From the Department of Biophysics, Graduate School of
Science and the
Department of Cell and Developmental
Biology, Graduate School of Biostudies, Kyoto University, Sakyo-ku,
Kyoto 606-8502, Japan
Received for publication, September 20, 2000, and in revised form, October 17, 2000
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ABSTRACT |
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The MAPK cascades regulate a wide variety
of cellular functions, including cell proliferation, differentiation,
and stress responses. Here we have identified a novel MAP kinase kinase
kinase (MAPKKK), termed MLTK (for MLK-like
mitogen-activated protein triple kinase), whose
expression is increased by activation of the ERK/MAPK pathway. There
are two alternatively spliced forms of MLTK, MLTK The mitogen-activated protein kinase
(MAPK)1 pathways function in
a variety of physiological aspects in yeast to human cells (1-5). So
far, at least four independent MAPK pathways have been identified. They
include the extracellular signal-regulated kinase (ERK) pathway, the
c-Jun N-terminal kinase (JNK)/stress-activated protein kinase (SAPK)
pathway, the p38 pathway, and the ERK5/Big MAPK1 (BMK1) pathway. The
ERK pathway regulates cell proliferation, cell differentiation, and
developmental processes (3, 6). The JNK/SAPK and p38 pathways are
involved in cellular stress responses and apoptosis (7, 8). The
ERK5/BMK1 pathway has been reported to participate in EGF
(epidermal growth
factor)-stimulated growth control (9, 10). These MAPKs are
activated through phosphorylation of threonine and tyrosine residues by
MAPK kinases (MAPKKs or MAPK/ERK kinases (MEKs)), which include
MAPKK1/MEK1 and MAPKK2/MEK2 as ERK activators, SEK1/MKK4 and
MKK7 as JNK/SAPK activators, MKK3 and MKK6 as p38 activators, and MEK5
as an ERK5 activator (8, 11). The substrate specificity of MAPKKs for MAPKs is relatively strict, and these MAPKK/MAPK cascades function as
independent signaling units.
MAPKK kinases (MAPKKKs) are a family of serine/threonine protein
kinases that can activate one or several of the MAPKK/MAPK cascades
(8). MAPKKKs phosphorylate two serine/threonine residues in the
activation phosphorylation sites of MAPKKs. The identified MAPKKKs
include the Raf family, the MEK kinase (MEKK) family, and the
mixed-lineage kinase (MLK) family. The Raf family kinases selectively
activate the ERK pathway. On the other hand, MEKK1, MEKK2, and MEKK3
activate both the ERK pathway and the JNK/SAPK pathway (12). MLK3,
MUK, TAK1, and ASK1 activate the JNK/SAPK and p38 pathways (8).
Recently, Cot/Tpl-2 was reported to activate all known MAPK pathways:
the ERK, JNK/SAPK, p38 (p38 Actin reorganization underlies changes in cell morphology and is
essential for cell migration and motility. The Rho family GTPases are
known to regulate particular types of actin reorganization (17, 18).
Rho and Rac promote stress fiber formation and membrane ruffling,
respectively (19, 20). Their effects on the actin cytoskeleton are at
least partially mediated by protein kinases such as p160 Rho
kinase and p21-activated protein kinase (17), which are reported to
affect actin dynamics through regulation of the phosphorylation state
of myosin light chain. However, there is no MAPKKK that has been shown
to participate in actin reorganization.
In this study, we have identified a novel MAPKKK, designated MLTK for
MLK-like mitogen-activated protein
triple kinase, whose expression is up-regulated
by activation of the ERK pathway in Swiss 3T3 cells. We show that MLTK
can be activated by osmotic shock with hyperosmolar media and induce
the disruption of actin stress fibers and dramatic changes in cell morphology.
cDNA Subtraction--
Swiss 3T3 cells were stably
transfected, first with p3'SS (Stratagene) and then with
HA-LASDSE MEK1 (21) subcloned into pOPI3 (Stratagene). Clones
were selected using hygromycin B (200 µg/ml; Life Technologies, Inc.)
and Geneticin (400 µg/ml; Life Technologies, Inc.) as the selection
drugs. A clone (3L-9) that showed a high inducibility of MEK1 was
obtained and used for the experiment. 5 mM IPTG was added
to the medium to induce MEK1 expression (LacSwich system, Stratagene).
cDNA subtraction was performed using a PCR-Selected cDNA
Subtraction kit (CLONTECH). The mRNA from
untreated 3L-9 was used as the "driver." The mRNA from 3L-9
cultured for 6 h after the addition of IPTG and that from 3L-9
cultured for 18 h were mixed, and the mixture was used as the
"tester." The subtractive screening was carried out according to
the manufacturer's instructions. A 1022-base pair fragment of MLTK was
obtained as a candidate positive cDNA clone.
Cloning of MLTK and DNA Construction--
5'- and 3'-RACE
analyses for the cDNA fragment obtained were performed using mouse
heart Marathon Ready cDNA (CLONTECH) as a
template. The internal primers used were 5'-GTCCTGTGATATCCATTTGGCTC-3' (antisense for 5'-RACE) and 5'-CGGGAGAGACGTCTCAAGATGTGGG-3' (sense for
3'-RACE). Each RACE product was cloned into pCR2.1-TOPO (Invitrogen) and sequenced with an ABI 377 sequencer. Full-length mouse MLTK
A kinase-negative form of MLTK was produced using a QuickChange
site-directed mutagenesis kit (Stratagene) with the mutagenic primers 5'-GGACAAGGAGGTGGCTGTAATGAAGTTACTCAAAATAGAG-3'
(sense) and 5'-CTCTATTTTGAGTAACTTCATTACAGCCACCTCCTTGTCC-3'
(antisense) (the mutated bases are underlined) by PCR and cloned
into pSR
Expression vectors of the dominant-negative form of MAPKKs are
described elsewhere (MEK1-SASA (22), MKK6-SATA (23), MKK7-KL (24), and
MEK5-SATV (10)). cDNA of Cell Culture and Transfection--
COS-7 and Swiss 3T3 cells
were maintained in Dulbecco's modified Eagle's medium supplemented
with 10% fetal calf serum and antibiotics (100 units/ml penicillin and
0.2 mg/ml kanamycin). Transfection of the expression vectors was
performed using LipofectAMINE and LipofectAMINE PLUS (Life
Technologies, Inc.) according to the manufacturer's protocol.
