From the Department of Immunology, The Scripps Research Institute, La Jolla, California 92037
Received for publication, October 25, 2002
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ABSTRACT |
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The mitogen-activated protein kinases
(MAPKs) play an important role in a variety of biological processes.
Activation of MAPKs is mediated by phosphorylation on specific
regulatory tyrosine and threonine sites. We have recently found that
activation of p38 The mitogen-activated protein kinase
(MAPK)1 signaling pathways
have been shown to function in a wide variety of cellular processes, including cell growth, cell migration, cell differentiation, cell death, and immune cell activation (1-8). To date four distinct subfamilies of MAPKs have been identified: extracellular
signal-regulated kinases (ERKs), c-Jun N-terminal or stress-activated
protein kinases (JNK/SAPK), ERK5/big MAPK1 (BMK1), and p38 MAPKs (3,
9-14). Whereas the ERK family is activated by growth factors and is
involved in cell proliferation, JNK and p38 are predominantly activated in response to pro-inflammatory cytokines and various types of environmental stresses (1, 3-5, 15).
Activation of MAPK is mediated through phosphorylation of specific
tyrosine and threonine residues by upstream MAPK kinase (MAPKK), which
is activated through phosphorylation of serine/threonine residues by
MAPKKK further upstream (4, 16-18). ERK family kinases are activated
by the MEK1 and MEK2, JNK by MKK4 and MKK7, and p38 by MKK3 and MKK6
(3, 4, 10, 19). Further upstream, Raf-1, A-Raf, B-Raf, and MOS
function as MAPKKKs in the ERK activation pathway (20, 21). A subgroup
of MAPKKK, including MEKK1-4, MTK1, MLK1-3, DLK/MUK, ASK1, Tpl-2/Cot,
and TAK1, activates the MAPKKs that phosphorylate JNK and perhaps p38
as well (4, 7, 17, 22). Biochemical and genetic evidence have supported
the above three-kinase module of MAPK activation (1, 7, 18). In
addition, the MAPK cascades are evolutionarily conserved (1, 18).
However, we have recently demonstrated an alternative activation pathway of p38 In this report we describe the characterization of a splicing variant
of TAB1, TAB1 cDNA of TAB1 Construction of Expression Vectors--
The TAB1 RT-PCR Analysis--
Total RNA was isolated from HEK293 (293),
HPEG2, MDA-231, and RPMI8226 cells using the RNeasy Mini kit (Qiagen,
Valencia, CA) following the manufacturer's instructions and treated
with RNase-free DNase I. Reverse transcription was performed using SuperScript II RT (Invitrogen, Rockville, MA) according to the supplier's specifications. cDNA (1 µl) was amplified by PCR
using the Taq Clontech 50xAdvantage 2 DNA polymerase (94 °C for 2 min, 58 °C for 10 s, 68 °C
for 30 s, 45 cycles). The upstream oligonucleotide primer
used for TAB1 and TAB1 Transfection of Cells--
HEK293 or MDA231 cells were
maintained in Dulbecco's modified Eagle's medium supplemented with
5% fetal bovine serum, 2 mM glutamine, and 100 µg/ml
penicillin and streptomycin. Cells on six-well plates were transiently
transfected with 1 µg (total) of plasmid DNA using LipofectAMINE.
After 36 h, the cells were treated with stimuli as described in
the text. The plasmid pCMV Western Blot and Immunoprecipitation Analysis--
Total cell
lysates were prepared using a lysis buffer A: 20 mM
Tris-HCl, pH 7.5, 120 mM NaCl, 10% glycerol, 1 mM Na3VO4, 2 mM EDTA, 1 mM phenylmethanesulfonyl fluoride, 1% Triton X-100. Equal protein loading of cell extracts in SDS-PAGE was determined by
Bio-Rad protein assay solution (Bio-Rad, Hercules, CA) and by staining
the transferred nitrocellulose membrane with Ponceau S solution (Sigma,
St. Louis, MO). Standard Western blot methods were used (35). Rabbit
polyclonal antibodies raised against bacterially expressed recombinant
His-p38 protein, rabbit polyclonal antibodies raised against
bacterially expressed recombinant His-TAB1 Reporter Gene Assay--
Cells were grown on 35-mm-diameter
multiwell plates and transiently transfected with GAL4-responsive
luciferase plasmid or the NF- Preparation of Recombinant Proteins--
GST fusion proteins of
TAB1, TAB1 Protein Kinase Assays--
In vitro kinase assays
were carried out at 37 °C for 30 min using ~0.2 µg of
recombinant kinase or recombinant protein, 5 µg of kinase substrate,
250 µM ATP, and 10 µCi of [ RNAi--
RNAi constructs were constructed using pSuper vector
(38). The hairpin RNA encoded by R1 and R1(M) constructs is shown in Fig. 2C. The sequences of other RNAi constructs, which
cannot decrease in endogenous genes, are available upon request.
Each of the pSuper constructs was co-transfected with pSVNeo into
breast cancer cell line MDA231, and stable lines were selected by G418 at 1 mg/ml concentration.
Matrigel Invasion Assay--
Cell invasion was analyzed using
BIOCOAT Matrigel invasion chambers (BD Biosciences) according to the
manufacturer's instructions. Briefly, 2 × 105 cells
in 300 µl were added to each chamber and allowed to invade Matrigel
for 20 h at 37 °C and 5% CO2 atmosphere. The
non-invading cells on the upper surface of the membrane were removed
from the chambers, and the invading cells on the lower surface of
membrane were stained with a Diff-Quick stain kit (BD Biosciences).
