FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2005440054> ?p ?o ?g. }

• W2005440054 endingPage "35070" @default.
• W2005440054 startingPage "35060" @default.
• W2005440054 abstract "A previously unidentified Rho GTPase-activating protein (GAP) domain-containing protein was found in a yeast two-hybrid screen for cDNAs encoding proteins binding to the Src homology 3 domain of Cdc42-interacting protein 4 (CIP4). The protein was named RICH-1 (RhoGAP interacting withCIP4 homologues), and, in addition to the RhoGAP domain, it contained an N-terminal domain with endophilin homology and a C-terminal proline-rich domain. Transient transfections of RICH-1 indicated that it bound to CIP4 in vivo, as shown by co-immunoprecipitation experiments, as well as co-localization assays. In vitro assays demonstrated that the RhoGAP domain of RICH-1 catalyzed GTP hydrolysis on Cdc42 and Rac1, but not on RhoA. Ectopic expression of the RhoGAP domain as well as the full-length protein interfered with platelet-derived growth factor BB-induced membrane ruffling, but not with serum-induced stress fiber formation, further emphasizing the notion that, in vivo, RICH-1 is a GAP for Cdc42 and Rac1. A previously unidentified Rho GTPase-activating protein (GAP) domain-containing protein was found in a yeast two-hybrid screen for cDNAs encoding proteins binding to the Src homology 3 domain of Cdc42-interacting protein 4 (CIP4). The protein was named RICH-1 (RhoGAP interacting withCIP4 homologues), and, in addition to the RhoGAP domain, it contained an N-terminal domain with endophilin homology and a C-terminal proline-rich domain. Transient transfections of RICH-1 indicated that it bound to CIP4 in vivo, as shown by co-immunoprecipitation experiments, as well as co-localization assays. In vitro assays demonstrated that the RhoGAP domain of RICH-1 catalyzed GTP hydrolysis on Cdc42 and Rac1, but not on RhoA. Ectopic expression of the RhoGAP domain as well as the full-length protein interfered with platelet-derived growth factor BB-induced membrane ruffling, but not with serum-induced stress fiber formation, further emphasizing the notion that, in vivo, RICH-1 is a GAP for Cdc42 and Rac1. GTPase-activating protein Cdc42-interacting protein 4 Cdc42/Rac interactive binding fluorescein isothiocyanate hemagglutinin platelet-derived growth factor Src homology 3 tetramethyl rhodamine isothiocyanate Wiskott-Aldrich syndrome protein base pair(s) glutathioneS-transferase dithiothreitol phosphate-buffered saline enhanced green fluorescent protein The cytoskeleton is a major determinant for eukaryotic cell function. The cytoskeleton is formed by three distinct filament systems, the microfilament system, the intermediate filament system, and the microtubule system, which act in concert to orchestrate processes such as cell locomotion, changes in cell morphology, and intracellular transport (1Mitchison T.J. Cramer L.P. Cell. 1996; 84: 371-379Abstract Full Text Full Text PDF PubMed Scopus (1308) Google Scholar, 2Schmidt A. Hall M.N. Annu. Rev. Cell Dev. Biol. 1998; 14: 305-338Crossref PubMed Scopus (371) Google Scholar). Cytoskeletal elements, in particular, the microfilament system, are under a constant reconstruction in response to external stimuli. There exists a close correlation between the activation of transmembrane receptors as well as cell adhesion molecules and the mobilization of the microfilament system (1Mitchison T.J. Cramer L.P. Cell. 1996; 84: 371-379Abstract Full Text Full Text PDF PubMed Scopus (1308) Google Scholar, 2Schmidt A. Hall M.N. Annu. Rev. Cell Dev. Biol. 1998; 14: 305-338Crossref PubMed Scopus (371) Google Scholar). Actin monomers, which polymerize into asymmetric filaments, form the core of the microfilament system. In addition, a large number of actin-binding proteins assist in organization of actin filaments in a variety of supramolecular structures. A family of signaling intermediates, the Rho GTPases, has been shown to be a pivotal regulator of the microfilament system and thereby of the morphogenetic and motile properties of mammalian cells. Each member of the archetypal trio of Rho GTPases, RhoA, Rac1, and Cdc42, has been found to regulate distinct actin filament-containing structures. Rho regulates the formation of focal adhesions and the subsequent assembly of stress fibers, and Rac regulates the formation of membrane lamellae, whereas Cdc42 triggers the outgrowth of peripheral spike-like protrusions called filopodia (3Hall A. Science. 