The RhoA-binding protein, Rhophilin-2, Regulates Actin Cytoskeleton Organization
2002; Elsevier BV; Volume: 277; Issue: 46 Linguagem: Inglês
10.1074/jbc.m203569200
ISSN1083-351X
AutoresJeremy W. Peck, Michael D. Oberst, Kerrie B. Bouker, Emma T. Bowden, Peter D. Burbelo,
Tópico(s)Cellular transport and secretion
ResumoThe Rho GTPases regulate the actin cytoskeleton through interactions with various downstream effector molecules. Here we have identified a ubiquitously expressed human RhoA-binding protein, designated Rhophilin-2. Rhophilin-2 shows 40% amino acid similarity to human Rhophilin-1 and contains an N-terminal Rho-binding, a central Bro1-like, and a C-terminal PDZ domain. Glutathione S-transferase-capture experiments revealed that Rhophilin-1 and Rhophilin-2 interacted with both GDP- and GTP-bound RhoA in vitro. Despite the ability of Rhophilin-1 and Rhophilin-2 to interact with RhoA in a nucleotide-independent fashion, Rho-induced serum response element transcriptional activity was not altered by expression of either of these molecules. Although Rhophilin-2-expressing HeLa cells showed a loss of actin stress fibers, Rhophilin-1 expression had no noticeable effect on the actin cytoskeleton. Coexpression of Rhophilin-2 with a constitutively active Rho mutant reversed the disassembly phenotype, in which the coexpressing cells were more spread and less contracted than Rho alone-expressing cells. Expression of various Rhophilin-2 deletion and point mutants containing the N-terminal RhoA-binding domain but lacking other regions suggested that the disassembly of F-actin stress fibers was not simply caused by Rho sequestration. In addition, the Bro1 and PDZ domains of Rhophilin-2 were required for disassembly. RhoA activity assays also revealed that Rhophilin-2-expressing cells showed increased levels of RhoA-GTP suggesting that the Rhophilin-2-induced disassembly of stress fibers was not mediated by decreased RhoA activity. Based on the biochemical and biological activity, Rhophilin-2 may function normally in a Rho pathway to limit stress fiber formation and/or increase the turnover of F-actin structures in the absence of high levels of RhoA activity. The Rho GTPases regulate the actin cytoskeleton through interactions with various downstream effector molecules. Here we have identified a ubiquitously expressed human RhoA-binding protein, designated Rhophilin-2. Rhophilin-2 shows 40% amino acid similarity to human Rhophilin-1 and contains an N-terminal Rho-binding, a central Bro1-like, and a C-terminal PDZ domain. Glutathione S-transferase-capture experiments revealed that Rhophilin-1 and Rhophilin-2 interacted with both GDP- and GTP-bound RhoA in vitro. Despite the ability of Rhophilin-1 and Rhophilin-2 to interact with RhoA in a nucleotide-independent fashion, Rho-induced serum response element transcriptional activity was not altered by expression of either of these molecules. Although Rhophilin-2-expressing HeLa cells showed a loss of actin stress fibers, Rhophilin-1 expression had no noticeable effect on the actin cytoskeleton. Coexpression of Rhophilin-2 with a constitutively active Rho mutant reversed the disassembly phenotype, in which the coexpressing cells were more spread and less contracted than Rho alone-expressing cells. Expression of various Rhophilin-2 deletion and point mutants containing the N-terminal RhoA-binding domain but lacking other regions suggested that the disassembly of F-actin stress fibers was not simply caused by Rho sequestration. In addition, the Bro1 and PDZ domains of Rhophilin-2 were required for disassembly. RhoA activity assays also revealed that Rhophilin-2-expressing cells showed increased levels of RhoA-GTP suggesting that the Rhophilin-2-induced disassembly of stress fibers was not mediated by decreased RhoA activity. Based on the biochemical and biological activity, Rhophilin-2 may function normally in a Rho pathway to limit stress fiber formation and/or increase the turnover of F-actin structures in the absence of high levels of RhoA activity. Members of the Rho family of GTPases, including Cdc42, Rac, and Rho, are key regulators of the actin cytoskeleton (1Hall A. Science. 