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INTRODUCTION |
The c-Jun N-terminal kinase
(JNK)1 or stress-activated
protein kinase MAPK cascade is a major mammalian MAPK cascade
activated by physical stresses, inflammatory cytokines, T-cell
costimulation, and growth factors (1-3). Like all other MAPKs, JNK is
activated by phosphorylation of the conserved Thr and Tyr residues by
its activating MAPK kinases (MAPKKs). Two MAPKKs, JNKK1/MKK4 and
JNKK2/MKK7, were identified as participating in JNK activation
(4-13). Multiple MAPKK kinases (MAPKKKs) have been shown to utilize
JNKK1/MKK4 and JNKK2/MKK7 to activate the JNK cascade (14-17). These
MAPKKKs have in common conserved kinase domains with substantial
homology to the yeast MAPKKK STE11 (18-24). However, other than the
conserved catalytic domains, these kinases in general are not similar
and are believed to be involved in transducing different upstream signals to downstream targets (14, 25-28).
It has been shown that during T-cell activation, activation of the JNK,
but not of the Erk MAPK, cascade is dependent on costimulation of
T-cell receptor (TCR) and the costimulatory receptor CD28 (29). Activation of c-Jun/AP-1, the nuclear target of the JNK MAPK cascade, is also dependent on costimulation of TCR and CD28 in naïve
CD4+ T-cells (30). More recently, the role of the JNK MAPK
cascade in T-cells was studied in gene-knockout mice, which showed that JNK1 and JNK2 play a crucial role in T-cell activation and
differentiation (31-33).
The signal transduction pathways that lead to JNK activation in T-cells
remain unknown. Studies in fibroblasts indicated that members of the
mitogen-activated protein kinase/extracellular signal-regulated kinase
kinase (MEK) kinase (MEKK) gene family are the major MAPKKKs for the
JNKs (1, 15, 18, 19, 28, 34). MEKK1, the first MEKK identified in
mammalian cells, was suggested to be involved in TCR-mediated
JNK activation and cytokine gene expression (35). Interestingly, a
recent study suggested that MEKK2 rather than MEKK1 was involved in
transducing TCR signals in T-cells (36). This study showed that murine
MEKK2 but not MEKK1 is translocated to the T-cell/antigen-presenting
cell interface during antigen stimulation (36). Whether other members
of the MEKK gene subfamily are involved in T-cell signal transduction is not clear. So far, no lymphocyte-specific MAPKKKs for the JNK cascade have been identified.
In this study, we described the cloning and characterization of human
MEKK2, a potent JNK MAPK activator, in T-cells. We found that MEKK2 was
a specific JNK upstream-activating kinase in T-cells and was involved
in transducing TCR/CD3-mediated T-cell signals for JNK MAPK activation
and T-cell-specific gene expression.
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EXPERIMENTAL PROCEDURES |
Isolation of Full-length Human MEKK2 cDNA Clone--
The
degenerate oligonucleotides TTAATGGCNGTNAA(a/g)CA and
TTNGCNCC(t/c)TTNAC(a/g)TCNC(g/t)(a/g)TG, which corresponded to
sequences in kinase subdomains II and VII, respectively, of the
yeast MAPKKK STE11 (37) were used to amplify related gene fragments by
reverse transcription-polymerase chain reaction (PCR) with mRNA
from Jurkat T-cells. A mixture of cDNA fragments of about 300 base
pairs was amplified and subcloned into the TA cloning vector
(Invitrogen, San Diego, CA) for sequencing. We identified a cDNA
fragment encoding a polypeptide 47% similar to STE11, 49% similar to
murine MEKK1, and 99% similar to murine MEKK2 at the predicted kinase
subdomains II-VII. This cDNA fragment was used as a probe to
further screen a Jurkat cDNA library. We obtained four cDNA
clones that contained a complete putative kinase domain, but all lacked
the complete 5' coding sequence. Full-length cDNA clones were
isolated by further screening a human lymphocyte cDNA library
(Invitrogen) with PCR with primers that matched the most 5'
sequence of the partial cDNA clones isolated from the Jurkat
cDNA library. Ten individual clones were isolated, and the longest
one, clone 2 g, was completely sequenced and used in this study.
