely
understood. Furthermore, it is currently not known whether the competence of PKC
to deliver activation signals is controlled by its
intracellular localization, interaction with binding partners, or both.
Because overexpression of the PKC
-interacting protein, PKC-interacting cousin of thioredoxin, inhibits PKC
-mediated activation of JNK and NF-
B (42), this protein seems to be involved in the first signaling step prior to the separation of the two signaling pathways. Given the importance of PKC
for the activation of NF-
B and JNK, selective inhibition of this kinase might be a
useful strategy to interfere with several activation pathways and
thereby modulate T-cell costimulatory signals in inflammatory diseases.
 |
ACKNOWLEDGEMENTS |
We are grateful to Dr. Stephan Urban
(Heidelberg) for help with gel filtration, Dr. Susanne Bacher and
Ingrid Fryson for helpful comments on the manuscript, Tarik Hamid for
technical assistance, and colleagues who generously provided plasmids.
 |
FOOTNOTES |
*
This work was supported by grants from the Land
Baden-Württemberg, European Union (QLK3-2000-00463), Fonds
der Chemischen Industrie, Deutsche Forschungsgemeinschaft (Schm
1417/3-1), and Deutsche Krebshilfe.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. Tel.: 49-6221-423725;
Fax: 49-6221-423746; E-mail: L.Schmitz@DKFZ.de.
Published, JBC Papers in Press, March 23, 2001, DOI 10.1074/jbc.M011139200
 |
ABBREVIATIONS |
The abbreviations used are:
JNK, c-Jun
N-terminal kinase;
NF-
B, nuclear factor-
B;
IL, interleukin;
PKC, protein kinase C;
JNKK, JNK kinase;
DN, dominant negative;
EMSA, electrophoretic mobility shift assay;
HA, hemagglutinin;
GST, glutathione S-transferase.
 |
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