1998; 279: 509-514Crossref PubMed Scopus (5196) Google Scholar). In mammalian cells, Rho regulates the formation of focal complexes and stress fibers (2Paterson H.F. Self A.J. Garrett M.D. Just I. Aktories K. Hall A. J. Cell Biol. 1990; 111: 1001-1007Crossref PubMed Scopus (569) Google Scholar, 3Ridley A.J. Hall A. Cell. 1992; 70: 389-399Abstract Full Text PDF PubMed Scopus (3806) Google Scholar, 4Nobes C.D. Hall A. Cell. 1995; 81: 53-62Abstract Full Text PDF PubMed Scopus (3711) Google Scholar). Rho is also involved in the dynamics of other actin-based structures and processes, including phagocytosis (5Caron E. Hall A. Science. 1998; 282: 1717-1721Crossref PubMed Scopus (801) Google Scholar), the cleavage furrow associated with cytokinesis during mitosis (6Kishi K. Sasaki T. Kuroda S. Itoh T. Takai Y. J. 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Biochemical studies using the Rho-specific inhibitor C3 toxin, demonstrate that Rho signaling is involved in tyrosine kinase phosphorylation of both focal adhesion kinase and paxillin (12Kumagai N. Morii N. Fujisawa K. Nemoto Y. Narumiya S. J. Biol. Chem. 1993; 268: 24535-24538Abstract Full Text PDF PubMed Google Scholar, 13Rankin S. Morii N. Narumiya S. Rozengurt E. FEBS Lett. 1994; 354: 315-319Crossref PubMed Scopus (131) Google Scholar, 14Seckl M.J. Morii N. Narumiya S. Rozengurt E. J. Biol. Chem. 1995; 270: 6984-6990Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). One likely mechanism for focal adhesion assembly involves an early step in which Rho induces stress fiber formation. The Rho-induced stress fibers generate membrane tension that induces the aggregation of integrins, which in turn recruit other components of focal adhesions and promote their tyrosine phosphorylation (15Chrzanowska-Wodnicka M. Burridge K. J. Cell Biol. 1996; 133: 1403-1415Crossref PubMed Scopus (1395) Google Scholar). The diversity of Rho-regulated cytoskeletal effects stems from the ability of Rho to interact with a large number of downstream effector proteins (16Bishop A.L. Hall A. Biochem. J. 2000; 348 (Pt. 2,): 241-255Crossref PubMed Scopus (1666) Google Scholar). The most well studied class of these RhoA effector proteins is the family of Rho-associated kinases (ROCKs) (otherwise known as ROKs or Rho-kinases). ROCKs regulate stress fiber formation (17Leung T. Manser E. Tan L. Lim L. J. Biol. Chem. 1995; 270: 29051-29054Abstract Full Text Full Text PDF PubMed Scopus (633) Google Scholar, 18Matsui T. Amano M. Yamamoto T. Chihara K. Nakafuku M. Ito M. Nakano T. Okawa K. Iwamatsu A. Kaibuchi K. EMBO J. 1996; 15: 2208-2216Crossref PubMed Scopus (935) Google Scholar, 19Ishizaki T. Naito M. Fujisawa K. Maekawa M. Watanabe N. Saito Y. Narumiya S. FEBS Lett. 1997; 404: 118-124Crossref PubMed Scopus (454) Google Scholar, 20Amano M. Chihara K. Kimura K. Fukata Y. Nakamura N. Matsuura Y. Kaibuchi K. Science. 1997; 275: 1308-1311Crossref PubMed Scopus (946) Google Scholar), cytokinesis (21Kosako H. Yoshida T. Matsumura F. Ishizaki T. Narumiya S. Inagaki M. Oncogene. 2000; 19: 6059-6064Crossref PubMed Scopus (187) Google Scholar), and myosin-based contractility (22Amano M. Ito M. Kimura K. Fukata Y. Chihara K. Nakano T. Matsuura Y. Kaibuchi K. J. Biol. Chem. 1996; 271: 20246-20249Abstract Full Text Full Text PDF PubMed Scopus (1665) Google Scholar). In cell culture studies, expression of a constitutively active ROCK mutant induces the formation of thick actin fibers and the assembly of focal adhesions (20Amano M. Chihara K. Kimura K. Fukata Y. Nakamura N. Matsuura Y. Kaibuchi K. Science. 