Plasmids, Proteins, and Antibodies--
SR
RasL61,
SR
RafBxB, SR
MEKK2, SR
HA-JNK1, SR
HA-Erk2, SR
HA-MEKK2,
-73Col-Luc, -79Jun-Luc, IL-2-Luc, and pActin-
-Gal were described
previously (2, 28, 29). The hemagglutinin (HA)-tagged MEKK2(NT)
expression vector Sr
HA-MEKK2(NT) was constructed by introducing an
NcoI site into the first Met codon of MEKK2 by a PCR-based
method and subcloning the NcoI-XhoI fragment that encodes amino acids 1-359 into the expression vector pSR
3HA. MEKK2(KM) is a MEKK2 mutant with a Lys-385
Met mutation that was generated by PCR-directed mutagenesis. The MEKK2(KM) mutant cDNA was subcloned into SR
HA as described for SR
HA-MEKK2
(28). Expression and purification of the bacterial fusion protein
glutathione S-transferase (GST)-c-Jun-(1-79) was
previously described (6, 38). Anti-HA antibody 12CA5 was prepared from
the hybridoma 12CA5 (39). MEKK2 antibody 1128 was described previously
(28), and MEKK2 antibody 1129 was prepared by immunizing rabbits with GST human MEKK2 N-terminal region (amino acids 1-359) fusion protein. Anti-phosphotyrosine antibody PY20 was purchased from Pharmingen (San
Diego, CA).
Northern Blot Analysis--
A multitissue poly(A)+
blot was purchased from CLONTECH (Palo Alto, CA)
and probed with a 1-kilobase HindIII-XhoI
cDNA probe from the N-terminal region of human MEKK2 using the
manufacturer's suggested protocol.
Cell Culture and Transfection Procedures--
Jurkat T-cells
were cultured in RPMI medium supplemented with 10% fetal bovine serum,
1 mM glutamate, 100 units/ml penicillin, and 100 mg/ml
streptomycin. COS-1 cells were cultured in Dulbecco's modified
Eagle's medium supplemented with 5% fetal bovine serum, 100 units/ml
penicillin, and 100 mg/ml streptomycin. Jurkat T-cells were transfected
by electroporation as described previously (29). COS-1 cells were
transfected with LipofectAMINE (Life Technologies, Inc.).
In Vitro Kinase Assay--
Cell lysates were prepared 40 h
after transfection as previously described (29) and incubated with
appropriate antibodies for 4 h at 4 °C in a rotator. Then,
protein A-Sepharose beads were added, and the mixture was incubated for
another 45 min. The beads were washed four times with lysis buffer (20 mM Tris, pH 7.5; 0.5% Nonidet P-40; 250 mM
NaCl; 3 mM EDTA; 3 mM EGTA; and 100 mM Na3VO4) and twice with kinase
reaction buffer (20 mM HEPES, pH 7.6; 1 mM
p-nitrophenyl phosphate; 20 mM
MgCl2; 2 mM EDTA; 2 mM EGTA; and
100 µM Na3VO4). The
immunoprecipitates were subjected to kinase assays in 30 µl of kinase
buffer with appropriate substrates in the presence of 0.5 µl of
[
-32P]ATP and 20 µM cold ATP. After 20 min at 30 °C, the reactions were terminated with SDS-polyacrylamide
gel electrophoresis loading buffer and boiled for 5 min. The proteins
were separated by SDS-polyacrylamide gel electrophoresis, and
32P incorporation was determined with a Bio-Rad FX
phosphorimaging apparatus (Bio-Rad).
Luciferase Reporter Gene Assay--
Luciferase assay was
described previously (13), and luciferase activity was measured with a
luminometer TD-20/20 from Promega (Madison, WI).
