From the Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9041
Received for publication, January 24, 2001, and in revised form, February 16, 2001
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ABSTRACT |
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Previous studies demonstrated that in
vitro the protein kinase TAO2 activates MAP/ERK kinases (MEKs) 3, 4, and 6 toward their substrates p38 MAP kinase and c-Jun N-terminal
kinase/stress-activated protein kinase (JNK/SAPK). In this study, we
examined the ability of TAO2 to activate stress-sensitive MAP kinase
pathways in cells and the relationship between activation of TAO2 and
potential downstream pathways. Over-expression of TAO2 activated
endogenous JNK/SAPK and p38 but not ERK1/2. Cotransfection experiments
suggested that TAO2 selectively activates MEK3 and MEK6 but not MEKs 1, 4, or 7. Coimmunoprecipitation demonstrated that endogenous TAO2 specifically associates with MEK3 and MEK6 providing one mechanism for
preferential recognition of MEKs upstream of p38. Sorbitol, and to a
lesser extent, sodium chloride, Taxol, and nocodazole increased TAO2
activity toward itself and kinase-dead MEKs 3 and 6. Activation of
endogenous TAO2 during differentiation of C2C12 myoblasts paralleled
activation of p38 but not JNK/SAPK, consistent with the idea that TAO2
is a physiological regulator of p38 under certain circumstances.
TAO1 and TAO2 are closely related protein kinases whose cDNAs
were originally isolated from rat based on sequence similarity to the
yeast p21-activated protein kinase Ste20p (1, 2). The domain
organization and regulation of TAOs is distinct from yeast
p21-activated protein kinases. The kinase domain is at the TAO N
terminus, and neither TAO1 nor TAO2 contains consensus motifs for
activation by small G proteins. Each TAO is greater than a thousand
amino acids in length; thus, each contains a regulatory domain of over
700 amino acids. A kinase that is over 90% identical to TAO2 was also
identified in a screen for RNAs overexpressed in human prostate
carcinoma; in this context it was named PSK for prostate-derived
STE20-like kinase (3). A third kinase, named JIK for
JNK1 inhibitory kinase, has a
similar organization and size and is over 60% identical to TAOs (4). A
chicken TAO-like kinase has also been found and is called KFC for
kinase from chicken (5).
TAO1 and TAO2 activate stress-sensitive MAP kinase cascades in
vitro by phosphorylating the upstream MAP/ERK kinases (MEKs), MEKs
3, 4, 6, and 7 (1, 2). Phosphorylation by TAOs increases their
activities toward the downstream MAP kinases, p38 MAP kinase, and the
c-Jun N-terminal kinases/stress-activated protein kinases (JNK/SAPKs).
These in vitro activities of TAO1 and TAO2 suggest that they
function in stress responsive pathways within the cell as MEKK-level
kinases. In vitro studies revealed a MEK binding domain just
C-terminal to the TAO catalytic domain (2). This domain binds MEK3 and
MEK6 with selectivity over other MEK family members; these MEKs are
directed toward p38 family members. Both prostate-derived STE20-like
kinase and kinase from chicken were reported to activate JNK/SAPKs in
cotransfected cells (3, 5). JNK inhibitory kinase, in contrast, was
reported to inhibit JNK/SAPK activity (4). Effects of these TAO-related
kinases on p38 were not reported.
Numerous MEKK-level kinases have been implicated in regulating the
stress-responsive JNK/SAPK and p38 pathways (reviewed in 6, 7). We
wished to determine the behavior of TAOs in cells to clarify their
roles in regulating these pathways. Thus, TAO2 was coexpressed in cells
with various MEKs and MAP kinases, and endogenous MAPKs were
immunoprecipitated from TAO2-transfected cells to examine effects of
TAO2 expression on their activities. We also developed antibodies that
could immunoprecipitate native TAO2 to determine whether TAO2 and MEKs
could be coimmunoprecipitated and tested the capacities of various
stimuli to activate TAO2. Finally, we examined the relationship between
TAO2, p38, and JNK/SAPK activities during differentiation of C2C12 myoblasts.
