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• W2027449551 abstract "RhoA, Cdc42, and Rac1 are small GTPases that regulate cytoskeletal reorganization leading to changes in cell morphology and cell motility. Their signaling pathways are activated by guanine nucleotide exchange factors and inactivated by GTPase-activating proteins (GAPs). We have identified a novel RhoGAP, BPGAP1 (for BNIP-2 and Cdc42GAP Homology (BCH) domaincontaining, Proline-rich and Cdc42GAP-like protein subtype-1), that is ubiquitously expressed and shares 54% sequence identity to Cdc42GAP/p50RhoGAP. BP-GAP1 selectively enhanced RhoA GTPase activity in vivo although it also interacted strongly with Cdc42 and Rac1. “Pull-down” and co-immunoprecipitation studies indicated that it formed homophilic or heterophilic complexes with other BCH domain-containing proteins. Fluorescence studies of epitope-tagged BPGAP1 revealed that it induced pseudopodia and increased migration of MCF7 cells. Formation of pseudopodia required its BCH and GAP domains but not the prolinerich region, and was differentially inhibited by coexpression of the constitutively active mutant of RhoA, or dominant negative mutants of Cdc42 and Rac1. However, the mutant without the proline-rich region failed to confer any increase in cell migration despite the induction of pseudopodia. Our findings provide evidence that cell morphology changes and migration are coordinated via multiple domains in BPGAP1 and present a novel mode of regulation for cell dynamics by a RhoGAP protein. RhoA, Cdc42, and Rac1 are small GTPases that regulate cytoskeletal reorganization leading to changes in cell morphology and cell motility. Their signaling pathways are activated by guanine nucleotide exchange factors and inactivated by GTPase-activating proteins (GAPs). We have identified a novel RhoGAP, BPGAP1 (for BNIP-2 and Cdc42GAP Homology (BCH) domaincontaining, Proline-rich and Cdc42GAP-like protein subtype-1), that is ubiquitously expressed and shares 54% sequence identity to Cdc42GAP/p50RhoGAP. BP-GAP1 selectively enhanced RhoA GTPase activity in vivo although it also interacted strongly with Cdc42 and Rac1. “Pull-down” and co-immunoprecipitation studies indicated that it formed homophilic or heterophilic complexes with other BCH domain-containing proteins. Fluorescence studies of epitope-tagged BPGAP1 revealed that it induced pseudopodia and increased migration of MCF7 cells. Formation of pseudopodia required its BCH and GAP domains but not the prolinerich region, and was differentially inhibited by coexpression of the constitutively active mutant of RhoA, or dominant negative mutants of Cdc42 and Rac1. However, the mutant without the proline-rich region failed to confer any increase in cell migration despite the induction of pseudopodia. Our findings provide evidence that cell morphology changes and migration are coordinated via multiple domains in BPGAP1 and present a novel mode of regulation for cell dynamics by a RhoGAP protein. Cells undergo dynamic changes as part of their adaptation and response to stimuli. These include their abilities to proliferate, differentiate, or execute death. Many of these processes are controlled by a series of signaling events relayed via a cascade of molecular interaction that are normally associated with the enzymatic or structural modifications of target proteins. Furthermore, there exist various checkpoints that serve to fine-tune the amplitude, duration, as well as the integration of such circuitry response. One of the relatively well characterized signaling circuits in eukaryotic system is the Ras small GTP-binding protein (GTPase) superfamily (1Etienne-Manneville S. Hall A. Nature. 