Suprachiasmatic Nucleus Circadian Oscillatory Protein, a Novel
Binding Partner of K-Ras in the Membrane Rafts, Negatively Regulates
MAPK Pathway*
Kimiko
Shimizu
,
Masato
Okada§,
Katsuya
Nagai§, and
Yoshitaka
Fukada
¶
From the
Department of Biophysics and Biochemistry,
Graduate School of Science, The University of Tokyo and Japan Science
and Technology Corporation, Core Research for Evolutional Science and
Technology, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-0033, Japan and the
§ Division of Protein Metabolism, Institute for Protein
Research, Osaka University, 3-2 Yamada-Oka, Suita,
Osaka 565-0871, Japan
Received for publication, December 27, 2002
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ABSTRACT |
Suprachiasmatic nucleus circadian oscillatory
protein (SCOP) is a member of the leucine-rich repeat (LRR)-containing
protein family. In addition to circadian expression in the rat
hypothalamic suprachiasmatic nucleus, SCOP is constitutively expressed
in neurons throughout the rat brain. Here we found that a
substantial amount of SCOP was localized in the brain membrane rafts,
in which only K-Ras was abundant among Ras isoforms. SCOP interacted
directly through its LRR domain with a subset of K-Ras in the guanine
nucleotide-free form that was present in the raft fraction. This
interaction interfered with the binding of added guanine nucleotide to
K-Ras in vitro. A negative regulatory role of SCOP for
K-Ras function was examined in PC12 cell lines stably overexpressing
SCOP or its deletion mutants. Overexpression of full-length SCOP
markedly down-regulated ERK1/ERK2 activation induced by depolarization
or phorbol ester stimulation, and this inhibitory effect of
overexpressed SCOP was dependent on its LRR domain. These
results strongly suggest that SCOP negatively regulates K-Ras signaling
in the membrane rafts, identifying a novel mechanism for regulation of
the Ras-MAPK pathway.
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INTRODUCTION |
SCOP1 (suprachiasmatic
nucleus (SCN) circadian oscillatory protein) was originally identified
in differential display screening of genes whose expression is
regulated in a circadian manner within the rat hypothalamic SCN
(1). The mRNA and protein levels of SCOP in the rat SCN
increase during subjective night with a peak at midnight under constant
dark conditions (1). In addition to circadian expression in the SCN,
SCOP is expressed at a constant level in neurons throughout the rat
brain (1), but its function in neurons including the SCN is yet to be
elucidated. SCOP is a large polypeptide (1,696 amino acids) containing
a pleckstrin homology (PH) domain, leucine-rich repeat (LRR), protein
phosphatase 2C (PP2C)-like domain and glutamine (Q)-rich region (see
Fig. 2), suggesting its unique role in intracellular signaling. The LRRs are characterized by multiple repeats of a sequence harboring leucine residues at invariable positions (2), and LRR-containing proteins are thought to contribute to diverse biological functions through the LRR-mediated protein-protein interactions (2). The
LRR in SCOP is composed of 18-fold repeats of a short stretch unit with
a relatively conserved but variable sequence,
LXXLXLXXNXLXXLPXXAXXL, where L, N, P, A, and X denote leucine, asparagine, proline,
aliphatic, and any amino acid, respectively (1). Yeast
Saccharomyces cerevisiae adenylate cyclase contains 26-fold
repeats of a similar unit (3), to which Ras binds for
Ras-dependent enzymatic activation (4, 5). The homologous
LRR is also found in human and Caenorhabditis elegans SUR-8
(6), which interacts directly with Ras to regulate its signaling (6,
7). It is thus predicted that SCOP may also interact with Ras to
regulate Ras-mediated signaling pathway.
