From the Department of Biochemistry, Hong Kong University of Science & Technology, Clear Water Bay, Kowloon, Hong Kong
Received for publication, October 30, 2002, and in revised form, January 9, 2003
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Mammalian STE20-like kinase 2 (MST2), a member of
the STE20-like kinase family, has been shown in previous studies
to undergo proteolytic activation by caspase-3 during cell apoptosis. A
few studies have also implicated protein phosphorylation reactions in
MST2 regulation. In this study, we examined the mechanism of MST2
regulation with an emphasis on the relationship between caspase-3 cleavage and protein phosphorylation. Both the full-length MST2 and the
caspase-3-truncated form of MST2 overexpressed in 293T cells exist in a
phosphorylated state. On the other hand, the endogenous full-length
MST2 from rat thymus or from proliferating cells is mainly
unphosphorylated whereas the caspase-3-truncated endogenous MST2 from
apoptotic cells is highly phosphorylated. Cell transfection
studies using mutant MST2 constructs indicate that MST2 depends on the
autophosphorylation of a unique threonine residue,
Thr180, for kinase activity. The autophosphorylation
reaction shows strong dependence on MST2 concentration suggesting that
it is an intermolecular reaction. While both the full-length MST2 and the caspase-3-truncated form of MST2 undergo autophosphorylation, the
two forms of the phosphorylated MST2 display marked difference in
susceptibility to protein phosphatases. The full-length phospho-MST2 is
rapidly dephosphorylated by protein phosphatase 1 or protein phosphatase 2A whereas the truncated MST2 is remarkably resistant to
the dephosphorylation. Based on the present results, a novel molecular
mechanism for MST2 regulation in apoptotic cells is postulated. In
normal cells, because of the low concentration and the ready reversal
of the autophosphorylation by protein phosphatases, MST2 is present
mainly in the unphosphorylated and inactive state. During cell
apoptosis, MST2 is cleaved by caspase-3 and undergoes irreversible
autophosphorylation, thus resulting in the accumulation of active MST2.
Mammalian STE20-like kinases comprise an extended protein kinase
subfamily whose kinase domains show strong homology to that of STE20
kinase of the budding yeast (1, 2). Among 30 members of the protein
subfamily known to date, mammalian STE20 kinase 1 (MST1)1 and 2 (MST2) share
the most extensive sequence similarity (3-5). Apoptotic agents
including staurosporine, Fas antigen/CD95, okadaic acid, synthetic
apoptotic compound MT-21, anti-cancer drug cytotrienin A and
bisphosphonates, as well as environmental stresses such as heat shock
and high concentrations of sodium arsenite have been shown to activate
MST1 or MST2 in cultured mammalian cells (6-13). The activation of
MST1 or MST2 during apoptosis is generally attributed to the production
of an active fragment of the kinase in a caspase-3-catalyzed reaction
(7-15). Both MST1 and MST2 undergo phosphorylation in cells but the
mechanism of the enzyme regulation by protein phosphorylation is not
entirely clear. A number of studies have suggested that protein
phosphorylation is accompanied with an activation of the kinase. Using
a phosphorylation site-specific antibody, Lee and Yonehara (16) have
shown that MST in okadaic acid-treated cells is phosphorylated at a
site in the kinase activation loop. The observation suggests that the
kinase is activated upon protein phosphorylation. In agreement with
such a suggestion, Graves et al. (17) have shown that
treatment of the extracts of anti-Fas-treated BJAB cells by protein
phosphatase 2A markedly reduced the kinase activity of MST1. On the
other hand, evidence suggesting an inhibitory protein phosphorylation
mechanism for MST1 has also been reported (3).
The present study examines the regulation of the MST2 kinase activity
in detail. Both the native form and the caspase-3-truncated form of
MST2 depend on autophosphorylation of a unique threonine residue,
Thr180, for kinase activity. The autophosphorylation
is an intermolecular reaction, showing strong dependence on the enzyme
concentration. Whereas the phosphorylation of the native MST2 can be
readily reversed by protein phosphatases, the truncated phospho-MST2 is resistant to protein phosphatases. On the basis of these results, a
novel molecular mechanism involving caspase-3 cleavage, MST2 autophosphorylation, and protein phosphatase reaction is postulated to
account for the regulation of MST2 activity in normal and apoptotic cells.
Cloning and Site-directed Mutagenesis--
Expression constructs
of rat MST2 were generated as NH2-terminal Myc-tagged forms
using pCMV-Myc vector obtained from Clontech (BD
Biosciences). Caspase-3-truncated MST2 (TF-MST2) was constructed by
introducing a stop codon after amino acid 322 using PCR amplification. Substitution of Thr180, Thr117, and
Thr384 by alanine as well as Lys56 by arginine,
was performed with a QuikChange site-directed mutagenesis kit
(Stratagene) and confirmed by DNA sequencing.
Cell Culture and Transfection--
HeLa and 293T cells were
maintained in Dulbecco's modified Eagle's medium supplemented with
10% fetal bovine serum and 100 unit/ml penicillin-streptomysin
(Invitrogen) at 37 °C in a humidified atmosphere with 5%
CO2. Transient transfection was carried out using
LipofectAMINE 2000 reagent (Invitrogen). Cells were incubated for
24 h before harvesting. The cells were washed twice with
phosphate-buffered saline and lysed with cold lysis buffer (50 mM Hepes, pH 7.2, 150 mM NaCl, 1 mM
EDTA, 1 mM EGTA, 5 mM NaF, 2 mM
DTT, 0.1% Nonidet P-40, 0.1 µM microcystin-LR (MCT)
(Calbiochem), 20 mM Cross-linking MST2 Antibody to Protein A/G-agarose Beads--
2
ml of MST2 antibodies were dialyzed against the cross-linking buffer
(0.15 M NaCl, 0.1 M
Na2HPO4, pH 7.2) overnight. Then the antibodies
were diluted with the antibody binding/washing buffer (50 mM sodium borate, pH 8.2) in a 1:1 ratio and incubated with
1 ml of pre-equilibrated protein A/G-agarose beads (A/G in 1:1 ratio,
50% slurry, Invitrogen) for 1 h at room temperature. After
extensive washing with the antibody binding/washing buffer, the beads
were incubated with 10 mg of disuccinimidyl suberate (Pierce) for
1 h, which was dissolved in 1 ml of Me2SO then diluted with 1.5 ml of cross-linking buffer. After extensive washing with the
antibody binding/washing buffer, 2 ml of blocking buffer (0.1 M ethanolamine, pH 8.2) was applied to the beads and mixed
for 15 min. The beads were then washed with 5 ml of elution buffer (0.1 M glycine-HCl, pH 2.8) to remove the uncross-linked
antibodies, and further washed with the binding/washing buffer. Finally
the protein A/G beads cross-linking with MST2 antibodies were
equilibrated with lysis buffer for later MST2 immunoprecipitation.