Plasmids, Mutagenesis, and Proteins--
A fragment encoding
TAO2-(1-451) was amplified by polymerase chain reaction with a
BamHI site and EcoRI site incorporated at its 5'
and 3' ends, respectively. The BamHI- and
EcoRI-digested polymerase chain reaction product was ligated
into BamHI- and EcoRI-digested pGEX-KG to create
the pGEX-KG-TAO2-(1-451) plasmid. A
BamHI/SalI-digested fragment from
pGEX-KG-TAO2-(1-451) was ligated to
BglII/SalI-digested pCMV5-Myc or
BglII/SalI-digested pCMV5-HA to create
pCMV5-Myc-TAO2-(1-451) or pCMV5-HA-TAO2-(1-451), respectively. The
resulting plasmids were analyzed by EcoRI digestion and
confirmed by sequencing. The D169A mutation was introduced into both
plasmids with the QuikChange kit (Stratagene) according to the
manufacturer's recommendations. The K82M and K64M mutations were
created in the original pNPT7-5-MEK6 and pNPT7-5-MEK3 plasmids
(kindly provided by Signal Pharmaceuticals and Kunliang Guan,
respectively) also using the QuikChange kit. Escherichia
coli strain BL21-DE3 transformed with either
His6-MEK6KM or His6-MEK3KM was induced with 100 µM isopropyl-1-thio- Cell Culture, Cell Lysates, and Transfections--
293 cells and
Neuro2A cells were grown in Dulbecco's modified Eagle's medium
supplemented with 10% fetal bovine serum, 1% L-glutamine,
and 100 units/ml penicillin/streptomycin. ATT20, GC-B6, PC12, and PC12M
were cultured under similar conditions. C2C12 cells were purchased from
ATCC. To prepare whole cell lysates, cells were washed once with cold
phosphate-buffered saline and lysed with Triton lysis buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.5% Triton
X-100, 80 mM Antibodies, Immunoblots, and Immunoprecipitation--
A
polyclonal antiserum (U2253) was raised against the antigenic peptide
MPAGGRAGSLKDPDVAELFFK (residues 1-21 of TAO2 protein) in rabbits. This
peptide was conjugated to Limulus hemocyanin (14) and
dialyzed into phosphate-buffered saline. A total of five boosts were
performed. The final bleed of U2253 was used in all subsequent studies
involving TAO2 antibodies. For immunoblot analysis, recombinant
proteins or cell lysates were subjected to SDS-PAGE and then
transferred to nitrocellulose membranes. The membranes were blocked
with 5% milk in TBST (20 mM Tris, pH 8.0, 500 mM NaCl, and 0.05% Tween20) overnight and then incubated with primary antibody diluted in TBST plus 0.5% milk at a 1:10,000 dilution for 1 h. After three washes with TBST, the membranes were
incubated with secondary antibody diluted in TBST plus 0.5% milk for
30 min. Membranes were washed again with TBST three times and
Tris-buffered saline once and then developed using enhanced chemiluminescence. Polyclonal anti-ERK2 A249 and anti-ERK1/2 Y691 were
used to immunoprecipitate and to immunoblot endogenous ERKs, respectively (14). Polyclonal anti-p38 P287 (12) or C-20 (from Santa
Cruz) was used to immunoprecipitate or to immunoblot endogenous p38,
respectively. C-20 recognizes both Differentiation of C2C12 Cells--
Mouse myocyte C2C12 cells
were purchased from ATCC. Cells were grown in Dulbecco's modified
Eagle's medium with 10% fetal bovine serum to 80% confluence and
then placed in Dulbecco's modified Eagle's medium with 2% horse
serum for 4 days to allow differentiation essentially as described (16,
17). Half of this medium was replaced with fresh medium daily. During
differentiation, cells were treated daily with either dimethyl
sulfoxide (Me2SO) diluent or 15 µM SB203580
in Me2SO. At the indicated times, cells were harvested in
detergent lysis buffer as described, and enzymes were isolated and
assayed as described in the figure legend.
Stress-sensitive MAPKs Are Activated by Cotransfected TAO2--
To
investigate TAO2 selectivity in intact cells, cotransfection
experiments were performed. In a cotransfection assay, an epitope-tagged MAPK construct was transfected into 293 cells with either the empty vector control or with a differently tagged TAO2 construct. The overexpressed MAPK was immunoprecipitated from the cells
and assayed using transcription factor substrates (Fig. 1). HA-JNK1, when cotransfected with
various TAO2 proteins, became much more active toward its substrate
ATF2 (Fig. 1A). Similarly, HA-SAPK
TAO2-(1-451) appeared to be generally more active than TAO2-(1-320)
or TAO2-(1-993). TAO2-(1-993) is probably less active because earlier
studies with protein produced in Sf9 cells, suggested that it
contains a negative regulatory domain that inhibits its activity (2).
Studies on kinase from chicken also indicated an inhibitory effect of
the C terminus on its catalytic domain (5). The difference in action
between TAO2-(1-320) and TAO2-(1-451) is likely due to the absence
and presence, respectively, of the MEK3/6 binding domain. This domain
of TAO2 has been localized to residues 314-451 (2). Thus,
TAO2-(1-320), the TAO2 minimal catalytic domain, lacks the MEK3/6
binding site and, thus, displays little selectivity among
stress-sensitive MEKs.
TAO2 Activates Endogenous, Stress-sensitive MAPKs--
The above
studies demonstrated that JNK/SAPK and p38 can be activated by TAO2 in
overexpression systems. Therefore, we examined effects of TAO2 on
endogenous MAPKs. Empty vector control, TAO2-(1-451), or kinase-dead
TAO2-(1-451)DA were transfected into 293 cells, and endogenous p38,
JNK/SAPK, and ERK1/2 were immunoprecipitated from transfected cells and
assayed using transcription factor substrates. Both endogenous p38 and
endogenous JNK/SAPK were significantly activated by TAO2-(1-451)
toward their substrate ATF2 (Fig. 2, A and B), whereas the activity of endogenous
ERK1/2 was not stimulated to an appreciable extent (Fig.
2C). The full-length TAO2 protein, TAO2-(1-1235), due to
its poor expression level, did not appear to activate stress-sensitive
MAPKs in similar assays. Full-length TAO1 also failed to express or
activate well in mammalian cells (1). Nevertheless, the kinase activity
of full-length TAO2 can be detected by autophosphorylation or by
reporter assays (data not shown).
