Usage of Tautomycetin, a Novel Inhibitor of Protein Phosphatase 1 (PP1), Reveals That PP1 Is a Positive Regulator of Raf-1 in
Vivo*
Shinya
Mitsuhashi
,
Hiroshi
Shima
,
Nobuhiro
Tanuma
,
Nobuyasu
Matsuura§,
Mutsuhiro
Takekawa¶
,
Takeshi
Urano**,
Tohru
Kataoka
,
Makoto
Ubukata§, and
Kunimi
Kikuchi
§§
From the
Division of Biochemical Oncology and
Immunology, Institute for Genetic Medicine, Hokkaido University,
Kita-15, Nishi-7, Kita-ku, Sapporo 060-0815, Japan, the
§ Laboratory of Biofunctional Chemistry, Biotechnology
Research Center, Toyama Prefectural University, Kosugi, Toyama
939-0398, Japan, the ¶ Division of Molecular Cell Signaling,
Institute of Medical Science, University of Tokyo, 4-6-1 Shiroganedai,
Minato-ku, Tokyo 108-8639, Japan,
PRESTO, Japan Science and
Technology Corporation (JST), Kawaguchi, Saitama, 332-0012, Japan,
** Department of Biochemistry II, Nagoya University School of
Medicine, 65 Tsurumai-machi, Showa-ku, Nagoya 466-0065, Japan, and

Division of Molecular Biology, Department
of Molecular and Cellular Biology, Kobe University Graduate School of
Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan
Received for publication, August 30, 2002, and in revised form, September 27, 2002
 |
ABSTRACT |
Protein phosphatase type 1 (PP1), together with
protein phosphatase 2A (PP2A), is a major eukaryotic serine/threonine
protein phosphatase involved in regulation of numerous cell functions. Although the roles of PP2A have been studied extensively using okadaic
acid, a well known inhibitor of PP2A, biological analysis of PP1
has remained restricted because of lack of a specific inhibitor. Recently we reported that tautomycetin (TC) is a highly specific inhibitor of PP1. To elucidate the biological effects of TC, we demonstrated in preliminary experiments that treatment of COS-7 cells
with 5 µM TC for 5 h inhibits endogenous PP1
by more than 90% without affecting PP2A activity. Therefore, using TC
as a specific PP1 inhibitor, the biological effect of PP1 on MAPK
signaling was examined. First, we found that inhibition of PP1 in COS-7 cells by TC specifically suppresses activation of ERK, among three MAPK
kinases (ERK, JNK, and p38). TC-mediated inhibition of PP1 also
suppressed activation of Raf-1, resulting in the inactivation of the
MEK-ERK pathway. To examine the role of PP1 in regulation of Raf-1, we
overexpressed the PP1 catalytic subunit (PP1C) in COS-7 cells and found
that PP1C enhanced activation of Raf-1 activity, whereas
phosphatase-dead PP1C blocked Raf-1 activation. Furthermore, a physical
interaction between PP1C and Raf-1 was also observed. These data
strongly suggest that PP1 positively regulates Raf-1 in
vivo.
 |
INTRODUCTION |
Protein phosphatases regulate numerous cellular functions and
signal transduction pathways in cooperation with protein kinases (1,
2). Protein phosphatase types 1 and 2A, known as
PP11 and PP2A, are two of
four major protein serine/threonine phosphatases (PPs) that regulate
diverse cellular events such as cell division, transcription,
translation, muscle contraction, glycogen synthesis, and neuronal
signaling (3-5).
Okadaic acid (OA), a polyether fatty acid from the marine black sponge
Halichondria okadai, was first identified as a small molecular weight inhibitor of PP and has been studied extensively (6).
More than 40 compounds that inhibit PP1 as well as PP2A have been
identified. Using these natural compounds, numerous experiments have
been performed to analyze the roles of PPs in various cellular events
(6, 7). The IC50 values of such phosphatase inhibitors are
almost identical for PP1 and PP2A, with the exception of compounds such
as OA, TF-23A, and fostriecin (8-10). PP2A is selectively inhibited by
OA, TF-23A, and fostriecin, and this selectivity has made it possible
to analyze PP2A function in living cells. However, no known inhibitor
inhibits PP1 specifically.
Oikawa et al. (11) reported the total chemical synthesis of
tautomycin (TM), a small molecular weight PP inhibitor originally isolated from Streptomyces spiroverticillatus. Using the
synthesized TM and related compounds, we previously examined the
structure-function relationship of TM and found that the left- and
right-hand moieties of TM are required for inhibition of PP and
induction of apoptosis, respectively (12). We also reported that the
spiroketal structure in the right-hand moiety of tautomycin has nothing
to do with phosphatase inhibition but rather induces apoptosis (13).
These results strongly suggest that tautomycetin (TC), an
antifungal antibiotic originally isolated from Streptomyces
griseochromogenes (14), could be a potent PP1-specific inhibitor
that would likely exhibit few nonspecific effects because it is
structurally similar to TM but lacks a spiroketal structure (15).