Immunoprecipitation and Western Blotting--
For Myc-tagged
protein immunoprecipitation, the transfected cell lysates (30 µg)
were incubated with anti-Myc (9E10) monoclonal antibody at 4 °C for
2 h or overnight in the presence of protein A/G-Sepharose beads
(A/G in 1:1 ratio). For MST2 immunoprecipitation, thymus extracts or
cell lysates were incubated with MST2 antibody cross-linked to protein
A/G-agarose beads. After extensive washing, the immunoprecipitates
were resolved by SDS-PAGE and transferred to polyvinylidene difluoride
membrane (Bio-Rad). The membrane was blocked in TBST buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, and 0.1%
Tween 20) with 5% skim milk or 3% bovine serum albumin at room
temperature for 1 h. The membrane was incubated with the indicated
primary antibodies for 2 h at room temperature or overnight at
4 °C. After extensive washing, the membrane was incubated with horseradish peroxidase-conjugated secondary antibody for 2 h. After further washing with TBST buffer, the blot was visualized by
enhanced chemiluminescence according to the manufacturer's instruction
(Amersham Biosciences). The following phospho-antibodies were purchased
from Cell Signaling Technology: anti-phosphothreonine polyclonal
antibody (Anti-P-Thr Ab, catalog number 9381), phospho-(Ser/Thr) kinase
substrate antibody sampler kit (catalog number 9920), including phospho-(Ser/Thr) Akt substrate antibody, phospho-(Ser) PKC substrate antibody, phospho-(Ser/Thr) PKA substrate antibody, phospho-(Thr) MAPK/CDK substrate monoclonal antibody, and phospho-(Thr) PDK1 substrate antibody. Anti-poly(ADP-ribose) polymerase (H-250) polyclonal antibody, anti-Myc monoclonal antibody (9E10), and polyclonal antibody
(A-14) were purchased from Santa Cruz Biotechnologies. Anti-MST2
antibody was raised in rabbit against an NH2-terminal peptide (EQPPAPKSKLKKLSCys) of rat MST2 by our laboratory.
In Vitro Kinase Assay--
The MST2 kinase was assayed using
cdc2(6-20)F15K19 synthetic peptide as a substrate. The reaction
mixture comprised 0.1 µM MCT, 0.1 mM ATP, and
0.3 µCi of [ Protein Phosphatase 1 Treatment--
Myc-tagged FL-MST2 and
TF-MST2 were immunoprecipitated by anti-Myc (9E10) antibody. After
extensive washing with the dephosphorylation buffer (50 mM
Tris-HCl, pH 7.5, 1 mM DTT, 1 mM
MnCl2), the immunoprecipitates were incubated with PP1 (0.2 unit/reaction, Calbiochem) in dephosphorylation buffer for 30 min at
30 °C, then washed by phosphate-buffered saline and subjected to
Western blotting analysis. The immunoprecipitates incubated with
dephosphorylation buffer without PP1 served as control.
Purification of the Endogenous MST2 Kinase--
All of the
purification procedures were carried out at 4 °C. Rat thymus (5 g)
was homogenized with 25 ml of buffer A (50 mM Hepes, pH
7.2, 150 mM NaCl, 1 mM EDTA, 1 mM
EGTA, 5 mM NaF, 20% glycerol, 2 mM DTT, 0.1 µM MCT, 20 mM MST2 Overexpressed in 293T Cells Is Phosphorylated and
Active--
The full-length (FL-MST2) and the caspase-3-truncated form
of MST2 (TF-MST2) were subcloned into pCMV-Myc vector and transfected into 293T cells, the cell lysates were analyzed by Western blotting using an anti-Myc antibody, an anti-MST2 antibody, or an
anti-phosphothreonine antibody. As shown in Fig.
1A (left panel),
the two forms of MST2 were expressed to similar levels in 293T cells.
Using an anti-MST2 antibody, we found that the levels of the
transfected enzymes were at least 100-fold higher than that of the
endogenous MST2 (results not shown). Both the transfected FL-MST2 and
TF-MST2 existed in a phosphorylated state as judged by the
anti-phosphothreonine blot (Fig. 1A, right
panel). The phosphorylation of MST2 underwent rapid
dephosphorylation upon cell lysis unless a phosphatase inhibitor, MCT was added to the lysis buffer, which preserved the
phosphorylated MST2 for more than 6 h after cell lysis (Fig.
1B). In a dose-dependent assay, 0.1 µM MCT was found to be effective in keeping MST2 in a
phosphorylated form (Fig. 1C). For experiments described in this report, the lysis buffer routinely contained 0.1 µM
MCT unless indicated otherwise.
To determine the kinase activity of the full-length and the
caspase-3-truncated forms of MST2 overexpressed in the cells, cell
lysates containing equal amounts of the two forms of MST2, as judged by
Western blotting analysis, were subjected to immunoprecipitation using
an anti-Myc antibody. The immunoprecipitates were then assayed for MST2
kinase activity. Fig. 1D shows that both the full-length and
the truncated MST2 were active when the kinase dead mutant K56R served
as an inactive control. However, the truncated form consistently
displayed 2-3-fold higher kinase activity than the full-length MST2.
In addition to MST2, a few other phosphorylated bands were revealed in
the lysates of MST2-transfected cells by the anti-phosphothreonine immunoblot (Fig. 1A, right panel). For example,
two phosphorylated bands of 46 and 48 kDa were consistently observed in
the lysates of cells transfected with either the full-length MST2 or
the truncated MST2. Another phosphorylated band migrating at 90 kDa was
only observed in lysates of cells transfected with the truncated MST2 but not the full-length MST2. These proteins may represent downstream targets of MST2.
