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INTRODUCTION |
Mitogen-activated protein kinases
(MAPKs)1 are highly conserved
mediators of signal transduction that are present in all eukaryotes and
play essential roles in regulating cell differentiation, cell proliferation, and cell death. The core of the cascade consists of
three sequentially acting protein kinases: a MAPK, a MAPK kinase, and a
MAPK kinase kinase (1). In yeast, there are five MAPK cascades that
have pivotal roles in regulating sporulation, cell wall remodeling,
osmolyte synthesis, filamentation, and mating (2, 3). In mammalian
systems, the most thoroughly studied MAPKs are ERK1/2, JNK, and p38
MAPK modules. The ERK1/2 cascade consists of Raf as the MAPK kinase
kinase, MEK as the MAPK kinase, and ERK1/2 as the MAPK. This cascade is
involved in regulation of cell growth and differentiation, whereas the
JNK and p38 cascades have been implicated in several stress responses
including apoptosis and inflammation (4-9).
In budding yeast, the serine/threonine kinase Ste20 activates the MAPK
kinase kinase Ste11, an upstream activator of the MAPK kinase Ste7.
Ste7 in turn activates two MAPKs, FUS and KSS1, in the mating pathway
of budding yeast (10, 11). The homologous family of mammalian
Ste20-like kinases can be divided into two subfamilies based on their
regulation and structure and is rapidly growing. p21
Rac/Cdc42-activated kinases (PAKs) (12) represent the first subfamily
of kinases, containing a C-terminal Ste20-like kinase domain and a
N-terminal regulatory domain with Cdc42 and Rac binding regions. Like
Ste20, PAKs can be activated by binding to the GTP-bound form of
Rac/Cdc42 (12-15). In cotransfection experiments, constitutively
active mutants of PAK1 or PAK3 can activate the JNK and p38 pathways
(14, 16). PAKs have also recently been shown to directly phosphorylate
c-Raf at serine 338, enhancing Raf activity (17), and to potentiate
activation of ERKs (18). Thus, PAKs can act as upstream activators of
the MAPK cascades in mammalian cells.
The second subfamily of Ste20-like kinases, in which germinal center
kinase is the prototype (19), is characterized by an N-terminal kinase
domain followed by a C-terminal regulatory domain. This subfamily also
includes HPK1 (20, 21), GLK (22), NIK( (23), KHS (24), HGK (25), LOK
(26), SLK (27), SOK1 (28), MST1 and MST2 (also known as Krs) (29-31),
and MST3 and MST3b (32, 33). Germinal center kinase, GLK, HGK,
HPK, NIK, and KHS have been shown to activate the JNK pathway at the
level of MAPK kinase kinase (21-24, 28, 34). MST1, MST2, MST3, MST3b,
and SOK1 have extensive homology in their C-terminal tail but have not
been demonstrated to directly activate any of the known MAPK cascades (28). However, there are reports that MST1 can activate JNK and p38
(35), and MST3 can potentiate ERK activation under certain conditions
(33).
In the present study, using a screen for Raf-interacting proteins, we
cloned and characterized MST4, a new member of the mammalian Ste20
family that has a strong homology to mammalian Ste20-like kinases MST1,
MST2, MST3, and SOK1. The existence of a new member of the MST family,
as well as an alternatively spliced, inactive isoform, suggests that
these enzymes may play discrete and specific roles in regulating MAPK
signaling pathways.
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MATERIALS AND METHODS |
Mutagenesis of PEG202-c-Raf--
The catalytic domain of Raf was
amplified by PCR with primer 1 (5'-gacggatccgttcacagccgaaaacccccgtgcc-3') and primer 2 (5'-cgaccatggctagaagacaggcagcctcgg-3'), digested with XhoI
and BamHI, and subcloned into PEG202 to construct PEG202-c-Raf. MEK1 was amplified by PCR with primer 3 (5'-gcctcgagcccaagaagaagccgacg-3') and primer 4 (5'-gcctcgagtcagatgctggcagcgtg-3'), digested with XhoI, and
subcloned into PJG4-5 to construct PJG-4-5-MEK. The PEG202-Raf was
transformed into Epicurian coli XL1-red cells (Stratagene), which are
deficient in three of the primary DNA repair pathways. After recovery
in SOC medium (GIBLO BRL) at 37 °C for 1 h, the culture
was diluted to 10 ml of SOC for further incubation overnight. The next day, 200 µl of the culture was diluted with a fresh 10 ml of
SOC and grown overnight, while the remaining culture was used
for plasmid DNA preparation. The second overnight culture was further
diluted for another overnight incubation and plasmid DNA preparation. A
third overnight culture was also used for preparation of plasmid DNA.