As a molecular switch, Ras cycles between a GTP-bound active state and
a GDP-bound inactive state via GTP hydrolysis and GDP/GTP exchange
steps. Two major types of Ras-interacting molecules have been described
that modulate the Ras GTP-GDP cycle through regulation of either the
activation or inactivation step. The former is mediated by a class of
proteins, guanine nucleotide-exchange factors (GEFs), that interact
with the GDP-bound form of Ras to reduce its affinity for GDP. This
facilitates formation of the guanine nucleotide-free form of Ras, to
which GTP binds for its activation. The latter is accelerated by GTPase
activating proteins (GAPs), which increase the intrinsic GTPase
activity of Ras and thereby promote the inactivation of Ras. Although
the regulatory elements of Ras proteins may not be fully understood
yet, these steps should provide a mechanism that manipulates the timing
and strength of Ras signaling to ensure appropriate signaling output
(8). In addition to these regulatory steps, the presence of Ras
isoforms may contribute to a well ordered framework of Ras-mediated
signaling. Despite a high degree of sequence similarity among Ras
isoforms, accumulating evidence points to a preferential activation of
specific effectors by each Ras isoform (9, 10). This issue seems to be
associated with protein clustering in membrane microdomains such as
rafts (11, 12). Membrane rafts are small platforms composed of
sphingolipids, cholesterol, and a given set of proteins and are
important for regulation of cellular signaling (11, 12). Selective
localization of the Ras isoforms at different membrane microdomains
reinforces the idea of distinct functionality and regulation of these
proteins (13). Knockout mouse for each of the Ras isoforms showed that K-Ras, but not H-Ras or N-Ras, is essential for development (14-16), and these results further support the idea that Ras isoforms play distinct roles.
The mitogen-activated protein kinase (MAPK) pathway is one of the
best-characterized effector systems downstream of Ras (17, 18).
Ras-MAPK-mediated signaling plays critical roles in cell proliferation,
differentiation, and migration in response to extracellular signals.
Recent studies have demonstrated an important contribution of Ras-MAPK
cascade to the circadian clock system in the rodent SCN (19) and the
chick pineal gland (20, 21). Here we show that SCOP functions as a
negative regulator of the Ras-MAPK pathway by interacting with the
nucleotide-free form of K-Ras in the membrane rafts to down-regulate
the nucleotide binding for its activation. The results presented in
this study illustrate a novel type of regulatory mechanism for Ras
signaling and suggest a contribution of SCOP to a variety of cellular functions.
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EXPERIMENTAL PROCEDURES |
Preparation of Detergent-insoluble Membrane Raft
Fraction--
The raft fraction was prepared as described (22) with
modifications. Whole brains from adult male rats (7 weeks) were
homogenized in buffer A (50 mM Tris-HCl, 1 mM
EDTA, 50 mM NaCl, 2.5 mM
-mercaptoethanol, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 1 mM
phenylmethanesulfonyl fluoride, pH 7.4) with a 10× volume of the brain
mass. The resulting homogenate was centrifuged at 1,000 × g for 15 min to sediment tissue debris. The unprecipitated
material was centrifuged at 100,000 × g for 1 h.
The resulting membrane pellet was suspended in buffer A with 1% (v/v)
Triton X-100 at a volume equal to that used for the initial homogenate
and then incubated for 1 h on ice and combined with an equivalent
volume of buffer A with 80% (w/v) sucrose. The mixture was divided
into 3-ml samples. Each sample was overlaid sequentially with buffer A
with 35% (w/v) sucrose (5 ml) and 5% (w/v) sucrose (2 ml),
respectively. After centrifugation at 35,000 rpm for 20 h at
4 °C in a Beckman SW41 swing type rotor, the Triton X-100 insoluble
cloudy material located at the interface between 5 and 35% sucrose
solutions was collected ("raft fraction").
Preparation of Fusion Proteins--
Various SCOP fragments (see
Fig. 2A) were produced in Escherichia coli as GST
fusion proteins. These include (i) GST·PH/LRR containing the
PH domain and LRR (amino acids 420-1241 in SCOP), (ii)
GST·LRR/PHOS/Q containing the LRR, PP2C-like, and Q-rich domains
(amino acids 588-1696), (iii) GST·LRR containing a part (the 7th
through the 18th repeat) of LRR (amino acids 735-1027), and (iv)
GST·PHOS containing the PP2C-like domain (amino acids 1082-1384).
The GST fusion proteins were purified with glutathione-Sepharose 4B
(Amersham Biosciences) according to the manufacturer's
protocol. The GST and MBP fusion proteins of full-length K-Ras
were produced in E. coli. The latter was purified by
amylose resin (New England Biolabs) according to the manufacturer's protocol.
