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1 School of Pharmacy, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan
2 Department of Molecular Pathology, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
3 Department of Biochemistry 1, Hamamatsu University School of Medicine, 1-20-1 Handayama, Hamamatsu, 431-3192, Japan
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Abstract |
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Bs and ß-catenin. Protein substrates of FWD1/ß-TrCP contain a consensus DSG
XS motif (where represents a hydrophobic residue and X represents any amino acid). Recognition by FWD1/ß-TrCP requires phosphorylation of the conserved serines in that motif. Here we show that FGD1, a Cdc42 guanine nucleotide exchange factor (GEF), is a novel target of the SCFFWD1/ß-TrCP ubiquitin ligase. A mutant FGD1 protein, FGD1(SA), in which both of the critical serine residues in the DSGXS motif have been replaced by alanines, does not interact with FWD1/ß-TrCP and exhibits increased stability. Morphological changes induced by wild-type FGD1 (FGD1(WT)) are reduced by the co-expression of SCFFWD1/ß-TrCP whereas those induced by FGD1(SA) are not affected. FGD1(SA)-expressing cells show a higher level of cell motility than FGD1(WT)-expressing cells. We present a novel ‘turning off’ mechanism for the inactivation of FGD1, an upstream regulator for Cdc42. |
Introduction |
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E3s are classified into two groups: the HECT (homologous to E6-AP carboxy terminus) domain-containing E3s and the RING finger-containing E3s. HECT domain proteins, such as E6-AP, are characterized by the ability to form a thiolester intermediate with the activated ubiquitin before transfer of ubiquitin to substrates (Hershko & Ciechanover 1998). In contrast, the RING finger-containing E3s do not form a covalent bond with the activated ubiquitin and instead catalyze ubiquitination by association with substrate (Pickart 2001). RING finger-containing E3s come in two varieties: the single-subunit RING E3s and the multisubunit RING E3s (Pickart 2001). Among the latter, one of the better-defined E3s is SCFFWD1/ß-TrCP(Karin & Ben-Neriah 2000).
SCF-type E3s are assemblies of several common (Skp1, Cul1, and Roc1/Rbx1/Hrt1) and single-variable (F-box protein) protein components. The F-box protein FWD1/ß-TrCP has been implicated in the ubiquitination of CD4 (through HIV protein Vpu), IBs, ß-catenin, and hDlg. All these proteins share similar phosphorylation sites with the consensus sequence of DSGXS ( represents a hydrophobic residue and X represents any amino acid) (Karin & Ben-Neriah 2000; Mantovani & Banks 2003).
Database searches show that this consensus sequence is conserved in FGD1. FGD1 is implicated as a critical participant in mammalian development because disruption of this gene results in the disease Faciogenital Dysplasia (FGDY; Aarskog syndrome), a skeletal dysplasia and multiple congenital anomaly syndrome (Pasteris et al. 1994). FGD1 contains (sequentially from N-terminus) a proline-rich domain, a Dbl homology (DH) domain arranged in tandem with a pleckstrin homology (PH) domain, a FYVE domain, and a second C-terminal PH domain (Pasteris et al. 1994; Schmidt & Hall 2002). The DH domain was first identified in the Dbl oncogene product as the region required for mediating guanine nucleotide exchange with the Rho family GTPase Cdc42 (Hart et al. 1991). This domain has since been found in a large family of proteins. In each case, a PH domain immediately follows the DH domain. Most of the proteins possessing this tandem DH-PH module are now recognized as guanine nucleotide exchange factors (GEFs) which function as upstream activators for Rho family GTPases (Schmidt & Hall 2002). However, very little is known about how GEFs are inactivated.
Here we show that FGD1, containing the FWD1/ß-TrCP recognition sequence [DS283GIDS287] between the proline-rich domain and the DH domain, is polyubiquitinated by SCFFWD1/ß-TrCP. FGD1(SA), the mutant form of FGD1, in which S283 and S287 have been replaced by alanine, is resistant to ubiquitination and degradation by the proteasome. When expressed in cells, FGD1(SA) induces stronger biological responses than wild-type FGD1, suggesting that the phosphorylation-coupled ubiquitination system acts as a negative regulator to turn off the GEF function of FGD1.