After two washes with water, the chambers were allowed to air dry. The number of invading cells was then counted using a phase-contrast microscope.
Identification of TAB1
To determine whether TAB1 and TAB1 Expression of TAB1
To examine the protein expression of TAB1 and TAB1
RNA interference (RNAi) is the process of gene silencing whereby
double-stranded RNA induces the homology-dependent
degradation of its cognate mRNA. We designed RNAi constructs to
target TAB1 and TAB1 TAB1
To investigate the specificity of the interaction between TAB1
TAB1 was originally identified as a binding protein of TAK1. It
promotes the TAK1 kinase activation through the binding with TAK1. The
sequence data shown in Fig. 1A suggest that TAB1 Activation of p38
It is known that p38 TAB1 but Not TAB1
It was reported that TAB1-mediated TAK1 activation could dramatically
induce NF- TAB1 Extracellular Stimuli Affects TAB1 Reduction of TAB1 TAB1 MAPK can be carried out not only by its upstream
MAPK kinases (MKKs) but also by p38
autophosphorylation. p38
autoactivation requires an interaction of p38
with TAB1
(transforming growth factor-
-activated protein kinase 1-binding
protein 1). The autoactivation mechanism of p38
has been found to be
important in cellular responses to a number of physiologically relevant
stimuli. Here, we report the characterization of a splicing variant of
TAB1, TAB1
. TAB1 and TAB1
share the first
10 exons. The 11th and 12th exons of TAB1 were spliced out
in TAB1
, and an extra exon, termed exon
, downstream
of exons 11 and 12 in the genome was used as the last exon in
TAB1
. The mRNA of TAB1
was expressed in all cell lines examined. The TAB1
mRNA encodes a protein with an
identical sequence to TAB1 except the C-terminal 69 amino acids were
replaced with an unrelated 27-amino acid sequence. Similar to TAB1,
TAB1
interacts with p38
but not other MAPKs and stimulates p38
autoactivation. Different from TAB1, TAB1
does not bind or activate
TAK1. Inhibition of TAB1
expression with RNA interference in
MDA231 breast cancer cells resulted in the reduction of basal activity
of p38
and invasiveness of MDA231 cells, suggesting that
TAB1
is involved in regulating p38
activity in
physiological conditions.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
MAPK that did not require upstream kinases. We found
that TAB1 interacts with p38
and leads to the activation of p38
(23). TAB1 is a protein that was initially described as an activator of
a member of MAPKKK, TAK1, in response to stimulation of transforming
growth factor-
(24). When ectopically expressed together with TAK1,
TAB1 interacts with TAK1 and leads to enhancement of TAK1 activation
(24-29). The C-terminal 68-amino acid portion of TAB1 is sufficient
for binding to and activation of TAK1 (24, 25, 28). Activation of TAK1
by TAB1 is mediated via autophosphorylation of Ser-192 within its
kinase activation loop (26). We found that TAB1-mediated p38
activation also occurs through an autoactivation mechanism (23).
However, the portion of the TAB1 protein that is responsible for
p38
interaction and activation resides within amino acids 373-418,
which is N-terminal to the TAK1 binding site (23, 24).
. TAB1
is an alternative splicing product of the
TAB1 gene. TAB1
and TAB1 have identical amino acid
sequences with the exception of the C terminus. Similar to TAB1,
TAB1
interacts with p38
and induces p38
autoactivation.
Different from TAB1, TAB1
did not bind with TAK1 and had no effect
on TAK1 activation. The present findings suggest that the alternative
splicing of TAB1 has a role in intracellular signaling transduction.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
--
We completely sequenced two EST clones
that showed homologues to TAB1 (clone identification numbers: 50210 and
3049027). One of the clones showed 100% homology with the 5'-end
portion of TAB1 cDNA but different 3'-end sequence. This cDNA
was termed as TAB1
, and the cDNA sequence has been deposited in
GenBankTM (AF425640).
coding
region was subcloned from a cDNA clone (clone number 50210) into
the mammalian expression vector pcDNA3 (Invitrogen, San Diego, CA)
and the pET14 vector (Novagen, Madison, WI), respectively. Other
expression vectors, including FLAG-p38, -p38
, -p38
, -p38
, -p38(KM), -JNK1, -ERK2, MKK6(E), MKK6(A), MKK3(A), and His-p38, were
constructed as described previously (23, 30-34).
was: 5'-CAATCATCGCAGAGCCAGAAATC-3' located in
exon-8. The downstream primers were: TAB1,
5'-ACGCTCCAGAGGCGGTAAAACTC-3' in exon-11; TAB1
,
5'-CACTGAGGGATGGCTGTCAAATC-3' in exon-
(Fig.1). The obtained
RT-PCR fragments for TAB1 and TAB1
were purified and sequenced to
confirm their identity. Semi-quantitative RT-PCR was performed using
competitive quantitative RT-PCR kits from Ambion (Austin, TX).
(Clontech, Palo Alto,
CA) was co-transfected, and transfection efficiency was normalized by
quantifying
-galactosidase activity.
protein, polyclonal goat
anti-TAB1 (N-19) (Santa Cruz Biotechnology, Santa Cruz, CA), anti-FLAG
M2 mAb (Sigma, St. Louis, MO), anti-HA mAb (12CA5) (Roche Molecular
Biochemicals, Indianapolis, IN), and anti-phospho p38 (New England
BioLabs, Beverley, MA) were used in immunoblotting. Selective viewing
of ectopically overexpressed TAB1 and TAB1
by Western blotting was
achieved by adjusting exposure time. For co-immunoprecipitation, cell
lysates prepared as described above were incubated with anti-FLAG M2
beads (Sigma) and gently shaken for 4 h at 4 °C. The beads were
washed three times with lysis buffer and 1 time with 50 mM
Tris, pH 6.8. Then, the SDS sample buffer (50 µl) was added and
heated for 5 min at 100 °C. The supernatant was applied to SDS-PAGE
and detected by immunoblotting.