These three plasmid DNA preparations were pooled together as the
mutated PEG202-c-Raf library, which was transformed into yeast EGY48
along with PJG-MEK in order to screen for a mutated PEG202-Raf protein
that has a high affinity for MEK. A mutant construct was isolated
(PEG202-Raf-M), and we confirmed that the mutant Raf protein had an
enhanced affinity for MEK by retransforming the PEG202-c-Raf-M
construct back into yeast EGY48 with PJG4-5-MEK and testing its
interaction with MEK. Sequencing the c-Raf insertion in PEG202 revealed
a mutation at residue Ser610 that introduced a new stop
codon at this site, resulting in a truncated C-terminal Raf protein.
Yeast Two-hybrid Screen--
The LexA yeast two-hybrid system
was kindly provided by Dr. Roger Brent. A human fetal library, made
from a 22-week-old human fetal frontal cortex, was used to search for
proteins interacting with the kinase domain of mutant PEG202-Raf-M (see
above). Two clones showing strong interaction with Raf were obtained.
Routine yeast work and yeast transformation were performed as
described (36).
PCR Analysis of cDNA Libraries--
cDNA from two human
fetal brain libraries, a human testes library, and multiple human
tissue cDNA panels from CLONTECH were analyzed
by polymerase chain reaction using primer MST-761
(5'-ctactaagattccgaatcagagccctc-3') and primer MST-1
(5'-atggcccactcgccggtggctgtc-3').
Tissue Northern Blot--
A ~1-kilobase pair DNA
fragment was amplified by PCR, purified using a Qiagen PCR purification
kit, and labeled with 32P using the Megaprime DNA labeling
kit (Amersham Pharmacia Biotech). The probe was hybridized to a
poly(A)+ RNA human multiple tissue Northern blot
(CLONTECH) following the user manual.
Cell Culture, Transfection, and Lysis--
COS and 293 cells
were grown in a 95% air, 5% CO2 incubator at 37 °C.
Dulbecco's modified Eagle's medium supplemented with antibiotics (50 units/ml penicillin and 50 µg/ml streptomycin) and 10% fetal bovine
serum was used for cell growth. Transfection of COS and 293 cells was
performed using the TransITTM polyamine transfection
reagent (Panvera, Madison, WI). For lysis, cultured cells were washed
twice with ice-cold phosphate-buffered saline and lysed in 1%
Triton-based lysis buffer containing 1% Triton X-100, 100 mM NaCl, 50 mM Tris-HCl, pH 7.5, 50 mM NaF, 40 mM
-glycerophosphate, 2 mM EDTA, 1 mM sodium vanadate, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin, 1 µg/ml
leupeptin, and 20 mM
-nitrophenyl phosphate.
Site-directed Mutagenesis--
A primer
(5'-gtcgttgctattcgaatcatagac-3') was used for unique site elimination
site-directed mutagenesis of MST4a to generate the kinase inactive
mutant MST4a-K53R. The critical lysine in the kinase domain (VAIKIID)
was mutated to arginine. The mutagenesis was performed according to the
manual for the Amersham Pharmacia Biotech U.S.E. mutagenesis kit. The
smaller BamHI/BsmI DNA fragment from
PCR3.1-MST4a-K53R was ligated into the larger
BamHI/BsmI DNA fragment from PCR3.1-MST4 to
construct PCR3.1-MST4-K53R.
