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INTRODUCTION |
Apoptosis, or programmed cell death, is an active process
fundamental to the development and homeostasis of multicellular organisms (for review, see Ref. 1). For example, apoptosis of
lymphocytes is essential for both eliminating self-reactive cells
during development and regulating clonal populations during and after
an immune response (for review, see Ref. 2). The decision made by a
cell to undergo apoptosis is influenced by many factors. Signals from
death receptors such as CD95/Fas and chemical stressors such as
staurosporine induce apoptosis, whereas growth factor and cytokine
receptors, such as the cytokine interleukin-2 (IL-2)1 promote cell survival
(for review, see Refs. 3 and 4).
The critical effectors of the apoptotic response are a family of
aspartate-specific cysteine proteases, called caspases (for review, see
Ref. 5). Caspases exist as inactive zymogens and are activated in a
proteolytic cascade. Recently, it has become clear that caspases target
critical components of signaling pathways which normally regulate cell
growth and development (for review, see Ref. 6). It is these
substrates, and not caspases themselves, that are the true apoptotic
effectors. Thus, identifying caspase targets and determining the effect
of caspase-mediated cleavage on their activity will be critical in
understanding the biochemical mechanisms of apoptosis. Among the
caspase substrates that have been identified are several protein
kinases, suggesting that phosphorylation/dephosphorylation mechanisms
may play an important role in the induction or progression of
apoptosis. However, very little is known about protein kinases as
potential apoptotic effectors. Even though some protein kinases have
been shown to be capable of inducing apoptosis upon overexpression, the
precise mechanism by which these kinases induce apoptosis is unclear
(for review, see Refs. 7 and 8).
Mst1 (mammalian sterile 20-like kinase 1) is a ubiquitously expressed
serine/threonine kinase that belongs to a growing family of protein
kinases that are homologous to yeast Ste20 and which regulate diverse
cellular processes such as morphogenesis, stress responses, and
proliferation (9-11; for review, see Ref. 12). One subfamily of
Ste20-like kinases comprises the mammalian p21-activated protein
kinases (PAKs), which directly interact with and are regulated by the
small GTPases Rac and Cdc42. The other subgroup, which includes Mst1,
Mst2, Khs, Gck, Sok1, Nik, Hpk1, and Sps1, lack a domain for
interacting with GTPases (for review, see Ref. 13). The carboxyl
terminus of Mst1 mediates dimerization and appears to serve an
inhibitory function (14). Despite considerable effort on the part of
several laboratories, the only stimuli that have been shown to regulate
Mst1 are cellular stresses and apoptotic stimuli.
Mst1 is cleaved and activated during apoptosis induced by stimuli that
include ligation of CD95/Fas and treatment with staurosporine or
genotoxic agents (15-17). We have also shown that Mst1 can induce caspase activity and apoptosis upon overexpression. Mst1 activity, although not required for apoptosis induced by ligation of CD95/Fas, is
required for apoptosis in response to certain genotoxic agents (15).
Collectively, these findings suggest that Mst1 might be an important
caspase target that functions to amplify the apoptotic response. Other
members of the Ste20 family which have been shown to be caspase targets
include PAK-2 (18, 19), Hpk (20), and Slk (21). In this study, we
investigated the relative contributions of proteolysis and
phosphorylation to Mst1 activation during apoptosis. We conclude that
Mst1 contains two cleavage sites that may be targeted by different
caspases, that Mst1 requires prior phosphorylation for full activity
after proteolysis, and that the state of Mst1 phosphorylation
influences its sensitivity to caspase-mediated cleavage. In addition,
Mst1 apparently functions upstream of MEKK1 in the SAPK pathway. These
findings provide an example of the close relationship between
proteolysis and phosphorylation in the regulation of apoptosis.
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EXPERIMENTAL PROCEDURES |
Reagents and Cells--
The anti-Fas monoclonal antibody
IPO-4 (22) was kindly provided by Dr. Svetlana Sidorenko. Staurosporine
and histone H1 were obtained from Sigma. Myelin basic protein
(MBP) was prepared from bovine brain as described previously (23).