B-dependent luciferase
reporter plasmid (36, 37). A
-galactosidase expression plasmid
(pCMV-
-gal, Clontech, Palo Alto, CA) was used to
control for transfection efficiency. The total amount of DNA for each
transfection was kept constant by using the empty vector pcDNA3.
Cell extracts were prepared 36 h later, and
-galactosidase and
luciferase activities were measured.
, and MKK4 were expressed in Escherichia coli
strain BL21 and purified using glutathione-Sepharose 4B beads (Amersham
Biosciences, Uppsala, Sweden). All of the His6-tagged recombinant proteins were expressed in the BL21(DE3) strain and purified using the nickel-nitrilotriacetic acid purification system (Qiagen, Valencia, CA). Myelin basic protein (MBP) was purchased from Sigma.
-32P]ATP in
20 µl of kinase reaction buffer. Reactions were terminated by the
addition of Laemmli sample buffer. Reaction products were resolved by
12% SDS-PAGE, and the extent of protein phosphorylation was visualized
by autoradiography. For the transiently expressed protein, FLAG-p38
protein in 293 cells was immunoprecipitated with anti-M2 beads. After
washing three times with lysis buffer and one time with kinase reaction
buffer, the beads were further used in the analysis of kinase activity.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
--
In our study of TAB1, we have
sequenced a couple of EST clones that have sequence homology with TAB1.
The cDNA of one of the EST clones appeared to be a splicing variant
of TAB1; its sequence was 100% identical to TAB1 in the 5'-portion but
different at the 3'-end. The protein encoded by this cDNA was
termed TAB1
and shown is in Fig.
1A in comparison with TAB1.
The N-terminal sequence of amino acids 1-435 of TAB1
is identical
to TAB1. Different from TAB1, TAB1
lacks the
C-terminal 69 amino acids found in TAB1 and has an unrelated 29-amino
acid sequence instead (Fig. 1A). Because the C-terminal
68-amino acid portion of TAB1 was mapped as a TAK1 binding domain (25,
28), TAB1
does not have the TAK1 binding domain.
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Fig. 1.
TAB1 is a splicing
isoform of TAB1. A, amino acid sequences of TAB1
and
TAB1. B, structure of exon and intron in TAB1
and
TAB1.
are splicing variants, we
analyzed the gene structure in the TAB1 locus. We found
that the TAB1 gene is composed of 12 exons.
TAB1
shares 1-10 exons with TAB1. Exons 11 and 12 of TAB1 were spliced out in TAB1
, and
an exon further downstream termed exon
was the 11th exon of
TAB1
(Fig. 1B). Thus, the difference between
TAB1 and TAB1
is indeed generated by alternative splicing.
--
To investigate the expression of
TAB1
in different cells, total RNAs were extracted from HEK293,
HPEG2, MDA-231, and RPMI 8226 cells. The existence of TAB1
and TAB1
transcripts was examined by RT-PCR using their specific primers. Both
TAB1 and TAB1
transcripts were found in all cell lines tested (Fig.
2A), suggesting that TAB1 and
TAB1
are concurrently expressed.
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Fig. 2.
Expression of
TAB1 . A, expression of TAB1
and TAB1 mRNA in different cells. Total RNA was isolated from
different cells, and RT-PCR was performed using a common 5' primer and
TAB1
- or TAB1-specific 3' primer. B, proteins expression
of TAB1 isoforms in different cells. Total cell lysates of different
cells as indicated were resolved by SDS-PAGE and immunoblotted with
antibody against the N terminus of TAB1 and TAB1
. The ectopically
overexpressed TAB1 and TAB1
in 293 cells are shown in the
right panel. A short exposure time was used to selectively
show the overexpressed TAB1 and TAB1
. C, the 19-bp
hairpin RNAs (hpRNA) encoded by R1 and R1(M) RNAi
constructs. D, the expression of TAB1
protein in MDA231
cells that were stably transfected with RNAi constructs for TAB1
.
Lysates of the cells stably transfected with nothing (
), empty pSuper
vector, R1, R1(M), R2, or R3 were subjected to immunoblotting using
antibody against the N terminus of TAB1 and TAB1
. E, the
expression of TAB1 and TAB1
mRNA in the stably transfected
MDA231 cell lines. Competitive quantitative RT-PCR was performed using
the total RNA isolated from the cell lines indicated.
