From the Institute for Virus Research, Kyoto
University, Kyoto 606-8507 and § Institute of Physical and
Chemical Research (RIKEN), Saitama 351-0198, Japan
Received for publication, June 13, 2000, and in revised form, March 5, 2001
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ABSTRACT |
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The human serine/threonine kinase, mammalian
STE20-like kinase (MST), is considerably homologous to the budding
yeast kinases, SPS1 and STE20, throughout their kinase domains. The
cellular function and physiological activation mechanism of MST is
unknown except for the proteolytic cleavage-induced activation in
apoptosis. In this study, we show that MST1 and MST2 are direct
substrates of caspase-3 both in vivo and in
vitro. cDNA cloning of MST homologues in mouse and nematode
shows that caspase-cleaved sequences are evolutionarily conserved.
Human MST1 has two caspase-cleavable sites, which generate
biochemically distinct catalytic fragments. Staurosporine activates MST
either caspase-dependently or independently, whereas Fas
ligation activates it only caspase-dependently.
Immunohistochemical analysis reveals that MST is localized in the
cytoplasm. During Fas-mediated apoptosis, cleaved MST translocates into
the nucleus before nuclear fragmentation is initiated, suggesting it
functions in the nucleus. Transiently expressed MST1 induces striking
morphological changes characteristic of apoptosis in both nucleus and
cytoplasm, which is independent of caspase activation. Furthermore,
when stably expressed in HeLa cells, MST highly sensitizes the cells to
death receptor-mediated apoptosis by accelerating caspase-3 activation.
These findings suggest that MST1 and MST2 play a role in apoptosis both
upstream and downstream of caspase activation.
Apoptosis or programmed cell death is a normal cell
suicide mechanism that is highly conserved from nematode to human (1, 2). This regulated process plays a critical role during embryogenesis, tissue homeostasis, and remodeling and serves to remove unwanted or
deleterious cells such as self-reactive lymphocytes, tumor cells, or
virus-infected cells. Deregulation of apoptosis thus contributes to the
pathogenesis of cancer, autoimmune disease, and sustained viral
infection, whereas excessive apoptosis results in inappropriate cell
loss and consequently degenerative disorders (3).
The Fas-mediated apoptosis has been extensively investigated as a model
system of mammalian apoptotic cell death (4). Stimulation of Fas
induces the translocation and recruitment of FADD together with a
complex of pro-caspase-8 and FLASH to the cytoplasmic tail of Fas
(5-7), forming the death-inducing signaling complex (8). Then
pro-caspase-8 becomes proteolytically autoactivated by oligomerization, whereupon it stimulates other caspases, including caspase-3, caspase-6, and caspase-7, by cleaving them (9-12). These downstream caspases cleave the death substrates that are central to apoptotic events such as morphological changes and DNA fragmentation (12, 13).
MST11 and MST2 belong to a
mammalian SPS1/STE20-like kinase family, which is rapidly increasing in
number (14-18). However, the cellular function of MST as well as that
of most other SPS1/STE20-like kinases is largely unknown. MST1 and MST2
have an N-terminal catalytic domain and C-terminal regulatory region,
whereas other subfamily members such as p21-activated kinase (PAK) have
a long N-terminal regulatory domain containing GTPase binding domain
that confers the ability to bind to activated Cdc42 and/or Rac1 (17,
19, 20). Notably, PAK2 is proteolytically activated by caspase in Fas-mediated apoptosis and has been reported to induce morphological changes of apoptosis (21, 22). In contrast, the C-terminal regulatory
region of MST does not have any known interaction motif, although this
region is required for dimerization (23). Some of the MST subfamily is
responsive to cellular stress; inflammatory cytokines such as tumor
necrosis factor (TNF) Previously we identified a protein kinase that is strongly activated in
Fas-mediated apoptosis. Biochemical purification of its activity
revealed the kinase to be a catalytic fragment of MST1 (27).
Fas-mediated activation of MST resulted from specific cleavage at the
caspase recognition site and was inhibited by caspase inhibitor,
suggesting the direct cleavage of MST by caspase. These studies
suggested that MST and other SPS1/STE20 family kinases are involved in
apoptosis. In the present study, we investigated the involvement of MST
in apoptosis. We clearly show that both MST1 and MST2 are cleaved by
caspase in vivo and in vitro. The proteolytic
cleavage reveals the activation mechanism of MST and causes its
cellular translocation. Most important, we show that MST can
caspase-independently induce morphological changes characteristic of
apoptosis and enhances the sensitivity for death receptor-mediated apoptosis in epithelial cells.
Materials--
GFP expression vector pCMX-SAH/Y145F,
Expression and Purification of His-tagged
Proteins--
His-tagged MST was expressed as a kinase-deficient
protein because kinase-active MST was poorly expressed in bacteria.
Human MST cDNA with a mutation at catalytic amino acid residue
(MST1K59R or MST2K56R) was subcloned in frame
into His tag expression vector, pQE-30 (Qiagen). For C-terminal
His-tagging, MST1K59R,D326N was subcloned into pET29a
(Novagen). To obtain active His-tagged caspase-3, cDNA of human
caspase-3 lacking N-terminal 28 amino acid residues was amplified by
PCR using 5'-AGGGATCCTCTGGAATATCCCTGGAC-3' and
5'-TGAGCCTTTGACCATGCCCACAGA-3' as primers (underline indicates BamHI site). PCR product was double-digested with
BamHI and PstI and subcloned into pQE30 vector.
His-tagged proteins were expressed in BL21(DE3)lysS bacteria and
purified using His-Trap affinity column as described previously
(28).
Mammalian Expression Constructs--
Expression constructs of
MST were prepared as an N-terminal FLAG tag. We introduced FLAG tag
after the initiation codon in pME18S vector (27) and generated a
FLAG-tag expression vector, pME18S-FL. The cDNA encoding
full-length MST1, MST1 Preparation of Anti-MST Monoclonal Antibody--
For
immunization, affinity-purified His-MST1K59R and
His-MST2K56R were further loaded onto Mono-Q anionic
exchange column (Amersham Pharmacia Biotech) and eluted with a linear
NaCl gradient of 0.1 to 0.5 M as described (28).
Purified fraction, dialyzed against 20 mM Tris·HCl
(pH 7.5) containing 150 mM NaCl, was used for immunization of Balb/c mice. Establishment and cloning of hybridoma cells were performed as described previously (28). Monoclonal antibodies were
purified from hybridoma culture with protein G-affinity column as
described (Amersham Pharmacia Biotech) (28).