2002; 420: 629-635Crossref PubMed Scopus (3839) Google Scholar, 2Bar-Sagi D. Hall A. Cell. 2000; 103: 227-238Abstract Full Text Full Text PDF PubMed Scopus (702) Google Scholar, 3Mackay D.J. Hall A. J. Biol. Chem. 1998; 273: 20685-20688Abstract Full Text Full Text PDF PubMed Scopus (567) Google Scholar) that binds and slowly hydrolyzes GTP to GDP, which is still bound to the proteins. The GTP-bound form assumes an active conformation that allows interaction with downstream effectors, thus the “on-switch,” whereas its conversion to the GDP-bound form keeps the proteins in an “off-switch” mode and renders the GTPase inactive. The balance of these two forms determines the final execution of the pathway. This is regulated by two other important classes of proteins, one that helps enhance its GTPase activity, termed GTPase-activating proteins (GAPs) 1The abbreviations used are: GAPs, GTPase-activating proteins; EST, expressed sequence tag; HA, hemagglutinin; ANOVA, analysis of variance; LD, longest diameter; SD, shortest diameter; IP, immunoprecipitation; IB, immunoblotting; FL, full-length; GFP, green fluorescent protein; GST, glutathione S-transferase; WCL, whole cell lysate.1The abbreviations used are: GAPs, GTPase-activating proteins; EST, expressed sequence tag; HA, hemagglutinin; ANOVA, analysis of variance; LD, longest diameter; SD, shortest diameter; IP, immunoprecipitation; IB, immunoblotting; FL, full-length; GFP, green fluorescent protein; GST, glutathione S-transferase; WCL, whole cell lysate. and the other, termed guanine nucleotide exchange factors (GEFs) that activate the protein by catalyzing its exchange of GDP for GTP. Many members of the small GTPases have already been identified, and they can further be subdivided into various families or subfamilies according to the similarities in their primary sequences. Members from different families exhibit diverse functions ranging from the control of intracellular trafficking to cytoskeletal rearrangements and cell cycle progression. The degree of specificity is further extended to even closely related members within the same families. For example, in the Rho family, the Cdc42 plays an important role in the formation of filopodia, whereas RhoA and Rac1 activation results in the formation of stress fibers and membrane ruffles respectively (4Hall A. Science. 1998; 279: 509-514Crossref PubMed Scopus (5216) Google Scholar). In addition, there is a hierarchy of network in certain cell types where activation of one member can affect the activity of another. For example, activation of Cdc42 leads to filopodia formation, which could in turn activate Rac1 (5Kozma R. Ahmed S. Best A. Lim L. Mol. Cell Biol. 1995; 15: 1942-1952Crossref PubMed Scopus (881) Google Scholar, 6Nobes C.D. Hall A. Biochem. Soc. Trans. 1995; 23: 456-459Crossref PubMed Scopus (302) Google Scholar), whereas Rac1 activation leads to inactivation of RhoA in NIH3T3 resulting in the epithelioid phenotype (7Sander E.E. ten Klooster J.P. van Delft S. van der Kammen R.A. Collard J.G. J. Cell Biol. 1999; 147: 1009-1022Crossref PubMed Scopus (737) Google Scholar, 8Evers E.E. Zondag G.C. Malliri A. Price L.S. ten Klooster J.P. van der Kammen R.A Collard J.G. Eur. J. Cancer. 2000; 36: 1269-1274Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar, 9Zondag G.C. Evers E.E. ten Klooster J.P. Janssen L. van der Kammen R.A. Collard J.G. J. Cell Biol. 2000; 149: 775-782Crossref PubMed Scopus (251) Google Scholar). In contrast, in Swiss 3T3 fibroblasts, Rac1 activates RhoA instead (10Ridley A.J. Paterson H.F. Johnston C.L. Diekmann D. Hall A. Cell. 1992; 70: 401-410Abstract Full Text PDF PubMed Scopus (3071) Google Scholar). With an increasing number of known GTPases, there remain key questions as to how each one of them can be regulated by their GEFs, GAPs, or other regulators in vivo. The human genome is predicted to encode at least 50 members of the GAP family (11Moon S.Y. Zheng Y. Trends Cell Biol. 2003; 13: 13-22Abstract Full Text Full Text PDF PubMed Scopus (486) Google Scholar, 12Peck J. Douglas G. Wu C.H. Burbelo P.D. FEBS Lett. 2002; 528: 27-34Crossref PubMed Scopus (140) Google Scholar). Current data show that various GAP domains exhibit overlapping substrate specificity both in vitro and in vivo but all involve a common mechanism of action by utilizing an “arginine-finger” motif in trans to stabilize the transition state of GTP hydrolysis (13Nassar N. Hoffman G.R. Manor D. Clardy J.C. Cerione R.A. Nat. Struct. Biol. 1998; 5: 1047-1052Crossref PubMed Scopus (176) Google Scholar, 14Gamblin S.J. Smerdon S.J. Curr. Opin. Struct. Biol. 1998; 8: 195-201Crossref PubMed Scopus (65) Google Scholar). For example, the p50RhoGAP (also known as Cdc42GAP) (15Barfod E.T. Zheng Y. Kuang W.J. Hart M.J. Evans T. Cerione R.A. Ashkenazi A. J. Biol. Chem. 1993; 268: 26059-26062Abstract Full Text PDF PubMed Google Scholar, 16Lancaster C.A. Taylor-Harris P.M. Self A.J. Brill S. van Erp H.E. Hall A. J. Biol. Chem. 1994; 269: 1137-1142Abstract Full Text PDF PubMed Google Scholar) and p122RhoGAP (17Homma Y. Emori Y. EMBO J. 1995; 14: 286-291Crossref PubMed Scopus (189) Google Scholar) bind and inactivate mainly Cdc42 and RhoA, respectively. In comparison, p200RhoGAP targets RhoA and Rac1 but not Cdc42 (18Moon S.Y. Zang H. Zheng Y. J. Biol. Chem. 2003; 278: 4151-4159Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar) while p115RhoGAP confines its action mainly to RhoA (19Christerson L.B. Gallagher E. Vanderbilt C.A. Whitehurst A.W. Wells C. Kazempour R. Sternweis P.C. Cobb M.H. J. Cell. Physiol. 