Recently, we demonstrated that TC is a specific PP1 inhibitor in
vitro and proposed that TC may be used as a novel powerful
probe to elucidate the physiological roles of PP1 in various biological
events (16).
PP1 is composed of the catalytic subunit (PP1C) and a wide variety of
targeting/regulatory subunits (4, 5). Thus far four PP1C isoforms,
,
1,
2 and
, have been identified that are widely expressed in
mammalian tissues (17-21). Biochemical analysis using bacterially
expressed PP1C isoforms of all four types has shown that they have
similar properties (22). PP1C may be regulated by its interaction with
a variety of subunits that appear to target PP1C to specific
subcellular locations and define substrate specificity.
For three decades, we have extensively investigated neoplastic
alterations in hepatomas of enzymes involved in glycogen metabolism and
containing protein phosphatases. We first found that PP1
activity was markedly elevated in rat ascites hepatomas (23, 24). Then we observed that levels of both PP1
mRNA and PP1
protein were irreversibly increased in hepatomas and that PP1
protein accumulated in the non-nuclear membrane fraction and the nuclei (19, 25-27). The
increase in PP1
mRNA expression seen in rat ascite hepatoma cells was due to the enhanced promoter activity of the PP1
gene (28,
29). In contrast to increases in PP1 expression seen in rat ascite
hepatoma cells, PP2A and PP2C expression was not increased but rather
was down-regulated (26). These results strongly suggested a positive
involvement of PP1 in regulating cell growth of tumor cells. However,
the mechanism underlying this role of PP1 remained unknown.
Activation of mitogen-activated protein kinase (MAPK) cascades plays a
key role in transducing various extracellular signals to the nucleus
(30-32). Three distinct MAPK cascades have been described:
extracellular signal-regulated kinases (ERK), c-Jun NH2 kinases (JNK), and homologues of the budding yeast HOG1
protein (p38). Activation of MAPKs requires phosphorylation of
conserved threonine and tyrosine residues by dual-specificity MAPK
kinases, which in turn are activated by the phosphorylation of two
serine residues by upstream MAPK kinase kinases. The ERK pathway
(Raf-MEK1,2-ERK1,2) is activated by mitogen via Ras and by phorbol
esters via protein kinase C. The stress-activated MAPK pathways JNK
(MEK kinase 1,3-SEK1,2-JNK1,2,3) and p38 (ASK1,
TAK1-MKK3,6-p38
,
,
,
) are activated by cellular stress,
e.g. UV light, osmotic and oxidative stress, and
inflammatory cytokines (30-32). Phosphorylation of MAPKs results in
their translocation to the nucleus, where they activate transcription
factors by phosphorylation. Activities of MAPKs, MAPK kinases, and MAPK
kinase kinases are also regulated by dephosphorylation at serine,
threonine, and tyrosine residues by serine/threonine, tyrosine, and
dual-specificity phosphatases, respectively (32-37). Numerous
observations suggest that PP2A plays a major role in the
down-regulation of JNK, MEK, and ERK activities. Therefore, the
inhibition of intracellular PP2A by OA leads to the activation of these
enzymes (6, 7, 35). However, the involvement of PP1 in MAPK pathways
has not been studied because of the lack of a PP1-specific inhibitor.
In the present study, we found that treatment of COS-7 cells with 5 µM TC selectively inhibited PP1 activity by more than 90% without affecting PP2A activity, demonstrating that TC is a useful
tool for analysis of the biological function of PP1. Using TC, we then
examined the involvement of PP1 in regulating MAPKs. These results are
summarized as follows: (i) TC specifically inhibits activation of ERK
among three MAPKs (ERK, JNK, and p38) upon treatment of cells with TPA
and EGF; (ii) TC treatment suppresses activation of Raf-1 activity,
which results in inactivation of the MEK-ERK pathway; (iii)
overexpression of phosphatase-dead mutants of PP1C on the Raf-1-MEK
pathway results in effects similar to those found in TC treatment of
cells; (iv) PP1C physically interacts with Raf-1. These results are the
first demonstration that PP1 is a positive regulator of the Raf-MEK-ERK pathway.
 |
EXPERIMENTAL PROCEDURES |
Reagents--
TC was prepared from S. griseochromogenes as described previously (14, 15). OA and TC were
dissolved in dimethyl sulfoxide (Me2SO) and were
stored at
80 °C. Rabbit skeletal muscle glycogen phosphorylase and
epidermal growth factor (EGF) were purchased from Sigma.
Purified protein phosphatase type 2B (PP2B)/calcineurin from bovine
brain was purchased from Upstate Biotechnology. Phosphorylase kinase,
recombinant GST-I-2 (a specific inhibitor of PP1), GST-PP5 (protein
phosphatase type 5), GST-MEK-Hisx6, GST-KNERK, and GST-LDP-1 (low
molecular weight dual-specificity phosphatase (DSP-1)) were prepared as
described previously (22, 38, 39). An anti-MEK-1 antibody, H-8, was
purchased from Santa Cruz Biotechnology. Other reagents were purchased
from Wako (Osaka, Japan).