Epitope Tagging--
Primer FLAG-MST-5'
(5'-gccgccatggactacaaggacgacgatgacaaggcccactcgccggtggctg-3') and primer
MST-3'-1165 (5'-ccatcgatcatggagctcatgggttaag-3') were used to clone the
MST4a from PJG-MST4 by PCR and clone MST4 from
Marathon-readyTM cDNA by PCR with a
CLONTECH PCR Advantage 2 kit. The PCR products were
purified by the Qiagen PCR purification kit and subcloned into the TA
cloning vector PCR3.1 (Invitrogen) to construct PCR3.1-MST4a and
PCR3.1-MST4. Primer FLAG-MST-5' and primer MST-761
(5'-ctactaagattccgaatcagagccctc-3') were used to clone the MST4-NT from
Placenta Marathon-ready cDNA by PCR. The PCR products were ligated
into the TA cloning vector PCR3.1 (Invitrogen) to construct the
N-terminal construct PCR3.1-MST4-NT. The FLAG-MST-5' primer
contained the FLAG epitope right after the ATG code and the 19 nucleotides in the 5'-end of MST4. The plasmids were sequenced by the
interdisciplinary center for biotechnology research sequencing core
laboratory at the University of Florida or by the University of Chicago
Cancer Research Center DNA sequencing facility.
Immunoprecipitation and in Vitro Kinase Assay--
For MST4,
FLAG-MST4a, FLAG-MST4, FLAG-MST4-K53R, or FLAG-MST4-NT were transfected
into cells, grown in regular medium for 24 h, and starved in
serum-free medium or grown in regular medium for 48 h. Following
lysis in 1% Triton X-100 buffer, the cell extracts were incubated with
protein G beads coated with anti-FLAG M5 antibody for 3 h. The
immune complex was washed three times with lysis buffer and two times
with kinase buffer (20 mM Hepes, pH 7.4., 10 mM
MgCl2, 1 mM MnCl2, 1 mM
dithiothreitol, 0.2 mM sodium vanadate, 10 mM
-nitrophenyl phosphate). 5 µg of MBP substrate was used per
reaction in kinase buffer containing 5 µCi of
[
-32P]ATP. The kinase reaction was incubated for 25 min at 30 °C and then boiled with sample buffer for 5 min. The
reaction products were separated on 12% SDS-polyacrylamide gels and
transferred to a nitrocellulose membrane. Western blot analysis was
performed as described previously (37). For MAPK assays, either
HA-ERK2, HA-JNK, or HA-p38 was expressed in cells along with FLAG-MST4 expression vectors and/or Myc-Raf-1 expression vectors as
indicated and immunoprecipitated with anti-HA antibodies as above.
Kinase activities were monitored by immunoblotting with the appropriate anti-phospho-pTXpY antibodies (New England Biolabs, Boston).
Fluorescence in Situ Chromosomal Hybridization--
Human
metaphase cells were prepared from phytohemagglutinin-stimulated
peripheral blood lymphocytes. The MST4 probe was a cDNA
probe containing full-length MST4a. Fluorescence in
situ hybridization was performed as described previously (38).
Biotin-labeled probes were prepared by nick translation using
Bio-16-dUTP (Enzo Diagnostics). Hybridization was detected with
fluorescein-conjugated avidin (Vector Laboratories), and chromosomes
were identified by staining with 4,6-diamidino-2-phenylindole dihydrochloride.
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RESULTS |
Molecular Cloning of MST4 and MST4a, New Members of the Mammalian
Ste20-like Kinase Family--
A human fetal brain library was employed
in a LexA-based yeast two-hybrid screen to identify proteins that
interact with Raf. A modified Raf bait was created by mutagenizing the
yeast plasmid PEG202 containing the catalytic domain of Raf and
selecting for enhanced interaction with MEK1 (see "Materials and
Methods"). Using the modified Raf bait plasmid, we obtained two
cDNA clones that coded for the same previously uncharacterized
cDNA. Sequence analysis showed that both clones encoded a
polypeptide of 354 amino acids, which was later termed MST4a (Fig.
1).

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Fig. 1.
Sequence comparison of MST4 and MST4a.
Shown are the predicted amino acid sequences of MST4a, isolated
by a yeast two-hybrid screen, and the full-length MST4 generated by
PCR. Subdomains characteristic of protein kinases are indicated by
Roman numerals. The kinase subdomains IX, X, and XI are
underlined. Sequences in common between MST4 and MST4a are
shaded.
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Eukaryotic protein kinases have a common catalytic core structure in
their kinase domain, which typically contains 11 conserved subdomains
(39). Comparison of the MST4a protein sequence with that of MST family
members revealed that MST4a lacked part of the kinase domain. In order
to see if MST4a might be a splicing variant of a full-length MST
cDNA, the kinase subdomains I-XI in MST4 cDNAs from three
separate libraries were examined by PCR analysis. As shown in Fig.
