FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2069945609> ?p ?o ?g. }

• W2069945609 endingPage "23375" @default.
• W2069945609 startingPage "23371" @default.
• W2069945609 abstract "The Rho GDP dissociation inhibitor (GDI) forms a complex with the GDP-bound form of the Rho family small G proteins and inhibits their activation. The GDP-bound form complexed with Rho GDI is not activated by the GDP/GTP exchange factor for the Rho family members, suggesting the presence of another factor necessary for this activation. We have reported that the Rho subfamily members regulate the ezrin/radixin/moesin (ERM)-CD44 system, implicated in reorganization of actin filaments. Here we report that Rho GDI directly interacts with ERM, initiating the activation of the Rho subfamily members by reducing the Rho GDI activity. These results suggest that ERM as well as Rho GDI and the Rho GDP/GTP exchange factor are involved in the activation of the Rho subfamily members, which then regulate reorganization of actin filaments through the ERM system. The Rho GDP dissociation inhibitor (GDI) forms a complex with the GDP-bound form of the Rho family small G proteins and inhibits their activation. The GDP-bound form complexed with Rho GDI is not activated by the GDP/GTP exchange factor for the Rho family members, suggesting the presence of another factor necessary for this activation. We have reported that the Rho subfamily members regulate the ezrin/radixin/moesin (ERM)-CD44 system, implicated in reorganization of actin filaments. Here we report that Rho GDI directly interacts with ERM, initiating the activation of the Rho subfamily members by reducing the Rho GDI activity. These results suggest that ERM as well as Rho GDI and the Rho GDP/GTP exchange factor are involved in the activation of the Rho subfamily members, which then regulate reorganization of actin filaments through the ERM system. The small G proteins of the Rho family, consisting of the Rho, Rac, and Cdc42 subfamilies, are implicated in various cell functions, such as cell shape change, cell motility, and cytokinesis, through reorganization of actin filaments (for reviews, see Refs. 1Hall A. Annu. Rev. Cell Biol. 1994; 10: 31-54Crossref PubMed Scopus (768) Google Scholar and 2Takai Y. Sasaki T. Tanaka K. Nakanishi H. Trends Biochem. Sci. 1995; 20: 227-231Abstract Full Text PDF PubMed Scopus (367) Google Scholar). Rho GDI 1The abbreviations used are: GDI, GDP dissociation inhibitor; GEF, GDP/GTP exchange factor; ERM, ezrin/radixin/moesin; GST, glutathione S-transferase; GDP-RhoA, the GDP-bound form of RhoA; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; HA, hemagglutinin; PBS, phosphate-buffered saline; GTPγS, guanosine 5′-O-(3-thiotriphosphate). is a general regulator that forms a complex with the GDP-bound inactive form of the Rho family members and inhibits their activation (2Takai Y. Sasaki T. Tanaka K. Nakanishi H. Trends Biochem. Sci. 1995; 20: 227-231Abstract Full Text PDF PubMed Scopus (367) Google Scholar). The GDP-bound form complexed with Rho GDI is not activated by Rho GEFs, such as Dbl and Rom1/2, in a cell-free system (2Takai Y. Sasaki T. Tanaka K. Nakanishi H. Trends Biochem. Sci. 1995; 20: 227-231Abstract Full Text PDF PubMed Scopus (367) Google Scholar, 3Hart M.J. Eva A. Evans T Aaronson S.A. Cerione R.A. Nature. 1991; 354: 311-314Crossref PubMed Scopus (338) Google Scholar, 4Yaku H. Sasaki T. Takai Y. Biochem. Biophys. Res. Commun. 1994; 198: 811-817Crossref PubMed Scopus (74) Google Scholar, 5Ozaki K. Tanaka K. Imamura H. Hihara T. Kameyama T. Nonaka H. Hirano H. Matsuura Y. Takai Y. EMBO J. 1996; 15: 2196-2207Crossref PubMed Scopus (185) Google Scholar), suggesting the presence of another factor necessary for this activation in intact cells. We have recently reported that the Rho subfamily members regulate the ezrin/radixin/moesin (ERM)-CD44 system (6Takaishi K. Sasaki T. Kameyama T. Tsukita Sa. Tsukita Sh. Takai Y. Oncogene. 