From the Laboratory of Molecular and Genetic
Information, Institute for Molecular and Cellular Biosciences, The
University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan and
§ Second Department of Surgery, Gunma University School of
Medicine, 3-39-22 Showa-machi, Maebashi, Gunma 371, Japan
Received for publication, August 29, 2002, and in revised form, January 14, 2003
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ABSTRACT |
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Cadherin adhesion molecules are believed
to be important for synaptic plasticity. Activity-induced changes in synaptic transmission efficacy, such
as long term potentiation
(LTP)1 and long term
depression, have been postulated to be involved in information storage
during learning. Many studies have revealed that synaptic remodeling
and plastic changes in dendritic spine morphology play a role in
synaptic plasticity (1-6). Furthermore, changes in synaptic strength
have been shown to involve the structural and functional modifications
of the molecules present in the postsynaptic density (PSD) (7-10), an
electrondense structure containing various structural and signaling
molecules such as ion channels, scaffolding proteins, protein
kinases, small G-proteins, cell adhesion proteins, and cytoskeletal
proteins (11, 12).
The cadherins are a family of single-pass transmembrane proteins that
mediate Ca2+-dependent, homophilic
intercellular adhesion (13). Some members of the cadherin family of
adhesion proteins are localized to synaptic junctions and have been
implicated in synaptic plasticity (14, 15). For example, inhibitory
antibodies to the first extracellular domain of N-cadherin, one of the
classical cadherins enriched in neural cells, have been shown to
attenuate the induction of LTP (16, 17). Also, antagonistic peptides
containing the consensus sequence for cadherin dimer formation prevent
the induction of LTP (16). Furthermore, inhibition of cadherin activity
by a dominant-negative N-cadherin, as well as by mutation of
N-Methyl-D-aspartate (NMDA) receptors, which
play a central role in synaptic plasticity, are heteromeric ion
channels consisting of essential NR1 subunits and one or more of
the modulatory NR2 subunits, NR2A-D (22-24). The NMDA receptors are
associated with the PSD-95 family of proteins, the most abundant
constituents of PSD, via its NR2 subunits (25-28). This interaction is
important for specific localization of NMDA receptors in the PSD and
its coupling to cytoskeletal networks and signaling molecules (29-33). To further elucidate the role of Plasmid Construction--
The human RICS cDNA clone KIAA0712
was provided by T. Nagase (Kazusa DNA Research Institute) and subcloned
into the mammalian expression vector pcDNA3.1(+) (Invitrogen).
Mutant RICSs were generated by PCR. The authenticity of all mutants was
verified by DNA sequencing. For retrovirus-mediated expression of RICS, the RICS cDNA was inserted into pMX-puro, provided by T. Kitamura.
Two-hybrid Screening--
Two-hybrid screening was performed as
described previously (43).
In Vitro Binding
Assay--
[35S]Methionine-labeled RICS was synthesized
by in vitro transcription-translation using the
TNTTM-coupled reticulocyte lysate system (Promega).
Proteins fused to glutathione S-transferase (GST) were
synthesized in Escherichia coli and isolated by
absorption to GSH-Sepharose (Amersham Biosciences). GST fusion
proteins immobilized to beads were incubated with in vitro
translation products in Buffer A (0.1% Triton X-100, 10 mM
Tris-HCl, 140 mM NaCl, 1 mM EDTA, 1 mM sodium vanadate, 50 mM NaF, 10 µg/ml
aprotinin, 10 µg/ml leupeptin, 1 mM
p-amidinophenylmethanesulfonyl fluoride hydrochloride,
pH 8.0) and then washed extensively with Buffer A. Proteins adhering to
the beads were analyzed by SDS-PAGE followed by autoradiography.
Preparation of PSDs from Mouse Brain--
The PSD fractions were
isolated from mouse brain following the protocols described previously
(76, 77). Briefly, adult mouse brains were homogenated in an ice-cold
solution containing 0.32 M sucrose, 1 mM
HEPES-KOH, pH 7.4, 1 mM NaHCO3, 1 mM MgCl2, 0.1 mM
phenylmethanesulfonyl fluoride, 10 µg/ml aprotinin, 10 µg/ml
leupeptin, 1 mM NaF, and 1 mM
Na3VO4 and centrifuged at 1,000 × g and then 13,800 × g each for 10 min. The
resulting pellet was further fractionated by discontinuous sucrose
density gradient centrifugation at 82,500 × g for
2 h to obtain the synaptosome fraction. The synaptosome fraction
was solubilized with 0.5% Triton X-100, and the PSD (One
Triton) pellet was collected by centrifugation at 82,500 × g for 30 min. The PSD (One Triton) pellet was re-extracted in 0.5% Triton X-100 and centrifuged at 201,800 × g
for 1 h to obtain the PSD (Two Triton) pellet. Alternatively, the
PSD (One Triton) pellet was treated with 3%
N-lauroylsarcosinate and then centrifuged at 201,800 × g for 1 h to obtain the PSD (One Triton + Sarcosyl)
pellet. All pellets were resuspended in 40 mM Tris-HCl, pH
8.0. To dissolve the PSD (Two Triton) pellet, 0.3% SDS was added.
Isolated PSD fractions were checked for the absence of synaptophysin, a
presynaptic protein, and concentration of PSD-95 by immunoblotting
analysis prior to use in other experiments.