2 (left panel), two
PCR products with different sizes were amplified from a fetal human
brain (CLONTECH) cDNA library that was
different from the human brain cDNA library used in the original
yeast two-hybrid screen as well as from a testes and a placenta
library. Sequence analysis indicated that the smaller PCR product had
the same sequence as the kinase domain of MST4a, and the larger PCR
product had an additional cDNA insert. Subcloning the full-length
fragments into an expression vector and subsequent sequencing revealed
that the larger PCR product corresponded to the full-length MST
cDNA containing the missing IX, X, and XI kinase domains (MST4,
Fig. 1). The deduced protein sequence of MST4, consisting of 416 amino
acids (Fig. 3A), has a kinase
domain at the N terminus and a regulatory domain at the C terminus.
Comparison of the protein sequence of MST4 with other enzyme sequences
(Fig. 3B) indicated that the kinase domain is most closely
related to the catalytic domains of Mammalian Ste20-like kinases MST1
(51% identity), MST2 (54% identity), MST3 (65% identity), and SOK1
(63% identity). The C-terminal regulatory domain of MST4 is most
similar to the C-terminal domains of MST3 and SOK1.

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Fig. 2.
Amplification of MST4 and MST4a kinase
domains by PCR from different cDNA libraries. Left
panel, the PCR products were amplified from placenta and testis
Marathon ready cDNA and fetal brain cDNA. Right
panel, PCR products were amplified from
CLONTECH multiple human tissue cDNA panels.
Samples were subjected to electrophoresis in a 1.2% agarose gel as
described under "Materials and Methods."
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Fig. 3.
Nucleotide and predicted amino acid sequence
of human MST4 and sequence alignment between MST4 and other mammalian Ste20-like kinases. A, cDNA and
predicted protein sequence of the full-length MST4. B,
sequence comparison between MST4 and other members of the MST family.
The predicted amino acid sequence of MST4 was compared with that of the
mammalian Ste20-like kinases MST1, MST2, MST3, and SOK1 by Geneworks.
Amino acids conserved in all protein are shaded.
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Most of the genomic DNA sequence of human MST4 has been
mapped. Interestingly, there is a putative 186-base pair exon
corresponding to kinase subdomains IX-XI that, if spliced out of the
MST4 mRNA transcript, could account for the sequence found in the
truncated MST4a variant. These results suggest that MST4a is
likely to be an alternatively spliced form of MST4. When we
did a more comprehensive analysis of the tissue distribution of MST4
and MST4a by PCR, we were able to detect both forms in cDNAs from a
variety of other adult and fetal human tissues
(CLONTECH) including kidney, lung, liver, and
pancreas as well as fetal thymus, spleen, muscle, liver, and kidney
(Fig. 2, right panel). In most tissues, the major
expressed form is MST4, but it appears that MST4a is more highly
expressed in the brain.
Tissue Distribution and Chromosomal Localization of MST4--
A
human multiple tissue Northern blot was used to identify the expression
pattern of MST4. Only one mRNA band with a size of 3.6 kilobases was detected. The results showed that MST4
is ubiquitously expressed, with strong expression in placenta, weak expression in skeletal muscle and pancreas, and moderate expression in
brain, heart, lung, liver, muscle, and kidney. It was not possible to
determine the difference in expression of MST4 versus MST4a by this approach, since the difference in size between the two mRNAs is too small to be detected by Northern analysis (Fig.
4). To map the MST4 gene, we
performed fluorescence in situ hybridization using a
biotin-labeled MST4 probe on normal human metaphase
chromosomes. Hybridization of the MST4 cDNA probe
resulted in specific labeling only of the X chromosome (Fig.
5). Labeling of Xq25-27 was observed on
four (eight cells), three (16 cells), or two (one cell) chromatids of
the X chromosome homologues in 25 cells examined from
mitogen-stimulated lymphocytes isolated from a healthy female. Of 82 signals observed, one signal (1.2%) was located at Xq25, 67 signals
(82%) were located at Xq26, and 14 signals (17%) were located at
Xq27. No background signals were observed at other chromosomal sites.
We also observed a specific signal at Xq26 in an additional
hybridization experiment using this probe (data not shown). These
results indicate that the MST4 gene is localized to Xq26.
This result was later verified by examination of the relative location
of the MST4 gene in the X chromosome genomic map.

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Fig. 4.
