COMMUNICATION
Frabin, a Novel FGD1-related Actin Filament-binding Protein Capable of Changing Cell Shape and Activating c-Jun N-terminal Kinase*

Hiroshi ObaishiDagger , Hiroyuki NakanishiDagger , Kenji MandaiDagger , Keiko SatohDagger , Ayako SatohDagger , Kenichi TakahashiDagger , Masako MiyaharaDagger , Hideo NishiokaDagger , Kenji Takaishi§, and Yoshimi TakaiDagger §

From the Dagger  Takai Biotimer Project, ERATO, Japan Science and Technology Corporation, c/o JCR Pharmaceuticals Co., Ltd., 2-2-10 Murotani, Nishi-ku, Kobe 651-2241, Japan and the § Department of Molecular Biology and Biochemistry, Osaka University Medical School, Suita 565-0871, Japan

    ABSTRACT
Top
Abstract
Introduction
Procedures
Results & Discussion
References

We purified from rat brain a novel F-actin-binding protein with a Mr of about 105,000 (p105), which was estimated by SDS-polyacrylamide gel electrophoresis. We cloned its cDNA from a rat brain cDNA library and characterized it. p105 was a protein of 766 amino acids and showed a calculated Mr of 86,449. p105 consisted of one F-actin-binding domain at the N-terminal region, one Dbl homology domain and one pleckstrin homology domain at the middle region, and one cysteine-rich domain at the C-terminal region. This domain organization of p105 was similar to that of FGD1, which has been determined to be the genetic locus responsible for faciogenital dysplasia or Aarskog-Scott syndrome. We therefore named p105 frabin (FGD1-related F-actin-binding protein). Frabin bound along the sides of F-actin and showed F-actin-cross-linking activity. Overexpression of frabin in Swiss 3T3 cells and COS7 cells induced cell shape change and c-Jun N-terminal kinase activation, respectively, as described for FGD1. Because FGD1 has been shown to serve as a GDP/GTP exchange protein for Cdc42 small G protein, it is likely that frabin is a direct linker between Cdc42 and the actin cytoskeleton.

    INTRODUCTION
Top
Abstract
Introduction
Procedures
Results & Discussion
References

Dynamic reorganization of the actin cytoskeleton is essential for many cell functions, including cell shape change, adhesion, and motility (for reviews see Refs. 1-3). Evidence is accumulating that the Rho family small G proteins are important regulators of these actin-dependent cell functions (for reviews see Refs. 4 and 5). The Rho family is divided into three subfamilies: the Cdc42, Rac, and Rho subfamilies (Cdc42, Rac, and Rho, respectively). In fibroblasts, Cdc42 induces filopodium formation; Rac induces lamellipodium and membrane ruffle formation: and Rho regulates assembly of stress fibers and focal adhesions. In addition to these functions, the Rho family members are involved in the regulation of cell growth: Cdc42 and Rac, but not Rho, induce mitogen-activated protein kinase-mediated JNK1 activation (6, 7); and Cdc42, Rac, and Rho all induce transcriptional activation of the genes containing the serum response element (8).

The Rho family members cycle between the GDP-bound inactive and GTP-bound active forms (4, 5). The conversion from the GDP-bound form to the GTP-bound form is stimulated by a GEP. Many GEPs for the Rho family members have thus far been identified and shown to share two conserved domains: a DH domain of about 250 aa and a PH domain of about 100 aa. The majority of the GEPs have been isolated as oncogenes (4, 5), but FGD1, a GEP specific for Cdc42, has been determined by positional cloning to be the genetic locus responsible for faciogenital dysplasia or Aarskog-Scott syndrome (9, 10).

In preceding papers, we purified from rat brain novel F-actin-binding proteins, neurabin-I (11) and -II (12) and lafadin (13). During the purification of lafadin, we identified another F-actin-binding protein with a Mr of about 105,000. Molecular cloning of this protein revealed that it showed a significant homology to FGD1. We named this protein frabin (FGD1-related F-actin-binding protein). Frabin bound to F-actin in vivo and in vitro and showed activities similar to those of FGD1.

    EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results & Discussion
References

Materials-- Rabbit skeletal actin monomer (14), F-actin (11), and 125I-labeled F-actin (11) were prepared as described. Various GST fusion proteins of full-length and truncated forms of frabin were expressed in Escherichia coli and purified (11). Various Myc-tagged proteins of full-length and truncated forms of frabin were expressed in COS7 cells as described (11). 125I-Labeled F-actin blot overlay, immunofluorescence microscopy, gel filtration, cosedimentation of various GST fusion proteins of full-length and truncated forms of frabin with F-actin, and transmission electron microscopy of negatively stained specimens were done as described (11). Protein concentrations were determined with bovine serum albumin as a reference protein (15).

Purification and Molecular Cloning of Frabin-- Fetal brains from 420 mother rats (17-day gestation) were homogenized with a solution containing 10 mM Tris/Cl at pH 8.0, 2 mM EDTA, 5 mM EGTA, and a protease inhibitor mixture (1 mM phenylmethylsulfonyl fluoride, 20 µg/ml of leupeptin, and 1 µg/ml of pepstatin A). The homogenate was mildly stirred for 1 h and centrifuged at 200,000 × g for 1 h. The supernatant was stored at -80 °C until use. One-seventh of the supernatant (515 ml, 1.9 g of protein) was applied on a Q-Sepharose FF column (2.6 × 34 cm; Amersham Pharmacia Biotech) equilibrated with Buffer A (20 mM Tris/Cl at pH 8.0, 0.5 mM EDTA, and 1 mM dithiothreitol). Elution was performed with a 800-ml linear gradient of NaCl (0-0.5 M) in Buffer A. Fractions of 10 ml each were collected. Frabin appeared in Fractions 35-55. These fractions (210 ml, 300 mg of protein) were collected. Half of the sample was diluted with a 2-fold volume of Buffer A and applied on a Mono Q HR 10/10 (Amersham Pharmacia Biotech) equilibrated with Buffer A. Elution was performed with a 48-ml linear gradient of NaCl (0-0.3 M) in Buffer A, followed by 34 ml of Buffer A containing M NaCl. Fractions of 2 ml each were collected. Frabin appeared in Fractions 20-22. These fractions (6 ml, 19 mg of protein) were collected. The other half of the sample of the Q-Sepharose FF column chromatography was also subjected to the Mono Q column chromatography in the same manner as described above. The active fractions of the two Mono Q column chromatographies were combined, and solid (NH4)2SO4 was added to give a final concentration of 20% saturation. The sample was centrifuged at 200,000 × g for 20 min, and the supernatant was applied on a TSKgel phenyl-5PW column (2.15 × 15 cm, Tosoh) equilibrated with Buffer A containing 0.9 M (NH4)2SO4. Elution was performed with a 210-ml linear gradient of (NH4)2SO4 (0.9-0 M) in Buffer A, followed by 80 ml of Buffer A. Fractions of 4 ml each were collected. Frabin appeared in Fractions 18-21. These fractions (16 ml, 6.3 mg of protein) were collected and applied on a TSKgel phenyl-5PW RP column (0.75 × 7.5 cm, Tosoh) equilibrated with 0.05% trifluoroacetic acid. Elution was performed with a 88-ml linear gradient of acetonitrile (20-52.5%) in 0.05% trifluoroacetic acid, followed by 27 ml of 100% acetonitrile in 0.05% trifluoroacetic acid. Fractions of 2.7 ml each were collected. Frabin appeared in Fraction 23 (see Fig. 1). This fraction (2.7 ml, 54 µg of protein) was collected. The rest of the supernatant fraction from the brain homogenate was also subjected to the successive column chromatographies in the same manner as described above. The active fractions of the seven pheny-5PW RP column chromatographies were combined, lyophilized, and stored at -80 °C.