Immunoblotting--
Transfected COS-7 cells were lysed in
extraction buffer (20 mM Tris (pH 7.5), 1% Triton X-100, 5 mM EGTA, 12 mM Immunoprecipitation and Kinase Assay--
COS-7 cells
transfected with HA-MLTK
For the coupled kinase assay, the immunoprecipitates were first
incubated with His-MEK1, His-SEK1, or His-MKK6 recombinant protein (0.2 µg each/reaction) (26) at 30 °C for 15 min in kinase reaction
buffer without the radiolabeled ATP. The reactions were then added with
the kinase-negative form of GST-ERK, GST-SAPK, or GST-p38 (27) together
with 2 µCi of [ Cell Staining--
Cells were fixed and permeabilized as
described previously (22). HA-tagged proteins were detected with
anti-HA antibody 12CA5. Actin filaments were visualized using
tetramethylrhodamine B isothiocyanate (TRITC)- or fluorescein
isothiocyanate (FITC)-labeled phalloidin. Fluorescence images were
observed using a Zeiss Axiophoto or a confocal microscope
(Bio-Rad).
Identification of a Novel Protein Kinase, MLTK--
To isolate
genes whose expression is increased after activation of the ERK/MAPK
pathway, we established a stable Swiss 3T3 cell line in which
expression of an active MEK1 mutant, LASDSE MEK1 (21), was induced by
the addition of IPTG to the culture medium (Fig.
1A, upper). In one
such cell line (clone 3L-9), expression of LASDSE MEK1 induced by IPTG
treatment caused morphological transformation of the cells (Fig.
1A, lower). We performed a subtractive cDNA
screening using mRNAs from untreated and IPTG-treated 3L-9 cells.
Among the isolated cDNA clones, we found a cDNA fragment that
encodes a novel protein kinase that appears to be a member of the
MAPKKK family. RT-PCR analysis revealed that the corresponding mRNA
existed in unstimulated cells and was being up-regulated at least
18 h after the induction of active MEK1 (Fig. 1B).
Northern blot analysis of the cDNA fragment obtained showed that
the transcript (~7.7 kilobases) was expressed ubiquitously,
with higher expression levels in heart and skeletal muscle (Fig.
2D). There were two minor
bands of ~3.3 and 1.6 kilobases in human heart and skeletal muscle
(Fig. 2D).
The 5'- and 3'-RACE analyses showed that at least two mRNA variants
with different 3'-sequences are expressed in mouse heart. cDNA
cloning of the two variants and the open reading frame prediction revealed that the two proteins have identical amino acid sequences in
the N-terminal region (residues 1-311), which contains a kinase domain
and a leucine zipper motif (Fig. 2, A and B). The
kinase domain shows the highest homology to that of MLK2 with 44.7%
identity and is 32.7% identical to that of TAK1 (Fig. 2C).
Thus, we termed this kinase MLTK for MLK-like
mitogen-activated protein triple kinase. The
two isoforms, designated MLTK
Each MLTK contains two putative nuclear export signal (NES)-like
sequences, which are characterized by four hydrophobic residues with
appropriate spacing (Fig. 2E, upper). We
expressed HA-tagged MLTK MLTK Acts as a MAPKKK and Is Activated by Osmotic Shock through
Autophosphorylation--
We examined whether MLTK
We then examined whether the activity of MLTK was up-regulated by
extracellular stimuli. COS-7 cells were transfected with HA-MLTK
We then produced and expressed kinase-negative mutants of MLTK,
MLTK
It has been reported that autophosphorylation of MEKK1, TAK1, or
Ssk2 is achieved through an intramolecular reaction (30-32). To
examine whether the autophosphorylation of MLTK occurs through an
inter- or intramolecular reaction, we tested if the autophosphorylation occurs between two isoforms of MLTK. COS-7 cells were transfected with
HA-tagged MLTKs and either left untreated or stimulated by osmotic
shock (0.5 M NaCl). Both wild-type MLTK MLTK
To examine whether or not other MAPKKKs also cause these changes in
stress fibers and cell morphology, we tested MLTK
To test if activation of the p38 pathway is sufficient for the
MLTK In this study, we have identified and characterized a novel member
of the MAPKKK family (designated MLTK) that can be activated by osmotic
shock with hyperosmolar media and that can regulate actin organization
and cell morphology. MLTK contains a leucine zipper motif just after
the N-terminal kinase domain in the primary structure (Fig.
2B). All of the members of the MLK subfamily of MAPKKKs have
leucine zipper motifs (usually two). Moreover, the kinase domain of
MLTK is most similar to that of MLK2. Thus, MLTK may be a close
relative of the MLK subfamily of MAPKKKs and was therefore termed MLTK
for MLK-like mitogen-activated protein
triple kinase, although MLTK seems to have only
one leucine zipper motif. MLTK Activation of MLTK--
MLTKs are specifically activated by
osmotic shock with hyperosmolar media. In budding yeast, Ssk2, a
MEKK family MAPKKK, is shown to be activated by Ssk1, a regulatory
protein in the two-component osmosensor whose binding to Ssk2 induces
the autophosphorylation of Ssk2 (32). MLTKs might also lie downstream
of a mammalian osmosensor system whose molecular identity is unknown.
As for some of the MAPKKKs, their possible activation mechanisms have
been analyzed (28, 31, 37, 38). For example, MLK3 has a CRIB
(Cdc42 and Rac interactive
binding) domain, and binding of activated Rac/Cdc42 to MLK3
enhances dimerization of MLK3. Dimerization of MLK3 is achieved through
its leucine zippers and is required for both its autophosphorylation
and kinase activity for downstream targets (28). TAK1 undergoes
autophosphorylation in response to upstream stimuli by an unknown
mechanism (which may involve its association with adapter proteins TAB1
and TAB2). This autophosphorylation of TAK1 is required for activation
of TAK1 activity for downstream targets (31).
