Skip to main page content
U.S. flag
See More

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2000 Jul 10;150(1):177-91.
doi: 10.1083/jcb.150.1.177.

Ankyrin-Tiam1 interaction promotes Rac1 signaling and metastatic breast tumor cell invasion and migration

L Y Bourguignon  1 , H Zhu, L Shao, Y W Chen
Affiliations

Ankyrin-Tiam1 interaction promotes Rac1 signaling and metastatic breast tumor cell invasion and migration

L Y Bourguignon et al. J Cell Biol. .

Abstract

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 ((717)GEGTDAVKRS(727)L) 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.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Detection of Tiam1 expression in SP-1 cells or COS-7 transfectants. SP-1 and COS-7 cells, which were transfected with the full-length Tiam1 cDNA (FL1591) or NH2 terminally truncated C1199 Tiam1 cDNA or vector alone, were solubilized in SDS sample buffer and analyzed by SDS-PAGE and immunoblot as described in Materials and Methods. (lane 1) Anti-Tiam1–mediated immunoblot of SP-1 cells. (lane 2) Anti-Tiam1–mediated immunoblot of COS-7 cells transfected with the full-length Tiam1 cDNA (FL1591). (lane 3) Anti-Tiam1–mediated immunoblot of COS-7 cells transfected with the NH2 terminally truncated C1199 Tiam1 cDNA. (lane 4) Immunoblot of SP-1 cells with preimmune rabbit serum. (lane 5) Immunoblot of COS-7 cells, which were transfected with Tiam1 cDNA [FL1591], with preimmune rabbit serum. (lane 6) Immunoblot of COS-7 cells, which were transfected with the NH2 terminally truncated C1199 Tiam1 cDNA, with preimmune rabbit serum.
Figure 2
Figure 2
Tiam1-mediated GDP/GTP exchange for Rho GTPases. Purified E. coli-derived GST-tagged GTPases (e.g., Rac1, Cdc42, or RhoA) were preloaded with GDP. First, 2 pmol Tiam1 that was isolated from SP1 cells or COS-7 transfectants was added 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-γ–35S (∼1,250 Ci/mmol). Subsequently, 2.5 pmol GDP-loaded GST-tagged Rho GTPases (e.g., Rac1, RhoA, Cdc42, or GST alone) were mixed with the reaction buffer containing Tiam1 and GTP-γ–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 as described in Materials and Methods. The termination reactions were filtered immediately through nitrocellulose filters, and the radioactivity associated with the filters were measured by scintillation fluid. The amount of GTP-γ–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%. (A) Kinetics of GTP-γ–35S bound to GDP-loaded GST-Rac1 (a), GST-Cdc42 (b), or GST-RhoA (c), or GST alone (d) in the presence of Tiam1 (isolated from SP-1 cells). (B) The maximal level of GTP-γ–35S bound to GST-Rac1 in the presence of Tiam1 isolated from SP1 grown in 5% FCS (a, shaded bar) or 20% FCS (a, blank bar); or the full-length Tiam1 (1,591) isolated from COS-7 transfectants grown in 5% FCS (b, shaded bar) or 20% FCS (b, blank bar); or the C1199 Tiam1 isolated from COS-7 transfectants grown in 5% FCS (c, shaded bar), or 20% FCS (c, blank bar); or Tiam1 isolated from vector-transfected COS-7 cells grown in 5% FCS (d, shaded bar) or 20% FCS (d, blank bar).
Figure 3
Figure 3