TAO2 Selectively Activates MEK3 and MEK6 in Cotransfected
Cells--
A cotransfection approach was also employed to examine
substrate specificity of TAO2 for downstream MEKs. The overexpressed MEK proteins were immunoprecipitated from the cells and assayed using
the corresponding MAPK as substrate. The MAPK substrates were defective
in autophosphorylation to eliminate background phosphorylation.
RasVal12 was included as a positive control for MEK1, and
MEKK1 was included as a positive control for MEK4 and MEK7. Activity of
MEK1 toward its substrate ERK2KR was not enhanced by cotransfected TAO2
(Fig. 3A, lane 2),
nor were MEK4 and MEK7 activated by TAO2 toward their substrate
SAPK Overexpressed TAO2 Preferentially Phosphorylates MEK3 and
MEK6--
pCMV5-Myc-TAO2-(1-451) and TAO2-(1-451)DA were transfected
into 293 cells (Fig. 4A).
Overexpressed TAO2 proteins were then immunoprecipitated from the cells
through their Myc epitope and assayed using catalytically defective
MEKs 3, 4, and 6 as substrates. Kinase reactions were resolved on
SDS-PAGE and subjected to autoradiography (Fig. 4B). MEK3KM
and MEK6KM were phosphorylated by TAO2-(1-451) to different extents
(lanes 1 and 5), whereas MEK4KM, which should migrate right
above the autophosphorylated TAO2-(1-451) band, was barely
phosphorylated by TAO2 (lane 3). TAO2-(1-451)DA was unable
to phosphorylate any of the substrates, consistent with its lack of
catalytic activity (lanes 2, 4, and
6). The inability of the immunoprecipitated protein to
phosphorylate MEK4 under these conditions is probably a result of its
weaker affinity for MEK4 relative to MEK3 and MEK6.
Endogenous TAO2 Forms a Complex with MEK3 and MEK6 in Vivo--
A
polyclonal TAO2 antiserum was generated against an N-terminal peptide
from TAO2. The antibody was preincubated with antigenic peptide to
demonstrate that the peptide blocked recognition of the TAO2 band (Fig.
5A). We examined several cell
lines of neuronal origin because Northern analysis indicated TAO2
mRNA is most highly expressed in brain (1). Using the antibody, we
found that TAO2 is expressed in numerous cell lines including 293 fibroblasts (Fig. 5B). By fractionation TAO2 is found in
both cytosol and particulate fractions of 293 cells but is not
detectable in nuclei (Fig. 5C).
Given the apparent importance of the MEK3/6 binding site for TAO2
specificity, we determined if endogenous TAO2 associates with MEK3
in vivo. 293 cells were transfected with Myc-MEK3.
Endogenous TAO2 was immunoprecipitated from the cells with the
anti-TAO2 antibody and blotted with anti-Myc antibody. Myc-MEK3 was
detected in the TAO2 immune complexes (Fig.
6A). To investigate whether coprecipitation can be demonstrated with both endogenous proteins, TAO2
was immunoprecipitated from proliferating 293 cells with the anti-TAO2
antibody, and the precipitates were then blotted with a goat anti-MEK6
antibody. Endogenous MEK6 was found in immune complexes with endogenous
TAO2 but not with Raf, the MEKK-level activator of the ERK pathway
(Fig. 6B), indicating that this interaction is specific. No
such interactions could be observed between TAO2 and MEK4 or MEK7 (data
not shown), consistent with results above and in vitro
binding studies (2).
Sorbitol Stimulates the Activity of Endogenous TAO2--
We wished
to determine whether TAO2 itself can be activated by stress stimuli
that reportedly activate JNK/SAPK or p38. To this end, 293 cells were
serum-starved for 24 h and then exposed to osmotic stresses, NaCl,
sorbitol, or the microtubule-directed drugs, Taxol and nocodazole.
Because TAO2 contains many sites of autophosphorylation,
autophosphorylation is an easy method to detect its activity (1, 2).
Endogenous TAO2 was immunoprecipitated from the cells and its ability
to autophosphorylate was measured. TAO2 autophosphorylation was most
highly stimulated by sorbitol and slightly elevated by NaCl, taxol, and
nocodazole (Fig. 7A). Endogenous TAO2 immunoprecipitated from untreated or sorbitol-treated cells was then assayed using kinase-dead MEK3 and MEK6 as substrates to
confirm that enhanced autophosphorylation is a reflection of enhanced
TAO2 activity under these circumstances. Phosphorylation of both MEK3
and MEK6 by TAO2 was enhanced by sorbitol treatment (Fig.
7B). Thus, sorbitol, and to a lesser extent several other cell stresses, increased TAO2 activity.
Activities of Stress-sensitive Kinases and TAO2 during
Differentiation of C2C12 Myoblasts--
C2C12 myoblasts can be induced
to differentiate and form myotubes in culture (16-18). p38 is
activated during the differentiation process, and its activation is
required for differentiation induced by either insulin or growth factor
withdrawal. In contrast, JNK/SAPK activities have been reported to
change little, and AP-1 activity, which is enhanced by JNK/SAPKs, is
suppressed during differentiation consistent with no role of JNK/SAPKs
in differentiation (16-18). We wished to determine whether changes in
TAO2 activity occurred during differentiation. If so, that would
support the idea that activation of endogenous TAO2 parallels
activation of p38 but not the JNK/SAPK pathway. C2C12 cells were
maintained in differentiation medium with or without the p38 inhibitor
SB203580 for 4 days (Fig. 8A).