Mammalian Expression Vectors--
pSR
-HA-ERK2,
pSR
-HA-JNK1, pMT3-HA-p38
, pEBG-MEK1 (GST-MEK1), pEBG-SEK1
(GST-SEK1), pEBG-MKK6 (GST-MKK6), pH8-Flag-Raf-1, pcDNA3-Myc-PP1
1, and pcDNA3-Myc-PP1
1(H125A) were
described previously (36-38, 40).
Cell Culture--
COS-7 and 293-T cells were maintained in
Dulbecco's modified Eagle's medium (Sigma) or RPMI 1640 medium
(Invitrogen) containing 10% fetal bovine serum, 1.9 g/liter sodium
bicarbonate, 100 µg/ml streptomycin, and 20 units/ml penicillin G
(termed "complete medium") at 37 °C under 5%
CO2.
Cell Treatment and Phosphatase Assays--
8-9 × 105 cells were cultured in 1 ml of complete medium in 35-mm
dishes. Five µl of Me2SO or Me2SO plus OA
or/and TC was added, and the cells were incubated at 37 °C for
5.5 h. Adherent cells were washed with 1 ml of phosphate-buffered
saline (divalent, cation-free) on ice and scraped in 100 µl of
hypotonic buffer (50 mM Tris-HCl (pH 7.5), 1 mM
EGTA, 0.1%
-mercaptoethanol, 1 mM benzamidine, 10 µg/ml leupeptin, 10 µg/ml soybean trypsin inhibitor, and 10 µg/ml
aprotinin) at 4 °C. Suspensions were subjected to three cycles of
freezing in liquid nitrogen followed by thawing at 30 °C and were
then centrifuged at 4 °C for 10 min at 15,000 rpm. The resulting
supernatants were used as enzymes.
Phosphorylase was 32P-labeled by phosphorylase kinase to 1 mol of phosphate/mol of phosphorylase and used at 5 µM in
the assay (16, 41). PP1 and PP2A activities were measured as described previously (16, 41) with a slight modification. Briefly, 10 µl of
cell-free extracts were diluted with solution A (50 mM
Tris-HCl (pH 7.5), 0.1 mM EGTA, and 0.1% (v/v)
-mercaptoethanol) containing 1 mg/ml bovine serum albumin. The
diluted enzymes were then preincubated with 30 µl of solution A
containing 0.33 mg/ml bovine serum albumin and 0.02% (w/v) Brij-35
with or without the inhibitor for 15 min at 30 °C. The reaction was
initiated with 32P-labeled substrate in 20 µl of solution
A containing 15 mM caffeine. After 10 min at 30 °C, the
reaction was stopped by adding 50 µl of 10 mM
H2SO4 acid solution containing 20 mM silicotungstic acid, and the solution was centrifuged.
Subsequent procedures were essentially the same as those described
previously (16, 41). One unit of the enzyme was defined as the amount
of enzyme required to catalyze the release of 1 µmol of
phosphate/min. The activity of calcineurin was measured as described
previously (42).
Transient Transfection and Stimuli--
COS-7 and 293-T cells in
35-mm dishes were co-transfected with 1 µg of pEBG-MEK1, pEBG-SEK1,
or pEBG-MKK6 together with 1 µg of pSR
-HA-ERK2, pSR
-HA-JNK1, or
pMT3-HA-p38
, respectively. For transient assays, cells were
transfected using Fugene-6 (Roche Diagnostics Inc., Mannheim, Germany)