The Relationship of MST2 Phosphorylation and Kinase Activity, and
Determination of the Phosphorylation Site--
Although
phosphorylation of MST1 and MST2 has been shown previously, the protein
kinase responsible for the phosphorylation is not known. In an attempt
to determine the kinase catalyzing MST2 phosphorylation, a set of
antibodies against the phosphorylated substrate sites of various
protein kinases including PKA, Akt, PKC, MAPK/CDK, and PDK1 were tested
for the ability to react with MST2. Among the antibodies tested, the
anti-phospho-PKA substrate and anti-phospho-Akt substrate antibodies
reacted with MST2 (results not shown). However, co-transfection of a
constitutively active Akt with MST2 or treating the MST2-transfected
cell culture with dibutyryl-cAMP had no effect on the phosphorylation
of MST2 suggesting that neither kinase catalyzes the phosphorylation of
MST2 in the cells (data not shown).
Although neither PKA nor Akt was the kinase catalyzing the
phosphorylation of Myc-MST2, the recognition of MST2 by antibodies specific for phospho-substrates of PKA and Akt suggested that MST2 was
phosphorylated at a site conforming to the phosphorylation motifs of
these protein kinases. Three threonine residues, Thr117,
Thr180, and Thr384, in MST2 appeared to reside
at sites conforming to the consensus phosphorylation motifs of PKA or
Akt. To investigate whether or not they are the true phosphorylation
sites in MST2, we generated mutant constructs of both the full-length
and the caspase-3-truncated MST2 with three sites individually mutated:
T117A, T180A, and T384A. The mutant constructs were transfected into
293T cells and the cell lysates were analyzed by Western blot using the
anti-phosphothreonine antibody. Fig.
2A shows that the
phosphorylation was markedly reduced in the full-length MST2 (T180A)
mutant and it was completely eliminated in the truncated MST2 (T180A)
mutant. In contrast, mutating Thr117 had little or no
effect on MST2 phosphorylation. Direct kinase assays using the anti-Myc
immunoprecipitates also showed that T180A mutants were inactive whereas
T117A mutant had approximately the same kinase activity as the wild
type MST2 (Fig. 2B). Similar to the T117A mutant, mutating
T384A mutant had little effect on MST2 phosphorylation and kinase
activity (results not shown). These results suggest that MST2 depends
on the phosphorylation of Thr180 for kinase activity. The
observation that the full-length MST2 (T180A) but not the truncated
MST2 (T180A) was still phosphorylated, albeit at a much lower level
than the wild type MST2, suggests the existence of additional
phosphorylation site(s) in the caspase-3-cleaved carboxyl-terminal
region of the kinase.
To test the possibility that the phosphorylation of MST2 is an
autocatalytic reaction, a kinase dead mutant construct, MST2 (K56R),
was transfected into 293T cells and the phosphorylation of the
overexpressed protein analyzed by Western blot. As shown in Fig.
2A, the phosphorylation of the mutant MST2 was much lower than that of the wild type MST2. Significantly, the levels of phosphorylation of MST2 (T180A) and MST2 (K56R) were almost identical. The results suggest that the phosphorylation of Thr180 of
MST2 is catalyzed by an autophosphorylation reaction whereas the
phosphorylation of the additional site(s) in the carboxyl-terminal region of MST2 was catalyzed by another protein kinase(s).
Characterization of MST2 Autophosphorylation Reaction--
To
find out whether MST2 undergoes autophosphorylation and autoactivation,
we have undertaken a detailed characterization of the MST2
phosphorylation reaction. Cultured 293T cells with overexpressed
FL-MST2 were lysed in the lysis buffer containing no MCT to allow
dephosphorylation of the phospho-Myc-MST2 (see Fig. 1B).
After immunoprecipitation of MST2 using an anti-Myc antibody, the
protein phosphorylation state and the kinase activity of the enzyme
were examined. Western blot analysis indicated that MST2 in the
immunoprecipitates was almost totally devoid of reactivity toward the
anti-phosphothreonine antibody. However, the enzyme exhibited
significant kinase activity (Fig.
3A). The observation suggests
that the unphosphorylated MST2 may undergo autophosphorylation during
the kinase assay.
To determine whether or not MST2 is phosphorylated during the course of
the kinase assay, aliquots of the MST2 reaction were withdrawn at
intervals and analyzed for both the substrate phosphorylation (i.e. the kinase activity) and MST2 phosphorylation. Fig.
3B shows that MST2 was indeed phosphorylated during the
course of the kinase reaction. The increase in MST2 phosphorylation was
obvious right from the beginning of the time course and continued
throughout the entire course of 20 min. As a control, phospho-MST2
immunoprecipitated from the cell lysates containing MCT was also
analyzed and the result indicates that it could be further
phosphorylated. The time course of the substrate phosphorylation (Fig.
3A) by dephosphorylated MST2 (in lysates without MCT)
displays an upward curvature with a pronounced initial lag suggesting
an increase in enzyme activity during the course of the reaction. A
slight curvature can also be detected in the time course of the control
reaction, but the initial lag is not apparent. These results provide
strong support to the suggestion that MST2 depends on phosphorylation
for kinase activity.
To further test whether the MST2 autophosphorylation was an
intermolecular or intramolecular reaction, the dependence of the protein phosphorylation on MST2 concentration was characterized. Cultured 293T cells containing overexpressed MST2 were lysed in the
absence of MCT, the lysates were further incubated at 4 °C for
1 h to ensure that MST2 was mostly dephosphorylated. The cell lysates were then diluted with the lysis buffer to different protein concentrations, and the phosphorylation was started at 22 °C with the supplement of ATP and Mg2+. Three minutes after the
start of the phosphorylation reaction, SDS loading buffer was added to
stop the reaction. The samples were then analyzed by Western blot using
the anti-phosphothreonine antibody. Lysates with a wide range of
cellular protein concentrations, from 0.03 to 1 mg/ml, were used in the
phosphorylation reactions. A very short reaction time, 3 min, was used
in hoping that the phosphorylation could reflect the initial rate of
the reaction. As shown in Fig. 4,
although equal amounts of cellular proteins were subjected to Western
blot analysis, the level of MST2 phosphorylation differed depending on
the protein concentration in the phosphorylation reaction. The level of
MST2 phosphorylation increased with the protein concentration in the
phosphorylation reaction. Densitometric analysis of the immunoblot
indicates that the level of MST2 phosphorylation at 0.03 mg/ml was less
than 1% that at 1 mg/ml. The observation suggests that
autophosphorylation of MST2 is through intermolecular but not
intramolecular reaction.
