From the Department of Pharmacology, Kyoto University Faculty of Medicine, Sakyo-ku, Kyoto 606, Japan and the ¶ CRC Oncogene and Signal Transduction Group, MRC Laboratory for Molecular Cell Biology and Department of Biochemistry, University College London, Gower Street, London WC1E 6BT, United Kingdom
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ABSTRACT |
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Based on their Rho binding motifs several Rho target molecules can be classified into three groups; class I includes the protein kinase PKN, rhophilin, and rhotekin, class II includes the protein kinases, Rho-associated coiled-coil containing protein kinases, ROCK-I and ROCK-II, and class III includes citron. Taking advantage of the selectivity in recognition by these targets between Rho and Rac, we examined the regions in Rho required for selective binding of each class of Rho target molecules. Yeast two-hybrid assays were performed using Rho/Rac chimeras and either rhophilin, ROCK-I, or citron. This study showed the existence of at least two distinct regions in Rho (amino acids 23-40 and 75-92) that are critical for the selective binding of these targets. The former was required for binding to citron, whereas the latter was necessary for binding to rhophilin. On the other hand, either region showed affinity to ROCK-I. This was further confirmed by ligand overlay assay using both recombinant ROCK-I and ROCK-II proteins. Consistently, Rho/Rac chimeras containing either region can induce stress fibers in transfected HeLa cells, and this induction is suppressed by treatment with Y-27632, a specific inhibitor of ROCK kinases. These results suggest that the selective binding of different classes of Rho targets to Rho is determined by interaction between distinct Rho-binding motifs of the targets and different regions of Rho.
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INTRODUCTION |
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The small GTPase Rho functions as molecular switches in various cellular processes. Rho mediates the integrin-dependent cell adhesion of a variety of cells to the substratum, which is typically found as the formation of focal adhesions and stress fibers in fibroblasts. Another member of the Rho family proteins, Rac, induces the assembly of small focal complexes at the periphery of cells, and controls the production of lamellipodia and membrane ruffles (1-4). Rho is required for cytokinesis, inducing and maintaining the contractile ring (5, 6). Rho is also critical in actomyosin-based contractility as it increases calcium sensitivity in smooth muscles (7) and mediates stimulus-evoked neurite retraction in N1E-115 neuroblastoma cells (8). Furthermore, it has been reported that Rho is involved in transcriptional activation through the serum response element (9), and in cell growth and transformation (10-12).
These various activities of Rho appear to be regulated by multiple
signaling pathways, which may be driven by multiple effector molecules.
Based on selective binding to the GTP-bound form of Rho, we recently
isolated several potential Rho target proteins. Using yeast two-hybrid
system with Rho as a bait, we isolated a protein serine/threonine
kinase PKN and two nonkinase molecules, rhophilin and rhotekin (13,
14). Interestingly, these proteins possess Rho-binding domains of 70 amino acid-stretch in their N termini, which show 27-40% identity.
This conserved Rho-binding motif is called class I to distinguish it
from other Rho-binding motifs. We also isolated another
serine/threonine protein kinase, p160ROCK, as a
[35S]GTPS-Rho-binding protein using the ligand overlay
assay (15). This molecule has a serine/threonine kinase domain in its
N-terminal region, followed by an approximately 600-amino acid-long
-helix capable of forming a coiled-coil structure, a cysteine-rich
zinc finger-like motif and a pleckstrin homology region in the C
terminus. An isozyme of p160ROCK was also isolated and
called ROCK-II/ROK
/Rho kinase (16-18). We and others have reported
that the ROCK1 isozymes are
Rho effectors that induce formation of focal adhesions and stress
fibers (19-21). This has been confirmed recently by the use of a
specific ROCK inhibitor, Y-27632 (22). The Rho-binding domain of
p160ROCK is 81-amino acid-long (aa 934-1015) and is
located between a coiled-coil structure and a pleckstrin homology
domain (23). This region is conserved in ROCK-II/ROK
/Rho kinase
(16-18). This Rho-binding motif has no homology with the class I
motif, thus defining a distinct class of binding motifs for the
GTP-bound form of Rho (class II). Using the two-hybrid system, we also
isolated a GTP-Rho-interacting molecule defining a third class of
Rho-binding motif. This molecule, citron, like ROCK-I and -II, contains
a long coiled-coil structure followed by a pleckstrin homology domain and a cysteine-rich region (24). The Rho-binding domain of this protein
has been mapped within the coiled-coil region, where no significant
homology could be found with the class I or II motifs.