ZVAD-fmk was purchased from the Kamiya Biomedical Company (Tukwila,
WA). Rabbit polyclonal antibodies specific for the amino terminus of
Mst1 have been described previously (15). The 9E10 monoclonal antibody specific for the Myc epitope tag was obtained from the American Type
Culture Collection (Rockville, MD). The anti-hemagglutinin (HA)
monoclonal antibody 12CA5 was obtained from Roche Molecular Biochemicals. The BJAB human B lymphoma line was generously provided by
Dr. Vishva Dixit (Genentech, South San Francisco, CA). The 293T
cell line was kindly provided by by Dr. Jonathan Cooper (FHCRC, Seattle, WA).
Plasmids--
Myc-tagged Mst1 constructs in the pCMV5M
expression vector were constructed as described (9, 14, 15). Mst1
D326N,
326, D349E, D326N/D349E, S327E, and S327G were constructed by
polymerase chain reaction mutagenesis. pSR
456-HA-SAPK
, Ask1, and
Tak1 were constructed as described previously (24).
MBP In-gel Kinase Assays--
After incubation with the
indicated stimuli, 5-10 × 106 cells were lysed for
15 min on ice in 1 ml of lysis buffer (20 mM Hepes pH 7.4, 2 mM EGTA, 50 mM
-glycerophosphate, 1%
Triton X-100, 10% glycerol, 1 mM dithiothreitol (DTT), 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 1 mM Na2VO4). Cell debris was removed by microcentrifugation at 14,000 × g for 10 min at 4 °C prior to the addition of Laemmli
sample buffer. 10-20 µg of cell extract was used for both Western
blotting and MBP in-gel kinase assays. Cell extract was loaded onto a
10% SDS-polyacrylamide gel that had been polymerized in the presence
of 0.2 mg/ml bovine brain MBP. After running, the gel was washed twice
at room temperature for 30 min with 100 ml of buffer A (50 mM Hepes pH 7.6, 5 mM 2-mercaptoethanol) containing 20% isopropyl alcohol. The gel was then washed twice at room temperature with buffer A and twice with buffer A containing 6 M urea. Renaturation was achieved by sequentially washing
the gel three times for 15 min at 4 °C with buffer A containing 3 M urea, 1.5 M urea, 0.75 M urea,
and buffer A alone. After an overnight wash in buffer A with 0.05%
Tween 20 (v/v), the gel was washed twice and equilibrated at 30 °C
for 30 min in kinase buffer (20 mM Hepes pH 7.6, 20 mM MgCl2, 2 mM DTT) prior to the addition of 20 µM MgATP and 100 µCi of
[32P]ATP and incubation for at 30 °C for 30 min. The
reaction was stopped, and unincorporated [32P]ATP was
removed by washing 10 times for 30 min at room temperature with 100 ml
of 5% trichloroacetic acid (w/v) and 1% NaPPi
(w/v). After staining and destaining the gel was dried prior to autoradiography.
Apoptosis Assays--
Apoptotic cells were quantified using
annexin V flow cytometry as described previously (15). Briefly, cells
were incubated with fluorescein isothiocyanate-conjugated annexin V to
allow necrotic cells to be excluded (CLONTECH, Palo
Alto, CA). The cells were subsequently analyzed using a Becton
Dickinson FACStar Plus flow cytometer.
In Vitro Protease Assays--
Mst1 and various Mst1 mutants were
translated in vitro using a coupled transcription and
translation system with T7 polymerase (Promega, Madison, WI). Apoptotic
cell extract was prepared as follows. After stimulation, cells were
washed once in phosphate-buffered saline and resuspended at 2 × 108/ml in hypotonic lysis buffer (50 mM NaCl,
40 mM
-glycerophosphate, 10 mM Hepes pH 7.0, 5 mM EGTA, 2 mM MgCl2). The lysate
was then subjected to four freeze-thaw cycles prior to centrifugation
at 10,000 × g for 10 min. Recombinant caspases
were purchased from R&D systems (Minneapolis, MN). 10 µg of the
indicated caspase, in 5 µl of hypotonic lysis buffer, was incubated
for 1 h at 37 °C with 5 µl of 35S-labeled
in vitro translated Mst1, Mst1 D326N, Mst1 D349E, or Mst1
D326N/D349E in caspase assay buffer (100 mM Hepes pH 7.5, 10% sucrose, 0.1% CHAPS, 10 mM DTT, 0.1 mg/ml ovalbumin).
The reaction was stopped by the addition of 4 × Laemmli sample
buffer and subjected to SDS-polyacrylamide gel electrophoresis prior to
drying and autoradiography.