1998; 279: 509-514Crossref PubMed Scopus (5216) Google Scholar, 4Van Aelst L. D'Souza-Schorey C. Genes Dev. 1997; 11: 2295-2322Crossref PubMed Scopus (2094) Google Scholar). The potential of the Rho GTPases to function as signaling switches resides in their ability to cycle between active, GTP-bound states and inactive, GDP-bound states. This cycling is orchestrated by guanine nucleotide exchange factors, GTPase-activating proteins (GAPs),1 and guanine nucleotide dissociation inhibitors. Guanine nucleotide exchange factors stimulate the replacement of GDP by GTP, whereas GAPs stimulate the intrinsic GTP hydrolysis of the GTPase (5Whitehead I.P. Campbell S. Rossman K.L. Der C.J. Biochim. Biophys. Acta. 1997; 1332: F1-F23Crossref PubMed Scopus (334) Google Scholar, 6Lamarche N. Hall A. Trends Genet. 1994; 10: 436-440Abstract Full Text PDF PubMed Scopus (210) Google Scholar, 7Zalcman G. Dorseuil O. Garcia-Ranea J.A. Gacon G. Camonis J. Prog. Mol. Subcell. Biol. 1999; 22: 85-113Crossref PubMed Scopus (32) Google Scholar). Guanine nucleotide dissociation inhibitors act by blocking GDP dissociation, and, in resting cells, the Rho GTPases are thought to reside in an inactive complex with guanine nucleotide dissociation inhibitor proteins (7Zalcman G. Dorseuil O. Garcia-Ranea J.A. Gacon G. Camonis J. Prog. Mol. Subcell. Biol. 1999; 22: 85-113Crossref PubMed Scopus (32) Google Scholar, 8Sasaki T. Takai Y. Biochem. Biophys. Res. Commun. 1998; 245: 641-645Crossref PubMed Scopus (167) Google Scholar). Recent work has revealed that, in mammals, the Rho family consists of at least 16 distinct members that can be further divided into six subgroups: Cdc42 (Cdc42, TC10, TCL, and Chp), Rac (Rac1–3 and RhoG), Rho (Rho A-C), Rnd (Rnd1–2 and RhoE), RhoD, and RhoH (9Aspenström P. Exp. Cell Res. 1999; 246: 20-25Crossref PubMed Scopus (102) Google Scholar, 10Vignal E. De Toledo M. Comunale F. Ladopoulou A. Gauthier-Rouvière C. Blangy A. Fort P. J. Biol. Chem. 2000; 275: 36457-36464Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). Moreover, the Rho GTPases have been shown to regulate several vital cellular processes in addition to cytoskeletal rearrangements. Rac and Cdc42 participate in transcriptional control via the Jun N-terminal kinase/stress-activated protein kinase and p38MAPKsignaling cascades, Rho has a role in serum response factor-regulated gene transcription, and all three contribute to transcriptional activation via the nuclear factor κB signaling pathway (4Van Aelst L. D'Souza-Schorey C. Genes Dev. 1997; 11: 2295-2322Crossref PubMed Scopus (2094) Google Scholar, 10Vignal E. De Toledo M. Comunale F. Ladopoulou A. Gauthier-Rouvière C. Blangy A. Fort P. J. Biol. Chem. 2000; 275: 36457-36464Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 11Kjøller L. Hall A. Exp. Cell Res. 1999; 253: 166-179Crossref PubMed Scopus (342) Google Scholar, 12Mackay D.J.G. Hall A. J. Biol. Chem. 1998; 273: 20685-20688Abstract Full Text Full Text PDF PubMed Scopus (567) Google Scholar, 13Gómez J. Martı́nez-A C. González A. Rebollo A. Immunol. Cell Biol. 1998; 76: 125-134Crossref PubMed Scopus (33) Google Scholar, 14Aspenström P. Curr. Opin. Cell Biol. 1999; 11: 95-102Crossref PubMed Scopus (286) Google Scholar, 15Bishop A.L. Hall A. Biochem. J. 2000; 348: 241-255Crossref PubMed Scopus (1672) Google Scholar). Furthermore, the Rho GTPases are also participants in signaling leading to cell cycle entry and apoptosis (4Van Aelst L. D'Souza-Schorey C. Genes Dev. 1997; 11: 2295-2322Crossref PubMed Scopus (2094) Google Scholar, 13Gómez J. Martı́nez-A C. González A. Rebollo A. Immunol. Cell Biol. 1998; 76: 125-134Crossref PubMed Scopus (33) Google Scholar). The identification of binding partners for the Rho GTPases has resulted in insights into the mechanisms by which these proteins mobilize the microfilament system (4Van Aelst L. D'Souza-Schorey C. Genes Dev. 1997; 11: 2295-2322Crossref PubMed Scopus (2094) Google Scholar, 14Aspenström P. Curr. Opin. Cell Biol. 1999; 11: 95-102Crossref PubMed Scopus (286) Google Scholar, 15Bishop A.L. Hall A. Biochem. J. 2000; 348: 241-255Crossref PubMed Scopus (1672) Google Scholar). A major advancement in our understanding originates from the work on the Wiskott-Aldrich syndrome protein (WASP) family of proteins, which have emerged as important regulators of actin assembly in eukaryotic cells (16Pollard T.D. Blanchoin L. Mullins R.D. Annu. Rev. Biophys. Biomol. Struct. 