Phosphorylation State of the Endogenous MST2--
The observation
that autophosphorylation of MST2 shows strong dependence on the enzyme
concentration has raised the possibility that the state of
phosphorylation of the overexpressed MST2 did not reflect the
phosphorylation state of the endogenous MST2. To address such a
possibility, we analyzed the phosphorylation status of the endogenous
MST2 from rat thymus. Rat thymus contains relatively high amounts of
MST2 that could be enriched readily on a DEAE ion exchange
chromatography column. The endogenous MST2 in rat thymus was purified
using DEAE-Sepharose Fast Flow column chromatography followed by FPLC
Superose 6 gel filtration column chromatography. Fractions eluted from
the gel filtration column were pooled and subjected to
immunoprecipitation by an anti-MST2 antibody, which was cross-linked to
protein A/G-agarose beads by disuccinimidyl suberate as described under
"Experimental Procedures." The cross-linked antibodies could not be
released from the agarose beads during boiling in the SDS loading
buffer, thus the immunoprecipitated MST2 that has a similar mobility as
IgGs could be detected clearly by Western blot analysis. As shown in
Fig. 5A, the
immunoprecipitates of partially purified thymus MST2 fractions
displayed intense immunostain by the anti-MST2 antibody, but very faint
signals by the anti-phosphothreonine antibody. The lack of staining
could not be attributed to low reactivity to the anti-phosphothreonine antibody because the control sample of Myc-MST2 from cell lysates with
MCT was intensely stained. Like the recombinant MST2 from the
transfected 293T cells, the rat thymus MST2 has kinase activity (data
not shown). Analysis of MST2 in the kinase reactions by Western blot
showed that MST2 phosphorylation was significantly increased at the end
of the reaction (Fig. 5B). Therefore the kinase activity may
be attributed to the enzyme autoactivation during the course of the
enzyme assay. The observation indicates the endogenous MST2 is capable
of autophosphorylation and autoactivation. Similar to thymus MST2, the
endogenous MST2 from 293T cells exists mainly in the unphosphorylated
state (Fig. 5A).
Because MST1/2 has been documented to undergo activation during cell
apoptosis, we examined the phosphorylation status of MST2 in
anisomycin-induced apoptotic HeLa cells. Anisomycin, a protein
synthesis inhibitor, strongly activates JNK/SAPK and p38 MAPK and it is
also known to induce apoptosis in several mammalian cells (19). HeLa
cells were treated with 20 µg/ml anisomycin for 6 h to render a
portion of cells detached from the plates. The attached and detached
cells, referred to as the adherent and floating cells, respectively,
were separately collected and lysed as described in the legend of Fig.
6. Western blotting analysis of the cell
lysates showed that a significant portion of poly(ADP-ribose) polymerase, a known substrate of caspase-3, was converted to the capase-3-cleaved form, an 85-kDa protein species. Although the cleaved
poly(ADP-ribose) polymerase could be detected in the anisomycin-treated adherent HeLa cells, the majority of the cleaved PAPR was found in the
floating cells (Fig. 6A). The observation indicates that anisomycin treatment could induce the caspase-3-mediated apoptosis in HeLa cells and the floating cells comprise mainly
apoptotic cells.
To determine the phosphorylation states of MST2 in anisomycin-induced
apoptotic cells, MST2 in the cell lysates were immunoprecipitated using
anti-MST2 antibody and the immunoprecipitates were subjected to Western
blotting analysis using both the anti-MST2 antibody and the
anti-phosphothreonine antibody. Western blotting of MST2 showed that a
significant amount of MST2 in lysates of anisomycin-treated floating
cells was cleaved and displayed as two closely spaced SDS-PAGE bands
corresponding to ~34- or 36-kDa proteins. Both cleaved MST2 bands
reacted with anti-phosphothreonine antibody, whereas the full-length
MST2 displayed no such reactivity (Fig. 6B). The full-length
and the cleaved MST2 species were present predominantly in the adherent
and floating cells, respectively (Fig. 6B). The observation,
in agreement with early studies (7, 8, 11-13), suggests that MST2 is
cleaved by caspase-3 in apoptotic cells. Although the same amount of
protein lysates from the adherent and floating cells was used for
Western blotting analysis, the amount of MST2 was significantly lower
in the floating cells than in the adherent cells. Unlike MST1, there is
only one caspase-3 cleavage site in MST2 (15, 17); the reason for the
existence of two SDS-PAGE bands of the caspase-3-cleaved MST2 remains
unclear. From the relative intensity of the anti-MST2 and
anti-phosphothreonine immunostain of the truncated MST2, it may be
suggested that the enzyme was highly phosphorylated. Kinase activity
determination of the immunoprecipitates indicated that the truncated
MST2 was much more active than the full-length MST2 (Fig.
6C). Thus, MST2 in apoptotic cells appears to exist mainly
in a caspase-3-cleaved, phosphorylated and highly active form.
Differential Susceptibility of the Full-length and the
Caspase-3-truncated MST2 to Protein Phosphatases--
Previous studies
showed that MST2 was cleaved by caspase-3 and activated during cell
apoptosis. Because both the full-length MST2 and caspase-3-truncated
MST2 appear to depend on phosphorylation of Thr180 for
kinase activity, we have investigated the possibility that the two
forms of MST2 are differentially regulated by
phosphorylation/dephosphorylation mechanisms. A caspase-3-truncated
MST2 construct was transfected into 293T cells to overexpress the
truncated MST2. In an attempt to generate the dephosphorylated TF-MST2,
the cells were lysed in buffer without MCT. To our surprise, the
TF-MST2, unlike the FL-MST2, was found not to require MCT for the
preservation of the protein phosphorylation. The enzyme in lysates with
and without MCT displayed essentially identical reactivity toward the
anti-phosphothreonine antibody on the Western blot. Fig.
7A shows that the
phosphorylation state of the overexpressed TF-MST2 was not
significantly changed even after 9 h of incubation in cell
lysates. In contrast, greater than 90% loss of FL-MST2 phosphorylation
occurred after 30 min of incubation (see Fig. 1B). The
observation suggests that the phosphorylation of TF-MST2 is almost
irreversible whereas the phosphorylation of FL-MST2 is readily reversed
by protein phosphatases.
As described in an early section, overexpressed FL-MST2 in 293T cells
was partially phosphorylated because it could be further phosphorylated
in the cell lysates with MCT upon ATP/Mg2+ supplements (see
Fig. 3B). In contrast, overexpressed TF-MST2 in 293T cells
appears to be fully phosphorylated. 293T cell lysates with MCT
containing overexpressed TF-MST2 was supplemented with ATP/Mg2+ and incubated at 30 °C for 30 min. The sample,
along with the control sample without the ATP/Mg2+
supplements, was analyzed for protein phosphorylation by Western blot.
