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INTRODUCTION |
MAP1 kinases have been
known to be activated by various stresses and growth stimuli and to be
involved in transmitting signals from outside of cells into cell nuclei
(1-8). In mammalian systems, three major types of MAP kinases, namely
ERK, p38, and JNK/SAPK, have been identified and characterized
extensively. Recently, striking progress in molecular cloning
techniques and in bioinformatics has greatly facilitated homology-based
discoveries of novel protein kinases that are more or less closely
related to conventional MAP kinases (9). Among them, protein kinase
catalytic domains of ERK5 (10, 11) and ERK7 (12) are highly homologous
to those of conventional MAP kinases, and activation mechanisms and downstream targets of ERK5 have been described (for review, see Ref.
13).
We have recently identified and cloned a novel member of the MAP kinase
superfamily, MOK, by a computer search of the expressed sequence tags
database followed by screening of human and mouse cDNA (14). MOK is
most similar to previously identified protein kinases, male germ
cell-associated kinase (MAK) (15) and MAK-related kinase (MRK) (16).
The kinase catalytic domains of MOK, MAK, and MRK are highly homologous
to that of conventional MAP kinases, especially at their activation
loops where all of them encode the MAP kinase characteristic
TXY motifs (9). MRK is abundantly expressed in ovary,
whereas MAK and MOK are abundant in testis; hence they are postulated
to play roles in regulating the meiotic cell division in reproductive
tissues. Previously we observed that MAP kinase-stimulating treatment
of cells including serum addition, anisomycin treatment, and osmotic
shock did not significantly activate MOK (14), suggesting that specific
upstream signal-transducing molecules are involved in the activation
processes of MOK. Treatment of cells with tumor promoter
12-O-tetradecanoylphorbol-13-acetate activated MOK,
and the activity of MOK was dependent on its phosphorylation status
(14), but the precise physiological functions and cellular regulatory
factors of MOK remain unknown.
Catalytic subunits of certain protein kinases are associated with
cellular proteins. cAMP-dependent protein kinase catalytic subunit is complexed with a regulatory subunit (for a review, see Ref.
17). CK2 (casein kinase II) is composed of catalytic
and
'
subunits and a regulatory
subunit (18, 19). Cell cycle-regulated
Cdk protein kinases are accompanied by their specific activating
subunits, cyclins (20-22). MAP kinases are known to physically
associate with upstream activating kinases either directly (23, 24) or
via common tethering proteins called molecular scaffold (reviewed in
Refs. 25 and 26). The specific associations of noncatalytic proteins
are important for catalytic subunits in their regulation such as
inhibitory control, signal-dependent activation, substrate
recognition, or cellular localization.
More generally, a number of protein kinases are known to be associated
with cellular molecular chaperones. Among many molecular chaperones,
HSP90 and Cdc37 are often observed to be complexed with
signal-regulated protein kinases such as pp60v-src
(27, 28), Raf1 (29), Cdk4 (30-32), and CK2 (33-35). HSP90 is
ubiquitously and abundantly expressed in all organisms and tissues and
associates with various kinds of important cellular proteins including
transcription factors and protein kinases, thus assisting correct
folding and functions of target proteins (36-39). On the other hand,
Cdc37 is thought to be a unique molecular chaperone that is involved
specifically in the folding and regulation of protein kinases (30, 35,
40-42).
To elucidate the physiological function and regulation of MOK, we have
biochemically searched for cellular binding proteins of MOK in this
report and identified several cellular MOK-associating proteins. The
results have revealed that the associated proteins consist of a
specific set of molecular chaperones including HSP90 and Cdc37.
Functional importance of the association of molecular chaperones on the
stability of MOK has been presented and discussed.
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EXPERIMENTAL PROCEDURES |
Plasmids--
Plasmids encoding HA-tagged MOK (wild type and
kinase-dead) were described previously (14). Plasmids encoding
FLAG-tagged MOK were constructed by inserting a BamHI
fragment including the entire coding region of MOK into a
BamHI site of pFLAG-CMV2 (Kodak Scientific Imaging Systems).
MRK cDNA was described previously (16), and mammalian expression
vectors with or without tags (C-terminal Myc tag or N-terminal HA tag)
were constructed and kindly provided by Drs. S. Abe and H. Ellinger-Ziegelbauer. Expression vectors encoding two alternative
splicing forms of rat MAK (MAK2.6 and MAK3.8) were described (15).
Mammalian expression vectors encoding conventional MAP kinases
(pSR
-HA-MAP kinase for HA-tagged ERK-type MAP kinase from
Xenopus, pSR
-HA-p38 from human, and pSR
-HA-SAPK/JNK
from rat) are described previously (43, 44). Rat Cdc37 cDNA was
originally isolated by Dr. S. Sakiyama and colleagues (45). An
expression plasmid of rat Cdc37 in pSR
-HA1 was kindly provided by
Dr. S. Matsuda (46). A BglII fragment encoding the Cdc37
coding region was excised and inserted into a BamHI site of
pFLAG-CMV2 to make mammalian FLAG-Cdc37 expression vector.
Antibodies--
Anti-HA (clone 12CA5), anti-FLAG (clone M2), and
anti-Myc (clone 9E10) monoclonal antibodies were from Roche Molecular
Biochemicals, Kodak Scientific Imaging Systems, and Santa Cruz
Biotechnology, Inc., respectively. Anti-MOK (14), anti-HSP90 (47),
anti-GRP94 (48), and anti-FKBP52 (49, 50) rabbit antibodies were
described previously. Anti-HSP60 (clone LK-1) was obtained from
StressGen. Anti-Cdc37 (C-19), anti-HSP/HSC70 (clone W27 that recognizes
both HSP70 and HSC70), anti-HSP70 (goat polyclonal, specific for
HSP70), anti-HSC70 (goat polyclonal, specific for HSC70), and anti-ERK2 (clone D-2) antibodies were purchased from Santa Cruz. Anti-mammalian Hop/STI1 (clone 28) was obtained from Transduction Laboratories. Horseradish peroxidase-conjugated secondary antibodies were from Amersham Pharmacia Biotech (against rabbit and mouse primary
antibodies) and from Santa Cruz (against goat primary antibodies).
