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Original Article |
Correspondence to: Lilly Y.W. Bourguignon, Department of Cell Biology and Anatomy, University of Miami Medical School, 1600 N.W. 10th Avenue, Miami, FL 33101. Tel:(305) 547-6691 Fax:(305) 545-7166 E-mail:Lbourgui{at}med.miami.edu.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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Tiam1 (T-lymphoma invasion and metastasis 1) is one of the known guanine nucleotide (GDP/GTP) exchange factors (GEFs) for Rho GTPases (e.g., Rac1) and is expressed in breast tumor cells (e.g., SP-1 cell line). Immunoprecipitation and immunoblot analyses indicate that Tiam1 and the cytoskeletal protein, ankyrin, are physically associated as a complex in vivo. In particular, the ankyrin repeat domain (ARD) of ankyrin is responsible for Tiam1 binding. Biochemical studies and deletion mutation analyses indicate that the 11–amino acid sequence between amino acids 717 and 727 of Tiam1 (717GEGTDAVKRS727L) is the ankyrin-binding domain. Most importantly, ankyrin binding to Tiam1 activates GDP/GTP exchange on Rho GTPases (e.g., Rac1).
Using an Escherichia coli–derived calmodulin-binding peptide (CBP)–tagged recombinant Tiam1 (amino acids 393–728) fragment that contains the ankyrin-binding domain, we have detected a specific binding interaction between the Tiam1 (amino acids 393–738) fragment and ankyrin in vitro. This Tiam1 fragment also acts as a potent competitive inhibitor for Tiam1 binding to ankyrin. Transfection of SP-1 cell with Tiam1 cDNAs stimulates all of the following: (1) Tiam1–ankyrin association in the membrane projection; (2) Rac1 activation; and (3) breast tumor cell invasion and migration. Cotransfection of SP1 cells with green fluorescent protein (GFP)–tagged Tiam1 fragment cDNA and Tiam1 cDNA effectively blocks Tiam1–ankyrin colocalization in the cell membrane, and inhibits GDP/GTP exchange on Rac1 by ankyrin-associated Tiam1 and tumor-specific phenotypes. These findings suggest that ankyrin–Tiam1 interaction plays a pivotal role in regulating Rac1 signaling and cytoskeleton function required for oncogenic signaling and metastatic breast tumor cell progression.
Key Words: Tiam1, ankyrin, Rac1 signaling, invasion/migration, metastatic breast tumor cells
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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Members of the Rho subclass of the ras superfamily (small molecular masses GTPases, e.g., Rac1, RhoA, and Cdc42) are known to be associated with changes in the membrane-linked cytoskeleton (
Several guanine nucleotide exchange factors (GEFs,1 the dbl or DH family) have been identified as oncogenes because of their ability to upregulate Rho GTPase activity during malignant transformation (
Overexpression of both NH2 and COOH terminally truncated as well as full-length Tiam1 proteins induces the invasive phenotype in otherwise noninvasive lymphoma cell lines (
Ankyrin belongs to a family of cytoskeletal proteins that mediate linkage of integral membrane proteins with the spectrin-based skeleton in regulating a variety of biological activities (
89–95 kD, also called the ankyrin repeat domain [ARD]; Davis and Bennet, 1990; 62 kD;
In this study, we have focused on the regulatory aspect of Tiam1-Rac1 signaling in metastatic breast tumor cells (SP-1 cell line). Our results indicate that Tiam1 interacts with ankyrin in vivo and in vitro. In particular, the ankyrin repeat domain (ARD) is directly involved in Tiam1 binding. Biochemical analyses show that the Tiam1 fragment (amino acids 393–738) contains an ankyrin-binding site and competes for Tiam1 binding to ankyrin. Most importantly, the binding of ankyrin, in particular, the ankyrin repeat domain (ARD), to Tiam1 activates Rho-like GTPases such as Rac1. Overexpression of Tiam1 in SP-1 cells by transfecting Tiam1 cDNA induces Tiam1–ankyrin association in the cell membrane, Rac1 signaling, and metastatic phenotypes. Both Tiam1–ankyrin interaction and tumor-specific behaviors are significantly inhibited by cotransfecting SP-1 cells with the Tiam1 (amino acids 393–738) fragment cDNA and Tiam1 cDNA. Our observations suggest that Tiam1 interaction with ankyrin promotes Rho GTPase activation and cytoskeletal changes required for metastatic breast tumor cell invasion and migration.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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Cell Culture