, total cell
lysates were prepared from HEK293, HEPG2, MDA231, RPMI8226, and MCF-7
cells. The expression of TAB1 or TAB1
was analyzed by Western
blotting using anti-TAB1 antibodies. The antibody against a peptide,
found in the N terminus of both TAB1 and TAB1
, detected two major
protein bands in Western blotting analysis (Fig. 2B, left panel). The same result was obtained with an antibody
that was raised by using recombinant TAB1
protein (data not shown). We overexpressed TAB1 and TAB1
in 293 cells and detected the ectopically expressed TAB1 and TAB1
by Western blotting (Fig. 2B, right panel). Comparison of the migrations of
endogenous proteins detected by the anti-TAB1/TAB1
antibodies with
the ectopically expressed TAB1 and TAB1
gives no indication which
protein band or bands are TAB1 or TAB1
, because there was a higher
molecular weight band of endogenous protein that could be
post-translationally modified TAB1 or TAB1
or could even be another
TAB1 isoform. We have tried to raise TAB1- and TAB1
-specific
antibodies using C-terminal peptides from TAB1 and TAB1
. Because the
difference between TAB1 and TAB1
is very limited (29 and 69 amino
acids), there were not many peptide sequences that could be chosen to generate antibodies. As a result, we have not generated any antibodies that can selectively detect TAB1 or TAB1
. We cannot formally conclude whether TAB1 and/or TAB1
were expressed as protein in cells
by the Western blotting analysis shown in Fig. 2B, but the protein expression of at least one of TAB1 isoforms was confirmed.
using the method of Brummelkamp et
al. (38) and stably transfected these constructs into MDA231
cells. Stable transfection of the RNAi constructs, which target the
common region of TAB1 and TAB1
, and the specific region of TAB1 did
not affect the expression of TAB1s in MDA231 cells (data not shown).
Transfection of R1 (Fig. 2C), one of the RNAi constructs
that were designed to target TAB1
, resulted in reduction of the
expression of the upper-band protein detected by anti-TAB1 antibody
(Fig. 2D). In contrast, transfection of R1(M), which had a
single point mutant in comparison with R1 (Fig. 2C), did not
produce any effect, indicating the specificity of the RNAi effect. The
other two RNAi constructs that were designed to target TAB1
also had
no effect on TAB1
expression. These results indicated that the upper
band is TAB1
. Because the apparent molecular weight of endogenous
TAB1
is higher than ectopically expressed TAB1
(Fig. 2,
B and D), the endogenous TAB1
is most likely
being post-translationally modified. The reduction of TAB1
protein
by R1 is specific, because the protein level of the lower band, which
is most likely to be TAB1, and p38
were not affected by R1 (Fig.
2D). To further confirm that R1 selectively targeted TAB1
mRNA, we examined mRNA expression of TAB1 and TAB1
by
competitive quantitative RT-PCR using their specific primers. The cells
stably transfected with R1, but not with R1(M) or empty vector, had
reduced TAB1
mRNA (Fig. 2E), confirming that R1
specifically mediated a reduction of TAB1
mRNA. Thus, TAB1
protein is expressed in cells.
Interacts with p38
but Not with TAK1--
Our previous
studies have demonstrated that the TAB1 protein interacts with p38
(23). To find out if the TAB1
interacts with p38
,
expression plasmids encoding TAB1
, TAB1, and FLAG-p38
were
co-transfected into 293 cells. The FLAG-p38
was precipitated with
agarose beads conjugated with anti-FLAG antibody (M2), and the
existence of TAB1 in the immunoprecipitation complexes was detected by
immunoblotting with anti-TAB1 antibody. The results showed that both
TAB1
and TAB1 bind with p38
(Fig.
3A).
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Fig. 3.
TAB1 interacts with
p38
but not TAK1. A,
FLAG-p38
was co-expressed with TAB1 or TAB1
in 293 cells. Cells
were lysed 24 h after transfection, and immunoprecipitation was
performed with ant-FLAG antibody. The immunoprecipitates and cell
lysates were analyzed by immunoblotting with antibody against TAB1 or
FLAG as indicated. TAB1
was immunoprecipitated with p38
.
B, the interaction of TAB1
with p38
, p38
, or p38
was examined as in A. These three p38 family members had no
or very weak interaction with TAB1
. C, the interaction of
TAB1
with JNK1 or ERK2 was examined as in A. No
interaction was detected. D, TAB1 or TAB1
was
co-expressed with HA-TAK1. TAK1 was immunoprecipitated with anti-HA
antibody. The immunoprecipitates and cell lysates were analyzed by
immunoblotting with antibody to TAB1 or HA as indicated. TAB1
was
not co-immunoprecipitated with TAK1. The experiments were performed
three times with comparable results.
and
p38
, we first tested if TAB1
could interact with three other p38
isoforms. As shown in Fig. 3B, a small amount of TAB1
was
pulled down by p38
but not by p38
or p38
. The expression level
of p38
was higher than any other p38 subfamily members (Fig.
3B, middle panel, and data not shown), but the
amount of TAB1
pulled down with p38
was very little in comparison
with p38
(Fig. 3, A and B), so the affinity of
TAB1
and p38
should be much smaller than TAB1
and p38
. We
next evaluated whether there is interaction between TAB1
with the
other two MAPKs, JNK1 and ERK2. The results demonstrated that TAB1
bound strongly with p38
but not with ERK2 or JNK1 (Fig.
3C).
does not
have a TAK1 binding domain, however, whether TAB1
can interact with
TAK1 needs to be examined. We transfected the HA-TAK1, TAB1, and
TAB1
expression plasmids into 293 cells. Immunoprecipitation was
performed with agarose beads conjugated with anti-HA antibody. The
immunoprecipitates were immunoblotted with anti-TAB1 antibody. We found
that immunoprecipitation of TAK1 pulled down TAB1 but not TAB1
(Fig.
3D), indicating that there was no interaction between
TAB1
and TAK1. These data agree with domain mapping of TAB1 reported
by other groups and indicate that TAB1
only interacts with p38
and not TAK1.
by TAB1
--
It has been shown that
interaction of TAB1 with p38
resulted in autoactivation of p38
(23). We next evaluated the ability of TAB1
to activate p38
. The
expression plasmids of TAB1
, TAB1, and p38
were co-transformed
into 293 cells in different combinations, and the extent of p38
dual
phosphorylation was examined by immunoblotting using anti-phospho-p38
antibody. As we reported previously, co-expression of TAB1 with p38
in cells led to p38
phosphorylation; expression of TAB1
also led
to the phosphorylation of p38
(Fig.