1997; 275: 1308-1311Crossref PubMed Scopus (946) Google Scholar, 23Watanabe N. Kato T. Fujita A. Ishizaki T. Narumiya S. Nat Cell Biol. 1999; 1: 136-143Crossref PubMed Scopus (724) Google Scholar). The non-kinase Rho effector protein, mDia1, also regulates stress fiber formation, apparently by recruiting, among others, the actin-monomer-binding protein profilin (24Satoh S. Tominaga T. J. Biol. Chem. 2001; 16: 16Google Scholar, 25Watanabe N. Madaule P. Reid T. Ishizaki T. Watanabe G. Kakizuka A. Saito Y. Nakao K. Jockusch B.M. Narumiya S. EMBO J. 1997; 16: 3044-3056Crossref PubMed Scopus (683) Google Scholar). Similar to ROCK, expression of constitutively active mutants of mDia1 also induces the formation of actin fibers; however, these actin fibers are thin compared with those induced by ROCK (23Watanabe N. Kato T. Fujita A. Ishizaki T. Narumiya S. Nat Cell Biol. 1999; 1: 136-143Crossref PubMed Scopus (724) Google Scholar). Coexpression of constitutively active mutants of ROCK and mDia1 induces stress fibers and focal adhesions that closely resemble those caused by constitutively active RhoA (23Watanabe N. Kato T. Fujita A. Ishizaki T. Narumiya S. Nat Cell Biol. 1999; 1: 136-143Crossref PubMed Scopus (724) Google Scholar). Like ROCK, mDia1 may also function in cytokinesis and can be detected at the cleavage furrow in mitotic Swiss-3T3 cells (26Kato T. Watanabe N. Morishima Y. Fujita A. Ishizaki T. Narumiya S. J. Cell Sci. 2001; 114: 775-784Crossref PubMed Google Scholar). Another RhoA effector protein, Citron kinase, also accumulates at the cleavage furrow and participates in the regulation of cytokinesis (27Madaule P. Eda M. Watanabe N. Fujisawa K. Matsuoka T. Bito H. Ishizaki T. Narumiya S. Nature. 1998; 394: 491-494Crossref PubMed Scopus (327) Google Scholar). Constitutively active Citron mutants block cytokinesis of HeLa cells resulting in multinucleated cells (27Madaule P. Eda M. Watanabe N. Fujisawa K. Matsuoka T. Bito H. Ishizaki T. Narumiya S. Nature. 1998; 394: 491-494Crossref PubMed Scopus (327) Google Scholar). These observations indicate that multiple Rho effector proteins may be involved in the regulation of stress fibers, focal adhesions, and cytokinesis induced by RhoA. Much of what is known about how the effector proteins in Rho signaling pathways function have relied upon the generation and expression of constitutively active and dominant negative mutants. Biochemical and biological analyses of several Rho effector proteins, including ROCK (20Amano M. Chihara K. Kimura K. Fukata Y. Nakamura N. Matsuura Y. Kaibuchi K. Science. 1997; 275: 1308-1311Crossref PubMed Scopus (946) Google Scholar), Citron (27Madaule P. Eda M. Watanabe N. Fujisawa K. Matsuoka T. Bito H. Ishizaki T. Narumiya S. Nature. 1998; 394: 491-494Crossref PubMed Scopus (327) Google Scholar), and mDia1 (23Watanabe N. Kato T. Fujita A. Ishizaki T. Narumiya S. Nat Cell Biol. 1999; 1: 136-143Crossref PubMed Scopus (724) Google Scholar), demonstrate that they exist in an inactive state unless they are bound to Rho. The inactive state of these effectors is maintained by intramolecular autoinhibitory interactions, which are interrupted by the binding of GTP-bound Rho, thus inducing conformational changes that expose their active domains. Deletion analysis has identified regions within these Rho effectors that are essential for this autoinhibition. For example, deletion of the N-terminal, Rho-binding domain of ROCK (20Amano M. Chihara K. Kimura K. Fukata Y. Nakamura N. Matsuura Y. Kaibuchi K. Science. 1997; 275: 1308-1311Crossref PubMed Scopus (946) Google Scholar) and C-terminal, Rho-binding domain of Citron (27Madaule P. Eda M. Watanabe N. Fujisawa K. Matsuoka T. Bito H. Ishizaki T. Narumiya S. Nature. 1998; 394: 491-494Crossref PubMed Scopus (327) Google Scholar) generates mutants with constitutively active kinase activity. Similarly, deletion of the Rho-binding domain within the N terminus of mDia1 results in constitutive activation of this non-kinase effector resulting in the formation of thin actin stress fibers (23Watanabe N. Kato T. Fujita A. Ishizaki T. Narumiya S. Nat Cell Biol. 1999; 1: 136-143Crossref PubMed Scopus (724) Google Scholar). Interestingly, overexpression of the C-terminal autoinhibitory domain of mDia1 alone in mammalian cells can induce stress fiber formation by activating endogenous mDia1 (28Alberts A.S. J. Biol. Chem. 2001; 276: 2824-2830Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar). These results suggest that disruption of autoinhibitory interactions by the binding of GTP-bound Rho is a common mechanism of regulation of these effector proteins. Here we have identified and characterized a novel human RhoA-binding protein, related to mouse Rhophilin-1 (29Watanabe G. Saito Y. Madaule P. Ishizaki T. Fujisawa K. Morii N. Mukai H. Ono Y. Kakizuka A. Narumiya S. Science. 