The TF-MST2 that had been subjected to the phosphorylation condition
displayed essentially identical reactivity toward the anti-phosphothreonine antibody as the control enzyme (data not shown).
To further test the suggestion that the phospho-TF-MST2 cannot be
readily dephosphorylated, both the overexpressed phospho-FL-MST2 and
phospho-TF-MST2 were immunoprecipitated using the anti-Myc (9E10)
antibody, and then tested for their dephosphorylation by protein
phosphatases. Western blot analysis of the immunoprecipitates showed
that the full-length MST2 could be readily dephosphorylated by protein
phosphatase 1, whereas under the same condition, the truncated MST2 was
not dephosphorylated significantly (Fig. 7B). A similar
result was obtained if protein phosphatase 2A instead of protein
phosphatase 1 was used (data not shown).
To ensure that the differential response of FL-MST2 and TF-MST2 to
protein phosphatases is also manifested in the kinase activity, the
effect of dephosphorylation on the activities of phospho-FL-MST2 and
phospho-TF-MST2 were determined and compared. 293T cells overexpressing FL- or TF-MST2 were lysed in the buffer with or without 0.1 µM MCT. The protein kinases were then immunoprecipitated
with an anti-Myc antibody (9E10) and analyzed for kinase activities.
The activity of FL-MST2 from lysates with 0.1 µM MCT was
about double of lysates without MCT, whereas TF-MST2 samples obtained
from the different lysis conditions displayed identical kinase activity (Fig. 7C). From these results, we suggest that FL-MST2 and
TF-MST2 are differentially regulated by protein
phosphorylation/dephosphorylation mechanisms. Both forms of MST2 depend
on autophosphorylation for kinase activity but only the FL-MST2
activation can be reversed by protein phosphatases. As a result,
TF-MST2 in cells is predominantly in the phosphorylated state, and
therefore "constitutively active."
In this study, we used deletion and site-directed mutant
constructs of MST2, in combination with in vitro
characterization of the enzyme activation and enzyme
phosphorylation/dephosphorylation, to explore the mechanism of MST2
regulation. Our results showed that the phosphorylation of MST2 at a
threonine residue, Thr180, in the kinase activation loop
was crucial for MST2 kinase activity. Substitution of
Thr180 by an alanine residue completely abolished the
activity of either the full-length or the truncated form of MST2
suggesting that both forms of MST2 depend on Thr180
phosphorylation for kinase activity. The suggestion was further substantiated by the observation that dephosphorylation of MST2 caused
a significant decrease in kinase activity. A number of studies have
suggested that MST2 (or MST1) is activated by a protein phosphorylation
mechanism (16, 17). Amino acid sequence at Thr180 of MST2,
KRXTXXGTP, is conserved in several MST kinase
family members including The mechanism of MST2 phosphorylation is complex, and
Thr180 is likely not the sole phosphorylation site in the
enzyme. For example, the MST2 mutant T180A overexpressed in 293T cells
still showed immunoreactivity toward anti-phosphothreonine antibody,
indicating the existence of the phosphorylation site threonine
residue(s) in addition to Thr180. The observation that the
truncated MST2 (T180A) mutant was completely devoid of immunoreactivity
toward the anti-phosphothreonine antibody indicates that the additional
threonine phosphorylation site(s) is localized in the caspase-3-cleaved
carboxyl-terminal fragment. The regulatory significance of the
additional threonine phosphorylation is not known but it is clearly not
essential for the kinase activity. In addition, MST2 may contain
phosphoserine residues, which would have eluded detection by the
anti-phosphothreonine antibody used in this study. It should also be
noted that MST1/2 appears to contain phosphorylation sites that are
involved in kinase inhibition; Creasy and Chernoff (3) showed that MST1
from epidermal growth factor-treated COS cells could be activated by
protein phosphatase 2A. Graves et al. (17) have identified
Ser326 as a major phosphorylation site in MST1, the
phosphorylation at this site regulates the caspase-3 cleavage of the
enzyme. This serine residue is conserved in MST2 as Ser323,
its phosphorylation, however, was not investigated in this study.
Several lines of evidence suggest that the phosphorylation of
Thr180 in MST2 is an autocatalytic reaction. The strongest
evidence is that the kinase dead mutant MST2 (K56R), in contrast to
wild type MST2 or active MST2 mutants, was poorly phosphorylated when overexpressed in 293T cells. The observation that the dephosphorylated and immunoprecipitated MST2 could be phosphorylated in the presence of
ATP/Mg2+, with accompanying kinase activation, further
supports such a suggestion. The time course of the MST2-catalyzed
reaction showed upward curvature, also characteristic of an
autoactivating reaction.
One important feature of the MST2 autophosphorylation reaction is that
the reaction showed a strong dependence on the enzyme concentration.
The result indicates that the autophosphorylation reaction is an
intermolecular rather than intramolecular reaction. It is well
established that both MST1 and MST2 could form homodimers. The protein
domain responsible for MST2 dimerization locates in the extreme
COOH-terminal 57 amino acids (5). We observed that a
dimerization-deficient mutant, MST2 (L448P), had essentially the same
level of phosphorylation as wild type MST2 in 293T
cells.2 The result suggests
that the enzyme concentration dependence of the autophosphorylation
reaction is not related to the dimerization of the enzyme, rather it
may be attributed to the interaction between MST2 dimers.
During the preparation of the manuscript, a paper (22) on
the regulation of MST1 by protein phosphorylation was published. Many
of the findings on the regulatory phosphorylation of MST1 are the same
as those we have reported in this study for MST2. They include the
identification of the activation loop threonine, Thr183, as
the essential phosphorylation site and demonstrated that this threonine
phosphorylation is via an intermolecular autophosphorylation reaction.
Interestingly, the approaches used in the two studies are significantly different.
The strong protein concentration dependence of the autophosphorylation
reaction appears to have important regulatory significance for MST2. In
contrast to the transfected MST2, endogenous MST2 in cultured cells or
in rat thymus had essentially no reactivity toward the
anti-phosphothreonine antibody. This may be attributed to the low
cellular MST2 concentration, which can support only very slow
autophosphorylation reactions. Thus, one potential mechanism for MST2
activation is to facilitate the autophosphorylation reaction by
increasing the enzyme concentration. Lee and Yonehara (16) have
observed the shuttling of MST between nuclear and cytoplasmic compartments, and Khokhlatchev et al. (23) have demonstrated the membrane recruitment of MST1 by the Ras effector protein NORE. These results suggest that MST1/2 may be induced to translocate to
specific cellular compartments. It is conceivable that
compartmentalization of MST1/2 in the cells could markedly increase the
local enzyme concentration so as to facilitate the
autophosphorylation/autoactivation reaction.