Mouse breast tumor cells (e.g., SP1 cell line; provided by Dr. Bruce Elliott, Department of Pathology and Biochemistry, Queen's University, Kingston, Ontario, Canada) were used in this study. Specifically, the SP1 cell line was derived from a spontaneous intraductal mammary adenocarcinoma that arose in a retired female CBA/J breeder in the Queen's University animal colony. These cells were capable of inducing lung metastases by sequential passage of SP1 cells into mammary gland (
Antibodies and Reagents
For the preparation of polyclonal rabbit anti-Tiam1 antibody, specific synthetic peptides (15–17 amino acids unique for the COOH-terminal sequence of Tiam1) were prepared by the Peptide Laboratories of the Department of Biochemistry and Molecular Biology using an automatic synthesizer (model ACT350; Advanced Chemtech). These Tiam1-related polypeptides were conjugated to polylysine and subsequently injected into rabbits to raise the antibodies. The anti-Tiam1–specific antibody was collected from each bleed and stored at 4°C containing 0.1% azide. The anti-Tiam1 IgG fraction was prepared by conventional DEAE-cellulose chromatography. Mouse monoclonal anti-hemagglutinin (HA epitope) antibody (clone 12 CA5) and mouse monoclonal anti–green fluorescent protein (GFP) antibody were purchased from Boehringer Mannheim and PharMingen, respectively. Escherichia coli (E. coli)–derived GST-tagged Rac1/Cdc42 and GST-tagged RhoA was provided by Dr. Richard A. Cerione (Cornell University, Ithaca, NY) and Dr. Martin Schwartz (Scripps Research Institute, La Jolla, CA), respectively. Mouse monoclonal erythrocyte ankyrin (ANK1) and ANK3 antibodies were prepared as described previously (
Cloning, Expression, and Purification of GST-tagged Ankyrin Repeat Domain (GST-ARD) and GFP-tagged Spectrin Binding Domain (GFP-SBD) of Ankyrin
pGEX-2TK recombinant plasmid expressing GST-ARD (NH2-terminal portion of ankyrin, residues 1–834) was constructed as follows. Two pGEX-2TK recombinant plasmids pA3-79 (expressing epithelial Ank3 NH2-terminal 1–455 amino acids) and pA3-88 (expressing epithelial Ank3 NH2-terminal 317–834 amino acids; 
F'-competent cells. The obtained clones were sequenced to verify the correct generation of the full-length ARD.
Spectrin binding domain (SBD) cDNA of human erythrocyte ankyrin was cloned into the eukaryotic expression vector, GFPN1 (CLONTECH Laboratories, Inc.) using the PCR-based cloning strategy. Ankyrin's SBD cDNA was amplified by PCR with two specific primers (left, 5'-CGCTCGAGATGAAGGCTGAGAGGCGGGATTCC-3' and right, 5'-ATAAGCTTCAGGGGCGTCGGGGTCCTTCT-3') linked with specific enzyme digestion site (XhoI and HindIII). The PCR product, which was digested with XhoI and HindIII, was purified with QIAquick PCR purification kit (QIAGEN). Ankyrin's SBD cDNA fragment was cloned into GFPN1 vector digested with XhoI and HindIII. The cDNA sequence was confirmed by nucleotide sequencing analysis. The GFP-tagged spectrin binding domain (GFP-SBD) of ankyrin is expressed as an 89-kD polypeptide in SP1 or COS-7 cells by SDS-PAGE and immunoblot analyses. The 89-kD GFP-SBD (but not ARD) displays specific spectrin binding property as described previously (
Expression Constructs
Both the full-length mouse Tiam1 cDNA (FL1591) and the NH2 terminally truncated Tiam1 cDNA (C1199) were provided by Dr. John G. Collard (The Netherlands Cancer Institute, The Netherlands). Specifically, the full-length Tiam1 (FL1591) cDNA was cloned into the eukaryotic expression vector, pMT2SM. The truncated C1199 Tiam1 cDNA (carrying a hemagglutinin epitope [HA] tag at the 3' end) was cloned in the eukaryotic expression vector, pUTSV1 (Eurogentec, Belgium).
The deletion construct, HA-tagged C1199 Tiam1
717-727 (deleting the sequence between amino acids 717 and 727 of Tiam1) was derived from C1199 Tiam1 using QuickChangeTM site-directed mutagenesis kit (Stratagene). In brief, two complimentary mutagenic oligonucleotide primers containing the desired deletion (5'-CCCAACCATCAACCAGGTGTTTGAGGGAATATTTGATG-3') was designed and synthesized. First, the cycling reaction, using 30-ng double-stranded DNA template of C1199 Tiam1 plasmid and two complimentary primers, was performed to produce mutated cDNA according to the manufacturer's instruction. Subsequently, 1 µl of the DpnI restriction enzyme (10 U/µl) was added directly to the cycling reaction products to digest the parental supercoiled double-stranded DNA. This DpnI-treated cDNA was used to transform supercompetent cells (e.g., Epicurian coli XL 1-blue). Finally, the deletion construct was confirmed by DNA sequencing.