Transfections--
BJAB cells were transfected by
electroporation in a Bio-Rad Gene Pulser II. Briefly cells were washed
twice and resuspended at 2.8 × 107 cells/ml in
electroporation buffer (RPMI 1640 with the addition of 1 mM
sodium pyruvate, 2 mM L-glutamine, 1 x
nonessential amino acids, 15% fetal calf serum v/v). 10 µg of pCMV
vector containing the appropriate Myc-tagged Mst1 construct was added
to 350 µl of cells (1 × 107) in a 0.4-cm cuvette
and incubated on ice for 10 min. The cells were then resuspended prior
to electroporation (230 volts, 960 microfarads). After electroporation
the cells were incubated on ice for a further 10 min before being
resuspended in 20 ml of electroporation buffer and allowed to recover
for 3 h at 37 °C in a humidified incubator. After the removal
of cell debris by Ficoll density gradient centrifugation, the cells
were resuspended at 5 × 105/ml in RPMI and 10% fetal
calf serum and returned to the incubator. At the indicated time cells
were harvested for the preparation of cell extract for Western
blotting, as described above, or were prepared for immunohistochemical analysis.
Cotransfection and Kinase Assays--
293T cells were
transfected in 60-mm dishes with 4 µg of the appropriate plasmids
using 24 µl of LipofectAMINE (Life Technologies, Inc.) for 10 h
according to the manufacturer's instructions. 24 h after
transfection, cells were harvested in lysis buffer (20 mM
Tris-Cl, pH 7.5, 10 mM
-glycerophosphate, 5 mM EGTA, 10 mM NaF, 1 mM
NaPPi, 150 mM NaCl, 1% Nonidet P-40, 4 mM DTT, 1 mM sodium vanadate, 1 mM
phenylmethylsulfonyl fluoride, and 20 mg/ml aprotinin). After being
clarified by centrifugation at 15,000 × g for 30 min,
the supernatants were incubated with anti-HA or anti-Myc antibody and
protein G-Sepharose for 3 h. The beads were washed four times with
lysis buffer and once with wash buffer (20 mM Tris, pH 7.5, 2 mM EGTA, 1 mM DTT). Kinase assays were carried out by incubating immunoprecipitates with the appropriate substrate in kinase buffer (20 mM Tris, pH 7.5, 10 mM MgCl2, and 100 µM
[
-32P]ATP, 0.1 µCi) for 10 min at 30 °C. After 20 min the reactions were stopped by the addition of 4 × Laemmli
sample buffer and subjected to SDS-polyacrylamide gel electrophoresis
prior to drying and autoradiography.
Data Presentation--
All data shown are representative of at
least three separate experiments.
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RESULTS |
Mst1 Has Two Caspase Sites--
We and others have shown that
Mst1, which normally migrates at 61 kDa, is cleaved in response to a
variety of apoptotic stimuli to generate a 36-kDa catalytically active
fragment (15-17 and Fig. 1, left
panels). However, in response to withdrawal of IL-2 from
factor-dependent CTLL-2 cells, two Mst1 cleavage products were observed (Fig. 1, right panels). A 41-kDa band was
detected after 3 h of withdrawal, followed by the previously
characterized 36-kDa band by 6 h. A small amount of the 41-kDa
Mst1 species was visible at later time points in cells treated with
staurosporine (Fig. 1, center panels). The appearance of the
36-kDa Mst1 cleavage product correlated with induction of apoptosis
upon withdrawal of IL-2, but the 41-kDa band was first detected before
significant apoptosis was observed. Although the majority of kinase
activity was attributable to the 36-kDa form of Mst1, MBP in-gel kinase assays indicate that the 41-kDa form may be catalytically active (Fig.
1, lower panels). These findings suggest that Mst contains two caspase cleavage sites that generate 36-kDa or 41-kDa cleavage products.

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Fig. 1.
BJAB cells were treated with either 1 µg/ml
anti-CD95/Fas (left panels) or 100 nM
staurosporine (middle panels). CTLL-2 cells were
washed and resuspended in medium without IL-2 (right
panels). At the indicated times cell extract was prepared, and
Western blotting with Mst1 antiserum (upper panels) or MBP
in-gel kinase assays (lower panels) was performed as
described under "Experimental Procedures." The percentage of cells
undergoing apoptosis was determined at each time point by annexin V
binding assay.