Antibodies--
Antibodies to RICS were prepared by immunizing
rabbits with peptides containing amino acids 670-735, 933-1009,
1518-1577 and 1675-1738, respectively. Antibodies were purified by
affinity chromatography using columns to which the antigens used for
immunization had been linked. Antibodies to N-cadherin, PSD-95 (used
for immunoprecipitation and immunoblotting), NR2B (for
immunocytochemistry), and Rac1 were from Transduction Laboratories.
Antibody to NR2A/2B (for immunoprecipitation and immunoblotting) was
obtained from Chemicon and Upstate Biotechnology. Antibodies to
synaptophysin and MAP2 were obtained from Sigma. Antibody to
NR2B (for immunoprecipitation) and PSD-95 (for immunocytochemistry) was
from Upstate Biotechnology. Antibodies to Immunoprecipitation and Immunoblotting--
Immunoprecipitation
and immunoblotting were performed as described elsewhere (49, 50).
Briefly, the PSD (One Triton) (50 µg) was first solubilized in 2%
SDS in IP buffer (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, 1.4 mM
KH2PO4, 5 mM EDTA, 5 mM
EGTA, 1 mM Na3VO4, 50 mM NaF, 0.1 mM phenylmethanesulfonyl fluoride, 10 µg/ml aprotinin, 10 µg/ml leupeptin) and then diluted with 5 volumes of IP buffer containing 2% Triton X-100. The lysates were
incubated with 10 µg of anti-RICS antibody and 30 µl of protein G-Sepharose (1:1 slurry) overnight at 4 °C. Immunocomplexes were washed once with IP buffer containing 1% Triton X-100, once with IP
buffer containing 1% Triton X-100 plus 500 mM NaCl, and
finally three times with IP buffer. Immunoprecipitates were subjected to immunoblotting analysis. The blots were probed with the indicated antibodies and then visualized with alkaline phosphatase-conjugated secondary antibodies (Promega).
Primary Neuron Culture and Immunostaining--
Hippocampal
primary neuronal cultures prepared from embryonic day 18-19 rat
embryos (78) were plated on coverslips coated with
poly-L-lysine (50 µg/ml) (5 × 104
cells/well). Cultures were grown in Neurobasal medium (Invitrogen) supplemented with B27 (Invitrogen) and 0.5 mM glutamine.
After 3 weeks, cultured hippocampal neurons were fixed with 2%
paraformaldehyde in phosphate-buffered saline for 10 min at room
temperature and then ice-cold methanol for 10 min at GAP Assay--
The GAP domains of RICS (amino acids 1-263) and
p50RhoGAP (amino acids 189-439) fused to GST were expressed in
E. coli. His6-tagged Rho GTPases (0.1 µg of
each protein) generated by the baculovirus system were preincubated
with [ Phosphatase Assay--
Lysate were prepared from mouse brain,
and immunoprecipitation experiments were performed as described above.
The immunoprecipitates were washed three times with BAP buffer (20 mM Tris-HCl, pH 8.0, 1 mM MgCl2)
and incubated at 30 °C for 5 min and then bacterial alkaline
phosphatase (TAKARA) was added to the reaction mixtures and incubated
for 5 min before termination by SDS-PAGE sample buffer.
RBD and PBD Assay--
The Rho-binding domain (RBD; amino acids
2-89) of mouse rhotekin and p21-binding domain (PBD; amino acids
65-136) of mouse PAK-3 were prepared as GST fusion proteins. NIH3T3
cells infected with Ret-RICS were lysed with lysis buffer (1% Triton
X-100, 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10 mM MgCl2, 1 mM
phenylmethanesulfonyl fluoride, 10 µg/ml aprotinin, 10 µg/ml
leupeptin, 0.5% sodium deoxycholate, 0.1% SDS). The GTP-bound form of
endogenous Rho GTPases was affinity-purified from the clarified lysates
containing 700 µg (for RhoGAP), 100 µg (for RacGAP), or 200 µg
(for Cdc42GAP) of protein with 20 µg of GST-RBD or GST-PBD. Bound
Rho, Rac1, and Cdc42 were detected by immunoblotting with antibodies
against RhoA, Rac1, and Cdc42, respectively (41, 42).
Phosphorylation of RICS by Purified CaMKII--
PSD fractions
were solubilized with IP buffer containing 1% Triton X-100, 0.2% SDS,
and 0.5% sodium deoxycholate for 30 min at room temperature and
centrifuged at 201,800 × g for 1 h. The supernatant was subjected to immunoprecipitation with anti-RICS antibody. Immunoprecipitated RICS (about 1 µg/ml) in 50 mM Tris-HCl, pH 8.0, 10 mM MgCl2,
10 mM dithiothreitol, and 30 µM
[ Molecular Cloning of the RICS cDNA--
We screened a mouse
17-day-embryo cDNA library by the yeast two-hybrid system using the
armadillo repeats 10-12 of mouse Expression of RICS mRNA and Protein--
Northern blot
analysis detected a doublet 9.8- and 10-kb mRNA. Both bands were
detected at high levels in mouse kidney, brain, testis, and heart and
at low levels in skeletal muscle, liver, lung, and spleen (Fig.
2A).