1995; 11: 39-48PubMed Google Scholar, 7Hirao M. Sato N. Kondo T. Yonemura S. Monden M. Sasaki T. Takai Y. Tsukita Sh. Tsukita Sa. J. Cell Biol. 1996; 135: 37-51Crossref PubMed Scopus (512) Google Scholar), which has also been implicated in reorganization of actin filaments (8Arpin M. Algrain M. Louvard D. Curr. Opin. Cell Biol. 1994; 6: 136-141Crossref PubMed Scopus (162) Google Scholar, 9Tsukita Sa. Yonemura S. Tsukita Sh. Curr. Opin. Cell Biol. 1997; 9: 70-75Crossref PubMed Scopus (312) Google Scholar, 10Tsukita Sa. Yonemura S. Tsukita Sh. Trends Biochem. Sci. 1997; 22: 53-58Abstract Full Text PDF PubMed Scopus (274) Google Scholar). ERM are intracellular proteins with at least two functionally different domains, the N-terminal plasma membrane-interacting and C-terminal actin filament-interacting domains (8Arpin M. Algrain M. Louvard D. Curr. Opin. Cell Biol. 1994; 6: 136-141Crossref PubMed Scopus (162) Google Scholar, 9Tsukita Sa. Yonemura S. Tsukita Sh. Curr. Opin. Cell Biol. 1997; 9: 70-75Crossref PubMed Scopus (312) Google Scholar, 10Tsukita Sa. Yonemura S. Tsukita Sh. Trends Biochem. Sci. 1997; 22: 53-58Abstract Full Text PDF PubMed Scopus (274) Google Scholar). ERM are translocated to the plasma membrane probably through the interaction with the cytoplasmic domain of integral plasma membrane proteins, such as CD44, providing the actin filament association sites (11Underhill C.B. J. Cell Sci. 1992; 103: 293-298Crossref PubMed Google Scholar, 12Lesley J. Hyman R. Kincade P.W. Adv. Immunol. 1993; 54: 271-335Crossref PubMed Scopus (1032) Google Scholar, 13Tsukita Sa. Oishi K. Sato N. Sagara J. Kawai A. Tsukita Sh. J. Cell Biol. 1994; 126: 391-401Crossref PubMed Scopus (685) Google Scholar). When cells are treated with agonists or Ca2+, the GDP-bound form of RhoA, a member of the Rho subfamily, staying in the cytosol in complex with Rho GDI is activated to the GTP-bound form and translocated to the same areas as ERM are translocated (6Takaishi K. Sasaki T. Kameyama T. Tsukita Sa. Tsukita Sh. Takai Y. Oncogene. 1995; 11: 39-48PubMed Google Scholar). We have recently found that Rho GDI and CD44 are co-immunoprecipitated with moesin when the crude lysate of baby hamster kidney cells is treated with an anti-moesin monoclonal antibody and that the Rho subfamily members stimulate the interaction of ERM with the plasma membrane (7Hirao M. Sato N. Kondo T. Yonemura S. Monden M. Sasaki T. Takai Y. Tsukita Sh. Tsukita Sa. J. Cell Biol. 1996; 135: 37-51Crossref PubMed Scopus (512) Google Scholar). These results have raised a possibility that ERM or CD44 has an activity to make the GDP-bound form of the Rho subfamily members complexed with Rho GDI sensitive to the action of Rho GEFs. We have examined this possibility using RhoA as a substrate for Rho GDI and Dbl as a Rho GEF. A series of truncated mouse radixin fusion proteins were expressed and purified fromEscherichia coli. Nr-Fragment (amino acids 1–280), Nr1-Fragment (amino acids 1–318), Cr-Fragment (amino acids 281–584), and full-length radixin (amino acids 1–584) were expressed as GST fusion proteins from pGEX vectors. The fusion proteins were purified using glutathione-Sepharose 4B columns, and the GST carrier was cleaved off by digestion with thrombin (14Tanaka K. Sasaki T. Takai Y. Methods Enzymol. 1995; 256: 41-49Crossref PubMed Scopus (8) Google Scholar). Bovine Rho GDI was also expressed and purified as a GST fusion protein from E. coli, and the GST carrier was cleaved off by digestion with thrombin. The C-terminal fragment of Dbl containing the catalytic domain was expressed and purified as a GST fusion protein from E. coli (4Yaku H. Sasaki T. Takai Y. Biochem. Biophys. Res. Commun. 