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Io test whether two caspase sites exist in Mst1, we transfected BJAB B
cells with either wild-type Mst1 or mutants in which the site
responsible for generation of the 36-kDa species had been mutated to
render it resistant to caspase-mediated cleavage (Mst1 D326N, Fig.
2A). Fig. 2B shows
that after transfection of wild-type Mst1, both full-length Mst1 and a
band corresponding to the 36-kDa species were observed. Treating the
cells with anti-Fas increased cleavage of Mst1. In contrast,
transfection of Mst1 D326N yielded a band corresponding to the 41-kDa
species. This suggests that although the Mst1 D326N mutant was
resistant to cleavage at the DEMD326S site, it was still cleaved at a
second caspase site. Based on molecular mass we predicted that a
likely cleavage site for generation of the 41-kDa band was aspartate 349 within the potential caspase consensus sequence of
TMTD349G (Fig. 2A). To test this hypothesis we
generated mutants of Mst1 in which this site was mutated alone (Mst1
D349E) or in combination with the first site (Mst1 D326N/D349E). As
predicted, the Mst1 D349E mutant behaved like wild-type Mst1, yielding
a 36-kDa cleavage product that was stimulated by anti-Fas (Fig.
2B). However, the Mst1 D326N/D349E double mutant was
completely resistant to caspase-mediated cleavage in either the absence
or presence of anti-Fas. These results confirm that Mst1 contains two
caspase cleavage sites at DEMD326S and TMTD349G
(Fig. 2A). The fact that Mst1 wild-type, D326N, and D349E
are equally sensitive to cleavage argues against a model in which proteolysis at one site is required before cleavage at the other.

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Fig. 2.
A, structural organization of Mst1
indicating the positions of the two caspase cleavage sites, the
autophosphorylation site, and the various mutants of Mst1 employed in
these studies. B, BJAB cells were transiently transfected
with 10 µg of Myc-tagged Mst1, Mst1 D326N, Mst1 D349E, or Mst1
D326N/D349E. 12 h after transfection, the cells were either
untreated or treated with 1 µg/ml anti-CD95/Fas for a further 10 h. Cell extract was prepared and exogenous Mst1 visualized by Western
blotting with anti-Myc. wt., wild-type.
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Differential Caspase Sensitivity of Mst1 Cleavage Sites--
The
fact that Mst1 contains two caspase cleavage sites raised the
possibility that each site is cleaved by different caspases. To test
this possibility we examined the ability of recombinant purified
caspase 3, 6, 7, or 9 to cleave in vitro translated, [35S]methionine-labeled Mst1 at either site in
vitro (Fig. 3). To control for
differences in specific activity among the different caspase
preparations, we carried out preliminary dose-response experiments to
establish conditions under which each caspase had similar activity with
respect to wild-type Mst1 (data not shown). All four caspases cleaved
wild-type Mst1 and Mst1 D349E to generate a 36-kDa band. However, under
these conditions, only caspase 6 and 7 could cleave the Mst1 D326N
mutant. Consistent with our expression studies, Mst1 D326N/D349E was
completely resistant to cleavage in vitro. These data
suggest that caspases differ in their preferences for cleavage at the
two sites in Mst1 and support the idea that these sites might be
targeted by different caspases in vivo.

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Fig. 3.
Mst1, Mst1 D326N, Mst1 D349E, or Mst1
D326N/D349E was transcribed and translated in vitro in
the presence of [35S]methionine. Labeled Mst1 was
then incubated for 60 min with recombinant, purified caspase 3, 6, 7, 9 or buffer alone. The reactions were stopped by the addition of SDS
sample buffer, and Mst1 was visualized by radiography after
SDS-polyacrylamide gel electrophoresis. wt.,
wild-type.
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Role of Phosphorylation in Regulating Mst1 Activity during
Apoptosis--
We next wished to ascertain the relative contributions
of caspase cleavage and phosphorylation/dephosphorylation to the
activation of Mst1 during apoptosis. To determine whether cleavage was
sufficient to activate Mst1, we immunoprecipitated endogenous Mst1 from
unstimulated BJAB cells, incubated the immunoprecipitate with
recombinant caspase 3 in vitro, and performed an immune
complex kinase assay with histone H1 as substrate. No significant
increase in kinase activity was detected after incubation with caspase
3 despite significant cleavage of Mst1 (Fig.