The Tiam1 (amino acids 393–728) fragment was cloned into calmodulin-binding peptide (CBP)–tagged vector (pCAL-n; Stratagene) using the PCR-based cloning strategy. Using human Tiam1 cDNA as a template, the Tiam1 fragment was amplified by PCR with two specific primers (left, 5'-AACTCGAGATGAGTACCACCAACAGTGAG-3' and right, 5'-AAAAAGCTTTCAGCCATCTGGAACAGTGTCATC-3') linked with a specific enzyme digestion site (XhoI or HindIII). The PCR product, which was digested with XhoI and HindIII, was purified with QIAquick PCR purification kit (QIAGEN). The Tiam1 fragment cDNA was cloned into pCAL-n vector digested with XhoI and HindIII. The inserted Tiam1 fragment sequence was confirmed by nucleotide sequencing analyses. The recombinant plasmids were transformed to BL21-DE3 to produce CBP-tagged Tiam1 fragment fusion protein. This fusion protein was purified from bacteria lysate by calmodulin affinity resin column (Sigma Chemical Co.).
The Tiam1 fragment cDNA was also cloned into pEGFPN1 vector (CLONTECH Laboratories, Inc.) digested with XhoI and HindIII to create GFP-tagged Tiam1 fragment cDNA. The inserted Tiam1 fragment sequence was confirmed by nucleotide sequencing analyses. This GFP-tagged Tiam1 fragment cDNA was used for transient expression in SP1 cells as described below. The GFP-tagged Tiam1 fragment is expressed as a 68-kD polypeptide in SP1 or COS-7 cells by SDS-PAGE and immunoblot analyses.
Cell Transfection
To establish a transient expression system, cells (e.g., SP-1 or COS-7 cells) were transfected with various plasmid DNAs including Tiam1 cDNAs (e.g., the full-length mouse Tiam 1 cDNA [FL1591], or HA-tagged C1199 Tiam1 cDNA, or HA-tagged C1199 Tiam1717-727 cDNA, or GFP-tagged Tiam1 fragment cDNA, or HA-tagged C1199 Tiam1 cDNA plus GFP-tagged Tiam1 fragment cDNA (cotransfection), or vector control constructs) using electroporation methods. In brief, cells (e.g., SP-1 or COS-7 cells) were plated at a density of 106 cells per 100-mm dish, and were transfected with 25 µg/dish plasmid DNA using electroporation at 230 V and 960 µFD with a gene pulser (Bio-Rad). Transfected cells were grown in 5 or 20% FCS-containing culture medium for at least 24–48 h. Various transfectants were analyzed for the expression of Tiam1 or HA-tagged (or GFP-tagged) Tiam1 mutant proteins by immunoblot, immunoprecipitation, and functional assays as described below.
Immunoprecipitation and Immunoblotting Techniques
SP-1 cells or COS cells (e.g., untransfected or transfected by various Tiam1 cDNAs including the full-length mouse Tiam1 cDNA [FL1591] or HA-tagged C1199 Tiam1 cDNA) were first extracted with a solution containing 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, and 1% NP-40 buffer, followed by solubilizing in SDS sample buffer, and analyzed by SDS-PAGE (with 7.5% gel). Separated polypeptides were transferred onto nitrocellulose filters. After blocking nonspecific sites with 3% BSA, the nitrocellulose filters were incubated with 5 µg/ml either of rabbit anti-Tiam1 or mouse anti-HA (or preimmune serum) plus peroxidase-conjugated goat anti–rabbit IgG or goat anti–mouse IgG (1:10,000 dilution), respectively. In controls, peroxidase-conjugated normal mouse IgG or preimmune rabbit IgG was also incubated with anti-Tiam1–mediated immunocomplex. The blots were developed using ECL chemiluminescence reagent (Amersham Life Science) according to the manufacturer's instructions.
In some cases, SP-1 cells (transfected with HA-tagged C1199 Tiam1 cDNA, or HA-tagged C1199 Tiam1717-727 cDNA, or GFP-tagged Tiam1 fragment cDNA, or cotransfected with HA-tagged C1199 Tiam1 cDNA and GFP-tagged Tiam1 fragment cDNA) were immunoblotted with anti-HA antibody (5 µg/ml) or anti-GFP antibody (5 µg/ml), respectively, followed by incubation with HRP-conjugated goat anti–mouse IgG (1:10,000 dilution) at room temperature for 1 h.