To identify the gene product, we generated antibodies to the
carboxyl-terminal portion (amino acids 1518-1578) of RICS and confirmed that the antibody reacts specifically with RICS generated by
in vitro translation (Fig. 2B). The antibody also
recognized Myc-tagged RICS that was exogenously expressed in 293 cells
and immunoprecipitated with anti-Myc antibody (Fig. 2B).
Immunoblotting analysis of a lysate from mouse brain and human
colorectal tumor DLD-1 cells with anti-RICS antibody but not total IgG
from nonimmunized rabbit detected a doublet of 250 and 210 kDa (see
Fig. 2B and Fig. 3C). Also,
immunoprecipitation-immunoblotting experiments with anti-RICS antibody
but not total IgG of nonimmunized rabbit detected both 250- and 210-kDa
proteins (Fig. 2B), and precipitation of these proteins was
inhibited by preincubation of the antibody with the antigen used for
immunization (Fig. 3C).
Furthermore, three other antibodies raised against different epitopes
also detected the 250- and 210-kDa proteins (data not shown). These results suggest that the 250- and 210-kDa proteins identified by the
anti-RICS antibodies are the RICS gene products. The nature of the
doublet is currently under investigation. In addition, the migration of
the 250- and 210-kDa RICS proteins was accelerated by phosphatase
treatment of RICS immunoprecipitates (Fig. 2B). However, the
migration of these proteins was not increased by phosphatase treatment
in the presence of a phosphatase inhibitor, RICS Is Present in the PSD Fraction--
We next
examined the subcellular localization of RICS by subcellular
fractionation of mouse brain (Fig. 2C). Similar to PSD-95, RICS was found to be concentrated in the synaptosomes and in the Triton
X-100 insoluble fraction (One-Triton and Two-Triton PSD; see
"Experimental Procedures"). Most of RICS was present in the PSD
fraction even after extraction with N-lauroylsarcosinate, suggesting that RICS is tightly bound to the core structure of the
PSD.
RICS Binds to RICS Interacts with The RICS-
The RICS Is Localized to the Synapse in Cultured Hippocampal
Neurons--
Immunofluorescent staining of rat hippocampal neuronal
cultures was performed to examine the colocalization of RICS and
PSD-95. When hippocampal cells were cultured for 3 weeks, RICS was
expressed in MAP2-positive neurons and glial fibrillary acidic
protein-positive glial cells. In neuronal cells, dendrites were
brightly labeled by anti-RICS antibody and appeared as dots (Fig.
4, A-D). This punctate staining for RICS coincided with those observed for NR2B and
PSD-95, although some fraction of NR2B and PSD-95 puncta did not
costain for RICS (Fig. 4, H-M). Numerous RICS
clusters colocalized with RICS Possesses GAP Activity for Rho GTPases in Vitro--
Because
the amino acid sequence of RICS has homology to the GAPs for Rho
GTPases, we examined whether RICS also functions as a GAP for Rho,
Rac1, and/or Cdc42. When the GAP domain of RICS fused to GST was
incubated with RhoA, Rac1, or Cdc42 bound to [ RICS Possesses GAP Activity for Cdc42 and Rac1 in Vivo--
We
next examined whether RICS exhibits GAP activity toward Rho GTPases
in vivo. NIH3T3 cells were infected with a retrovirus encoding Myc-tagged RICS (Ret-RICS), and GAP activity of RICS was
examined by RBD or PBD assay (41, 42). We found that expression of RICS
resulted in decreases in the levels of the active forms of Cdc42 and
Rac1, whereas expression of the inactive mutants RICS-R58I or -R58M did
not (Fig. 5B). On the other hand, RICS induced a slight
reduction in the level of the active form of RhoA. Under these
experimental conditions, the total amount of Cdc42, Rac1, and RhoA
remained constant. These results suggest that RICS possesses GAP
activity for Cdc42 and Rac1 in vivo.
RICS Is Phosphorylated by CaMKII--
RICS possesses 24 consensus
sites (RXX(S/T)) for phosphorylation by CaMKII. We therefore
examined whether RICS is phosphorylated by purified CaMKII in the
presence of Ca2+ and/or calmodulin. As shown in Fig.
6A, RICS immunoprecipitated from the PSD fraction of mouse brain lysate was phosphorylated by
purified CaMKII in a Ca2+- and
calmodulin-dependent manner (Fig. 6A).
Phosphorylation of RICS was inhibited by the addition of the CaMKII
inhibitor KN-93 to the kinase reaction mixture (Fig. 6A).
These results suggest that RICS is phosphorylated by CaMKII.
The GAP Activity of RICS Is Inhibited by Phosphorylation by
CaMKII--
We next investigated whether phosphorylation by CaMKII
affects the GAP activity of RICS against Cdc42. RICS immunoprecipitated from the PSD fraction of mouse brain lysate was phosphorylated by
purified CaMKII, and its GAP activity against Cdc42 was measured. The
GAP activity of RICS was inhibited following phosphorylation in the
presence of Ca2+ and calmodulin (Fig. 6B).
Inhibition of GAP activity was blocked when the phosphorylation
reaction was performed in the presence of KN-93 or a nonhydrolyzable
ATP analog, AMP-PNP. Furthermore, the GAP activity of RICS was not
inhibited by phosphorylation in the absence of Ca2+ and
calmodulin. These results imply that the GAP activity of RICS is
inhibited by CaMKII-mediated phosphorylation.