4A). Mst1 was then
immunoprecipitated from BJAB cells that had been treated with
staurosporine for 30 min. Under these conditions, in-gel kinase assays
and Western blotting indicated that Mst1 was activated but not cleaved.
Although Mst1 immunoprecipitated from staurosporine-treated cells was
activated by 4-fold compared with untreated cells, in vitro
cleavage with caspase 3 further stimulated Mst1 activity by 2-3-fold.
These results suggest that to be fully stimulated by proteolysis, Mst1 must first be activated by a mechanism distinct from proteolysis.

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Fig. 4.
A, Mst1 was immunoprecipitated
(IP) from BJAB cells that were either untreated or treated
with 1 µM staurosporine for 15 min. The immunoprecipitate
was incubated for 60 min with either recombinant purified caspase 3 or
control buffer. Mst1 kinase activity was then determined by immune
complex kinase assay using histone H1 as substrate (upper
panel). Mst1 cleavage was confirmed by Western blotting with Mst1
antiserum (lower panel). B, Mst1 was
immunoprecipitated from BJAB cells that were untreated, treated with 1 µg/ml anti-Fas for 8 h, or treated with 1 µM
staurosporine for either 30 min or 8 h. The immunoprecipitate was
incubated for 60 min with either recombinant purified protein
phosphatase 2A (PP2A) or control buffer. Mst1 kinase
activity was then determined by immune complex kinase assay using
histone H1 as substrate.
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A mechanism consistent with these results is that Mst1 must be
phosphorylated prior to cleavage. To determine whether phosphorylation was required for activation, Mst1 was immunoprecipitated from cells
treated with anti-Fas or staurosporine. The immune complexes were then
incubated with purified recombinant protein phosphatase 2A prior
to performing an immune complex kinase assay. To prevent protein
phosphatase 2A from directly dephosphorylating histone H1, okadaic acid
was included in the kinase assay buffer. Incubation with protein
phosphatase 2A almost completely abolished the increase in kinase
activity observed in Mst1 immunoprecipitates from cells treated with
either anti-Fas or staurosporine for long or short time points (Fig.
4B). In contrast, protein phosphatase 1 had no effect on
Mst1 activity, although it did inactivate SAPK in immune complex kinase
assays (data not shown). These findings indicate that phosphorylation
is absolutely required for Mst1 activity regardless of its state of proteolysis.
To explore further the relationship among cleavage, phosphorylation,
and activation of Mst1 during apoptosis, we transiently transfected
BJAB cells with various Mst1 mutants and measured their kinase activity
in vitro in the presence or absence of anti-Fas. Comparable
expression levels of wild-type Mst1, truncated Mst1 D326, Mst1 D326N,
Mst1 D349E, and protease-resistant Mst1 D326N/349E were evident by
Western blotting (Fig. 5A,
upper panel). Treatment with anti-Fas increased the kinase
activity of wild-type Mst1 by about 4-fold (Fig. 5A,
lower panel). In contrast, Mst1 D326 exhibited a high basal
activity that was not enhanced significantly by Fas ligation. However,
the activity of the protease-resistant Mst1 D326N and D349E mutants was
stimulated by 3-fold after treatment with anti-Fas. In contrast, the
kinase activity of the double protease-resistant form of Mst1
(D326N/349E) was only weakly activated upon anti-Fas treatment (Fig.
5). These results indicate that cleavage is required for the full
activation of Mst1 during apoptosis.

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Fig. 5.
BJAB cells were transiently transfected with
10 µg of Myc-tagged Mst1, Mst1 D326, Mst1
D326N, Mst1 D349E, or Mst1 D326N/D349E. 12 h after
transfection, the cells were either untreated or treated with anti-Fas
for a further 10 h. A, cell extract was prepared and
exogenous Mst1 visualized by Western blotting with anti-Myc
(upper panel). wt., wild-type. Mst1 was also
immunoprecipitated (IP) with anti-Myc and subjected to
immune complex kinase activity with histone H1 as substrate
(lower panel). B, Mst1 autophosphorylation upon
immune complex kinase assay.
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Autophosphorylation of Mst1--
During these experiments we
observed autophosphorylation of Mst1 (Fig. 5B).