2000; 29: 545-576Crossref PubMed Scopus (1176) Google Scholar, 17Zigmond S.H. J. Cell Biol. 2000; 150: 117-120Crossref PubMed Google Scholar). This family of proteins also includes N-WASP and Scar/WAVE 1–3 (18Snapper S.B. Rosen F.S. Annu. Rev. Immunol. 1999; 17: 905-929Crossref PubMed Scopus (193) Google Scholar). WASP was originally identified as the gene defective in the rare immunodeficiency disorder Wiskott-Aldrich syndrome (19Derry J.M. Ochs H.D. Francke U. Cell. 1994; 78: 635-644Abstract Full Text PDF PubMed Scopus (828) Google Scholar, 20Ochs H.D. Springer Semin. Immunopathol. 1998; 19: 435-458Crossref PubMed Scopus (31) Google Scholar). WASP is a multidomain adapter protein that contains a phosphoinositide-binding domain, a Cdc42/Rac interactive binding (CRIB) domain that specifically binds Cdc42 (21Burbelo P.D. Drechsel D. Hall A. J. Biol. Chem. 1995; 270: 29071-29074Abstract Full Text Full Text PDF PubMed Scopus (557) Google Scholar), and an extended proline-rich domain that binds SH3 domain-containing proteins such as Nck, Src, and Btk/Tec (18Snapper S.B. Rosen F.S. Annu. Rev. Immunol. 1999; 17: 905-929Crossref PubMed Scopus (193) Google Scholar). Moreover, it was recently found that the members of the WASP family bind directly to actin and to the so-called Arp2/3 complex. This multisubunit protein complex has an essential role in regulating actin polymerization in cells. It is suggested that Arp2/3 is needed for both the formation of new actin filaments and binding to the sides of actin filaments, thereby forming branched actin filament networks (16Pollard T.D. Blanchoin L. Mullins R.D. Annu. Rev. Biophys. Biomol. Struct. 2000; 29: 545-576Crossref PubMed Scopus (1176) Google Scholar, 17Zigmond S.H. J. Cell Biol. 2000; 150: 117-120Crossref PubMed Google Scholar, 18Snapper S.B. Rosen F.S. Annu. Rev. Immunol. 1999; 17: 905-929Crossref PubMed Scopus (193) Google Scholar). Several other Cdc42-binding proteins have been found to affect the organization of the actin cytoskeleton, although the mechanisms by which they act are less well known. A common denominator for most of these proteins is the presence of a CRIB domain (21Burbelo P.D. Drechsel D. Hall A. J. Biol. Chem. 1995; 270: 29071-29074Abstract Full Text Full Text PDF PubMed Scopus (557) Google Scholar). For instance, MSE55 has been shown to induce filopodia-like protrusions when overexpressed in fibroblasts (22Burbelo 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), whereas some of the five known MSE55-related Borg (binders of Rho GTPases) proteins have been suggested to function as negative regulators of Rho (23Joberty G. Perlungher R.R. Macara I.G. Mol. Cell. Biol. 1999; 19: 6585-6597Crossref PubMed Scopus (105) Google Scholar). The SPEC-1/2 (small protein effectors of Cdc42) proteins induce an accumulation of polymerized actin in peripheral membrane protrusions in fibroblasts (24Pirone 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). Cdc42-interacting protein 4 (CIP4), however, binds Cdc42 via a domain motif unrelated to the CRIB domain (25Aspenström P. Curr. Biol. 1997; 7: 479-487Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar). Overexpression of CIP4 in fibroblasts leads to the disappearance of filamentous actin bundles in these cells. Moreover, simultaneous expression of activated Cdc42 and CIP4 results in a relocalization of the uniformly distributed CIP4 into peripheral and dorsal clusters or villi-like structures, which might represent precursors of filopodia (25Aspenström P. Curr. Biol. 1997; 7: 479-487Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar). To elucidate the mechanisms by which CIP4 acts, we sought to identify binding partners for the protein. A yeast two-hybrid screen identified a previously uncharacterized RhoGAP domain-containing protein that specifically interacted with the SH3 domain of CIP4. We named this protein RICH-1 (RhoGAP interacting withCIP4 homologues). The N-terminal portion of RICH-1, including the RhoGAP domain, was shown to be homologous to a protein of unknown function, KIAA0672, which we propose should be renamed RICH-2. The RhoGAP domain of RICH-1 was furthermore shown to be similar to the analogous domain of the Abl-binding protein 3BP-1 (26Cicchetti P. Mayer B.J. Thiel G. Baltimore D. Science. 