1996; 271: 645-648Crossref PubMed Scopus (348) Google Scholar), which we have named Rhophilin-2. Structurally, Rhophilin-2 contains at least three distinct regions that are homologous to the previously characterized Rho-binding, Bro1, and PDZ (PS.D.-95,Disc-large, ZO-1) domains. Northern analysis revealed that Rhophilin-2 mRNA was expressed in all human tissues examined. Studies in NIH-3T3 fibroblasts showed that expression of Rhophilin-2 or the related molecule, Rhophilin-1, did not alter Rho-induced SRE 1The abbreviations used are: SRE, serum response element; EST, expressed sequence tag; FITC, fluorescein isothiocyanate; GEF, guanine exchange factors; GST, glutathioneS-transferase; RACE, rapid amplification of cDNA ends 1The abbreviations used are: SRE, serum response element; EST, expressed sequence tag; FITC, fluorescein isothiocyanate; GEF, guanine exchange factors; GST, glutathioneS-transferase; RACE, rapid amplification of cDNA ends transcriptional activity. Expression of Rhophilin-2, but not Rhophilin-1 in HeLa cells, caused the loss of stress fibers. This disassembly phenotype induced by Rhophilin-2 was reversed by coexpression with RhoA. Examination of HeLa cells overexpressing various deletion mutants of Rhophilin-2 showed that the Rho-binding, Bro1, and PDZ domains are required for the disassembly of F-actin. RhoA activity assays also revealed that Rhophilin-2-expressing cells showed increased levels of RhoA-GTP suggesting that the Rhophilin-2-induced disassembly of stress fibers was not mediated by decreased RhoA activity. Taken together these results suggest that Rhophilin-2 may act as a scaffold protein that limits stress fiber formation or increases the turnover of F-actin in the absence of high levels of RhoA signaling activity. A human melanocyte cDNA clone encoding a protein with a potential Rho-binding domain was identified by a TBLASTN search of the expressed sequence tag (EST) database at the National Center for Biotechnology Information using the 13-amino acid Rho-binding domain of PKN1 (ELKLKEGAENLRR) as the query. A 1.7-kb human cDNA clone containing this EST (unique IMAGE Consortium identifier 249775 and GenBankTM entryH85494) was obtained and sequenced using a combination of manual sequencing with Sequenase (United States Biochemicals, Cleveland, OH) and automated fluorescent sequencing using sequence-deduced oligonucleotide primers. Using the entire 1.7-kb nucleotide sequence as query, a more abundant, second set of distinct EST clones was identified by additional database searching. DNA sequencing of one of these clones (unique IMAGE Consortium identifier 724324 and GenBankTM entry AA410792) revealed that it was identical to the first clone except for a C-terminal 534-bp segment. We designated the larger clone Rhophilin-2, because it encodes a protein similar to mouse Rhophilin over its entire coding sequence, whereas the shorter form was designated Rhophilin-2β. Because both Rhophilin-2 isoforms lacked the 5′-end, a rapid amplification of cDNA ends (RACE) PCR strategy was employed. The upstream primer (5′-GTTGAACAGGACACTGGCCTTCTC C-3′) and the downstream adaptor primer AP1 (5′-CCATCCTAATACGACTCACTATAGGG C-3′) were used in the first round of PCR using cDNA from kidney (Clontech Laboratories, Palo Alto, CA). The conditions of PCR were 30 cycles of 94 °C for 45 s, 65 °C for 45 s, and 72 °C for 2 min. A second round of nested PCR used 1/50 of the first round reaction product as a template. The new upstream primer (5′-ACAAAGTCCAGCTGGATG-3′) and second adaptor primer AP2 (5′-ACTCACTATAGGGCTCGAGCGGC-3′) were used with a PCR program of 30 cycles consisting of 94 °C for 45 s, 58 °C for 45 s, and 