From the Department of Physiological Chemistry and the
Department of Molecular Neurobiology, Faculty of
Pharmaceutical Sciences, Kyoto University, Sakyo-ku,
Kyoto 606, Japan
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ABSTRACT |
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We previously reported that the activation of
prostaglandin E receptor EP3 subtype caused neurite retraction via
small GTPase Rho in the EP3B receptor-expressing PC12 cells (Katoh, H.,
Negishi, M., and Ichikawa, A. (1996) J. Biol. Chem.
271, 29780-29784). However, a potential downstream effector of
Rho that induces neurite retraction was not identified. Here we
examined the morphological effect of p160 RhoA-binding kinase ROK, a
target for RhoA recently identified, on the nerve growth
factor-differentiated PC12 cells. Microinjection of the catalytic
domain of ROK
rapidly induced neurite retraction similar to that
induced by microinjection of a constitutively active Rho,
RhoV14, whereas microinjection of the kinase-deficient
catalytic domain of ROK
did not induce neurite retraction. This
morphological change was observed even though C3 exoenzyme, which was
known to inactivate Rho, had been preinjected. On the other hand,
microinjection of the Rho-binding domain or the pleckstrin homology
domain of ROK
inhibited the EP3 receptor-induced neurite retraction.
These results demonstrate that ROK
induces neurite retraction acting downstream of Rho in neuronal cells.
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INTRODUCTION |
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Rho, a member of the Ras superfamily of small GTPases, is
implicated in various cellular morphological functions, such as formation of stress fibers and focal adhesion (1), cell motility (2),
cytokinesis (3), cell aggregation (4), and smooth muscle contraction
(5). When cells are activated by extracellular stimuli, inactive
GDP-bound Rho is converted to active GTP-bound Rho. Once activated, Rho
probably interacts with its specific targets, leading to a variety of
biological functions (6). Recently, several target proteins that
interact only with GTP-bound Rho have been identified, including p128
protein kinase N (7, 8), p160 RhoA-binding kinase ROK (9) also known
as its bovine counterpart Rho-kinase (10) or its mouse counterpart ROCK-II (11), rhophilin (7), rhotekin (12), and p140mDia (13). Among
them, ROK
has been reported to be involved in several functions of
Rho: the regulation of myosin phosphorylation (14, 15), the formation
of stress fibers and focal adhesions (16, 17), and probably the
regulation of cytokinesis (18).
Rho has also been implicated in the control of neuronal cell morphologies. The activation of a certain heterotrimeric GTP-binding protein (G-protein)-coupled receptor,1 such as lysophosphatidic acid and thrombin receptors, caused the rapid retraction of extended neurites in several neuronal cell lines (19-21). Clostridium botulinum C3 exoenzyme, which specifically ADP-ribosylates Rho and suppresses the actions of Rho (22, 23), inhibits the receptor-mediated neurite retraction (24, 25), indicating that Rho activity is required for this morphological change. Although this effect appears to be induced by the contractility of the actin-based cytoskeleton (24, 26), a downstream effector of Rho that induces neurite retraction has not yet been identified.
We previously reported that the activation of prostaglandin EP3
receptor caused Rho-dependent neurite retraction in the
NGF-differentiated PC12 cells expressing the EP3B receptor (27), one of
the EP3 receptor isoforms isolated from bovine adrenal medulla (28). In
non-neuronal cells, the activation of EP3 receptor stimulates the
Rho-mediated formation of stress fibers (29), indicating that EP3
receptor is a potent activator of Rho in various cell types. In this
report, we have examined the putative role of ROK in the EP3
receptor-mediated neurite retraction in the NGF-differentiated PC12
cells. We show that ROK
is involved in the EP3 receptor-mediated neurite retraction and that the activation of ROK
is sufficient for
inducing neurite retraction.
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EXPERIMENTAL PROCEDURES |
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Materials-- M&B28767 was a generous gift from Dr. M. P. L. Caton of Rhone-Poulene Ltd. NGF 2.5S was purchased from Promega Corporation, and C. botulinum C3 exoenzyme was from Seikagaku Kogyo (Tokyo, Japan). The sources of the other materials are shown in the text.