1992; 257: 803-806Crossref PubMed Scopus (420) Google Scholar). The high degree of similarity between the proteins suggested that RICH-1, RICH-2, and 3BP-1 form a closely related family of RhoGAPs.In vitro assays demonstrated that the RhoGAP domains of RICH-1 and RICH-2 specifically activate the GTP hydrolysis of Rac1 and Cdc42, but not of RhoA. Ectopic expression of the RhoGAP domain of RICH-1 abrogated PDGF-BB-induced membrane ruffles but not the serum-induced stress-fibers, further emphasizing the notion that RICH-1 is a Rac and Cdc42-specific GAP. TheSaccharomyces cerevisiae strain Y190 (genotype, MATa,gal4–542, gal80–538, his3,trp1–901, ade2–101, ura3–52,leu2–3, 112, URA3::GAL1-LacZ, Lys2::GAL1-HIS3cyhr) was transformed with a cDNA encoding the SH3 domain of CIP4 (amino acid residues 489–545) fused to the GAL4 DNA-binding domain in the pYTH6 vector. This GAL4 DNA-binding domain-CIP4:SH3-expressing yeast strain was used to screen a cDNA library from Epstein-Barr virus-transformed human B cells fused to the GAL4 activation domain in the pACT vector as described previously (25Aspenström P. Curr. Biol. 1997; 7: 479-487Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar, 27Aspenström P. Olson M.F. Methods Enzymol. 1995; 256: 228-241Crossref PubMed Scopus (34) Google Scholar). Thirteen clones encoding potential CIP4-interacting proteins were isolated, one of which encoded a partial cDNA for a previously uncharacterized RhoGAP domain-containing protein, which was subsequently named RICH-1. This yeast two-hybrid clone represented a fragment that was later found to encode amino acid residues 387–803 of the full-length polypeptide. The partial clone was used to screen a human brain λ ZAP II cDNA library following standard procedure (28Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Clones (9 × 106) were screened, and three positive clones were isolated after rescreening. These clones were recovered as phagemid Bluescript SK−constructs by excision from the λ vector with helper phage R408 as described in Ref. 29Short J.M. Fernandez J.M. Sorge J.A. Huse W.D. Nucleic Acids Res. 1988; 16: 7583-7600Crossref PubMed Scopus (1080) Google Scholar. One of the clones contained a putative initiator codon; however, the predicted amino acid sequence stopped prematurely before the RhoGAP domain, resulting in an open reading frame of 226 amino acid residues. Polymerase chain reaction was employed to screen various cDNA libraries for splice variants of RICH-1 that encoded a RhoGAP domain-containing protein. The presence of a splice variant encoding an insert of an extra 82 bp was detected in human B cells as well as in HeLa cells. This splice variant encoded a protein with an open reading frame of 803 amino acid residues. We designated the long form RICH-1 and the short form RICH-1B. The nucleotide sequences of RICH-1 and RICH-2 have been deposited in the EMBL data base with the accession numbers AJ306731 (RICH-1) and AJ306732 (RICH-1B). Fragments of RICH-1, RICH-1B, and RICH-1 RhoGAP domain were generated as BamHI-EcoRI polymerase chain reaction fragments and inserted into the pRK5myc vector. The C-terminal domain of RICH-1 (amino acids 384–803) was subcloned into theHindIII and EcoRI sites of the pEGFP-C3 vector. The SH3 domains of CIP4 (amino acid residues 489–545), forming-binding protein 17 (amino acid residues 550–617), and syndapin (amino acid residues 376–441) were subcloned into the pGEX-2T vectors. DNA sequencing on a Perkin Elmer Genetic Analyzer 310 confirmed the fidelity of the nucleotide sequence. The DNA work followed standard procedures (28Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Sequential deletion mutants of RICH-1 lacking the proline-rich motives were generated by polymerase chain reaction (see Fig. 5 for a detailed description) and subcloned into the pRK5myc vector. The arginine to alanine