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Results |
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Through the use of a BLAST database search, we found that FGD1, the Cdc42 GEF responsible for Faciogenital Dysplasia, contains the sequence DS283 GIDS287, which matches the consensus sequence recognized by FWD1/ß-TrCP, the subunit of SCFFWD1/ß-TrCP ubiquitin ligase (Fig. 1A). In order to clarify whether or not the function of FGD1 is regulated by SCFFWD1/ß-TrCP ubiquitin ligase, we established stable cell lines in which expression of either a wild-type of FGD1 (FGD1(WT)) or a mutant FGD1 encoding the sequence of DA283 GIDA287 (FGD1(SA)) is regulated by a tetracycline derivative, doxycycline (Dox) (Fig. 1B,C). As shown in Fig. 1C, Dox treatment led to the significant induction of FGD1(WT) or FGD1(SA) in the cell lines derived from HeLa Tet-On cells and the induction levels of FGD1(WT) and FGD1(SA) were comparable.
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Once phosphorylated, the known [DSGXS] proteins with the exception of Vpu are recognized as a substrate for polyubiquitination by SCFFWD1/ß-TrCP. In the case of Vpu, it is not targeted for polyubiquitination but instead acts as an adaptor that directs SCFFWD1/ß-TrCP complex to an associated protein, CD4 (Margottin et al. 1998). To examine whether or not FGD1 undergoes polyubiquitination, cells transfected with HA-tagged ubiquitin cDNA were lyzed and the levels of polyubiquitination in FGD1 immunoprecipitates were examined. As shown in Fig. 3A, a remarkable polyubiquitination ladder was observed in the FGD1(WT) immunoprecipitates (lane 2) but not in FGD1(SA) immunoprecipitates (lane 5). When proteasomal activity was inhibited by MG132, the density of the ladder was significantly increased in FGD1(WT) immunoprecipitates (lane 3). To confirm that the polyubiquitination ladder was due to ubiquitinated forms of FGD1(WT) and not SCFFWD1/ß-TrCP or other interacting proteins, the FLAG epitope immunoprecipitates were boiled in SDS-PAGE sample buffer, then diluted and immunoprecipitated again with anti-FLAG antibody. The reimmunoprecipitated FGD1(WT) but not FGD1(SA) contained HA-tagged ubiquitin conjugates (Fig. 3B), demonstrating that FGD1 is a substrate for SCFFWD1/ß-TrCP. Without transfecting HA-ubiquitin cDNA, similar results were obtained using anti-ubiquitin antibody to detect endogenous ubiquitin conjugates (Fig. 3C).
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Since FGD1 is reported as a Cdc42 GEF (Benard & Bokoch 2002), we examined the amount of active form of Cdc42 in cells expressing FGD1(WT) or FGD1(SA) by a pull-down assay using GST-PAK CRIB fusion protein. As shown in Fig. 5, FGD1(SA) as well as FGD1(WT) induced a significant increase in the amount of active Cdc42, suggesting that mutation in the SCFFWD1/ß-TrCP recognition motif does not interfere with the Cdc42 GEF function of FGD1. When cells were subjected to immunostaining analyses, FGD1(SA)-expressing cells induced prominent morphological changes as represented by the filopodia formation that is induced by Cdc42, as did FGD1(WT)-expressing cells (Fig. 6A). Over-expression of SCFFWD1/ß-TrCP reduced the immunofluorescence signal from the FLAG epitope and inhibited the filopodia formation in FGD1(WT)-expressing cells (Fig. 6B). In contrast, even in cells over-expressing SCFFWD1/ß-TrCP, FGD1(SA) induced significant filopodia formation (Fig. 6B), suggesting that SCFFWD1/ß-TrCP-mediated degradation negatively regulates the Cdc42 GEF activity of FGD1.