These findings suggest that there are at least three classes of Rho targets with distinct Rho-binding motifs. This raises a question about the domains of Rho that interact with these targets; do different classes of Rho-binding motifs bind to an identical effector domain of Rho or does each motif recognize a different region of Rho? To solve this issue, we have determined the regions of Rho that are required for selective binding of each class of Rho target molecules to Rho.
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EXPERIMENTAL PROCEDURES |
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Materials--
[35S]GTPS (1,000 Ci/mmol) was
purchased from NEN Life Science Products. Mouse monoclonal
anti-vinculin antibody (V-4505), mouse monoclonal anti-Flag antibody
(M2), and rabbit polyclonal anti-Flag antibody (D-8) were purchased
from Sigma, Eastman Kodak Company, and Santa Cruz Biotechnology,
respectively. fluorescein isothiocyanate-conjugated goat anti-mouse IgG
and rhodamine-conjugated horse anti-rabbit IgG were obtained from
Vector Laboratories, and rhodamine-conjugated phalloidin was from
Molecular Probes. Plasmids of pGEX-Val14-Rho73Rac
(N-terminal 75 residues from Rho and the remainder from Rac),
pGEX-Val12-Rac73Rho, pGEX-Val12-Rac117Rho,
pGEX-L63-Rho73Rac90Rho, pGEX-L61-Rac73Rho90Rac,
and pGEX-rhoGAP were described previously (25). As indicated above, the
amino acid numbers in Rho/Rac chimeras used in this study are based on
those of Rac and not of Rho (Fig. 1).
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Construction of Rho/Rac Chimeras and Yeast Two-hybrid Assay with Various Rho-binding Proteins-- Rho/Rac chimeras used in the present study are shown in Fig. 2. cDNA constructs of these chimeras were prepared as follows. cDNA fragments corresponding to aa 1-24, 22-42, and 40-62 of RhoA, and corresponding to aa 1-22, 20-40, and 38-60 of Rac1 were amplified by polymerase chain reaction with Val14-RhoA (23) and Val12-Rac117Rho cDNA as templates, respectively, using primer pairs as shown in Table I. Polymerase chain reaction conditions were 95 °C for 3 min, 25 cycles of 1 min at 95 °C, 1 min at 58 °C and 1 min at 72 °C, and 72 °C for 2 min. The products were then blunted and ligated into the HincII site of pBluescript SK+ (pSK+-Rho1-24, -Rho22-42, -Rho40-62, -Rac1-22, -Rac20-40, and -Rac38-60, respectively). To combine Rho1-24 or Rac1-22, and Rho22-42 or Rac20-40, the PstI-NdeII fragment of pSK+-Rho1-24/-Rac1-22 and the NdeII-PstI fragment of pSK+-Rho22-42/-Rac20-40 were ligated into the PstI site of pSK+ to generate pSK+-Rho42, -Rho21Rac40, and -Rac21Rho42. To combine Rho42, Rho21Rac40, or Rac21Rho42, and Rho40-62 or Rac38-60, the plasmids pSK+-Rho40-62 and -Rac38-60 were digested with EcoRI and blunted, then digested with TspEI. Each of these fragments and the TspEI-PstI fragment of Rho42, Rho21Rac40, or Rac21Rho42 were inserted into the PstI and EcoRV sites of pSK+ to generate pSK+-Rho38Rac60, -Rho21Rac60, -Rho21Rac38Rho62, -Rac21Rho62 and -Rac21Rho38Rac60. Then, to make full-length chimeras, each of these BamHI-PvuII fragments of the above plasmids and each PvuII-EcoRI fragment of Val14-Rho or Val14-Rho73Rac were ligated into the BamHI and EcoRI sites of pSK+ to generate pSK+-Rho38Rac, Rho21Rac, Rho21Rac38Rho, Rac21Rho73Rac and Rac21Rho38Rac. For the yeast two-hybrid system, the BamHI-EcoRI fragments of each pSK plasmid DNA (Rho38Rac, Rho21Rac, Rho21Rac38Rho, Rac21Rho73Rac, and Rac21Rho38Rac) were inserted into modified pBTM116 (24). The BamHI-PvuII fragment of pGEX-Rho73Rac90Rho and the PvuII-EcoRI fragment of pGEX-Rac73Rho90Rac were inserted into the BamHI and EcoRI sites of modified pBTM116 to generate Rho90Rac. Similarly the BamHI-PvuII fragment of pGEX-Rac73Rho90Rac and the PvuII-EcoRI fragment of pGEX-Rho73Rac90Rho were ligated into the BamHI and EcoRI sites of modified pBTM116 (Rac90Rho). Finally the BamHI-PvuII fragment of pGEX-Rac73Rho and the PvuII-EcoRI fragment of pGEX-Rho73Rac were inserted into the BamHI and EcoRI sites of modified pBTM116 to generate pBTM116-Rac.