according to the manufacturer's recommendation. Forty hours after
transfection, cells were maintained with or without phosphatase
inhibitors for 4.5-5.5 h and then stimulated with either
12-O-tetradecanoyl-13-phorbol acetate (TPA) or EGF for ERK2
activation. For JNK1 and p38
activation, cells were stimulated with
0.4 M sorbitol for 30 min.
Detection of Activated MAPKs and MEK by
Immunoblot--
Transfected cells were washed with 1 ml of
phosphate-buffered saline on ice and lysed by sonication in MAPK lysis
buffer (20 mM Tris-HCl (pH 7.5), 1% Triton X-100, 0.5%
deoxycholate, 10% glycerol, 137 mM NaCl, 5 mM
EDTA, 50 mM
-glycerophosphate, 2 mM
orthovanadate, 20 mM NaF, 1 mM dithiothreitol,
0.5 mM benzamidine, 10 µg/ml leupeptin, and 10 µg/ml
aprotinin). Extracts were prepared by centrifugation at 20,000 × g for 10 min. Each sample was separated by
SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to
nitrocellulose membranes (Amersham Biosciences). The phosphorylation status of activated MAPKs, MEK1, and SEK1 was monitored by
anti-phospho-ERK antibodies (New England Biolabs (NEB)), anti-ACTIVE
JNK antibody (Promega), an anti-phospho p38 antibody (NEB), an
anti-phospho MEK1/2 antibody (NEB), or an anti-phospho SEK1/MKK4
antibody (NEB) followed by horseradish peroxidase-conjugated donkey
anti-rabbit IgG secondary antibody (Chemicon International, Temecula,
CA). The expression levels of HA-tagged MAPKs and GST-tagged MAPK
kinases were monitored by anti-HA (12CA5) monoclonal (Roche
Diagnostics) anti-GST monoclonal antibodies (CG1B), respectively,
followed by horseradish peroxidase-conjugated rabbit anti-mouse IgG
secondary antibody (Kirkegaard & Perry Laboratories, Gaithersburg, MD). Signals were detected using the enhanced chemiluminescence reagent (ECL, Amersham Biosciences).
Assay for Raf Kinase Activity--
Transfected or treated cells
on 35-mm dishes were lysed by sonication in MAPK lysis buffer. After
centrifugation of cell lysates at 20,000 × g
for 10 min, the resulting supernatant was used as a cellular extract.
FLAG epitope-tagged Raf-1 in extracts was immunoprecipitated with an
anti-FLAG M2 monoclonal antibody (Sigma). Raf kinase activity was
determined by incubating immunoprecipitates in the presence of GST-MEK
(0.1 µg) and GST-KNERK (1.5 µg) in 40 µl of kinase reaction
mixture (20 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 20 mM
-glycerophosphate, 0.4 mM benzamidine, 5% glycerol, and 100 µM ATP)
for 8-25 min at 30-35 °C. After incubation, proteins in the
reaction mixture were separated by SDS-PAGE. Phospho-ERK and Raf-1 were
detected by immunoblotting with anti-phosphoERK antibody,
anti-Raf-1 antibody C12, or anti-Raf-1 antibody E10 (Santa Cruz
Biotechnology). The amounts of phospho-ERK and Raf-1 were quantified
using a luminescent image analyzer, LAS-1000 Plus (Fujifilm, Tokyo, Japan).
Co-immunoprecipitation--
Transfected cells were lysed in 300 µl/100-mm plate co-immunoprecipitation buffer (50 mM
Tris-HCl, pH 7.5, 30 °C, 4 mM EDTA, 5% glycerol, 0.1%
Triton X-100, 1 mM benzamidine, 50 mM
-glycerophosphate, 2 mM orthovanadate, 0.1%
-mercaptoethanol) containing 10 µg/ml leupeptin and 10 µg/ml
aprotinin. Cell lysates were centrifuged at 20,000 × g
for 10 min. 150 µl of supernatant was incubated with anti-Raf-1
antibody (1.6 µg) or anti-Myc antibody (14 µg). After rotation for
30 min at 4 °C, 5 µl of protein G-Sepharose 4 fast flow (Amersham
Biosciences) was added to the mixture. After rotation for 1.5 h at
4 °C, the beads were washed with 1 ml of co-immunoprecipitation
buffer. The immunoprecipitates were resuspended in 25 µl of 1.25×
Laemmli's SDS sample buffer, boiled for 5 min, separated by SDS-PAGE
on 9% gels, and transferred to a nitrocellulose membrane (Amersham
Biosciences). Raf-1 or Myc-tagged proteins were detected by
immunoblotting with the respective antibodies.
Protein Measurement--
The protein concentration was measured
by a modification of the method of Bradford using bovine serum albumin
as a standard (43). Briefly, cell lysates were diluted with 0.01%
Triton X-100. 50 µl of diluted lysate was added to 0.95 ml of a
mixture of 0.2 ml of dye reagent (Bio-Rad) and 0.75 ml of 0.01% Triton
X-100. After incubation for 10 min at room temperature, the sample was read against appropriate solvent dye blanks at 595 nm.
 |
RESULTS |
Tautomycetin Specifically Inhibits PP1 Activity in COS-7
Cells--
We recently demonstrated that tautomycetin is the most
specific PP1 inhibitor among more than 40 phosphatase inhibitors
assayed in vitro; however, its biological effect remained to
be examined (16). To do so, we first measured the activity levels of
PP1 and PP2A in extracts prepared from COS-7 cells pretreated with TC
or okadaic acid, an inhibitor of PP2A (Fig.
1). From preliminary experiments
of dose responsivity, the concentrations required for complete
inhibition of PP1 and PP2A in COS-7 cells were 5 µM TC
and 100 nM OA, respectively (data not shown). To
differentiate between residual phosphatase activity in cell extracts,
phosphatase activity levels were measured using phosphorylase a
as a substrate in the presence or absence of 1 nM OA to
specifically inhibit PP2A or 167 nM I-2 to specifically
inhibit PP1. Total phosphatase activities in extracts of OA-treated
cells were resistant to 1 nM OA, and ~90% of the
phosphatase activity was inhibited by 167 nM I-2, showing
that OA treatment specifically inhibits intracellular PP2A. Residual
phosphatase activity in extracts from TC-treated cells was resistant to
167 nM I-2 and completely inhibited by 1 nM OA.