Myotubes were obvious in the day 4 cultures maintained in the absence
of SB203580 but were largely absent if cells had been constantly
exposed to SB203580. The amount of p38 protein did not change, but, as
expected, its activity increased on or before day 2 and remained
elevated through day 4 (Fig. 8, B and C).
SB203580 suppressed the elevated activity. The amount of TAO2 protein
in the cells decreased significantly over the differentiation time course in a manner largely or entirely insensitive to SB203580 (Fig.
8B). However, TAO2-specific activity increased over the time
course of differentiation also in an SB203580-independent manner
despite the decrease in its mass (Fig. 8D). The amount of
JNK/SAPK protein in the cells increased; nevertheless, its specific
activity decreased over the time course (Fig. 8, B and E). In contrast to p38 and TAO2, JNK/SAPK activity in cells
measured on day 0 was substantial perhaps due to the effects of serum
growth factors. Because JNK/SAPK activity is not required for
differentiation, its activation may be suppressed during this process.
Thus, TAO2 activity parallels p38 activity but not JNK/SAPK activity.
This is consistent with the idea that TAO2 is coupled to p38 but not JNK/SAPKs in cells.
Recent molecular cloning studies have revealed the existence of a
multitude of MEKK-level kinases in mammalian systems including TAO1 and
TAO2 (1, 2, 19-21). This diversity may allow multiple mechanisms for
activation of MAP kinase subgroups in a ligand- and cell
type-dependent manner. In several cases, MAPK regulation by
MEKK-level kinases correlates well with the ability of MEKKs to
phosphorylate and activate corresponding MEK proteins in MAPK modules.
Thus, we previously examined the substrate specificity of TAOs in
vitro and, in this report, TAO2 in cell culture systems. In
vitro, the purified, recombinant, catalytic domains of TAO1 and
TAO2 activated MEKs 3, 4, and 6 toward their substrates, p38 and
JNK/SAPK (1, 2). In contrast, when an active TAO2 truncation mutant was
cotransfected with various MEK proteins only MEK3 and MEK6 of the p38
pathway were activated. TAO2 truncation mutants activated both
cotransfected and endogenous p38 and JNK/SAPKs. By monitoring TAO2,
p38, and JNK/SAPK activities during C2C12 differentiation, we were able
to show that conditions that lead to activation of endogenous TAO2 are
associated with increases in p38 but not JNK/SAPK activity. This sort
of evidence supports the conclusion that TAO proteins are primarily
coupled to the p38 pathway and have little role in regulating
JNK/SAPK.
The specificity for MEKs 3 and 6 may reflect the importance of the MEK
binding domain of TAO2. Earlier we identified this TAO domain
immediately C-terminal to its kinase domain and found that it
recognized an N-terminal motif in MEKs 3 and 6 (1, 2). Thus far, we
have not found a direct site for p38 binding on TAO2. Because the
JNK/SAPK activators, MEK4 and MEK7, neither bind TAO2 nor are activated
by TAO2 in cells, activation of JNK/SAPK by TAO may be due in part to
TAO2 overexpression and to the relaxed specificity of MEK6. Indeed,
in vitro kinase studies have shown that MEK6 can
phosphorylate JNK/SAPK to an appreciable extent, although the specific
activity is lower than that of MEK4 toward JNK/SAPK (data not shown).
Enzyme-substrate interactions have received much attention in studying
molecular determinants of pathway specificity. Evidence for the
importance of these interactions comes from studies of cAMP-dependent protein kinase and the ERK and JNK/SAPK MAPK
modules among others (22-25). A docking site for p38 in MEK6 and
MEK3b, an alternatively spliced isoform of MEK3, appears to contribute to specificity of p38 signaling (26). The association of TAO with
MEK3/6 and p38 indirectly through the MEK in signaling complexes provides compelling evidence for their interrelated functions even in
the absence of information regarding their physiological roles. By
selectively interacting with and phosphorylating MEK3 and MEK6 in
vivo, TAO2 works in spatial proximity to these immediate upstream
regulators of p38, thereby facilitating signal transduction from TAO2
to MEK3/6 and subsequently to p38.