72 °C for 2 min. Positive clones potentially encoding the 5′-end of Rhophilin-2 were identified using restriction enzyme digestion and confirmed through DNA sequencing. The sequence of human Rhophilin-2 and Rhophilin-2β are available from GenBankTMunder accession number AF268032. A multiple tissue Northern blot containing immobilized poly(A+) RNA from several human tissues was obtained from Clontech Laboratories (Palo Alto, CA). A probe common to both isoforms derived from the 3′-end of Rhophilin-2 was linearized and used in a T3 RNA polymerase transcription reaction with [32P]UTP (3000 Ci/mmol). The resulting high specific activity, antisense riboprobe was hybridized at 65 °C under stringent conditions. Following hybridization, the blot was washed and exposed to x-ray film for 72 h. Rhophilin-1, Rhophilin-2, and Rhophilin-2 mutant proteins were expressed in mammalian cells using the pCAF expression vector (30Pirone D.M. Fukuhara S. Gutkind J.S. Burbelo P.D. J. Biol. Chem. 2000; 275: 22650-22656Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar) containing the FLAG epitope tag (DYKDADDDK) at the N terminus. Full-length cDNAs encoding human Rhophilin-1 were amplified by PCR with appropriate adapter primers from a corresponding IMAGE cDNA clone (IMAGE Consortium identifier 2155941), subcloned into theBamH1-XhoI sites of pCAF, and also sequenced (GenBankTM accession number AY082588). Similarly, Rhophilin-2 was subcloned into the XbaI-NotI sites of the pCAF vector. Deletions of Rhophilin-2 were generated by PCR and/or by restriction enzyme digestion: Rhophilin-2-Δ1 (missing amino acids 1–122 including the putative Rho-binding domain), pCAF-Rhophilin-2-Δ2 (missing amino acids 588–685), and pCAF-Rhophilin-2-N1 (containing just the N terminus and missing amino acids 158–685) (see Fig. 8). The Rhophilin-2-ΔBro1 mutant contains an in-frame deletion of amino acids 185–302. Rhophilin-2 point mutants in the Rho-binding site (Rhophilin-2-E55A,N56A) and in the PDZ domain (Rhophilin-2-R517A,L525A,G526A) were generated from the epitope-tagged constructs by using two Rhophilin-2 sequence-specific oligonucleotides and the QuickChange mutagenesis kit (Stratagene). The integrity of all deletion and point mutant constructs were confirmed by DNA sequencing. Details of how each of the plasmids was constructed are available upon request. GST-capture experiments were performed as described previously (31Burbelo P.D. Snow D.M. Bahou W. Spiegel S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 9083-9088Crossref PubMed Scopus (29) Google Scholar). Briefly, GST or GST-RhoA fusion proteins were expressed in Escherichia coli, purified on glutathione-agarose resin, and loaded with guanosine 5′-3-O-(thio)triphosphate or GDP. N-terminal epitope-tagged mammalian expression vectors for Rhophilin-1, Rhophilin-2, and Rhophilin-2 mutants were transfected into Cos1 cells using FuGENE 6 transfection reagent (Roche Molecular Biochemicals) as described by the manufacturer. Forty-eight h later, cell lysates were prepared and used in binding experiments with immobilized GST recombinant proteins. Following washing, bound proteins were eluted and separated using an 8% SDS-PAGE gel. Proteins were then transferred electrophoretically to nitrocellulose and probed with anti-FLAG M2 monoclonal antibody. The FLAG-tagged Rhophilin proteins were then detected by incubating with rabbit anti-mouse horseradish peroxidase followed by incubation with enhanced chemiluminescence reagents (Pierce) and exposure to x-ray film. NIH-3T3 fibroblasts were maintained in Dulbecco's modified Eagle's medium supplemented with 10% calf serum and seeded to a density of 80,000 cells per well into 12-well culture dishes. NIH-3T3 fibroblasts were transfected with a serum response element luciferase reporter plasmid (Stratagene) containing four SRE sites. SRE-luciferase reporter plasmid (500 ng) was cotransfected with the plasmid for cytomegalovirus-Renillaluciferase (1 ng) and with 500 ng of pCAF parental vector, Rhophilin-1, or Rhophilin-2. In the RhoA-V14 cotransfection experiments, SRE-luciferase reporter plasmid (500 ng) was cotransfected with the plasmid for cytomegalovirus-Renilla luciferase (1 ng) and with 250 ng of pCAF, Rhophilin-1, or Rhophilin-2 and 250 ng of either pcDNA or pcDNA-RhoA-V14. The same total amount of DNA was used in each transfection, which were performed using the LipofectAMINE Plus reagent (Invitrogen) according to the manufacturer's instructions. 