These results strongly suggest that TC penetrates cell membranes and
binds to and specifically inhibits PP1, and the binding between TC and
PP1C is tight enough to resist dissociation during preparation of cell
extracts. We then examined the effects of TC on other phosphatases. TC
had no effect on purified calcineurin and LDP-1 up to 1 µM. The IC50 for PP5 is ~1
µM higher than for PP2A (data not shown). From these
results, we concluded that TC could be used as a tool to investigate
the biological function of PP1.

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Fig. 1.
Inhibition of phosphatase activities in COS-7
cells by OA and TC. COS-7 cells were treated for 5.5 h with
vehicle (None), 100 nM OA, 5 µM
TC, or 100 nM OA and 5 µM TC (OA + TC). The lysates were assayed for phosphatase activity using
32P-labeled phosphorylase a as a substrate. Assays
were carried out without inhibitors or with 1 nM OA, 167 nM GST-I-2, or 1 nM OA plus 167 nM
GST-I-2. The data shown represent the mean ± S.D. of duplicate
assays from three separate experiments.
|
|
Tautomycetin Inhibits Activation of ERK in Cells--
It is known
that PP2A inhibits the activation of ERK and JNK, based on observations
that treatment of cells with OA results in
hyperphosphorylation/activation of ERK and JNK (6, 35). Because we were
interested in the role of PP1 in the regulation of MAPK activation, we
compared the effects of OA and TC on activation of HA-ERK2, HA-JNK1, or
HA-p38
expressed in COS-7 and 293T cells (Fig.
2). As expected, OA at 100 nM
increased the phosphorylation levels of HA-ERK2 in unstimulated COS-7
and 293T cells (Fig. 2A, lanes 3 and
9). However, in contrast, TC at 5 µM
dramatically decreased the phosphorylation levels of HA-ERK2 activated
by TPA in COS-7 and 293T cells (Fig. 2A, lanes 6 and 12). On the other hand, TC and OA showed similar
positive effects on JNK activation (Fig. 2B), but neither TC
nor OA showed any effect on p38 activation (Fig. 2C). These
results suggest that inhibition of PP1 by pretreatment of cells with TC
specifically blocks ERK activation.

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Fig. 2.
Distinct effects of OA and TC on three MAPKs
activation. COS-7 (lanes 1-6) and 293-T (lanes
7-12) cells were transfected with pSR -HA-ERK2 plus pEBG-MEK1
(A), pSR -HA-JNK1 plus pEBG-SEK1 (B), or
pMT-HA-p38 plus pEBG-MKK6 (C). Transfected cells were
incubated for 4.5 h with vehicle (None: lanes
1, 2, 7, and 8), 100 nM OA
(lanes 3, 4, 9, and 10), or 5 µM TC (lanes 5, 6, 11, and
12). Cells were then stimulated with either 4 ng/ml phorbol
12-myristate 13-acetate for 30 min (for ERK2 activation) or 0.4 M sorbitol for 30 min (for JNK1 and p38 activation).
Following cell lysis, immunoblots were performed as described under
"Experimental Procedures." Similar results were obtained in three
separate experiments.
|
|
Tautomycetin Inhibits Activation of MEK in Cells--
ERK is
phosphorylated directly by MEK. To clarify whether inhibition of ERK
activation is due to suppression of MEK activation, we compared the
effect of TC on GST-MEK activation with that on HA-ERK upon stimulation
at various concentrations of TPA in COS-7 cells. As shown in Fig.
3A, TC decreased
phosphorylation/activation of GST-MEK substantially. The rates of
decrease in phospho-GST-MEK by TC treatment were similar to those seen
with phospho-GST-MEK (Fig. 3A, compare lanes
14 and 15 with lanes 4 and 5),
suggesting that inhibition of ERK activation results from inhibition of
MEK activation. Under these conditions, OA enhanced phosphorylation of
MEK as well as ERK (6, 7, 32).

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Fig. 3.
Effects of OA and TC on activation of ERK and
MEK in COS-7 cells. COS-7 cells were transfected with
pSR -HA-ERK2 and pEBG-MEK1. The transfected cells were incubated for
5.5 h with vehicle (None: lanes 1-6), 100 nM OA (lanes 7-12), or 5 µM TC
(lanes 13-18). Cells were then stimulated with either TPA
for 10 min (A) or EGF for 4 min (B) at the
indicated concentrations. Immunoblots were performed as described under
"Experimental Procedures." Similar results were obtained in four
separate experiments.
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|
To clarify whether inhibition of MEK activation is a TPA-specific
event, we analyzed the effect of TC on MEK and ERK activation by EGF.