Pull-down Assay of Ras Proteins with SCOP Fragments--
The
raft fraction (30 µl) was mixed with 1 ml of buffer A containing 60 mM n-octyl-
-D-glucoside (nOG) and
incubated for 1 h at 4 °C. In the presence or absence of
guanine nucleotide, the nOG-solubilized raft fraction was incubated for
4 h at 4 °C with each GST fusion protein of the SCOP fragments
(0.5 µg) premixed with glutathione-Sepharose beads (10 µl). The
beads were then washed three times with buffer A containing 60 mM nOG. Bound proteins were separated by
SDS-PAGE, followed by immunoblot analysis with anti-K-Ras monoclonal
antibody (1:400, Santa Cruz Biotechnology). In experiments with
recombinant K-Ras, each GST fusion protein of the SCOP fragments (0.5 µg) pre-bound to glutathione-Sepharose beads was incubated with
purified MBP·K-Ras (1 µg) for 4 h at 4 °C in a binding
buffer (20 mM Tris-HCl, 200 mM NaCl, 5 mM EDTA, 1% (v/v) Nonidet P-40, 5% (v/v) glycerol, 2 mg/ml bovine serum albumin, 5 mM
-mercaptoethanol, 10 µg/ml aprotinin, 10 µg/ml leupeptin, pH 7.4). The beads were washed
three times with the binding buffer, once with the binding buffer in
the absence of bovine serum albumin, and treated with SDS sample buffer
for the immunoblot analysis.
Detection of Active Form of K-Ras--
The raft fraction (30 µl) was mixed with 1 ml of buffer A containing 60 mM nOG
and 10 mM MgCl2 and incubated for 1 h at
4 °C. The raft fraction thus solubilized was incubated with 10 µl of GST·RBD (Ras-binding domain of Raf-1) coupled to
glutathione-agarose beads (Upstate Biotechnology). The beads were
washed three times with buffer A, and the bound proteins were subjected
to SDS-PAGE and immunoblot analysis with the anti-K-Ras antibody.
GTP
S Binding Assay--
The guanine nucleotide binding
activity of GST·K-Ras was evaluated at 4 °C by incubating with 20 µM [35S]GTP
S (3 Ci/mmol, PerkinElmer
Life Sciences) and the raft fraction (1 µl) in the presence or
absence of GST·LRR or GST in a total of 30 µl of buffer B (25 mM Tris-HCl, 100 mM NaCl, 1 mM
dithiothreitol, 10 mM MgCl2, 60 mM
nOG, pH 7.4). The GTP
S binding reaction was quenched by the addition
of 170 µl of a washing buffer (100 mM Tris-HCl, 1 mM MgCl2, pH 7.4) containing 3 mM
GTP. The reaction mixture was transferred to a multiscreen filter cup
(Millipore, 0.45-µm cellulose membranes, type HA) for filtering. The
filter was immediately washed five times with 0.2 ml of the washing
buffer, dried, and solubilized by adding 100 µl of 2-methoxyethanol
for scintillation counting with 800 µl of scintillation cocktail
ACSII (Amersham Biosciences).
Plasmid Construction and Functional Analysis of SCOP in PC12
Cells--
The cDNA encoding mutant SCOP devoid of either the LRR
(
586-1096 in amino acids, termed SCOP-
LRR) or PP2C-like and
Q-rich domain (
1097-1696, termed SCOP-
PHOS/Q) was cloned into
the pAP3neo mammalian expression vector. The nucleotide sequences of
these clones were confirmed by sequencing.
PC12 cells were maintained in Dulbecco's modified Eagle's medium
containing 10% horse serum and 5% fetal bovine serum. The cultured
cells were transfected with the expression plasmid using LipofectAMINE
Plus (Invitrogen). The transfected cells were selected in the
presence of G418 (0.5 mg/ml) as a selective antibiotic to obtain
several lines of stable transformants. The expression level of SCOP
protein in each cell line was evaluated by immunoblot analysis with
EC and
CB antibodies that recognize the LRR- and PP2C-like
domains of SCOP, respectively (1). Wild-type PC12 cells or the
stable transformants thus established were plated on 35-mm culture
dishes at a density of 8 × 104 cells/cm2
and cultured for 20 h at 37 °C. Cells were exposed to 50 mM KCl or 200 nM
o-tetradecanoylphorbol-13-acetate (TPA, from Sigma) and collected at the indicated time point in 100 µl of ice-cold RIPA
buffer (20 mM Tris-HCl, 150 mM NaCl and 1%
(v/v) Nonidet P-40, 1% (w/v) deoxycholate, 0.1% SDS, 5 mM
-mercaptoethanol, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 1 mM phenylmethanesulfonyl fluoride, 50 mM NaF, 1 mM Na3VO4, pH 7.4). After a 30-min
incubation on ice for solubilization, the lysate was centrifuged at
15,000 × g for 20 min at 4 °C. Supernatant
was subjected to immunoblot analysis using anti-P-MAPK antibody (New
England Biolabs) or Pan-ERK antibody (Transduction Laboratories) with
the aid of a chemiluminescence detection system (PerkinElmer Life Sciences).