4A). To ensure that the
phosphorylation of p38
detected by anti-phospho-p38 antibody indeed
represented p38
activity, FLAG-p38
protein was immunoprecipitated
by using anti-FLAG M2 beads and further analyzed by in vitro
kinase assay using MBP as a substrate. The increase of p38
dual
phosphorylation by TAB1 or TAB1
was correlated with the increase of
p38
activity (Fig. 4B).
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Fig. 4.
TAB1 promotes
p38
activation. A, FLAG-p38
was co-expressed with TAB1
or TAB1 in 293 cells. FLAG-p38
was
immunoprecipitated with anti-FLAG antibody. The immunoprecipitates and
cell lysates were analyzed by immunoblotting with anti-phospho-p38,
anti-FLAG, or anti-TAB1 as indicated. Co-expression of TAB1
or TAB1
increased p38
phosphorylation. B, the experiments were
performed as in A, and the immunoprecipitates were used as
kinase in the in vitro kinase assay using MBP as a
substrate. TAB1
and TAB1 increased p38
kinase activity.
C, expression vectors of a luciferase reporter gene under
the control of 5xGal4 binding site (5xGAL), GAL4 binding
domain fused with ATF2 activation domain (GAL-ATF2),
TAB1
, TAB1, and FLAG-p38
were transfected into 293 cells in
different combinations as indicated. Luciferase activity was measured
24 h later. TAB1 and TAB1
enhanced ATF2-dependent
luciferase expression when co-expressed with p38
. Experiments were
performed three times with comparable results.
activation has an effect on expression of a
number of genes. Previous studies showed that activation of p38
by
MKK6 or MKK3 augments the transcriptional activity of ATF2 in transient
transfection assays (19). To determine whether activation of p38
by
TAB1
interaction had a similar effect on ATF2, we used fusion
proteins containing the transactivation domain of ATF2 fused with GAL4
DNA binding domain. Whether TAB1
-mediated p38
phosphorylation can
transduce signaling downstream was determined by whether
TAB1
-mediated activation of p38
could enhance the transcriptional
activity of GAL4-ATF2 protein. The GAL4-driven luciferase
reporter construct, the expression plasmid of GAL-ATF2 fusion protein,
and the expression plasmids of p38
, TAB1, or TAB1
were
transfected into 293 cells in different combinations. Luciferase
activity was examined 48 h after transfection. Luciferase expression was significantly increased in the cells co-expressing p38
and TAB1 or TAB1
(Fig. 4C). Thus, TAB1
-mediated
p38
activation can transduce signaling downstream.
Activates TAK1--
Because TAB1 activates
TAK1 (24), we investigated whether the TAB1
had effect on TAK1. The
expression vectors of TAB1
or TAB1 were transfected into 293 cells
with HA-TAK1. HA-TAK1 was immunoprecipitated by anti-HA beads, and
kinase activity of TAK1 was examined by a coupled in vitro
kinase assay using the GST-MKK4 and His-p38
(M) as substrates (Fig.
5A). The increase in kinase
activity of TAK1 can be determined by activation of MKK4, which in turn
phosphorylates p38
(M). As reported by others, co-expression of TAB1
increased TAK1 activity. In contrast, expression of TAB1
had no
effect TAK1 activity (Fig. 5A). These data are consistent
with the data in Fig. 3D, showing that TAB1
did
interact with TAK1.
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Fig. 5.
TAB1 does not
activate TAK1. A, TAB1 or TAB1
were co-expressed
with HA-TAK1 in 293 cells. TAK1 was immunoprecipitated, and in
vitro coupled kinase assay was performed using immunoprecipitates
as kinase, GST-MKK4 as a substrate for TAK1, and His-p38
(M) as a
substrate for MKK4. Only TAB1 increased TAK1 activity. B,
NF-
B reporter was co-expressed with TAK1, TAB1, or TAB1
in 293 cells as indicated. Luciferase activity was measured 24 h later.
TAK1 and TAB1
co-expression did not enhance
NF-
B-dependent gene expression. The experiments were
performed two times with comparable results.
B reporter gene expression. We were able to reproduce the
result of NF-
B activation by TAK1 and TAB1 co-expression (Fig.
5B). In contrast to TAB1 co-expression, TAB1
co-expression did not have any effect on TAK1-mediated
NF-
B-dependent reporter gene expression (Fig.
5B). These data further support the notion that TAB1
does
not have any effect on TAK1.
-induced p38
Activation Is Dependent on p38
Autophosphorylation--
We uncovered recently that TAB1-mediated
p38
activation is through p38
autophosphorylation (23). The same
approaches were used here to determine whether TAB1
-mediated p38
phosphorylation is also autophosphorylation. First, we examined whether
TAB1
-mediated p38
phosphorylation can be inhibited by dominant
negative MKK6 or TAK1, the known upstream kinases of p38
. Mutation
of regulatory phosphorylation sites in MKK6 to alanine (MKK6(A))
resulted in the loss of kinase activity, and this mutant has been
successfully used as a dominant negative mutant in a number of studies
(19, 31, 39). Mutation in the ATP binding site of TAK1 (TAK1(K63W)) leads to kinase death, and this mutant can be used as a dominant negative mutant (24, 27). We co-transfected MKK6(A) or TAK1(K63W) with
TAB1
and p38
and compared the level of p38
phosphorylation. Expression of MKK6(A) or TAK1(K63W) did not interfere with
TAB1
-induced p38
activation (Fig.