Cell Culture, Transfection, and Induction of
Apoptosis--
Human thymoma-derived HPB-ALL, human acute T cell
leukemia-derived Jurkat, and murine lymphoma-derived WR19L12a cells
were cultured in RPMI 1640 with 10% fetal calf serum, 20 mM Hepes (pH 7.3), 50 µM Preparation of Cell Lysate, Immunoprecipitation, and Immunoblot
Analysis--
Cells were washed once with PBS and suspended in cold
lysis buffer (40 mM Hepes (pH 7.4) with 10% glycerol, 1%
Triton X-100, 0.5% Nonidet P-40, 150 mM NaCl, 50 mM NaF, 20 mM In Vitro Cleavage Assay and Cleavage Site Mapping--
Purified
His-MST1K59R or His-MST2K56R was incubated with
the purified recombinant active caspase-3 at 30 °C for 30 min. The
reaction was stopped by adding Laemmli's sample buffer, resolved on
10% SDS-polyacrylamide gel, and immunoblotted with anti-MST mAb. For purification of C-terminal His-tagged protein,
MST1K59R,D326N was subcloned into pET29a (Novagen) and
transformed into BL21(DE3)lysS bacteria. Jurkat cells were exposed
to CH-11 for 4 h, and lysate was prepared in lysate buffer
containing protease inhibitor mixture. Cell lysate (107
cell equivalents) was incubated with 50 µg of purified
MST1K59R,D326N-His at 30 °C for 1 h, and the
reaction was terminated by adding 50 µM Z-VAD-fmk. The
sample was mixed with nickel-nitrilotriacetic acid-agarose (Qiagen) and
incubated at 4 °C for 2 h. The resin was washed with 10 volumes
of lysis buffer containing 5 mM imidazole. Laemmli's
sample buffer was directly added to the resin, and cleaved proteins
were separated on 15% SDS-polyacrylamide gel and transferred to
polyvinylidene difluoride membrane. The membrane was immunoblotted or
stained with Coomassie Brilliant Blue. The fragment was cut and
analyzed by N-terminal peptide sequencing (27, 28).
Cloning of Murine and C. elegans MST Homologues--
MST
homologues of mouse and C. elegans were identified by
homology search against public data bases and cloned by PCR-based methods as follows. BLAST search identified overlapping EST murine cDNA clones homologous to 3' regions of human
MST1 gene. To obtain a complete cDNA sequence,
5'-rapid amplification of cDNA ends PCR was carried out using a
cDNA library of mouse lymphoma WR19L12a cells as template. For
cloning of murine MST2, primers were designed based on the reported
nucleotide sequence (GenBankTM accession number U28726),
5'-CAGGATCCGCCATGGAGCAGCCGC-3' and 5'-AACTGCAGTCAGAAATTCTGCTGCCTCC-3'.
The cDNA was amplified from the WR19L12a cDNA library. The
C. elegans homologue was found by a BLAST search against EST
or genome data base (ACeDB). C. elegans MST was obtained
from a strain N2 cDNA library by PCR using
5'-atgccaccgtctacagacag-3' and 5'-cgaagccgtcattgaagtcg-3' as primes.
The cloned cDNA was compared with a genome data base (ACeDB) and
matched completely with the predicted sequences by Gene Finder, a gene
structure prediction program. The cDNAs of murine and C. elegans MST were subcloned pME18S-FL and pCMX-SAH/Y145F vector, respectively.
MST Kinase Assay--
Endogenous MST or various forms of
FLAG-tagged MST were immunoprecipitated with 2 µg of antibody, and
the same amounts of immunoprecipitates as analyzed by immunoblot were
used for in-gel phosphorylation assay (27) or immune complex kinase
assay. For immune complex kinase assay, immunoprecipitates were
incubated with 2 µg of histone H2B in 20 µl of kinase reaction
buffer (40 mM Hepes (pH 7.5) with 20 mM
MgCl2, 20 mM Fluorescence Microscopy of Cultured Cells--
NIH 3T3 or HeLa
cells on glass culture slides were washed with PBS, fixed in 3.7%
formaldehyde in PBS containing 0.1% Triton X-100 for 10 min, and
blocked in 2% bovine serum albumin in PBS for 10 min. Anti-FLAG mAb (1 µg/ml) was treated for 1 h and then washed in PBS. The secondary
antibody, fluorescein isothiocyanate-conjugated anti-mouse antibody
(Cappel), was used at a 1:500 dilution for 45 min. Cells were washed
with PBS, incubated with Hoechst 33342 (0.2 µg/ml), and washed again
in PBS. For fluorescence microscopy of live cells, Hoechst 33342 (0.2 µg/ml) was added in culture medium, and the cells were incubated for
30 min at 37 °C before microscopic observation. Cells were washed
with PBS and observed under a 40× water immersion lens (Carl Zeiss).
Samples were examined, and images were collected using Axioplan2
microscope (Carl Zeiss) connected to a CCD camera. The figures were
prepared using Photoshop software (Adobe).
Fluorometric Caspase Activity Assay--
Cell lysate (100 µl)
was incubated with 50 µM
DEVD-7-amido-4-trifluoromethylcoumarin in reaction buffer, 10 mM Tris·HCl (pH 7.5), and 1 mM EDTA and
incubated at 37 °C for 30 min. The reaction was terminated by adding
an equal volume of 50 mM glycine HCl (pH 2.3), and
fluorescence emission at 460 nm (excitation at 370 nm) was measured.
Proteolytic Cleavage and Activation of Both MST1 and MST2 by
Caspase in Fas-mediated Apoptosis--
We established several
hybridoma cell lines producing anti-MST mAbs, and purified mAbs were
characterized by immunoblot analyses for HeLa cells transfected with
FLAG-MST1 or FLAG-MST2 (Fig.
1A). Monoclonal antibody G2B
equally recognized both MST1 and MST2, whereas A8C selectively
recognized only MST1. When apoptosis was induced, G2B and A8C could
detect a cleaved 34-kDa fragment of MST. On the contrary, J7B detected
the C-terminal 21-kDa fragment of MST2. Other mAbs, DIH, F5C, and
H3B, recognized N-terminal 34-kDa fragment of MST1 and
MST2.2
Immunoprecipitation analysis showed that A8C and D1H selectively
immunoprecipitated FLAG-MST1, whereas J7B immunoprecipitated only
FLAG-MST2 (Fig. 1B). A8C could not immunoprecipitate
N-terminal 34-kDa fragment although A8C recognized it in
immunoblot.2 Both FLAG-MST1 and FLAG-MST2 were efficiently
immunoprecipitated by G2B or H3B. With G2B and A8C anti-MST mAbs, we
examined the cleavage of endogenous MST1 and MST2 during Fas-mediated
apoptosis in HPB-ALL cells, which expressed both MST1 and MST2. G2B
recognized a 34-kDa fragment of MST at 2 h after the stimulation
of Fas (Fig. 1C). This fragment was also confirmed by
immunoprecipitation (Fig. 2A).
The cleavage of MST was inhibited when apoptosis was blocked by the
pretreatment with Z-VAD-fmk, a broad spectrum caspase inhibitor (Fig.
1C and 2A). The molecular weight of cleaved MST
coincided with that of the previously purified protein kinase from
apoptotic HPB-ALL lysate, confirming our result that MST is
specifically cleaved to a 34-kDa catalytic fragment in apoptosis (Fig.
2A) (27). A8C efficiently precipitated MST1 when apoptosis
was not induced or Z-VAD-fmk was pretreated. However, A8C could not
immunoprecipitate MST1 when apoptosis was induced (Fig. 2B).
These results suggest that MST1 was completely cleaved to a fragment
that could not be precipitated by A8C because A8C could
immunoprecipitate only uncleaved MST1.2
Next, we examined whether the cleavage of MST induces its activation by
immune complex kinase assay using histone H2B as substrate. As depicted
in Fig. 2, A-D, the phosphorylation of H2B was
substantially increased by Fas ligation with approximately the same
kinetics as processing of MST and DNA ladder formation. Preincubation
with Z-VAD-fmk effectively prevented MST activation for up to 3 h
after Fas ligation. Bacterially expressed His-tagged MST1 and MST2 were purified and incubated with active caspase-3 in vitro.