Certain MEKKs implicated in regulating JNK/SAPK and p38 are reportedly
responsive to cellular stresses. For example, MEKK1 can be activated by
cross-linking of high affinity IgE receptors or microtubule-directed
drugs (15, 27, 28) and TAK1 is sensitive to UV, sorbitol, and Fas
receptor ligation (29). To identify stimuli or ligands that regulate
TAO2, we generated a high-titer TAO2 antibody that enabled us to
immunoprecipitate endogenous TAO2 proteins from cells treated with a
wide array of stimuli. Of the more than twenty stimuli tested, most had
no detectable effect on TAO2 activity (data not shown). The osmotic
stress, sorbitol, as well as some other stresses, activated TAO2
modestly suggesting that TAO2 may indeed be involved in
stress-responsive pathways. Kinase-dead TAO2 did not block activation
of p38 by sorbitol (data not shown). However, many MEKKs are activated
by sorbitol, and all may contribute to p38 activation. If this is correct, it will be difficult to identify the individual contributions of these MEKKs to sorbitol-induced p38 stimulation. Importantly, we
have recently been able to show that kinase-dead TAO2 was
effective in blocking activation of p38 by
carbachol2. Thus, the
aggregate of our studies link TAO1/2 to a subset of conditions
regulating the p38 MAP kinase cascade.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-galactopyranoside
after growth to an outer diameter of 0.8 and grown at room
temperature for another 7 h before harvesting. Purification
procedures were as described (8). MEK4KM and SAPK
KA protein
preparations were kindly provided by Mahesh Karandikar. ATF2-(1-254)
and Myc-(1-103) protein preparations were kindly provided by Don
Arnette. pCEP4-HA-ERK2, pSR
-HA-JNK1, pSR
-HA-SAPK
, and
pCEP4-HA-p38
were as described (9, 10). pCMV5-HA-MEK1,
pCMV5-HA-MEK4, pCMV5-Myc-MEK3, pSR
-HA-MEK6, pCMV5-MEKK1, and
RasVal12 were kindly provided by Jeff Frost and Jennifer
Swantek and were described elsewhere (11-13).
pCS3+-MT-Myc-MEK7 was provided by Shuichan Xu.
-glycerophosphate, 0.5 mM
sodium orthovanadate, 1 mM EDTA, 20 µg/ml aprotinin, 10 µg/ml pepstatin A, 10 µg/ml leupeptin, and 1 mM
phenylmethylsulfonyl fluoride). Insoluble material was pelleted by
centrifugation, and supernatants were removed, snap frozen in liquid
nitrogen, and stored at
80° C. For cell fractionation, cells were
washed once with phosphate-buffered saline and resuspended in hypotonic
buffer (10 mM Hepes, pH 7.6, 1.5 mM
MgCl2, 10 mM NaCl, 1 mM EDTA, 1 mM EGTA, and supplemented with protease inhibitors and
phosphatase inhibitors as above). Cells were lysed with a Dounce
apparatus, and the nuclei were collected by sedimentation at 800 × g for 5 min. A particulate fraction was collected by
sedimenting the supernatant at 100,000 × g for 30 min.
The nuclear pellet was washed with 20 mM Hepes, pH 7.6, 2.5% glycerol, 0.42 M NaCl, 1.5 mM
MgCl2, 1 mM EDTA, and 1 mM EGTA for
30 min. Both the washed nuclear pellet and particulate fraction were
lysed with 1% SDS, 10 mM Tris-HCl, pH 7.6, 1 mM EDTA, 1 mM EGTA, and supplemented with
protease inhibitors and phosphatase inhibitors as above. Three µg of
various MEK or MAPK constructs were cotransfected with 2 µg of either
empty vector control or TAO2 constructs in 293 cells grown to 80%
confluence on 60-mm dishes using the calcium phosphate method. Cells
were serum-starved for 24 h beginning the second day and then
harvested. Alternatively, cells were transfected with 5 µg of either
empty vector control or TAO2 constructs to examine the activities of endogenous MAPKs.
and
isoforms of p38. Endogenous JNK/SAPK and MEK6 were immunoprecipitated and immunoblotted with Santa Cruz antibodies, rabbit C-17 and goat N-19, respectively. Anti-Raf1 (C-12) was also purchased from Santa Cruz. Monoclonal anti-HA
antibody (12CA5) and anti-Myc antibody (9E10) were obtained from
Berkeley Antibody Company and the Cell Culture Center, respectively. MEKs or MAPKs were immunoprecipitated from 0.3 mg of whole cell lysate
protein via the above antibodies and 30 µl of protein A-Sepharose slurry. Beads were washed three times with 1 ml of 500 mM
NaCl and 20 mM Tris-HCl, pH 7.4, washed one time with 1 ml
of 20 mM Tris, pH 8.0, and 10 mM
MgCl2, and subjected to in vitro kinase assays
(2). Endogenous TAO2 was immunoprecipitated from 1.5 mg lysate protein
as described in (15) with 5 µl of TAO2 antibody (U2253) and 30 µl
of protein A-Sepharose slurry. Beads were washed three times with 1 ml
of 750 mM NaCl, 20 mM Tris-HCl, pH 7.4, and
0.1% Triton X-100, washed one time with 1 ml of 20 mM
Tris, pH 8.0, and 10 mM MgCl2, and subjected to
in vitro kinase assays. Kinase assay conditions were as
before, except that the ATP concentration was 10 µM. Fold
changes were calculated as the ratio of the cpm in the substrate in the
experimental sample and the control sample. Phosphorylation of
substrate bands was quantified by liquid scintillation counting.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
was activated by the
TAO2 constructs toward ATF2 (Fig. 1B). The activity of
HA-p38 on its substrate ATF2 was also greatly enhanced by cotransfected
TAO2-(1-320) or TAO2-(1-451) (Fig. 1C). In contrast to
these stress-activated MAPKs, ERK2 was not activated toward its
substrate Myc-(1-103) by any of the cotransfected TAO2 plasmids (Fig.