In the present study, we found that We demonstrated that RICS possesses GAP activity for Ccd42 and Rac1.
This result is consistent with the observation that the GAP domain of
RICS is highly homologous to that of CdGAP, which also possesses GAP
activity for Cdc42 and Rac1 (35). The Rho GTPases regulate the actin
cytoskeleton (55, 56) and play an important role in the maintenance and
reorganization of dendritic spines (57, 58). Rac1 is required for the
maintenance of dendritic spines, whereas elevation of RhoA activity
leads to pronounced simplification of dendritic branch patterns (59).
Furthermore, Karilin-7, a guanine nucleotide exchange factor for Rac1,
has been reported to be a regulator of the postsynaptic actin
cytoskeleton (51). On the other hand, it has been reported that
overexpression of either an active or dominant-negative form of Cdc42
does not induce significant changes in the maintenance of spine density or morphology (60). Thus, RICS may be involved in the maintenance and
reorganization of dendritic spines through its GAP activity toward Rac1.
It has been shown that NMDA receptor signaling influences dendritic
branching, and this regulation involves Rho GTPases that are contained
in the NMDA receptor complex (61). In addition, it has been reported
that PSD-95 is involved in regulating spine structure and number;
overexpression of PSD-95 in cultured hippocampus neurons leads to
increases in the size and number of dendritic spines (62). Therefore,
the fact that RICS is contained in the NMDA receptor-PSD-95 complex
raises the possibility that activation of NMDA receptors leads to
RICS-mediated regulation of Rho GTPase. In this regard, it is
interesting that RICS was found to be a substrate for CaMKII, and its
GAP activity is inhibited by phosphorylation by CaMKII.
CaMKII is highly concentrated in the PSD and plays a key role in LTP
(34, 63-67). CaMKII is activated following Ca2+ influx
through the NMDA receptors during LTP induction and potentiates synaptic efficacy (68-70). Stimulation by Ca2+/calmodulin
induces binding of CaMKII to the cytoplasmic carboxyl-terminus of NR1,
NR2A, and NR2B (71-73), and the interaction with NR2B locks CaMKII
into an active conformation (74). Hence, it is interesting to speculate
that CaMKII activated by Ca2+ entry through NMDA receptors
inactivates RICS, which in turn increases the active GTP-bound forms of
Cdc42 and Rac1. This would thereby induce, for example, remodeling of
dendritic spines. Thus, RICS may play a role in the NMDA
receptor-mediated signal transduction pathway and be involved in the
regulation of synaptic plasticity.
The adhesion proteins are not simply basic structural scaffolds that
hold the synapse and its components together. For example, N-cadherin
distribution can be dynamically regulated by neuronal activity, in a
manner requiring the NMDA receptors (7). It has been postulated that
the function of N-cadherin is to transiently change spine morphology.
Indeed, inhibition of cadherin activity has been shown recently (18) to
alter dendritic spine morphology, suggesting that cadherins function as
regulators of synaptic plasticity by modulating this morphology. On the
other hand, it has been reported that cadherin-mediated junction
formation activates Rac1 (75) and that Rac1 is required for the
maintenance of dendritic spines (59). These findings raise the
possibility that the cadherin system regulates spine morphology by
regulating Rho GTPases. Hence, it is possible that RICS contained in
the N-cadherin- It has been reported recently (10) that neural activity induces
redistribution of In conclusion, our results suggest that RICS is involved in the
synaptic adhesion- and NMDA receptor-mediated organization of
cytoskeletal networks and signal transduction. We speculate that RICS
may regulate dendritic spine morphology and strength by regulating
Cdc42 and Rac1.
During the revision of this paper, Nakamura et al. (79) and
Moon et al. (80) reported the identification of Grit and
p200RhoGAP, respectively, which are identical to RICS. Nakamura
et al. (79) have shown that Grit may regulate neurite
extension through association with the TrkA receptor and N-Shc and
CrkL/Crk adapter molecules. Moon et al. (80) have
demonstrated that p200RhoGAP may be involved in the regulation of
neurite outgrowth, and its activity may be regulated through an
interaction with Src-like tyrosine kinases. It has been shown that Grit
prefers RhoA and Cdc42 to Rac1 whereas p200RhoGAP prefers RhoA and
Rac1. Discrepancies between three papers (Refs. 79 and 80 and
this article) are currently under investigation in our laboratory.
-Catenin, which links
cadherins and the actin cytoskeleton, is a modulator of cadherin
adhesion and regulates synaptic structure and function. Here we show
that
-catenin interacts with a novel GTPase-activating
protein, named RICS, that acts on Cdc42 and Rac1. The
RICS-
-catenin complex was found to be associated with N-cadherin,
N-methyl-D-aspartate receptors, and postsynaptic density-95, and localized to the postsynaptic density. Furthermore, the GTPase-activating protein activity of RICS was inhibited by phosphorylation by
Ca2+/calmodulin-dependent protein kinase II.
These results suggest that RICS is involved in the synaptic adhesion-
and N-methyl-D-aspartate-mediated organization
of cytoskeletal networks and signal transduction. Thus, RICS may
regulate dendritic spine morphology and strength by modulating Rho GTPases.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
N-catenin, has been shown to alter dendritic spine
morphology (18), suggesting that cadherins function as regulators of
synaptic plasticity by modulating spine morphology.