Affinity-purified rabbit anti-MAK polyclonal antibody was described
previously (15).
Other Reagents--
Geldanamycin was purchased from Life
Technologies, Inc., and stock solution was prepared at 10 mM in Me2SO. MG-132 was purchased from
Calbiochem, and stock solution was made at 10 mM in
Me2SO. Tran35S-label (mixture of
[35S]Met and [35S]Cys) was obtained from
ICN Biomedicals.
Series of Deletion Mutants of MOK--
A series of deletion
mutants of MOK was produced by in vitro mutagenesis of
plasmids as described below. C-terminal deletion mutants were made by
introducing stop codons at the indicated sites to make truncated
proteins. For example, MOK(N107) encodes amino acids 1-107 of MOK.
N-terminal deletion mutants were made by introducing a new
MfeI restriction enzyme site by in vitro mutagenesis at the indicated positions. The wild type mouse MOK coding
region possesses a unique MfeI site near its N-terminal end
(amino acid 7); thus MfeI treatment and self-ligation of
plasmids after introducing an additional MfeI site resulted
in the N-terminal deletion mutants. For example, MOK(D8-195) encodes a
protein with amino acids 1-7 fused to the sequence from amino acid 196 to the end of MOK, lacking amino acids 8-195.
Site-directed in vitro mutagenesis was performed using
mutagenic primers and DpnI digestion essentially as
described (14). The sense sequences of mutagenic primers used were
5'-GAGAAAAAGATTATGCTCTAGATGTATCAGCTG-3' for MOK(N107),
5'-CACCGACGGGTTCTAGACATACAAGATGGACC-3' for MOK(N178), 5'-CAGCACCCCTATTTCTAGATGCAGAGGGCAGC-3' for MOK(N285),
5'-CAGAAAATCTGGTTCAATTGCACTAATATG-3' for MOK(D8-078),
5'-CTGCGTGTTCTACGCAATTGCCAGCCTGCAGC-3' for MOK(D8-195), and
5'-GTCCCAAGTTCTCAATTGTGCCAGAATCATC-3' for MOK(D8-309).
Expression in Mammalian Cells and Immunoprecipitation of Tagged
MOK--
COS7 cells were cultured in Dulbecco's modified Eagle's
medium with 10% fetal calf serum. Mammalian expression plasmids
encoding various MOK constructs were transfected into COS7 cells by
electroporation as described previously (14). Cell lysates were
prepared, and the tagged protein was immunoprecipitated as described
(14). For affinity purification of FLAG-tagged proteins, beads that had
been covalently conjugated with anti-FLAG antibody were used. For large
scale affinity purification of FLAG-MOK and its associated proteins,
anti-FLAG affinity beads were packed into a column (inner diameter = 0.5 cm), and cell extract was applied to the column three times
followed by extensive washing with cell lysis buffer before elution.
Pulse-Chase Experiments with Geldanamycin Treatment of
Cells--
COS7 cells were transfected with HA-MOK plasmid as above.
48 h later, the culture medium was replaced with Met- and Cys-free Dulbecco's modified Eagle's medium containing 10% fetal calf serum, and cells were incubated with or without geldanamycin (5 µM) for 2 h. Then cells were labeled with
[35S]Met/[35S]Cys in fresh Met- and
Cys-free medium with or without geldanamycin for 2 h. Next,
radioactive medium was removed, and cells were further incubated for
chase in complete medium containing 10 µg/ml cycloheximide for
various times indicated before lysis. When indicated, geldanamycin (5 µM) was also included during the chase period. The amount
of radiolabeled MOK was determined by immunoprecipitation and
SDS-PAGE followed by fluorography of dried gels.
Treatment of Cells with Geldanamycin and
MG132--
MOK-transfected COS7 cells were treated with MG132
(25 µM) or vehicle (Me2SO) for 3 h then
with geldanamycin (5 µM) or control Me2SO for
2 or 4 h. MG132 was also included in the medium during the
incubation with geldanamycin. Cells were collected as described above
and then centrifuged at 17,000 × g for 60 min. The
supernatants were recovered as soluble fractions, and the precipitates
were solubilized by adding diluted (5:7) SDS sample buffer and then boiling for 5 min to obtain insoluble fractions.
Purification of MOK-associated Proteins--
MOK-associated
proteins were isolated by using anti-FLAG affinity beads as described
above. The associated proteins were eluted by treating the beads twice
with a high salt buffer containing 1.5 M NaCl, 2 mM EDTA, 50 mM Tris-HCl, pH 7.4. Eluted
proteins were diluted 10× in a buffer consisting of 50 mM
Tris-HCl, 2 mM EDTA, pH 7.4, and applied on a MonoQ ion
exchange column. The column was washed extensively with 100 mM NaCl, 2 mM EDTA, 50 mM Tris-Cl,
pH 7.4, and bound proteins were eluted and purified by a liner salt
gradient (100 mM to 1.5 M NaCl) in 2 mM EDTA, 50 mM Tris-Cl, pH 7.4. Finally protein
samples were separated by SDS-PAGE, and protein bands were cut out from
the gel. The gel pieces were equilibrated in a buffer (50 mM Tris-Cl, 150 mM NaCl, pH 8.0) and
homogenized in diluted (5:7) SDS sample buffer, and then the gel
suspensions were applied to a second SDS-PAGE for analysis.
Expression and Purification of HSP90 and Cdc37--
Human
HSP90
cDNA was originally from Dr. K. Yokoyama (51). The coding
region was inserted into the EcoRI site of pMAL-cRI (New
England Biolabs) by blunt ligation. The resulting expression plasmid
encodes human HSP90
with maltose-binding protein in the N termini.