1D) further supporting the conclusion that TAO2 is not
an upstream activator of the classical ERK pathway.
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Fig. 1.
Stress-sensitive MAPKs are activated by
cotransfected TAO2. A, HA-JNK1 was transfected into 293 cells with empty vector or with various Myc-tagged TAO2 constructs.
HA-JNK1 was immunoprecipitated from the cells and assayed with ATF2 as
substrate. Top panel, autoradiogram showing phosphorylation
of ATF2 by JNK1. Bottom panel, immunoblot of lysates with
anti-HA antibody, indicating that equal amounts of JNK1 were expressed.
B, experiments as in A with HA-SAPK .
C, experiments as in A with HA-p38. D,
experiments as in A with HA-ERK2 except that Myc-(1-103)
was utilized as the substrate. One of three similar experiments.
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Fig. 2.
TAO2 activates endogenous, stress-sensitive
MAPKs. A, 293 cells were transfected with pCMV5-Myc,
pCMV5-Myc-TAO2-(1-451), or pCMV5-Myc-TAO2-(1-451)DA. Endogenous p38
was immunoprecipitated from the cell lysates and assayed using ATF2 as
substrate. Top panel, autoradiogram of phosphorylation of
ATF2. Bottom panel, anti-p38 immunoblot of lysates. Fold
phosphorylation of the substrate was indicated. B,
endogenous JNK/SAPK was immunoprecipitated from the same lysates and
assayed with ATF2. C, endogenous ERK1/2 was
immunoprecipitated from the same lysates and assayed with Myc-(1-103).
One of four similar experiments.
KA (lanes 5 and 8). In comparison,
activities of MEK3 and MEK6 toward p38 were significantly stimulated by
cotransfected TAO2 (lanes 11 and 13) suggesting
that TAO2 selectively activates MEK3 and MEK6 of the p38 pathway in
intact cells. A kinase-dead construct, TAO2-(1-451)DA, failed to
activate cotransfected MEK6 (lane 14) indicating that TAO2
kinase activity is essential.
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Fig. 3.
TAO2 selectively activates MEK3 and MEK6 in
cotransfected cells. A, various MEK constructs
were cotransfected into 293 cells with TAO2 or control constructs.
Lanes 1-3: MEK1 with empty vector, TAO2-(1-451), or
RasVal12, respectively; lanes 4-6: MEK4 with
empty vector, TAO2-(1-451), or MEKK1, respectively; lanes
7-9: MEK7 with empty vector, TAO2-(1-451), or MEKK1,
respectively; lanes 10 and 11: MEK3 with empty
vector or TAO2-(1-451), respectively; lanes 12-14: MEK6
with empty vector, TAO2-(1-451), or TAO2-(1-451)DA, respectively.
Overexpressed MEK proteins were immunoprecipitated from the cell
lysates through their epitope tags, and subjected to kinase assays
using corresponding MAPKs as substrates. Shown in the autoradiogram is
phosphorylation of MAPKs by MEKs. B, immunoblots of the cell
lysates showing comparable amounts of MEKs were expressed. One of four
similar experiments.
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Fig. 4.
Overexpressed TAO2 preferentially
phosphorylates MEK3 and MEK6. A, 293 cells were
transfected with pCMV5-Myc-TAO2-(1-451) or pCMV5-Myc-TAO2-(1-451)DA.
Cells were harvested in detergent lysis buffer. The cell lysates were
resolved by SDS-PAGE and subjected to immunoblotting with the anti-Myc
antibody to detect TAO2 proteins. B, TAO2 proteins were
immunoprecipitated from 0.25 mg of lysate protein and assayed using 0.5 µg of purified recombinant MEK proteins as substrates. Kinase
reactions were resolved by SDS-PAGE and subjected to autoradiography.
One of three similar experiments.
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Fig. 5.
Endogenous TAO2 can be detected in numerous
cell lines and in both membrane and cytosolic fractions.
A, 10 µg of lysate protein from various cells were
resolved by SDS-PAGE and immunoblotted using either anti-TAO2 antiserum
without (upper panel) or with antigenic peptide preincubated
overnight at a concentration of 20 µg of peptide/µl of serum
(bottom panel). B, 30 µg of protein from
lysates of the indicated cell lines were resolved by SDS-PAGE and
immunoblotted with the anti-TAO2 antibody. N2A refers to neuro2A cells.
PC12M is a line selected from PC12 because it is a better transfection
recipient. PC12M cells differentiate more poorly and are
less sensitive to NGF. ATT20 and GC-B6 are pituitary cell lines; NG108
is a neuroblastoma × glioma hybrid. C, 293 cells were
fractionated into soluble, particulate, and nuclear fractions as
described under "Experimental Procedures." 30 µg of protein from
each fraction were subjected to SDS-PAGE and immunoblotted with the
anti-TAO2 antibody.
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Fig. 6.