As shown in Fig. 3B, TC also suppressed phosphorylation of
GST-MEK and HA-ERK induced by EGF. We also examined the effect of TC on
SEK1 and MKK6. The effects of TC on SEK1 and MKK6 activation were
similar to that of JNK and p38, respectively (data not shown). These
results indicate that TC specifically blocks MEK activation.
Dose-dependent Inhibition of MEK by
Tautomycetin--
Dose-dependent inhibition of EGF- and
TPA-induced MEK activation by TC was analyzed. As shown in Fig.
4, TC decreased phosphorylation levels of
MEK induced by EGF and TPA in a dose-dependent manner. It
is noteworthy that 5 µM TC, a concentration sufficient
for complete inhibition of intracellular PP1 (Fig. 4), is enough to reduce the phosphorylation level to background (compare lanes 17 and 26 to lane 1 in Fig. 2). Under
these conditions, OA increased phosphorylation levels of MEK in a
dose-dependent manner with a plateau at 50-100
nM, which is enough to inhibit PP2A in vivo, as
shown in Fig. 1. These results suggest that inhibition of PP1 causes
suppression of MEK activation.

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Fig. 4.
Dose-dependent effects of OA and
TC on MEK activation in COS-7 cells. COS-7 cells were transfected
with pSR -HA-ERK2 and pEBG-MEK1. Transfected cells were incubated for
5.5 h with vehicle (None: lanes 1,
10, and 19), OA (lanes 2-5,
11-14, and 20-23), or TC (lanes
6-9, 15-18, and 24-27) at the indicated
concentrations. Cells were then stimulated for 4 or 10 min with 1 ng/ml
EGF (lanes 10-18) or 5 ng/ml TPA (lanes 19-27),
respectively. Immunoblots were performed as described under
"Experimental Procedures." Similar results were obtained in four
separate experiments.
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|
Tautomycetin Prevents Raf-1 Activation--
To clarify how
activation of MEK is inhibited by TC, we analyzed the effect of TC on
the activation of Raf-1, which is a major MEK kinase in
vivo. As shown in Fig.
5A, Flag-Raf-1 activity was increased with a peak at 5-10 min upon EGF treatment. Under these conditions, treatment of cells with 5 µM TC inhibited
FLAG-Raf-1 activation by 71% at 5 min, whereas treatment with 100 nM OA inhibited FLAG-Raf-1 by 95% at 5 min. We then
compared the time courses of Raf-1 activity (Fig. 5A) and
MEK phosphorylation (Fig. 5B) with or without inhibitors
upon EGF treatment. As shown in Fig. 5, TC inhibition of activation of
Raf-1 and MEK was almost the same in terms of the time course and rate
of inhibition, suggesting that inhibition of PP1 by TC negatively
regulates Raf-1 activation. Inhibition of PP2A by OA was shown to
affect Raf-1 negatively and MEK positively.

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Fig. 5.
Effects of OA and TC on activation of Raf-1
and MEK in COS-7 cells. A, COS-7 cells were transfected with
pH8-FLAG-Raf-1 and then incubated for 5 h with vehicle (open
circles), 100 nM OA (closed circles), or 5 µM TC (crosses). Cells were then stimulated
with 1 ng/ml EGF for the indicated time periods. After extraction of
the cells, Raf-1 kinase assays were performed as described under
"Experimental Procedures." Similar results were obtained in three
separate experiments. B, COS-7 cells were incubated for
4.5 h with vehicle (None: lanes 1-6,
open circles), 100 nM OA (lanes
7-12, closed circles), or 5 µM TC
(lanes 13-18, crosses). Cells were stimulated
for the indicated periods with 1 ng/ml EGF. Immunoblots were performed
as described under "Experimental Procedures." Arrows
indicate endogenous phospho-MEK and MEK. Similar results were obtained
in three separate experiments.
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The Catalytic Subunit of PP1 Inhibits Activation of MEK and Raf-1
in Vivo--
To elucidate a physiological role of PP1 in Raf-1
activation, wild type and a catalytically inactive form (H125A) of PP1C (Myc-tagged) were transiently expressed in COS-7 cells. Cell extracts were then prepared, and the phosphorylation level of GST-MEK upon EGF
activation was examined by immunoblot (Fig.
6A). Compared with extracts
from control cells transfected with the empty vector (mock), extracts
from cells transfected with pcDNA3/Myc-PP1
1 exhibited an
increase in the phosphorylation level of GST-MEK with or without EGF
stimulation. By contrast, extracts from cells transfected with
pcDNA3/Myc-PP1
1(H125A) showed marked decreases in the
phosphorylation level of GST-MEK. In order to examine effect of PP1 on
Raf-1 activation, FLAG-Raf-1 was co-expressed with Myc-PP1C or
Myc-PP1C(H125A). Compared with extracts from control cells, extracts
expressing Myc-PP1C exhibited marked increases in Raf-1 activity with
or without EGF stimulation. By contrast, extracts from cells expressing
Myc-PP1C(H125A) showed a marked decrease in Raf-1 activity, suggesting
that Myc-PP1C(H125A) works as a dominant negative in Raf-1 activation.