SP-1 cells were also immunoprecipitated with rabbit anti-Tiam1 (5 µg/ml) or mouse antiankyrin antibodies (e.g., 5 µg/ml of either mouse anti-ANK3 antibody or mouse anti-ANK1 antibody), followed by immunoblotting/reblotting with ankyrin antibodies (e.g., 1 µg/ml mouse anti-ANK3 antibody, or 5 µg/ml mouse anti-ANK1 antibody, or 1 µg/ml rabbit anti-Tiam1), respectively, followed by incubation with HRP-conjugated goat anti–mouse IgG or goat anti–rabbit IgG (1:10,000 dilution) at room temperature for 1 h. In reblotting controls, both peroxidase-conjugated normal mouse IgG or rabbit preimmune IgG was also used. The blots were developed using ECL chemiluminescence reagent (Amersham Life Science) according to the manufacturer's instructions.
Effects of Synthetic Peptides on Ankyrin-Tiam1 Interaction
Nitrocellulose discs (1-cm diam) were coated with 1 µg of a panel of synthetic peptides including the ankyrin-binding region peptide (717GEGTDAVKRS727L), a scrambled peptide (GRATLEGSDKV) and another Tiam1-related peptide (399GTIKRAPFLG409P; synthesized by Dr. Eric Smith, University of Miami). After coating, the unoccupied sites on the discs were blocked by incubation with a solution containing 20 mM Tris-HCl, pH 7.4, and 0.3% BSA at 4°C for 2 h. The discs were incubated with various concentration of 125I-labeled cytoskeletal proteins (erythrocyte ankyrin/ARD/ankyrin's SBD/spectrin; 3000 cpm/ng) at 4°C for 2 h in 1 ml binding buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.2% BSA).
In some experiments, 125I-labeled Tiam1 (3,000 cpm/ng) was incubated with ankyrin-coated beads in the presence of various concentrations (10-10–10-6 M) of unlabeled synthetic peptide (e.g., 717GEGTDAVKRS727L or the scrambled sequence, GRATLEGSDKV, or another Tiam1-related peptide, 399GTIKRAPFLG409P) at 4°C for 2 h in 1 ml binding buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.2% BSA). 125I-labeled Tiam1 fragment (3,000 cpm/ng) was also incubated with beads containing 1.0 µg of each of the following four proteins: intact ankyrin, ARD, or spectrin binding domain of ankyrin (GFP-SBD), or GFP alone. After binding, the peptide-coated discs (or cytoskeletal protein–conjugated beads) were washed three times in the binding buffer, and the radioactivity associated with the peptide-coated discs (or cytoskeletal protein–conjugated beads) was estimated. As a control, the ligands were also incubated with uncoated nitrocellulose discs (or beads) to determine the binding observed because of the stickiness of various ligands. Nonspecific binding was observed in these controls. In the peptide competition assay, the specific binding observed in the absence of any of the competing peptides is designated as 100%. The results represent an average of duplicate determinations for each concentration of the competing peptide used.
Binding of Ankyrin or ARD to Tiam1 In Vitro
Aliquots (0.5–1.0 µg of protein) of purified Tiam1 (e.g., intact Tiam1, or C1199 Tiam1, or Tiam1 fragment)–conjugated beads were incubated in 0.5 ml of binding buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.1% BSA, and 0.05% Triton X-100) containing various concentrations (10–800 ng/ml) of 125I-labeled intact ankyrin (purified from human erythrocytes; 5,000 cpm/ng protein) or 125I-labeled recombinant ARD fragment at 4°C for 4 h. Specifically, equilibrium binding conditions were determined by performing a time course (1–10 h) of 125I-labeled ankyrin (or ARD) binding to Tiam1 at 4°C. The binding equilibrium was found to be established when the in vitro ankyrin (or ARD)-Tiam1 binding assay was conducted at 4°C after 4 h. After binding, beads were washed extensively in binding buffer, and the bead-bound radioactivity was counted.
As a control, 125I-labeled ankyrin or 125I-labeled ARD was also incubated with uncoated beads to determine the binding observed because of the nonspecific binding of various ligands. Nonspecific binding, which represented 20% of the total binding, was always subtracted from the total binding. Our binding data are highly reproducible. The values expressed in the Results represent an average of triplicate determinations of three to five experiments with an SD less than ± 5%. In some cases, 125I-ankyrin (1–10 ng) was incubated with a polyacrylamide gel containing purified Tiam1 (obtained from anti-Tiam1 affinity column chromatography) in the absence or the presence of 100-fold excess amount of unlabeled ankyrin/spectrin (in the same binding buffer as described above) for 1 h at room temperature. After incubation, the gel was washed five times with the same binding solution and analyzed by autoradiographic analyses.