His-tagged MST1 and MST2 were completely cleaved to the 34-kDa
fragment, and the cleavage was blocked by Z-VAD-fmk (Fig.
2E). These results suggest that both human MST1 and MST2 are
physiological substrates of caspase-3 and are proteolytically activated
by caspase in Fas-mediated apoptosis.
Evolutionary Conservation of the Cleavage Site in MST--
To
address the functional importance of cleavage sequences in human MST1
and MST2 (hMST1 and hMST2), we cloned murine and C. elegans
homologues (mMST and cMST, respectively) and compared the polypeptide
sequences of hMST1, hMST2, mMST1, mMST2, rat MST2 (rMST2,
GenBankTM accession number AJ001529), and cMST (Fig.
3). Murine MST1 and MST2, which show 75%
identity to each other, had 97 and 96% homology to human MST1 and
MST2, respectively (Fig. 3A). Previously reported cDNA
(GenBankTM accession number U28726) that encodes a murine
MST2-like polypeptide deleted the C-terminal region, which is caused by
single nucleotide deletion. However, we could not obtain MST2 cDNA
with nucleotide deletion or alternative splicing from a murine lymphoma
WR19L12a cDNA library (28). We obtained only one MST homologue in
C. elegans, which showed similar homology (~50%) to
mammalian MST1 and MST2. The kinase domain was most conserved, with
95-100% homology between mammalian homologues. Even the kinase domain
of cMST showed 75% homology to mammalian MST1 or MST2. The C-terminal
60 amino acids required for dimerization were also highly conserved in all homologues (23) (Fig. 3B). The putative
caspase-cleavable sites (Fig. 3B, indicated by
arrow) were conserved in all MST homologues, and these
homologues were actually cleaved when apoptosis was
induced.2 Thus, the functional cleavage sequence for
caspase is evolutionarily conserved in MST homologues.
Two Activation Mechanisms of MST1 following Treatment with
Staurosporine--
Previously, Taylor et al. (16)
identified a protein kinase responsive to stress (KRS), which was
activated by treatment with staurosporine but not by Fas ligation.
Subsequent biochemical purification revealed it as MST (16). However,
since our result showed that Fas ligation activated MST (27) (Fig. 2),
we compared the MST activation by staurosporine treatment and that by
Fas ligation in Jurkat cells that undergo apoptosis following these stimulations. Both staurosporine treatment and Fas ligation effectively induced the cleavage of MST1 to 34 kDa (Fig.
4). Unexpectedly, MST1D326N
in which the site of cleavage (Asp326) by caspase-3 is
mutated to Asn (27) was also processed to a slower migrating fragment
of 40 kDa. In addition, the mobility of the 34-kDa fragment was
slightly but significantly different in staurosporine- and Fas-induced
apoptosis. The reason for the different mobility might be that
staurosporine treatment caused a change of phosphorylation state in
MST1, which was not caused by Fas stimulation. We investigated the
kinase activity of each MST1 fragment by in-gel phosphorylation assay
using histone as substrate. Phosphorylation at 55 kDa corresponding to
full-length MST1 was greatly increased at 0.5 h by staurosporine
treatment and peaked at 1 h (Fig. 4, lanes 1-4). In
contrast, Fas ligation could not induce phosphorylation of full-length
MST1 up to 3 h (Fig. 4, lanes 5 and 6).
Phosphorylation at 40 kDa was also rapidly induced at 0.5 h by
staurosporine, although this fragment was not detected in immunoblot
analysis. Fas stimulation did not induce a 40-kDa fragment in
immunoblot or kinase activity of 40-kDa in-gel kinase assay. The 34-kDa
fragment with histone kinase activity was generated from FLAG-MST1
(Fig. 4, lanes 1-6) but not from FLAG-MST1D326N
within 3 h by staurosporine and Fas ligation (Fig. 4, lanes
7-12). These results indicate that staurosporine can activate MST
by two mechanisms, caspase-independent early activation and
caspase-dependent late activation, whereas Fas ligation
activates MST only in a caspase-dependent pathway.
Cleavage of MST1D326N to a 40-kDa fragment indicated that
another cleavage site exists in the C-terminal region of
Asp326, and this cleavage is also the biochemical event
involved in apoptosis. His-tagged MST1K59R,D326N was
effectively cleaved to 40 kDa in apoptotic Jurkat cell
lysate.2 To determine the site of this cleavage, we
purified a C-terminal 15-kDa fragment derived from
MST1K59R,D326N and performed N-terminal peptide sequencing.
It was revealed that Asp349 was the cleavage site, and the
peptide sequence surrounding Asp349 (TMTD349G)
was similar to the identified caspase recognition sequence of MST
homologue, particularly that of cMST (Fig. 3B). We mutated both Asp326 and Asp349 to Asn
(MST1D326N,D349N) and tested whether
MST1D326N,D349N is resistant to proteolytic cleavage during
Fas-mediated apoptosis. MST1D326N,D349N was cleaved
to neither the 40- nor to the 34-kDa fragment (Fig. 5). MST2D322N was not cleaved
to a smaller fragment indicating that MST2 is cleaved only at
Asp322 during apoptosis. In contrast, wild type MST and
MST1D326N were cleaved to the 34- and 40-kDa fragment,
respectively (Fig. 5). Therefore, MST1 is cleaved at two different
sites resulting in a 34- or 40-kDa fragment, and the 40-kDa fragment is
further cleaved to 34 kDa.
Apoptotic Morphological Changes Induced by MST1--
To
investigate the possible function of MST in apoptosis, we transfected
NIH 3T3 cells with FLAG-tagged MST constructs (Fig. 6A). Expression of the various
MST1 constructs was confirmed by immunoblotting with anti-FLAG mAb
(Fig. 6B, top). Overexpression did not induce MST1 cleavage.
The expression level of MST1
Cells expressing active MST1 showed marked changes resulting in
round morphology (Fig. 6C). The morphological change induced by MST1 was indistinguishable from that induced by overexpression of
caspase-8, an initiator caspase. However, the round morphology induced
by MST1 was not inhibited by the treatment with Z-VAD-fmk, although
that by caspase-8 was inhibited by Z-VAD-fmk.2 We compared
the morphological change induced by MST1 with that caused by PAK2 (21).
Similar change was observed when PAK2 was transfected into NIH 3T3
cells. Neither kinase-negative PAK2K299R,
MST1K59R, nor MST1
We investigated MST-induced morphological changes in detail. In
MST1-expressing HeLa cells at 16 h post-transfection, the cytoplasm was remarkably reduced compared with untransfected cells or
transfected cells with MST1K59R (Fig. 7B, top
and middle). The morphological change of chromosome was not
observed, although the nuclei were slightly reduced and rounded. MST1
and MST1K59R localized in the cytoplasm. At 48 h
post-transfection, the cytoplasmic space was completely lost, and cells
were eventually detached from the plate (Fig. 7B, bottom).