Activation rates of FLAG-Raf-1 and GST-MEK by Myc-PP1C were similar,
and inhibition rates of FLAG-Raf-1 and GST-MEK by Myc-PP1C(H125A) were
also similar, indicating that the target of PP1 is not MEK but Raf-1 or
an effector upstream of Raf-1. Similar results were obtained using
other isoforms of PP1C such as PP1
and PP1
(data not shown).

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Fig. 6.
Effects of over-expressed PP1C on Raf-1
activity in COS-7 cells. COS-7 cells were transfected with
pEBG-MEK1, pH8-FLAG-Raf-1, and either pcDNA3-Myc-PP1 1
(wt) or pcDNA3-Myc-PP1 1/H125A (H125A). As
a control, pEBG-MEK1, pH8-FLAG-Raf-1, and pcDNA3 were transfected
(Control). The transfected cells were stimulated with 0.5 ng/ml EGF for 4 min (A) or 1 ng/ml EGF for 2.5 min
(B). Following preparation of cell lysates, immunoblots
(A) and Raf-1 kinase assay (B) were performed as
described under "Experimental Procedures." Values are means ± S.D. (n = 3).
|
|
Interaction of PP1
1 and Raf-1--
Because PP1 was shown to be
a positive regulator of Raf-1 activity, we examined whether PP1
physically associates with Raf-1 (Fig.
7). FLAG-Raf-1 was detected in Myc-PP1C
immunoprecipitates, and Myc-PP1C was detected in FLAG-Raf-1
immunoprecipitates, demonstrating that PP1C interacts physically with
Raf-1 in vivo.

View larger version (30K):
[in this window]
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|
Fig. 7.
PP1C interacts with Raf-1 in COS-7
cells. COS-7 cells were transfected with either 1 µg of
pH8-FLAG-Raf-1 or pH8 (empty vector) and either 1 µg of
pcDNA3-Myc-PP1 1 or pcDNA3 (empty vector). Following
immunoprecipitation (IP), immunoblots (WB,
Western blot) were performed as described under "Experimental
Procedures." Similar results were obtained in three separate
experiments.
|
|
 |
DISCUSSION |
The present study demonstrates that using TC and OA, the functions
of PP1 and PP2A can be differentiated in vivo. We recently reported that TC has a 40-fold preference for purified PP1 compared with purified PP2A in vivo, suggesting that TC is a useful
tool in elucidating the physiological function of PP1 in various
biological events (16). Here we have demonstrated that treatment of
cells with 5 µM TC causes a complete inhibition of PP1
activity without affecting PP2A activity. TM has been used to analyze
PP1 in vivo because of its relatively high affinity for PP1
among PP inhibitors. However, 10 µM TM, a minimum
concentration sufficient to induce complete inhibition of PP1, also
induced inhibition of PP2A by 50% (44). Therefore, it is a great
advantage for in vivo analysis of PP1 and PP2A that 5 µM TC and 100 nM OA can differentiate between the functions of PP1 and PP2A in cells, respectively. This method could be used widely to reveal the unknown functions of PP1
in vivo.
The Ras-Raf-MEK-ERK cascade plays a central role in mediating various
extracellular stimuli into the nucleus. The duration and magnitude of
ERK activation are regulated at multiple points in the signaling
cascade. The activity of ERK reflects a balance between the activities
of upstream activating kinases and protein phosphatases. Previously,
the involvement of PP1 in the ERK pathway had not been established,
whereas investigations of the role of PP2A in the ERK pathway have
produced contradictory results. In vitro, PP2A
dephosphorylates and inactivates MEK and ERK, and both MEK and ERK are
activated by OA treatment in vivo (6). Expression of SV40
small T antigen, which inhibits PP2A, results in activation of MEK and
ERK (45). On the other hand, genetic analysis suggests a positive role
of PP2A in the regulation of the ERK pathway during
Drosophila photoreceptor development (46). To dissect PP1
and PP2A functions in the ERK pathway, we compared the PP1 and PP2A
functions on Raf-1, MEK, and ERK by differential usage of TC and OA.
The present data suggest that PP1 is required for Raf-1 activation but
is not involved in dephosphorylation of MEK or ERK in vivo
(Fig. 5). The role of PP2A in Raf-MEK-ERK signaling is complex. PP2A is
required for Raf-1 activation but is involved in dephosphorylation of
MEK and ERK. We conclude that PP1 is a positive regulator of Raf-1,
whereas PP2A can function as a positive regulator of Raf-1 but a
negative regulator of MEK and ERK. It is of note that activation of
HA-ERK by OA is less than activation of GST-MEK (Fig. 3A).
It is possible that ERK is dephosphorylated not only by PP2A but also
by dual-specificity or tyrosine phosphatases activated by OA.