The purified phenyl-5PW RP sample (300 µg of protein) was subjected to SDS-PAGE (10% polyacrylamide gel). The protein band corresponding to a protein with a Mr of about 105,000 was processed as described (11). The aa sequences of the peptides derived from the protein band were determined with a peptide sequencer. A rat brain cDNA library in lambda ZAPII (Stratagene) was screened using the oligonucleotide probes designed from the partial aa sequences.

Assay for JNK Activity-- Assay for JNK activity was done as described (16). Briefly, pSRalpha -HA-JNKalpha was transfected with pCMV-Myc-full-length frabin, pEF-BOS-Myc-V12Cdc42 (17), or pEF-BOS-Myc-Dbl (18) in COS7 cells. After incubation for 16 h in serum-starved Dulbecco's modified Eagle's medium, the cell lysates were subjected to immunoprecipitation with an anti-HA antibody. Each immunoprecipitate was then washed and used for assay with GST-c-Jun as a substrate.

Microinjection-- Swiss 3T3 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. The subconfluent cells were serum-starved for 24 h before microinjection. pCMV-Myc-full-length frabin or pEF-BOS-Myc-V12Cdc42 (0.05 mg/ml each) was then microinjected into the nuclei of the cells, and 18 h later the cells were subjected to immunofluorescence microscopy as described (11).

    RESULTS AND DISCUSSION
Top
Abstract
Introduction
Procedures
Results & Discussion
References

During the purification of lafadin from rat brain using a blot overlay method with 125I-labeled F-actin (13), we detected another protein band of 125I-labeled F-actin binding activity with a Mr of about 105,000. We purified it by chromatographies of Q-Sepharose, Mono Q, phenyl-5PW, and phenyl-5PW RP columns. On the final phenyl-5PW RP column chromatography, the 125I-labeled F-actin-binding protein band coincided well with a protein with a Mr of about 105,000 (p105), which was identified by protein staining (Fig. 1).


View larger version (64K):
[in this window]
[in a new window]
 
Fig. 1.   Phenyl-5PW RP column chromatography of p105 (frabin). A, absorbance at 280 nm. B, protein staining with silver. An aliquot (160 µl) of each fraction was concentrated and subjected to SDS-PAGE (10% polyacrylamide gel), followed by protein staining with silver. C, 125I-labeled F-actin blot overlay. An aliquot (160 µl) of each fraction was concentrated and subjected to SDS-PAGE (10% polyacrylamide gel), followed by 125I-labeled F-actin blot overlay. Arrows, p105 (frabin).

We determined the partial aa sequences of p105. The aa sequences of p105 were not found in current protein data base. On the basis of this information, we isolated a p105 cDNA from a rat brain cDNA library and determined its nucleotide sequence. The encoded protein consisted of 766 aa and showed a calculated Mr of 86,449 (GenBankTM accession number AF038388) (Fig. 2A). This Mr value was less than that estimated by SDS-PAGE. To confirm whether this clone contained a full-length cDNA of p105, we constructed the eukaryotic expression vector with this cDNA and expressed the protein in COS7 cells. The expressed protein showed the mobility similar to that of native p105 on SDS-PAGE and had the 125I-labeled F-actin binding activity (Fig. 2B). Therefore, we concluded that this clone encoded the full-length cDNA of p105.


View larger version (58K):
[in this window]
[in a new window]
 
Fig. 2.   Molecular characterization of frabin. A, deduced amino acid sequence of frabin. Underlines, aa sequences of 14 peptide peaks derived from the purified sample of frabin. B, 125I-labeled F-actin binding activity of recombinant frabin. pCMV-frabin was transfected to COS7 cells, and the cell extract was subjected to SDS-PAGE (10% polyacrylamide gel), followed by 125I-labeled F-actin blot overlay. Lane 1, native frabin; lane 2, pCMV-frabin; lane 3, mock. Arrow, frabin; asterisks, radioactive bands of 125I-labeled F-actin binding activity of endogenous proteins of COS7 cells. C, 125I-labeled F-actin binding activity of various truncated forms of frabin. GST fusion proteins (0.2 µg of protein each) were subjected to SDS-PAGE (12% polyacrylamide gel), followed by 125I-labeled F-actin blot overlay. Lane 1, GST-frabin-1 (aa 1-150); lane 2, GST-frabin-2 (aa 1-208); lane 3, GST-frabin-3 (aa 209-520); lane 4, GST-frabin-4 (aa 521-766). D, comparison of domain organization between frabin and FGD1. ABD, F-actin-binding domain; PRD, proline-rich domain; CRD, cysteine-rich domain.