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Discussion |
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Bs and ß-catenin (Karin & Ben-Neriah 2000). As a common feature, these proteins contain the consensus sequence, DSGXS, which links upstream kinase signaling to the downstream ubiquitin/proteasome system. However, distinct kinases are responsible for serine phosphorylation of IBs and ß-catenin within that consensus sequence. The phosphorylation of IBs does not occur in resting cells. Once cells are stimulated with proinflammatory cytokines, such as TNF, rapid activation of the IB kinase (IKK) results in the phosphorylation of the two serine residues in the consensus sequence and leads to almost complete degradation of IBs within minutes, allowing NF-B to translocate into nuclei (Karin & Ben-Neriah 2000). The activation of IKK is transient and the NF-B signal is eventually terminated through cytoplasmic resequestration of NF-B, which depends on IB
synthesis, a process requiring NF-B transcriptional activity, as well as IB-dependent nuclear export.
The phosphorylation of ß-catenin is regulated in a different manner than that of IBs. Cells have two pools of ß-catenin: a membrane-associated pool of stable protein that participates in formation of adherence junctions and a soluble pool of highly unstable protein that functions as the signal transducer/transcription factor to promote proliferation, an activity that is tightly controlled by a destruction complex containing adenomatous polyposis coli (APC) tumor suppressor protein, Axin, and GSK3ß (Willert & Nusse 1998; Polakis 2000). In the absence of Wnt, GSK3ß is constitutively active and promotes degradation of ß-catenin through the phosphorylation of two serine residues in the N-terminal DSGXS motif.
The interaction of FGD1(WT) with FWD1/ß-TrCP was observed in resting cells and was inhibited by the treatment of cells with GSK3ß inhibitor (Fig. 2). This suggests that GSK3ß, and not the extracellular stimuli-regulated IKK, may be the kinase responsible for the phosphorylation of FGD1 leading to FWD1/ß-TrCP binding. Interestingly, GSK3ß has been reported to be inactivated at the leading edge of migrating cells as a result of phosphorylation of serine 9 by protein kinase C zeta (PKC
) (Etienne-Manneville & Hall 2003). Importantly, Cdc42, which is activated transiently at the leading edge of migrating cells, is responsible for this inhibitory phosphorylation of GSK3ß by PKC (Etienne-Manneville & Hall 2003). FGD1 is thought to be targeted to specific intracellular compartments such as polyphosphoinositides-enriched membrane subdomains through PH and/or FYVE domains (Estrada et al. 2001), although the precise mechanism has not been fully elucidated. Phosphatidylinositol (3, 4, 5)-trisphosphate, which is known to bind PH domains, accumulates at the leading edges of migrating cells (Meili et al. 1999; Haugh et al. 2000; Servant et al. 2000). Therefore, it is likely that the Cdc42 activation that occurs at the leading edge of migrating cells is due to the action of recruited FGD1. Cdc42 activation causes inactivation of GSK3ß, resulting in the accumulation of DSGIDS-unphosphorylated forms of FGD1. This stabilized FGD1 then leads to the sustained activation of Cdc42. When Cdc42 is inactivated by specific GAPs, the level of active GSK3ß increases, resulting in DSGIDS phosphorylation and the subsequent destruction of FGD1 through the ubiquitin-proteasome system, thereby turning off FGD1-mediated signal transduction (Fig. 8).
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Recently, the discs large (Dlg) tumor suppressor was shown to be degraded by the proteasome through ubiquitination coupled to phosphorylation of the DSGXS motif (Mantovani & Banks 2003). Dlg is involved in the control of cell contact, polarity, and proliferation by interacting with several components of the junctional complex and with APC (Matsumine et al. 1996; Mantovani & Banks 2003). In differentiated epithelial cells, localization of Dlg to the basolateral membrane junctions leads to its stabilization, which appears to be promoted by cell-cell contact. In contrast, when cells do not engage in stable junctions, Dlg is degraded rapidly (Mantovani et al. 2001). It is quite interesting that the key molecules that regulate cell adhesion, cell growth, and cell movement, such as ß-catenin, FGD1, and Dlg, may be under the control of the same destruction system composed of GSK3ß, SCFFWD1/ß-TrCP, and proteasome.