We showed that MLTK forms a dimer; both MLTK
As overexpressed MLTK has kinase activity for substrates under
unstimulated conditions, MLTK seems to be able to undergo
autophosphorylation when overexpressed. Our data show that
overexpressed MLTK MLTK-induced Activation of the MAPK Pathways--
MLTKs are
capable of activating the four major MAPK pathways: ERK, JNK/SAPK, p38,
and ERK5/BMK1. The kinase domain of MLTK MLTK
The present observation that several MAPKKKs other than MLTK
It has been reported that the low molecular weight GTPase Rho regulates
the formation of actin stress fibers and the assembly of focal
adhesions and that the inhibition of Rho function by C3 toxin blocks
stress fiber formation (19, 43). It is possible that MLTK and MLTK
. When
overexpressed in cells, both MLTK
and MLTK
are able to activate
the ERK, JNK/SAPK, p38, and ERK5 pathways. Moreover, both MLTK
and
MLTK
are activated in response to osmotic shock with hyperosmolar
media through autophosphorylation. Remarkably, expression of
MLTK
, but not MLTK
, in Swiss 3T3 cells results in the disruption
of actin stress fibers and dramatic morphological changes. A
kinase-dead form of MLTK
does not cause these phenomena. Inhibition
of the p38 pathway significantly blocks MLTK
-induced stress fiber
disruption and morphological changes. These results suggest that MLTK
is a stress-activated MAPKKK that may be involved in the regulation of
actin organization.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
isoform), and ERK5/BMK1 pathways (13).
Some of the MAPKKKs were shown to be activated by particular
extracellular stimuli. For example, Raf-1 is activated by mitogens and
phorbol esters, TAK1 by transforming growth factor-
(TGF-
) and
cytokines (14, 15), and ASK1 by tumor necrosis factor-
(TNF
)
(16). Thus, various MAPKKKs appear to respond to a variety of
extracellular stimuli and activate a limited number of the MAPKK/MAPK cascades.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and
MLTK
were obtained by PCR using mouse heart Marathon Ready cDNA
(CLONTECH) as a template. Full-length mouse MLTK
and MLTK
with BamHI sites at both ends of the cDNA
were cloned into pSR
-HA. For human MLTKs, a human expressed sequence
tag data base was searched using the mouse MLTK sequence
obtained in the subtraction. Based on the corresponding human sequence,
RACE analyses and full-length cloning were done using a Superscript
human fetal brain cDNA library (Life Technologies, Inc.) as a
template for PCR.
-HA and pSR
-Myc.
N-MEKK1, which contains the C terminus,
the kinase domain of mouse MEKK1, was obtained by PCR using the
RT-PCR product of NIH3T3 cell mRNA as a template and then subcloned
into pSR
-HA. Expression vectors of HA-ASK1 and HA-
N-TAK1 are
described previously (14, 25).
-glycerophosphate, 5 mM NaF, 1 mM sodium PPi, 1 mM Na2VO3, 2 mM
dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, and
0.5% aprotinin). Cell lysates were subjected to immunoblotting using
mouse monoclonal anti-HA antibody (12CA5), rabbit anti-HA antibody
(Y-11, Santa Cruz Biotechnology), mouse monoclonal anti-Myc antibody
(9E10, Santa Cruz Biotechnology), rabbit anti-Myc antibody (A-14, Santa
Cruz Biotechnology), mouse anti-phosphorylated ERK antibody (E10, New
England Biolabs Inc.), rabbit anti-phosphorylated JNK antibody (New
England Biolabs Inc.), and rabbit anti-phosphorylated p38 antibody (New
England Biolabs Inc.) as the primary antibodies. The reacted antibodies
were detected by horseradish peroxidase-conjugated secondary
antibodies, followed by visualization using an ECL chemiluminescence
kit (PerkinElmer Life Sciences).
or HA-MLTK
were exposed to the various
stimuli for 10 min. Cells were lysed, and HA-MLTKs were
immunoprecipitated by anti-HA antibody 12CA5 (3 µg/300 µl of
lysate) at 4 °C for 2 h. The precipitates were washed three times with phosphate-buffered saline (pH 7.4) containing 0.05% Tween
20 and added to 15 µl of kinase reaction buffer (20 mM
Tris (pH 7.5), 10 mM MgCl2, 100 µM ATP, and 2 µCi [
-32P]ATP)
containing 5 µg of His-tagged MKK6 generated in Escherichia coli. The mixtures were incubated at 25 °C for 5 min and
stopped by adding SDS sample buffer. Samples were subjected to
SDS-PAGE, and 32P incorporated into His-MKK6 was detected
by autoradiography. A portion of each precipitate was subjected to
immunoblotting using anti-HA antibody.
-32P] ATP and further incubated at
30 °C for 7 min. The reactions were stopped by adding SDS sample
buffer, and 32P incorporated into each MAPK protein was
detected by autoradiography. Coimmunoprecipitation assay for
detecting dimerization of MLTK was performed essentially as described
(28) using lysis buffer containing 50 mM Hepes (pH 7.5),
150 mM NaCl, 1.5 mM MgCl2, 1 mM EGTA, 10% glycerol, 2 mM dithiothreitol, 10 mM
-glycerophosphate, 1 mM NaF, 1 mM Na2VO3, 1 mM
phenylmethylsulfonyl fluoride, and 1% aprotinin.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Identification of MLTK as a molecule whose
expression is increased by ERK activation. A, cloning
of a stable Swiss 3T3 cell line (3L-9) in which expression of HA-tagged
LASDSE MEK1 was induced by the addition of IPTG. The cloned cell line
(3L-9) was treated with 5 mM IPTG for the indicated times,
and the cell lysates were subjected to immunoblotting using anti-HA
antibody (upper). After 24 h, morphological
transformation of the cells was observed (lower).