-Catenin interacts with the cytoplasmic domain of classical
cadherins and links cadherins and the actin cytoskeleton (19, 20).
-Catenin and N-cadherin are present in axons and dendrites prior to
synapse formation and then cluster at developing synapses and form a
symmetrical adhesion structure in synaptic junctions (21). Moreover, it
has been reported recently (10) that neural activity induces
redistribution of
-catenin from dendritic shafts into spines, where
it interacts with cadherin to influence synaptic size and strength.
-Catenin is also known to be localized in PSD as a component of the
NMDA receptor multiprotein complex (12). Furthermore, the
-catenin
redistribution induced by depolarization is completely blocked by NMDA
receptor antagonists, indicating that the redistribution is caused by
synaptic activation of NMDA receptors (10). Also it has been reported
that stimulation of NMDA receptors by the agonist NMDA induces
molecular modification of N-cadherin, i.e. increased trypsin
resistance, as well as pronounced dimerization of N-cadherin (7). Thus,
-catenin and N-cadherin are structurally and functionally linked to
NMDA receptors.
-catenin in synaptic function, we
attempted to identify novel binding partners of
-catenin. In the
present study, we show that
-catenin interacts with a novel
GTPase-activating protein for Cdc42 and Rac1, termed RICS (RhoGAP involved in the
-catenin-N-cadherin and NMDA receptor signaling). RICS was found to localize to the PSD and to be
associated with N-cadherin, PSD-95, and NMDA-R, in addition to
-catenin. Furthermore, we demonstrate that its GAP activity is
down-regulated through phosphorylation by
Ca2+/calmodulin-dependent protein kinase II
(CaMKII), a kinase critical for synaptic plasticity that is activated
by NMDA receptor-mediated influx of Ca2+ (34).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-catenin were from Upstate
Biotechnology and Transduction Laboratories. Antibodies to RhoA and
Cdc42 were from Santa Cruz Biotechnology, Inc.
20 °C. Cells
were then double-stained with antibodies to RICS and PSD-95, antibodies
to RICS and NR2B, antibodies to RICS and
-catenin, or antibodies to
RICS and MAP2. The staining patterns obtained with antibody to RICS
were visualized with fluorescein isothiocyanate-labeled anti-rabbit
antibodies (ICN Biomedicals); those obtained with antibodies to MAP2,
PSD-95, NR2B, and
-catenin were visualized with RITC-labeled
anti-mouse antibody (ICN Biomedicals). The cells were photographed with
a Carl Zeiss LSM510 laser scanning microscope.
-32P]GTP (3 µCi, 6000 Ci/mmol) in a mixture
(20 µl) containing 20 mM Tris-HCl, pH 7.5, 25 mM NaCl, 5 mM EDTA, 0.1 mM
dithiothreitol for 10 min at 30 °C. After the addition of
MgCl2 (final concentration 20 mM), 10 nM GST, GST-RICS-GAP, or GST-p50RhoGAP in GAP buffer (20 mM Tris-HCl, pH 7.5, 0.1 mM dithiothreitol, 1 mM GTP, 0.86 mg/ml bovine serum albumin) was added to the
mixture and incubated at room temperature, and 10-µl samples were
removed at 0, 2, 4, and 6 min, diluted in 1 ml of ice-cold Buffer B (50 mM Tris-HCl, pH 7.5, 50 mM NaCl, 5 mM MgCl2), and filtered through nitrocellulose membranes prewetted with Buffer B. After washing twice with 10 ml of
ice-cold Buffer B, the radioactivity remaining on the filter was determined.
-32P]ATP was incubated at 30 °C for 1 h with
gentle agitation in the presence or absence of purified rat brain
CaMKII (1 µg/ml) (Calbiochem), Ca2+ (0.4 mM
EGTA/0.7 mM CaCl2 or 0.4 mM EGTA),
1 µg of bovine brain calmodulin (Chemicon), and 30 µM
CaMKII inhibitor KN-93 (Calbiochem) in the total volume of 100 µl.
For the GAP assay, phosphorylation of immunoprecipitated RICS was
performed at 30 °C for 2 h using 2 mM ATP.
Phosphorylated RISC was washed three times with GAP buffer and
subjected to in vitro GAP assay as described above.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-catenin as bait and obtained a
cDNA fragment of a novel gene, which we have designated
RICS (Fig. 1). Addition
of the nucleotide sequence of the upstream portion of the clone
obtained by the 5'-rapid amplification of cDNA ends system revealed
a composite sequence containing a 5,220-bp-long open reading frame,
encoding a predicted protein of 1740 amino acids. Its human homolog
corresponds to KIAA0712 and contains a 5214-bp-long open reading frame,
encoding a predicted protein of 1738 amino acids (Fig. 1A).
The predicted amino acid sequence of RICS revealed that its
amino-terminal region possesses striking amino acid homology to several
GAPs that are modulators of the Rho family of small G-proteins (Fig. 1,
B and C). In particular, the GAP domain of RICS
shares 67.9% identity with that of CdGAP (35) (Fig. 1, B
and C). In addition, the central region of RICS contains
three Pro-rich sequences, each of which conforms to the Src homology
3-binding motif (Fig. 1A).