An in vitro binding assay designed to measure the stoichiometry of GST-ARD fusion protein and C1199 Tiam1 was also carried out. Specifically, in each reaction, 15–60 µl of glutathione-Sepharose bead slurry containing GST-ARD or GST alone was suspended in 0.5 ml of binding buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.1% BSA, and 0.05% Triton X-100). Purified C1199 Tiam1 (0.5–1.0 µg) was added to the bead suspension in the absence or the presence of an excess amount of CBP-tagged Tiam1 fragment (100 µg) at 4°C for 4 h. After binding, the GST fusion protein was eluted with its associated C1199 Tiam1 using 150 µl of 50 mM Tris-HCl, pH 8.0, buffer containing 30 mM glutathione. The amount of eluted GST fusion protein and C1199 Tiam1 was determined by SDS-PAGE and Coomassie blue staining followed by densitometric scanning using a software NIH Image V1.54. The amount of ARD (mol) per C1199 Tiam1 (mol) was calculated. Values represent relative binding abilities averaged from three experiments ± SEM.
Binding of 125I-Labeled Ankyrin to C1199 Tiam1 and the Mutant Protein
SP1 cells were transfected with HA-tagged C1199 Tiam1 cDNA, or HA-tagged C1199 Tiam1717-727 cDNA, or vector alone. These transfectants were extracted with a solution containing 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, and 1% NP-40, and immunoprecipitated with anti-HA immunoaffinity beads. Subsequently, aliquots (50 ng proteins) of these beads were incubated with 0.5 ml of a binding buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.1% BSA, and 0.05% Triton X-100) in presence of various concentrations (10–400 ng/ml) of 125I-labeled ankyrin (5,000 cpm/ng protein) at 4°C for 5 h. After binding, beads were washed extensively in binding buffer and the bead-bound radioactivity was counted.
As a control, 125I-labeled ankyrin was also incubated with uncoated beads to determine the binding observed because of the nonspecific binding of the ligand. Nonspecific binding, which represented 15–20% of the total binding, was always subtracted from the total binding. The values expressed in the Results represent an average of triplicate determinations of three to five experiments with an SD less than ± 5%.
Tiam1-mediated GDP/GTP Exchange for Rho GTPases
Purified E. coli–derived GST-tagged GTPases (e.g., Rac1, Cdc42, or RhoA; 20 pmol) were preloaded with GDP (30 µM) in 10 µl buffer containing 25 mM Tris-HCl, pH 8.0, 1 mM DTT, 4.7 mM EDTA, 0.16 mM MgCl2, and 200 µg/ml BSA at 37°C for 7 min. To terminate preloading procedures, additional MgCl2 was added to the solution (reaching a final concentration of 9.16 mM) as described previously (
Subsequently, 2 pmol of Tiam1, isolated from untransfected or transfected cells according to the procedures described above, was preincubated with no ankyrin or ankyrin (e.g., 1 µg/ml of either intact ankyrin or ARD), followed by adding to the reaction buffer containing 20 mM Tris-HCl, pH 8.0, 100 mM NaCl, 10 mM MgCl2, 100 µM AMP-PNP, 0.5 mg/ml BSA, and 2.5 µM GTP-
–35S (1,250 Ci/mmol). Subsequently, 2.5 pmol GDP-loaded GST-tagged Rho GTPases (e.g., Rac1, RhoA, or Cdc42) or GDP-treated GST were mixed with the reaction buffer containing Tiam1 and GTP-–35S to initiate the exchange reaction at room temperature. At various time points, the reaction of each sample was terminated by adding ice-cold termination buffer containing 20 mM Tris-HCl, pH 8.0, 100 mM NaCl, and 10 mM MgCl2 as described previously (–35S bound to Tiam1 or control sample (preimmune serum–conjugated Sepharose beads) in the absence of Rho GTPases (e.g., Rac1, Cdc42, or RhoA) was subtracted from the original values. Data represent an average of triplicates from three to five experiments. SD < 5%.