Endogenous TAO2 forms a complex with MEK3 and
MEK6 in intact cells. A, 293 cells were transfected
with pCMV5-Myc-MEK3. Endogenous TAO2 was immunoprecipitated from the
lysates. As a control proteins immunoprecipitated with preimmune serum
were also collected. The immunoprecipitates were immunoblotted with
anti-Myc (upper panel). Anti-Myc blot of the lysates
(bottom panel). B, proteins were
immunoprecipitated from 293 cells with anti-TAO2 antibodies, the
preimmune serum, or anti-Raf antibodies. Endogenous MEK6 was detected
in the immunoprecipitates via a MEK6 antibody (upper panel).
Equal amounts of MEK6 were present in the lysates (bottom
panel). One of two similar experiments.
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Fig. 7.
Sorbitol stimulates the activity of
endogenous TAO2. A, cultured 293 cells were
serum-starved for 24 h and then untreated or stimulated with 0.5 M NaCl or sorbitol for 30 min or with 5 µM
Taxol or nocodazole for 1 h. Endogenous TAO2 was
immunoprecipitated from the lysates and allowed to autophosphorylate.
Upper panel, autoradiogram of TAO2 autophosphorylation.
Bottom panel, anti-TAO2 immunoblot of lysates. B,
endogenous TAO2 was immunoprecipitated from lysates of
sorbitol-stimulated or unstimulated cells and autophosphorylated
(top) or assayed with MEK3KM (second panel) or
MEK6KM (third panel) as substrates. Bottom panel,
immunoblots of TAO2 in lysates. One of three similar experiments.
View larger version (42K):
[in a new window]
Fig. 8.
Activities of endogenous p38 and TAO2 were
increased during C2C12 muscle differentiation. A, C2C12
cells were cultured and induced to differentiate on day 0 as under
"Experimental Procedures." Cells were treated with
Me2SO diluent as a control or with SB203580 daily during
differentiation. Morphologies of the cells on differentiation day 0 (upper panels) and day 4 (lower panels) were
visualized. B, 15 µg of lysate protein collected on each
day were resolved by SDS-PAGE and immunoblotted with anti-p38
(top panel), anti-TAO2 (middle panel), or
anti-JNK/SAPK (bottom panel) antibodies. C,
endogenous p38 was immunoprecipitated from 0.1 mg of lysate protein and
assayed with ATF2. Half of the reactions were resolved on SDS-PAGE and
analyzed by autoradiography (top panel). 1/10 of the
reactions were immunoblotted for immunoprecipitated protein to
demonstrate that amounts were roughly equal (bottom panel).
D, endogenous TAO2 was immunoprecipitated from various
amounts of total proteins (day 0, 0.1 mg; day 1, 0.25 mg; day 2, 0.3 mg; day 3, 0.7 mg; day 4, 0.8 mg) so that comparable amounts of
immunoreactive TAO2 were allowed to autophosphorylate. Upper
panel, autophosphorylation of TAO2 in half of the reactions.
Bottom panel, anti-TAO2 immunoblot of 1/10 of the reactions
indicating equal amounts of TAO2 were assayed. E, endogenous
JNK/SAPK was immunoprecipitated from various amounts of total proteins
(day 0 and day 1, 0.7 mg; day 2, 0.6 mg; day 3, 0.15 mg; day 4, 0.1 mg)
and assayed with ATF2. Upper panel, autoradiogram showing
ATF2 phosphorylation by JNK/SAPK in half of the reactions. Bottom
panel, anti-JNK/SAPK immunoblot of 1/10 of the reactions
indicating comparable amounts of JNK/SAPK were assayed. One of three
representative experiments is shown.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank Bing-e Xu, Tara Beers Gibson, and Gray Pearson (UT Southwestern) for critical reading of the manuscript, Signal Pharmaceuticals for the MEK6 cDNA, Kunliang Guan for the MEK3 cDNA, members of the Cobb laboratory for proteins and constructs, and Dionne Ware for administrative assistance.
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FOOTNOTES |
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* This work was supported by Grant GM53032 from the National Institutes of Health (to M. H. C.).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.
Submitted in partial fulfillment of the requirements for the Ph.D. degree.
§ To whom correspondence should be addressed:Dept. of Pharmacology, Univ. of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75390-9041. Tel.: 214-648-3627; Fax: 214-648-3811; Email: mcobb@mednet.swmed.edu.
Published, JBC Papers in Press, March 8, 2001, DOI 10.1074/jbc.M100681200
2 Z. Chen, L. Chen, A. G. Gilman, and M. Cobb, manuscript in preparation).
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ABBREVIATIONS |
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The abbreviations used are: JNK, c-Jun N-terminal kinase; MAP, mitogen-activated protein; MEK, MAP/ERK kinases; ERK, extracellular signal-regulated protein kinase; JNK/SAPK, c-Jun N-terminal kinases/stress-activated protein kinases; MEKK, MEK kinase; PAGE, polyacrylamide gel electrophoresis; HA, hemagglutinin.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. |
Hutchison, M.,
Berman, K.,
and Cobb, M. H.
(1998)
J. Biol. Chem.
273,
28625-28632 |
2. |
Chen, Z.,
Hutchison, M.,
and Cobb, M. H.
(1999)
J. Biol. Chem.
274,
28803-28807 |
3. |
Moore, T. M.,
Garg, R.,
Johnson, C.,
Coptcoat, M. J.,
Ridley, A. J.,
and Morris, J. D.