mutation of RICH-1 in codon 288 (RICH-1R288A), which rendered the protein catalytically inactive, was generated by the QuikChange site-directed mutagenesis kit (Stratagene). Probes representing DNA fragments encoding amino acid residues 387–803 of RICH-1, amino acid residues 1–818 of RICH-2, and the 82-bp insert uniquely present in RICH-1 were labeled with [32P]CTP using the rediprime labeling kit (Amersham Pharmacia Biotech). The probes were then hybridized to hybridization-ready Northern blots (Human Multiple Tissue Northern blot; CLONTECH) according to the ExpressHyb (CLONTECH) protocol provided by the manufacturer. Glutathione S-transferase (GST) fusion proteins of Rac1, Cdc42 (brain isoform), and RhoA were expressed in Escherichia coli, purified on glutathione-Sepharose beads (Amersham Pharmacia Biotech), and isolated from GST fusion proteins by thrombin cleavage as described previously (25Aspenström P. Curr. Biol. 1997; 7: 479-487Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar, 30Self A.J. Hall A. Methods Enzymol. 1995; 256: 3-10Crossref PubMed Scopus (167) Google Scholar). GST fusion proteins of the RhoGAP domains of RICH-1 (amino acid residues 221–489), RICH-2 (KIAA0672 obtained from the Kazusa DNA Research Institute, Chiba, Japan; amino acids residues 217–469), and p50RhoGAP (amino acid residues 230–439) were expressed in E. coli. The bacteria were lysed in a buffer containing 50 mm Tris-HCl, pH 7.5, 5 mmMgCl2, 50 mm NaCl, 10% glycerol, 0.1% Triton X-100, 1 mm phenylmethylsulfonyl fluoride, 1% aprotinin (Trasylol; Beyer), and 1 mm dithiothreitol (DTT). The proteins were then eluted from the glutathione-Sepharose beads with 5 mm reduced glutathione, desalted on PD10 prepacked chromatography columns (Amersham Pharmacia Biotech), equilibrated in 20 mm Tris-HCl, pH 7.5, 10% glycerol, and 1 mmDTT, and concentrated using Centricon-10 (Millipore). Protein concentrations were determined according to the Bradford method. GST fusion proteins of the SH3 domains described in Fig. 4 A were prepared as described above but were retained on the glutathione-Sepharose beads. The procedure we used essentially followed the procedure described by Self and Hall (31Self A.J. Hall A. Methods Enzymol. 1995; 256: 67-76Crossref PubMed Scopus (115) Google Scholar). Briefly, 0.1 μg of recombinant wild-type Rac, Rho, or Cdc42 was preloaded with 10 μCi of [γ-32P]GTP (Amersham Pharmacia Biotech) in 20 μl of 20 mm Tris-HCl, pH 7.5, 25 mm NaCl, 5 mm EDTA, and 0.1 mm DTT. The mixture was incubated for 10 min at 30 °C, and then the reaction was terminated by adding 5 μl of 0.1m MgCl2, and the resulting [γ-32P]GTP-loaded GTPase solutions were stored on ice. For the GAP assays, equimolar amounts of the GTPases and the GST-GAP domains were used. Three μl of the [γ-32P]GTP-loaded GTPase was added to a 30-μl mixture of 20 mm Tris-HCl, pH 7.5, 1 mm nonradioactive GTP, 0.87 mg/ml bovine serum albumin, and 0.1 mm DTT with the GST-RhoGAP domains of either RICH-1, RICH-2, or p50RhoGAP. The mixture was incubated at 30 °C, 5-μl aliquots were removed after 0, 3, 6, 9, and 15 min, and the reaction was stopped by the addition of 1 ml of ice-cold buffer A (50 mm Tris-HCl, pH 7.5, 50 mm NaCl, and 5 mm MgCl2). The samples were collected on nitrocellulose filters and washed with 10 ml of ice-cold buffer A, and the portion of [γ-32P]GTP remaining bound to the GTPases was determined by scintillation counting. For the exchange assays, a similar protocol was employed, but 0.5 mCi of [8-3H]GTP (Amersham Pharmacia Biotech) was used instead. Cos-1, Swiss 3T3, and NIH 3T3 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum (10% calf serum for NIH 3T3 cells) and penicillin/streptomycin at 37 °C in an atmosphere of 5% CO2. Porcine aortic endothelial cells stably expressing the PDGF-β receptor (clone PAE/PDGFRβ) were cultured in Ham's F-12 medium supplemented as described above. For the characterization of antisera, transiently transfected Cos-1 cells were metabolically labeled for 4 h at 37 °C with 15 μCi/ml Easy Tag EXPRESS35S protein labeling mix in MCDB 104 medium lacking cysteine and methionine and supplemented with HEPES. Cos-1 cells were transfected according to the DEAE-dextran method (32Kriegler M. Gene Transfer and Expression: A Laboratory