Detection of Tiam1–ankyrin complex in SP1 cells. SP1 cells (5 × 105 cells) were solubilized by 1% NP-40 buffer and processed for antiankyrin or anti-Tiam1–mediated immunoprecipitation, followed by immunoblotting with anti-Tiam1 or anti-ANK1 (or anti-ANK3) antibody, respectively, as described in Materials and Methods. (A) Analysis of Tiam1–ANK1 complex (lane 1). Anti-ANK1–mediated immunoprecipitation followed by immunoblotting with rabbit preimmune serum. (lanes 2 and 3) Detection of Tiam1 in the complex by mouse anti-ANK1–mediated immunoprecipitation, followed by immunoblotting with rabbit anti-Tiam1 antibody (lane 2) or reblotting with mouse anti-ANK-1 antibody (lane 3). (lanes 4–7) Detection of ANK1 in the complex by rabbit anti-Tiam1–mediated immunoprecipitation, followed by immunoblotting with mouse anti-ANK1 antibody (lane 4) or reblotting with rabbit anti-Tiam1 antibody (lane 5), or peroxidase-conjugated normal mouse IgG (lane 6) or peroxidase-conjugated rabbit preimmune IgG (lane 7). (B) Analysis of Tiam1-ANK3 complex: (lane 1) anti-ANK3-mediated immunoprecipitation followed by immunoblotting with rabbit preimmune serum. (lanes 2 and 3) Detection of Tiam1 in the complex by mouse anti-ANK3-mediated immunoprecipitation, followed by immunoblotting with rabbit anti-Tiam1 antibody (lane 2) or reblotting with mouse anti-ANK-3 antibody (lane 3). (lanes 4–7) Detection of ANK3 in the complex by rabbit anti-Tiam1–mediated immunoprecipitation, followed by immunoblotting with mouse anti-ANK3 antibody (lane 4) or reblotting with rabbit anti-Tiam1 antibody (lane 5), or peroxidase-conjugated normal mouse IgG (lane 6), or peroxidase-conjugated rabbit preimmune IgG (lane 7).
Figure 3
Figure 3
Detection of Tiam1–ankyrin complex in SP1 cells. SP1 cells (5 × 105 cells) were solubilized by 1% NP-40 buffer and processed for antiankyrin or anti-Tiam1–mediated immunoprecipitation, followed by immunoblotting with anti-Tiam1 or anti-ANK1 (or anti-ANK3) antibody, respectively, as described in Materials and Methods. (A) Analysis of Tiam1–ANK1 complex (lane 1). Anti-ANK1–mediated immunoprecipitation followed by immunoblotting with rabbit preimmune serum. (lanes 2 and 3) Detection of Tiam1 in the complex by mouse anti-ANK1–mediated immunoprecipitation, followed by immunoblotting with rabbit anti-Tiam1 antibody (lane 2) or reblotting with mouse anti-ANK-1 antibody (lane 3). (lanes 4–7) Detection of ANK1 in the complex by rabbit anti-Tiam1–mediated immunoprecipitation, followed by immunoblotting with mouse anti-ANK1 antibody (lane 4) or reblotting with rabbit anti-Tiam1 antibody (lane 5), or peroxidase-conjugated normal mouse IgG (lane 6) or peroxidase-conjugated rabbit preimmune IgG (lane 7). (B) Analysis of Tiam1-ANK3 complex: (lane 1) anti-ANK3-mediated immunoprecipitation followed by immunoblotting with rabbit preimmune serum. (lanes 2 and 3) Detection of Tiam1 in the complex by mouse anti-ANK3-mediated immunoprecipitation, followed by immunoblotting with rabbit anti-Tiam1 antibody (lane 2) or reblotting with mouse anti-ANK-3 antibody (lane 3). (lanes 4–7) Detection of ANK3 in the complex by rabbit anti-Tiam1–mediated immunoprecipitation, followed by immunoblotting with mouse anti-ANK3 antibody (lane 4) or reblotting with rabbit anti-Tiam1 antibody (lane 5), or peroxidase-conjugated normal mouse IgG (lane 6), or peroxidase-conjugated rabbit preimmune IgG (lane 7).
Figure 4
Figure 4
Binding interaction between Tiam1 and the cytoskeletal protein ankyrin. (A) Tiam1 (isolated from SP-1 cells) bound to the anti-Tiam1 immunobeads were incubated with 125I-labeled ankyrin (5,000 cpm/ng protein) in the absence (a) or the presence of 100-fold excess of unlabeled ankyrin (b) or spectrin (c). After binding, the immunobeads were washed extensively in binding buffer and the bead-bound radioactivity was estimated. (B) Autoradiogram of 125I-labeled ankyrin binding to a polyacrylamide gel containing purified Tiam1 (isolated from SP-1 cells) in the absence (a) or the presence of 100-fold excess of unlabeled ankyrin (b) or spectrin (c).
Figure 5
Figure 5