B, the MLTK transcript is increased by activation of the ERK
pathway. The 3L-9 clone was treated with 5 mM IPTG for 0, 6, or 18 h. The total RNA was collected and then subjected to
reverse transcription reaction using an oligo(dT) primer. Part of the
cDNA obtained was used for RT-PCR analysis using a sense primer
(5'-CTGCTGACGGAGTGCTGAAG-3') and an antisense primer
(5'-GTCGCTCAAGGGTTGCCTCA-3'). Note that the amplified PCR fragment
corresponds to nucleotides 464-916 of MLTK, which is common to MLTK
and MLTK
. Glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) was adopted as a control for RT-PCR.
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Fig. 2.
Cloning of MLTK. A,
sequences of mouse MLTK and MLTK
. The cDNA sequences of
MLTK
and MLTK
and the predicted amino acid sequences are shown.
Nucleotides 1-1025 of MLTK
are identical to those of MLTK
. The
fragment obtained in the subtraction corresponds to nucleotides
2-1023. Our 5'-RACE analysis revealed two different upstream
nucleotide sequences, both of which contain two and three in-frame stop
codons, respectively. Therefore, we conclude that translation starts at
nucleotides 38-40 (ATG). B, schematics of MLTK
and
MLTK
. S/T kinase, serine/threonine kinase domain
(residues 16-277); L-zip, leucine zipper motif (residues
287-322); SAM, sterile
-motif (residues 337-408).
C, comparison of the kinase domain sequences of MLTK, MLK2,
and TAK1. Identical residues are shown in dark gray,
and homologous residues are shown in light gray. The
homology of the kinase domain between MLTK and MLK2 is 64.6%, and that
between MLTK and TAK1 is 51.8%. D, Northern blot analysis.
The 1022-base pair fragment of mouse MLTK cDNA (obtained in the
subtraction) was labeled with digoxigenin using a DIG High Prime
kit (Roche Molecular Biochemicals). The probe (25 ng/ml) was then
hybridized to human multiple-tissue Northern membranes
(CLONTECH). The bands were detected by alkaline
phosphatase-conjugated anti-digoxigenin antibody (Roche Molecular
Biochemicals) according to the manufacturer's protocol.
Arrowheads indicate the detected bands. E,
nuclear export of MLTK. Upper, the candidate NES sequences
in MLTK are aligned with the NES sequence in MEK1 (44). Both MLTK
and MLTK
have two NES-like sequences. The presumably important
hydrophobic residues are shown in boldface.
Lower, leptomycin B (LMB) treatment resulted in
the nuclear accumulation of MLTK. HA-MLTK
and HA-MLTK
were
expressed in COS-7 cells, and the cells were either left untreated
(
LMB) or treated with leptomycin B (2 ng/ml)
for 2 h (+LMB). The subcellular localization of MLTKs
was determined by immunostaining using anti-HA antibody. Experiments
were repeated twice and gave similar results.
and MLTK
, respectively, may be
alternatively spliced forms. The C-terminal region of MLTK
(residues
268-395) shows homology to the C-terminal region of TAK1 (42.5%
identity). The C-terminal region of MLTK
has a sterile
-motif
that has been shown to be involved in the protein-protein interactions
and dimer formation of several signaling molecules and transcription
regulators (29). The calculated molecular masses of MLTK
and MLTK
are 91.7 and 51.3 kDa, respectively.
and MLTK
in COS-7 cells and determined
their subcellular localization. Both HA-MLTK
and HA-MLTK
were
present mainly in the cytoplasm (Fig. 2E, lower).
When the cells were treated with leptomycin B, which inhibits the
function of CRM1, an NES receptor, HA-MLTK
accumulated in the nuclei
(Fig. 2E, lower). HA-MLTK
also tended to
appear in the nucleus after leptomycin B treatment, but nuclear
accumulation of HA-MLTK
was observed in ~10% of the expressing
cells (Fig. 2E, lower). These results suggest
that the cytoplasmic localization of both MLTK
and MLTK
may be
maintained by their NESs and that both molecules may be shuttling
between the nucleus and the cytoplasm. However, we could not detect
significant changes in the subcellular distribution of MLTK
or
MLTK
after activation of these kinases by stimulation with osmotic
shock (data not shown; see below).
and MLTK
could
activate the MAPK pathways in cotransfection assays in COS-7 cells.
Both MLTK
and MLTK
activated any of the four coexpressed MAPKs:
ERK2, JNK/SAPK (SAPK
), p38 (p38
), and ERK5/BMK1 (Fig.
3A). When MAPKKs were cotransfected with MLTKs, MEK1, SEK1, MKK7, MKK3, and MKK6 were activated markedly by either of the MLTKs (Fig. 3B and data
not shown). In in vitro coupled kinase assays, both MLTK
and MLTK
activated MEK1, SEK1, and MKK6 directly, although MLTK
was slightly stronger than MLTK
in activating SEK1 and MKK6 (Fig.
3C). These results suggest that both MLTKs can activate all
known MAPK pathways by phosphorylating and activating the respective
MAPKKs. Thus, both MLTK
and MLTK
are members of the MAPKKK
family.
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Fig. 3.
Activation of the MAPK pathways by MLTK.
A, COS-7 cells were cotransfected with HA-MLTKs and
Myc-tagged ERK (Xenopus ERK2), JNK/SAPK (rat SAPK ), or
p38 (human p38
) and incubated for 20 h (ERK) or 8 h
(JNK/SAPK and p38). The lysates were subjected to immunoblotting with
the antibodies against the phosphorylated (P) forms of ERK,
JNK/SAPK, and p38. Expression of MLTKs and each MAPK was detected with
anti-HA antibody and anti-Myc antibody, respectively. COS-7 cells were
also cotransfected with Myc-MLTKs and HA-ERK5 (mouse) and incubated for
16 h. The lysates were then incubated with anti-HA antibody, and
immunoprecipitated HA-ERK5 was subjected to the in vitro
kinase assay using myelin basic protein (MBP) as an
exogenous substrate. Experiments were performed twice with similar
results. B, COS-7 cells were cotransfected with HA-MLTK
or HA-MLTK
and Myc-tagged MEK1 (Xenopus) or MKK7 (mouse).
Myc-MAPKKs were immunoprecipitated with anti-Myc antibody and then
subjected to in vitro kinase assays using GST-MAPKs as
exogenous substrates (GST-ERK for MEK1 and GST-SAPK for MKK7).