By use of fusion proteins of several truncated forms of p105 with GST, we determined the minimum 125I-labeled F-actin-binding domain to be aa 1-150 (Fig. 2C). Computer homology search revealed that the aa sequence of this domain showed no significant homology to any protein in current protein data base. However, the aa sequence following the F-actin-binding domain showed a significant homology to that of FGD1 (Fig. 2D). p105 had one DH domain and one PH domain the aa sequences of which showed 71% and 57% identity to those of FGD1, respectively. p105 also had one cysteine-rich domain the aa sequence of which showed 65% identity to that of FGD1. We therefore named p105 frabin.

When GST-full-length frabin was incubated with F-actin followed by ultracentrifugation, the fusion protein was recovered with F-actin in the pellet (Fig. 3A). The stoichiometry of the binding of GST-frabin to actin was 1 GST-frabin molecule/~14 actin molecules. The Kd value was about 9 × 10-7 M. Similar results were obtained with GST-frabin-1 (aa 1-150) and GST-frabin-2 (aa 1-208) (data not shown). The binding of native frabin to 125I-labeled F-actin was inhibited by an excessive amount of myosin S1, a protein that binds along the sides of F-actin (19, 20) (Fig. 3B). This inhibition was reversed by the addition of MgATP because MgATP dissociates the actin-myosin complex (21), indicating that frabin bound along the sides of F-actin. The F-actin-cross-linking activity of GST-full-length frabin and GST-frabin-1 (aa 1-150) was examined by transmission electron microscopy of negatively stained specimens. GST-full-length frabin caused F-actin to associate into bundles, whereas GST-frabin-1 or GST alone did not (Fig. 3C).


View larger version (65K):
[in this window]
[in a new window]
 
Fig. 3.   Biochemical and biophysical properties of frabin. A, binding of GST-full-length frabin to F-actin. Various amounts of GST-full-length frabin were mixed with F-actin, followed by ultracentrifugation. Amounts of the free and bound GST fusion protein were calculated by determining the protein amounts from the supernatant and pellet fractions with a densitometer. Inset, cosedimentation of GST-full-length frabin with F-actin. GST-full-length frabin (3 µg of protein) was mixed with F-actin (30 µg of protein), followed by ultracentrifugation. Lane 1, supernatant fraction in the absence of F-actin; lane 2, pellet fraction in the absence of F-actin; lane 3, supernatant fraction in the presence of F-actin; lane 4, pellet fraction in the presence of F-actin. Arrow, actin; arrowhead, GST-full-length frabin. B, inhibition by myosin S1 of the binding of frabin to 125I-labeled F-actin. The purified, native sample of frabin (3 µg of protein) was subjected to SDS-PAGE (10% polyacrylamide gel), followed by the blot overlay with 125I-labeled F-actin pretreated with myosin S1 in the presence or absence of ATP. Lane 1, control; lane 2, with myosin S1 in the absence of ATP; lane 3, with myosin S1 in the presence of ATP. C, F-actin-cross-linking activity of frabin estimated by electron microscopy. a, GST-full-length frabin; b, GST-frabin-1 (aa 1-150); c, GST alone. Bars, 200 nm. D, localization of frabin and F-actin in COS7 cells. Full-length and truncated forms of frabin were expressed as Myc-tagged proteins in COS7 cells. The cells were doubly stained with an anti-Myc antibody and rhodamine-phalloidin. The anti-Myc antibody was visualized with an fluorescein isothiocyanate-conjugated anti-mouse antibody. a-c, Myc-tagged protein; d-f, F-actin. a and d, Myc-full-length frabin; b and e, Myc-frabin-1 (aa 1-150); c and f, Myc-frabin-2 (aa 151-766). Bars, 20 µm.