2002; 192: 200-208Crossref PubMed Scopus (26) Google Scholar). Therefore, it appears that there is no specific GAP for a single GTPase. Instead, there exists a GAP that recognizes more than one GTPase, and a single GTPase can be a target of multiple GAPs. The molecular basis for such distinctive or overlapping recognition profile remains to be understood. Furthermore, most of these GAPs possess multiple signaling modules that could couple their activities to other signaling pathways. This could have far reaching consequences for the regulation of Rho and other small GTPase signals, and remains to be seen how, where, and when any subsets or combinations of these cellular counterparts will co-exist and exert their effects. In order to understand the specificity versus redundancy nature of the RhoGAPs as well as the roles of their various signaling modules, we have set out to study novel proteins that harbor the GAP domain together with other protein domains. Bioinformatic searches through the human genome public databases revealed a striking number of sequences that encode putative GAP proteins and with various arrays of domain organizations. One of the family proteins that we are interested in has the organization that is similar to that of the Cdc42GAP, yet exhibiting diversed sequences in other regions. Here we report the cloning and functional characterization for such a member in this family that harbors (from the proximal N terminus) a BNIP-2 and Cdc42GAP Homology (BCH)/Sec14p-like domain that we first described (20Low B.C. Lim Y.P. Lim J. Wong E.S. Guy G.R. J. Biol. Chem. 1999; 274: 33123-33130Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 21Low B.C. Seow K.T. Guy G.R. J. Biol. Chem. 2000; 275: 14415-14422Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, 22Low B.C. Seow K.T. Guy G.R. J. Biol. Chem. 2000; 275: 37742-37751Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 23Zhou Y.T. Soh U.J. Shang X. Guy G.R. Low B.C. J. Biol. Chem. 2002; 277: 7483-7492Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar), a proline-rich sequence, and a functional GAP domain. We showed that BP-GAP1 differentially modulates RhoA, Cdc42, and Rac1 signaling pathway by a mechanism that required cooperation between the BCH and GAP domain. When expressed in non-metastatic human breast epithelial cancer cell lines MCF7 cells, BPGAP1 induced cell protrusions/pseudopodia that required its GAP activity as well as the BCH domain, but not the proline-rich sequence. However, the proline-rich region was required for ensuring cell migration following the morphological changes induced by both GAP and BCH domains. These results indicate the unique interplay by different domains of BPGAP1 in exerting cell dynamics and confirm that changes in cell morphology is a prerequisite but not necessarily the only determinant for cell migration, it requires the input of other factor(s) as well. Our findings also emphasize the need to address functions of distinct protein domains in various RhoGAP families in order to have a better understanding of their physiological functions and regulation. Bioinformatics—To identify novel proteins containing GAP domains, the peptide sequence of the RhoGAP domain of p50RhoGAP/Cdc42GAP (GenBank™ accession number: Q07960; residues: 260–439) was used as query sequence in the “position-specific iteractive BLAST” against the current non-redundant sequence as well as human and mouse EST databases (www.ncbi.nlm.nih.gov/). Progress of the identification was described in the text. Multiple sequence alignments were generated using Vector NTI suite (InforMax, Inc.). RT-PCR Cloning of BPGAP1 Isoforms and Plasmid Constructions—To obtain the full-length cDNA of BPGAP1, total RNA was isolated from MCF7 cells using the RNeasy kit (Qiagen) according to the manufacturer's instructions. 