Upper, autoradiography of GST-MAPKs; lower,
immunoblotting with anti-Myc antibody showing the immunoprecipitated
Myc-MAPKKs. COS-7 cells were also cotransfected with MLTK
or MLTK
and HA-MKK3 (human) or HA-MKK6 (human). Cell lysates were then
subjected to immunoblotting with anti-phospho-MKK3/MKK6 antibody
(upper) or anti-HA antibody (lower).
C, shown are the results form the in vitro
coupled kinase assay. His-tagged MEK1, SEK1 (mouse), or MKK6 was
incubated with or without immunoprecipitated HA-MLTK
(+
) or HA-MLTK
(+
) in the presence of
ATP (non-radiolabeled). Then, the GST-fused, kinase-negative form of
ERK, JNK/SAPK, or p38 and 2 µCi of [
-32P]ATP were
added to the reactions and further incubated. The reactions were
subjected to SDS-PAGE, and autoradiography was performed. HA-MLTK
and HA-MLTK
phosphorylated none of the GST-MAPKs directly
(data not shown). Experiments were repeated twice and gave similar
results.
and
exposed to various stimuli for 10 min. HA-MLTK
was then
immunoprecipitated and subjected to the in vitro kinase
assay using His-MKK6 as an exogenous substrate. MLTK
had a
relatively high activity when overexpressed in the cells (Fig.
4A). Treatment with fetal calf
serum (15%), insulin (5 µg/ml), TGF-
(20 ng/ml), 12-O-tetradecanoylphorbol-13-acetate (100 ng/ml),
lysophosphatidic acid (100 ng/ml), UV (80 J/m2), and
anisomycin (100 µg/ml) did not activate MLTK
(Fig. 4A) or MLTK
(data not shown). On the other hand, treatment with NaCl (0.5 M) significantly activated MLTK
(Fig. 4,
A and B) and MLTK
(data not shown). The NaCl
treatment caused a slight mobility retardation of MLTK
in SDS-PAGE
(Fig. 4A). Treatment with sorbitol (0.5 M) or
KCl (0.5 M) as well as NaCl (0.5 M) also caused
a mobility shift of MLTK
and MLTK
(Fig. 4B,
lower) and induced an increase in their kinase activity (the
activity of MLTK
for MKK6 and the autophosphorylation activity of
MLTK
) (Fig. 4B, upper). The in vitro treatment of immunoprecipitated HA-MLTK
with calf
intestine alkaline phosphatase increased its mobility in SDS-PAGE and
decreased its kinase activity (Fig. 4C). Thus, the mobility
retardation correlates with the kinase activity of MLTK.
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Fig. 4.
MLTK is activated by osmotic shock.
A, COS-7 cells transfected with HA-MLTK were either left
untreated (NT, no treatment) or exposed to various stimuli
(15% fetal calf serum (FCS), 5 µg/ml insulin, 100 ng/ml
12-O-tetradecanoylphorbol-13-acetate (TPA), 20 ng/ml TGF-
, 100 ng/ml lysophosphatidic acid (LPA), 0.5 M NaCl, 80 J/m2 UV, and 100 µg/ml anisomycin)
for 10 min. HA-MLTK
was immunoprecipitated (IP) and
subjected to the in vitro kinase assay at 25 °C for 5 min
using His-MKK6 as the exogenous substrate. The results from
autoradiography are shown (upper). A portion of the immune
complex was analyzed by immunoblotting with anti-HA antibody
(lower). Experiments were repeated twice with similar
results. The mobility retardation of HA-MLTK
, which correlated with
its activation, was observed. B, shown is the activation of
MLTK
by osmotic shock. COS-7 cells transfected with either MLTK
(left) or MLTK
(right) were exposed to the
indicated osmotic stimuli (0.5 M each). The phosphorylation
activity of MLTK
for His-MKK6 in vitro was measured
(left), as was the autophosphorylation activity of MLTK
(right). Immunoprecipitated MLTKs were also subjected to
immunoblotting with anti-HA antibody (lower). In the
upper left panel, -fold increases in phosphate
incorporation into MKK6 are shown at the bottom of each lane.
C, overexpressed MLTK
undergoes autophosphorylation in
the cells. HA-MLTK
was immunoprecipitated in the presence of
phosphatase inhibitors (12 mM
-glycerophosphate, 5 mM NaF, 1 mM sodium PPi, and 1 mM Na2VO3). Immunoprecipitated
HA-MLTK
was washed and either left untreated or treated with calf
intestine alkaline phosphatase (+PPase) or a control buffer
(+mock). Then, the kinase activity for MKK6 was measured
(upper), and the mobility in SDS-PAGE was analyzed as
described for A (lower).
Immunoprecipitation in the absence of phosphatase inhibitors abrogated
both the kinase activity and mobility retardation of MLTK
(data not
shown). Note that in this autoradiography (upper), the
exposure time was longer than that in A and B.
Experiments were repeated twice with similar results. D,
shown are the effects of the kinase-negative mutation of MLTK.
Left, COS-7 cells transfected with the HA-tagged wild-type or
kinase-negative mutant of MLTK
or MLTK
were either left untreated
or stimulated with 0.5 M NaCl for 15 min. HA-MLTKs were
then immunoprecipitated and subjected to in vitro kinase
assays using His-MKK6 as an exogenous substrate. We could not detect
marked activation of wild-type MLTKs by osmotic shock in this
experiment, in which the kinase assay was performed at a higher
temperature (30 °C) and for longer times (15 min) than in
A or B to detect the possible low kinase activity
of MLTK
-KM and MLTK
-KM. Right, lysates of COS-7 cells
cotransfected with HA-MLTKs and HA-ERK were subjected to immunoblotting
with anti-phosphorylated (P) ERK antibody as in the legend
to Fig. 3A.