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Expression and Purification of Recombinant Proteins and Ligand
Overlay Assay--
The BamHI-EcoRI fragments of
each pBTM116 plasmid DNA coding for each Rho/Rac chimera were inserted
into pGEX-2T. pQE constructs for the Rho-binding domains of ROCK-I and
-II were described previously (23, 18). Escherichia coli
(DH5) was transformed with the pGEX plasmids or pQE plasmids described
above and grown. Isopropyl--D-thiogalactoside was added
to the culture when A600 of the suspension was
0.8, and the culture was continued for another 20 h at 30 °C.
Cells were collected and lysed, and the expressed proteins were
purified as described previously (23). The purity of the preparations was examined by SDS-polyacrylamide gel electrophoresis. Each
preparation showed a single major protein band at the expected size
(data not sown).
Analysis for Rho and Rac Phenotypes in HeLa Cells--
The
BamHI-EcoRI fragments from pBTM116 plasmids
containing each Rho/Rac chimera cDNA was subcloned into the
BamHI and EcoRI sites of a pCMX-flag
vector such that the insert would come in-frame to a Flag epitope at
the N terminus. A HindIII-EcoRI fragment was
excised from each resultant plasmid, blunted, and inserted into the
blunted XhoI site of a pCAG-GS to generate a mammalian expression construct driven by a chick -actin promoter (28). A
mammalian expression construct for botulinum C3 exoenzyme
was prepared to inhibit the action of endogenous Rho. pET-C3 (29) was
digested with NdeI and BamHI, blunted, and
inserted into the blunted BstXI site of pEF-BOS (30) to
yield pEF-C3.
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RESULTS |
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We have recently identified the Rho-binding domain of
p160ROCK (ROCK-I) and found that the amino acid sequence of
this domain has no homology with those of rhophilin, PKN, or rhotekin,
which have the class I Rho-binding motif (14), nor to the Rho-binding domain of another Rho-target protein, citron (23, 24). This suggested
that Rho-target molecules, all showing the selective binding to the
GTP-bound form of Rho, could be separated into at least three classes
based on the Rho-binding motifs. We designate here the motifs of the
Rho-binding domains of ROCK-I and -II as class II and that in citron as
class III. This poses a question as to the binding sites of Rho
responsible for interaction with each class of target molecules. To
investigate which region(s) of Rho determines binding of each class of
the Rho targets, we have prepared various chimeras between dominant
active mutant forms of Rho and Rac (Fig. 2), and analyzed their binding
to various Rho targets in a yeast two-hybrid analysis. Active Rho was
foiled with active Rac at about 20-amino acid intervals so that we
could identify the Rho region conferring specific binding to Rho. We have chosen the Rho-binding domains of rhophilin, ROCK-I, and citron as
representatives of the class I, II, and III domains, respectively, and
expressed each as a fusion protein to the VP16 activation domain in the
yeast strain AMR70. These transformants were mated with a yeast strain
L40 expressing various Rho/Rac chimeras fused to the LexA DNA-binding
domain. Interaction was estimated by detection and quantification of
-galactosidase activity. To test that all the Rho/Rac chimeras used
here were expressed in yeast and translocated to the nucleus, we used
the Rho/Rac-binding domains of p120rhoGAP and kinectin as
positive controls (25, 26).