 |
RESULTS |
Colocalization and Interaction of SCOP with K-Ras in Membrane
Rafts--
Functional studies on the LRRs in yeast adenylate cyclase
(3-5) and SUR-8 (6, 7) suggested the possibility that SCOP interacts
with Ras through the LRR domain. On the other hand, the PH domain has
been implicated in membrane localization of proteins (23-26), and it
seems to confer raft association in some cases (27, 28). We therefore
paid special attention to the LRR and PH domains and explored possible
localization of SCOP with Ras protein in the membrane rafts. Rat brain
membranes were treated with 1% (v/v) Triton X-100 and subjected to a
discontinuous sucrose gradient centrifugation. The raft fraction was
recovered as Triton X-100-insoluble materials at the interface between
5 and 35% (w/v) sucrose solutions (29). This fraction contained a
substantial amount of SCOP and K-Ras, whereas the majority of H-Ras and
N-Ras proteins were detected in the other fractions (Fig.
1). We examined the interaction of SCOP
fragments with K-Ras recovered in the raft fraction. The raft fraction
was treated with 60 mM nOG for solubilization and mixed
with a GST·SCOP fragment fusion protein (Fig.
2, A and B) that
was pre-bound to glutathione-Sepharose beads. Immunoblot analysis of
the precipitated proteins demonstrated that GST·PH/LRR and GST·LRR
associated with K-Ras but neither GST·PHOS nor GST alone did (Fig.
2C). Neither H-Ras nor N-Ras was detected in the same blot
by the specific antibodies (data not shown). Similar experiments were
performed with 1% Triton X-100-soluble fraction (100,000 × g supernatant prepared from the rat brain homogenate; see
"Experimental Procedures"), and we found that none of the Ras
isoforms coprecipitated with any of the SCOP fragments examined (data
not shown). These results demonstrate the LRR mediated interaction of
SCOP with K-Ras only in the membrane rafts. This is indicative of
either a difference in the state of K-Ras protein between the raft and
non-raft fractions or of alternate involvement of raft-specific
component(s) in this interaction. The latter possibility was excluded
by in vitro pull-down experiments in which both
GST·LRR/PHOS/Q and GST·LRR were shown to precipitate MBP·K-Ras
(but not MBP) expressed in E. coli (Fig. 2D).

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Fig. 1.
SCOP and K-Ras in the raft fraction. The
raft fraction was separated by ultracentrifugation as described under
"Experimental Procedures." After centrifugation of the rat brain
homogenate in a discontinuous sucrose gradient, the top 1.0 ml was
discarded and then 1.0-ml fractions were collected from the top to the
bottom (Fr. 1-9). An aliquot (10 µl) of each fraction was
subjected to immunoblot analysis by using antibodies against SCOP
( EC, Ref. 1), K-Ras, H-Ras, and N-Ras. Fr. 1 and Fr. 2 correspond to
the interface between 5 and 35% sucrose solutions (raft
fraction). Fr. 6-9 correspond to Triton X-100-soluble membrane
fractions.
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Fig. 2.
Pull-down assay of K-Ras with SCOP
fragments. A, structures of full-length SCOP and
various SCOP fragments fused to GST: PH plus LRR domain
(GST·PH/LRR), LRR plus PP2C-like and Q-rich
domain (GST·LRR/PHOS/Q), a part of
the LRR domain (GST·LRR), and PP2C-like domain
(GST·PHOS). The numbers indicate positions of the amino
acid residues in full-length SCOP. B, the GST fusion
proteins and GST were electrophoresed and stained with Coomassie
Brilliant Blue. C, pull-down experiments. Each GST
fusion protein, GST (each 0.5 µg), or buffer alone (Beads)
was pre-mixed with glutathione-Sepharose beads (10 µl) and incubated
with the nOG-solubilized raft fraction. Proteins bound to the beads
were blotted with K-Ras antibody. One-sixth volume of the raft fraction
used in this assay was electrophoresed and blotted in the lane
designated Raft. D, each GST fusion protein or
GST alone (each 0.5 µg) was pre-mixed with glutathione-Sepharose
beads (10 µl) and incubated with either MBP·K-Ras (upper
panel) or MBP (lower). Proteins bound to the beads were
blotted with anti-MBP antibody. MBP·K-Ras and MBP used in this assay
were included (input).