24 h post-transfection the medium was removed and incubated in Dulbecco's modified Eagle's medium containing 0.5% fetal bovine serum. After 18 h, designated cells were pulsed for 3 h with Dulbecco's modified Eagle's medium containing 10% calf serum. Luciferase activity was measured using the Dual LuciferaseTM reporter assay system (Promega) and measured on a LB9501 Berthold luminometer. Transfection experiments were performed in triplicate, and SRE-luciferase activity is shown from two independent experiments. Immunofluorescence of HeLa cells was performed essentially as described previously (30Pirone D.M. Fukuhara S. Gutkind J.S. Burbelo P.D. J. Biol. Chem. 2000; 275: 22650-22656Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar) except that FuGENE 6 transfection reagent was used. At 24 h post transfection, the cells were fixed, permeabilized, and immunostained with either monoclonal or polyclonal antibodies to detect the FLAG epitope in Rhophilin-2-transfected cells. Following washes in phosphate-buffered saline, the coverslips were incubated with secondary antibodies as indicated in the figure legends. F-actin staining in permeabilized cells was performed using Texas Red-conjugated phalloidin (Sigma). Cotransfection experiments were also performed with Myc-tagged RhoA-V14. Confocal microscopy was performed with an Olympus Fluoview confocal microscope (Olympus America, Inc., Melville, NY) attached to an Olympus 1 × 70 inverted fluorescent scope equipped with a 60× oil immersion lens. Digitalized images were captured using Fluoview software (Olympus America, Inc.). RhoA activity assays were conducted as described previously (32Ren X.D. Kiosses W.B. Schwartz M.A. EMBO J. 1999; 18: 578-585Crossref PubMed Scopus (1359) Google Scholar, 33Ren X.D. Schwartz M.A. Methods Enzymol. 2000; 325: 264-272Crossref PubMed Google Scholar). Briefly, Cos1 cells were transfected with the appropriate vector using the FuGENE 6 transfection reagent (Roche Molecular Biochemicals) as described by the manufacturer. Twenty-four h post-transfection cells were lysed with buffer containing 50 mm Tris, pH 7.5, 500 mm NaCl, 10 mm MgCl2, 1% Triton X-100, 0.1% SDS, 10 μg/ml each of leupeptin and aprotinin, and 1 mmphenylmethylsulfonyl fluoride. Cell lysates were clarified by centrifugation at 13,000g at 4 °C for 10 min and then incubated immediately with 30 μl (20 μg) of GST-Rhotekin-RBD (Upstate Biotechnology, Lake Placid, NY) for 45 min at 4 °C. A 20-μl aliquot of supernatant was also saved for determination of total RhoA levels in each sample. Following incubation, the beads were spun down and washed three times in wash buffer containing 0.1% Triton X-100 and 5 mm MgCl2 in phosphate-buffered saline. Following washing, bound proteins were eluted and separated using a 4–20% SDS-PAGE gel. Proteins were then transferred electrophoretically to nitrocellulose and probed with anti-RhoA polyclonal antibody (Upstate Biotechnology, Lake Placid, NY) at 3 μg/ml overnight at 4 °C. RhoA protein were then detected by incubating with a goat anti-rabbit horseradish peroxidase followed by incubation with enhanced chemiluminescence reagents (Pierce) and exposure to x-ray film. Several well characterized Rho effector proteins contain a short amino acid sequence that enables them to bind directly to GTP-bound Rho (34Reid T. Furuyashiki T. Ishizaki T. Watanabe G. Watanabe N. Fujisawa K. Morii N. Madaule P. Narumiya S. J. Biol. Chem. 1996; 271: 13556-13560Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar). In an effort to identify additional Rho effector proteins, we searched the human EST database for sequences similar to the Rho-binding domain of human PKN1 (29Watanabe G. Saito Y. Madaule P. Ishizaki T. Fujisawa K. Morii N. Mukai H. Ono Y. Kakizuka A. Narumiya S. Science. 