A summary of these analyses are shown in Fig. 2. All the chimeras shown
in Fig. 2 interacted with both rhoGAP and kinectin. As both Rho and Rac
bind to kinectin, failure of a given chimera in binding to kinectin may
indicate that normal conformation is altered in this chimera. Among
chimeras tested in the two-hybrid system, four chimeras, Rac90Rho,
Rac117Rho, Rho73Rac90Rho and Rac73Rho90Rac, did not show positive
binding to kinectin. Their analyses were therefore not included in Fig.
2. Significant interactions with rhophilin were found for Rho, Rho90Rac
and Rac73Rho, all of which contained aa 75-92 of Rho. However, under
the same conditions, -galactosidase signals were not detected in the
diploids containing chimeras that lacked aa 75-92 of Rho such as Rac
or Rho73Rac. These results suggested that aa 75-92 of Rho is necessary
for rhophilin to bind to Rho.
As for citron, interactions were strong with Rho, Rho90Rac, Rho73Rac,
Rho38Rac, Rac21Rho73Rac and Rac21Rho38Rac, all of which contained aa
23-40 of Rho. Only a weak signal was observed with Rho21Rac (Fig. 2).
A weak interaction was also found with Rac. This is consistent with our
previous finding that Rac-GTPS binds weakly to citron in a membrane
overlay assay (24). These results imply that aa 23-40 of Rho is
critical for high affinity interaction between Rho and citron. However,
unlike rhophilin, no requirement of aa 75-92 was observed. These
results indicate that the high affinity interactions between Rho-GTP
and two different classes of effectors, rhophilin and citron, are
determined by two independent target recognition sequences, aa 75-92
and aa 23-40 of Rho.
We next examined the target binding determinant of Rho required for binding of the class II Rho target, ROCK-I. Strong color development was found with Rho, Rho90Rac, and Rho73Rac. The interaction was reduced but still significant with Rho38Rac and was abolished with Rho21Rac and Rac (Fig. 2). These results clearly suggested the importance of aa 23-40 of Rho in ROCK binding, similar to the case with citron. Consistently, the binding domain of ROCK-I could bind to Rac21Rho38Rac. A reduction in the interaction was noted when the interaction with Rac21Rho38Rac was compared with that with Rac21Rho73Rac, suggesting that aa 41-75 of Rho may also contribute to some extent. On the other hand, ROCK-I also interacted with Rac73Rho, which did not contain aa 23-75 (Fig. 2), indicating that another binding recognition site to class II molecules was present in a region C-terminal of aa 75. This was consistent with the positive interaction of ROCK-I with Rho21Rac38Rho.
To confirm this possibility and identify a region responsible for this
interaction, we examined the ROCK-I interaction with Rac73Rho90Rac and
compared it with that of Rho73Rac90Rho. The ligand overlay assay was
performed, because these two chimeras did not bind to kinectin in the
two-hybrid assay. As shown in Fig.
3A,
[35S]GTPS-bound Rho but not Rac bound to the
Rho-binding domain of ROCK-I renatured on the membrane. The binding
similar in extent to Rho was found with both Rac73Rho90Rac and
Rho73Rac90Rho. These results supported the above idea and revealed that
in addition to aa 23-40, aa 75-92 of Rho could provide the binding
determinant for ROCK-I.
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Using the ligand overlay assay, we also examined the binding determinants for ROCK-II. Radioactive binding was seen only in lanes that were overlaid with the chimeras containing either aa 23-40 or aa 75-92 of Rho such as Rac21Rho38Rac and Rac73Rho90Rac (Fig. 3B). No binding was detected with Rac117Rho, indicating that the binding determinants for ROCK-II, a class-II molecule, were localized to a Rho region N-terminal of aa 119. Thus, the results obtained by the ligand overlay analysis were consistent with those of the two-hybrid system, and indicated that aa 23-40 and aa75-92 of Rho were important in selective binding to both ROCK-I and -II. It remains unclear whether aa 93-119 of Rho plays any role in this binding.