In addition, condensation and fragmentation of chromosome was obvious
by Hoechst staining. MST1 appeared to localize in the nucleic area but
not overlapped with chromosome. However, these cells were not positive
for terminal deoxynucleotidyltransferase-mediated dUTP nick
end-labeling staining, and the characteristic DNA ladder in apoptosis
was not detected on agarose gel electrophoresis.2 These
observations indicate that chromosome was not cleaved to much smaller
fragments generated by caspase-activated nuclease, such as CAD. We
conclude that part of apoptosis-related morphological changes are
induced by overexpression of MST, which mimics the caspase-dependent activation of MST in apoptosis.
Nuclear Translocation of Caspase-cleaved MST--
MST1 and
MST1K59R were exclusively localized in the cytoplasm when
expressed as FLAG-tagged or GFP fusion forms (Fig. 7 and
8A). MST1
To test whether the localization of MST is changed in
Fas-mediated apoptosis, we established stable HeLa cell lines
expressing caspase-cleavable and uncleavable MST1. MST1 was fused at
the C-terminal end of GFP for rapid detection of morphological change and translocation. Established cell lines expressing wild type MST did
not show significant morphological differences with control cell lines,
although transiently expressed MST induced morphological change (Fig.
7). In the stable cell lines, cleavable and uncleavable MST1 showed a
similar cytoplasmic localization before induction of
apoptosis.2 However, in early apoptotic cells that showed
blebbing of membrane but retained their nuclear structure, cleavable
MST1 and MST1K59R translocated into the nucleus although
MST1 and MST1K59R were still detected in the cytoplasm
(Fig., 8B, top and middle rows). In contrast,
uncleavable MST1K59R,D326N,D349N (Fig. 5) remained in the
cytoplasm although the apoptosis was apparently occurring as judged by
the apoptotic blebbing of membrane (Fig. 8B, bottom row). In
later apoptotic cells with nuclear fragmentation, even
MST1K59R,D326N,D349N completely lost cytoplasmic
localization and was detected in the fragmented nuclei,2
suggesting the translocation after the breakdown of the nuclear envelope. We conclude that MST1 is cytoplasmic protein and the cleaved
MST1 translocates into the nucleus before the nuclear fragmentation is initiated.
MST Sensitizes Fas- and TNF
Interestingly, GFP-MST1 greatly facilitated the activation of
caspase-3-like protease in Fas-mediated apoptosis (Fig. 9C) suggesting that MST1 can accelerate caspase-3 activation. To test this
possibility, we investigated the cleavage kinetics of caspase-3 in
detail. As shown in Fig. 9D, the p17 subunit of caspase-3
was detected at 1.5-2 h in MST1-expressing cell lines (HL-WT1 and HL-WT2) but p17 was detected after 3 h in control cells.
Furthermore, the cleavage kinetics of GFP-MST1 was correlated with that
of caspase-3 supporting the accelerated activation of caspase-3, because MST1 was shown to be cleaved by activated caspase-3 (Fig. 2).
GFP-MST1 did not accelerate the onset of other apoptosis induced by
pharmaceutical drugs such as ceramide, staurosporine, and
etoposide.2 We conclude that stably expressed MST1
sensitizes death receptor-mediated apoptosis through the accelerated
onset of caspase-3 activation.
Cleavage and Activation of MST in Apoptosis--
We previously
reported that during Fas-mediated apoptosis MST1 and MST2 are
proteolytically activated by specific cleavage. However, it remained
unclear how MST1 and MST2 are involved in apoptosis. In this study, we
clearly show that MST1 and MST2 are physiologically activated by
caspase-3-like protease and involved in apoptosis. Caspase cleavage
sequences of MST1 (Asp-Glu-Met-Asp326-Ser) and MST2
(Asp-Glu-Leu-Asp322-Ser) are more conserved and optimal
than those of other protein kinases known to be activated
proteolytically by caspase-3-like proteases including caspase-6 and
caspase-7 (27, 36). This probably explains why only MST was purified as
a major protein kinase that was activated in Fas-mediated apoptosis
(27). Caspase cleavage sequences in MST1 and MST2 are conserved not
only in their mammalian but in the nematode homologue, strongly
suggesting their biological significance including a role in apoptotic
cell death signaling. The cleavage of MST may be a common molecular event in apoptosis since various apoptotic signals induce
caspase-dependent MST cleavage, including signals from Fas
and TNF
The cleavage of MST is closely coupled with its activation. Although
MST has basal kinase activity in non-apoptotic cells, caspase-dependent MST cleavage induces significant and
strong activation of the catalytic fragment. Furthermore, truncated
MST, MST1 Apoptotic Cleavage of MST Induces Nuclear Translocation--
We
show that MST is localized exclusively in the cytoplasm. Deletion
analysis shows that the C-terminal region of MST is sufficient for
cytoplasmic localization that is lost on treatment with leptomycin. Cytoplasmic localization of MST may be regulated by nuclear export signal although the possibility remains that other nuclear export signal-containing proteins or cytoplasmic anchor protein regulates MST
localization. MST cleavage by caspase results in the subcellular relocalization of catalytic fragment. In Fas-mediated apoptosis, cleaved MST1 translocates into the nucleus before nuclear fragmentation is initiated. This result suggests additional nuclear functions of
cleaved MST1 in apoptosis. Histone H2B is selectively phosphorylated in
mammalian apoptotic cells and is associated with chromatin condensation
in apoptosis (39). It is of interest to test whether cleaved MST
phosphorylates H2B in apoptosis because H2B is an excellent substrate
in vitro (Fig. 2 and 6). Caspase-dependent cleavage might affect other biochemical functions of MST. MST1 and MST2
were reported to form homo- or heterodimers through the C-terminal
region (23), and cleavage of MST naturally leads to the dissociation of
N-terminal catalytic fragments. Therefore, cleavage of MST during
apoptosis results in at least three coupled events, activation,
monomerization, and translocation of catalytic fragment into the
nucleus. These representative regulatory mechanisms, observed in many
signaling molecules, may be involved in the apoptotic function of
MST.
MST Is Involved in Apoptotic Events--
Overexpression of MST or
truncated MST induces morphological changes in cells. These changes are
at least partly related to the characteristic morphology of apoptotic
cells such as cell rounding, detachment from substratum, and the
fragmentation and condensation of nucleus. However, we could not detect
other apoptotic characteristics such as externalization of
phosphatidylserine, DNA laddering, or formation of apoptotic bodies.
These morphological changes induced by MST are similar to those induced
by PAK2 (21, 22). Overexpression of each kinase-negative,
caspase-resistant form of MST1 and MST2 does not inhibit Fas-mediated
apoptosis.2 This suggests that other SPS1/STE20 family
members are also involved in apoptosis. Recently, STE20-related kinase,
SLK, was reported to be cleaved by caspase-3 and to induce cytoskeletal
rearrangement (40-42). Caspase-dependent cleavage and
effects on apoptotic morphology of MST, PAK2, and SLK suggest that
SPS1/STE20 family kinases are closely involved in apoptosis, probably
targeting similar molecules.
Previously it was suggested that overexpression of MST1 activates
caspase through the JNK/SAPK kinase pathway (43). However, others (37)
could not detect the SAPK/JNK activation. We detected neither the JNK
activation nor the caspase activation by overexpression of MST, and
PARP, caspase substrate, was not cleaved in our system.2 In
addition, the MST1-induced morphological change of the cell was
unaffected by preincubation with caspase inhibitor, Z-VAD-fmk (Fig. 6,
C and D). Caspase-resistant
MST1D326N,D349N still induced similar morphological changes
in the presence of caspase inhibitor. Thus, we conclude that
morphological changes induced by transient overexpression of MST do not
require the activation of caspase, and SAPK/JNK might not be involved
in the morphological change.