Northern blot analysis of MST4 in various
human tissues. A human multiple tissue Northern blot
(CLONTECH) was probed with labeled full-length
MST4a generated by PCR. kb,
kilobases.
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Fig. 5.
In situ hybridization of a
biotin-labeled MST4 probe to human metaphase cells
from phytohemagglutinin-stimulated peripheral blood lymphocytes.
The X chromosome homologues are identified with arrows;
specific labeling was observed at Xq26. The inset shows
partial karyotypes of two X chromosome homologues illustrating specific
labeling at Xq26 (arrows). Images were obtained using a
Zeiss Axiophot microscope coupled to a cooled charge-coupled device
(CCD) camera. Separate images of 4,6-diamidino-2-phenylindole
dihydrochloride-stained chromosomes and the hybridization signal were
captured and merged using image analysis software (IP
LabSpectrum).
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Catalytic Activity of MST4 and MST4a--
In order to determine
whether MST4 or its variant is an active kinase, the N-terminal domains
of MST4 and MST4a were tagged with a FLAG epitope, and the cDNAs
were expressed in 293 cells using a cytomegalovirus promoter (Fig.
6A). To control for
nonspecific kinase activity, a kinase-inactive mutant of MST4,
MST4-K53R, was also transiently transfected into 293 cells. Following
transfection, 293 cells were lysed, and the cell lysates were subjected
to immunoprecipitation with anti-FLAG antibody (Fig. 6B).
The immunoprecipitates were then assayed for in vitro kinase
assay using myelin basic protein as a substrate. The results indicate
that MST4 is an active kinase, whereas MST4a did not exhibit kinase
activity (Fig. 6B). Interestingly, the kinase activity of
MST4 isolated from cells grown in serum-free or serum-containing medium
was comparable, indicating that MST4 possesses a high basal kinase
activity (Fig. 6C). Under the same conditions, a
phosphoprotein with the same size as MST4 (46 kDa) was also detected in
an in vitro kinase reaction when immunoprecipitates containing MST4 but not kinase-inactive MST4 (MST4-K53R) were used, suggesting that MST4 also functions as an autophosphorylating kinase (Fig. 6C).

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Fig. 6.
Kinase activity and autophosphorylation of
MST4 and MST4a. A, expression of the FLAG-MST and
FLAG-MST4a proteins. 293 cells were transfected with expression vectors
for FLAG-MST4, FLAG-MST4a, FLAG-MST4-K53R, or the control vector. The
cell lysates were collected after 24 h. Expressed proteins were
detected by Western blot analysis with anti-FLAG antibody.
B, catalytic activity of MST4. COS cells were transfected
with either the control vector or expression vectors for FLAG-MST4,
FLAG-MST4a, or FLAG-MST4-K53R. The cells were collected 40 h after
transfection, and the cell lysates were immunoprecipitated
(IP) with anti-FLAG antibody and analyzed by Western
blotting with anti-FLAG antibody (top panel).
Duplicate samples were subjected to an in vitro kinase assay
using MBP as a substrate as described under "Materials and Methods"
(bottom panel). C, comparison of
kinase activity of MST4 in cells grown with or without serum. 293 cells were transfected with FLAG-MST4,
FLAG-MST4-K53R, or control vector and grown in serum-containing medium
for 24 h. Cells were then transferred to serum-free medium
(S ) or serum-containing medium (S+) for another
24 h, and the cell lysates were immunoprecipitated with anti-FLAG
antibody and analyzed by Western blotting with anti-FLAG antibody.
Duplicate samples were subjected to an in vitro kinase
reaction using MBP as substrate. D, co-immunoprecipitation
of MST4 and c-Raf-1. COS cells were transfected with expression vectors
for FLAG-MST4, FLAG-MST4a, Myc-Raf-1 or green fluorescent protein as a
control vector. Lysates were collected after 48 h
post-transfection, and proteins were immunoprecipitated with either
anti-FLAG or anti-Myc antibodies. Samples were then analyzed by Western
blotting with either anti-FLAG or anti-Myc antibodies as
indicated.