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Using RBD, a specific probe for the GTP-bound active form of Ras, we
then determined whether the nucleotide binding state of K-Ras affected
its interaction with SCOP LRR. First we measured the amount of
active form of K-Ras in the nOG-solubilized raft fraction and found an
intermediate level of K-Ras capable of binding with GST·RBD (Fig.
3A, top panel,
none). The amount of K-Ras precipitated with GST·RBD
markedly increased after incubation with 10 µM GTP
S (Fig. 3A, top panel), indicating that a certain
amount of K-Ras in the fraction can be converted to the active
GTP
S-bound form only by incubation with GTP
S. On the other hand,
K-Ras in the GTP-bound form in the original fraction was converted to a
form incapable of binding with RBD (probably the GDP-bound form) only by incubation with 10-100 µM GDP (Fig. 3A,
top panel). These preparations of K-Ras were then tested for
binding selectivity of SCOP LRR by incubating with GST·LRR/PHOS/Q or
GST·LRR. We found that a certain amount of K-Ras in the original
nOG-solubilized raft fraction bound with both GST·LRR/PHOS/Q and
GST·LRR (Fig. 3A, panels 2 and 3,
none). This interaction was abrogated when the raft fraction was preincubated not only with GDP but also with GTP
S (Fig.
3A, panels 2 and 3). These results
strongly suggest that SCOP LRR has no or very low affinity for both
GDP- and GTP
S-bound forms of K-Ras. It is most probable that the LRR
domain binds to the nucleotide-free form of K-Ras that is probably
present in the membrane rafts.

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Fig. 3.
GDP and GTP S inhibit
binding of K-Ras to SCOP LRR. A, the nOG-solubilized
raft fraction was incubated for 1 h at 4 °C in the presence of
GTP S (10 µM) or GDP (10,
50, 100 µM) or in the absence
(none) of the guanine nucleotides and then mixed with one of
the GST fusion proteins, GST·RBD, GST·LRR/PHOS/Q, GST·LRR, or GST
alone (indicated on the left of each panel). The proteins
precipitated with the beads were subjected to immunoblot analysis with
K-Ras antibody. B, the nOG-solubilized raft fraction was
preincubated for 1 h at 4 °C in the presence of 10 µM GDP (lane 2), 10 µM GTP S
(lane 5), or in the absence of nucleotides (lanes
1, 3, 4, and 6-8), and then
mixed with 0.5 µg of GST·LRR pre-bound to glutathione-Sepharose
beads (10 µl). All the mixtures were further incubated for 4 h
at 4 °C. During the last 1 h (lanes 3 and
6) or 2 h (lanes 4 and 7), the
mixtures were incubated in the presence of 10 µM GDP
(lanes 3 and 4) or 10 µM GTP S
(lanes 6 and 7) or in the absence of nucleotides
(lanes 1, 2, 5 and 8). As a
control, the nOG-solubilized raft fraction was mixed with GST·PHOS
pre-bound to glutathione-Sepharose beads (10 µl) and incubated for
4 h at 4 °C (lane 8). Proteins bound to the beads
were analyzed by immunoblot with K-Ras antibody.
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SCOP LRR Is a Negative Regulator for Guanine Nucleotide Binding to
K-Ras--
We then asked whether the interaction of K-Ras with the LRR
domain may affect its guanine nucleotide binding by examining the
effect of postincubation of the LRR·K-Ras complex with GDP or
GTP
S. Once bound to GST·LRR in the absence of guanine nucleotides, most of the K-Ras continued to be associated with LRR even after postincubation with 10 µM GDP (Fig. 3B,
lanes 3 and 4). This contrasted sharply with the
effect of the preincubation (lane 2). Similar results were
obtained in pre- and postincubation experiments with 10 µM GTP
S (Fig. 3B, lanes 5-7),
although a minimal amount of K-Ras was dissociated from GST·LRR after
the postincubation (lanes 6 and 7). These
observations suggest the LRR-dependent inhibition of
guanine nucleotide binding to the nucleotide-free form of K-Ras in the
raft fraction.