When stably expressed in the GFP fusion form, MST1 highly sensitizes
HeLa cells to Fas- or TNF
Our result provides some clue to the regulation and function of MST in
non-apoptotic cell signaling. Although no physiological activators are
known, the rapid activation of MST by staurosporine indicates that
there exists a caspase-independent mechanism of activation, which is
regulated by upstream kinase(s) or phosphatase(s). Another critical
clue to understanding the function of MST may be obtained from the
analysis of subcellular localization since some protein kinases are
tightly regulated in intracellular spaces (44-46). We propose the
regulation of subcellular localization as a possible mechanism
regulating cellular function of MST. The identification of
physiological substrate will undoubtedly lead to a better understanding
of MST function in apoptosis or other cell signaling.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
can trigger activation of GCK, and SOK is
responsive to oxidative stress (24-26). However, MST1 is only
activated by non-physiological stresses such as high temperature heat
shock and high concentrations of sodium arsenite or staurosporine
(16).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-galactosidase expression vector pJ7
-LacZ, and
Caenorhabditis elegans N2 cDNA library were kindly
provided by Dr. K. Umezono (Kyoto University, Japan), Dr. A. Murakami
(Kyoto University, Japan), and Dr. A. Sugimoto (Tokyo University,
Japan), respectively. Leptomycin was a generous gift from Dr. M. Yoshida (Tokyo University). Anti-FLAG antibody and cycloheximide were
obtained from Sigma; agonistic anti-Fas antibody (CH-11) and anti-GFP
antibody were from MBL Inc. (Japan); anti-caspase-3 antibody was from
PharMingen; Z-VAD-fmk, DEVD-CHO, and
DEVD-7-amido-4-trifluoromethylcoumarin were from Peptide Institute Inc.
(Japan); human recombinant TNF was from Calbiochem.
327-487 (deleting aa 327-487), MST1
1-326
(deleting aa 1-326), full-length MST2, MST2
323-491 (deleting aa
323-491), and MST2
1-322 (deleting aa 1-322) were amplified by PCR
and inserted in frame into pME18S-FL. For GFP fusion, cDNA of MST
was subcloned in frame into pCMX-SAH/Y145F. Mutagenesis of the amino
acid residue was performed with Quick-Change site-directed mutagenesis
kit (Stratagene) and confirmed by DNA sequencing. Human PAK2 cDNA
and caspase-8 cDNA were amplified by PCR from an HPB-ALL cDNA
library (29) and inserted into pME18S vector.
-mercaptoethanol,
50 units/ml penicillin, and 50 µg/ml streptomycin. Murine fibroblast
NIH 3T3, human adenocarcinoma-derived HeLa, and human embryonic
kidney-derived 293T cells were maintained in Dulbecco's modified
Eagle's medium supplemented with 10% fetal calf serum and 100 µg/ml
kanamycin. Jurkat cells were transfected by electroporation at 300 V
with capacitance of 960 microfarads. NIH 3T3 or HeLa cells were seeded
on glass culture slides (Nalge Nunc) or 60-mm culture plates 24 h
before transfection and then transfected with LipofectAMINE (Life
Technologies, Inc.) in accordance with the manufacturer's suggestion.
To establish stable HeLa cell lines, pME18S-Neo vector harboring
neomycin-resistant gene was co-transfected with expression vectors
encoding GFP, GFP-MST1, or MST1K59R. The transfectants were
grown and selected in the presence of 1 mg/ml G418. To induce
apoptosis, cells were treated with anti-Fas antibody, CH-11 (0.1 µg/ml) (27, 30), TNF
(50 ng/ml), or staurosporine (0.5 µM). Where indicated, cells were co-treated with
cycloheximide (10 µg/ml).
-glycerol phosphate, 1 mM EDTA, 1 mM EGTA, 1 mM
phenylmethylsulfonyl fluoride, and 0.1 mM vanadate) with
protease inhibitor mixture (1 µg/ml aprotinin, leupeptin, and
pepstatin). Cell lysates were cleared by centrifugation at 15,000 rpm
for 20 min. For immunoprecipitation, cell lysate was incubated with 2 µg of anti-FLAG mAb or anti-MST mAb for 2 h at 4 °C and
precipitated with protein G-Sepharose (Amersham Pharmacia Biotech).
Cell lysate or immunoprecipitates were resolved by SDS-PAGE and
transferred to polyvinylidene difluoride membrane (Millipore). The
membrane was blocked in TBST buffer (20 mM Tris·HCl (pH
7.5) containing 150 mM NaCl and 0.1% Tween 20) with 5%
skim milk at room temperature for 1 h. The membrane was incubated
with anti-FLAG mAb or anti-MST mAb for 1 h, washed in TBST, and
then incubated for 1 h with horseradish peroxidase-conjugated
anti-mouse IgG (Amersham Pharmacia Biotech). After further washing with
TBST, peroxidase activity was detected on x-ray films using an enhanced chemiluminescence detection system (DuPont).
-glycerol phosphate, and 0.1 mM vanadate) containing 25 µM ATP and 2.5 µCi of [
32P]ATP for 20 min at 30 °C. Reactions
were terminated by adding 7 µl of Laemmli's sample buffer and
boiling for 5 min. A portion of the sample (15 µl) was separated on a
15% SDS-polyacrylamide gel and autoradiographed or analyzed by
PhosphorImage analyzer.
-Galactosidase Staining--
NIH 3T3 or HeLa cells were
cultured on 6-well plates and transfected with 1 µg of MST kinase
expression vector and 0.2 µg of a
-galactosidase expression vector
(pLacZ). After 48 h, the cells were washed once with PBS and fixed
with 2% formaldehyde and 0.2% glutaraldehyde in PBS for 5 min at
4 °C. After being further washed with PBS, the cells were
overlaid with X-gal staining solution (5 mM potassium
ferricyanide, 5 mM potassium ferrocyanide, 1 mM
MgCl2, and 1 mg/ml X-gal in PBS) for 1 h at 37 °C
and photographed.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Characterization of anti-MST mAbs.
A, FLAG-MST1 or FLAG-MST2 was expressed in HeLa cells on
6-well plates, and apoptosis was induced by 4 h of incubation
with CH-11 (0.1 µg/ml) and cycloheximide (10 µg/ml). Cell lysate
(20 µg) was resolved on 12% SDS-PAGE gel and immunoblotted with
anti-FLAG or anti-MST mAbs as indicated. B, FLAG-MST1 and
FLAG-MST2 were immunoprecipitated with anti-FLAG or the indicated
anti-MST mAbs. Immunoprecipitates (IP) were resolved on
SDS-PAGE gel and immunoblotted with anti-FLAG mAb. C,
HPB-ALL cells (2 × 107) were preincubated without
( ) or with (+) 25 µM Z-VAD-fmk for 1 h, and
apoptosis was induced by incubation with CH-11 (0.1 µg/ml) for the
indicated times. Cell lysates (50 µg) were analyzed by immunoblotting
with anti-MST mAb, G2B.
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Fig. 2.