5 μg of this RNA was subjected to the first-strand cDNA synthesis with Expand Reverse Transcriptase Kit (Roche Applied Science) primed with oligo(dT) (Operon) for 60 min at 42 °C in a total volume of 20 μl. 0.5 μg of this cDNA was then amplified by the high fidelity, long-template Taq polymerase enzyme (Roche Applied Science) using specific primers corresponding to the putative sequence BAA91614. PCR conditions were: initial denaturation 94 °C, 2 min; subsequent cycling (30 cycles) at 94 °C, 10 s; annealing at 50 °C, 30 s; extension at 68 °C, 2 min; and final extension at 68 °C, 7 min. These PCR primers contained HindIII and XhoI restriction sites on the forward and reverse primers, respectively, to facilitate their subsequent cloning. The full-length PCR products were gel-purified (Qiagen) and cloned into a FLAG epitope-tagged or GFP-tagged expression vector, pXJ40 (Dr. E. Manser, Institute of Molecular and Cell Biology, Singapore). Sequence unique to BPGAP1 was obtained (GenBank™ AF544240), and fragments encoding its various domains were generated from the full-length template using specific primers in a standard PCR and then gel-purified for cloning. For each construct, several clones were chosen and sequenced entirely in both directions using the ABI PRISM BigDye Terminator Cycle Sequencing kit (Applied Biosystem). All plasmids were purified using Qiagen miniprep kit for subsequent use in transfection experiments. For generation of deletion mutants, inverse-PCR was carried out to exclude region of interest whereas point mutation R232A was performed by site-directed mutagenesis as previously described (21Low B.C. Seow K.T. Guy G.R. J. Biol. Chem. 2000; 275: 14415-14422Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). Escherichia coli strain DH5α was used as host for the propagation of the clones. Reagents used were of analytical grade, and standard protocols for molecular manipulations and media preparation were as described (24Sambrook J. Russell D.W. Molecular Cloning, A Laboratory Manual. 3rd ed. Cold Spring Harbor Press, NY2001: 1.109-1.110Google Scholar). Semi-quantitative RT-PCR—To distinguish the mRNA expression level of BPGAP1 and Cdc42GAP in various cells and tissues, RT-PCR using the oligo-dT primers was employed. Total RNA was isolated using the RNeasy kit (Qiagen) from either various cultured cell lines or from various organs obtained from a 2-week-old male mouse and primed for the first-strand cDNA synthesis as described above. Equal amounts of the reverse transcription product were then subjected to PCR amplification for BPGAP1 and Cdc42GAP. The full-length PCR products of BPGAP1 were then subjected to internal amplification using primers that encompass BPGAP1-specific BCH region that contained the unique insertion (see text). The housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used to normalize the level of expression. The results were verified in at least two independent experiments with varying numbers of PCR cycles to ensure near-linear amplification. Cell Culture and Transfection—Human breast cancer MCF7, human embryonic kidney epithelial cells 293T, human stomach cancer lines MCN45 and KMN74 were all grown in RPMI 1640 medium supplemented with 10% (v/v) fetal bovine serum, 2 mm l-glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin (all from Hyclone), and maintained at 37 °C in a 5% CO2 atmosphere. Human cervical cancer epithelial HeLa cells were grown in Dulbecco's modified Eagle's medium (high glucose), whereas human colon epithelial HT29 and HCT116 were grown in McCoy's medium (Sigma). Cells at 90% confluence in 100-mm plates or 6-well plates were transfected with 5 or 2 μg of indicated plasmids using FuGENE 6 transfection reagent, according to the manufacturer's instructions (Roche Applied Science). Precipitation/Pull-down Studies and Western Blot Analyses—Control 293T cells or cells transfected with expression plasmids were lysed in 1 ml of lysis buffer (150 mm sodium chloride, 50 mm Tris, pH 7.3, 0.25 mm EDTA, 1% (w/v) sodium deoxycholate, 1% (v/v) Trition X-100, 50 mm sodium fluoride, 5 mm sodium orthovanadate, and a mixture of protease inhibitors (Roche Applied Science)). The lysates were