Western Blot--
Western blot with anti-HA antibody 12CA5 and
anti-MEKK2 antibodies 1128 and 1129 was performed as previously
described using an ECL kit (Amersham Pharmacia Biotech)
(28).
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RESULTS |
Cloning of Human MEKK2--
To isolate a potential MAPKKK gene
upstream of the JNK cascade from T-cells, we made degenerated
oligonucleotides that corresponded to the sequences of the kinase
subdomains II and VII of yeast MAPKKK STE11 (37), because MAPKKK genes
were highly conserved from yeast to mammals, and used them to amplify
related gene fragments by reverse transcription-PCR of mRNA from
Jurkat T-cells. A cDNA fragment with substantial homology to the
catalytic domain of STE11, MEKK1, and murine MEKK2 was identified and
used as a probe to clone a full-length cDNA from a human lymphocyte
cDNA library as described under "Experimental Procedures." This
clone, clone 2 g, was completely sequenced and found to contain an
open reading frame of 619 amino acids. A homology search of the data
base (NCBI-nr) revealed a high homology to murine MEKK2, suggesting
that this clone was the human counterpart of murine MEKK2 (18).
This clone was also highly homologous to human MEKK3 (71%), murine
MEKK3 (71%), and, to a lesser extent, human MEKK1 in the putative
catalytic domain (58%). Thus we named the clone human MEKK2
(GenBankTM accession number AF111105). A comparison of the
sequence of human and murine MEKK2 and human and murine MEKK3 is shown
in Fig. 1A. Northern blot
analysis showed that human MEKK2 mRNA was strongly expressed in
brain, heart, muscle, spleen, kidney, liver, placenta, and blood
leukocytes but expressed at low levels in thymus, lung, small
intestine, and colon (Fig. 1B). We used MEKK2-specific antibody 1128 to immunoprecipitate the endogenous MEKK2 from Jurkat T-cells and found that the endogenous MEKK2 had an apparent molecular mass of about 70 kDa (Fig. 1C).

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Fig. 1.
Cloning of human MEKK2. A,
alignment of the complete human MEKK2 amino acid sequence (AF111105) to
the sequences of murine MEKK2 (NM011946), human MEKK3 (NM002401), and
murine MEKK3 (Q61084). Shading indicates identical amino
acids, and boxes indicate homologous amino acids.
B, Northern blot analysis of human MEKK2 gene expression.
C, Western blot analysis of the endogenous human MEKK2 in
Jurkat T-cells. Whole-cell extracts were prepared from 107
Jurkat T-cells and immunoprecipitated with either pre-immune serum or
MEKK2-specific antibody 1128 as indicated. The immunocomplex was
separated by SDS-polyacrylamide gel electrophoresis and analyzed by
Western blotting with MEKK2-specific antibody 1129. PBL, peripheral blood lymphocyte; kb,
kilobases; Kd, kilodaltons.
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MEKK2 Activates the JNK Cascade in T-cells--
We recently
demonstrated that in fibroblasts human MEKK2 is an important activating
kinase of JNK and acts through formation of a specific MAPK module with
JNKK2 and JNK1 (28). To investigate whether MEKK2 is also involved in
transducing T-cell-activating signals and JNK activation, we expressed
full-length human MEKK2 with HA-tagged JNK1 in Jurkat T-cells and then
determined the effect on JNK1 enzymatic activity. As shown in Fig.
2A, expression of the
full-length MEKK2 cDNA in T-cells led to strong JNK1 activation. In
contrast, it had no effect on Erk2, a JNK-related MAPK in T-cells. Expression of RasL61 and RafBxB, which are active forms of the Erk
upstream activators Ras and Raf, respectively, led to Erk2, but not
JNK1, activation in T-cells (Fig. 2A). This result suggested that MEKK2 was a specific JNK, but not Erk, activator in T cells. Using
the same protocol, we found that MEKK2 activated JNK2 similarly in
T-cells (data not shown), suggesting that MEKK2 regulates both JNK1 and
JNK2.