In another example of the involvement of the ubiquitin ligase in the regulation of the Rho family of GTPases, Wang et al. (2003) showed that the HECT domain-containing E3 ubiquitin ligase, Smurf1, ubiquitinates RhoA at the restricted cellular loci where protrusions are actively formed. These studies revealed that PKC, an effector of the Cdc42/PAR6 polarity complex, recruits Smurf1 to protrusions. Those results and our present results suggest that there is crosstalk between Cdc42 and RhoA to control cell shape, polarity, and motility and that this crosstalk is regulated by distinct types of ubiquitin ligase, i.e., SCF-type, SCFFWD1/ß-TrCP and HECT-type, Smurf1. Further insights into various signaling pathways will highlight the ubiquitin ligases as the molecular switches which turn off the signal in a temporally and spatially coordinated manner.
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Experimental procedures |
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Doxycycline (Dox) and anti-FLAG M2 antibody (Ab) were purchased from Sigma-Aldrich. Anti-HA (12CA5) and anti-Myc (9E10) Abs were from Roche Applied Science. Proteasome inhibitor MG132 and GSK3ß inhibitor [3-(3-carboxy-4-chloroanilino)-4-(3-nitrophenyl)maleimide] were purchased from Calbiochem. Anti-ubiquitin (P4D1) and anti-HA (Y-11) Abs were from Santa Cruz Biotechnology. Anti-ß-catenin antibody (clone 14) was from BD Pharmingen and Transduction Laboratories.
cDNA construction and plasmids
A cDNA encoding full-length mouse FGD1 was generated by RT-PCR based on the reported nucleotide sequence (GENBANK accession number U22325). Briefly, poly (A+) RNAs were isolated from mouse L929 cells using QuickPrep Micro mRNA Purification Kit (Amersham-Pharmacia) and then reverse-transcribed by the use of Ready-To-Go-You-Prime First-Strand Beads (Amersham-Pharmacia) according to the manufacturer's instructions. FGD1 cDNA was amplified with TaKaRa LA Taq (TAKARA SUZO Co., Ltd). A FLAG tag was created by PCR. In order to replace Ser283 and Ser287 with alanines, site-directed mutagenesis was carried out by PCR using the following primers which were designed in inverted tail-to-tail direction to amplify the vector together with the target sequence: 5'-ATTGACGCCATCAGCAGCTCGCCA-3' (sense); 5'-GCCGGCGTCCCGGTTAGGCAC-3' (anti-sense). FLAG-FGD1 and its S/A mutant cDNAs were subcloned into pTRE vector (Clontech) and resultant plasmids were termed pTRE-FLAG-FGD1(WT) and pTRE-FLAG-FDG1(SA), respectively. The plasmid pT7blue2-Cdc42 was generously provided by Dr Hirofumi Tanaka and was used as a template to generate N-terminal Myc-tagged Cdc42. The resultant amplified cDNA was subcloned into pcDNA3. Other plasmids used in this study were previously described (Hayakawa et al. 2003). All the amplified sequences were verified by dideoxy sequencing.
Transfection and establishment of stable cell lines
Transfection was carried out using FuGENE 6 (Roche Applied Science) according to the manufacturer's instructions. HeLa Tet-On cells (LOT 710056) purchased from Clontech were co-transfected either with pTRE-FLAG-FGD1(WT) and pTK-Hyg or with pTRE-FLAG-FGD1(SA) and pTK-Hyg. The hygromycin-resistant clonal cell lines were tested for Dox-regulated FLAG-tagged FGD1 expression.