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Since MST4a was isolated by association with the Raf kinase domain in a
yeast two-hybrid system, we determined whether MST4 or MST4a associate
with c-Raf-1 in cells. Therefore, COS cells were co-transfected with
expression vectors for Myc-Raf-1 and FLAG-MST4 or FLAG-MST4a. Raf was
then immunoprecipitated with an anti-Myc antibody and analyzed for MST4
association by immunoblotting with an anti-FLAG antibody. Conversely,
MST4 or MST4a was immunoprecipitated with an anti-FLAG antibody and
analyzed for Raf association with an anti-Myc antibody. As shown in
Fig. 6D, no association of c-Raf-1 with MST4 or MST4a was
observed. We also determined whether co-expression of Myc-Raf-1 with
FLAG-MST4 altered MST4 kinase activity. Consistent with the
co-immunoprecipitation results, no effect of Raf-1 on MST4 kinase
activity was detected (data not shown). These results suggest that MST4
is not stably associated with or modulated by c-Raf-1.
C-terminal Regulatory Domain of MST4 Enhances but Is Not Essential
for Its Kinase Activity--
Removal of the C-terminal regulatory
domain of MST1 and MST2 by caspases results in a significant increase
in MST1 or MST2 kinase activity (35, 40). To address the function of
the C-terminal tail of MST4, a cDNA encoding the N-terminal kinase
domain of MST4 but lacking the C terminus of the protein (FLAG-MST4-NT) was transfected into COS cells. For comparison, COS cells were also
transfected with expression vectors for FLAG-MST4 or FLAG-MST4-K53R. Following immunoprecipitation with anti-FLAG antibodies, the MST4 proteins were assayed for kinase activity using MBP as a substrate. As
shown in Fig. 7, FLAG-MST4-NT has reduced
kinase activity compared with MST4, indicating that the C-terminal
domain acts to enhance MST4 activity. Interestingly, MST4-NT is no
longer autophosphorylated, suggesting that the site of
autophosphorylation may be in the C-terminal tail of the protein (Fig.
7).

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Fig. 7.
Kinase activity of the catalytic
domain of MST4 (MST4-NT). Lysates from 293 cells transfected with
FLAG-MST4, FLAG-MST4-K53R, FLAG-MST4-NT, or control vector were
immunoprecipitated (IP) with anti-FLAG antibody and analyzed
by Western blotting with anti-FLAG antibody (top
panel). Duplicate samples were subjected to an in
vitro kinase assay using MBP as substrate (bottom
panel).
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MST4 Does Not Activate ERK2 in 293 Cells--
MST4a was isolated
by interaction with the Raf kinase domain and is most closely related
to MST3, an enzyme that has been reported by one group to potentiate
the Raf/MEK/ERK signaling cascade. To determine whether overexpression
of MST4 might also activate ERK1 or ERK2, vectors expressing MST4,
MST4-K53R or control vector were co-transfected with HA-ERK2 into 293 cells. The tagged ERK2 was immunoprecipitated from the lysates of
transfected cells using an anti-HA antibody, and its kinase activity
was assayed by Western blotting with anti-phospho-pTEpY MAPK antibody.
A sample from each lysate was assayed directly by Western blotting with anti-FLAG antibody to confirm the expression of MST4 (data not shown).
As shown in Fig. 8, overexpression of
MST4 does not significantly affect the activation of ERK2 in cells
grown either in serum or under serum-free conditions. Furthermore, we
have also conducted similar experiments involving co-expression of MST4
(or MST4a or MST4-K53R), ERK2, and Myc-Raf-1 to determine whether MST4
can potentiate or inhibit Raf activation of ERK. Again, we could not detect any effect of MST4 on Raf-mediated ERK activity (data not shown), consistent with the lack of association with c-Raf-1 in cells.
Thus, despite the interaction of MST4a with the kinase domain of Raf in
the yeast two-hybrid system, we have no evidence to date that MST4
either stimulates or potentiates ERK2 activity in 293 cells.

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Fig. 8.
Overexpression of MST4 does not activate,
potentiate or inhibit the ERK pathway in 293 cells. 293 cells were
co-transfected with either control vector or HA-ERK and either control
vector, FLAG-MST4, or FLAG-MST4-K53R. Cells were growth in
serum-containing medium for 24 h and then incubated in serum-free
medium for another 24 h. The cells were then left untreated
( Ser) or stimulated with 20% serum (+Ser).
Following treatment, cell lysates were immunoprecipitated with anti-HA
antibody, resolved by SDS-polyacrylamide gel electrophoresis (10%),
and immunoblotted with anti-HA antibody. The blot was then stripped and
reprobed with anti-phospho-pTEpY-ERK antibody.