Interestingly, whereas full-length forms of Mst1 and the 41-kDa species
strongly autophosphorylated, the 36-kDa form of Mst1 autophosphorylated
very weakly. The 36-kDa species did not autophosphorylate after
phosphatase treatment, suggesting that the differential ability of the
36-kDa and 41-kDa species could not be explained by prior
phosphorylation of the 36-kDa form (data not shown). It is unclear
whether Mst1 autophosphorylates on residues that regulate its kinase
activity or on residues that influence some other aspect of its
function. Collectively, this evidence points to the possibility that
Mst1 autophosphorylates on serine residues located outside the
activation loop and possibly in the regulatory domain between the two
caspase cleavage sites (amino acids 326-349).
To determine whether autophosphorylation of Mst1 correlated with
increased in vitro kinase activity, we allowed Mst1 in
immune complexes to autophosphorylate for various periods of time and then measured kinase activity against exogenous histone H1. Under the
conditions employed, Mst1 autophosphorylated to saturation by around
1 h (Fig. 6A, upper
panel). The effects of alternative divalent cations, such as
manganese, on Mst1 autophosphorylation have not been tested. Regardless
of the extent of autophosphorylation, Mst1 activity against histone H1
remained constant (Fig. 6A, lower panel). The
autoradiogram has been overexposed to emphasize the basal activity of
Mst1.

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Fig. 6.
A, Mst1 was immunoprecipitated
(IP) from BJAB cells and incubated in the presence or
absence of MgATP for the indicated times. Mst1 kinase activity
was then determined by immune complex kinase assay using histone H1 as
substrate. wt., wild-type. B, BJAB cells were
transiently transfected with 10 µg of Myc-tagged Mst1, Mst1 D327G, or
Mst1 D327E. 12 h after transfection, Mst1 was immunoprecipitated
with anti-Myc and incubated in the presence or absence of MgATP
for 60 min. The immunoprecipitates were then incubated for 60 min with
recombinant purified caspase 3. Mst1 autophosphorylation was then
determined by immune complex kinase assay using histone H1 as substrate
(upper panel). Mst1 cleavage was confirmed by Western
blotting with Mst1 antiserum (lower panel).
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One possible autophosphorylation site is serine 327. Because this
residue is adjacent to the DEMD326S cleavage site, its
state of phosphorylation might influence Mst1 cleavage. To test this
possibility we constructed mutants of Mst1 in which serine 327 was
mutated to either a glycine (Mst1 S327G) or a glutamic acid (Mst1
S327E). Relative to wild-type Mst1, these mutants displayed a reduced
ability to autophosphorylate in vitro (Fig. 6B,
upper panel). Wild-type Mst1 that had been allowed to
autophosphorylate was less sensitive to cleavage in vitro by
caspase 3 than Mst1 that was not autophosphorylated (Fig. 6B). In contrast, Mst1 S327G retained its sensitivity to
cleavage, whereas Mst1 S327E was constitutively resistant to cleavage.
These findings suggest that serine 327 is a major in vitro
Mst1 autophosphorylation site and that its state of phosphorylation may
influence the sensitivity of Mst1 to cleavage at the
DEMD326S site. Interestingly, the recently identified Ste20
homolog SPAK, which is a close relative of Mst1, contains a putative
caspase cleavage site in which the amino acid following the critical
aspartate is a glutamic acid (25). Whether SPAK is a less efficient
caspase target as a consequence of there being a glutamic acid residue at this position remains to be determined.
MEKK1 Is a Downstream Target of Mst1--
Another important
question concerns the downstream targets of Mst1. Because other
mammalian Ste20 homologs have been shown to function as MAPKKKKs in
MAPK cascades, we tested the ability of Mst1 to activate components of
the SAPK pathway in a transient coexpression system. We have shown
previously that coexpression of wild-type Mst1 activated SAPK and the
MAPKK MKK7 (15). To localize more closely the position in the
SAPK pathway at which Mst1 functions, we cotransfected Mst1 with SAPK
and kinase-dead mutants of several MAPKKKs known to function in this
pathway. Interestingly, kinase-dead MEKK1, but not kinase-dead Ask1 or Tak1, blocked the ability of Mst1 to activate SAPK (Fig.
7). These results are consistent with
Mst1 functioning as a MAPKKKK, upstream of MEKK1, in the SAPK
pathway.

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Fig. 7.