The alpha -actinin/spectrin family members have most extensively been studied as F-actin-binding proteins having F-actin-cross-linking activity (22, 23). They usually form oligomers and thereby show the F-actin-cross-linking activity. When frabin was subjected to gel filtration, it appeared at a position corresponding to a Mr of about 250,000 (data not shown). Because the Mr values of frabin estimated by SDS-PAGE and calculated from its predicted aa sequence were about 105,000 and 86,000, respectively, these results suggest that frabin forms an oligomer with multiple F-actin-binding heads and thereby shows the F-actin-cross-linking activity.

To further obtain the evidence that frabin is an F-actin-binding protein, we expressed Myc-tagged full-length frabin or a Myc-tagged mutant, which lacked the F-actin-binding domain, in COS7 cells and compared the localization of each expressed protein with that of endogenous F-actin. Frabin having the F-actin-binding domain was colocalized with F-actin, but frabin lacking the domain was not (Fig. 3D). On the basis of these observations, we have concluded that frabin serves as an F-actin-binding protein in intact cells and that the F-actin-binding domain is located at the N-terminal region (aa 1-150).

Of the activities of FGD1 thus far reported (10), we examined whether frabin is also capable of changing cell shape and activating JNK. When frabin or a dominant active mutant of Cdc42 (V12Cdc42) was expressed in Swiss 3T3 cells, they similarly induced cell shape change characterized by microspike formation (Fig. 4). Although we did not determine whether the microspikes were filopodia or retraction fibers, the former is likely because Cdc42 has been shown to induce filopodium formation (24). Frabin or V12Cdc42 was expressed in COS7 cells together with HA-tagged JNK. The JNK was immunoprecipitated, and its kinase activity toward GST-c-Jun was determined. Frabin as well as V12Cdc42 similarly induced JNK activation (Fig. 5). These results indicate that frabin shows at least the two activities similar to those of FGD1. We have not determined here whether frabin shows GEP activity to Cdc42. However, because the DH and PH domains of frabin are highly homologous to those of FGD1, it is likely that frabin serves as a GEP for Cdc42 and induces cell shape change and JNK activation through Cdc42 activation as described for FGD1 (10).


View larger version (98K):
[in this window]
[in a new window]
 
Fig. 4.   Frabin-induced cell shape change of Swiss 3T3 cells. pCMV-Myc-full-length frabin or pEF-BOS-Myc-V12Cdc42 was microinjected into the nuclei of serum-starved Swiss 3T3 cells. The cells were doubly stained with an anti-Myc antibody and rhodamine-phalloidin. The anti-Myc antibody was visualized with an fluorescein isothiocyanate-conjugated anti-mouse antibody. A-C, F-actin; D-F, Myc-tagged protein. A and D, cells microinjected with pCMV-Myc vector alone; B and E, cells microinjected with pEF-BOS-Myc-V12Cdc42; C and F, cells microinjected with pCMV-Myc-full-length frabin. Bars, 20 µm. 40 cells were microinjected with pCMV-Myc vector alone. Of the 84 cells microinjected with pEF-BOS-Myc-V12Cdc42, 15 cells expressed the Myc-tagged protein. Of the 105 cells microinjected with pCMV-Myc-full-length frabin, 12 cells expressed the Myc-tagged protein. Microspike formation was observed in more than 90% of the cells expressing the respective Myc-tagged proteins.


View larger version (22K):
[in this window]
[in a new window]
 
Fig. 5.   Frabin-induced JNK activation in COS7 cells. HA-tagged JNK was expressed in COS7 cells along with Myc-full-length frabin, Myc-V12Cdc42, or Myc-Dbl and then immunoprecipitated with an anti-HA antibody. Comparable amounts of each immunoprecipitate were subjected to the assay for JNK activity using GST-c-Jun as a substrate and Western blot analysis using an anti-JNK antibody (Santa Cruz). A, JNK activity; B, Western blot analysis. Lane 1, control; lane 2, V12Cdc42; lane 3, Dbl; lane 4, frabin. Arrowhead, GST-c-Jun.