The LRR-mediated negative regulation of K-Ras activation (GTP binding)
was investigated directly by measuring the rate of GTP
S binding to
recombinant GST·K-Ras protein. The GTP
S binding to GST·K-Ras
absolutely required the presence of the rafts (Fig. 4A), which likely provide a
guanine nucleotide exchange factor activity for recombinant K-Ras. In
the presence of a constant amount of the raft fraction, GTP
S binding
to GST·K-Ras showed a nearly linear relationship with the amount of
GST·K-Ras protein at any time point of incubation (Fig.
4A, solid symbols). We investigated the effect of
GST·LRR and found that the addition of increasing amounts of
GST·LRR reduced the amount of GTP
S binding (Fig. 4B). The presence of a 10-fold molar excess of GST·LRR (60 pmol) over GST·K-Ras (6 pmol) inhibited the binding to a level of ~40% of that observed in the presence of an equivalent amount of GST
(Fig. 4B).

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Fig. 4.
The effect of SCOP LRR on
GTP S binding to K-Ras. A,
the time course of the GTP S binding reaction to various amounts of
GST·K-Ras (0-15 pmol) in the presence (solid symbols) or
absence (open symbols) of the raft fraction (1 µl).
B, a fixed amount of GST·K-Ras (6 pmol) was mixed
with the raft fraction (1 µl), and the reaction was started by the
addition of [35S]GTP S (final 20 µM) in
the presence or absence of GST·LRR (30 or 60 pmol) or GST (60 pmol)
in a total of 30 µl (in buffer B). The mixture was incubated at
4 °C for 30 min. [35S]GTP S bound to GST·K-Ras
is shown as the mean with actual values of duplicate samples. The data
are representative results of four independent experiments with similar
results.
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SCOP Suppresses the Activation of MAPK Pathway--
To explore the
function of SCOP in vivo, we established several lines of
PC12 cells stably overexpressing full-length SCOP (Fig.
5A, SCOP) and
examined the effect of Ras-stimulating treatment on extracellular
signal-regulated kinase 1 and 2 (ERK1/2) that are well established
targets downstream of Ras. In PC12 cells, membrane depolarization
elicits voltage-sensitive calcium channel-dependent calcium ion
influx that triggers ERK phosphorylation/activation via activation of
Ras (30). Treatment of wild-type PC12 cells with 50 mM KCl
stimulated phosphorylation of ERK1/2, which reached a maximal level 5 min after KCl application (Fig. 5B, WT).
Phosphorylation of ERK decreased to an intermediate level that was
sustained for at least 60 min after the onset of the stimulation. In
SCOP-overexpressing cells, KCl treatment also elevated ERK1/2
phosphorylation (Fig. 5B, SCOP). However, with
the exception of the 2-min time point, the level of phosphorylation
observed was significantly lower than that seen in KCl-stimulated
wild-type cells. Moreover the kinetics for phosphorylation of ERK1/2 in
SCOP-overexpressing cells lacked the sharp peak seen in WT cells upon
KCl treatment (Fig. 5B, SCOP). To determine
whether these differences were dependent on the LRR domain of SCOP, we
established two other PC12 cell lines (Fig. 5A), each
overexpressing a SCOP deletion mutant devoid of either the LRR
(SCOP-
LRR) or PP2C-like plus Q-rich domain (SCOP-
PHOS/Q). The KCl-stimulated
PC12 cells overexpressing SCOP-
LRR showed kinetics for ERK1/2
phosphorylation very similar to wild-type cells (Fig. 5B,
compare SCOP-
LRR with WT). By
contrast, the PC12 cells overexpressing SCOP-
PHOS/Q exhibited a weak
sustained response of ERK1/2 phosphorylation (Fig. 5B,
SCOP-
PHOS/Q), as was observed in
cells overexpressing full-length SCOP. This LRR-dependent
inhibition of ERK1/2 phosphorylation by SCOP was reproduced for each
construct in two other cell lines overexpressing each protein at
different levels (data not shown). No significant change in ERK2
protein level was observed, not only among samples from different time points (< 3 h) but also among cell lines (Fig. 5B,
lower panel in each pair).

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Fig. 5.
The effects of overexpressed SCOP and its
deletion mutants on ERK phosphorylation in PC12 cells.