293T cells were transfected with HA-tagged
SAPK, Mst1, and kinase dead mutants of MEKK1, TAK1, or ASK1. SAPK
activity was determined by immune complex kinase assay using
glutathione S-transferase (GST)-Jun as substrate.
IP, immunoprecipitation; wt., wild-type.
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DISCUSSION |
Previous studies have identified a single caspase cleavage site in
Mst1 (DEMD326S) which yielded a 36-kDa catalytically active
fragment (15-17). More recently, several lines of evidence led us to
conclude that a second site closer to the carboxyl terminus of Mst1
also exists. For example, withdrawal of IL-2 from CTLL-2 cells, which
are dependent on this growth factor for their survival, results in the
appearance of a 41-kDa species that preceded that of the 36-kDa
cleavage product by about 2-3 h (Fig. 1). A 41-kDa band was also
detected at later time points in staurosporine but not anti-Fas-treated BJAB cells. Consistent with a second cleavage site in Mst1 is the
observation that a mutant of Mst1 resistant to cleavage at the primary
cleavage site (Mst1 D326N) is proteolyzed to generate a band that
corresponds to the 41-kDa Mst1 species in response to anti-Fas
treatment. Further mutational analyses identified the second cleavage
site as being 23 amino acids carboxyl-terminal of the
DEMD326S site at a TMTD349G sequence (see Fig.
2A). Specifically, a double Mst1 D326N/D349E mutant was
found to be completely resistant to cleavage upon expression in and
anti-Fas treatment of BJAB cells.
To reconcile these findings with previous results as well as to analyze
the caspase specificity of the two sites, we subjected various Mst1
mutants to cleavage by caspase 3, 6, 7, or 9 in vitro. Caspases 3 and 9 efficiently cleaved Mst1 at the DEMD326S
site but were relatively inefficient at cleaving Mst1 at
TMTD349G (Fig. 3). In contrast, caspases 6 and 7 cleaved
Mst1 at either site. The TMTD349G site is closer in
sequence to the caspase 6 consensus sequence than the caspase 3 consensus predicted by combinatorial peptide analysis (26). These
findings raise the possibility that the two sites in Mst1 may be
targeted by different caspases. In addition, the resistance of the
TMTD349G site to proteolysis by caspase 3 provides an
explanation for our original identification of a single cleavage site.
These results raise the issue of why, if Mst1 contains two caspase
sites, is only one cleavage product observed in response to
anti-CD95/Fas? There are several possible explanations for this.
Because the two cleavage sites may be targeted by different caspases,
anti-CD95/Fas may lead to activation of caspases that preferentially
generate the 36-kDa species while stimuli such as IL-2 withdrawal may
induce a different pattern of caspase specificities. A variation on
this hypothesis is that there may be a kinetic difference between
cleavage at the two sites such that the 41-kDa species is a transient
intermediate. Although the time course of appearance of the 41-kDa band
relative to the 36-kDa band in response to withdrawal of IL-2 is
consistent with the 41-kDa form being an intermediate cleavage product,
we have no definitive evidence that ordered proteolysis occurs at these
two sites in response to other stimuli such as anti-CD95/Fas. In
addition, the fact that this site is not conserved in Mst2 raises the
possibility that Mst1 and Mst2 are targeted differentially during apoptosis.
The carboxyl terminus of Mst1 has been shown to exert a negative
regulatory influence upon the kinase domain (14). Although removal of
this domain by caspase-mediated cleavage correlates with increased
activation of Mst1, we wanted to understand the relative contributions
that caspase-mediated proteolysis and phosphorylation/dephosphorylation might have to the activation of Mst1 during apoptosis. This objective was complicated by the fact that no physiological stimulus regulating Mst1 in the absence of caspase cleavage has been identified. However, because Mst1 is activated but not proteolyzed upon short term treatment
with staurosporine, we used staurosporine as a pharmacological activator of Mst1. Cleavage with recombinant caspase 3 in
vitro enhanced the activity of Mst1 from staurosporine-treated
cells but not untreated cells (Fig. 4). These findings suggest that in
addition to caspase-mediated cleavage, Mst1 may also require phosphorylation on critical regulatory residues to be fully activated. Consistent with this hypothesis, Mst1 activated by either staurosporine or anti-Fas treatment can be inactivated by treatment with protein phosphatase 2A. Our transfection studies with various Mst1 mutants provide further support for this model. The kinase activities of
wild-type Mst1, Mst1 D326N, and Mst1 D349E were all stimulated by
anti-Fas. However, the cleavage-resistant mutant (Mst1 D349E) was only
slightly stimulated. Thus, both phosphorylation and caspase cleavage
contribute to the activation of Mst1 during apoptosis. A similar
requirement for both phosphorylation and proteolysis has been shown for
-PAK/PAK-2 (19).