Double Immunofluorescence Staining
SP1 cells (untransfected or transfected with various plasmid DNAs such as HA-tagged C1199 Tiam1 cDNA, GFP-tagged Tiam1 fragment cDNA, or HA-tagged C1199 Tiam1 cDNA plus GFP-tagged Tiam1 fragment cDNA [as a cotransfection], or vector alone) were first washed with PBS (0.1 M phosphate buffer, pH 7.5, and 150 mM NaCl) buffer and fixed by 2% paraformaldehyde. Subsequently, cells were rendered permeable by ethanol treatment followed by staining with different immunoreagents. Specifically, untransfected cells were incubated with rhodamine (Rh)-conjugated mouse anti-ANK3 (50 µg/ml) and fluorescein (FITC)-conjugated rabbit anti-Tiam1 (50 µg/ml), respectively. HA-tagged C1199 cDNA–transfected cells were stained with Rh-conjugated mouse anti-ANK3 antibody (50 µg/ml) and FITC-conjugated mouse anti-HA IgG (50 µg/ml), respectively. GFP-tagged Tiam1 fragment cDNA–transfected cells were labeled with Rh-conjugated anti-ANK3 (50 µg/ml). Some SP1 transfectants (cotransfected with Tiam1 fragment cDNA and HA-tagged C1199 Tiam1 cDNA) were stained with Rh-conjugated anti-HA (50 µg/ml) or Rh-conjugated anti-ANK3 (50 µg/ml), respectively. To detect nonspecific antibody binding, vector-transfected cells were labeled with Rh-conjugated anti-ANK3 (50 µg/ml) followed by incubating with FITC-conjugated anti-HA (50 µg/ml). No anti-HA labeling was observed in such control samples. In some experiments, GFP-tagged Tiam1 fragment cDNA–transfected cells were also incubated with Rh-labeled rabbit preimmune IgG (50 µg/ml). No nonspecific rhodamine staining was detected in these samples. The FITC- and Rh-labeled samples were examined with a confocal laser scanning microscope (MultiProbe 2001 inverted CLSM system; Molecular Dynamics).
Tumor Cell Migration and Invasion Assays
24 transwell units were used for monitoring in vitro cell migration and invasion as described previously (10-4 cells/well in PBS, pH 7.2, untreated or treated with cytochalasin D [20 µg/ml] or DMSO alone) were placed in the upper chamber of the transwell unit. The growth medium containing high glucose DME supplemented by 10% FBS was placed in the lower chamber of the transwell unit. After an 18-h incubation at 37°C in a humidified 95% air/5% CO2 atmosphere, cells on the upper side of the filter were removed by wiping with a cotton swap. Cell migration and invasion processes were determined by measuring the cells that migrate to the lower side of the polycarbonate filters by standard cell number counting methods as described previously (
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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Identification of the GEF, Tiam1 in Breast Tumor Cells (SP-1 Cells)
Rho GTPases such as Rac1 become activated when bound GDP is exchanged for GTP by a process catalyzed by GEFs such as Tiam1 (200 kD; Fig 1, lane 1). This 200-kD Tiam1-like molecule, expressed in SP-1 cells, is very similar to the Tiam1 detected in COS-7 cells that were transiently transfected with the full-length Tiam1 cDNA (Fig 1, lane 2) or NH2 terminally truncated C1199 Tiam1 cDNA (Fig 1, lane 3 revealing primarily C1199 Tiam1 [160 kD] and a low level of endogenous Tiam1 [200 kD]). We believe that the Tiam1 detected in SP-1 cells or COS-7 transfectants, revealed by anti-Tiam1–mediated immunoblot, is specific since no protein is detected in these cells using preimmune rabbit IgG (Fig 1, lanes 4–6).
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To confirm that the Tiam1-like molecule functions as a GDP/GTP exchange factor (or a GDP-dissociation stimulator protein) for Rac1, we have isolated Tiam1 from SP-1 cells using anti-Tiam1–conjugated Sepharose beads. Our results indicate that SP1's Tiam1 activates GDP/GTP exchange on GST-Rac1 (Fig 2 A, a) and, to a lesser extent, on GST-Cdc42 (Fig 2 A, b) and GST-RhoA (Fig 2 A, c). The initial onset of the exchange reaction on GST-Rac1 occurs within 0.5–1 min after the addition of Tiam1, and the reaction reaches its maximal level 16 min after Tiam1 addition (Fig 2 A, a). In contrast, the initial rate of Tiam1-catalyzed GDP/GTP exchange on Cdc42 (Fig 2 A, b) and RhoA (Fig 2 A, c) appears to be significantly lower than that detected on Rac1 (Fig 2 A, a). In the control samples, the amount of [35S]GTP-–S associated with GST alone is found to be significantly decreased (Fig 2 A, d). Further analysis indicates that the ability of Tiam1 isolated from SP-1 cells to promote GDP/GTP exchange on Rac1 (Fig 2 B, a) is identical to that carried out by the Tiam1 isolated from COS-7 transfected with the full-length Tiam1 cDNA (Fig 2 B, b) or NH2 terminally truncated C1199 Tiam1 cDNA (Fig 2 B, c). Therefore, we believe that the Tiam1 in SP-1 cells clearly functions as a GDP/GTP exchange factor for Rho-like GTPases such as Rac1 GTPase.