In addition to raising local MST2 concentration, the
autophosphorylation reaction in the cells may be enhanced by inhibiting protein phosphatases. It has been reported that MST phosphorylation in
the activation loop could be induced by introducing the phosphatase inhibitor, okadaic acid, or calyculin A into cultured cells or neutrophils (16, 20). However, physiological stimulus that can activate
MST1/2 by inhibiting protein phosphatases has not been reported. In
this respect, the observation that the autophosphorylated TF-MST2 is
highly resistant to protein phosphatase reactions is significant. We
have found, in this study, that the autophosphorylated TF-MST2, in
contrast to the autophosphorylated FL-MST2, is highly resistant to
dephosphorylation by both protein phosphatase 1 and protein phosphatase
2A. Our result differs from the finding of Graves et al.
(17) that MST1 from apoptotic cells could be inactivated by protein
phosphatase 2A. The discrepancy may be attributed to a difference in
the regulatory properties of MST1 and MST2. The possibility that the
discrepancy arises from the differences in experimental conditions and
that factors in addition to the molecular properties of TF-MST2
contribute to the remarkable phosphatase resistance cannot be excluded.
It is well documented that MST1 and MST2 are activated and cleaved by
caspase-3 during cell apoptosis. Because both the full-length and the
caspase-3-truncated forms of MST depend on the phosphorylation of
Thr180 for kinase activity, the question arises as to how
the caspase-3 action contributes to the activation of MST2 during
apoptosis. Based on results of the present study, we have proposed a
molecular model to address this question. As schematically shown in
Fig. 8, the model suggests that at the
prevailing cytoplasmic MST2 and protein phosphatase concentrations in
growing cells, MST2 exists mainly in the unphosphorylated and inactive
state. During cell apoptosis, caspase-3 is activated resulting in the
conversion of MST2 to the truncated form. Both the full-length and the
truncated MST2 can undergo autophosphorylation and autoactivation. The
autophosphorylation of the full-length MST2 is readily reversed by
protein phosphatases while that of the truncated form is remarkably
resistant to protein phosphatases. Thus, through the caspase-3 action,
MST2 is converted into a constitutively active kinase. Although, the
proposed model of MST2 regulation is far from proven, it is supported
by the finding that truncated MST2 in anisomycin-induced apoptotic HeLa cells is present in an active and highly phosphorylated form whereas the full-length MST2 is not phosphorylated and displays very low kinase
activity.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-glycerolphosphate, 1 mM sodium orthovanadate, and the protease inhibitor
mixture, CompleteTM (Roche Molecular Biochemicals)) for 15 min at 4 °C. Cell lysates were cleared by centrifugation at 14,000 rpm for 10 min. Protein concentration was determined by the Bradford
assay (Bio-Rad).
-32P]ATP and 0.5 mM
synthetic peptide substrate in 1× kinase assay buffer (50 mM MOPS, pH 7.4, 10 mM MgCl2, 10 mM
-glycerolphosphate, 2 mM sodium
fluoride). The kinase reaction was started by the addition of ATP and
carried out for 30 min at 30 °C except for indications in specific
experiments. The reaction was terminated by the addition of a
half-assay volume of 50% trichloroacetic acid. The mixture was
centrifuged at 13,000 rpm for 5 min and 2/3 volume of the supernatant
was analyzed for phosphate incorporation using P81 paper as described
(18).
-glycerolphosphate, 1 mM sodium orthovanadate, and 2× CompleteTM
protease inhibitor mixture). The homogenate was first centrifuged at
3000 rpm for 10 min, and the supernatant was further centrifuged at
100,000 × g for 1 h. The 100,000 × g supernatant was diluted with a double volume of buffer B
(20 mM Tris-HCl, pH 7.4, 1 mM EDTA, 1 mM EGTA, 5 mM NaF) with inhibitor mixture (1×
CompleteTM protease inhibitor mixture, 0.1 µM
MCT, 20 mM
-glycerolphosphate, 1 mM sodium
orthovanadate, 2 mM DTT, and 10% glycerol), and then applied to a 35-ml DEAE-Sepharose Fast Flow column pre-equilibrated with buffer B. The column was washed followed by elution with 200 ml of
buffer B with 0-0.5 M linear NaCl gradient. MST2 was eluted in fractions with 0.075-0.1 M NaCl gradient (total
5 fractions, 5 ml/fraction), and each fraction (~9 mg of protein) was
concentrated with Centricon 30 (Amincon) to 0.2 ml and loaded onto the
FPLC Superose 6 HR 10/30 gel filtration column, respectively (Amersham Biosciences). The column was pre-equilibrated and run with buffer C (50 mM Tris-HCl, pH 7.5, 1 mM DTT, 1 mM
EDTA, 0.01 µM Microcystin-LR, 0.1×
CompleteTM protease inhibitor mixture). The kinase
containing fractions were analyzed by Western blotting using anti-MST2 antibody.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (36K):
[in a new window]
Fig. 1.
The full-length and the caspase-3-truncated
forms of MST2 overexpressed in cells are phosphorylated and
active. A, 293T cells were singly transfected with
control vector pCMV-Myc, the full-length MST2 (FL-MST2), or the
caspase-3-truncated form of MST2 (TF-MST2) as indicated. Cells were
harvested after 24 h and cell lysates were analyzed by
immunoblotting with an anti-Myc (A-14) antibody (left panel)
or an anti-phosphothreonine antibody (right panel).
B, FL-MST2-transfected 293T cells were harvested in lysis
buffers with or without the presence of 0.1 µM MCT.
Prolonged incubation was carried out from 30 min up to 9 h and the
incubation was stopped at various times indicated with the addition of
SDS loading buffer. The phosphorylation level of MST2 was analyzed with
anti-phosphothreonine antibody. C, FL-MST2-transfected 293T
cells were harvested in lysis buffers with different concentrations of
MCT as indicated. The protection effect of different MCT concentrations
on MST2 phosphorylation was analyzed by immunoblotting with the
anti-phosphothreonine antibody, and the MST2 level in each cell lysate
was demonstrated by the anti-Myc (A-14) antibody. D, cell
lysates containing equal amounts of FL-MST2 and TF-MST2 were
immunoprecipitated by anti-Myc (9E10) antibody and subjected to
in vitro kinase assays as described under "Experimental
Procedures."