A, the RIPA-solubilized lysate of wild-type PC12 cells
(WT) or its stable cell lines transformed with full-length
SCOP (SCOP), LRR-deleted SCOP
(SCOP- LRR), and PP2C-like and Q-rich
domain-deleted SCOP (SCOP- PHOS/Q)
were subjected to immunoblot analysis with a mixture of anti-SCOP
antibodies (Ref. 1, EC and CB). B and
C, the cultured PC12 cells were exposed to 50 mM KCl (B) or 200 nM TPA
(C) at time 0 and collected at the indicated time point for
preparing soluble lysate in RIPA buffer. These samples were subjected
to immunoblot analysis with anti-P-MAPK (upper panel in each
pair) or Pan-ERK antibody (lower panels). The latter
antibody displayed a low sensitivity to ERK1, and therefore an ERK1
protein band is not evident in the blot as compared with its
immunoreactivity to anti-P-MAPK.
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In another experiment, the PC12 cells were treated with TPA, which
induces PKC activation (31). This treatment also stimulates phosphorylation and activation of MAPK in a Ras-dependent
manner (32,33). Wild-type PC12 cells responded to TPA treatment with a
sharp increase in ERK1/2 phosphorylation, peaking at 5 min, which then
declined gradually over 180 min (Fig. 5C, WT).
This sharp response of ERK1/2 phosphorylation to TPA treatment was blunted not only in the cells overexpressing full-length SCOP (Fig.
5C, SCOP) but also in
SCOP-
PHOS/Q-overexpressing cells (Fig. 5C,
SCOP-
PHOS/Q). On the other hand,
the sharp response of ERK1/2 to TPA treatment was observed in PC12
cells overexpressing SCOP-
LRR, again indicating the importance of
the SCOP LRR domain for negative regulation of the MAPK. No significant
change in ERK2 protein level was observed among the various samples
(Fig. 5C, lower panels in each pair).
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DISCUSSION |
LRRs have been found in a variety of proteins with diverse
functions and cellular locations (2). We first predicted the interaction of SCOP LRR (18 repeats) with Ras because of its similarity in sequence to the LRR of yeast adenylate cyclase (26 repeats) (3) and
of SUR-8 (18 repeats) (6), both of which are known to bind with Ras for
functional regulation. The present study provides evidence for the
direct interaction of Ras with SCOP LRR, but the LRR domain of SCOP was
functionally different from those of yeast adenylate cyclase and SUR-8
in terms of Ras regulation. Yeast adenylate cyclase interacts with the
GTP-bound form of Ras and is thereby activated (4, 34), whereas the
interaction of SUR-8 with the GTP-bound form of Ras results in
activation of MAPK pathway (7). In contrast, SCOP associates with the nucleotide-free form of K-Ras in the membrane rafts to down-regulate the Ras-MAPK pathway. This is a novel mechanism for negative regulation of Ras protein function through interaction with LRRs.
Despite the high sequence similarity among Ras isoforms, SCOP
selectively associates with K-Ras, a unique isoform that is abundant in
the raft fraction of the rat brain homogenate (Fig. 1). It has been
recognized that activation of different Ras isoforms results in
distinct signaling outputs (9, 14, 15, 35-37), and the membrane
microdomains such as rafts and caveolae most likely contribute to the
segregated signaling (11, 12). It should be noted that in some cell
lines established from non-neural tissues H-Ras operates in membrane
rafts, whereas K-Ras seems to be located outside rafts (13, 38), in
contrast with the selective enrichment of K-Ras in the rafts of the rat
brain membranes (Fig. 1). This may be ascribed to cell type-specific
compositions of rafts (for example, the existence of neuron-specific
proteins such as SCOP), and hence neural membrane rafts might be unique because they lack caveolin (11).
Although SCOP and K-Ras were also present in the Triton X-100-soluble
(non-raft) fraction, we observed no significant binding of SCOP LRR to
K-Ras present in non-raft fractions. This observation supports the idea
that SCOP and K-Ras may become associated only in the membrane rafts or
alternatively that their interaction may induce the rafts formation.