Immunoblotting, immunoprecipitation, and immunostaining
Cells were washed with PBS and cell extracts were prepared using either SDS-PAGE sample buffer or IP buffer as described below. After normalization of protein content by the protein assay kit (Bio-Rad), samples were resolved by SDS-PAGE and subjected to immunoblotting analyses. Immunocomplexes on PVDF membranes were visualized using enhanced chemiluminescence detection (Amersham Bioscience). For immunoprecipitation analyses, cells were lyzed in IP buffer (25 mM HEPES/NaOH pH 7.4, 0.3 M NaCl, 10% glycerol, 0.5% NP-40, 0.4 mM EDTA, 2 mM DTT, 20 mMß-glycerophosphate, 10 mMp-nitrophenyl phosphate, 20 mM NaF, 0.5 mM Na-O-vanadate, 1 mM PMSF, 10 µg/mL leupeptin, 10 µg/mL aprotinin, 3 µg/mL pepstatin, and 15 µg/mL bestatin) and protein concentrations of cell extracts were normalized. In experiments to detect the interaction of FGD1 with FWD1/ß-TrCP, DTT was excluded from the IP buffer. Cell extracts were incubated with relevant Abs and protein G-Sepharose beads and immunoprecipitates were resolved by SDS-PAGE followed by immunoblotting. To confirm that FGD1 was ubiquitinated, re-immunoprecipitation was carried out as follows; after the initial immunoprecipitation with anti-FLAG Ab, the resultant immunoprecipitates were boiled in SDS-PAGE sample buffer containing 2% SDS and 5% 2-mercaptoethanol, and the supernatant was diluted with IP buffer to reduce the SDS concentration to less than 0.1%. These samples were then immunoprecipitated with anti-FLAG Ab and the resultant immunoprecipitates were resolved by SDS-PAGE followed by immunoblotting. Immunofluorescence staining for the FLAG epitope and HA epitope was carried out as follows. Cells cultured on glass coverslips were fixed with 4% paraformaldehyde, then permeabilized with methanol, and blocked with 10% skim milk in PBS. The cells were incubated with primary Abs (mouse anti-FLAG M2 Ab and rabbit anti-HA Ab) for 2 h, followed by incubation with secondary Abs (FITC-conjugated AffiniPure goat anti-mouse IgG and Texas Red dye-conjugated AffiniPure goat anti-rabbit IgG purchased from Jackson ImmunoReseach Laboratories) for 1 h. Nuclei were counterstained with Hoechst 33258. Fluorescence images were captured on a CCD camera (MicroMax system; Princeton Instruments) mounted on a Leica DM IRB microscope using IP Laboratory imaging software (Scanalytics).
Pulse-chase metabolic labeling experiments
HeLa Tet-On-derived sublines were transfected with plasmids encoding Myc-tagged Cul1, HA-tagged FWD1/ß-TrCP, Skp1, ROC1, and ubiquitin. After 24 h, cells were treated or left untreated with Dox for 20 h. The cells were then washed three times with methionine- and cysteine-free DMEM (Met/Cys(–) DMEM) and incubated for 45 min in Met/Cys(–) DMEM containing 5% dialyzed FCS, 0.1 mCi/mL of [35S] Express Protein Labeling Mix (PerkinElmer), and 10 µM MG132. The medium was then removed, and the cells were either immediately lyzed in the IP buffer described above (for non-induced and t = 0 time points) or ‘chased’ in complete DMEM containing 10% FCS, 2.5 mM methionine, 10 µM cysteine, and 10 µg/mL cycloheximide for the indicated times. Cells were then lyzed in IP buffer and protein concentrations of cell extracts were normalized. These lyzed samples were precleared with protein G-Sepharose and the resultant supernatants were immunoprecipitated with anti-FLAG M2 Ab and collected with protein G-Sepharose. The beads were washed four times with RIPA buffer (IP buffer supplemented with 0.1% SDS and 0.5% deoxycholate) and subjected to SDS-PAGE followed by fluorography. Quantification of the bands was done by densitometric analysis (NIH Image 1. 63).
GST-CRIB pull-down assay
The Cdc42/Rac interacting binding (CRIB) domain were prepared as a GST fusion protein. A fragment encoding amino acids 57–141 of PAK1B was generated by PCR using the following primers: 5'-AGCTGGATCCATTTTACCTGGAGAT-3' (sense); 5'-AGCTGAATTCATTTCTGGCTGTTGGATGTC-3' (anti-sense). The resultant amplified cDNA was digested with BamHI/EcoRI and inserted between the BamHI and EcoRI sites of pGEX4T-1 to yield GST-CRIB domain (Sander et al. 1998). HeLa Tet-On-derived sublines were transfected with pcDNA3-Myc-Cdc42. After 6 h, cells were treated or left untreated with Dox for 48 h. Then cells were lyzed in lysis buffer (50 mM HEPES/NaOH pH 7.4, 5 mM MgCl2, 100 mM NaCl, 5% glycerol, 1% NP-40, 1 mM Na-O-vanadate, 1 mM PMSF, 10 µg/mL leupeptin, 10 µg/mL aprotinin, 3 µg/mL pepstatin, and 15 µg/mL bestatin) and the resultant cell extracts (200 µL) were combined with 400 µL of binding buffer (25 mM HEPES/NaOH pH 7.4, 40 mM NaCl, 1 mM DTT, 30 mM MgCl2, and 0.5% NP-40) and then the protein concentrations were normalized. Each sample was then incubated with GST-CRIB (20 µg) bound to glutathione-Sepharose for 1 h at 4 °C, followed by three washes with washing buffer (25 mM HEPES/NaOH pH 7.4, 40 mM NaCl, 1 mM DTT, 30 mM MgCl2, and 1% NP-40). The resultant beads were subjected to SDS-PAGE followed by immunoblotting to detect the Myc epitope.