directly analyzed, either as whole cell lysates (25 μg) or aliquots (500 μg) used in affinity precipitation/pull-down experiments with various GST fusion proteins (5 μg), as previously described (21Low B.C. Seow K.T. Guy G.R. J. Biol. Chem. 2000; 275: 14415-14422Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). Samples were run in SDS-PAGE gels and analyzed by Western blotting with FLAG monoclonal antibody (Sigma). Immunofluorescence—Cells were seeded on coverslips in 6-well plates, transfected with various expression constructs for 16–20 h, and then stained for immunofluorescence detection as previously described (25Lim J. Wong E.S. Ong S.H. Yusoff P. Low B.C. Guy G.R. J. Biol. Chem. 2000; 275: 32837-32845Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Fluorescent images were taken with a confocal laser microscopy system (Fluoview, FV300, Olympus). FLAG-tagged proteins were detected with monoclonal anti-FLAG, followed by Texas Red® dye-conjugated rabbit anti-mouse IgG (Jackson ImmunoResearch). For cells co-expressing GFP-tagged recombinants and HA-tagged proteins, HA-tagged constructs were detected with polyclonal anti-HA, followed by Texas Red® dye-conjugated goat anti-rabbit IgG. For cells expressing only GFP-tagged recombinants, the morphology of cells was examined directly under a fluorescent microscope after the transfection for 16–20 h as previously described (23Zhou Y.T. Soh U.J. Shang X. Guy G.R. Low B.C. J. Biol. Chem. 2002; 277: 7483-7492Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). Preparation of GST Fusion Proteins—GST fusion proteins were purified using glutathione-agarose beads. In brief, E. coli cells were lysed by sonication in a HEPES buffer, pH 7.5, 150 mm NaCl2, 1 mm EDTA, multiple protein inhibitors (Roche Applied Science), 0.1% (w/v) β-mercaptoethanol, and 0.1% (w/v) Triton-100). Following centrifugation (10,000 rpm, 30 min, 4 °C), the supernatants of lysates were mixed with glutathione-agarose beads (Amersham Biosciences) and incubated at 4 °C for overnight. Beads were washed three times with 10 ml of HEPES buffer. When needed, fusion proteins were eluted with 10 mm glutathione solution in the HEPES buffer. Protein concentrations were measured by using Bradford assay (Bio-Rad). In Vitro GTPase Activity Assay—GTPase activity assays were performed with the Enz-check™ Phosphate Assay kit (E-6646, Molecular Probes) to monitor the rate of phosphate release from GTP hydrolysis catalyzed by recombinant Cdc42, RhoA, or Rac1 (pGEX plasmids of these and Cdc42GAP are gifts from Dr. A. Hall, University College London, United Kingdom) in the presence of GST control or GST-BPGAP1 full-length, domains, or its mutant. For these assays, we used a previously described protocol (26Wu G. Li H. Yang Z. Plant Physiol. 2000; 124: 1625-1636Crossref PubMed Scopus (97) Google Scholar) with some modifications. In brief, 0.5 nmol of purified GST-BPGAP1 full-length, domains, or mutant proteins (in a volume of 15 μl), was mixed in a cuvette with 10 μl of 0.2 mm GTP, 0.2 ml of 2-amino-6-mercapto-7-methylpurine ribonucleoside, 10 μl (1 unit) of purine nucleotide phosphorylase, and 0.78 ml of HEPES buffer (pH 7.5). The cuvette was immediately placed in the spectrophotometer to monitor absorbance at 360 nm (A 360). 10 μl of 1 m MgCl2 solution was added to 0.25 nmol of eluted GST, GST-Cdc42, GST-RhoA, or GST-Rac1 fusion proteins and incubated for 10 min at room temperature. When the first multiple turnover reached an equilibrium at A 360, the second mixture of small GTPase solution was added to initiate the reaction. The reading at A 360 was recorded every 10 s. In Vivo GTPase Activity and Binding Assay—GTP-bound Cdc42, Rac1, or RhoA was determined by specific binding to the p21-binding domain of PAK1 (GST-PBD) (27Bagrodia S. Taylor S.J. Creasy C.L. Chernoff J. Cerione R.A. J. Biol. Chem. 1995; 270: 22731-22737Abstract Full Text Full Text PDF PubMed Scopus (331) Google Scholar) or rhotekin (GST-RBD) (28Ren X.D. Kiosses W.B. Schwartz M.A. EMBO J. 1999; 18: 578-585Crossref PubMed Scopus (1361) Google Scholar) (all kindly provided by Dr. Simone Schoenwaelder; Monash University, Australia). In brief, cell lysates expressing HA-tagged wild-type small GTPases (Cdc42, Rac1, or RhoA) with or without FLAG-tagged BPGAP1 were incubated with 5 μg of recombinant GST-PBD or