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Fig. 1.
Structure of RICS. A,
predicted amino acid sequence of RICS. Human RICS is a 1738-amino acid
protein that contains a RhoGAP domain (box) and three
proline-rich motifs (bold). The region required for binding
to -catenin (amino acids 1182-1371) is underlined. The
axonal sorting motifs have dashed underlines. B,
alignment of the amino acid sequences of the RhoGAP domains of RICS,
CdGAP, p50RhoGAP, and p190RhoGAP. Identical residues are shown in
bold type. The residues that participate in catalysis (*)
and in the interaction with Rho family GTPase (+) are indicated.
C, schematic representation of RICS and the Rho
GTPases.
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Fig. 2.
RICS mRNA and
protein. A, expression of RICS mRNA in
mouse adult tissues. Multiple tissue northern (MTN) blots of
mouse adult tissues (Clontech) were probed with a
cDNA encoding RICS. As a control, expression of
actin mRNA was also examined. The positions of
the RICS and actin mRNAs are indicated by
arrowheads. B, identification and
characterization of the RICS protein. Left panel,
lanes 1 and 2, in vitro translated
RICS was subjected to immunoblotting with total IgG from nonimmunized
rabbit (lane 1) or anti-RICS antibody (lane 2).
Lanes 3 and 4, Myc-tagged RICS exogenously
expressed in 293 cells was immunoprecipitated with nonimmune IgG
(lane 3) or anti-Myc antibody (lane 4) and then
immunoblotted with anti-RICS antibody. Lanes 5 and
6, a lysate from DLD-1 cells was subjected to immunoblotting
with anti-RICS antibody (lane 5) or nonimmune IgG
(lane 6). Right panel, lane 1, a
lysate from mouse brain was subjected to immunoblotting with anti-RICS
antibody. Lanes 2-6, lysates from mouse brain were
subjected to immunoprecipitation followed by immunoblotting with
anti-RICS antibody. Lane 2, total IgG of nonimmune rabbit
instead of anti-RICS antibody was used for immunoprecipitation.
Lanes 4 and 5, the RICS immunoprecipitates were
treated with bacterial alkaline phosphatase in the absence (lane
4) or presence of -glycerophosphate (10 µM)
(lane 5). C, RICS is enriched in the PSD in mouse
brain. Mouse brain was fractionated as described under "Experimental
Procedures." Mouse brain homogenate (25 µg), the synaptosome
fraction (25 µg), PSD extracted once (One Triton; 2.5 µg) or twice with Triton X-100 (Two Triton; 2.5 µg), and
PSD extracted once with Triton X-100 followed by
N-lauroylsarcosinate (One Triton + Sarcosyl; 2.5 µg) were subjected to immunoblotting analysis with antibodies to
RICS, PSD-95, or synaptophysin.
-glycerophosphate. These
results suggest that RICS is phosphorylated in living cells.
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Fig. 3.
Association of RICS with
-catenin, N-cadherin, NMDA receptors, and
PSD-95. A, mapping of the regions in RICS required for
interaction with
-catenin. Deletion constructs of RICS were analyzed
for their ability to interact with
-catenin in the two-hybrid
system. Schematic representation of RICS deletion mutants and
corresponding
-catenin-binding activities are shown. +, detectable
activity; ±, very weak activity;
, no detectable activity.
B, association of RICS with
-catenin in vitro.
In vitro translated [35S]methionine-labeled
full-length RICS or a RICS fragment lacking amino acids 1182-1371 were
incubated with GST-
-catenin-Sepharose. The bound proteins were
analyzed by SDS-PAGE followed by autoradiography. Ten % of in
vitro translated proteins used for pull-down assays were applied
to each Input lane. WT, wild-type. C,
association of RICS with
-catenin in vivo. Lysates
prepared from mouse brain were subjected to immunoprecipitation
(IP) with the antibodies indicated, fractionated by
SDS-PAGE, and immunoblotted with the antibodies indicated.
Ag+, antibodies were preincubated with antigens before use
in immunoprecipitation. In lanes labeled Lysate,
10% of lysates used for immunoprecipitation were applied.
Control IgG, preimmune control serum was used for
immunoprecipitation. WB, Western blot. D,
the RICS-
-catenin complex is associated with N-cadherin, NMDA-R, and
PSD-95 in vivo. Lysates prepared from the PSD of mouse brain
were subjected to immunoprecipitation with the indicated antibodies,
fractionated by 6% SDS-PAGE, and immunoblotted with the indicated
antibodies. Ag+, antibodies were preincubated with antigens
before use in immunoprecipitation. In lanes labeled
Lysate, 5% of lysates used for immunoprecipitation were
applied.
-Catenin in Vitro--
The minimal fragment of
RICS obtained in the two-hybrid screen contained the carboxyl-terminal
557 amino acids. Two-hybrid assays using deletion fragments of RICS
further confirmed that a fragment containing amino acids 1182-1371 was
positive for interaction with
-catenin, whereas a fragment lacking
this region was not (Fig. 3A). A fragment containing amino
aids 1182-1303 was weakly positive. Consistent with these results,
RICS generated by in vitro translation was found to
specifically interact with
-catenin fused to GST (GST-
-catenin),
whereas RICS lacking amino acids 1182-1371 did not interact with
GST-
-catenin (Fig. 3B).