Manual. W. H. Freeman and Company, NY1990Crossref Google Scholar). Transfected cells were harvested 48 h after transfection, washed once in ice-cold PBS, and lysed on ice in Nonidet P-40 buffer (20 mm HEPES, pH 7.5, 150 mm NaCl, 10% glycerol, 1% Nonidet P-40, 1 mm phenylmethylsulfonyl fluoride, 1% aprotinin (Trasylol; Beyer) and 1 mm DTT). Lysed cells were collected in Eppendorf tubes and centrifuged for 15 min. The resulting supernatants were subjected to immunoprecipitation experiments or GST pull-down assays. For co-immunoprecipitations of HA epitope-tagged CIP4 and myc epitope-tagged RICH-1, supernatants were incubated together with antibodies as indicated in the figure legends for 1 h at 4 °C, after which immunoprecipitates were collected on protein G-Sepharose 4 Fast Flow (Amersham Pharmacia Biotech). The beads were washed three times with Nonidet P-40 buffer, and SDS-polyacrylamide gel electrophoresis sample buffer was added to each sample. The immunoprecipitates as well as control cell lysates were subjected to SDS-polyacrylamide gel electrophoresis, and the proteins were then transferred to Immobilon-P filters (Millipore). Western blots were detected by the BM chemiluminescence blotting substrate (Roche Molecular Biochemicals). The 35S-labeled RICH-1 immunoprecipitates were subjected to SDS-polyacrylamide gel electrophoresis, and the gels were fixed for 15 min in 25% methanol and 7.5% acetic acid and soaked in Amplify (Amersham Pharmacia Biotech) for 30 min. The gels were then dried and exposed on a phosphorimager (Fujix BAS 2000). For GST pull-down assays, supernatants from lysed RICH-1-transfected Cos-1 cells were incubated with GST fusion proteins of SH3 domains from a panel of proteins described in Fig. 4 A, and the presence of RICH-1 bound to the GST fusion protein was determined by Western blotting. For immunocytochemical analysis, Swiss 3T3 cells, NIH 3T3 fibroblasts, or PAE/PDGFRβ cells were seeded on coverslips and transfected by LipofectAMINE Plus (Life Technologies, Inc.) according to the protocols provided by the manufacturer. GST fusion proteins of the PAK-CRIB and WASP-CRIB domains encompassing amino acids 56–267 and 201–321 of PAK1B and WASP, respectively, were purified essentially as described in Ref. 33Ren X.-D. Schwartz M.A. Methods Enzymol. 2000; 325: 264-272Crossref PubMed Google Scholar. PAE/PDGFRβ cells were transiently transfected with pRK5mycCdc42 or pRK5mycRac1 in combination with the pRK5mycRICH-1 RhoGAP domain or pRK5myc encoding the catalytically inactive R288A mutant RICH-1 RhoGAP domain. Forty-eight h after transfection, the cells were washed with ice-cold PBS supplemented with 1 mm MgCl2. The cells were then lysed on ice in a cold room with a buffer containing 50 mm Tris-HCl, pH 7.5, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 0.5 m NaCl, 10 mmMgCl2, 1% aprotinin, and 1 mmphenylmethylsulfonyl fluoride. The cell lysates were immediately subjected to centrifugation. Active, GTP-bound Rac1 and Cdc42 were isolated from the supernatants by the addition of GST-PAK-CRIB and GST-WASP-CRIB, respectively, followed by a 10-min incubation. The beads were washed three times with a buffer containing 50 mmTris-HCl, pH 7.5, 1% Triton X-100, 150 mm NaCl, 10 mm MgCl2, 1% aprotinin, and 1 mmphenylmethylsulfonyl fluoride. Equal amounts of proteins were subjected to SDS-polyacrylamide gel electrophoresis, and the extent of GTP-bound Cdc42 and Rac1 was determined by Western blotting. Antisera were raised against peptides representing amino acid residues 53–67 (antiserum N) and 779–792 (antiserum C) of human RICH-1. These antisera, as well as mouse anti-myc (9E10; Santa Cruz Biotechnology), mouse anti-HA (12CA5;Roche Molecular Biochemicals), rabbit anti-green fluorescent protein (CLONTECH), and tetramethyl rhodamine isothiocyanate (TRITC)- and fluorescein isothiocyanate (FITC)-conjugated anti-rabbit and anti-mouse antibodies (DAKO), were used for immunoprecipitations and to determine the subcellular localization of CIP4 and RICH-1 mutants. Filamentous actin was visualized by TRITC- or FITC-conjugated phalloidin (Sigma). For immunocytochemistry assays, the cells were grown on coverslips and fixed in 2% paraformaldehyde in PBS for 20 min. The cells were washed with PBS and permeabilized in 