We have isolated here a novel F-actin-binding protein named frabin that has a structure homologous to FGD1 and shows activities similar to those of FGD1. The aa sequences of the DH, PH, and cysteine-rich domains are well conserved between frabin and FGD1, but the nucleotide sequence differences were not clustered but distributed throughout the sequences. Therefore, although we have not determined the genetic locus of frabin, it is likely that frabin is not a splicing variant but an isoform of FGD1 derived from a different gene. The aa sequence of the N-terminal region of frabin, which is responsible for the F-actin binding activity, is not conserved in FGD1, and it remains unknown whether the N-terminal proline-rich domain of FGD1 binds to F-actin.

The physiological significance of frabin binding to F-actin and showing activities similar to those of FGD1 has not been clarified here, but there are two possible roles of the F-actin binding activity of frabin: one is to be involved in Cdc42 activation, and the other is to be involved in Cdc42 action. It has been shown that a GEP specific for Rac, Tiam1, is localized at the membrane through its N-terminal PH domain, but not its PH domain adjacent to the DH domain, and that this domain is required for the Rac-induced membrane ruffling and JNK activation (25). On the basis of these observations, it has been suggested that the localization of Tiam1 at the membrane is an important factor for Rac activation (25). By analogy, it is possible that the localization of frabin at the actin cytoskeleton through its F-actin-binding domain is important for Cdc42 activation.

Mutations to the FGD1 gene, either by interruption of the gene by chromosomal translocation or by insertion of a single base pair causing premature translational termination within the DH domain, have been detected in faciogenital dysplasia or Aarskog-Scott syndrome (9). Mutations to the frabin gene may also cause multisystemic developmental diseases including faciogenital dysplasia. Further studies are necessary to understand the physiological and pathological roles of frabin.

    ACKNOWLEDGEMENT

We thank Dr. E. Nishida (Kyoto University, Kyoto, Japan) for providing us with the GST-c-Jun and pSRalpha HA-JNKalpha .

    FOOTNOTES

* 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 Molecular Biology and Biochemistry, Osaka University Medical School. Tel.: 81-6-879-3410; Fax: 81-6-879-3419; E-Mail: ytakai{at}molbio.med.osaka-u.ac.jp.

1 The abbreviations used are: JNK, c-Jun N-terminal kinase; GEP, GDP/GTP exchange protein; DH, Dbl homology; aa, amino acid(s); PH, pleckstrin homology; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis; HA, hemagglutinin; S1, subfragment 1.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results & Discussion
References