Expression and Purification of Recombinant Proteins--
The
coding region of human RhoA was generated by reverse
transcription-polymerase chain reaction (PCR) from HeLa cells using primers 5-CTGGACTCGAATTCGTTGCCTGAGCAATGG-3
and
5
-GCAAGATGAATTCTGATTTGTAATCTTAGG-3
. The PCR product was digested with
EcoRI, cloned into the pBluescript KS(+), and completely
sequenced. cDNAs for RhoAV14 and
RhoAV14A37 were generated by PCR-mediated mutagenesis (30),
subcloned into the BamHI/EcoRI sites of pGEX-4T-2
vector, and sequenced. Recombinant RhoAV14 and
RhoAV14A37 were expressed as glutathione
S-transferase (GST) fusion proteins in
Escherichia coli, and purified on glutathione-Sepharose
beads according to the method of Self and Hall (31). The sequence encoding ROK
containing the catalytic domain (CD-ROK
, amino acids
1-543), the Rho-binding domain (RBD-ROK
, amino acids 932-1065), and the pleckstrin homology (PH) domain (PHD-ROK
, amino acids 1116-1379) were generated by reverse transcription-PCR from PC12 cells, using primers 5
-ATGAGCGGATCCCCGCCGACGGGGAAA-3
and
5
-ACCTTCTGAATTCATATCTGAGAGCTCTGG-3
for CD-ROK
,
5
-GACGGATCCAAAGAGAAGATCATGAAAGAGC-3
and
5
-GTTGTGTGAATTCTTAACGTTCAG-3
for RBD-ROK
, and
5
-TCGCAGGGATCCGCCTTGCATATTGG-3
and 5
TCTTGTGGATGGAAGAATTCGATCACCTTC3
for PHD-ROK
, respectively. The kinase-deficient mutant of
CD-ROK
(CD-ROK
K112G) was generated by PCR-mediated
mutagenesis. All PCR products for each domain of ROK
were cloned
into the pCR2.1 vector and sequenced completely. The PCR products for
RBD-ROK
and PHD-ROK
were subcloned into the
BamHI/EcoRI sites of pGEX-4T-2 vector, and
recombinant proteins were expressed as GST fusion proteins in E. coli and purified on glutathione-Sepharose beads. The PCR products
for CD-ROK
and CD-ROK
K112G were subcloned into the
BamHI/EcoRI sites of pAcG2T vector, and recombinant proteins were expressed as GST fusion proteins in Sf9 cells with BaculoGoldTM system (PharMingen) and purified on glutathione-Sepharose beads according to the method of Matsui et
al. (10). All recombinant proteins were dialyzed with an injection
buffer (10 mM Tris-HCl, pH 7.6, 150 mM NaCl, 2 mM MgCl2, and 0.1 mM
dithiothreitol) at 4 °C overnight for microinjection. Protein
concentration was determined by comparing with bovine serum albumin
standards after electrophoresis on a SDS-polyacrylamide gel and
staining with Coomassie Brilliant Blue.
Cell Culture and Microinjection-- The EP3B receptor-expressing PC12 cells (27) were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, 5% horse serum, 4 mM glutamine, 100 units/ml penicillin, and 0.2 mg/ml streptomycin under humidified conditions in 95% air and 5% CO2 at 37 °C. For microinjection, cells were seeded on poly-D-lysine-coated (Sigma) 35-mm dishes, which were marked with a cross to facilitate the localization of injected cells. After the cells had been differentiated in serum-free Dulbecco's modified Eagle's medium containing 50 ng/ml NGF and 20 µM indomethacin for 3 days, microinjection was performed using an IMM-188 microinjection apparatus (Narishige, Tokyo, Japan). During microinjection, the differentiated cells were maintained in Hepes-buffered Dulbecco's modified Eagle's medium, pH 7.4, at 37 °C. Cells were photographed at × 200 magnification under a phase contrast microscope. For the quantitative examination shown in Fig. 3, neurite-retracted cells were defined as the cells that retracted by more than 10% of their original length within 30 min of the addition of the agonist or of the microinjection of recombinant proteins. The percentages of neurite-retracted cells were calculated by counting at least 30 cells in the same field. Data were obtained from triplicate experiments.