1994; 198: 811-817Crossref PubMed Scopus (74) Google Scholar). Human lipid-modified RhoA, Rac1, and Cdc42 were purified from the membrane fraction of Spodoptera frugiperda cells overexpressing each protein (4Yaku H. Sasaki T. Takai Y. Biochem. Biophys. Res. Commun. 1994; 198: 811-817Crossref PubMed Scopus (74) Google Scholar, 15Mizuno T. Kaibuchi K. Yamamoto T. Kawamura M. Sakoda T. Fujioka H. Matsuura Y. Takai Y. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 6442-6446Crossref PubMed Scopus (169) Google Scholar). [3H]GDP- or GDP-RhoA complexed with Rho GDI was obtained by first incubating GDP-RhoA with or without [3H]GDP, followed by incubation with Rho GDI for 30 min at 4 °C. The sample was then subjected to gel filtration using a Superdex 75 PC3.2/30 column (Pharmacia Biotech Inc.) equilibrated with 20 mm Tris-HCl (pH 7.5) containing 5 mm MgCl2, 1 mm EDTA, 1 mm dithiothreitol, and 0.1% CHAPS. [3H]GDP- or GDP-RhoA complexed with Rho GDI was detected by protein staining. The [3H]GDP- or GDP-bound form of Rac1 complexed with Rho GDI and the [3H]GDP- or GDP-bound form of Cdc42 complexed with Rho GDI were similarly prepared. Mammalian expression plasmids (pSRαneo-Myc and pEFBOS-HA) were generated to express fusion proteins with the N-terminal Myc and HA epitopes, respectively, as described (6Takaishi K. Sasaki T. Kameyama T. Tsukita Sa. Tsukita Sh. Takai Y. Oncogene. 1995; 11: 39-48PubMed Google Scholar, 16Orita S. Sasaki T. Komuro R. Sakaguchi G. Maeda M. Igarashi H. Takai Y. J. Biol. Chem. 1996; 271: 7257-7260Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). To generate pSRαneo-Myc-Rho GDI and pEFBOS-HA-Nr-Fragment, their cDNA constructs were made by the polymerase chain reaction using specific oligonucleotide primers and inserted into pSRαneo-Myc or pEFBOS-HA. Transient expression of Myc-Rho GDI with or without HA-Nr-Fragment was carried out using pSRαneo-Myc-Rho GDI and pEFBOS-HA or pEFBOS-HA-Nr-Fragment in COS-7 cells. The cells were plated at a density of 5 × 105 cells/60-mm dish and were incubated for 18 h. The cells were then cotransfected with 2 μg of pSRαneo-Myc-Rho GDI and 2 μg of pEFBOS-HA or pEFBOS-HA-Nr-Fragment using the DEAE-dextran method. Immunoprecipitation was performed at 48 h after the transfection. The cells were washed with PBS twice, lysed in lysis buffer (containing 20 mm Tris-HCl (pH 7.5), 5 mmMgCl2, 1 mm EDTA, 1 mmdithiothreitol, and 10 μm p-amidinophenylmethanesulfonyl fluoride), and sonicated. The cell lysate was centrifuged at 100,000 × g for 1 h to prepare the cytosol fraction. Myc-Rho GDI was precipitated with 3 μg of anti-Myc monoclonal antibody bound to 20 μl of protein A-Sepharose, followed by centrifugation and extensive washing with lysis buffer in the presence of 1% Nonidet P-40. Comparable amounts of the pellets were subjected to SDS-polyacrylamide gel electrophoresis, and the separated proteins were electrophoretically transferred to a nitrocellulose membrane sheet. The sheet was processed using the ECL detection kit (Pharmacia Biotech Inc.) to detect Myc-Rho GDI, RhoA, and HA-Nr-Fragment with the anti-Rho GDI polyclonal, anti-RhoA polyclonal, and anti-HA monoclonal antibodies as primary antibodies, respectively. Swiss 3T3 cells were plated at a density of 1 × 105 cells/35-mm grid dish in 1 ml of Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and were incubated for 3 days. Then, the medium was changed to a serum-free medium, and the cells were further incubated for 24 h. After the incubation, microinjection was performed. Nr-Fragment was expressed as a GST fusion protein and purified from E. coli. C3 was kindly supplied by S. Narumiya (Kyoto University, Kyoto, Japan). All proteins used were concentrated to 5 mg/ml with a Centricon-10 concentrator (Amicon, Inc.). Each sample to be tested was co-microinjected with 2.5 mg/ml rat IgG into cells as described (17Kotani H. Takaishi K. Sasaki T. Takai Y. Oncogene. 