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Fig. 2.
MEKK2 was a specific upstream activator of
JNK activation in T-cells. A, MEKK2 specifically
activated JNK but not Erk MAPK in T-cells. One µg of HA-JNK1 and
HA-Erk2 expression vectors was cotransfected with control empty vector
or expression vectors for active Ras mutant RasL61, active Raf mutant
RafBxB, and MEKK2 into Jurkat T-cells as indicated. Thirty-six h after
transfection, HA-JNK1 and HA-Erk2 were immunoprecipitated with anti-HA
antibody, and their activity was determined by in vitro
kinase assays (KA) using GST-c-Jun (for JNK) and myelin
basic protein (for Erk) as substrates. Expression of HA-JNK1 and
HA-Erk2 was measured by Western blot (WB). B,
effect of MEKK2 on AP-1 activation in Jurkat T-cells and COS-1 cells.
AP-1 reporter gene plasmid -73Col-Luc was cotransfected into Jurkat
T-cells (5 µg/transfection) and COS-1 cells (0.5 µg/transfection)
with the indicated amount of MEKK2 expression vector. pActin- -Gal
was included in each transfection to monitor the transfection
efficiency. The relative luciferase activity was measured 36 h
later and normalized to the -galactosidase activity. The results are
presented as the average of three independent experiments. The
error bars indicate the standard error.
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To investigate whether MEKK2 participates in JNK-dependent
gene expression, we used an AP-1 reporter plasmid, -73Col-Luc, to test
the effects of MEKK2 on AP-1 transcription activity in Jurkat T-cells
and COS-1 cells. Consistent with MEKK2 being an upstream activator for
JNK, we found that MEKK2 activated AP-1 reporter gene expression both
in T-cells and in COS-1 cells (Fig. 2B). This result
suggested that MEKK2 is an upstream activator of the JNK MAPK pathway
in T-cells.
Previously, we demonstrated that costimulation of Jurkat T-cells with
anti-CD3 and anti-CD28 antibodies led to strong synergistic JNK
activation (29). Because MEKK2 was able to activate JNK in T-cells
(Fig. 2A), we next examined whether MEKK2 could potentiate JNK1 activation by these T-cell stimuli. To do so, we transfected HA-JNK1 expression vector with increasing amounts of MEKK2 expression vector into Jurkat T-cells and measured the JNK activity without stimulation or with anti-CD3 or anti-CD28 antibody stimulation. As
shown in Fig. 3, whereas anti-CD28
stimulation had no effect on MEKK2-mediated JNK activation, anti-CD3
stimulation greatly enhanced the MEKK2-mediated JNK activation,
especially at low levels of MEKK2 expression. These results suggested
that MEKK2 might be involved in transducing TCR/CD3 signals during
T-cell antigen stimulation. The same conclusion was reached in a recent study on murine MEKK2 (36).

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Fig. 3.
Stimulation of T-cells with anti-CD3 antibody
potentiated MEKK2-mediated JNK1 activation. Two µg of HA-JNK1
expression vector was cotransfected with increasing amounts of MEKK2
expression vector into 107 Jurkat T-cells. Each
transfection mixture was divided equally into three parts at 36 h
and stimulated with either vehicle alone or anti-CD3 antibody OKT3 (10 µg/ml) or anti-CD28 antibody 9.3 (0.2 µg/ml) for 30 min before the
JNK activity was assayed as described in the legend to Fig.
2A. Relative JNK activity was determined with a Bio-Rad
phosphorimaging apparatus and plotted against the amounts of
MEKK2 expression vector in each transfection.