The plasmid was introduced into Escherichia coli host strain
TB1. After reaching an A600 level of 0.4, 1 mM isopropyl-1-thio-
-D-galactopyranoside was added, and the culture was further shaken for 2 h at 37 °C. E. coli cells were collected by centrifugation 3300 × g for 15 min at 2 °C, washed with ice-cold
phosphate-buffered saline, frozen at
80 °C, and then resuspended
in a lysis buffer (10 mM sodium phosphate, 30 mM NaCl, 0.25% Tween 20, 10 mM
2-mercaptoethanol, 10 mM EDTA, 10 mM
EGTA, pH 7.0) supplemented with 10 µg/ml leupeptin, 1.7 mM phenylmethylsulfonyl fluoride, and 0.1 trypsin
inhibitor unit/ml aprotinin. Then the cells were ruptured by repeated
freeze-thawing followed by sonication. NaCl concentration was adjusted
to 500 mM, and the solution was centrifuged at 11,000 × g for 30 min at 2 °C to recover the supernatant.
Purification of maltose-binding protein-HSP90 fusion protein by an
amylose column and cleavage with Factor Xa protease were performed
essentially as described in the manual of the manufacturer. Recombinant
HSP90 was further purified by MonoQ column chromatography using a
linear gradient (20-2000 mM NaCl in 50 mM
Tris-Cl, pH 7.4) and then desalted with PD10 column (Amersham Pharmacia
Biotech) in a buffer containing 50 mM Tris-Cl, 0.1 mM EGTA, 0.5 mM dithiothreitol, 10 mM sodium azide, 10% glycerol, pH 7.4. HSP90 from
mammalian sources was purified as described previously (33).
Rat Cdc37 BglII fragment described above was inserted into a
BamHI site of pGEX6P2 (Amersham Pharmacia Biotech). The
resulting plasmid encoding glutathione S-transferase-Cdc37
was introduced into E. coli strain BL21-CodonPlus (DE3)-RIL
(Stratagene). After reaching an A600 level of
0.6, 1 mM
isopropyl-1-thio-
-D-galactopyranoside was added for
induction, and the culture was further shaken for 4 h at 23 °C.
E. coli cells were collected by centrifugation at 3300 × g for 15 min at 2 °C, washed with ice-cold
phosphate-buffered saline, frozen at
80 °C, and resuspended in
B-PER solution (Pierce) supplemented with 10 µg/ml leupeptin, 1.7 mM 4-(2-aminoethyl)benzenesulfonyl fluoride, and 0.1 trypsin inhibitor unit/ml aprotinin. The extract was clarified
by centrifuging at 18,000 × g for 15 min at 2 °C. Glutathione-Sepharose (Amersham Pharmacia Biotech) was mixed with the
extract and rotated for 3 h at 4 °C. The beads were washed extensively with phosphate-buffered saline containing 1% Triton X-100
and then with cleavage buffer (50 mM Tris-Cl, 150 mM NaCl, 1 mM EDTA, 1 mM
dithiothreitol, pH 7.0). Suspension of beads in the cleavage buffer was
incubated with PreScission protease (Amersham Pharmacia Biotech) with
gentle agitation for 16 h at 4 °C. Cleaved recombinant Cdc37
was recovered by centrifugation and then dialyzed extensively in a
buffer (50 mM Tris-Cl, 150 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol, 10% glycerol, pH
7.4).
In Vitro Protein Kinase Assay--
MOK was expressed in COS7
cells and then immunoprecipitated as described (14). The
immunoprecipitates were mixed with 1 µg of substrate proteins and
incubated at 30 °C for 30 min with gentle mixing in phosphorylation
buffer containing [
-32P]ATP as described (14).
Other Procedures--
Silver staining of SDS-polyacrylamide gels
was performed using 2D-Silver Stain II kit (Daiichi Pure
Chemicals). Western blotting was performed using horseradish
peroxidase-conjugated secondary antibodies, and detection was performed
using the chemiluminescent system as described (14).
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RESULTS |
Identification of Proteins That Are Co-immunoprecipitated with
Transfected MOK--
COS7 cells were transfected with a plasmid
encoding HA-tagged MOK protein kinase, and cell lysates were prepared.
MOK protein was immunoprecipitated from the cell lysates with anti-HA
tag antibody, and the immunocomplexes were analyzed by SDS-PAGE and silver staining to visualize possible cellular MOK-associated proteins.
As shown in Fig. 1A, at least
one cellular protein with a molecular mass of ~90 kDa, marked
with an asterisk, was specifically co-immunoprecipitated
along with MOK (lane 4). Although a lot of nonspecifically
attached proteins can be seen because of the high sensitivity of silver
staining, specificity of the co-immunoprecipitation of this protein
with MOK was confirmed as follows. First, the 90-kDa band could not be
observed when lysates of control cells without HA-MOK transfection were
used (Fig. 1A, lanes 1 and 3). In
addition, the association of the 90-kDa protein was not observed when
nonimmune antibody was used for immunoprecipitation (Fig. 1A, lanes 1 and 2). In fact, we
confirmed immunoprecipitation of MOK only in lane 4 (Fig.
1B); thus MOK-associated proteins should also appear only in
lane 4 as was the case for the 90-kDa protein. The
molecular mass of expressed HA-MOK is ~47 kDa (Fig. 1B);
thus the 90-kDa protein cannot be a degradation product of expressed
MOK. Taken together, at least one (but not necessarily only one)
cellular protein with a molecular mass of 90 kDa associates with
MOK.

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Fig. 1.