GST-RBD conjugated with glutathione-Sepharose beads for 1 h at 4 °C, washed with buffer (50 mm HEPES, pH 7.4, 150 mm sodium chloride, 1.5 mm magnesium chloride, 5 mm EGTA, 10% (v/v) glycerol, 1% (v/v) Triton X-100, a mixture of protease inhibitors and 5 mm sodium orthovanadate) and separated on SDS-PAGE. Bound Cdc42, Rac1, or RhoA was analyzed by Western blotting using anti-HA antibodies (Roche Applied Science). Whole cell lysates were also analyzed for the presence of expressed Cdc42, Rac1, RhoA, and BPGAP1 for normalization. For detecting binding of endogeneous Rho GTPases, the following antibodies were used: polyclonal anti-Cdc42 (Santa Cruz Biotechnology), polyclonal anti-RhoA, and monoclonal anti-Rac1 (both from Upstate Biotechnology). Co-immunoprecipitation—293T cells were transfected with expression vectors for FLAG-BPGAP1 full length alone or together with either HA-BPGAP1, HA-Cdc42GAP, HA-BNIP-2 or HA-GTPases. Lysates were immunoprecipitated (IP) with anti-FLAG M2 beads (Sigma) and the associated proteins separated on SDS-PAGE, and probed with anti-Cdc42, RhoA, Rac1, or HA antibodies to reveal the binding of targets. Cell Measurement—MCF7 cells were transfected with GFP control or GFP-tagged BPGAP1 full-length, NP, and PC domains. After 20 h, the longest diameter (LD) and shortest diameter (SD) that bisected the center of cells and perpendicular to each were measured (29Maddox A.S. Burridge K. J. Cell Biol. 2003; 160: 255-265Crossref PubMed Scopus (228) Google Scholar). The total cell areas and the length of the cell protrusion (PT) were also measured after image capturing as previously described and analyzed using the Leica IM 1000 software. Measurements were means and S.D. from three separate experiments, each time with at least 30 different cells. Statistical comparison was made using ANOVA (StatsDirect). p values of <0.01 indicate significant difference compared with the vector control. Cell Migration Assay—The ability of cells to migrate through coated filters was measured with a modified Boyden chamber (24-well Transwell, Corning Costar; 8-μm pore size) as previous described (30Koo T.H. Lee J.J. Kim E.M. Kim K.W. Kim H.D. Lee J.H. Oncogene. 2002; 21: 4080-4088Crossref PubMed Scopus (106) Google Scholar). The lower surface of the filters was coated with 0.5-μg fibronectin (Sigma) as a chemoattractant. MCF7 cells transiently transfected with GFP vector, GFP-BPGAP1 full-length, different fragments, or mutants were seeded at a density of 3 × 105 cells in 100 μl of RPMI 1640 with 0.2% bovine serum albumin. The lower compartment was added with 600 μl of RPMI 1640 containing 10% fetal bovine serum. After incubation for 1 day at 37 °C in 5% CO2, the cells that did not penetrate the filters were completely wiped off with cotton swabs, and the cells that had migrated to the lower surface of the filter were fixed with methanol and counted. Three independent experiments were performed for each experimental condition. The data were represented as the means of three independent experiments with S.D. indicated. Statistical comparison was made using ANOVA (StatsDirect). p values of <0.01 indicate significant difference compared with the vector control. Identifying Novel GTPase-activating Proteins—To identify novel GTPase-activating proteins (GAPs) encoded in the human genome and to gain an insight on how they might regulate various cellular processes through their various protein modules, we undertook bioinformatics approach and employed the Conserved Domain Architecture Retrieval Tool (CDART) (www.ncbi.nlm.nih.gov/BLAST/) with the well characterized GAP domain of Cdc42GAP/p50RhoGAP as the query sequence. We have identified in silico many classes of proteins across species that harbor the homologous GAP domain together with other unique signaling protein domains. Some of them include the Pleckstrin homology domain, Src homology-3 domain, Fes/CIP4 homology domain, Rho guanine nucleotide exchange factor domain, and the p21 Rho binding domain. One of these classes is represented by several putative members that resemble the organization of the Cdc42GAP protein. They are typified by the presence, at the proximal N terminus, of the newly identified BNIP-2 and Cdc42GAP homology (BCH)/Sec14p-like domain that we first described in the BNIP-2 family (20Low B.C. Lim Y.P. Lim