-Catenin in Vivo--
To examine whether
RICS is associated with
-catenin in vivo, we subjected a
lysate from mouse brain to immunoprecipitation with anti-RICS antibody
and then immunoblotted with anti-
-catenin antibody. As shown in Fig.
3C, RICS was found to coprecipitate with
-catenin, and
this coprecipitation was inhibited by preincubation of anti-RICS
antibody with the antigen used for immunization. Also,
immunoprecipitation of the lysate with anti-
-catenin and subsequent
immunoblotting with anti-RICS antibody revealed an association between
RICS and
-catenin (Fig. 3C). Preincubation of the
anti-
-catenin antibody with the antigen prevented coprecipitation of
-catenin and RICS. These results suggest that RICS interacts with
-catenin in vivo.
-Catenin Complex Is Associated with N-Cadherin, NMDA
Receptors, and PSD-95 in Vivo--
Because
-catenin is known to be
associated with the cadherin family of proteins, we examined whether
the RICS-
-catenin complex is associated with E-cadherin and
N-cadherin. Immunoblot analysis of the RICS immunoprecipitates with
anti-E-cadherin and anti-N-cadherin antibodies revealed that E-cadherin
(data not shown) and N-cadherin (Fig. 3D) both coprecipitate
with RICS. Immunoblot analysis of the E-cadherin and N-cadherin
immunoprecipitates with anti-RICS antibody further confirmed that
E-cadherin and N-cadherin coprecipitate with RICS (data not shown).
Coprecipitation of these proteins was not observed when anti-RICS
antibody preincubated with the antigen was used for immunoprecipitation
(Fig. 3D). These results suggest that RICS,
-catenin, and
E-cadherin/N-cadherin are contained in the same complex in
vivo.
-catenin-cadherin complex has been reported to be linked to the
NMDA-R-PSD-95 complex (12). Therefore, we next examined whether RICS is
contained in the NMDA receptor-PSD-95 complex. When the RICS
immunoprecipitate was subjected to immunoblot analysis with
anti-NR2A/2B and anti-PSD-95 antibodies, respectively, both NR2A/2B and
PSD-95 were found to coimmunoprecipitate with RICS (Fig.
3D). Immunoblot analysis of the NR2A/2B and PSD-95
immunoprecipitates with anti-RICS antibody also showed that NR2A/2B and
PSD-95 coprecipitate with RICS (data not shown). However, the amounts
of RICS detected in the NR2A/2B and PSD-95 immunoprecipitates were
small, presumably because NR2A/2B and PSD-95 are present in a great
excess over RICS. On the other hand, focal adhesion kinase was not
coimmunoprecipitated with RICS (Fig. 3D). Taken together,
theses results suggest that RICS,
-catenin, N-cadherin, NMDA
receptor, and PSD-95 may be contained in the same complex in
vivo.
-catenin clusters (Fig. 4,
E-G). These staining patterns were not detected
when antibody was preadsorbed with an excess amount of the antigens
used for immunization.
View larger version (29K):
[in a new window]
Fig. 4.
Colocalization of RICS with
-catenin, PSD-95, and NMDA-R in cultured rat
hippocampal neurons. Hippocampal neurons cultured for 3 weeks were double-labeled with antibodies to RICS and MAP2
(A-D), antibodies to RICS and
-catenin
(E-G), antibodies to RICS and PSD-95 (H-J), or
antibodies to RICS and NR2B (K-M). C, control
experiments performed with anti-RICS antibody that had been preabsorbed
with antigen. Scale bars, 10 µm.
-32P]GTP, it was found to stimulate hydrolysis of GTP
to GDP (Fig. 5A). Arg-58 and
Lys-98 of the GAP domain of RICS correspond to the conserved amino acid
residues of RhoGAPs, which are known to be required for GAP activity
per se and for interaction with Rho GTPases, respectively
(36, 37). We therefore generated mutated versions of RICS, designated
RICS-R58A and -K98A, in which Arg-58 and Lys-98, respectively, were
replaced with Ala. Unexpectedly, RICS-R58A showed GAP activity against
RhoA, Rac1, and Cdc42, although RICS-K98A was inactive (Fig.
5A). After further considering the three-dimensional
structure of the Rho GTPases-GAP complexes (38-40), we then replaced
Arg-58 with Ile and Met, respectively, and generated RICS-R58I and
-R58M. As expected these mutants were found to possess no GAP activity
with respect to RhoA, Rac1, or Cdc42 (Fig. 5B; see below).
These results indicate that RICS possesses GAP activity toward RhoA,
Rac1, and Cdc42.
View larger version (25K):
[in a new window]
Fig. 5.
RICS possesses GAP activity for Rho
GTPases. A, time course of GTPase activity of
baculoviral His6-RhoA, -Rac1, and -Cdc42 in the presence or
absence of the GAP domain of RICS, -RICS-R58A, -RICS-K98A, or
-p50RhoGAP fused to GST. B, the levels of active GTP-bound
RhoA, Rac1, and Cdc42 were measured in NIH3T3 cells infected with a
control retrovirus or a retrovirus encoding either wild-type or mutant
RICS. Wild-type and mutant RICSs were expressed at comparable levels
(data not shown). GTPase activities of RhoA, Rac1, and Cdc42 were
assessed using a GST fusion protein derived from Rhotekin
(RBD), which selectively binds GTP-bound RhoA, or PAK-3
(PBD), which selectively binds GTP-bound Rac1 and Cdc42.