-KM and MLTK
-KM, in which Lys45 was replaced by
Met. COS-7 cells were transfected with each HA-tagged form of the MLTKs
and either left untreated or stimulated by osmotic shock (0.5 M NaCl). No kinase activity for MKK6 was detected in MLTK
-KM and MLTK
-KM. Moreover, although a clear mobility shift in
SDS-PAGE was seen in wild-type MLTK
and wild-type MLTK
in response to osmotic shock, no mobility shift took place in the case of
MLTK
-KM and MLTK
-KM (Fig. 4B, left),
indicating that the stimulus-dependent mobility shift of
MLTK is caused by autophosphorylation. It was confirmed that MLTK
-KM
and MLTK
-KM cannot activate any of the coexpressed MAPKs (Fig.
4D, right; and data not shown). Taken together,
these results suggest that both MLTK
and MLTK
are activated
through autophosphorylation in response to osmotic stress with
hyperosmolar media.
and wild-type MLTK
showed a mobility shift in SDS-PAGE in response to osmotic shock (Fig. 5, first through
sixth lanes), whereas MLTK
-KM and MLTK
-KM did not
(eleventh and twelfth lanes). When MLTK
-KM was coexpressed with wild-type MLTK
, however, MLTK
-KM showed a
relatively weak but significant mobility shift in response to osmotic
shock (Fig. 5, ninth and tenth lanes). In
contrast, MLTK
-KM did not show a mobility shift in response to
osmotic shock when coexpressed with wild-type MLTK
(Fig. 5,
seventh and eighth lanes). Therefore, part of the
autophosphorylation of MLTK
, but not MLTK
, may be achieved
through an intermolecular reaction. The stimulus-dependent mobility shift of kinase-negative MLTK
seems not to be complete compared with wild-type MLTK
, suggesting that both inter- and intramolecular reactions contribute to the full autophosphorylation of
MLTK
in response to osmotic shock. The autophosphorylation of
MLTK
may occur through an intramolecular reaction.
View larger version (29K):
[in a new window]
Fig. 5.
Inter- and intramolecular autophosphorylation
reactions of MLTK. COS-7 cells were transfected with the indicated
constructs. The cells were then either left untreated or stimulated
with 0.5 M NaCl for 15 min. Cell lysates were subjected to
immunoblotting with anti-HA antibody.
, but Not MLTK
, Causes Disruption of Actin Stress Fibers
and Changes in Cell Morphology--
We examined the effect of MLTK
expression on cell morphology and the cytoskeleton. HA-MLTK
or
HA-MLTK
was expressed in Swiss 3T3 cells, and actin filaments were
stained with FITC-labeled phalloidin. Expression of MLTK
, but not
MLTK
, caused disruption of actin stress fibers and cell shrinkage
(Fig. 6). The number of stress fibers or
actin bundles decreased, and each fiber became thin 3 or 5 h after
transfection of MLTK
(Fig. 6A, 3h and 5h panels). At 7-9 h, the stress fibers disappeared almost
completely, and the dot-like staining of actin, probably representing
amorphous aggregates of actin filaments, appeared in the cytoplasm
(Fig. 6A, 7h and 9h panels). At the
same time, the cells gradually shrunk, and several protrusions were
observed and became larger. At 18 h, cell shrinkage proceeded
dramatically, and the cells appeared to be on the point of being
detached from the culture dish (Fig. 6A, 18h
panel). Despite the dramatic change in cell shape, the nuclear
morphology was normal throughout (Fig. 6A,
MLTK
panels), which was also seen in the
4',6-diamidino-2-phenylindole (DAPI) staining of the nuclear DNA (data
not shown). After further incubation (30~40 h), the cells expressing
MLTK
often showed apoptotic nuclei and finally were detached from
the culture dish (data not shown). It can be speculated that the loss
of adequate attachment to the extracellular matrix might trigger
apoptosis. Expression of MLTK
also caused the diminution of thickly
bundled stress fibers to some extent. The effect was, however, much
weaker than that caused by MLTK
; expression of MLTK
did not cause
the complete disruption of stress fibers (Fig. 6, B and
C) or a dramatic change in cell morphology (B and
D).
View larger version (23K):
[in a new window]
Fig. 6.
MLTK , but not
MLTK
, induces the disruption of actin stress
fibers and changes in cell morphology. A and
B, expression of MLTK
causes the disruption of actin
stress fibers and morphological changes in Swiss 3T3 cells. Swiss 3T3
cells were transfected with HA-MLTK
(A) or HA-MLTK
(B) and then fixed at the indicated time points after
transfection. The cells were stained with anti-HA antibody and
FITC-labeled phalloidin to detect MLTK
and actin filaments,
respectively. Experiments were repeated several times and gave similar
results. Control, an untransfected control cell.
C and D, Swiss 3T3 cells were transfected with
pEGFP-C1 (GFP), HA-MLTK
(MLTK
), or
HA-MLTK
(MLTK
). 4 h (black bars) or
20 h (hatched bars) after transfection, the cells were
fixed and then stained with anti-HA antibody (for MLTKs) and
TRITC-labeled phalloidin. The percentages of cells in which stress
fibers were almost completely disrupted (C) and of cells
that shrank extremely (D) are shown under each condition.
100 cells were examined for each condition in one experiment, and shown
is the quantification of the data from two independent experiments.
E, Swiss 3T3 cells were transfected with HA-
N-MEKK1,
HA-ASK1, or HA-
N-TAK1. 20 h after transfection, the cells were
fixed and then stained with anti-HA antibody and FITC-labeled
phalloidin. Control, untransfected control cell.
N-MEKK1 (an activated
form of MEKK1) (33), ASK1 (16), and
N-TAK1 (an activated form of
TAK1) (14). Expression of any of these MAPKKKs did not cause apparent
changes in stress fibers or cell morphology within 4 h of
transfection (data not shown). After 20 h, in the cells expressing
any of the MAPKKKs, the stress fibers became significantly decreased
and thinner, and cell shrinkage took place (Fig. 6E).
Semiquantitative analysis showed that these MAPKKKs caused almost
complete loss of stress fibers in 20-30% of the cells and dramatic
cell shrinkage in 20-35% of the cells. Thus, all the MAPKKKs tested
can induce cellular changes similar to those caused by MLTK
.