1997; 14: 1705-1713Crossref PubMed Scopus (71) Google Scholar). GST-Nr-Fragment was microinjected at 4 mg/ml, and its intracellular concentration was ∼10 μm. C3 was microinjected at 40 μg/ml, and its intracellular concentration was ∼0.23 μm. The cells were fixed at 30 min after the microinjection with 3.7% paraformaldehyde in PBS for 10 min. The fixed cells were permeabilized with 0.2% Triton X-100 in PBS for 10 min. After being soaked in 10% fetal bovine serum/PBS for 1h, the cells were treated for 1h with fluorescein isothiocyanate-conjugated goat anti-rat IgG (Chemicon International, Inc., Temecula, CA) and rhodamine-labeled phalloidin (Molecular Probes Inc., Eugene, OR) in 10% fetal bovine serum/PBS for detection of the microinjected cells and actin filaments, respectively. After being washed with PBS three times, the cells were examined using an LSM 410 confocal laser scanning microscope (Carl Zeiss, Oberkochen, Germany). We first examined the direct physical interaction of Rho GDI with ERM or CD44 using the highly purified recombinant proteins. Rho GDI did not directly interact with full-length radixin or the cytoplasmic fragment of CD44 containing the ERM-interacting domain (amino acids 391–462) (data not shown), suggesting that Rho GDI indirectly interacts with these proteins through an unidentified protein or directly interacts with the specific region of ERM that is masked by folding of the proteins, because ezrin and radixin have been shown to be folded in a manner such that the N- and C-terminal regions mutually mask each other and prevent them from interacting with CD44 and actin filaments, respectively (7Hirao M. Sato N. Kondo T. Yonemura S. Monden M. Sasaki T. Takai Y. Tsukita Sh. Tsukita Sa. J. Cell Biol. 1996; 135: 37-51Crossref PubMed Scopus (512) Google Scholar, 18Martin M. Andréoli C. Sahuquet A. Montcourrier P. Algrain M. Mangeat P. J. Cell Biol. 1995; 128: 1081-1093Crossref PubMed Scopus (120) Google Scholar, 19Magendantz M. Henry M.D. Lander A. Solomon F. J. Biol. Chem. 1995; 270: 25324-25327Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 20Gary R. Bretcher A. Mol. Biol. Cell. 1995; 6: 1061-1075Crossref PubMed Scopus (377) Google Scholar). We therefore next examined whether Rho GDI interacts with the N-terminal fragment of radixin containing the CD44-interacting domain (Nr-Fragment) or the C-terminal fragment containing the actin filament-interacting domain (Cr-Fragment). Rho GDI interacted with Nr-Fragment, but not with Cr-Fragment or full-length radixin (Fig. 1 A). Rho GDI bound Nr-Fragment in a dose-dependent manner, and the value for the half-maximum binding of Rho GDI for Nr-Fragment was ∼0.6 μm (Fig. 1 B). This value was comparable to that for RhoA. We have previously shown that the GEF-independent GDP/GTP exchange reaction of RhoB at low Mg2+ concentrations (micromolar range) is much faster than that at high Mg2+ concentrations (millimolar range) (21Kuroda S. Kikuchi A. Takai Y. Biochem. Biophys. Res. Commun. 1989; 163: 674-681Crossref PubMed Scopus (12) Google Scholar), that Rho GDI inhibits the reactions both at low and high Mg2+ concentrations, but that the inhibitory effect of Rho GDI is apparently more obvious at low Mg2+concentrations than at high Mg2+ concentrations (14Tanaka K. Sasaki T. Takai Y. Methods Enzymol. 1995; 256: 41-49Crossref PubMed Scopus (8) Google Scholar). We first examined the effect of the interaction of Rho GDI with Nr-Fragment on its activity to inhibit the GDP/GTP exchange reaction of RhoA at low Mg2+ concentrations. This reaction was estimated by measuring the dissociation of [3H]GDP from [3H]GDP-RhoA complexed with Rho GDI and the binding of [35S]GTPγS to GDP-RhoA complexed with Rho GDI. Nr-Fragment reduced this Rho GDI activity in a dose-dependent manner (Fig. 2). Under comparable conditions, neither Cr-Fragment nor full-length radixin affected the Rho GDI activity. The same inhibitory effect of Nr-Fragment was also observed when Rac1 or Cdc42 was used as a substrate