J. Wong E.S. Guy G.R. J. Biol. Chem. 1999; 274: 33123-33130Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 21Low B.C. Seow K.T. Guy G.R. J. Biol. Chem. 2000; 275: 14415-14422Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, 22Low B.C. Seow K.T. Guy G.R. J. Biol. Chem. 2000; 275: 37742-37751Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 23Zhou Y.T. Soh U.J. Shang X. Guy G.R. Low B.C. J. Biol. Chem. 2002; 277: 7483-7492Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar) and a well conserved GAP domain at its distal C terminus. Present in between these two domains is a proline-rich moiety. Based on the predicted open reading frame from one of the putative sequences deposited, BAA91614, several conserved primers were designed and used in reverse-transcription-based PCR to isolate the full-length cDNA from human MCF7 cells. Interestingly, one unique sequence of cDNA was repeatedly identified (Fig. 1A), which codes for a protein that differs from BAA91614 by lacking 31 amino acids (Fig 1B , upper line). The protein also differs at the N terminus, from two putative proteins encoded from the same human ARHGAP8 locus (GenBank™ accession numbers: Q9NSG0 and AF195968). Despite using primers specific to those variants, we had not identified the full contigs for such transcripts in all samples examined thus far. Many classes of GAPs have been identified from the human genome and labeled ARHGAP1–12. However, they are not related to each other as each one carries different types and numbers of other associated protein domains. To provide meaningful reference to the specific subclass of GAP with its unique domain organization, we propose to name this family of proteins BPGAPs (for BCH domain-containing, proline-rich, and Cdc42GAP-like proteins) with their notable three-domain organization. We further sought to understand how one novel member we identified here, BPGAP1 (GenBank™ accession number: AF544240), regulates cellular processes via these protein domains. Efforts are underway to isolate the full contigs for other putative isoforms, BPGAP2 (represented by BAA91614), BPGAP3 (AF195968), and the longest subtype, BPGAP4 (Q9NSG0). It is believed that these isoforms could be derived from alternative RNA splicing of the same gene. A mouse homolog with 88% similarity to human BPGAP1 was also identified from the genome data base (encoded by accession NP_082731 or AI430858). Compared with Cdc42GAP, BPGAP1 displays unique divergence at various regions. Notably, the BPGAP1 has a much shorter sequence at the N terminus but a much longer C tail than Cdc42GAP (Fig. 2A). To understand the degree of similarity or divergence for the BCH and GAP domains, more detailed comparisons were made with similar domains found in other proteins. The BCH domain of BPGAP1 is more closely related to that of Cdc42GAP (84% similarity) (Fig. 2B) while its GAP domain also shares the highest degree of homology with that of Cdc42GAP (Fig. 2C). More importantly, BPGAP1 contains an invariant arginine at residue 232 (Fig. 2C, indicated by an arrow). This residue in other functional GAPs is known as an “arginine finger” and shown to be critical for acting as a catalytic residue in-trans (13Nassar N. Hoffman G.R. Manor D. Clardy J.C. Cerione R.A. Nat. Struct. Biol. 1998; 5: 1047-1052Crossref PubMed Scopus (176) Google Scholar, 14Gamblin S.J. Smerdon S.J. Curr. Opin. Struct. Biol. 1998; 8: 195-201Crossref PubMed Scopus (65) Google Scholar, 31Fidyk N.J. Cerione R.A. Biochemistry. 2002; 41: 15644-15653Crossref PubMed Scopus (29) Google Scholar). In addition, BPGAP1 possesses several more proline residues in the proline-rich sequence, which is very similar to those identified in RNB6, ena-VASP-like and cdc-related proteins (Fig. 2D). It could comprise more than o" @default.
• W2027449551 created "2016-06-24" @default.
• W2027449551 creator A5027749586 @default.
• W2027449551 creator A5055998574 @default.
• W2027449551 creator A5057348664 @default.
• W2027449551 date "2003-11-01" @default.
• W2027449551 modified "2023-10-14" @default.
• W2027449551 title "Concerted Regulation of Cell Dynamics by BNIP-2 and Cdc42GAP Homology/Sec14p-like, Proline-rich, and GTPase-activating Protein Domains of a Novel Rho GTPase-activating Protein, BPGAP1" @default.
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