Identification of a MOK-associated
protein. HA-tagged full-length mouse MOK was expressed by
transient transfection in COS7 cells and was immunoprecipitated with
anti-HA antibody. A, the immunoprecipitates were analyzed by
SDS-PAGE and silver staining. The position of a major 90-kDa
MOK-associated protein is indicated by an arrow with an
asterisk. B, MOK protein in the
immunoprecipitates was revealed by Western blotting with anti-MOK
antibody. The position of HA-MOK is indicated. Lane 1,
control immunoprecipitate with nonimmune antibody from nontransfected
cell lysate; lane 2, control immunoprecipitate with
nonimmune antibody from extract of cells transfected with HA-MOK;
lane 3, anti-HA immunoprecipitate from nontransfected cell
lysate; lane 4, anti-HA immunoprecipitate from
HA-MOK-transfected cell lysate.Imm.Prec.,
immunoprecipitate; N.I., nonimmune.
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Identification of 90-kDa MOK-associated Protein as a Major
Molecular Chaperone HSP90--
Several protein kinases including
pp60v-src, Raf1, Cdk4, and CK2 had been reported to
be associated with a 90-kDa cellular protein, and this protein had been
identified as a major cytosolic molecular chaperone/heat shock protein,
HSP90. Because the molecular mass of the MOK-associated protein
observed in Fig. 1A is similar to that of HSP90, we examined
the possibility of an association between MOK and HSP90 directly by
Western blotting. The same set of immunoprecipitates as in Fig. 1 was
probed with antibody specific for HSP90. As clearly shown in Fig.
2A, HSP90 was detected in the
immunocomplexes only in lane 4, which shows the anti-HA
immunoprecipitate from HA-MOK-expressing cell lysates. HSP90 was
not detected in control immunoprecipitates (lanes 1-3),
confirming specificity of the association between HSP90 and MOK. The
association of HSP90 with HA-MOK was not a result of binding of
HSP90 to the HA tag moiety of the fusion protein because we also
observed the specific co-immunoprecipitation between FLAG-MOK and HSP90
(Fig. 2B). Although these results may not rule out the
possibility of other 90-kDa MOK-associated proteins, they clearly
indicate that MOK is associated with HSP90 in mammalian cells and that
HSP90 can be isolated by co-immunoprecipitation with MOK.

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Fig. 2.
Identification of the 90-kDa
MOK-associated protein as HSP90. A, the same set of
immunoprecipitates as in Fig. 1 was examined for the existence of HSP90
by Western blotting. Lane 1, control (nonimmune)
immunoprecipitate from nontransfected cells; lane 2, control
(nonimmune) immunoprecipitate from extract of cells transfected with
HA-MOK; lane 3, anti-HA immunoprecipitate from
nontransfected cells; lane 4, anti-HA immunoprecipitate from
HA-MOK-transfected cell lysate. B, co-immunoprecipitation of
HSP90 with MOK was examined by using the FLAG tag system. COS7 cells
were transfected with FLAG-MOK expression vector as well as empty
vector, and immunoprecipitates were prepared with anti-FLAG antibody
from the cell lysates. The amounts of HSP90 (upper
panel) and MOK (lower panel) in the immunoprecipitates
were visualized by Western blotting with anti-HSP90 and anti-MOK
antibodies, respectively. Imm.Prec.,
immunoprecipitate; WB, Western blotting; IP,
immunoprecipitation; N.I., nonimmune.
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Treatment of Cells with a Specific HSP90 Function Inhibitor,
Geldanamycin, Induced Destabilization of MOK--
Geldanamycin, a low
molecular mass compound that is a derivative of ansamycin-type
benzoquinoid (52), has been shown to inhibit the molecular chaperone
function of HSP90 (53). After observing the physical association of
HSP90 with MOK as above, we examined the effect of geldanamycin
treatment of cells on MOK. COS7 cells had been transfected with HA-MOK
and treated with 5 µM geldanamycin for the indicated time
periods up to 6 h. Cell lysates were prepared, and changes of the
intracellular amount of MOK after geldanamycin treatment were examined
by Western blotting with antibody against MOK. As shown in Fig.
3A, the amount of MOK was
dramatically and rapidly decreased after the addition of geldanamycin.
An identical result was obtained when we used anti-HA antibody for
Western blotting (data not shown). This result strongly suggested that
MOK requires the molecular chaperone function of HSP90 for its
intracellular stability. As a control, ERK2, a conventional member of
the MAP kinases, did not respond to geldanamycin treatment of cells
(Fig. 3B), indicating that the sensitivity to geldanamycin
treatment is not a general feature of MAP kinase superfamily members.
The time course of the decrease of MOK was similar to that of
endogenous Raf1 (data not shown), a relatively unstable HSP90 target
(29). The decrease of HSP90-associated proteins with much longer
cellular half-lives such as SV40 large T antigen required a much longer
incubation with geldanamycin (54). These facts suggested that the
intracellular half-life of MOK should be relatively short in the
absence of HSP90 function.

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Fig. 3.
Inhibition of HSP90 function induced
destabilization of MOK in cells. COS7 cells transfected with wild
type MOK was treated with 5 µM geldanamycin for the time
periods indicated. A, cell extracts were prepared, and the
amounts of MOK were examined by anti-MOK Western blotting.
B, as a control, the amounts of endogenous ERK2 after
geldanamycin treatment were determined by Western blotting. The
positions of MOK and ERK2 are indicated. Lane 1, 0 h
(control); lane 2, 2 h; lane 3, 4 h;
lane 4, 6 h of geldanamycin treatment. C,
MOK protein stability was examined in the presence or the absence of
geldanamycin (5 µM) by pulse-chase analysis.
HA-MOK-expressing COS7 cells were radiolabeled for 2 h and then
chased in complete medium as described under "Experimental
Procedures." After the indicated time periods of chase (lane
1, 0 h; lane 2, 30 min; lane 3, 1 h; lane 4, 2 h), cell extracts were prepared, and
HA-MOK was immunoprecipitated. The remaining amounts of radiolabeled
MOK were analyzed by SDS-PAGE and fluorography. Upper panel,
immunoprecipitates from control cells; lower panel,
immunoprecipitates from geldanamycin-treated cells. IP,
immunoprecipitation.