for Rho GDI. The amino acid sequence of the N-terminal fragment is highly conserved within ERM (∼85% identical for any pair) (9Tsukita Sa. Yonemura S. Tsukita Sh. Curr. Opin. Cell Biol. 1997; 9: 70-75Crossref PubMed Scopus (312) Google Scholar, 10Tsukita Sa. Yonemura S. Tsukita Sh. Trends Biochem. Sci. 1997; 22: 53-58Abstract Full Text PDF PubMed Scopus (274) Google Scholar). Consistently, the N-terminal fragments of ezrin (amino acids 1–280) and moesin (amino acids 1–280) showed the same inhibitory effects on the Rho GDI activity for RhoA, Rac1, and Cdc42 (data not shown). These results indicate that the N-terminal region of ERM has a potency to directly interact with Rho GDI and to reduce its activity to inhibit the GDP/GTP exchange reactions of all the Rho GDI substrate small G proteins. We have shown that Rho GEFs, such as Dbl and Rom1/2, stimulate the GDP/GTP exchange reaction of GDP-RhoA free of Rho GDI, but not that of GDP-RhoA complexed with Rho GDI, at high Mg2+concentrations (4Yaku H. Sasaki T. Takai Y. Biochem. Biophys. Res. Commun. 1994; 198: 811-817Crossref PubMed Scopus (74) Google Scholar, 5Ozaki K. Tanaka K. Imamura H. Hihara T. Kameyama T. Nonaka H. Hirano H. Matsuura Y. Takai Y. EMBO J. 1996; 15: 2196-2207Crossref PubMed Scopus (185) Google Scholar). We therefore next examined the effect of Nr-Fragment on the Rho GDI activity to inhibit the Dbl-stimulated GDP/GTP exchange reaction of RhoA at high Mg2+concentrations. Dbl stimulated the dissociation of GDP from GDP-RhoA, but the dissociation of GDP from GDP-RhoA complexed with Rho GDI was markedly reduced, and this reaction was restored by Nr-Fragment (Fig. 3 A). This inhibitory effect of Nr-Fragment on the Rho GDI activity was dose-dependent and also observed in the Dbl-dependent binding of GTPγS to GDP-RhoA complexed with Rho GDI (Fig. 3 B). Nr-Fragment also reduced the Rho GDI activity to inhibit the Dbl-independent GDP/GTP exchange reaction of RhoA, but the level of this reduction was apparently small due to the slow rate of the GDP/GTP exchange reaction of RhoA at high Mg2+ concentrations. These results indicate that the interaction of Rho GDI with the N-terminal region of ERM reduces its activity in both the Rho GEF-independent and -dependent GDP/GTP exchange reactions of RhoA. Since the C-terminal fragment of ezrin has been shown to interact with the N-terminal fragment longer than amino acids 1–296 (20Gary R. Bretcher A. Mol. Biol. Cell. 1995; 6: 1061-1075Crossref PubMed Scopus (377) Google Scholar), and the C-terminal fragment of radixin has been shown to interact with the N-terminal fragment containing amino acids 1–318 (19Magendantz M. Henry M.D. Lander A. Solomon F. J. Biol. Chem. 1995; 270: 25324-25327Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar), Nr-Fragment used here was not expected to interact with Cr-Fragment. We therefore prepared a longer N-terminal fragment (Nr1-Fragment, amino acids 1–318). Nr1-Fragment also reduced the Rho GDI activity in a dose-dependent manner, with an efficacy similar to that of Nr-Fragment (data not shown). We then examined the effect of Cr-Fragment on the Nr-Frament and Nr1-Fragment activities to reduce the Rho GDI activity. Cr-Fragment reduced the Nr1-Fragment activity, but not the Nr-Fragment activity (Fig. 4). These results provide additional evidence for the specific physical and functional interaction of Rho GDI with ERM and moreover suggest that the region of the N-terminal half of ERM for the interaction with their C-terminal region is different from that for the interaction with Rho GDI. We next examined whether ERM indeed regulate the Rho GDI activity in intact cells. Endogenous RhoA was co-immunoprecipitated with Myc-tagged Rho GDI (Myc-Rho GDI) from the lysate of the COS-7 cells transiently expressing Myc-Rho GDI alone, suggesting that endogenous RhoA was complexed with exogenous Rho GDI in intact