Cell migration assay
HeLa Tet-On-derived sublines were treated or left untreated with Dox for 48 h, and then harvested in 5% FCS/DMEM. Harvested cells were washed once with DMEM containing 0.25% BSA (BSA/DMEM), and resuspended in BSA/DMEM at a density of 5 x 105 cells/mL. Cell migration was studied in blind-well chemotactic chambers (Neuroprobe) using polycarbonate filters (8-mm pores; Costar) precoated with 100 µg/mL type IV collagen. Assays were performed with 200 µL of cell suspension in the top wells and 1% FCS/DMEM in the bottom wells. After incubation for 4 h, filters were fixed with 4% paraformaldehyde, then permeabilized with methanol, and blocked with 10% skim milk in PBS. The cells on the filters were incubated with mouse anti-FLAG M2 Ab for 2 h, followed by the incubation with FITC-conjugated AffiniPure goat anti-mouse IgG for 1 h. Then nuclei were counterstained with Hoechst 33258. The non-migrating cells on the surface of the top part of filters were wiped off, and migrating cells remaining on the bottom part of filters were mounted on glass slides. Fluorescence images of migrating cells stained with anti-FLAG M2 Ab/FITC-conjugated AffiniPure goat anti-mouse IgG or with Hoechst 33258 were captured on a CCD camera. Five individual frames for each specimen were taken to count the numbers of FLAG-positive cells and Hoechst 33258-stained nuclei. The same cell suspensions used for the migration assays were loaded on to poly L-lysine-coated coverslips to determine the proportion of FGD1-expressing cells. Cells attached on coverslips were subjected to immunostaining with anti-FLAG Ab as described above and nuclei were counterstained with Hoechst 33258. The images of FLAG-positive cells and those of Hoechst 33258-stained nuclei were captured and the percentage of FGD1-expressing cells was calculated by dividing the numbers of FLAG-positive cells by the number of Hoechst 33258-stained nuclei. Data derived from five individual frames were averaged for each specimen.
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Acknowledgements |
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Footnotes |
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* Correspondence: E-mail: hayakawa{at}ps.toyaku.ac.jp
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References |
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Received: 26 November 2004
Accepted: 3 December 2004
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, ß-catenin, Vpu, Dlg, and FGD1. Black shading indicates sequence identity. (B) Schematic representation of the wild-type (FGD1(WT)) and mutant FGD1 (FGD1(SA)) used in this study. FGD1(SA) bears Ser to Ala amino acid substitutions at positions 283 and 287 within the FWD1/ß-TrCP putative consensus binding site. Domains shown are ‘Pro-rich’ (proline-rich domain), ‘DH’ (Dbl homology domain), ‘PH’ (pleckstrin homology domain), ‘FYVE’ (domain present in Fab1, YOTB, Vac1, and EEA1). (C) Hygromycin-resistant clonal cell lines were isolated from HeLa Tet-On cells transfected either with pTRE-FLAG-FGD1(WT) or with pTRE-FLAG-FGD1(SA), and designated as HeLa Tet-On-FGD1(WT) and HeLa Tet-On-FGD1(SA). Cells were treated or left untreated with 2 µg/mL Dox for 48 h, then lyzed in SDS-PAGE sample buffer and resultant whole cell lysates were subjected to immunoblotting analyses using anti-FLAG M2 antibody.