View larger version (33K):
[in a new window]
Fig. 2.
Various MST2 mutants show different
phosphorylation levels and kinase activities. A,
different forms of Myc-tagged MST2, including the wild-type FL-MST2,
truncated mutant (TF-MST2), FL-MST2 (T180A) mutant
(T180A), truncated MST2 (T180A) mutant (TF-MST2
T180A), FL-MST2 (T117A) mutant (T117A), and kinase dead
mutant (K56R), were transfected into 293T cells. The
expression levels and the phosphorylation levels of the Myc-tagged MST2
were detected by Western blot using the anti-Myc antibody (left
panel) and the anti-phosphothreonine antibody (right
panel). B, overexpressed Myc-tagged MST2 mutants were
immunoprecipitated by anti-Myc (9E10) antibody, and the
immunoprecipitates were subjected to in vitro kinase assays
as described under "Experimental Procedures."
View larger version (24K):
[in a new window]
Fig. 3.
The MST2 kinase activity is highly dependent
on MST2 phosphorylation. Two forms of FL-MST2 in transfected 293T
cell lysates were used in this experiment, including in vivo
phosphorylated FL-MST2 that was preserved in lysates with MCT and
in vitro dephosphorylated FL-MST2 in lysates without MCT.
A, cell lysates were immunoprecipitated using the anti-Myc
(9E10) antibody, and the immunoprecipitates were subjected to kinase
assays using cdc2(6-20)F15K19 peptide as a substrate at 30 °C, and
the reactions were stopped at various times as indicated. B,
the phosphorylation level of the immunoprecipitated MST2 during kinase
assay was detected by the anti-phosphothreonine antibody.
View larger version (18K):
[in a new window]
Fig. 4.
MST2 autophosphorylation is concentration
dependent. Two forms of FL-MST2 in transfected 293T cell lysates
were used in this experiment, including in vivo
phosphorylated FL-MST2 that was preserved in lysates with MCT and
in vitro dephosphorylated FL-MST2 in lysates without MCT.
Two samples were diluted with the lysis buffer (containing MCT) to
different concentrations from 0.03 to 1 mg/ml as indicated.
Phosphorylation was started with the supplement of ATP and
Mg2+ at 22 °C, the reaction lasted for 3 min and was
stopped with the supplement of SDS loading buffer. Equal amounts of the
lysates from each reaction were loaded onto SDS-PAGE, and the
phosphorylation of MST2 in each concentration was analyzed with an
anti-phosphothreonine antibody.
View larger version (22K):
[in a new window]
Fig. 5.
The endogenous MST2 is unphosphorylated.
A, rat thymus MST2 was serially purified using
DEAE-Sepharose Fast Flow column chromatography and FPLC Superose 6 gel
filtration chromatography as described under "Experimental
Procedures." One MST2-containing fraction (fraction 15, about 9 mg of
protein) from the DEAE-Sepharose Fast Flow column was concentrated to
200 µl and applied to the FPLC Superose 6 gel filtration column. 400 µl of pooled fractions or 293T cell lysates (1 mg of protein) were
immunoprecipitated, respectively, overnight by anti-MST2 antibody
cross-linked to protein A/G-agarose beads. The immunoprecipitates and
the control samples (Myc-FL-MST2 with or without the presence of MCT, 2 µg of cell lysates) were applied to a SDS-PAGE gel followed by
Western blot analysis using an anti-MST2 antibody and an
anti-phosphothreonine antibody. B, 200 µl of partially
purified thymus MST2 were immunoprecipitated overnight by cross-linked
MST2 antibodies. The immunoprecipitates were then subjected to in
vitro kinase assays as described under "Experimental
Procedures" in the absence of [ -32P]ATP and the
reaction was stopped with the addition of SDS loading buffer. The
immunoprecipitates in the beginning (0 min) and end (30 min) of the
kinase assay were subjected to Western blot analysis using an anti-MST2
antibody and an anti-phosphothreonine antibody.
View larger version (21K):
[in a new window]
Fig. 6.
The endogenous MST2 is cleaved and
phosphorylated in anisomycin-induced apoptosis. A,
exponentially growing HeLa cells were treated with 20 µg/ml
anisomycin (Calbiochem) for 6 h. Both the culture medium and the
washes were pooled and centrifuged at 2,000 rpm for 5 min, then the
cell pellets were collected as the floating cells (ANISO F)
followed by further washing and lysis. The adherent cells were scraped
by the rubber policeman and lysed and prepared as the adherent cell
lysates (ANISO A). The adherent HeLa cells without
anisomycin treatment were lysed and served as no treatment control
(No treat A). 30 µg of cell lysates were analyzed by
immunoblotting with an anti-poly(ADP-ribose) polymerase
(PARP) antibody. B, 150 µg of cell lysates of
adherent HeLa cells without treatment, anisomycin-treated adherent
cells, and anisomycin-treated floating cells were immunoprecipitated,
respectively, overnight with cross-linked MST2 antibodies. The
expression levels and the phosphorylation levels of the
immunoprecipitated endogenous MST2, which were compared with those of
the cell lysates (2 µg) containing transfected Myc-tagged MST2 with
or without the presence of MCT, were detected by Western blot using the
anti-MST2 antibody and the anti-phosphothreonine antibody.
C, 300 µg of cell lysates were immunoprecipitated
overnight by cross-linked MST2 antibodies and subjected to in
vitro kinase assays as described under "Experimental
Procedures," except using 0.05 mM ATP and 3 µCi of
[ -32P]ATP in each reaction.
View larger version (21K):
[in a new window]
Fig. 7.
FL-MST2 could be dephosphorylated by protein
phosphatases while TF-MST2 resists the dephosphorylation.
A, TF-MST2 transfected 293T cells were harvested in lysis
buffers with or without the presence of 0.1 µM MCT.