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Similar experiments were done to determine whether MST4 activates or
enhances JNK or p38, the other members of the MAPK superfamily. Thus,
FLAG-MST4, the kinase-dead mutant FLAG-MST4, or the control vector were
expressed along with HA-JNK in 293 cells. The cells were either
untreated or stimulated with sorbitol, and then JNK was
immunoprecipitated from the cell lysates with anti-HA antibody. As
illustrated in Fig. 9, JNK was stimulated
by sorbitol, and neither MST4 nor the kinase-dead MST4 mutant
significantly activated or suppressed JNK activity. In a parallel
experiment, 293 cells were transfected with expression vectors for
FLAG-MST4, the kinase-dead mutant FLAG-MST4, or the control vector
along with HA-p38. After further incubation of the cells or stimulation
with sorbitol, p38 was immunoprecipitated from the cell lysates with
anti-HA antibody. Like the results obtained for ERK and JNK, no effect of MST4 or the kinase-dead MST4 mutant on either p38 stimulation, potentiation, or inhibition was observed (Fig.
10).

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Fig. 9.
Overexpression of MST4 does not activate,
potentiate, or inhibit the JNK pathway in 293 cells. 293 cells
were co-transfected with either control vector or HA-JNK and either
control vector, FLAG-MST4, or FLAG-MST4-K53R. Cells were grown in
serum-containing medium for 24 h and then incubated in serum-free
medium for another 24 h. The cells were then left untreated
( Sor) or stimulated with 0.5 M sorbitol
(+Sor). Following treatment, cell lysates were
immunoprecipitated with anti-HA antibody, resolved by
SDS-polyacrylamide gel electrophoresis (10%), and immunoblotted with
anti-HA antibody. The blot was then stripped and reprobed with
anti-phospho-pTPpY-JNK antibody.
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Fig. 10.
Overexpression of MST4 does not activate,
potentiate or inhibit the p38 pathway in 293 cells. 293 cells were
co-transfected with either control vector or HA-p38 and either control
vector, FLAG-MST4, or FLAG-MST4-K53R. Cells were grown in
serum-containing medium for 24 h and then incubated in serum-free
medium for another 24 h. The cells were then left untreated
( Sor) or stimulated with 0.5 M sorbitol
(+Sor). Following treatment, cell lysates were
immunoprecipitated with anti-HA antibody, resolved by
SDS-polyacrylamide gel electrophoresis (10%), and immunoblotted with
anti-HA antibody. The blot was then stripped and reprobed with
anti-phospho-pTGpY-p38 antibody.
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DISCUSSION |
A novel member of mammalian Ste20-like kinase family, MST4, was
cloned and characterized. According to the primary sequence, MST4
belongs to the germinal center kinase subfamily because it has an
N-terminal kinase domain and a C-terminal regulatory domain. MST4 is
most highly related to MST3 (32), with 68% identity, and SOK (28),
with 63% identity within the kinase domain. An inactive mutant of
MST4, termed MST4a, was isolated by interaction with the Raf catalytic
domain in a yeast two-hybrid screen. Although Ste20-related kinases
activate the MAPK cascade in organisms ranging from yeast to mammals,
MST4 does not appear to activate or potentiate the ERK, JNK, or p38
MAPK in 293 cells.
Analysis of the genomic sequence indicates that MST4a is an
alternatively spliced isoform of MST4. MST4a is missing a 186-base pair
exon encoding kinase subdomains IX, X, and XI. Kinase subdomain IX has
been shown to be important for the kinase activity of protein kinase
C
, and it has been suggested that domains X and XI function to
stabilize the kinase-bound substrate (39). Consistent with this
possibility, the expressed MST4a kinase does not exhibit any kinase
activity toward MBP, a potent substrate of MST4 as well as other
members of the MST family. Since MST4a was independently cloned from
two different brain cDNA libraries at reasonably high expression
levels and MST4a transcripts can be translated into protein, it is
likely that MST4a represents an alternative isoform of MST4 in the
brain as well as other tissues.