To test whether the interactions observed in in vitro assays take place in signal transduction of Rho in situ in the intact cell, we expressed each Rho/Rac chimera in HeLa cells and examined their phenotypes. We and others (19-21) recently showed that the ROCK/ROK/Rho kinase family of kinases work as Rho effectors to induce the formation of focal adhesions and stress fibers in cultured cells. Furthermore, we have shown that Rho-dependent induction of stress fibers and focal adhesions is completely suppressed by the pyridine derivative Y-27632, a specific inhibitor of the ROCK isozymes, but that the treatment with this drug does not interfere with Rac-mediated formation of membrane ruffles (22). We therefore used the induction of stress fibers and focal adhesions and its suppression by Y-27632 as a marker of the in vivo interaction of Rho/Rac chimeras with the ROCK family of kinases and examined its correlation with binding in the in vitro systems. The results of these analyses are summarized in Figs. 4 and 5. Expression of active Rho in HeLa cells induced both the formation of stress fibers and the assembly of vinculin-containing focal adhesions, both of which were greatly attenuated by treatment with Y-27632 (Figs. 4 and 5 and data not shown). On the other hand, the formation of membrane ruffles mediated by active Rac was resistant to this treatment (Figs. 4 and 5). As shown in Fig. 4, Rho90Rac, Rho73Rac, Rho38Rac, and Rac73Rho, all of which bound to ROCK-I in the two-hybrid system, induced stress fibers, whereas these chimeras did not induce membrane ruffles. Stress fiber formation was also found on expression of Rac73Rho90Rac and Rho73Rac90Rho (data not shown). These results are consistent with the previous study by Diekmann et al. (25) using Rac73Rho and Rac73Rho143Rac chimeras. On the other hand, consistent with their lack of binding to ROCKs, Rho21Rac and Rac117Rho did not induce stress fibers or focal adhesions but induced membrane ruffles-like structures (Fig. 4 and data not shown). The stress fibers induced by Rho, Rho90Rac, Rho73Rac, and Rho38Rac were all suppressed by treatment with Y-27632 (Fig. 5) and focal adhesions were also reduced in number and size (data not shown). These results demonstrate that all chimeras showing binding to ROCK-I in in vitro induced stress fibers and focal adhesions and that this induction was mediated by activation of endogenous ROCK. Thus, the results obtained by the two-hybrid system and ligand overlay assay correlated well with the expression studies, supporting our proposal that Rho binding to ROCK is controlled through at least two distinct determinant sites, aa 23-40 and aa 75-92. Furthermore, another chimera, Rac90Rho, induced stress fibers in HeLa cells (Fig. 4) that could be suppressed by treatment with Y-27632 (Fig. 5), indicating that aa 93-119 of Rho may also contribute to activate endogenous ROCK as well as aa 75-92 of Rho.
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DISCUSSION |
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In this study, we examined the regions of Rho determining the selective binding of various Rho targets to Rho. Using the Rho-binding domains of citron and rhophilin, a critical interaction determinant for citron was mapped to aa 23-40 of Rho, whereas aa 75-92 of Rho was found to be essential for rhophilin binding. Interaction of another Rho target, ROCK, was mediated independently through either aa 23-40 or aa 75-92 of Rho. The bivalent nature of the Rho-ROCK interaction was confirmed in vivo by monitoring the stress fiber/focal adhesion-forming ability of various Rho/Rac chimeras. The in vivo experiments also suggested the presence of an additional binding determinant for ROCK in aa 93-119. These binding determinants are depicted in Fig. 6.
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The conformational change of small GTPases has been studied in Ras by
the x-ray analysis of crystal structures of the GTP- and GDP-bound
forms of Ras (31-34). These studies revealed that GTP induced a marked
conformational change in two regions, which are called "switch I"
and "switch II" regions. In Ras, the switch I region (aa 30-38)
overlaps with a domain (aa 32-40) that was identified by mutational
studies as a region essential for effector activation. This region
includes the interacting site for GAPs, Raf-1, and phosphatidylinositol
3-kinase (35-39). Switch II in Ras is located at aa 60-76, and this
region was suggested to be involved in the interaction with GAP (40).