6A). Similar results were
obtained when MKK3(A) or MKK4(A) was expressed (data not shown). These
results agreed with the report that TAB1-mediated p38
phosphorylation is independent from the known upstream kinases of
p38
and indicated that TAB1
-mediated p38
occurs most likely
through a similar mechanism. Second, we investigated whether
TAB1
-induced p38
activation is also dependent on p38
intrinsic
enzymatic activity. Two methods were used to diminish the p38
intrinsic activity. One was to use kinase-dead p38
mutants. A p38
mutant with an impaired ATP binding site (Lys-53 to Met mutant termed
p38
(M)) was used in the experiments. The other method was to treat
the cells with p38 inhibitor SB203580 (40, 41). As shown in Fig. 6B, phosphorylation of the kinase-dead mutant of p38
was
observed in the cells expressing MKK6(E) but not in the cells
expressing TAB1
; activation of p38
by TAB1
was inhibited by
treatment of cells with SB203580. These data indicate that
TAB1
-mediated p38
phosphorylation requires intrinsic kinase
activity of p38
. Third, we examined TAB1
-mediated p38
activation in vitro. p38
and TAB1
were synthesized in
and purified from bacteria as His-tagged and GST fusion proteins,
respectively. GST-TAB1
was incubated with His-p38
in kinase
reaction buffer with cold ATP. The activity of p38
was examined by
using myelin basic protein (MBP) as a substrate. TAB1
significantly
increased p38
activity toward MBP. Moreover, TAB1
-mediated p38
activity was reduced by SB203580 (Fig. 6C). Collectively,
our data demonstrate that TAB1
-mediated p38
activation is p38
autoactivation.
View larger version (25K):
[in a new window]
Fig. 6.
TAB1 -mediated
p38
dual phosphorylation is a
p38
autophosphorylation. A,
293 cells were co-transfected with different expression vectors as
indicated. 2- and 4-fold plasmid DNA of MKK6(A) and TAK1(K63W) were
used in some samples to increase the expression of these dominant
negative proteins. Immunoprecipitation and Western blotting were
performed as in Figs. 2 and 3. Overexpression of dominant negative MKK6
(MKK6(A)) or TAK1 (TAK1(K63W)) had no effect on
TAB1
-mediated p38
dual phosphorylation. B,
co-expression of FLAG-p38
or FLAG-p38
(M) with TAB1 or MKK6(E) was
performed as in A. SB203580 (5 µM) was added
into cell culture medium 4 h after transfection in the sample
indicated. Immunoprecipitation and Western blotting were performed as
in A. TAB1-mediated p38
dual phosphorylation requires
intrinsic p38
activity. C, TAB1-medtated p38
phosphorylation in vitro requires intrinsic p38
kinase
activity. GST-TAB1
, His-p38
, and MBP were incubated in kinase
reaction buffer containing [32P]ATP in the presence of
different concentrations of SB203580. The kinase reaction was stopped
by SDS-sample buffer, and phosphorylated proteins were resolved on
SDS-PAGE and exposed to x-ray film. SB203580 inhibited both
autophosphorylation and activity of p38
. Data shown are
representative of two to three independent experiments.
and p38
Association--
We reported previously that
TAB1-dependent activation of p38
was used differently by
different extracellular stimuli (23). Due to the identification
of the similar effects of TAB1
on p38
activation to those
of TAB1, we test whether TAB1
could selectively be involved in
p38
activation induced with different stimuli. The 293 cells were
transfected with TAB1
and stimulated with different stimuli. As
shown in Fig. 7A,
overexpression of TAB1
significantly enhanced peroxynitrite and
TNF-induced p38
phosphorylation. The effects of TAB1
on
anisomycin- and arsonite-induced p38
phosphorylation were modest.
Overexpression of TAB1
did not effect high osmolar sorbital-induced
p38
phosphorylation. The selective involvement of TAB1
in
enhancing p38
phosphorylation induced by different stimuli fit well
with the profiles of kinase cascade-independent p38
activation
reported previously (23). Because the association of TAB1
with
p38
can induce p38
activation, we next investigated whether the
extracellular stimuli selectively affected the association of TAB1
with p38
. 293 cells were transfected with expression plasmids of
TAB1 and p38
for 24 h, and then were stimulated with several
extracellular stimuli. The association of TAB1
with p38
was
examined by using a co-immunoprecipitation assay. We observed that TNF
and peroxynitrite significantly increased the binding of TAB1
with
p38
and anisomycin or arsonite also induced this association
but to a lesser extent. Sorbitol failed to induce this association
(Fig. 7B). These data correlated with the enhancement of
p38
phosphorylation by TAB1
overexpression in cells treated with
different stimuli (Fig. 7, A and B). The time
course of TAB1
-p38
interactions induced by TNF is shown in Fig.
7C. These results suggest that enhanced p38
and TAB1
interactions are differentially involved in different extracellular
stimuli-induced cellular activation.
View larger version (31K):
[in a new window]
Fig. 7.
TAB1 selectively
involves p38
activation induced by different
extracellular stimuli. A, FLAG-p38
was co-expressed
with or without TAB1
in 293 cells. 24 h later, the cells were
treated with TNF-
(100 ng/ml), peroxynitrite (500 µM),
anisomycin (50 ng/ml), arsonite (200 µM), and sorbitol
(0.4 M) for 30, 5, 30, and 30 min, respectively. The levels
of p38
and phospho-p38
were determined by immunoprecipitation and
immunoblotting as in Figs. 2 and 3. Expression of TAB1
enhanced
p38
phosphorylation induced by some stimuli but not others.