The lack of kinase activity in MST4a suggests that it may function as a
decoy or dominant-negative kinase, representing an alternative
mechanism for regulating MAPK signaling cascades. Several other kinases
and receptors appear to have similar splice variants. A 3' exon
encoding 10 amino acids corresponding to subdomains IX and X of MEK5
can be spliced out during transcription of MEK5 (41), and an
alternatively spliced exon also has been found in the comparable region
of JNK/stress-activated protein kinase. (42). Unlike MST4a, the MEK5
isoform that lacks the exon encoding subdomains IX and X has equivalent
in vitro protein kinase activity to the full-length MEK5.
Thus, it is possible that a large deletion of subdomains IX-XI, like
the spliced exon in MTS4a, could lead to loss of activity, while a
smaller deletion of subdomains IX-X, like the spliced exon in MEK5,
still enables the kinase to interact with the substrate. Recently, a
novel Ste20 kinase from chickens was cloned (KFC) that is also
expressed as a splice variant (43). The short form of KFC has a
69-amino acid deletion outside of the kinase domain and has no impact
on cell growth, but the longer version confers a growth advantage to
the cell. Thus, the alternative splicing of exons may provide another
mechanism for diversifying the function of protein kinases.
There are other examples of nonfunctional, decoy proteins that
have important physiological functions. Recently, a novel
phosphoserine/threonine-binding protein (STYX), related to dual
specificity protein-tyrosine phosphatase, was shown to contain a
naturally occurring Gly instead of Cys, the residue that is required
and conserved in dual specificity protein-tyrosine phosphatase
catalytic loops. The naturally occurring STYX protein does not have
phosphatase activity toward Tyr(P)-containing substrates;
however, the substitution of Gly to Cys in recombinant STYX protein
confers phosphatase activity, indicating that STYX is a
naturally occurring "dominant negative"
phosphotyrosine/serine/threonine-binding protein (44). Another example
is the TNF receptor superfamily that has two main subgroups of
receptors. The first group (DR) possess the death domain, which couples
the receptor to caspase cascade, whereas the second group consists of a
decoy receptor (DcR), which is structurally related to DR but lacks the
death domain and functions as an inhibitor (45). Taken together, these studies suggest that MST4a could be a naturally occurring
dominant-negative kinase that can act as an inhibitor of the signaling
pathway regulated by MST4.
Although other MSTs have been implicated as mediators of apoptosis, it
appears unlikely that MST4 plays a similar role. Interestingly, MST4
has a sequence (DESDS) that is very similar to the caspase 3 recognition sequence in MST1 (DEMDS) and MST2 (DELDS) at the junction
of the N-terminal catalytic and C-terminal domains. However, unlike
MST1 and MST2 (35, 40), MST4 was not cleaved in vivo or
in vitro by caspase 3 (data not shown). Loss of the
C-terminal regulatory domain in MST1 and MST2 results in an activation
of the catalytic activity, consistent with a positive feedback role in
apoptosis. In contrast, the C-terminal tail of MST4 has an activating
regulatory role, and loss of this domain by caspase cleavage would
result in decreased kinase activity. Thus, even if MST4 were a target
of cleavage by other caspases, the truncated enzyme would not function
as a direct downstream mediator of caspase-initiated cell death.
Further studies will be required to elucidate its true physiological role.
The role of MST4 in the activation or regulation of MAPK is also
unclear at this time. Unlike other MSTs, MST4 was not sufficient by
itself to activate ERKs, JNKs, or p38s or to potentiate or inhibit
serum or sorbitol stimulation of these enzymes in 293 cells. However,
it is possible that MST4 can modulate MAPK signaling in a tissue- or
cell-specific manner. Our isolation of MST4 via a Raf kinase
domain-based two-hybrid screen and the relationship of MST4 to MST3,
which has been implicated in ERK signaling in one report (33), suggest
a role for MST4 in the Raf-activated MAPK cascade. However, in a
different report, no ERK activation or potentiation by MST3 was
observed (32), and we have similarly failed to observe an interaction
of MST3 with ERKs. Thus, the relationship of both MST3 and MST4 to the
ERK kinase cascade must be considered unresolved. Although much remains
to be learned, the identification of yet another member of the
mammalian Ste20 family suggests that these enzymes may play an
important role in signaling specificity.
One potential approach toward elucidating MST4 function is genetic
analysis. MST4 has been mapped to a region of the chromosome implicated
in many disease states, particularly those involved in mental
retardation. With the complete elucidation of the human genomic map, it
should now be possible to determine whether MST4 plays a role in the
etiology of any of these brain-related diseases.