MST1 and MST2 are proteolytically activated
by caspase. A-D, HPB-ALL cells (5 × 107) were treated with Z-VAD-fmk and CH-11 as in the legend
of Fig. 1. Aliquots of lysate were immunoprecipitated with anti-MST
mAb, G2B (A and C), or A8C (B).
Immunoblot analysis was performed using G2B (A) or A8C
(B). As a control, purified MST from apoptotic HPB-ALL cells
(27) was also analyzed and indicated as purified 34-kDa MST
(A and B). An aliquot of immunoprecipitate was
subjected to in vitro kinase assay using histone H2B as
substrate (C). Cell lysates were analyzed for DNA
fragmentation as a marker of apoptosis (D). E,
bacterially expressed His-MST1 (1 µg) and His-MST2 (1 µg) were
incubated with (+) or without ( ) recombinant active caspase-3 (50 ng)
in the presence (+) or absence (
) of 25 µM
DEVD-CHO at 30 °C for 30 min. The samples were resolved on
SDS-PAGE gel and analyzed by immunoblotting with G2B.
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Fig. 3.
Evolutionary conservation of caspase cleavage
site in MST homologues. A, overall amino acid sequence
homology in MST homologues. B, alignment of C-terminal
regions. The conserved caspase recognition site is indicated by an
arrow. Arrowhead indicates the novel cleavage sequence in
human MST1 identified by peptide sequencing. h, human;
m, mouse; r, rat; c, C. elegans.
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Fig. 4.
Different activation mechanism of MST1 in
staurosporine-and Fas-induced apoptosis. Jurkat cells (1 × 107) were transfected with FLAG-MST1 (WT) or
FLAG-MST1D326N. After 48 h post-transfection,
apoptosis was induced with 0.5 µM staurosporine
(STR) or 0.1 µg/ml of CH-11. After immunoprecipitation
with anti-FLAG mAb, immunoblot analysis with anti-FLAG mAb
(top) or in-gel kinase assay using histone as substrate (0.2 mg/ml) (bottom) was performed.
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Fig. 5.
MST1 is cleaved at Asp326 and
Asp349 in apoptosis. HeLa cells on 6-well plate were
transfected with FLAG-MST1 or FLAG-MST2 constructs. After 48 h of
post-transfection, apoptosis was induced with of CH-11 (0.1 µg/ml)
and cycloheximide (10 µg/ml). Immunoblot analysis (20 µg) was
performed with anti-FLAG mAb. WT, wild type.
327-487, with a C-terminal truncation
corresponding to the active MST1 fragment generated by caspase, was
very low. In comparison with MST1, MST1
327-487 showed enhanced
histone phosphorylation activity but little autophosphorylation
activity (Fig. 6B, bottom). A similar result was obtained
when FLAG-tagged MST2 was expressed in NIH 3T3 cells.2
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Fig. 6.
MST kinase activity and its effect on
morphological change in NIH 3T3 cells. A, schematic
representation of MST1 constructs used in this study.
Arrowheads indicate caspase cleavage sites in human MST1.
B, FLAG-MST1 constructs were expressed in NIH 3T3 on a
6-well plate and immunoprecipitated with anti-FLAG mAb.
Immunoprecipitates were analyzed by immunoblotting with anti-FLAG mAb
(top) or in vitro kinase assay
(bottom). Kinase assay was performed using histone H2B as
substrate at 30 °C for 20 min. The phosphorylated proteins were
resolved on 15% SDS-PAGE gel and then analyzed by autoradiography.
Arrowhead indicates autophosphorylation activity of
MST1 327-487. C, pJ7
-LacZ (0.2 µg) was co-transfected with
vector (1 µg) expressing caspase-8, MST1K59R, MST1,
MST1K59R
327-487, MST1
327-487,
PAK2K299R, or PAK2. Cells transfected with MST1 were
cultured with or without Z-VAD-fmk (100 µM). At 48 h
post-transfection, cells were fixed and stained for
-galactosidase
expression. At least 100 blue cells/sample were counted, and the number
of round blue cells was expressed as a percentage of the total number
of blue cells. The means of three independent determinations ± S.D. are shown. WT, wild type.
1-326 deleting N-terminal kinase
domain evoked changes in cell morphology indicating that kinase
activity of MST1 or PAK2 is required for this morphological change.
MST-induced change of morphology was also observed in HeLa cells (Fig.
7). MST-expressing cells were rounded and
shrunken with associated destruction of cytoskeletal structure (Fig.
7A). Hyperactive MST1
327-487 showed more dramatic change
of morphology and resulted in the loss of MST1
327-487-expressing
cells by detachment from substrate. This result may elucidate why the
percentage of the round cells in MST1
327-487-transfected cells was
reduced in
-galactosidase assay (Fig. 6C). The enhanced
morphological change induced by MST1
327-487 may be the result
from the increased kinase activity because kinase-negative
MST1K59R
327-487 or MST1K59R did not induce
any morphological changes.
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Fig. 7.
Overexpression of MST induces cell
death with apoptosis-like morphology in HeLa cells. HeLa cells
cultured on glass chamber slides were transfected with indicated
constructs of GFP-MST1 and cultured for 48 h. A, cells
were fixed and then observed by green fluorescence microscopy.
Expression of kinase-active MST1 (WT) and MST1 327-487
but not kinase-negative MST1K59R and
MST1K59R
327-487 induced a round and shrunken
morphological appearance. B, cells transfected with
GFP-MST1K59R (top) or GFP-MST1 (middle,
bottom) were stained with Hoechst 33342 (0.2 µg/ml) at 16 (middle) or 48 h (top, bottom)
post-transfection. After washing with PBS, cells were photographed.
Representative cells with round morphological appearance are shown.
Green and blue indicates fluorescence of GFP and
nuclear staining of Hoechst 33342, respectively. DIC, differential
interference contrast.
1-326 deleting
N-terminal kinase domain was also localized in the cytoplasm, but
MST1
327-487 was distributed in both cytoplasm and nucleus. To test
whether the cytoplasmic localization of MST1 is controlled by active
nuclear export, HeLa cells expressing MST1 or MST1
1-326 were
treated with leptomycin, an inhibitor of CRM1/exportin (31, 32). MST1
or MST1
1-326 rapidly lost cytoplasmic localization (Fig.
8A). These results indicate that cytoplasmic localization of
MST1 is controlled by a C-terminal regulatory region through active
nuclear export by the CRM1/exportin (33-35).
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Fig. 8.
Nuclear translocation of caspase-cleavable
MST1 in Fas-mediated apoptosis. A, indicated FLAG-MST1
constructs were transfected into HeLa cells and cultured for 16 h.
Cells were fixed and immunostained with anti-FLAG mAb. Leptomycin
(LMB, 10 ng/ml) was treated to the cells transfected with
FLAG-MST1 (WT) and FLAG-MST1 1-326 and incubated for
1 h before fixation. B, apoptosis was induced in HeLa
cells stably expressing caspase-cleavable GFP-MST1 (top
row), GFP-MST1K59R (middle row), or
uncleavable GFP-MST1K59R,D326N,D349N (bottom
row) by treatment with CH-11 (0.1 µg/ml) and cycloheximide (10 µg/ml) for 2.5 h. Hoechst 33342 (0.2 µg/ml) was added for 30 min before observation. Cells were washed with PBS and observed by
fluorescence microscopy without fixation.