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To check directly whether the half-life of MOK in cells is shortened by
geldanamycin treatment, we performed pulse-chase experiments. COS7
cells transfected with HA-MOK were pulse-labeled with
[35S]Met/[35S]Cys for 2 h and then
chased in complete medium up to 2 h. Geldanamycin (5 µM) had been added to cells 2 h before the start of
pulse-labeling and also included during both the pulse and the chase
periods. Several time points after the start of chase, cell extracts
were prepared, MOK was immunoprecipitated, and the amounts of
radioactivity of MOK were analyzed by SDS-PAGE and fluorography. As
shown in Fig. 3C (upper panel), in the control
(treated with vehicle Me2SO) cells, MOK was relatively
stable, and only a minor decrease of synthesized MOK could be seen even
after 2 h. On the other hand, in the presence of geldanamycin, MOK
protein was very unstable, and the half-life of MOK in cells was
shortened to less than 1 h (Fig. 3C, lower
panel). In lane 1 of Fig. 3C, the amounts of radiolabeled MOK were almost equivalent between control and
geldanamycin-treated cells. Moreover, we included the protein synthesis
inhibitor cycloheximide during the chase period. Thus we concluded that
geldanamycin treatment enhanced the net degradation of synthesized MOK
without affecting the rate of protein synthesis of MOK. Taken together,
these results indicate that function of HSP90 was required for the
stable existence of MOK in cells after synthesis, and expressed MOK was
degraded rapidly in the absence of HSP90 function.
Role of Proteasome-dependent Pathways in the
Geldanamycin-induced Destabilization of MOK--
Protein degradation
can be achieved by several protease systems in cells. To examine the
roles of proteasome-dependent degradation pathways in the
instability of MOK in the absence of HSP90 function, we set up
experiments with a specific inhibitor of proteasome, MG132.
MOK-expressing COS7 cells were pretreated with MG132 (25 µM) for 3 h. Then cells were incubated with or
without geldanamycin for up to 4 h, and MG132 was also included
when indicated during geldanamycin treatment. After 2 or 4 h of
incubation, cells were ruptured, and the amounts of MOK in cell
extracts were determined by Western blotting with anti-HA antibody. We
anticipated that geldanamycin-induced degradation of MOK could be
blocked by MG132 treatment if the degradation is mediated by
proteasome-dependent pathways. Unexpectedly, the amounts of
MOK in soluble cellular extracts decreased almost in the same manner
even in the presence of MG132 as indicated in Fig.
4A (compare lanes
1-3 with lanes 4-6). Then we examined the amounts of
MOK in insoluble cell fractions recovered from centrifuge precipitates.
MOK in the insoluble fractions also decreased by geldanamycin treatment
(Fig. 4B, lanes 1-3) as in the case of soluble
fractions. Interestingly, in the presence of MG132, treatment of cells
with geldanamycin induced a significant increase and accumulation of
MOK in the insoluble fractions (Fig. 4B, lanes
4-6). This result indicates the essential role of
proteasome-dependent pathways in the degradation of MOK in
the presence of geldanamycin. In other words, loss of the HSP90
function induced destabilization and degradation of MOK, and if the
degradation was blocked by proteasome inhibitor, undegradable MOK was
aggregated and accumulated in the insoluble fraction.

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Fig. 4.
Involvement of
proteasome-dependent pathways in the
geldanamycin-stimulated MOK degradation. MOK-expressing COS7 cells
were incubated for 3 h with or without proteasome inhibitor MG132
(25 µM) and then further incubated with geldanamycin (5 µM) up to 4 h. The amounts of MOK in soluble
supernatants (A) and insoluble precipitates (B)
were determined by Western blotting with anti-HA antibody. Lanes
1-3, control cells treated with geldanamycin for 0, 2, and 4 h; lanes 4-6, MG132-treated cells further incubated with
geldanamycin for 0, 2, and 4 h. The positions of HA-MOK are
indicated.
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Determination of an HSP90-binding Region in MOK--
MOK consists
of a kinase catalytic domain in the N terminus and a C-terminal region
without known functional motifs that is not homologous to any other
protein. To determine an HSP90-binding region within MOK, we made a
series of deletion mutants of MOK. Wild type mouse MOK consists of 419 amino acids, and MOK(N107), MOK(N178), and MOK(N285) encode C-terminal
deletion mutants (the numbers in parentheses indicate the numbers of
amino acids from the N terminus). MOK(D8-078), MOK(D8-195), and
MOK(D8-309) encode N-terminal deletion mutants but contain the first 7 amino acids of the N-terminal end (the numbers in the parentheses
indicate the numbers of amino acids deleted; see scheme in Fig.
5D). We checked the expression
of the deletion mutants by Western blotting and found all of the
mutants expressed almost equally with expected molecular sizes (Fig.
5A, lanes 1 and 3-7), except a short
N-terminal region of MOK, MOK(N107) (lane 2, see details
below). These deletion mutants were examined for their HSP90 binding
activity by co-immunoprecipitation experiments. The
co-immunoprecipitation of HSP90 was revealed both by silver staining
(Fig. 5B) and by Western blotting (Fig. 5C).
Consistent results were obtained by silver staining and by Western
blotting, showing that C-terminal deletion mutants MOK(N178) (lane 3) and MOK(N285) (lane 4), as well as wild
type MOK(WT) (lane 1), could associate with HSP90 (Fig. 5,
B and C). On the other hand, N-terminal deletion
mutants MOK(D8-078) (lane 5), MOK(D8-195) (lane
6), and MOK(D8-309) (lane 7) could not associate with
HSP90 (Fig. 5, B and C). The relationship between
the HSP90 binding ability and the structures of the MOK deletion
mutants was summarized in Fig. 5D. The results clearly
indicate that the protein kinase consensus catalytic domain in the
N-terminal region of MOK is essential for the HSP90 binding, whereas
the nonconserved C-terminal tail is dispensable. More precisely, amino
acids 8-78 corresponding to kinase subdomains I-IV of MOK were very
important for the HSP90 binding.