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We have also noticed that Tiam1 isolated from nontransfected COS-7 cells grown in the presence of 20% FCS is capable of catalyzing GDP/GTP exchange on Rac1 at a much higher level (Fig 2 B, d, blank bar) than Tiam1 isolated from nontransfected COS-7 cells grown in the presence of 5% FCS (Fig 2 B, d, shaded bar). This observation is consistent with the previous findings that some serum components play an important role in upregulating the ability of Tiam1 to promote GDP/GTP exchange on Rac1 (
Interaction between Tiam1 and the Cytoskeletal Proteins, Ankyrin
Certain cytoskeleton proteins, such as ankyrin, are known to be involved in regulating a variety of cellular activities (
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Further analyses using an in vitro binding assay show that 125I-labeled ankyrin (i.e., erythrocyte ankyrin [ANK1]) binds Tiam1, which was isolated from SP1 cells, specifically (Fig 4 A, a). In addition, we have used 125I-labeled ankyrin to bind purified Tiam1 (isolated from SP-1 cells) on a gel (Fig 4 B, a). Our data indicate that Tiam1 binds to ankyrin (ANK1; Fig 4 B, a) directly. In the presence of an excess amount of unlabeled ankyrin, the binding between ankyrin and Tiam1 is greatly reduced (Fig 4A and Fig B, Fig b). Other cytoskeletal proteins, such as spectrin, do not interfere with ankyrin binding to Tiam1 (Fig 4A and Fig B, Fig c). However, the precise functional domain of ankyrin involved in Tiam1 binding remains to be determined.
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The NH2-terminal region of ankyrin's membrane binding domain (Fig 5 A, a) is comprised of a tandem array of 24 ankyrin repeats (so-called ankyrin repeat domain, ARD; Fig 5 A, b). The question of whether the membrane-binding domain of ankyrin (in particular, ARD) is involved in Tiam1 binding is now addressed in this study. First, the pGEX-2TK recombinant plasmid encoding ARD (NH2-terminal portion of ankyrin, from amino acids 1 to 834) was constructed with a GST tag and expressed in E. coli (
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Next, we have used the ARD fragment of ANK3 (GST-ARD) and purified Tiam1 to identify the exact Tiam1 binding site(s) on the ankyrin molecule. Specifically, we have tested the binding of Tiam1 to 125I-labeled intact erythrocyte ankyrin (ANK1), or 125I-labeled GST-ARD fragment of ANK3, under equilibrium binding conditions. Scatchard plot analyses indicate that intact erythrocyte ankyrin (ANK1) binds to Tiam1 at a single site (Fig 5 C) with high affinity (an apparent dissociation constant [Kd] of 0.72 nM). This ankyrin–Tiam1 binding interaction is comparable in affinity to Tiam1 binding (Kd 1.42 nM) to ANK3's ARD fragment (Fig 5 D). These findings strongly support the notion that ankyrin (in particular, the ARD) is involved in the Tiam1 binding site.
Determination of Tiam1's Ankyrin-binding Domain
Previous studies indicate that Tiam1's NH2-terminal pleckstrin homology (PHn) domain and an adjacent protein interaction domain (i.e., a sequence between amino acids 393 and 738 of Tiam1; Fig 6 A, a–c) is required for the activation of Rac1 signaling pathways leading to membrane ruffling and c-Jun NH2-terminal kinase activation (
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Furthermore, we have evaluated the binding interaction between GST-ARD fusion protein and the recombinant C1199 Tiam1 (NH2 terminally truncated Tiam1; Fig 6 D). First, glutathione-Sepharose beads containing GST-ARD were incubated with C1199 Tiam1 in the absence (Fig 6D and Fig E, lane 1) or the presence of an excess amount of Tiam1 fragment (Fig 6D and Fig E, lane 2). In controls, C1199 Tiam1 was also added to Sepharose beads containing GST alone (Fig 6D and Fig E, lane 3). After binding, the GST fusion protein was eluted with its associated C1199 Tiam1 using a buffer containing glutathione. The amount of eluted GST fusion protein and C1199 Tiam1 was determined by SDS-PAGE and Coomassie blue staining (Fig 6 E) followed by densitometric scanning analyses (Fig 6 D). Our results indicate that the stoichiometry of ARD–C1199 Tiam1 interaction is 1:1 (Fig 6 D, lane 1, and Fig 6 E, lane 1, a and b). In the presence of an excess amount (100-fold) of recombinant Tiam1 fragment, the binding between ankyrin ARD and C1199 Tiam1 is significantly reduced (Fig 6D and Fig E, lane 2, a and b). The control beads containing GST alone fail to bind C1199 Tiam1 (Fig 6 D, lane 3, and Fig 6 E, lane 3, a and b). These observations suggest that ankyrin ARD directly interacts with Tiam1, and that the ankyrin-binding domain (ARD)–containing Tiam1 fragment act as a potent competitive inhibitor of Tiam1 binding to ankyrin in vitro.