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Tyrosine Phosphorylation of MEKK2 by CD3 Stimulation--
The
above results suggested that MEKK2 may act downstream of the TCR/CD3
receptor. To further examine this issue, we tested whether MEKK2
could be directly modified following anti-CD3 antibody stimulation in T
cells. Recently, Schaefer et al. (36) found that murine
MEKK2 was translocated to the T-cell-antigen-presenting cell interface
after antigen stimulation, suggesting that MEKK2 may be an early target
of TCR signaling. Because stimulation of TCR rapidly activates several
protein tyrosine kinases, it is possible that human MEKK2 may be
regulated by tyrosine phosphorylation in T-cells. To test this
possibility, we transfected GST-tagged MEKK2 into Jurkat T-cells and
analyzed it with an anti-phosphotyrosine antibody after stimulation
with anti-CD3 antibody. As shown in Fig.
4, we found that there was a rapid
induction of MEKK2 tyrosine phosphorylation in response to CD3
stimulation. We also observed a similar pattern of tyrosine
phosphorylation of the endogenous MEKK2, and such phosphorylation was
not affected by addition of anti-CD28 antibody (data not shown). This
result indicated that MEKK2 was a substrate molecule for TCR/CD3
activated protein tyrosine kinases.

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Fig. 4.
Tyrosine phosphorylation of MEKK2 induced by
CD3 stimulation. Five µg of GST-MEKK2 expression vector was
transfected into Jurkat T-cells as described in the legend to Fig.
2A. Thirty-six h after transfection, the cells were
incubated with control antibody or anti-CD3 antibody OKT3 (10 µg/ml)
for 30 min at 4 °C. Free antibody was removed by washing two times
with cold phosphate-buffered saline, and the cells were further treated
with cross-linker antibody (Ab) goat anti-mouse IgG (10 µg/ml) for 20 min before harvesting. GST-MEKK2 was precipitated with
GST beads and subjected to Western blot (WB) analysis with
anti-phosphotyrosine (anti-PTy) antibody. MEKK2 expression
was determined by Western blotting with anti-MEKK2 antibody 1128.
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MEKK2 Is Required for JNK Activation by T-cell Costimulation and
IL-2 Gene Expression--
To further determine the role of MEKK2 in
T-cell stimulation, we constructed the expression vector
SR
HA-MEKK2(NT), which expresses a MEKK2 mutant, MEKK2(NT), which
lacks the entire catalytic domain. Expression of MEKK2(NT) in T-cells
inhibited MEKK2-induced JNK activation in a dose-dependent
manner (data not shown), suggesting that MEKK2(NT) is a dominant
negative mutant. To determine the effect of MEKK2(NT) on JNK activation
in T-cells, we transfected HA-JNK1 with increasing amounts of MEKK2(NT)
and determined the JNK activity 36 h later with or without T-cell
stimulation with anti-CD3 and anti-CD28 antibodies. As shown in Fig.
5A, JNK activation by T-cell
costimulation with anti-CD3 and anti-CD28 antibodies was strongly
inhibited by MEKK2(NT), suggesting that MEKK2 is required for JNK
activation by costimulation with anti-CD3 and anti-CD28 antibodies.
Interestingly, in a similar experiment, we found that costimulation of
JNK in T-cells by TPA and A23187 was not blocked by the mutant
MEKK2(NT) (Fig. 5B). Transfection of MEKK2(NT) into Jurkat
cells also did not inhibit the JNK activation in response to a combined
stimulation with TPA, A23187, and anti-CD28 antibody (data not shown).
Similar results were also reported with murine MEKK2 (36), suggesting
that MEKK2 may not be involved in JNK activation by TPA plus
Ca2+ plus CD28 in T cells.

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Fig. 5.
MEKK2 was required for JNK1 activation by CD3
and CD28 costimulation in T-cells. A, MEKK2(NT)
inhibited JNK activation by T-cell costimulation with anti-CD3 and
anti-CD28 antibodies. One µg of HA-JNK1 expression vector was
cotransfected with increasing amounts of MEKK2(NT) expression vector
into 107 Jurkat T-cells. Thirty-six h after transfection,
the cells were stimulated with 10 µg/ml anti-CD3 antibody OKT3 and
0.2 µg/ml anti-CD28 antibody 9.3 for 30 min before harvesting for the
JNK1 assay as described previously (29). Relative GST-c-Jun
phosphorylation was quantitated with a Bio-Rad phosphorimaging
apparatus. Relative JNK1 activity in each transfection is shown.