-mediated Apoptosis--
To address
further the function of MST in apoptosis, we investigated the
sensitivity of HeLa cell lines stably expressing MST1 in Fas- and
TNF
-mediated apoptosis. The expression of GFP-MST1 and
GFP-MST1K59R was confirmed by immunoblotting with G2B or
with anti-PARP antibody as a control for loading and transfer (Fig.
9A). All sub-cell lines
expressing GFP-MST1 were more sensitive to Fas- or TNF
mediated apoptosis than parental HeLa cells or
GFP-MST1K59R-expressing cells (Fig. 9B).
GFP-MST1K59R did not accelerate or inhibit Fas- or
TNF
mediated apoptosis.
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Fig. 9.
Stable expression of MST1 enhances Fas and
TNF -induced apoptosis. A, HeLa
cell lines (2 × 106) stably expressing GFP-MST1
(HL-WT1-4) or GFP-MST1K59R (HL-KR1-4) were analyzed for
GFP-MST1 expression by immunoblot with G2B. Expression of
poly(ADP-ribose) polymerase (PARP) was also measured with
anti-poly(ADP-ribose) polymerase antibody as a control for loading and
transfer. HL-Neo is a control neomycin-resistant cell line.
B, apoptosis was induced by incubation with CH-11 (0.1 µg/ml) or TNF
(50 ng/ml) together with cycloheximide (10 µg/ml)
for 4 h. The number of cells with apoptotic bodies and membrane
blebbing (200-300 cells) was expressed as a percentage of the total
number of cells. The means of three independent determinations ± S.D. are shown. C, cells (2 × 106) were
treated with CH-11 (0.1 µg/ml) together with cycloheximide (10 µg/ml) for 4 h, and then cell lysate was prepared.
Caspase-3-like activity was measured using 50 µM of
DEVD-7-amido-4-trifluoromethylcoumarin as substrate. D, two
MST1-expressing cell lines (HL-WT1 and HL-WT2) and control HL-Neo cell
lines were treated with CH-11 (0.1 µg/ml) and cycloheximide (10 µg/ml) for the indicated times. Cell lysate (50 µg) was analyzed by
immunoblot with anti-caspase-3 (top) or with G2B
(bottom). Full-length and cleavage products are indicated by
arrows and arrowheads, respectively. Other stable
cell lines showed essentially similar results.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
receptor, and chemical apoptotic inducers such as
staurosporine, etoposide, and ceramide. Recently bisphosphonates,
pharmacological drugs for osteoporosis, and anti-tumor drugs such as
cytotrienin A have been reported to induce
caspase-dependent cleavage of MST in osteoclast and
leukemia cells (37, 38).
327-487, has powerful kinase activity supporting the
activation by caspase-mediated cleavage. In human MST1, another
cleavage site (TMTDG349) was found in this report, which
generates a catalytic fragment of 40 kDa. The 40-kDa fragment is
detected early in the in-gel kinase assay, whereas the 34-kDa catalytic
fragment is detected only at a late time point by staurosporine
treatment or Fas stimulation (Fig. 4). Thus, cleavage at
Asp349 precedes that at Asp326 and
suggests rapid turnover of the 40-kDa fragment to the 34-kDa form.
Based on the cleavage kinetics, we conclude that human MST1 is
sequentially cleaved at Asp326 and Asp349 in
staurosporine-induced apoptosis. However, it is not likely that the
cleavage at Asp349 is a prerequisite for MST1 activation
because Asp349 is not conserved in murine MST1. In
addition, MST1D349N is processed to the 34-kDa fragment
with strong kinase activity. Nevertheless, a specific function of the
40-kDa fragment, particularly in early apoptotic events, cannot be
ruled out because it shows distinct catalytic activity from the 34-kDa fragment.
receptor-mediated apoptosis. Surprisingly, susceptibility to apoptosis was enhanced by accelerated activation of caspase. This result contradicts the finding of transient
expression (Fig. 6) that indicates caspase activation is not required
for the MST-induced apoptosis-like morphological changes. One possible
explanation is that MST has two distinctive functions, direct induction
of morphological changes and sensitization to death receptor-mediated
apoptosis. The limited apoptotic change induced by transiently
expressed MST may reflect MST function downstream of caspase. This
explanation is supported by the fact that MST1 is a substrate of
caspase, and truncated MST induces morphological changes that are more
dramatic. When stably expressed, MST may accelerate caspase activation
through the phosphorylation of pro- or anti-apoptotic regulators or
caspases. It cannot be ruled out that MST regulates the expression of
apoptotic regulators or caspases. Although it is largely unknown
how MST is involved in apoptosis, our result clearly shows that it acts
as both an upstream activator and a downstream effector of caspase; MST
regulates death receptor-induced caspase activation and amplifies
caspase-dependent morphological change.
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ACKNOWLEDGEMENT |
---|
We thank Masao Murakawa for construction of the FLAG-tagged MST vectors.
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FOOTNOTES |
---|
* This work was supported in part by a grant-in-aid for Scientific Research on Priority Areas from the Ministry of Education, Science, and Culture of Japan and a grant-in-aid for Scientific Research from Japan Society for the Promotion of Science.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/EMBL Data Bank with accession number(s) AF271359, AF271360, and AF271361.
¶ To whom correspondence should be addressed: Institute for Virus Research, Kyoto University, Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507. Japan. Tel.: 81-75-751-4783; Fax: 81-75-751-4784; E-mail: syonehar@virus.kyoto-u.ac.jp.
Published, JBC Papers in Press, March 7, 2001, DOI 10.1074/jbc.M005109200
2 K.-K. Lee, T. Ohyama, N. Yajima, S. Tsubuki, and S. Yonehara, unpublished observations.
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ABBREVIATIONS |
---|
The abbreviations used are:
MST, mammalian
STE20-like kinase;
GFP, green fluorescence protein;
PCR, polymerase
chain reaction;
mAb, monoclonal antibody;
PAK, p21-activated kinase;
TNF, tumor necrosis factor;
Z-, benzyloxycarbonyl-;
fmk, fluoromethylketone;
PBS, phosphate-buffered saline;
JNK, c-Jun
N-terminal kinase;
SAPK, stress-activated protein kinase;
PAGE, polyacrylamide gel electrophoresis;
aa, amino acids;
X-gal, 5-bromo-4-chloro-3-indolyl -D-galactopyranoside.
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REFERENCES |
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---|
1. | Vaux, D. L., Haecker, G., and Strasser, A. (1994) Cell 76, 777-779[Medline] [Order article via Infotrieve] |
2. | Steller, H. (1995) Science 267, 1445-1449[Medline] [Order article via Infotrieve] |
3. | Thompson, C. B. (1995) Science 267, 1456-1462[Medline] [Order article via Infotrieve] |
4. | Nagata, S. (1997) Cell 88, 355-365[Medline] [Order article via Infotrieve] |
5. | Boldin, M. P., Goncharov, T. M., Goltsev, Y. V., and Wallach, D. (1996) Cell 85, 803-815[Medline] [Order article via Infotrieve] |
6. | Muzio, M., Chinnaiyan, A. M., Kischkel, F. C., O'Rourke, K., Shevchenko, A., Ni, J., Scaffidi, C., Bretz, J. D., Zhang, M., Gentz, R., Mann, M., Krammer, P. H., Peter, M. E., and Dixit, V. M. (1996) Cell 85, 817-827[Medline] [Order article via Infotrieve] |
7. | Imai, Y., Kimura, T., Murakami, A., Yajima, N., Sakamaki, K., and Yonehara, S. (1999) Nature 398, 777-785[CrossRef][Medline] [Order article via Infotrieve] |
8. | Kischkel, F. C., Hellbardt, S., Behrmann, I., Germer, M., Pawlita, M., Krammer, P. H., and Peter, M. E. (1995) EMBO J. 14, 5579-5588[Abstract] |
9. | Nicholson, D. W., and Thornberry, N. A. (1997) Trends Biochem. Sci. 22, 299-306[CrossRef][Medline] [Order article via Infotrieve] |
10. |
Medema, J. P.,
Scaffidi, C.,
Kischkel, F. C.,
Shevchenko, A.,
Mann, M.,
Krammer, P. H.,
and Peter, M. E.