cells (Fig. 5 A). However, from the lysate of the cells transiently expressing both Myc-Rho GDI and HA-tagged Nr-Fragment (HA-Nr-Fragment), endogenous RhoA was not co-immunoprecipitated with Myc-Rho GDI, but HA-Nr-Fragment was co-immunoprecipitated with Myc-Rho GDI, suggesting that the RhoA complexed with Rho GDI was replaced by HA-Nr-Fragment to form the Rho GDI-HA-Nr-Fragment complex in intact cells. We further examined whether overexpression of Nr-Fragment mimics the functions of the Rho family members. For this purpose, we used Swiss 3T3 cells because the GTP-bound forms of RhoA, Rac1, and Cdc42 have been shown to induce the formation of stress fibers, lamellipodia, and filopodia, respectively, in this cell line (22Nobes C.D. Hall A. Cell. 1995; 81: 53-62Abstract Full Text PDF PubMed Scopus (3747) Google Scholar). The GTP-bound forms of Rac1 and Cdc42 are also known to induce membrane ruffling (22Nobes C.D. Hall A. Cell. 1995; 81: 53-62Abstract Full Text PDF PubMed Scopus (3747) Google Scholar). Serum-starved Swiss 3T3 cells had very few stress fibers, but microinjection of GST-Nr-Fragment into these cells induced the formation of prominent stress fibers (Fig. 5 B). This response was inhibited by co-microinjection with C3, which is known to ADP-ribosylate the Rho subfamily members and to inhibit their functions (1Hall A. Annu. Rev. Cell Biol. 1994; 10: 31-54Crossref PubMed Scopus (768) Google Scholar, 2Takai Y. Sasaki T. Tanaka K. Nakanishi H. Trends Biochem. Sci. 1995; 20: 227-231Abstract Full Text PDF PubMed Scopus (367) Google Scholar). However, microinjection of GST-Nr-Fragment did not induce the formation of lamellipodia and filopodia or membrane ruffling. Microinjection of GST did not show any effect (data not shown). These results have provided another line of evidence that ERM indeed initiate the activation of the Rho subfamily members through Rho GDI in intact cells. Moreover, these results, together with the in vitroresults described above that the N-terminal region of ERM has a potency to activate all the Rho, Rac, and Cdc42 subfamily members, have raised a possibility that there is a mechanism by which ERM induce the selective activation of each Rho GDI substrate small G protein in intact cells. This mechanism is currently unknown, but one possible factor involved in this mechanism is a Rho GEF specific for each Rho family member, such as Lbc, Tiam-1, and FGD1 (23Zheng Y. Olson M.F. Hall A. Cerione R.A. Toksoz D. J. Biol. Chem. 1995; 270: 9031-9034Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar, 24Michiels F. Habets G.G.M. Stam J.C. van der Kammen R.A. Collard J.G. Nature. 1995; 375: 338-340Crossref PubMed Scopus (509) Google Scholar, 25Zheng Y. Fischer D.J. Santos M.F. Tigyi G. Pasteris N.G. Gorski J.L. Xu Y. J. Biol. Chem. 1996; 271: 33169-33172Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). Elucidation of this mechanism is one of the important issues to be addressed next. ERM have been shown to be translocated from the cytosol to the plasma membrane at least partly through the interaction with CD44 in response to agonists (6Takaishi K. Sasaki T. Kameyama T. Tsukita Sa. Tsukita Sh. Takai Y. Oncogene. 1995; 11: 39-48PubMed Google Scholar, 13Tsukita Sa. Oishi K. Sato N. Sagara J. Kawai A. Tsukita Sh. J. Cell Biol. 1994; 126: 391-401Crossref PubMed Scopus (685) Google Scholar). This translocation has been shown to be accompanied by reorganization of actin filaments (8Arpin M. Algrain M. Louvard D. Curr. Opin. Cell Biol. 1994; 6: 136-141Crossref PubMed Scopus (162) Google Scholar, 9Tsukita Sa. Yonemura S. Tsukita Sh. Curr. Opin. Cell Biol. 1997; 9: 70-75Crossref PubMed Scopus (312) Google Scholar, 10Tsukita Sa. Yonemura S. Tsukita Sh. Trends Biochem. Sci. 1997; 22: 53-58Abstract Full Text PDF PubMed Scopus (274) Google Scholar). Our previous result, that CD44 and Rho GDI are co-immunoprecipitated with