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Fig. 5.
Identification of an HSP90-binding region of
MOK using a series of deletion mutants. A, expression
levels of various MOK deletion mutants were examined by anti-HA Western
blotting of the lysates of cells that had been transfected with
corresponding plasmids indicated above the lane
numbers. Associations of HSP90 with the same set of deletion
mutants of MOK as in A were examined by
co-immunoprecipitation experiments and revealed by silver staining
(B) or by Western blotting with anti-HSP90 antibody
(C). D, the relationship between structures and
the HSP90 binding abilities of MOK deletion mutants was schematically
illustrated. ++, bind strongly; +, bind well; , no binding.
WB, Western blotting; IP,
immunoprecipitation.
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Interestingly, mutant MOK(N107) with a large C-terminal deletion, which
was a short N-terminal segment of MOK, was hardly expressed in
COS7 cells (Fig. 5A, lane 2), despite the fact
that all of the mutants were in the same expression plasmid with the same promoter and 3'-untranslated sequences. We also tried to express
this fragment in several other expression plasmids and with various
lengths of the 3'-untranslated region, but we never succeeded in stably
expressing this fragment in mammalian cells (data not shown). We
postulate that this fragment requires HSP90 association but is not able
to bind intracellular HSP90 and therefore is quite unstable and rapidly
degraded in cells as in the case of wild type MOK in
geldanamycin-treated cells as shown in Fig. 3. On the other hand, three
C-terminal fragments were quite stably expressed even though they could
not associate with HSP90 (Fig. 5, A-C, lanes
5-7).
MAK and MRK Are Also Associated with HSP90--
We next examined
whether or not other related protein kinases are also associated with
HSP90. MOK, MRK, two splicing forms of MAK, ERK (Xenopus MAP
kinase), p38, and SAPK/JNK were expressed in COS7 cells (tagged or
without a tag) and immunoprecipitated with corresponding antibodies.
Then the immunoprecipitates were revealed by Western blotting with
anti-HSP90 antibody to examine the kinase-HSP90 association. As shown
in Fig. 6A, MRK (lanes 3 and 4), both forms of MAK (lanes 5 and
6), along with MOK (lane 2), clearly did
associate with HSP90. On the other hand, none of the conventional MAP
kinases (ERK, p38, and JNK/SAPK) was associated with HSP90 (Fig.
6A, lanes 7-9). The expression and
immunoprecipitation levels of these three conventional MAP kinases were
equal to or rather higher than that of MOK and MRK (Fig. 6B,
compare lanes 7-9 with lane 2 and 4),
confirming the specificity of the HSP90 binding only to MOK, MRK, and
MAK but not to conventional MAP kinases.

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Fig. 6.
Association of HSP90 with two closely related
kinases MAK and MRK. A, MOK, MRK, MAK, along with
conventional MAP kinases (ERK, p38, and SAPK/JNK) were expressed in
COS7 cells with either HA tag or Myc tag, or without tag, and
immunoprecipitated with corresponding antibodies. Association of HSP90
with these kinases was examined by Western blotting of the
immunoprecipitates with anti-HSP90 antibody. Lane 1, control
immunoprecipitates from nontransfected cell lysate; lanes
2-9, various kinases as indicated above the lane
numbers were expressed and immunoprecipitated; lane 10,
total cell lysate was examined to indicate the position of HSP90.
B, the same set of immunoprecipitates as in A was
probed with anti-HA antibody to show the nearly equivalent expression
and immunoprecipitation of various HA-tagged protein kinases. The
positions of HA-tagged kinases were shown by asterisks. Note
that in lanes 3, 5, and 6, expressed kinases were
without HA tag and thus not recognized by anti-HA antibody.
HC, heavy chain; LC, light chain.
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Association of Other Cellular Proteins with MOK--
We next
examined whether other proteins are included in the MOK-HSP90
complexes. We performed scale-up isolation of MOK-associated proteins
from lysates of FLAG-MOK-expressing cells by using affinity beads that
had been covalently conjugated with anti-FLAG antibody. Then we
precisely analyzed what kinds of other cellular proteins were isolated
along with MOK and HSP90. As indicated in Fig.
7A (lane 4), in
addition to the 90-kDa protein (shown as band 1), three
other proteins with approximate molecular masses of ~70 (doublet,
band 2), ~60 (band 3), and ~50 kDa
(band 4) were specifically isolated with MOK. All of these
proteins could not be isolated when control lysates were used (Fig.
7A, lanes 1 and 2) or when control
beads were used for affinity purification (Fig. 7A,
lanes 1 and 3), confirming the specificity of the
association of these proteins with MOK. As expected, the specific
isolation of MOK by anti-FLAG affinity beads only from
FLAG-MOK-transfected cell lysates (lane 4) was observed by
Western blotting with anti-MOK antibody (Fig. 7B).

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Fig. 7.
Association of other molecular chaperones
with MOK. FLAG-MOK was expressed in COS7 cells and isolated along
with its associated proteins by anti-FLAG affinity beads. The cells
without transfection and the beads without antibody conjugation were
used as controls. The isolated proteins were eluted by boiling the
beads in SDS sample buffer and separated by SDS-PAGE. Lane
1, purified by control beads from control lysate; lane
2, purified by anti-FLAG beads from control lysate; lane
3, purified by control beads from FLAG-MOK-expressed lysate;
lane 4, purified by anti-FLAG beads from FLAG-MOK-expressed
lysate. A, MOK-binding proteins were revealed by silver
staining. Specifically isolated MOK-binding proteins that appear only
in lane 4 were numbered (1-4) on the
right with their approximate molecular masses. B,
the specific isolation of MOK by affinity purification only from
FLAG-MOK-expressing cell lysate (lane 4) was shown by
Western blotting with anti-MOK antibody. C-E, MOK-binding
proteins were isolated as described above, and associations of
molecular chaperones with MOK were revealed by Western blotting with
antibodies against Cdc37 (C), HSP70 (D), and
HSP60 (E), respectively. The positions of endogenous Cdc37,
HSP70, HSP60, heavy chain (HC) and light chain
(LC) of immunoglobulin, and molecular mass markers were
shown in each panel.