B, TAB1
and FLAG-p38
were expressed in 293 cells, and
the cells were treated with different stimuli as indicated. FLAG-p38
was immunoprecipitated, and the co-precipitated TAB1
was determined.
The amount of TAB1
co-precipitated varied when the cells were
treated with different stimuli. C, TAB1
and FLAG-p38
were expressed in 293 cells, and the cells were treated with TNF for
different periods of time. FLAG-p38
was immunoprecipitated, and
co-precipitated TAB1
was determined. A time-dependent
co-precipitation of TAB1
was observed in TNF-treated cells. Data
shown are representative of two to three independent experiments.
by RNAi Reduced Basal Activity of p38
and
Invasiveness of MDA231 Cells--
MDA231 is an invasive breast cancer
cell line. We showed previously that the invasiveness of MDA231 is
dependent on, at least partially, the high basal activity of p38
(42). Because the RNAi construct R1 can specifically inhibit TAB1
expression in MDA231 cells (Fig. 2D), we evaluated the role
of TAB1
in p38
activity and invasion of MDA231 cells. As shown in
Fig. 8A, the basal level of
p38
phosphorylation was significantly reduced in R1 transfected
cells in comparison with non-transfected, vector-transfected, and
R1(M)-transfected cells. Therefore, TAB1
has a role in regulating basal activity of p38
. Stimulation of MDA231 cells with TNF can lead
to a 2- to 3-fold increase in p38
phosphorylation in vector- and
R1(M)-transfected cells (Fig. 8B). The p38
phosphorylation in R1-transfected cells was significantly lower in
comparison with controls before and after TNF stimulation (Fig.
8B), and the -fold induction of p38
phosphorylation by
TNF was also lower. We used Matrigel invasion assays to evaluate
whether reduction of TAB1
by RNAi had an effect on invasion of
MDA231 cells. As shown in Fig. 8C, invasion of MDA231 cells
was inhibited ~50% when TAB1
was knocked down by RNAi.
View larger version (13K):
[in a new window]
Fig. 8.
TAB1 RNAi reduces
basal activity of p38
and invasiveness of
MDA231 cells. A, basal level of p38
phosphorylation
in MDA231 cell lines stably transfected with TAB1
RNAi constructs.
Lysates of the cells were immunoblotted with anti-phospho-p38 and
anti-p38
antibody. B, p38
phosphorylation in the
stably transfected MDA231 cell lines stimulated with or without TNF.
C, Matrigel invasion assay of the stably transfected MDA231
cell lines. Quantification of cells invading the undersurface of the
Matrigel chamber is shown.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
is a splicing variant of TAB1 based on its gene structure
(Fig. 1B). The expression of TAB1
was demonstrated by
combined results from RT-PCR, Western blotting, and RNAi (Fig. 2).
TAB1
has a different C-terminal sequence from that of TAB1 and does not have a TAK1 interacting domain that is harbored at the C terminus of TAB1. TAB1
retained the p38
interacting domain located in the
N-terminal side to the TAK1 interacting domain in TAB1. In addition to
the interacting domains for p38
and TAK1, computer analysis
indicated that there is a protein phosphatase 2C (PP2C)-like domain in
the N-terminal of TAB1 and TAB1
. The domain structure of TAB1 and
TAB1
is shown in Fig. 9. Like TAB1,
TAB1
interacts with p38
, and this interaction leads to
autoactivation of p38
. Unlike TAB1, TAB1
cannot interact with
TAK1 and has no effect on TAK1 activity. Specific inhibition of TAB1
expression by RNAi reduced basal activity of p38
in MDA231 cells and
decreased the invasiveness of MDA231 cells, indicating that TAB1
is
involved in the regulation of p38
activity in a physiological
setting.
View larger version (10K):
[in a new window]
Fig. 9.
Domain structure of TAB1 and
TAB1 .
Our data show that TAB1- and TAB1-mediated p38
activation is an
autophosphorylation of p38
. However, how an interaction with TAB1 or
TAB1
leads to p38
autoactivation remains to be elucidated. It is
well established that activation of MAPKs by their corresponding MAPKKs
is through dual phosphorylation on specific threonine and tyrosine
residues (4, 18), and we have shown in our previous work (23) that
p38
autoactivation is also achieved by dual phosphorylation on the
regulatory sites. MAPKs are serine/threonine kinases, but low level
phosphorylation on their regulatory tyrosine residue has been observed
in ERK1, ERK2, and p38
in vitro (23, 43, 44). It was
demonstrated quite a long time ago that this phosphorylation is
mediated by autophosphorylation (43-45). These early works suggested
that autophosphorylation on one of the regulatory sites could occur in
MAPKs, although the efficiency was low. It is possible that the binding
of TAB1 or TAB1
to p38
changes the confirmation of p38
and
facilitates autophosphorylation of p38
on its regulatory sites.
p38
has a similar overall structure to the ERK2 MAPK (46). Analysis of crystal structures of inactive and active forms of the ERK2 has
indicated that phosphorylation of ERK2 results in a conformational change to an "energetically unfavorable state" (47). It is possible that autophosphorylation of p38
can be enhanced by stabilization of
p38
in an "energetically unfavorable transient confirmation" by
the binding of an "activator" protein like TAB1 or TAB1
(48, 49). A recent study by Emrick et al. (50) shows that a
double mutation of leucine 73 to proline and serine 151 to aspartic
acid in ERK2 leads to ERK2 autophosphorylation and the mutant is
constitutively active. By analyzing the three-dimensional structure of
ERK2, authors of this work suggested that mutations of L73P and S151D permit a regulatory phosphorylation site Y185 to move within hydrogen bonding distance of the catalytic aspartate Asp-147,
facilitating the phosphoryl transfer. This work supports the idea that
conformational changes can lead to MAPK autophosphorylation and
activation. The activation mechanism of p38
, after binding to TAB1
or TAB1
, should be similar to the constitutive active ERK2 mutant,
because both are activated by autophosphorylation. It is thus very
likely that TAB1 and p38
interaction leads to conformational changes that are similar to that in ERK2(L73P,S151D) and that such
changes relieve structural constraints in the catalytic site that
suppress autophosphorylation and autoactivation.