Expression of HA-JNK1 and MEKK2(NT) was measured by Western blotting.
B, MEKK2(NT) did not inhibit JNK activation by TPA and
A23187. One µg of HA-JNK1 expression vector was cotransfected with
increasing amounts of MEKK2(NT) expression vector into 107
Jurkat T-cells. The cells were stimulated with TPA (10 ng/ml) and
A23187 (1 µg/ml) for 30 min 36 h after transfection, and the
JNK1 activity was assayed as described for A. C,
MEKK2(KM) inhibited JNK1 activation by costimulation of T-cells with
anti-CD3 and anti-CD28 antibodies. One µg of HA-JNK1 expression
vector was cotransfected with increasing amounts of MEKK2(KM)
expression vector into 107 Jurkat T-cells. T-cell
stimulation and JNK1 activity assay were carried out as described in
A. D, MEKK2(KM) blocked -79Jun-Luc and IL2-Luc
reporter gene expression in T-cells. Two µg of -79Jun-Luc reporter
plasmid or 5 µg of IL2-Luc report plasmid was cotransfected with
increasing amounts of MEKK2(KM) expression vector. Twenty-four h after
transfection, the cells were stimulated with 10 µg/ml anti-CD3
antibody OKT3 and 0.2 µg/ml anti-CD28 antibody 9.3 for 12 h
before harvesting for the luciferase assay. Luciferase activity was
normalized to -galactosidase activity, and the results shown are the
average of three independent experiments. The error bars
indicate the standard error.
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Because MEKK2 was required for JNK activation in T-cells, we next
examined whether this effect was dependent on MEKK2 catalytic activity.
We constructed the expression vector SR
MEKK2(KM), which expresses a
MEKK2 mutant, MEKK2 (KM), with a point mutation of the conserved
Lys-385 to Met in the ATP binding motif. Such a mutation in the ATP
binding site has been shown to abolish the catalytic activity of many
serine/threonine kinases (14), and by itself MEKK2(KM) did not activate
JNK in T-cells (data not shown). We transfected this mutant kinase with
HA-JNK1 into Jurkat T-cells and determined the JNK1 activity with or
without anti-CD3 and anti-CD28 antibody costimulation. As shown in Fig.
5C, the MEKK2 mutant inhibited JNK activation by CD3 and
CD28 costimulation. This result showed that the MEKK2(KM) mutant also
had a dominant negative effect on JNK activation in T-cells.
It has been shown that the JNK MAPK cascade is crucial for AP-1 and
IL-2 gene expression in T-cells (29, 30). Because MEKK2 was involved in
regulating JNK activation in T-cells, it was likely that MEKK2 was also
required for the expression of these genes in T-cells. To investigate
this, we transfected MEKK2(KM) with the AP-1 reporter plasmid
-79Jun-Luc and the IL-2 reporter plasmid IL2-Luc into T-cells and
determined the effect on the reporter gene expression after T-cell
costimulation. As shown in Fig. 5D, expression of MEKK2(KM)
inhibited AP-1 and IL-2 reporter gene induction by T-cell
costimulation, suggesting that MEKK2 is crucial for T-cell gene expression.
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DISCUSSION |
A growing number of MAPKKKs, including MEKK1, MEKK2, MEKK3, MEKK4,
MEKK5, apoptosis signal-regulating kinase 1, transforming growth
factor-
-activated protein kinase 1, and tumor progression locus
2, have been shown to be capable of activating JNK (18, 20-24,
27, 34, 40). These kinases have a conserved kinase domain with
substantial homology to that of the yeast MAPKKK STE11. However, how
these MAPKKKs are involved in directing different cell surface signals
to the downstream cytoplasmic and nuclear effectors in different
tissues is still unknown.