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Association of Cdc37 with MOK--
Several HSP90-associated
protein kinases are known to be associated also with Cdc37, and
mobility of mammalian Cdc37 on SDS-PAGE is ~50 kDa. We postulated
that the MOK-associated 50-kDa protein shown in Fig. 7A
(band 4) can be Cdc37 and examined the possibility by
Western blotting with anti-Cdc37 antibody. The results in Fig. 7C clearly indicated the association of endogenous Cdc37
with affinity-purified MOK (lane 4). Cdc37 could not be
isolated by the control affinity purifications with nontransfected cell
lysates or with control affinity beads (Fig. 7C, lanes
1-3). In addition, we also detected co-immunoprecipitation of
HA-MOK with FLAG-Cdc37 when they were co-expressed in COS7 cells (data
not shown). Taken together, it is concluded that Cdc37 is specifically
associated with MOK-HSP90 complexes in cells.
Association of HSP70 with MOK--
The HSP70 family is another
major molecular chaperone that has been often observed in HSP90 target
protein complexes. We examined the association between HSP70 and MOK by
Western blotting with antibody recognizing both HSC70 (constitutive
form) and HSP70 (inducible form). MOK-associated proteins were isolated
by affinity purification as described above, and HSP70 binding was
detected (Fig. 7D, lane 4). The association of
HSP70 was not observed by control affinity purifications (Fig.
7D, lanes 1-3). We concluded that HSP70/HSC70 is
also a member of the MOK-HSP90-Cdc37 protein complexes. Because the
70-kDa protein in Fig. 7A (band 2) was observed
as a closely migrating doublet, we speculated that the doublet consists
of HSP70 and HSC70. We cut out separately the upper and lower migrating
70-kDa bands from the gel and examined the reactivity of eluted
proteins with two antibodies that recognize specifically either HSP70
or HSC70. The upper (slower) migrating band was recognized by
anti-HSC70 but not by anti-HSP70, whereas the lower (faster) migrating
band was recognized by anti-HSP70 but not by anti-HSC70 (data not
shown). The results clearly indicated that both HSC70 and HSP70 are
associated with MOK.
Association of Other Molecular Chaperones with MOK--
Next, the
same set of affinity-purified samples as in Fig. 7 (A-D)
was revealed by Western blotting with antibody specific for HSP60. The
result clearly indicated that endogenous HSP60 was co-purified with MOK
(Fig. 7E, lane 4). Again, HSP60 could not be
isolated by control affinity purifications (Fig. 7E,
lanes 1-3) showing the specificity of the association.
FKBP52 (FKBP59) and Hop (STI1) have been observed often in complexes of
HSP90-steroid hormone receptors, and both proteins possess molecular
masses of ~60 kDa; however, we could not detect FKBP52 or Hop as
MOK-associated proteins (data not shown).
Although we detected the above molecular chaperones in the
MOK-associated protein fraction, a possibility exists that molecular chaperones account for only a minor part of the isolated protein bands
seen in Fig. 7A. However, this possibility was excluded as
follows. As described under "Experimental Procedures," we purified all of the MOK-associated proteins (Fig. 7A, bands
1-4) by affinity purification and MonoQ ion exchange column
chromatography, and then finally the protein bands were excised after
SDS-PAGE. No other protein with the same molecular masses was observed
during the purification steps (data not shown). The purified (more than 95% pure) proteins were recognized by antibodies specific for HSP90,
HSP70, HSC70, Cdc37, and HSP60, respectively (data not shown),
indicating that the MOK-associated proteins observed in Fig.
7A were a set of molecular chaperones themselves and that no
other protein was hindered at the same molecular masses.
Cdc37, but Not HSP90, Was Phosphorylated by MOK--
As described
above, MOK was found to be associated with molecular chaperones; thus a
component(s) of the complexes could be phosphorylated by MOK protein
kinase. We immunoprecipitated MOK from transfected cells and examined
whether MOK phosphorylates purified HSP90 and/or Cdc37 in
vitro. Cdc37 and HSP90 were expressed as glutathione
S-transferase tag fusion proteins in E. coli and purified by tag-dependent affinity chromatography. A
kinase-dead version of MOK, MOK(KD), was used as a negative control. As
reported previously (14), myelin basic protein is a good substrate
in vitro for immunoprecipitated MOK (Fig.
8A, lane 4). Cdc37
was also significantly phosphorylated by immunoprecipitated MOK (Fig. 8B, lane 4). On the other hand, phosphorylation
of HSP90 was hardly observed in the same condition (Fig.
8C). We also checked MOK-dependent phosphorylation of purified HSP90 from several mammalian sources (COS7
cells, L5178Y cells, mouse liver, and porcine brain), but none of them
was phosphorylated significantly by MOK (data not shown). These results
indicated that Cdc37, but not HSP90, is an in vitro
substrate of MOK.

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Fig. 8.
Phosphorylation of Cdc37 by
immunoprecipitated MOK in vitro. FLAG-tagged wild
type MOK(WT) was expressed in COS7 cells and immunoprecipitated by
anti-FLAG antibody. The immunoprecipitates were incubated with myelin
basic protein (A), Cdc37 (B), or HSP90
(C) in the presence of [ -32P]ATP under
phosphorylation conditions. A kinase-dead mutant of MOK, MOK(KD), was
used as a negative control (lanes 1 and 3).