The present results obtained using TC, a novel PP1-specific
inhibitor, indicate that PP1 is a positive regulator of cell growth. The up-regulation of Raf-MEK-ERK by PP1 was observed not only in COS-7
and 293T cells but also in HeLa and HepG2 cells (data not shown),
suggesting a general role of PP1 as a positive regulator in cell growth
regulation. Through systematic experiments of protein phosphatase
activity, we concluded previously that PP1
in hepatoma cells is
irreversibly up-regulated at the transcriptional level (19, 25-29).
PP1 in hepatoma cells accumulates both in the non-nuclear particulate
fraction and in the nuclei. Our present results strongly suggest that
increased PP1 in hepatomas plays a role in their rapid growth rate,
whereas the significance of increased PP1 levels in the nucleus of
hepatomas remains unclear. Increased nuclear PP1 is thought to inhibit
rather than accelerate the cell cycle through dephosphorylation
of Rb (47, 48). We also previously reported that NIPP-1, a potent
nuclear inhibitor of PP1, is markedly increased in rapidly growing
hepatomas, suggesting that the increased activity of the nuclear PP1 in
hepatoma cells is suppressed (49).
Raf-1 is present in cells in a multiprotein complex containing 14-3-3, Ras, KSR (kinase suppressor of Ras), and Hsp90 (50, 51). Raf-1
contains several phosphorylation sites, and its activity and
localization has been reported to be dependent on the phosphorylation state of multiple sites (52, 53). Residues such as Ser-43, Ser-259, and Ser-621 were identified as inhibitory phosphorylation sites (52, 53), whereas phosphorylation of both Ser-338 and Ser-441 was
demonstrated to be required for Raf-1 activation (52, 53). Here we have
demonstrated that PP1 is present in a multicomplex with Raf-1 and
positively regulates Raf-1 activity. These data suggest that PP1
dephosphorylates Raf-1 or associated proteins such as KSR and
SOS (Son of Sevenless), which results in the activation of Raf-1.
Recently, it is reported that Ser-259 phosphorylation appears to be a
main target for the inhibitory phosphorylation of Raf-1 (52, 53). Thus
it is possible that Ser-259 on Raf-1 is a phosphorylation site targeted
by PP1. It is interesting that myosin-binding subunit (M110), a PP1
regulatory protein, was reported to interact with Raf-1 in
vivo and to be phosphorylated by Raf-1 in vitro (54).
Therefore, it is possible that PP1C associates with Raf-1 and that they
regulate each other in vivo. Further in vivo
analyses of the complex of PP1 and Raf-1 are necessary to clarify the
precise mechanism by which PP1 is targeted to Raf-1 and regulates its activity.
 |
ACKNOWLEDGEMENTS |
We are grateful to Dr. Hiroyuki Osada,
Institute of Physical and Chemical Research (RIKEN), for the gift of
S. griseochromogenes. We thank Dr. Toshiaki Koda (Institute
for Genetic Medicine, Hokkaido University) for providing the
anti-GST antibody, Dr. John M. Kyriakis for pMT3-HA-p38
, and Dr.
Michael Karin for pSR
-HA-ERK2 and pSR
-HA-JNK1. We thank Eiko
Yoshida and Yoshimi Saito for secretarial assistance. We are grateful
to Kaai Takaku (College of Medical Technology, Hokkaido University) for
technical assistance.
 |
FOOTNOTES |
*
This work was supported in part by a grant from the Hokkaido
Foundation for the Promotion of Scientific and Industrial Technology, Grants-in-aid for Scientific Research (B)(2) and (C)(2) provided by the
Japan Society for the Promotion of Science, and Grant-in-aid for
Scientific Research on Priority Areas (C)(2) provided by the Ministry
of Education, Culture, Sports, Science, and Technology of Japan.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. and Fax:
81-11-707-6839; E-mail: kikuchi@imm.hokudai.ac.jp.
Published, JBC Papers in Press, October 8, 2002, DOI 10.1074/jbc.M208888200
 |
ABBREVIATIONS |
The abbreviations used are:
PP1, serine/threonine protein phosphatase type 1;
PP2A, serine/threonine
protein phosphatase type 2A;
PP2B, serine/threonine protein phosphatase
type 2B;
PP, serine/threonine protein phosphatase;
OA, okadaic acid;
TC, tautomycetin;
MAPK, mitogen-activated protein kinase;
PP1C, PP1
catalytic subunit;
TM, tautomycin;
ERK, extracellular signal-regulated
kinase;
MEK, MAPK/ERK kinase;
JNK, c-Jun NH2 kinase;
p38, homologue of the budding yeast HOG1 protein;
Me2SO, dimethyl sulfoxide;
EGF, epidermal growth factor;
GST, glutathione
S-transferase;
I-2, protein phosphatase inhibitor-2;
TPA, 12-O-tetradecanoyl-13-phorbol acetate;
HA, hemagglutinin.
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