However, MLTK
was stronger than other MAPKKKs in causing these
changes in the cell. Especially, extraordinarily severe cell shrinkage
was seen only in the cells expressing MLTK
. It is worth mentioning
that the number of cells expressing
N-MEKK1 was drastically reduced
20 h after transfection, presumably due to cell death (data not shown).
-induced Stress Fiber Disruption and Cell Shrinkage Require
the p38 Pathway--
We expressed the kinase-negative form of MLTK
(MLTK
-KM) to determine whether the kinase activity of MLTK
is
required for causing these cellular changes. As shown in Fig.
7 (A and B), expression of MLTK
-KM did not cause stress fiber disruption or cell
shrinkage. Thus, the kinase activity of MLTK
is indispensable. As
MLTK
can activate four MAPK pathways (ERK, JNK/SAPK, p38, and
ERK5/BMK1), we then examined which pathway is important for causing
these changes. To do this, we expressed dominant-negative forms of each
MAPKK (MEK1-SASA, MKK6-SATA, and MEK5-SATV, in which two activation
phosphorylation sites were replaced by alanine and/or valine; and
MKK7-KL, in which a lysine residue involved in ATP binding was replaced
by leucine) together with MLTK
. It has previously been
demonstrated that each dominant-negative MAPKK specifically
inhibits activation of its own downstream MAPK. Expression of
MKK6-SATA, but not that of MEK1-SASA, MEK5-SATV, or MKK7-KL, inhibited
MLTK
-induced stress fiber disruption (Fig. 7A). Moreover, expression of MKP-5, a dual specificity phosphatase that strongly inactivates p38 (34), inhibited MLTK
-induced stress fiber disruption as well (data not shown). Thus, MLTK
-induced activation of the p38
pathway is required for MLTK
-induced stress fiber disruption. Similarly, expression of the dominant-negative form of MKK6 (Fig. 7B) or MKP-5 (data not shown) most severely impaired
MLTK
-induced cell shrinkage. Although it is possible that MAPK
pathways other than the p38 pathway are also involved in the
MLTK
-induced cellular changes by cooperating with the p38 pathway,
coexpression of other dominant-negative forms of MAPKKs with
dominant-negative MKK6 did not further enhance the inhibitory effect of
dominant-negative MKK6 (data not shown).
View larger version (14K):
[in a new window]
Fig. 7.
MLTK -induced
cellular changes require the kinase activity of MLTK
and activation of the p38 pathway. Swiss 3T3 cells were
transfected with the indicated constructs. 20 h after
transfection, the cells were fixed and then stained with anti-HA
antibody and TRITC-labeled phalloidin. The percentages of cells in
which stress fibers were almost completely disrupted (A) and
of cells that shrank extremely (B) are shown under each
condition. 100 cells were examined for each condition, and shown is the
quantification of the data from three independent experiments.
GFP, pEGFP-C1; DN, dominant-negative.
-induced cellular changes, we expressed MKK6, which is able to
specifically activate the p38 pathway (35). Expression of MKK6 did not
cause changes in stress fibers and cell morphology (Fig.
8). Coexpression of p38 and MKK6 also did
not cause these cellular changes (data not shown). Therefore, although
activation of the p38 pathway is necessary, it is not sufficient for
the MLTK
-induced cellular changes. It is possible that kinase
activity-independent actions of MLTK
might be involved in causing
these cellular responses. Then, to test this idea, we coexpressed the
kinase-negative form of MLTK
(MLTK
-KM) with MKK6, but
coexpression of MLTK
-KM and MKK6 did not cause these cellular
changes (Fig. 8). Expression of MKK7, an activator of JNK/SAPK, with or
without MLTK
-KM did not cause these cellular changes at all (Fig.
8). Expression of an active form of MEK5 (MEK5(D)) or coexpression of
MKK7 and MKK6 did not cause these responses, either. Thus, an MLTK
substrate(s) other than the known MAPKKs may cooperate with the
MLTK
-activated p38 pathway to cause such cellular responses. It
should be noted that the strong activation of the ERK pathway by
expression of a strongly active form of MEK1 (LASDSE), but not the
moderate activation of ERK by an active MEK (SDSE), caused stress fiber disruption and cell shrinkage (Fig. 8; data not shown), as previously reported (36).
View larger version (13K):
[in a new window]
Fig. 8.
Activation of the MAPK pathways is not
sufficient for the MLTK -induced cellular
changes. Swiss 3T3 cells were transfected with the indicated
constructs. 20 h after transfection, the cells were fixed and then
stained with anti-HA antibody and TRITC-labeled phalloidin. The
percentages of cells in which stress fibers were almost completely
disrupted (A) and of cells that shrank extremely
(B) are shown under each condition. 100 cells were examined
for each condition, and shown is the quantification of the data from
three independent experiments. GFP, pEGFP-C1.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, but not MLTK
, possesses a sterile
-motif just after the leucine zipper motif in the primary sequence.
Through these motifs, MLTKs might interact with other proteins that are
involved in the regulation of MLTK or with downstream targets of MLTK.
Interestingly, both MLTK
and MLTK
have NES sequences (Fig.
2E). Consistent with this, they are present mainly in the
cytoplasm; and after treatment with the NES inhibitor leptomycin B,
they come to localize also in the nucleus (Fig. 2E). The
physiological significance of the NES-dependent cytoplasmic
localization of MLTK should be examined in future studies.
and MLTK
were
able to form either a homodimer or a heterodimer, at least when they
were overexpressed in cells. However, dimerization of MLTK per
se might not be directly involved in the regulation of MLTK since
kinase-negative forms of MLTK (MLTK
-KM and MLTK
-KM) were able to
form dimers.2 On the other
hand, because phosphatase treatment of MLTK markedly decreased the
kinase activity of MLTK for substrates, phosphorylation of MLTK induces
its activation. Importantly, this stimulus-dependent phosphorylation of MLTK seems to be caused by autophosphorylation since
the mobility shift of MLTK in SDS-PAGE, which results from phosphorylation, was not observed at all for a kinase-negative form of
MLTK. Thus, MLTK may be activated by enhancement of the autophosphorylation activity of MLTK, the molecular mechanism of which
is not known at present.
migrates more slowly than a presumably
non-phosphorylated form of MLTK
that was obtained by phosphatase
treatment and that wild-type MLTK
migrates always more slowly than
kinase-negative MLTK
. Unlike MLTK
, however, a difference in
mobility was not detected between wild-type and kinase-negative
MLTK
. It is possible that the difference in their mobility was below
the limit of our observation, and/or there might be an additional
autophosphorylation site(s) in MLTK
that is not present in
MLTK
.
is identical to that of
MLTK
, and the activation profiles for various MAPKs are similar
between the two isoforms. So far, only two MAPKKKs, Cot/Tpl-2 and
MEKK3, have been reported to activate ERK5/BMK1 (13, 39). MLTKs have
been shown here to strongly activate the ERK5/BMK1 pathway, suggesting
that MLTK might be one of the important activators of the ERK5/BMK1
pathway in vivo.