0.2% Triton X-100 in PBS for 5 min. The cells were then washed again and incubated in the presence of 10 mm glycine in PBS for 1 h. Primary as well as secondary antibodies were diluted in PBS containing 5% fetal calf serum. Cells were incubated with primary antibodies followed by secondary antibodies for intervals of 1 h, with a washing step in between. The coverslips were mounted in Fluoromount-G (Southern Biotechnology Associates, Inc.) on object slides. Cells were photographed by a Hamamatsu ORCA charge-coupled device digital camera employing the QED Imaging System software using a Zeiss Axioplan2 microscope. The yeast two-hybrid system was used to identify binding partners for CIP4. In a screen, employing the SH3 domain of CIP4 as bait, 13 CIP4-interacting clones were isolated from a cDNA library from Epstein-Barr virus-transformed human B cells. Data base searches showed that one of the clones represented a partial cDNA of a previously unidentified RhoGAP domain-containing protein, which we named RICH-1. This clone lacked a putative transcriptional start site; therefore, the partial cDNA was used to screen a λ ZAP II human brain cDNA library. Three positive clones were recovered, one of which contained a putative initiator codon in agreement with the Kozak consensus (34Kozak M. J. Biol. Chem. 1991; 266: 19867-19870Abstract Full Text PDF PubMed Google Scholar). Surprisingly, the predicted open reading frame resulted in a protein of only 226 amino acids, terminating immediately upstream of the RhoGAP domain. This suggested the existence of alternative splice variants of RICH. The polymerase chain reaction revealed the occurrence of an 82-bp insert located upstream of the RhoGAP domain in RICH cDNAs from human B cells and HeLa cells. The presence of this insert resulted in a transcript with a considerably longer open reading frame of 803 amino acid residues (Fig. 1 A). The insert could not be detected in brain-derived cDNAs (see also Fig.2 B). We named the 803- and 226-amino acid residue splice variants RICH-1 and RICH-1B, respectively (Fig. 1 B).Figure 2Northern blot analysis of mRNA from human tissues as indicated. A, tissue distribution of RICH-1/RICH-1B. B, distribution of RICH-1 alone.C, distribution of RICH-2 (KIAA0672).View Large Image Figure ViewerDownload Hi-res image Download (PPT) RICH-1 exhibited extensive similarity to a protein with the annotation KIAA0672, identified by the Kazusa DNA Research Institute. Due to the similarity between the two proteins, we suggest that KIAA0672 should be renamed RICH-2. These two proteins also displayed a high degree of similarity to the c-Abl-interacting protein 3BP-1, suggesting that the three proteins form a closely related family of RhoGAP proteins. RICH-1 and RICH-2 both contain an N-terminal domain with homology to the endophilin family of proteins (Fig. 1, B and D) (35Ringstad N. Nemoto Y. De Camilli P. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8569-8574Crossref PubMed Scopus (328) Google Scholar). RICH-1B encompasses only the endophilin-like domain, suggesting that this part of the protein has a unique but as yet unknown function. Data base searches against the nucleotide sequences released by the Human Genome Project indicate that 3BP-1 also contains a similar domain, which was not reported in the original publication of the 3BP-1 amino acid sequence (36Cicchetti P. Ridley A.J. Zheng Y. Cerione R.A. Baltimore D. EMBO J. 1995; 14: 3127-3135Crossref PubMed Scopus (64) Google Scholar). 2P. Aspenström, unpublished observation. Multiple alignment of the central RhoGAP domains of RICH-1, RICH-2, and 3BP-1 demonstrated that they all contain the conserved arginine finger, which is present in this type of domain (Fig. 1 C) (6Lamarc" @default.
• W2005440054 created "2016-06-24" @default.
• W2005440054 creator A5024529237 @default.
• W2005440054 creator A5078666752 @default.
• W2005440054 date "2001-09-01" @default.
• W2005440054 modified "2023-10-14" @default.
• W2005440054 title "RICH, a Rho GTPase-activating Protein Domain-containing Protein Involved in Signaling by Cdc42 and Rac1" @default.
• W2005440054 cites W108705584 @default.
• W2005440054 cites W109074058 @default.
• W2005440054 cites W114522406 @default.
• W2005440054 cites W1540291954 @default.