Prolonged incubation was carried out from 30 min up to 9 h and the
incubation was stopped at various times as indicated with the addition
of SDS loading buffer. The phosphorylation level of MST2 was analyzed
with the anti-phosphothreonine antibody. B, Myc-tagged
FL-MST2 and TF-MST2 were immunoprecipitated by anti-Myc (9E10)
antibody, and the immunoprecipitates were subjected to PP1 treatment as
described under "Experimental Procedures." Anti-phosphothreonine
antibody was used for phosphorylation analysis and anti-Myc (A-14)
antibody was used to detect total MST2. C, FL-MST2 and
TF-MST2 transfected 293T cells lysed in the presence or absence of MCT
as indicated and immunoprecipitated by anti-Myc (9E10) antibody
were then subjected to in vitro kinase assay. The
kinase activity of FL-MST2 (left) or TF-MST2
(right) in the lysates with MCT was arbitrarily set at 100%
as control for comparing the kinase activity of FL-MST2 or TF-MST2 in
the lysates without MCT.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-PAK,
-PAK, and MST1, and
phosphorylation of this conserved threonine residue in these protein
kinases have been shown to result in kinase activation (20, 21). Thus, our result is in agreement with these early findings. On the other hand, Lee and Yonehara (16) suggested in a recent publication that the
activation of MST in apoptotic cells does not depend on protein
phosphorylation, because both the full-length MST and the
caspase-3-truncated form of MST in staurosporine-induced apoptotic cells existed in an unphosphorylated state. However, kinase activity of
MST in the staurosporine-treated cells was not determined.
View larger version (13K):
[in a new window]
Fig. 8.
Mechanism of MST2 kinase regulated by
phosphorylation, dephosphorylation, and caspase-3 cleavage.
Cellular MST2 undergoes intermolecular reversible autophosphorylation.
The balance of the autophosphorylation and protein phosphatase
reactions maintains the enzyme predominantly in the unphosphorylated
and inactive state. Under apoptotic conditions, caspase-3 is activated,
resulting in the conversion of full-length MST2 to the truncated form.
The autophosphorylated TF-MST2 is constitutively active because of its
resistance to protein phosphatases.
![]() |
ACKNOWLEDGEMENT |
---|
We are grateful to Dr. Zhenguo Wu for providing anisomycin and preparation of the manuscript.
![]() |
FOOTNOTES |
---|
* 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.
To whom correspondence should be addressed. Tel.: 852-2358-8701;
Fax: 852-2358-1552; E-mail: jerwang@ust.hk.
Published, JBC Papers in Press, January 27, 2003, DOI 10.1074/jbc.M211085200
2 Y. Deng, and J. H. Wang, unpublished results.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: MST, mammalian STE20-like kinase; MCT, microcystin-LR; PAK, p21-activated kinase; PKA, protein kinase A; PP1, protein phosphatase 1; PP2A, protein phosphatase 2A; FL-MST2, full-length MST2; TF-MST2, truncated form of MST2; Ab, antibody; DTT, dithiothreitol; JNK, c-Jun NH2-terminal kinase; MAPK, mitogen-activated protein kinase; SAPK, stress-activated protein kinase; MOPS, 4-morpholinopropanesulfonic acid.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Dan, I., Watanabe, N. M., and Kusumi, A. (2001) Trends Cell Biol. 11, 220-230[CrossRef][Medline] [Order article via Infotrieve] |
2. |
Kyriakis, J. M.
(1999)
J. Biol. Chem.
274,
5259-5262 |
3. |
Creasy, C. L.,
and Chernoff, J.
(1995)
J. Biol. Chem.
270,
21695-21700 |
4. | Creasy, C. L., and Chernoff, J. (1995) Gene (Amst.) 167, 303-306[CrossRef][Medline] [Order article via Infotrieve] |
5. |
Creasy, C. L.,
Ambrose, D. M.,
and Chernoff, J.
(1996)
J. Biol. Chem.
271,
21049-21053 |
6. |
Taylor, L. K.,
Wang, H.,
and Erikson, R.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
10099-10104 |
7. |
Graves, J. D.,
Gotoh, Y.,
Draves, K. E.,
Ambrose, D.,
Han, D. K. M.,
Wright, M.,
Chernof, J.,
Clark, E. A.,
and Krebs, E. G.
(1998)
EMBO J.
17,
2224-2234 |
8. | Lee, K. K., Murakawa, M., Nishida, E., Tsubuki, S., Kawashima, S., and Yonehara, S. (1998) Oncogene 16, 3029-3037[CrossRef][Medline] [Order article via Infotrieve] |
9. |
Kakeya, H.,
Onose, R.,
and Osada, H.
(1999)
Ann. N. Y. Acad. Sci.
886,
273-275 |
10. |
Reszka, A. A.,
Halasy-Nagy, J. M.,
Masarachia, P. J.,
and Rodan, G. A.
(1999)
J. Biol. Chem.
274,
34967-34973 |
11. | Watabe, M., Kakeya, H., and Osada, H. (1999) Oncogene 18, 5211-5220[CrossRef][Medline] [Order article via Infotrieve] |
12. | Kakeya, H., Onose, R., and Osada, H. (1998) Cancer Res. 58, 4888-4894[Abstract] |
13. |
Watabe, M.,
Kakeya, H.,
Onose, R.,
and Osada, H.
(2000)
J. Biol. Chem.
275,
8766-8771 |
14. |
Ura, S.,
Masuyama, N.,
Graves, J. D.,
and Gotoh, Y.
(2001)
Genes Cells
6,
519-530 |
15. |
Lee, K. K.,
Ohyama, T.,
Yajima, N.,
Tsubuki, S.,
and Yonehara, S.
(2001)
J. Biol. Chem.
276,
19276-19285 |
16. |
Lee, K. K.,
and Yonehara, S.
(2002)
J. Biol. Chem.
277,
12351-12358 |
17. |
Graves, J. D.,
Draves, K. E.,
Gotoh, Y.,
Krebs, E. G.,
and Clark, E. A.
(2001)
J. Biol. Chem.
276,
14909-14915 |
18. |
Litwin, C. M. E.,
Cheng, H.,
and Wang, J. H.
(1991)
J. Biol. Chem.
266,
2557-2666 |
19. |
Hazalin, C. A.,
Le Panse, R.,
Cano, E.,
and Mahadevan, L. C.
(1998)
Mol. Cell. Biol.
18,
1844-1854 |
20. |
Lian, J. P.,
Toker, A.,
and Badwey, J. A.
(2001)
J. Immunol.
166,
6349-6357 |
21. | Pombo, C. M., Bonventre, J. V., Molnar, A., Kyriakis, J., and Force, T. (1996) EMBO J. 15, 4537-4546[Abstract] |
22. |
Glantschnig, H.,
Rodan, G. A.,
and Reszka, A. A.
(2002)
J. Biol. Chem.
277,
42987-42996 |
23. | Kokhlatchev, A., Rabizadeh, S., Xavier, R., Nedwidek, M., Chen, T., Zhang, X. F., Seed, B., and Avruch, J. (2002) Curr. Biol. 12, 253-265[CrossRef][Medline] [Order article via Infotrieve] |