moesin (7Hirao M. Sato N. Kondo T. Yonemura S. Monden M. Sasaki T. Takai Y. Tsukita Sh. Tsukita Sa. J. Cell Biol. 1996; 135: 37-51Crossref PubMed Scopus (512) Google Scholar), indicates that these three proteins are able to form a ternary complex. Moreover, in a cell-free system, we have demonstrated that ERM directly interact with the cytoplasmic fragment of CD44 in the presence of phosphatidylinositol 4,5-bisphosphate at a physiological salt concentration (150 mm KCl), but that ERM directly interact with it in the absence of phosphatidylinositol 4,5-bisphosphate at a low salt concentration (40 mm KCl) (7Hirao M. Sato N. Kondo T. Yonemura S. Monden M. Sasaki T. Takai Y. Tsukita Sh. Tsukita Sa. J. Cell Biol. 1996; 135: 37-51Crossref PubMed Scopus (512) Google Scholar). We therefore finally examined whether full-length radixin complexed with CD44 at 40 mm KCl reduces the Rho GDI activity. The cytoplasmic fragment of CD44 did not make full-length radixin reduce the Rho GDI activity to inhibit the GDP/GTP exchange reaction of RhoA (data not shown). Our present results indicate that Rho GDI interacts with the N-terminal fragment of radixin, but not with full-length radixin, and suggest that full-length ERM should first open their folded structures for the interactions of Rho GDI and actin filaments at their N- and C-terminal regions, respectively. These results suggest that the interaction of radixin with CD44 does not change its configuration so that it interacts with Rho GDI and suggest that there is an unfolding mechanism of radixin that induces the interaction with Rho GDI. It is unknown whether the interaction of ERM with CD44 induces the interaction of ERM with actin filaments. Thus, the unfolding mechanism of ERM remains to be clarified, but our present results suggest that ERM are at least one of the factors of which direct interaction with Rho GDI initiates the activation of its substrate small G proteins. We have recently found that the Rho subfamily members stimulate the interaction of ERM with the plasma membrane when the membrane and cytosol fractions of baby hamster kidney cells were used (7Hirao M. Sato N. Kondo T. Yonemura S. Monden M. Sasaki T. Takai Y. Tsukita Sh. Tsukita Sa. J. Cell Biol. 1996; 135: 37-51Crossref PubMed Scopus (512) Google Scholar). However, the GTP-bound form of RhoA did not interact with radixin or CD44 when the highly purified recombinant proteins were used (data not shown), suggesting that the Rho subfamily members regulate the interaction of ERM with the plasma membrane through an unidentified downstream target molecule. Identification of this molecule is of crucial importance for our full understanding of the mode of action of the Rho subfamily members." @default.
• W2069945609 created "2016-06-24" @default.
• W2069945609 creator A5008221814 @default.
• W2069945609 creator A5012351376 @default.
• W2069945609 creator A5027773112 @default.
• W2069945609 creator A5041045423 @default.
• W2069945609 creator A5042331400 @default.
• W2069945609 creator A5043936847 @default.
• W2069945609 creator A5086548300 @default.
• W2069945609 creator A5087395205 @default.
• W2069945609 date "1997-09-01" @default.
• W2069945609 modified "2023-10-09" @default.
• W2069945609 title "Direct Interaction of the Rho GDP Dissociation Inhibitor with Ezrin/Radixin/Moesin Initiates the Activation of the Rho Small G Protein" @default.
• W2069945609 cites W1499601028 @default.
• W2069945609 cites W1499902954 @default.
• W2069945609 cites W1577146075 @default.
• W2069945609 cites W1656440259 @default.
• W2069945609 cites W1964707893 @default.
• W2069945609 cites W1982710998 @default.
• W2069945609 cites W1989143807 @default.
• W2069945609 cites W1992763171 @default.
• W2069945609 cites W1994139415 @default.
• W2069945609 cites W1995015322 @default.
• W2069945609 cites W1997224206 @default.
• W2069945609 cites W2001330472 @default.