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RESULTS AND DISCUSSION |
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In our previous study, we obtained evidence that M&B28767, an EP3 agonist, induced neurite retraction in the EP3B receptor-expressing PC12 cells and that this morphological change was completely inhibited when the cells were microinjected with C3 exoenzyme, which ADP-ribosylates and inactivates Rho (22, 23), indicating that the EP3B receptor induced neurite retraction through the activation of Rho (27). To determine whether activation of Rho is sufficient for inducing neurite retraction in the PC12 cells, we microinjected a constitutively activated recombinant RhoA, RhoAV14 into the NGF-differentiated PC12 cells and examined its effect. As shown in Fig. 1 (C and D), microinjection of RhoV14 into the cytoplasm caused retraction of the neurites within 30 min. More than 70% of the injected cells retracted their neurites (Fig. 3). This morphological change was similar to that stimulated by M&B28767 (Fig. 1, A and B). The neurite-retracted cells by microinjection of RhoAV14 was not stained with trypan blue (data not shown), indicating that they did not undergo cell death. On the other hand, RhoAV14 containing a T37A substitution in the effector region, RhoAV14A37, had no effect on the differentiated cells after microinjection (Fig. 1, E and F, and Fig. 3), suggesting that this mutation blocked the interaction of Rho with its target to induce neurite retraction. This result also indicated that there were not any nonspecific effects due to the microinjection itself.
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Previous studies suggested that the generation of actin-based
contractile forces was required for neurite retraction (24, 26). Among
several targets of Rho, ROK appears to participate in
Rho-dependent contractile events, such as the formation of stress fibers (16, 17) and the regulation of cytokinesis (18). By
Northern blot analysis, ROK
was expressed in the NGF-differentiated PC12 cells (data not shown). Therefore, we examined whether ROK
was
involved in the Rho-mediated neurite retraction in the
NGF-differentiated PC12 cells. ROK
contains the catalytic domain in
its amino terminus, the coiled-coil domain, the Rho-binding domain, and
the PH domain in its carboxyl terminus (9). It was recently shown that
the truncation mutant of ROK
containing the catalytic domain
displayed constitutive kinase activity without the addition of active
form of Rho, whereas the Rho-binding domain and the PH domain of ROK
served as dominant negative forms of the kinase (16, 17). Based on
these characters, we generated recombinant proteins containing these
domains: the catalytic domain of ROK
(CD-ROK
, amino acids 1-543), the kinase-deficient mutant of CD-ROK
(CD-ROK
K112G), the Rho-binding domain of ROK
(RBD-ROK
, amino acids 932-1065), and the PH domain of ROK
(PHD-ROK
, amino acids 1116-1379). To examine the effects of these
domains of ROK
on neurite retraction, we microinjected these
recombinant proteins into the NGF-differentiated cells and analyzed
their morphologies.
After the cells had been microinjected with CD-ROK, they rapidly
retracted their neurites within 30 min (Fig.
2, A and B, and
Fig. 3). This morphological change was
similar to that induced by microinjection of RhoAV14. The
neurite-retracted cells by microinjection of CD-ROK
was not stained
with trypan blue (data not shown), indicating that they did not undergo
cell death. On the other hand, microinjection of the kinase-deficient
mutant of CD-ROK
mutant CD-ROK
K112G had no effect
(Fig. 2, C and D, and Fig. 3), indicating that the kinase activity of CD-ROK
was required for inducing neurite retraction. When the cells were microinjected with C3 exoenzyme, the
M&B28767-induced neurite retraction was completely inhibited (Fig. 2,
E and F). However, the CD-ROK
-induced neurite
retraction was not inhibited after the cells had been microinjected
with C3 exoenzyme (Fig. 2, G and H), indicating
that CD-ROK
acted downstream of Rho.
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Next we microinjected RBD-ROK or PHD-ROK
, which served as
dominant negative forms of ROK
, into the differentiated cells and
examined each effect on the M&B28767-induced neurite retraction. When
the cells had been microinjected with RBD-ROK
or PHD-ROK
, the
M&B28767-induced neurite retraction was inhibited (Fig.