Coomassie Brilliant Blue (CBB) staining of the
phosphorylation mixtures (lanes 1 and 2) and the
corresponding autoradiogram (lanes 3 and 4) after
SDS-PAGE are shown. The positions of myelin basic protein, Cdc37,
HSP90, and heavy (HC)/light (LC) chains of
immunoglobulin are indicated.
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DISCUSSION |
In this report, we described identification of several cellular
MOK-binding proteins by using a mammalian expression system and
co-immunoprecipitation experiments. The data have clearly shown that
the majority of the MOK-associated proteins is a set of molecular
chaperones including HSP90, Cdc37, HSP70, HSC70, and HSP60, although we
do not exclude a possibility of other MOK-associated proteins in cells.
Previously, associations of HSP90 and Cdc37 with certain important
protein kinases such as pp60v-src (27, 28), Raf1
(29), and Cdk4 (30-32) have been described. Among a wide variety of
proteins that are associated with HSP90, only certain protein kinases
are associated with Cdc37 along with HSP90 (40). Thus the combination
of Cdc37 and HSP90 can be considered as a kinase-specific chaperone
complex (30, 35, 42). The results obtained here also support this idea.
It should be noted that not all of the protein kinases are associated
with HSP90-Cdc37 complexes, i.e. there is specificity for the binding. In fact, our results demonstrated that conventional MAP kinases (ERK, p38, and JNK/SAPK) did not bind HSP90 despite the
high homologies in the catalytic domains of conventional MAP kinases
and MOK. MOK is most intimately related to MAK and MRK, two closely
related protein kinases that show similarity to both the MAP kinases
and the cyclin-dependent protein kinases (9), and we
observed here that MAK and MRK also bind HSP90. It was reported that
HSP90-Cdc37 binds Cdk4 and Cdk6 but not Cdc2, Cdk2, Cdk3, Cdk5, or CAK
(31, 32, 55). Taken together, there seems to be a mechanism of
HSP90-Cdc37 that discriminates subtle differences between related kinases.
Our experiments with MOK deletion mutants clearly demonstrated that
HSP90 requires the protein kinase catalytic domain of MOK for binding.
It is also the case for other kinases such as pp60v-src (56), Raf (29), Plk (57), and CK2 (34).
Thus the HSP90 binding specificity seems to be determined by structures
of kinase domains, and it will be interesting to identify a region that is responsible for the HSP90 binding specificity. However, a
noncatalytic C-terminal part was reported to be important for HSP90
binding of Akt protein kinase (58); thus the rule may not be definitely generalized.
What is the physiological meaning of the association of these molecular
chaperones with MOK? Previously we observed that MOK is activated by
tumor promoter stimulation (14). In addition, most of the
HSP90-associated proteins are classified into signal switching
molecules (37). These facts imply that HSP90 is required for an
activable structure of MOK that is essential for its function. In this
article, we found that HSP90 is essential for the stability of MOK in
cells. In other words, MOK was not able to stably exist in the absence
of HSP90 and then rapidly degraded. Protein folding and protein
stability are intimately interrelated, and once a protein fails to be
correctly folded, the protein should be degraded. We have shown that
MOK degradation in the absence of HSP90 function was carried out by the
proteasome system and that MOK protein accumulated in the insoluble
fraction if both HSP90 and the proteasome system were inactivated. Thus
the balance between folding, degradation, and solubility of MOK should
be controlled by HSP90-Cdc37 molecular chaperones and proteasome
systems. This kind of link between protein folding and degradation is
not restricted only for MOK but for many other HSP90 target proteins
(59, 60). The pharmacological effect of the anti-cancer drug
geldanamycin was previously believed to be ascribed to the specific
inhibition of growth-related protein-tyrosine kinases. However, knowing
that inhibition of HSP90 function leads to degradation of certain other
kinases also as shown here, the effect of geldanamycin treatment should
be more complicated.
HSP90 often associates with target proteins not alone but with several
other molecular chaperones that are called "co-chaperones" (61) or
"HSP90 partner proteins" (38). In fact, we found here that HSP70
and HSC70 as well as Cdc37 are also included in the MOK-HSP90
complexes. For most of the steroid hormone receptors, high molecular
mass FK506-binding immunophilin FKBP52 is associated with the receptor
along with HSP90. Other co-chaperones such as p23, Hip, Hop
(STI1), and HSP40 (eukaryotic DnaJ) are also observed in certain HSP90
target complexes. However, we could not detect FKBP52, Hop, GRP94, or
HSP27 in the affinity-purified MOK-associated protein fraction (data
not shown). Thus it seems that specificity exists in the selection of
limited members of molecular chaperones for particular targets. Our
previous immunofluorescent study revealed that MOK localizes mostly in
the cytoplasm when expressed exogenously (14). On the other hand,
most of mammalian HSP60 resides within mitochondria, and we do not know
whether MOK associates with HSP60 within mitochondria or in the
cytoplasm. Mitochondria might be ruptured during the cell lysis
procedure; thus we cannot exclude the possibility that the HSP60
association with MOK may be observed only after cell lysis. In
addition, by our co-immunoprecipitation experiments it is not clear
whether MOK is associated with all of these molecular chaperones
cooperatively in the same complexes or each molecular chaperone
associates with MOK independently.
For efficient and specific signal transmission of the MAP kinase
cascades, tethering proteins (also called molecular glues or molecular
scaffolds) are often observed and thought to be important that
associate with each of the members of the cascades (25, 26). Thus a
certain HSP90-associated protein kinase may be an upstream activator
for MOK. Associated proteins can be also direct substrates for the
kinase, and in fact molecular chaperones including HSP90, Cdc37, and
FKBP52 have been reported to be phosphorylated by their associated
protein kinases (33, 35, 50). Although Cdc37 was found to be
phosphorylated by MOK in vitro, it is not obvious whether
the phosphorylation of Cdc37 transmits a downstream signal from
activated MOK.