-induced Stress Fiber Disruption and Cell
Shrinkage--
Expression of MLTK
in Swiss 3T3 cells causes the
disruption of actin stress fibers and cell shrinkage. A kinase-negative mutant of MLTK
did not cause these cellular changes, suggesting that
the kinase activity of MLTK
is essential. Our results also show that
the kinase activity of MLTK
is indispensable for MLTK
-induced activation of the MAPK pathways and that, among the MAPK pathways activated by MLTK
, the p38 pathway is specifically required for the
MLTK
-induced cellular changes. This is in line with previous studies
demonstrating the requirement of the p38 pathway for
stimulus-dependent rearrangement of the actin cytoskeleton
(40-42). Our results further show that activation of the p38 pathway
alone is not sufficient to cause these changes and that MLTK
may
have a substrate(s) other than the known MAPKK family molecules that
cooperates with the p38 pathway to cause these cellular changes. In
this regard, it is interesting to note that MLTK
, which has almost
the same capacity to activate the MAPK pathways as MLTK
, had little
effect on actin organization and cell morphology. As MLTK
has a long C-terminal region that is not present in MLTK
, it is possible that
this C-terminal region of MLTK
is important for the MLTK
-induced cellular responses. We speculate that the C-terminal region is involved
in docking to some substrate(s) whose phosphorylation is required for
the MLTK
-induced cellular changes. Moreover, to understand the
molecular mechanism of the cellular responses, it is important to
determine whether these cellular responses require de novo
protein synthesis. It should also be examined in future studies if
endogenous MLTK is significantly functional in regulating organization
of the actin cytoskeleton and cell morphology.
can also induce cellular changes similar to those caused by MLTK
is
surprising. As activation of the MAPK pathways alone appears not to be
sufficient to induce these effects, it is possible that these MAPKKKs,
like MLTK
, can have some substrates or activate some pathways (other
than the MAPK pathways) that affect the actin cytoskeleton and/or cell
morphology. Although activation of these different MAPKKKs would induce
apparently similar phenotypes (i.e. rearrangement of the
actin cytoskeleton), this would not necessarily imply that these
MAPKKKs have a common direct target.
modulates
the activity of Rho or other Rho family proteins. However, our
preliminary analysis with GST-PBD (p21Rac/Cdc42
binding domain) did not detect any marked
change in the activation state of Rac when coexpressed with MLTKs in
Swiss 3T3 or COS-7 cells.2 Conversely, it is also
possible that MLTK
is an effector of these GTPases. In our
preliminary experiments, however, expression of an active form of Ras,
Cdc42, Rac, or Rho did not activate coexpressed MLTK
in COS-7 cells.
Thus, MLTK
might not be an effector of these GTPases. In agreement
with this, we could not detect binding between MLTK
and these
GTPases (an activated form or a dominant-negative form of Cdc42, Rac,
or Rho) when they were coexpressed in COS-7 cells.2
Further detailed experiments are needed to clarify the relationship between MLTK
and the Rho family GTPases.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. M. Yoshida for kindly providing leptomycin B. We also thank K. Kaneshiro, K. Kawachi, and T. Moriguchi for help in sequencing, RT-PCR, and construction of several expression vectors and H. Ellinger-Ziegelbauer, H. Hanafusa, and T. Tanoue for helpful advice.
![]() |
FOOTNOTES |
---|
* This work was supported by grants-in-aid from the Ministry of Education, Science and Culture of Japan (to E. N.).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 sequences reported in this paper have been
submitted to the DDBJ/GenBankTM/EBI Data Bank
with accession numbers AB049731 (mouse MLTK), AB049732
(mouse MLTK
), AB049733 (human MLTK
), and
AB049734 (human MLTK
).
§ These two authors contributed equally to this work.
¶ Present address: Dept. of Cancer Cell Research, Inst. of Medical Science, University of Tokyo, Minato-ku, Tokyo 108-8639, Japan.
** To whom correspondence should be addressed: Dept. of Cell and Developmental Biology, Graduate School of Biostudies, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan. Tel.: 81-75-753-4230; Fax: 81-75-753-4235; E-mail: L50174@sakura.kudpc.kyoto-u.ac.jp.
Published, JBC Papers in Press, October 19, 2000, DOI 10.1074/jbc.M008595200
2 M. Adachi, I. Gotoh, and E. Nishida, unpublished data.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
MAPK, mitogen-activated protein kinase;
MAPKK, MAPK kinase;
MAPKKK, MAPK
kinase kinase;
ERK, extracellular signal-regulated kinase;
JNK, c-Jun
N-terminal kinase;
SAPK, stress-activated protein kinase;
BMK, Big MAPK;
MEK, MAPK/ERK kinase;
MEKK, MEK kinase;
MLTK, MLK-like
mitogen-activated protein triple kinase;
HA, hemagglutinin;
IPTG, isopropyl--D-thiogalactopyranoside;
RT-PCR, reverse transcription-polymerase chain reaction;
RACE, rapid
amplification of cDNA ends;
PAGE, polyacrylamide gel
electrophoresis;
GST, glutathione S-transferase;
NES, nuclear export signal;
TGF-
, transforming growth factor-
;
FITC, fluorescein isothiocyanate;
TRITC, tetramethylrhodamine
isothiocyanate.
![]() |
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