Protein sequence analyses show that Tiam1 contains the sequence 717GEGTDAVKRS727L (in mouse), or 717GEGTEAVKRS727L (in human) that shares a great deal of sequence homology with the ankyrin-binding domain of the cell adhesion receptor, CD44 family (
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We have also used the synthetic peptide corresponding to Tiam1's amino acid 717–727 sequence to compete for the binding of purified Tiam1 to ankyrin. As shown in Fig 7 A (c), the synthetic peptide competes effectively with Tiam1 to bind ankyrin with an apparent inhibition constant (Ki) 0.5 nM. However, control peptides such as GRATLEGSDKV (Fig 7 A, a) or GTIKRAPFLGP (Fig 7 A, b) do not compete at all with Tiam1 in ankyrin binding. These results suggest that the amino acid 717–727 sequence of Tiam1 is a critical part of the ankyrin-binding domain of Tiam1. Finally, we have constructed an HA-tagged C1199 Tiam1 deletion mutant lacking the ankyrin binding sequence, amino acids 717–727 (designated as C1199 Tiam1717-727; Fig 7 B, b). The truncated C1199 Tiam1 717-727 cDNA (Fig 7 B, b) and the wild-type C1199 Tiam1 (Fig 7 B, a) were transiently transfected into SP-1 cells. Our results indicate that both the C1199 Tiam1717-727 mutant (Fig 7 C, lane 3) and the wild-type C1199 Tiam1 (Fig 7 C, lane 2) are expressed as a 160-kD polypeptide in SP-1 transfectants using anti-HA–mediated immunoblotting. No protein band was detected in vector-transfected SP-1 cells (Fig 7 C, lane 1). In vitro binding data reveal that there is a strong binding interaction between ankyrin and HA-tagged C1199 (Fig 7 D, b). In contrast, the HA-tagged C1199 Tiam1717-727 mutant protein isolated from SP-1 transfectants displays a drastic reduction (90–95% inhibition) in ankyrin-binding ability (Fig 7 D, c) compared with the HA-tagged wild-type C1199 Tiam1 (Fig 7 D, b). No ankyrin binding is observed in materials associated with anti-HA beads using cell lysate isolated from vector-transfected cells (Fig 7 D, a). These findings suggest that the amino acid 717–727 region is critical for the interaction of Tiam1 with ankyrin.
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Most importantly, we have found that the binding of ankyrin (e.g., erythrocyte ankyrin [ANK1], Fig 8 A, or ANK3's ARD, Fig 8 B) to Tiam1 significantly increases the GDP/GTP exchange activity of Rac1 GTPase as compared with untreated Tiam1-mediated Rac1 activation (Fig 8 C). The SBD of ankyrin or other cytoskeletal proteins, such as spectrin, fails to stimulate Tiam1-mediated GDP/GTP exchange on Rac1 GTPase (data not shown). Therefore, we believe that ankyrin binding to Tiam 1 plays a pivotal role in the upregulation of Tiam 1-mediated GDP/GTP exchange activity of Rho-like GTPases (e.g., Rac1).
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Effect of Tiam1 or the Tiam1 Fragment on Rac1 Activation, Tumor Cell Invasion, and Migration
Previous studies have indicated that both ankyrin and Tiam1 are closely associated with certain tumor-specific behaviors, characterized by an invadopodia structure (or membranous projections) during epithelial tumor cell migration (
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Treatment of SP1 cells (e.g., untransfected or transfected cells) with certain agents (e.g., cytochalasin D, a microfilament inhibitor) causes a remarkable inhibition of tumor cell invasion (Table 2 A) and migration (Table 2 B). Tiam1-Rac1 signaling initiates oncogenic cascades including c-Jun kinase (JNK) activation, which triggers gene transcription through c-jun and promotes cell transformation (