• W2069945609 cites W2027810531 @default.
• W2069945609 cites W2042202484 @default.
• W2069945609 cites W2056918775 @default.
• W2069945609 cites W2061126410 @default.
• W2069945609 cites W2063949464 @default.
• W2069945609 cites W2072128091 @default.
• W2069945609 cites W2114964393 @default.
• W2069945609 cites W2115799757 @default.
• W2069945609 cites W2139570277 @default.
• W2069945609 cites W2144476583 @default.
• W2069945609 cites W2160905697 @default.
• W2069945609 cites W2176511881 @default.
• W2069945609 cites W2328730164 @default.
• W2069945609 doi "https://doi.org/10.1074/jbc.272.37.23371" @default.
• W2069945609 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/9287351" @default.
• W2069945609 hasPublicationYear "1997" @default.
• W2069945609 type Work @default.
• W2069945609 sameAs 2069945609 @default.
• W2069945609 citedByCount "414" @default.
• W2069945609 countsByYear W20699456092012 @default.
• W2069945609 countsByYear W20699456092013 @default.
• W2069945609 countsByYear W20699456092014 @default.
• W2069945609 countsByYear W20699456092015 @default.
• W2069945609 countsByYear W20699456092016 @default.
• W2069945609 countsByYear W20699456092017 @default.
• W2069945609 countsByYear W20699456092018 @default.
• W2069945609 countsByYear W20699456092019 @default.
• W2069945609 countsByYear W20699456092020 @default.
• W2069945609 countsByYear W20699456092021 @default.
• W2069945609 countsByYear W20699456092022 @default.
• W2069945609 countsByYear W20699456092023 @default.
• W2069945609 crossrefType "journal-article" @default.
• W2069945609 hasAuthorship W2069945609A5008221814 @default.
• W2069945609 hasAuthorship W2069945609A5012351376 @default.
• W2069945609 hasAuthorship W2069945609A5027773112 @default.
• W2069945609 hasAuthorship W2069945609A5041045423 @default.
• W2069945609 hasAuthorship W2069945609A5042331400 @default.
• W2069945609 hasAuthorship W2069945609A5043936847 @default.
• W2069945609 hasAuthorship W2069945609A5086548300 @default.
• W2069945609 hasAuthorship W2069945609A5087395205 @default.
• W2069945609 hasBestOaLocation W20699456091 @default.
• W2069945609 hasConcept C102931765 @default.
• W2069945609 hasConcept C12554922 @default.
• W2069945609 hasConcept C142669718 @default.
• W2069945609 hasConcept C147789679 @default.
• W2069945609 hasConcept C1491633281 @default.
• W2069945609 hasConcept C185592680 @default.
• W2069945609 hasConcept C196509110 @default.
• W2069945609 hasConcept C2776292417 @default.
• W2069945609 hasConcept C2780246058 @default.
• W2069945609 hasConcept C55493867 @default.
• W2069945609 hasConcept C86803240 @default.
• W2069945609 hasConcept C95444343 @default.
• W2069945609 hasConceptScore W2069945609C102931765 @default.
• W2069945609 hasConceptScore W2069945609C12554922 @default.
• W2069945609 hasConceptScore W2069945609C142669718 @default.
• W2069945609 hasConceptScore W2069945609C147789679 @default.
• W2069945609 hasConceptScore W2069945609C1491633281 @default.
• W2069945609 hasConceptScore W2069945609C185592680 @default.
• W2069945609 hasConceptScore W2069945609C196509110 @default.
• W2069945609 hasConceptScore W2069945609C2776292417 @default.
• W2069945609 hasConceptScore W2069945609C2780246058 @default.
• W2069945609 hasConceptScore W2069945609C55493867 @default.
• W2069945609 hasConceptScore W2069945609C86803240 @default.
• W2069945609 hasConceptScore W2069945609C95444343 @default.
• W2069945609 hasIssue "37" @default.
• W2069945609 hasLocation W20699456091 @default.
• W2069945609 hasOpenAccess W2069945609 @default.
• W2069945609 hasPrimaryLocation W20699456091 @default.
• W2069945609 hasRelatedWork W1965831400 @default.
• W2069945609 hasRelatedWork W2013376648 @default.
• W2069945609 hasRelatedWork W2024608966 @default.
• W2069945609 hasRelatedWork W2062892825 @default.

• next