4). All the cells microinjected with
RBD-ROK
or PHD-ROK
had no response to M&B28767. These results
suggest that ROK
is involved in the EP3 receptor-mediated neurite
retraction in the PC12 cells. Taken together, our results suggest that
ROK
induces neurite retraction acting downstream of Rho in the
NGF-differentiated PC12 cells.
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Recently, ROK was shown to be involved in Rho-induced formation of
stress fibers and focal adhesion in other cell types such as
fibroblasts. However, the organization of stress fibers induced by
constitutively active ROK
was apparently different from that induced
by lysophosphatidic acid or constitutively active Rho (16, 17),
suggesting that additional signals were required for Rho-induced stress
fiber formation. In this study, however, microinjection of CD-ROK
sufficiently induced neurite retraction similar to that induced by
RhoV14 even though C3 exoenzyme had been preinjected,
whereas CD-ROK
K112G failed to induce neurite retraction
(Figs. 2 and 3), suggesting that the increase in the kinase activity of
ROK
by Rho appears to be sufficient for inducing neurite retraction.
Because myosin-binding subunits of myosin phosphatase and myosin light
chain are known to be substrates for ROK
and activation of ROK
leads to phosphorylation and activation of myosin (14, 15), neurite
retraction may be induced by ROK
-mediated regulation of myosin
phosphorylation. In addition, it was recently reported that glial
fibrillary acidic protein, an intermediate filament protein expressed
in the cytoplasm of astroglia, was identified as another substrate for
ROK
(18). Therefore, we will consider substrate(s) of this kinase
for neurite retraction in future studies. Until now, we have obtained
evidence that the activation of EP3B receptor, coupling to Rho
activation, did not affect the NGF-induced mitogen-activated protein
kinase activation in the PC12 cells (data not shown), suggesting that the activation of Rho or ROK
did not inhibit the NGF-induced signaling to the Ras-mitogen-activated protein kinase pathway. To
examine the direct effect of Rho or ROK
on the NGF-induced differentiation, we are currently establishing PC12 cell lines that
express RhoAV14 or CD-ROK
under the control of an
inducible promoter.
As shown in Fig. 4, two fragments of ROK, the Rho-binding domain and
the PH domain, served as dominant negative forms of ROK
in the EP3
receptor-mediated neurite retraction, as reported for the formation of
stress fibers and focal adhesion (16, 17). ROK
has been shown to be
translocated to peripheral membranes upon transfection with
RhoV14 (9). Because PH domains are supposed to play a key
role in localization of molecules to the specific target regions in the membranes, the PH domain of ROK
may localize this kinase at the specified region in response to the EP3 receptor-induced activation of
Rho, and this translocation of ROK
to its target region seems to be
essential for inducing neurite retraction. On the other hand,
RBD-ROK
may block the interaction between endogenous Rho and ROK
.
We also showed that RhoAV14A37, a mutant at the effector
region, lost the ability to induce neurite retraction in the
differentiated PC12 cells (Fig. 1, E and F, and
Fig. 3). Indeed, RhoAV14 bound to the RBD-ROK
, but
RhoAV14A37 did not (data not shown). This defect in binding
to ROK
seems to be the reason for the inability of
RhoAV14A37 to induce neurite retraction.
In conclusion, we have here shown that ROK is an essential component
of Rho-mediated neurite retraction in neuronal cells. Considering that
ROK
is enriched in the brain (16), ROK
may play a critical role
in the regulation of neuronal cell morphology in the brain. However,
many questions have not yet been elucidated in this field, for example
how the G-protein coupled receptor activates Rho. Further
investigations are necessary to understand Rho-mediated signal
transduction in neuronal cells.
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FOOTNOTES |
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* This work was supported in part by Grants-in-aid for Scientific Research 09273105, 09259219, and 09307052 from the Ministry of Education, Science, and Culture 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.: 81-75-753-4547; Fax: 81-75-753-4557; E-mail: mnegishi{at}pharm.kyoto-u.ac.jp.
1 The abbreviations used are: G-protein, GTP-binding protein; PCR, polymerase chain reaction; GST, glutathione S-transferase; PH, pleckstrin homology; PHD, PH domain; NGF, nerve growth factor; CD, catalytic domain; RBD, Rho-binding domain.
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REFERENCES |
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