(2000)
J. Biol. Chem.
275,
4311-4322 |
4. |
Tassi, E.,
Biesova, Z.,
Di Fiore, P. P.,
Gutkind, J. S.,
and Wong, W. T.
(1999)
J. Biol. Chem.
274,
33287-33295 |
5. | Yustein, J. T., Li, D., Robinson, D., and Kung, H. J. (2000) Oncogene 19, 710-718[CrossRef][Medline] [Order article via Infotrieve] |
6. | Pearson, G., Gibson, T. B., Xu, B., Karandikar, M., Berman, K., and Cobb, M. H. (2001) Endocrine Reviews, in press |
7. | Davis, R. J. (2000) Cell 103, 239-252[Medline] [Order article via Infotrieve] |
8. |
Robbins, D. J.,
Zhen, E.,
Owaki, H.,
Vanderbilt, C.,
Ebert, D.,
Geppert, T. D.,
and Cobb, M. H.
(1993)
J. Biol. Chem.
268,
5097-5106 |
9. |
English, J. M.,
Pearson, G.,
Hockenberry, T.,
Shivakumar, L.,
White, M. A.,
and Cobb, M. H.
(1999)
J. Biol. Chem.
274,
31588-31592 |
10. |
Xu, S.,
Robbins, D. J.,
Christerson, L. B.,
English, J. M.,
Vanderbilt, C. A.,
and Cobb, M. H.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
5291-5295 |
11. |
Swantek, J. L.,
Christerson, L.,
and Cobb, M. H.
(1999)
J. Biol. Chem.
274,
11667-11671 |
12. | Swantek, J. L., Cobb, M. H., and Geppert, T. D. (1997) Mol. Cell. Biol. 17, 6274-6282[Abstract] |
13. |
Frost, J. A.,
Steen, H.,
Shapiro, P. S.,
Lewis, R.,
Ahn, J.,
Shaw, P. E.,
and Cobb, M. H.
(1997)
EMBO J.
16,
6426-6438 |
14. | Boulton, T. G., and Cobb, M. H. (1991) Cell Regul. 2, 357-371[Medline] [Order article via Infotrieve] |
15. |
Karandikar, M.,
Xu, S.,
and Cobb, M. H.
(2000)
J. Biol. Chem.
275,
40120-40127 |
16. |
Wu, Z.,
Woodring, P. J.,
Bhakta, K. S.,
Tamura, K.,
Wen, F.,
Feramisco, J. R.,
Karin, M.,
Wang, J. Y.,
and Puri, P. L.
(2000)
Mol. Cell. Biol.
20,
3951-3964 |
17. |
Cuenda, A.,
and Cohen, P.
(1999)
J. Biol. Chem.
274,
4341-4346 |
18. | Conejo, R., Valverde, A. M., Benito, M., and Lorenzo, M. (2001) J. Cell. Physiol. 186, 82-94[CrossRef][Medline] [Order article via Infotrieve] |
19. | Salmeron, A., Ahmad, T. B., Carlile, G. W., Pappin, D., Narsimhan, R. P., and Ley, S. C. (1996) EMBO J. 15, 817-826[Abstract] |
20. | Yamaguchi, K., Shirakabe, K., Shibuya, H., Irie, K., Oishi, I., Ueno, N., Taniguchi, T., Nishida, E., and Matsumoto, K. (1995) Science 270, 2008-2011[Abstract] |
21. | Wang, X. S., Diener, K., Tan, T. H., and Yao, Z. (1998) Biochem. Biophys. Res. Commun. 253, 33-37[CrossRef][Medline] [Order article via Infotrieve] |
22. |
Pawson, T.,
and Scott, J. D.
(1997)
Science
278,
2075-2080 |
23. | Tanoue, T., Adachi, M., Moriguchi, T., and Nishida, E. (2000) Nat. Cell Biol. 2, 110-116[CrossRef][Medline] [Order article via Infotrieve] |
24. |
Xu, B.,
Wilsbacher, J. L.,
Collisson, T.,
and Cobb, M. H.
(1999)
J. Biol. Chem.
274,
34029-34035 |
25. |
Yasuda, J.,
Whitmarsh, A. J.,
Cavanagh, J.,
Sharma, M.,
and Davis, R. J.
(1999)
Mol. Cell. Biol.
19,
7245-7254 |
26. |
Enslen, H.,
Brancho, D. M.,
and Davis, R. J.
(2000)
EMBO J.
19,
1301-1311 |
27. |
Ishizuka, T.,
Terada, N.,
Gerwins, P.,
Hamelmann, E.,
Oshiba, A.,
Fanger, G. R.,
Johnson, G. L.,
and Gelfand, E. W.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
6358-6363 |
28. |
Yujiri, T.,
Fanger, G. R.,
Garrington, T. P.,
Schlesinger, T. K.,
Gibson, S.,
and Johnson, G. L.
(1999)
J. Biol. Chem.
274,
12605-12610 |
29. |
Shirakabe, K.,
Yamaguchi, K.,
Shibuya, H.,
Irie, K.,
Matsuda, S.,
Moriguchi, T.,
Gotoh, Y.,
Matsumoto, K.,
and Nishida, E.
(1997)
J. Biol. Chem.
272,
8141-8144 |