Autophosphorylation is a common mechanism for the activation of a
number of different kinases such as receptor tyrosine kinases (51, 52)
and some of the MAPKKKs (53-56). TAB1-mediated TAK1 activation was
shown to be a result of autoactivation (26, 29). Because TAK1 and
p38 bind to different sites of TAB1 and both are activated by
autophosphorylation after binding to TAB1, one would assume that TAB1
has a structural feature that triggers autophosphorylation of these two
different kinases. Sequence analysis revealed that the N-terminal
two-thirds of TAB1 and TAB1
protein had low level sequence homology
with phosphatase PP2C. However, phosphatase activity has not been
detected in TAB1 or TAB1
so far. Whether this PP2C-like domain has a
function in regulating p38
and/or TAK1 activity is a subject for
future studies. Because the C-terminal 68-amino acid peptide of TAB1 is
sufficient to bind and activate TAK1, the N-terminal PP2C-like domain
is dispensable for TAK1 activation. To date we were unable to obtain a
protein that only contains the p38
interacting domain, so we were
unable to determine whether this domain alone can lead to p38
activation. A common docking domain has been identified in MAPKs as a
common site for binding to upstream MKK, downstream substrates, and
dual phosphatase (57, 58). A gain-of-function mutant of a
Drosophila melanogaster MAPK was found to be
resulting from disrupted interactions between MAPKs and phosphatase by
a mutation in a common docking domain (D334N) (59). It is possible that
TAB1 prevents p38
and phosphatase interactions to enhance p38
activity. However, this possibility was excluded, because purified TAB1
can cause p38
autophosphorylation in the absence of any phosphatase
in vitro (23). Furthermore, TAB1 did not compete with
PP2C-mediated inactivation of p38
in co-expression experiments (data
not shown). Genetic screening has isolated a gain-of-function mutant of
p38
(49). This mutant (F327L or S) can gain kinase activity in the absence of tyrosine phosphorylation, suggesting the driven force for
converting p38
to active conformation in this mutant is different from that of TAB1-mediated p38
activation.
The p38 MAPK is activated in response to a variety of extracellular
stimuli, including pro-inflammatory cytokines and environmental stresses (4, 60). How these different stimuli via different receptors
or other molecular sensors activate p38
is still not fully
understood. Previous work by a number of investigators, including us,
showed that there are at least two different mechanisms immediately
upstream of p38
in regulating p38
activity (19, 23). One is
dependent on upstream kinase MKK3 or MKK6; another is dependent on
TAB1-mediated p38
autophosphorylation. The data presented in this
report added TAB1
into the regulation network of p38
. To date we
were unable to generate isoform-specific antibodies for TAB1
and
TAB1, however, in combination with RNAi, we were able to identify the
protein band of TAB1
resolved on SDS-PAGE (Fig. 2D). The
role of TAB1
in controlling the basal activity of p38
has been
documented by our experiments (Fig. 8). Whether TAB1 and TAB1
have
different roles in mediating p38
activation in cells has not been
determined. Nevertheless, TAB1 and TAB1
are clearly different in
their functions, because TAB1 is capable of regulating both TAK1 and
p38
whereas TAB1
only activates p38
. The different functions
of TAB1 splicing variants may be a mechanism required for precise
regulation of intracellular signaling that controls p38
activation.
![]() |
ACKNOWLEDGEMENT |
---|
We thank Dr. Reuven Agami for the pSuper vector.
![]() |
FOOTNOTES |
---|
* This work was supported by United States Public Health Services Grant AI41637 from NIAID, National Institutes of Health.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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AF425640.
To whom correspondence should be addressed: Dept. of Immunology,
The Scripps Research Institute, 10550 North Torrey Pines Rd., La Jolla,
CA 92037. Tel.: 858-784-8704; Fax: 858-784-8665; E-mail: jhan@scripps.edu.
Published, JBC Papers in Press, November 11, 2002, DOI 10.1074/jbc.M210918200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
MAPK, mitogen-activated protein kinase;
MAPKK, MAPK kinase;
MAPKKK, MAPK
kinase kinase;
TAB1, transforming growth factor--activated protein
kinase 1-binding protein 1;
ERK, extracellular signal-regulated kinase;
MEK1, 2, MAPK/ERK kinase 1 and 2;
JNK, c-Jun N-terminal kinase;
SAPK, stress-activated protein kinase;
EST, expressed sequence tag;
RT, reverse transcription;
CMV, cytomegalovirus;
mAb, monoclonal antibody;
HA, hemagglutinin;
GST, glutathione S-transferase;
MBP, myelin basic protein;
RNAi, RNA interference;
TNF, tumor necrosis
factor;
PP2C, protein phosphatase 2C;
MKK, MAPK kinase;
BMK1, Big
MAPK1.
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