It is important to note that in addition to contributing to the
activation of Mst1, removal of the regulatory domain is likely to
influence other biochemical characteristics of the kinase. For example,
the carboxyl terminus of Mst1 contains a dimerization domain and a
putative nuclear exclusion sequence. Thus, protein-protein interactions, subcellular localization, and substrate specificity of
Mst1 are also likely to be altered upon cleavage by caspases. The
contribution of these factors to the ability of Mst1 to induce apoptosis is currently being investigated.
Mst1 autophosphorylates in vitro on a site within the
regulatory domain, located between the two caspase sites. This region of Mst1 contains several potential phosphorylation sites, including a
serine residue (serine 327) within the DEMD326S caspase
consensus site. Our analyses with S327E and S327G mutants of Mst1
indicate that serine 327 is a major in vitro
autophosphorylation site and that phosphorylation at this residue
renders Mst1 resistant to cleavage by caspase 3. A precedent for
phosphorylation of a caspase target regulating its cleavage is
provided by the inhibitor of nuclear factor-kB (I-
B
).
After phosphorylation at sites close to its caspase cleavage
site, I-
B
is resistant to caspase cleavage in vitro
(27). Although the physiological role of Mst1 phosphorylation at serine
327 is unclear, one possibility is that this might favor generation of
41-kDa Mst1 species rather than 36-kDa form. This may provide an
alternative explanation for why the 36 kDa form is observed in response
to IL-2 withdrawal but not anti-CD95/Fas treatment. We are currently
investigating whether Mst1 is phosphorylated on serine 327 in cells and
whether autophosphorylation is mediated in trans within Mst dimers.
Another important question concerns the identity of the downstream
targets of Mst1 under apoptotic and/or non-apoptotic circumstances. The ability of Mst1 to activate the SAPK and p38 MAPK pathways upon
coexpression suggests that components of these MAPK pathways might be
important effectors of Mst1. In this respect, our cotransfection studies indicate that Mst1 may function upstream of MEKK1 to activate SAPK. Interestingly, Cardone et al. (28) showed that caspase cleavage of MEKK1 from non-apoptotic cells did not activate MEKK1 to
the same extent as MEKK1 from cells that were undergoing apoptosis. These data are consistent with a requirement for activation of MEKK1 by
phosphorylation in addition to caspase-mediated cleavage. Taken
together with our previous findings, Mst1 may function as part of an
apoptotic Mst1/MEKK1/MAPKK7/SAPK signaling cassette. According to this
model, Mst1 might directly phosphorylate MEKK1 and thereby contribute
to its activation by caspase-mediated cleavage. Thus, rather than
rendering these protein kinases independent of their normal upstream
activators, caspases may exploit the amplification inherent within this
protein kinase cascade to transduce their apoptotic signal.
Although Mst1 is capable of inducing caspase activity and apoptosis
upon overexpression, its normal cellular role remains unclear. In this
respect, a recent study has reported a 33-kDa MBP-in gel kinase
activity, referred to as p33 quiescence-activated kinase
(p33QIK), which is active during
G0/G1 and inactivated as cells enter the cell
cycle (29). This kinase is recognized by anti-Mst antibodies and may be
either a novel proteolytic product of Mst1/2, an alternative splice
product of the Mst1/2 genes, or a separate gene product. The idea that
Mst1 is active in quiescent cells but inactivated during the
proliferative response is consistent with the fact that epidermal
growth factor treatment of NIH3T3 cells inhibits Mst activity (9). A
cytostatic role has also been suggested for the Ste20 homologies
-PAK/PAK-2, and Hpk1, which are also caspase targets (19, 30). One
possibility is that Mst1 transduces a quantitatively or qualitatively
different signal depending on its mechanism of activation. According to
this model Mst1 activated by phosphorylation/dephosphorylation in
response to non-apoptotic stimuli would regulate a specific cellular
process. However, in response to apoptotic stimuli involving both
cleavage and phosphorylation, Mst1 would contribute to the induction or
progression of apoptosis.