  1. Pollard, T. D., and Cooper, J. A. (1986) Annu. Rev. Biochem. 55, 987-1035[CrossRef][Medline] [Order article via Infotrieve]
  2. Gumbiner, B. M. (1996) Cell 84, 345-357[Medline] [Order article via Infotrieve]
  3. Lauffenburger, D. A., and Horwitz, A. F. (1996) Cell 84, 359-369[Medline] [Order article via Infotrieve]
  4. Takai, Y., Sasaki, T., Tanaka, K., and Nakanishi, H. (1995) Trends Biochem. Sci. 20, 227-231[CrossRef][Medline] [Order article via Infotrieve]
  5. Hall, A. (1998) Science 279, 509-514[Abstract/Free Full Text]
  6. Coso, O. A., Chiariello, M., Yu, J.-C., Teramoto, H., Crespo, P., Xu, N., Miki, T., and Gutkind, J. S. (1995) Cell 81, 1137-1146[Medline] [Order article via Infotrieve]
  7. Minden, A., Lin, A., Claret, F. X., Abo, A., and Karin, M. (1995) Cell 81, 1147-1157[Medline] [Order article via Infotrieve]
  8. Hill, C. S., Wynne, J., and Treisman, R. (1995) Cell 81, 1159-1170[Medline] [Order article via Infotrieve]
  9. Pasteris, N. G., Cadle, A., Logie, L. J., Porteous, M. E. M., Schwartz, C. E., Stevenson, R. E., Glover, T. W., Wilroy, R. S., and Gorski, J. L. (1994) Cell 79, 669-678[Medline] [Order article via Infotrieve]
  10. Zheng, Y., Fischer, D. J., Santos, M. F., Tigyi, G., Pasteris, N. G., Gorski, J. L., and Xu, Y. (1996) J. Biol. Chem. 271, 33169-33172[Abstract/Free Full Text]
  11. Nakanishi, H., Obaishi, H., Satoh, A., Wada, M., Mandai, K., Satoh, K., Nishioka, H., Matsuura, Y., Mizoguchi, A., and Takai, Y. (1997) J. Cell Biol. 139, 951-961[Abstract/Free Full Text]
  12. Satoh, A., Nakanishi, H., Obaishi, H., Wada, M., Takahashi, K., Satoh, K., Hirao, K., Nishioka, H., Hata, Y., Mizoguchi, A., and Takai, Y. (1998) J. Biol. Chem. 273, 3470-3475[Abstract/Free Full Text]
  13. Mandai, K., Nakanishi, H., Satoh, A., Obaishi, H., Wada, M., Nishioka, H., Itoh, M., Mizoguchi, A., Aoki, T., Fujimoto, T., Matsuda, Y., Tsukita, S., and Takai, Y. (1997) J. Cell Biol. 139, 517-528[Abstract/Free Full Text]
  14. Pardee, J. D., and Spudich, J. A. (1982) Methods Enzymol. 85, 164-181[Medline] [Order article via Infotrieve]
  15. Bradford, M. M. (1976) Anal. Biochem. 72, 248-254[CrossRef][Medline] [Order article via Infotrieve]
  16. Moriguchi, T., Kuroyanagi, N., Yamaguchi, K., Gotoh, Y., Irie, K., Kano, T., Shirakabe, K., Muro, Y., Shibuya, H., Matsumoto, K., Nishida, E., and Hagiwara, M. (1996) J. Biol. Chem. 271, 13675-13679[Abstract/Free Full Text]
  17. Komuro, R., Sasaki, T., Takaishi, K., Orita, S., and Takai, Y. (1996) Genes Cells 1, 943-951[Abstract/Free Full Text]
  18. Yaku, H., Sasaki, T., and Takai, Y. (1994) Biochem. Biophys. Res. Commun. 198, 811-817[CrossRef][Medline] [Order article via Infotrieve]
  19. Rayment, I., Holden, H. M., Whittaker, M., Yohn, C. B., Lorenz, M., Holmes, K. C., and Milligan, R. A. (1993) Science 261, 58-65[Medline] [Order article via Infotrieve]
  20. Schröder, R. R., Manstein, D. J., Jahn, W., Holden, H., Rayment, I., Holmes, K. C., and Spudich, J. A. (1993) Nature 364, 171-174[CrossRef][Medline] [Order article via Infotrieve]
  21. Fraser, A. B., Eisenberg, E., Kielley, W. W., and Carlson, F. D. (1975) Biochemistry 14, 2207-2214[Medline] [Order article via Infotrieve]
  22. Hartwig, J. H., and Kwiatkowski, D. J. (1991) Curr. Opin. Cell Biol. 3, 87-97[Medline] [Order article via Infotrieve]
  23. Matsudaira, P. (1991) Trends Biochem. Sci. 16, 87-92[CrossRef][Medline] [Order article via Infotrieve]
  24. Nobes, C. D., and Hall, A. (1995) Cell 81, 53-62[Medline] [Order article via Infotrieve]
  25. Michiels, F., Stam, J. C., Hordijk, P. L., van der Kammen, R. A., Stalle, L. R.-V., Feltkamp, C. A., and Collard, J. G. (1997) J. Cell Biol. 137, 387-398[Abstract/Free Full Text]


Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.