(1997)
EMBO J.
16,
2794-2804 |
11. |
Thornberry, N. A.,
and Lazebnik, Y.
(1998)
Science
281,
1312-1316 |
12. |
Wolf, B. B.,
and Green, D. R.
(1999)
J. Biol. Chem.
274,
20049-20052 |
13. | Enari, M., Sakahira, H., Yokoyama, H., Okawa, K., Iwamatsu, A., and Nagata, S. (1998) Nature 391, 43-50[CrossRef][Medline] [Order article via Infotrieve] |
14. | Creasy, C. L., and Chernoff, J. (1995) Gene (Amst.) 167, 303-306[CrossRef][Medline] [Order article via Infotrieve] |
15. |
Creasy, C. L.,
and Chernoff, J.
(1995)
J. Biol. Chem.
270,
21695-21700 |
16. |
Taylor, L. K.,
Wang, H. C.,
and Erikson, R. L.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
10099-10104 |
17. | Sells, M. A., and Chernoff, J. (1997) Trends Cell Biol. 7, 162-167[CrossRef] |
18. |
Kyriakis, J. M.
(1999)
J. Biol. Chem.
274,
5259-5662 |
19. |
Burbelo, P. D.,
Drechsel, D.,
and Hall, A.
(1995)
J. Biol. Chem.
270,
29071-29074 |
20. |
Tapon, N.,
Nagata, K.,
Lamarche, N.,
and Hall, A.
(1998)
EMBO J.
17,
1395-1404 |
21. |
Rudel, T.,
and Bokoch, G. M.
(1997)
Science
276,
1571-1574 |
22. |
Lee, N.,
MacDonald, H.,
Reinhard, C.,
Halenbeck, R.,
Roulston, A.,
Shi, T.,
and Williams, L. T.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
13642-13647 |
23. |
Creasy, C. L.,
Ambrose, D. M.,
and Chernoff, J.
(1996)
J. Biol. Chem.
271,
21049-21053 |
24. | Pombo, C. M., Kehrl, J. H., Sanchez, I., Katz, P., Avruch, J., Zon, L. I., Woodgett, J. R., Force, T., and Kyriakis, J. M. (1995) Nature 377, 750-754[CrossRef][Medline] [Order article via Infotrieve] |
25. | Pombo, C. M., Bonventre, J. V., Molnar, A., Kyriakis, J., and Force, T. (1996) EMBO J. 15, 4537-4546[Abstract] |
26. |
Pombo, C. M.,
Tsujita, T.,
Kyriakis, J. M.,
Bonventre, J. V.,
and Force, T.
(1997)
J. Biol. Chem.
272,
29372-29379 |
27. | Lee, K. K., Murakawa, M., Nishida, E., Tsubuki, S., Kawashima, S., Sakamaki, K., and Yonehara, S. (1998) Oncogene 16, 3029-3037[CrossRef][Medline] [Order article via Infotrieve] |
28. |
Lee, K. K.,
Murakawa, M.,
Takahashi, S.,
Tsubuki, S.,
Kawashima, S.,
Sakamaki, K.,
and Yonehara, S.
(1998)
J. Biol. Chem.
273,
19160-19166 |
29. | Sakamaki, K., Tsukumo, S., and Yonehara, S. (1998) Eur. J. Biochem. 253, 399-405[Abstract] |
30. | Yonehara, S., Ishii, A., and Yonehara, M. (1989) J. Exp. Med. 169, 1747-1756[Abstract] |
31. | Kudo, N., Wolff, B., Sekimoto, T., Schreiner, E. P., Yoneda, Y., Yanagida, M., Horinouchi, S., and Yoshida, M. (1998) Exp. Cell Res. 242, 540-547[CrossRef][Medline] [Order article via Infotrieve] |
32. |
Kudo, N.,
Matsumori, N.,
Taoka, H.,
Fujiwara, D.,
Schreiner, E. P.,
Wolff, B.,
Yoshida, M.,
and Horinouchi, S.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
9112-9117 |
33. | Fukuda, M., Asano, S., Nakamura, T., Adachi, M., Yoshida, M., Yanagida, M., and Nishida, E. (1997) Nature 390, 308-311[CrossRef][Medline] [Order article via Infotrieve] |
34. | Fornerod, M., Ohno, M., Yoshida, M., and Mattaj, I. W. (1997) Cell 90, 1051-1060[Medline] [Order article via Infotrieve] |
35. |
Ossareh-Nazari, B.,
Bachelerie, F.,
and Dargemont, C.
(1997)
Science
278,
141-144 |
36. |
Cryns, V.,
and Yuan, J.
(1998)
Genes Dev.
12,
1551-1570 |
37. |
Reszka, A. A.,
Halasy-Nagy, J. M.,
Masarachia, P. J.,
and Rodan, G. A.
(1999)
J. Biol. Chem.
274,
34967-34973 |
38. |
Watabe, M.,
Kakeya, H.,
Onose, R.,
and Osada, H.
(2000)
J. Biol. Chem.
275,
8766-8771 |
39. |
Ajiro, K.
(2000)
J. Biol. Chem.
275,
439-443 |
40. | Sabourin, L. A., and Rudnicki, M. A. (1999) Oncogene 18, 7566-7575[CrossRef][Medline] [Order article via Infotrieve] |
41. |
Sabourin, L. A.,
Seale, P.,
Wagner, J.,
and Rudnicki, M. A.
(2000)
Mol. Cell. Biol.
20,
684-696 |
42. | Yamada, E., Tsujikawa, K., Itoh, S., Kameda, Y., Kohama, Y., and Yamamoto, H. (2000) Biochim. Biophys. Acta 1495, 250-262[CrossRef][Medline] [Order article via Infotrieve] |
43. |
Graves, J. D.,
Gotoh, Y.,
Draves, K. E.,
Ambrose, D.,
Han, D. K.,
Wright, M.,
Chernoff, J.,
Clark, E. A.,
and Krebs, E. G.
(1998)
EMBO J.
17,
2224-2234 |
44. |
Engel, K.,
Kotlyarov, A.,
and Gaestel, M.
(1998)
EMBO J.
17,
3363-3371 |
45. | Mahanty, S. K., Wang, Y., Farley, F. W., and Elion, E. A. (1999) Cell 98, 501-512[Medline] [Order article via Infotrieve] |
46. |
Adachi, M.,
Fukuda, M.,
and Nishida, E.
(2000)
J. Cell Biol.
148,
849-856 |