• W2005440054 cites W1546570611 @default.
• W2005440054 cites W1596940979 @default.
• W2005440054 cites W1861604986 @default.
• W2005440054 cites W1903137736 @default.
• W2005440054 cites W1942356165 @default.
• W2005440054 cites W1964073548 @default.
• W2005440054 cites W1966178703 @default.
• W2005440054 cites W1967448808 @default.
• W2005440054 cites W1971377120 @default.
• W2005440054 cites W2010015418 @default.
• W2005440054 cites W2020157212 @default.
• W2005440054 cites W2021680936 @default.
• W2005440054 cites W2024927131 @default.
• W2005440054 cites W2034266654 @default.
• W2005440054 cites W2036123452 @default.
• W2005440054 cites W2037425715 @default.
• W2005440054 cites W2046345458 @default.
• W2005440054 cites W2053610637 @default.
• W2005440054 cites W2054327174 @default.
• W2005440054 cites W2054993296 @default.
• W2005440054 cites W2073891485 @default.
• W2005440054 cites W2074122548 @default.
• W2005440054 cites W2076255878 @default.
• W2005440054 cites W2077792958 @default.
• W2005440054 cites W2084900570 @default.
• W2005440054 cites W2088921403 @default.
• W2005440054 cites W2093315657 @default.
• W2005440054 cites W2093382854 @default.
• W2005440054 cites W2093396713 @default.
• W2005440054 cites W2096791952 @default.
• W2005440054 cites W2105746559 @default.
• W2005440054 cites W2121889667 @default.
• W2005440054 cites W2124009013 @default.
• W2005440054 cites W2128640800 @default.
• W2005440054 cites W2131411591 @default.
• W2005440054 cites W2135606345 @default.
• W2005440054 cites W2136611642 @default.
• W2005440054 cites W2148993806 @default.
• W2005440054 cites W2150125913 @default.
• W2005440054 cites W2155653297 @default.
• W2005440054 cites W2158044026 @default.
• W2005440054 cites W2159422521 @default.
• W2005440054 cites W2163627937 @default.
• W2005440054 cites W2166910344 @default.
• W2005440054 cites W32283874 @default.
• W2005440054 cites W4250102918 @default.
• W2005440054 cites W63752650 @default.
• W2005440054 doi "https://doi.org/10.1074/jbc.m103540200" @default.
• W2005440054 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/11431473" @default.
• W2005440054 hasPublicationYear "2001" @default.
• W2005440054 type Work @default.
• W2005440054 sameAs 2005440054 @default.
• W2005440054 citedByCount "114" @default.
• W2005440054 countsByYear W20054400542012 @default.
• W2005440054 countsByYear W20054400542013 @default.
• W2005440054 countsByYear W20054400542014 @default.
• W2005440054 countsByYear W20054400542015 @default.
• W2005440054 countsByYear W20054400542016 @default.
• W2005440054 countsByYear W20054400542017 @default.
• W2005440054 countsByYear W20054400542018 @default.
• W2005440054 countsByYear W20054400542019 @default.
• W2005440054 countsByYear W20054400542020 @default.
• W2005440054 countsByYear W20054400542021 @default.
• W2005440054 countsByYear W20054400542022 @default.
• W2005440054 countsByYear W20054400542023 @default.
• W2005440054 crossrefType "journal-article" @default.
• W2005440054 hasAuthorship W2005440054A5024529237 @default.
• W2005440054 hasAuthorship W2005440054A5078666752 @default.
• W2005440054 hasBestOaLocation W20054400541 @default.
• W2005440054 hasConcept C134306372 @default.
• W2005440054 hasConcept C163181125 @default.
• W2005440054 hasConcept C185592680 @default.
• W2005440054 hasConcept C207332259 @default.
• W2005440054 hasConcept C2779717556 @default.
• W2005440054 hasConcept C2780040266 @default.
• W2005440054 hasConcept C2994130171 @default.
• W2005440054 hasConcept C33923547 @default.
• W2005440054 hasConcept C36503486 @default.
• W2005440054 hasConcept C62478195 @default.
• W2005440054 hasConcept C80631254 @default.
• W2005440054 hasConcept C86803240 @default.
• W2005440054 hasConcept C95444343 @default.
• W2005440054 hasConceptScore W2005440054C134306372 @default.
• W2005440054 hasConceptScore W2005440054C163181125 @default.
• W2005440054 hasConceptScore W2005440054C185592680 @default.
• W2005440054 hasConceptScore W2005440054C207332259 @default.
• W2005440054 hasConceptScore W2005440054C2779717556 @default.

• next