1996; 271: 645-648Crossref PubMed Scopus (348) Google Scholar, 35Amano M. Mukai H. Ono Y. Chihara K. Matsui T. Hamajima Y. Okawa K. Iwamatsu A. Kaibuchi K. Science. 1996; 271: 648-650Crossref PubMed Scopus (395) Google Scholar). DNA sequencing of the single clone identified in this search (GenBankTM accession number H85494) revealed a 1.6-kb open reading frame. Subsequent searches of the EST database using the DNA sequence of this putative Rho-binding domain as the query identified multiple additional cDNA clones that could encode an alternatively spliced, longer isoform. DNA sequencing of several of these clones confirmed that they all encode a longer isoform, which we have designated Rhophilin-2, because it is highly homologous to mouse Rhophilin, which we now call Rhophilin-1 (29Watanabe G. Saito Y. Madaule P. Ishizaki T. Fujisawa K. Morii N. Mukai H. Ono Y. Kakizuka A. Narumiya S. Science. 1996; 271: 645-648Crossref PubMed Scopus (348) Google Scholar). The shorter isoform, designated Rhophilin-2β, is identical to Rhophilin-2 except that it is missing a 178-amino acid region in the C-terminal half of the larger form (Fig.1). A GenBankTM database search revealed that both isoforms are encoded in a bacterial artificial chromosome clone (CTC-263F14; GenBankTMaccession number AC011449) that maps to human chromosome 19. Sequence analysis of this bacterial artificial chromosome clone revealed that the 178 amino acids missing from the potential Rhophilin-2β isoform are encoded by five exons (data not shown). Because all of the identified cDNAs for Rhophilin-2 were missing part of the 5′-coding sequences, a 5′-RACE strategy was employed. Using human kidney cDNA as template, an additional 186 bp of sequence were cloned thereby producing full-length coding sequences for both Rhophilin-2 and Rhophilin-2β (data not shown; GenBankTMaccession number AF268032). Analysis of the deduced amino acid sequences revealed that Rhophilin-2 contains 685 amino acids, whereas Rhophilin-2β contains 507 amino acids (Fig. 1). We have also identified a second human Rhophilin cDNA by searching the human EST database with the mouse Rhophilin-1 cDNA (29Watanabe G. Saito Y. Madaule P. Ishizaki T. Fujisawa K. Morii N. Mukai H. Ono Y. Kakizuka A. Narumiya S. Science. 1996; 271: 645-648Crossref PubMed Scopus (348) Google Scholar). Sequencing (GenBankTM accession number AY082588) suggests that it encodes a human homologue of mouse Rhophilin-1 (29Watanabe G. Saito Y. Madaule P. Ishizaki T. Fujisawa K. Morii N. Mukai H. Ono Y. Kakizuka A. Narumiya S. Science. 1996; 271: 645-648Crossref PubMed Scopus (348) Google Scholar), because these proteins show 75% similarity with each other. Comparison between mouse and human Rhophilin-1 with human Rhophilin-2 revealed that they are similar in size and have three domains in common (Fig.2 A). Rhophilin-1 and Rhophilin-2 contain a single Rho-binding motif (Fig. 2 B), which is known to be capable of high affinity direct binding to Rho but not to other GTPases such as Cdc42, Rac, or Ras (34Reid T. Furuyashiki T. Ishizaki T. Watanabe G. Watanabe N. Fujisawa K. Morii N. Madaule P. Narumiya S. J. Biol. Chem. 1996; 271: 13556-13560Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar). Both human Rhophilin proteins also have a central domain of ∼200 amino acids that are significantly similar to one another (Fig. 2 A) and to Bro1 domains in other proteins (data not shown). Although the biological and biochemical functions of Bro1 domains are not known, the first Bro1-containing protein identified, from Saccharomyces cerevisiae, is linked genetically to a protein kinase C signaling pathway that regulates osmoregulation (36Nickas M.E. Yaffe M.P. Mol. Cell. Biol. 1996; 16: 2585-2593Crossref PubMed Scopus (65) Google Scholar). 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The greatest amino acid difference between Rhophilin-1
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