GST-RBD and -PBD precipitates were immunoblotted with antibodies to
RhoA, Rac1, and Cdc42.
View larger version (29K):
[in a new window]
Fig. 6.
The GAP activity of RICS is inhibited by
phosphorylation by CaMKII. A, phosphorylation of RICS
by purified CaMKII. RICS immunoprecipitated (IP) with
anti-RICS antibody (lanes 1-5) or preimmune IgG
(lanes 6 and 7) from the PSD fraction of mouse
brain lysate was phosphorylated in phosphorylation buffer containing
the indicated combinations of Ca2+, calmodulin, and the
CaMKII inhibitor KN-93 (30 µM). After incubation at
30 °C for 1 h, the samples were subjected to SDS-PAGE and
autoradiography. B, phosphorylation of RICS by purified
CaMKII inhibits its GAP activity against Cdc42. RICS was
immunoprecipitated with anti-RICS antibody (lanes 1-5) or
preimmune IgG (lanes 6 and 7) from the PSD
fraction of mouse brain lysate, phosphorylated by purified CaMKII as in
A at 30 °C for 2 h, and then subjected to an
in vitro GAP assay. Data were obtained from five independent
experiments. Phosphorylation conditions corresponding to each graph are
indicated in the right side.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-catenin interacts with
RICS via armadillo repeats 10-12. Furthermore, we showed that RICS is
enriched in the PSD fraction and coimmunoprecipitates with N-cadherin,
PSD-95, and NMDA receptors from the PSD of mouse brain. Consistent with
this result, RICS, PSD-95, and NMDA receptors colocalized at the
synapses in cultured hippocampal neurons. In addition, in
vitro pull-down assays showed that in vitro translated RICS weakly interacts with NR2A/2B and PSD-95 but not with NR1 (data
not shown). Although these weak interactions were reproducibly detected
in our experimental conditions, we do not know whether these
interactions are physiologically important. In any case, these results
suggest that RICS,
-catenin, N-cadherin, PSD-95, and NMDA receptors
are contained within the same complex in vivo. The PSD-95
family of proteins interacts with various proteins, including
DAP (hDLG-associated protein)/SAPAP (synapse-associated protein
90-associated protein)/GKAP (guanylate kinase-associated protein)
(43-46), SPAL/SPAR (47, 48), synGAP (49, 50), Kalirin-7 (51), CRIPT
(52), neuroligin (53), and nNOS (54). Thus, RICS may be contained in a
multicomponent complex consisting of adhesion proteins, NMDA receptors,
and other various structural and signaling molecules.
-catenin complex is involved in N-cadherin-mediated
regulation of spine morphology.
-catenin from dendritic shafts into spines and
increases its association with cadherins, thereby promoting changes in
synaptic structure and function (10). Thus, it would be interesting to
determine whether RICS is also induced to redistribute together with
-catenin into spines or if RICS associates with redistributed
-catenin in the PSD. The redistribution of
-catenin is mimicked
or prevented, respectively, by a tyrosine kinase or phosphatase
inhibitor (10). Furthermore, Murase et al. (10) demonstrated
that a point mutation in
-catenin Tyr-654 alters spine-shaft
distribution; a mutant
-catenin, in which Tyr-654 is replaced with
Phe, thereby abrogating phosphorylation, is concentrated in spines,
whereas a mutant
-catenin, in which Tyr-654 is replaced with Glu to
mimic phosphorylation, accumulates in dendritic shafts. Interestingly,
RICS interacts with the armadillo repeats 10-12 of
-catenin and
Tyr-654 resides in armadillo repeat 12. It would therefore be
interesting to examine whether phosphorylation of
-catenin Tyr-654
regulates its interaction with RICS.
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ACKNOWLEDGEMENTS |
---|
We thank T. Nagase for the KIAA0712 cDNA clone and C. Toyoshima for helpful discussion.
![]() |
FOOTNOTES |
---|
* This work was supported by grants-in-aid for Scientific Research on Priority Areas (C), Cancer and Advanced Brain Science Project, from the Ministry of Education, Culture, Sports, Science and Technology, 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.
¶ Contributed equally to this work.
To whom correspondence should be addressed. Tel.:
81-3-5841-8483; Fax: 81-3-3841-8482; E-mail:
akiyama@iam.u-tokyo.ac.jp.
Published, JBC Papers in Press, January 16, 2003, DOI 10.1074/jbc.M208872200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
LTP, long term
potentiation;
PSD, postsynaptic density;
NMDA, N-methyl-D-aspartate;
NMDA-R, NMDA receptor;
CaMKII, calmodulin-dependent protein kinase II;
GST, glutathione S-transferase;
IP, immunoprecipitation;
RBD, Rho-binding domain;
PBD, p21-binding domain;
AMP-PNP, adenosine
5'-(,
-imino)triphosphate or adenosine
5'-(
,
-iminotriphosphate);
GAP, GTPase-activating protein;
NR, NMDA receptor;
MAP, microtubules-associated protein.
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