However, direct involvement of a third (raft-specific) component is
inconsistent with the results of our pull-down experiments in which
recombinant SCOP and K-Ras proteins were both expressed in E. coli (Fig. 3D). Instead, SCOP LRR seems to associate
selectively with the nucleotide-free form of K-Ras present in the
membrane rafts but not with the GTP
S- nor GDP-bound forms that were
produced by adding the guanine nucleotides after solubilization of the
rafts. These data indicate that in the membrane rafts, just as in the
E. coli lysate, at least a part of K-Ras is kept in the
nucleotide-free form or in a form with an extremely low affinity for
the guanine nucleotides. The postincubation experiment with GTP
S or
GDP (Fig. 3B) strongly suggests that SCOP LRR "traps"
the nucleotide-free state (or a low affinity state) of K-Ras that is
generated by GDP release after receiving signals. This binding probably
down-regulates the K-Ras function (Fig. 5) by inhibiting the GTP
binding reaction (Fig. 4B). In this regard, the action of
SCOP provides a striking contrast with that of CDC25p, which is also
supposed to bind and stabilize the nucleotide-free state of Ras but
promotes its activation (39).
In PC12 cells, SCOP suppressed activation of ERK1 and -2 (Fig. 5),
which are well characterized effectors downstream of Ras. In both
cortical neurons and PC12 cells, calcium triggers ERK activation via
activation of Ras (30, 40). It is interesting to note that although KCl
and TPA treatment activate Ras through different pathways (30-33) the
effects of SCOP overexpression on the stimulus-dependent
change in ERK1/2 phosphorylation were similar to each other (Fig. 5).
This suggests that SCOP acts on Ras itself or on a step downstream of
Ras in the signaling pathway. The finding that SCOP binds directly to
K-Ras (Fig. 2B) supports K-Ras as the target site of
SCOP.
In rodent SCN, SCOP-mediated regulation of the K-Ras-MAPK pathway may
be important for the maintenance of circadian rhythm (19-21, 41). Both
the protein level of SCOP (1) and the phosphorylation (and activation)
of ERK1/2 (19) display a circadian variation in rodent SCN. MAPK
activity has its peak late in the subjective day when the protein level
of SCOP is low, whereas MAPK activity is minimum at mid-late subjective
night when SCOP expression is highest. Therefore, we hypothesize that
SCOP inhibition of ERK1/2 contributes to the circadian oscillation of
ERK1/2. In addition to the circadian expression in the SCN, a wide
distribution of SCOP in the rat brain (1) suggests that SCOP may play a
general role in regulation of the K-Ras-MAPK pathway and contribute to a variety of physiological functions such as neural plasticity and
neuronal survival. SCOP expression is not circadian-regulated in
non-SCN areas of the rat brain (1), and there must be an as yet
unidentified regulatory mechanism for SCOP function. A possible
regulatory step is the translocation of SCOP between the membrane rafts
and cytosol, which could be modulated by the PH domain and/or affected
by post-translational modifications such as phosphorylation. In
preliminary experiments, we detected phosphorylated SCOP in the brain
homogenate and 3T3 cell lysate. Future studies on each domain function
of SCOP would help to understand a new aspect of regulatory processes
of the K-Ras-MAPK pathway not only in the SCN but also in other brain areas.
 |
ACKNOWLEDGEMENTS |
We thank Maiko Katadae for excellent
technical assistance, Isaac Sterling, Prof. Daniel R. Storm, and Dr.
Guy C.-K. Chan for careful reading and many helpful comments on the
manuscript, and colleagues in the laboratories for technical support
and encouragement.
 |
FOOTNOTES |
*
This work was supported in part by grants-in-aid for
scientific research from the Japanese Ministry of Education, Culture, Sports, Science, and Technology.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: Dept. of
Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, Science Bldg. 3, Rm. 218A, Yayoi 2-11-16, Bunkyo-Ku, Tokyo
113-0032, Japan. Tel. and Fax: 81-3-5802-8871; E-mail:
sfukada@mail.ecc.u-tokyo.ac.jp.
Published, JBC Papers in Press, February 19, 2003, DOI 10.1074/jbc.M213214200
 |
ABBREVIATIONS |
The abbreviations used are:
SCOP, SCN circadian
oscillatory protein;
SCN, suprachiasmatic nucleus;
ERK, extracellular
signal-related kinase;
MAPK, mitogen-activated protein kinase;
PH, pleckstrin homology;
LRR, leucine-rich repeat;
PP2C, protein
phosphatase 2C;
GST, glutathione S-transferase;
nOG, n-octyl-
-D-glucoside;
RBD, Ras-binding
domain of Raf-1;
TPA, O-tetradecanoylphorbol-13-acetate;
WT, wild type;
MBP, maltose binding protein.
 |
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