In this study, we described the cloning and characterization of human
MEKK2, a full-length cDNA from Jurkat T-cells. The MEKK2 cDNA
described in this study encoded a serine/threonine kinase and is
probably the human counterpart of the reported murine MEKK2 (18),
because these two molecules are very similar (96% identical). Both
human and murine MEKK2 are much more similar to human and murine MEKK3
at the catalytic domain (94% identical) than are other members of the
MEKK gene subfamily, indicating that MEKK2 and MEKK3 are closely
related. Interestingly, Northern blot analysis showed that MEKK2 was
expressed at high levels in spleen and peripheral blood leukocytes but
at low levels in thymus, suggesting that MEKK2 may have an important
function in lymphocyte activation but not in normal T-cell development.
Expression of dominant negative MEKK2 mutants was found to block JNK
activation and AP-1 and IL-2 reporter gene induction by
costimulation of CD3 and CD28 during T-cell activation. Interestingly, we found that JNK activation in T-cells by TPA and Ca2+
ionophore was not inhibited by MEKK2 mutants. A similar result was
obtained by Schaefer et al. (36), who showed that expression of a similar murine MEKK2 dominant negative mutant could not inhibit JNK activation by TPA and ionomycine in murine T-cells. Therefore, these results indicated that MEKK2 is not involved in transducing TPA-
and Ca2+ ionophore-mediated T-cell-activating signals but
was required for transducing TCR/CD3-mediated activating signals.
Recently we demonstrated that MEKK2 is a major upstream activator of
the JNK MAPK through forming a triple molecular complex with JNKK2/MKK7
and JNK1 (28). Biochemical analysis of both human MEKK2 (28) and murine
MEKK2 (18, 41) showed that they are potent MAPKKKs for the JNK MAPK
module. Murine MEKK2 is also able to activate the Erk and p38 MAPKs in
addition to JNK MAPKs (18, 36, 41). However, we found that human MEKK2
activated JNK but not Erk in Jurkat T-cells. Because
stimulation of T-cells can activate both the JNK and Erk pathways, our
results suggested that MEKK2 might be involved in transducing TCR/CD3
signals to the JNK cascade but not to the Erk cascade.
How MEKK2 is involved in TCR/CD3 signal transduction in T-cells is
still unknown. Schaefer et al. (36) showed that murine MEKK2
is rapidly translocated to the T-cell-antigen-presenting cell interface
in response to antigen stimulation, suggesting that participation of
MEKK2 in TCR/CD3 was required for signaling to downstream targets. In
our study, we found that stimulation of CD3 in T-cells also resulted in
human MEKK2 tyrosine phosphorylation, suggesting that MEKK2 is a target
of the TCR/CD3-associated protein tyrosine kinases. It is possible that
MEKK2 tyrosine phosphorylation after TCR stimulation is involved in
regulating MEKK2 activity. Alternatively, tyrosine phosphorylation of
MEKK2 may provide additional docking sites for MEKK2 to interact with
TCR/CD3. Further studies on this issue should increase our
understanding of the general mechanism of MEKK2 activation.
Finally, transient expression of full-length MEKK2 in T-cells was able
to activate JNK constitutively in the absence of stimulation, but no
endogenous JNK activation was observed in unstimulated Jurkat T-cells
(Fig. 2A). This was not due to the absence of MEKK2 in
Jurkat T-cells, because by using antiserum raised against the N
terminus and C terminus, respectively, we easily detected MEKK2 protein in Jurkat T-cells (Fig. 1C). Therefore, the
endogenous MEKK2 in T-cells must be inactive before stimulation. It is
possible that MEKK2 is tightly regulated by inhibitors or by
compartmentalization, and such regulation could be disrupted in
transient transfection assay. In this regard, we recently isolated an
MEKK2-associated protein, and analysis of it will shed light on this issue.