Other regions called "activator domains" (approximately
corresponding to aa 26-28 and aa 40-49) of Ha-Ras seem to also be
required for effector binding. These motifs are exposed on the surface
of Ha-Ras, but their conformation is not significantly altered by
GDP/GTP exchange (41). Based on sequence alignments, we can map the
putative switch I region of Rho at aa 32-40 and the putative switch II
region at aa 62-78. Our results show that the aa 23-40 region of Rho
required for citron binding totally covers the switch I region, whereas
the aa 75-92 region essential for rhophilin binding partially overlaps with the switch II region and extends over its C terminus (Fig. 1). In
our preliminary yeast two-hybrid study, active Rho with a mutation in
switch II region (D76 Q) reduced the ability to bind,
but still bound to rhophilin significantly, suggesting that both switch
II and its neighboring C-terminal regions contribute to determination
of rhophilin binding. A previous study from our laboratory demonstrated
that Rho interaction with rhoGAP was inhibited by the Rho-binding
domain of rhotekin (14). We found that the interaction of Rho with
rhoGAP requires Asp65 and Asp67 in the putative
switch II region (42). Furthermore, a recent x-ray crystallography
analysis showed that the binding to rhoGAP of one of the Rho family
proteins, Cdc42, was indeed mediated through residue 63 (Gln) located
in the switch II region (43). These results are consistent with the
present findings and with the assumption that the binding of class I
molecules is determined by the region containing the switch II region
of Rho. Flynn et al. (44) found PKN contains three repeats
of the class I Rho-binding motif and homologous sequences in its N
terminus and called them HR1a, 1b and 1c. Using ligand overlay and
BIAcore, they found that HR1a interacted strongly with aa 75-145 of
Rho in a GTP-dependent manner, whereas HR1b interacted with
aa 1-75 of Rho with low affinity and in a GTP-independent manner.
Their results are consistent with the present findings because the Rho
binding region of rhophilin corresponds to HR1a of PKN.
It is evident from the present study that both aa 23-40 and aa 75-119
of Rho work as determinants in binding of the class II molecule, ROCK,
to Rho. Consistently, it was reported that Rho with a mutation in the
switch I region (T37 A) did not bind to the Rho-kinase
(ROCK-II) (17), and that other mutations in the switch I region
(F39
A, E40
L or E40
W)
also abolished the interaction between Rho and ROCK (27). The Rho
family GTPases contain unique sequences called the insert region in
their C-terminal half that is not found in Ras. Recently, it was found
that the insert region of Rac serves as a novel effector region for one
of its effectors, NADPH oxidase (45). In addition, Diekmann et
al. (25) showed that aa 143-175 of Rac is required for this
interaction. These studies suggest the importance of the C-terminal
half of Rac in the effector recognition. However, we have not found any
requirement of this part of Rho in interaction with the effectors
analyzed in this study.
In summary, we have identified Rho regions required for selective interaction of each class of Rho effector molecules. Modification of these regions and manipulation of their interactions may help to dissect the pleiotropic Rho-signaling pathways and to regulate each pathway separately.
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ACKNOWLEDGEMENTS |
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We are indebted to Stan Hollenberg, Rolf
Sternglanz, Stan Fields, and Paul Bartel for the gift of two-hybrid
strains, DNA, and detailed protocols, and to Erik Sahai for protocol of
the liquid -galactosidase assay of the yeast two-hybrid system. We are most grateful to K. Nonomura for skilled assistance, and to K. Okuyama and T. Arai for secretarial work.
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FOOTNOTES |
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* This work was supported in part by Grants-in-aid for Scientific Research from the Ministry of Education, Science and Culture of Japan (08102007), and by a grant from Human Frontier Science Program.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.
Japan Society for the Promotion of Science postdoctoral
fellow.
§ Present address: Dept. of Pharmacology, National Defense Medical College, Tokorozawa Namiki 3-2, Saitama 359-0042, Japan.
To whom correspondence should be addressed: Dept. of
Pharmacology, Kyoto University Faculty of Medicine, Yoshida, Sakyo-ku, Kyoto 606-8315, Japan. Tel.: 81-75-753-4392; Fax: 81-75-753-4693; E-mail, snaru{at}mfour.med.kyoto-u.ac.jp.
1 The abbreviations used are: ROCK, Rho-associated coiled-coil-containing protein kinase; aa, amino acid residue; PBS, phosphate-buffered saline.
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REFERENCES |
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