Ankyrin structure and ankyrin repeat domain (ARD) fusion protein. (A, a) Schematic illustration of functional domains in full-length ankyrin: ankyrin repeat domain (ARD), spectrin binding domain (SBD), and regulatory domain (RD). (A, b) ARD cDNA was constructed according to the strategy described in Materials and Methods. This ARD cDNA construct encodes for the NH2-terminal region of the ankyrin membrane binding domain with a tandem array of 24 ankyrin repeats. (B) A Coomassie blue stain of the 116-kD GST-ARD fusion protein purified by affinity column chromatography (lane 1), and the 89-kD ARD (lane 2) after the removal of GST by thrombin digestion. (C and D) Scatchard plot analyses of the equilibrium binding between 125I-labeled ankyrin and Tiam1. Various concentrations of 125I-labeled ankyrin (e.g., intact erythrocyte ankyrin [ANK1] or ARD) were incubated with purified Tiam1-coupled beads at 4°C for 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. Scatchard plot analysis of the equilibrium binding data between 125I-labeled intact erythrocyte ankyrin (ANK1) and Tiam1 (C); and Scatchard plot analysis of the equilibrium binding data between 125I-labeled ARD and Tiam1 (D).
Figure 6
Figure 6
Properties of Tiam1 and Tiam1 mutant proteins. (A, a) The full-length Tiam1 contains DH, dbl homology domain; DHR, discs-large homology domain; two pleckstrin homology (PH) domains (including the NH2-terminal PH [PHn] and the COOH-terminal PH [PHc]). (A, b) The NH2 terminally truncated C1199 Tiam1 encodes the COOH-terminal 1,199 amino acids. (A, c) The Tiam1 fragment encodes the sequence between amino acids 393 and 738. (B) Characterization of Tiam1 fragment (amino acids 393–738) fusion proteins. Coomassie blue staining of E. coli–derived CBP-Tiam1 fragment fusion protein purified by calmodulin affinity column chromatography (lane 1); and GFP-tagged Tiam1 fragment fusion purified by anti-GFP–conjugated affinity column chromatography (lane 2). (C, a) Binding of 125I-Tiam1 fragment to ankyrin. (C, b) Binding of 125I-Tiam1 fragment to ARD. (C, c) Binding of 125I-Tiam1 fragment to the spectrin binding domain of ankyrin. (C, d) Binding of 125I-Tiam1 fragment to spectrin. (D and E) Binding analysis between GST-ARD and the recombinant C1199 Tiam1 in vitro. In each reaction, glutathione-Sepharose bead slurry containing GST-ARD or GST alone was suspended in the 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 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. (D) The amount of C1199 Tiam1 (mol) associated with GST-ARD (mol) was measured in the absence (lane 1) or the presence of the recombinant Tiam1 fragment (lane 2) or C1199 Tiam1 associated with GST-coated beads (lane 3) using SDS-PAGE and Coomassie blue staining followed by densitometric analyses. (E) Coomassie blue staining of C1199 Tiam1 associated with GST-ARD in the absence (lane 1) or the presence of recombinant Tiam1 fragment (lane 2) or C1199 Tiam1 associated with GST-coated beads (lane 3).
Figure 7
Figure 7
Identification of the ankyrin binding domain of Tiam1. (A) 125I-labeled Tiam1 was incubated with ankyrin-coated beads in the presence of various concentrations of unlabeled synthetic peptide (GEGTDAVKRSL, corresponding to the sequence between amino acids 17 and 727 of Tiam1) (c), or the scrambled sequence (GRATLEGSDKV; a), or another Tiam1-related peptide (GTIKRAPFLGP, corresponding to the sequence between amino acids 399 and 409 of Tiam1; b) as described in Materials and Methods. 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. (B) Schematic illustration of the in vitro mutagenesis approach used in this study. Both C1199 Tiam1 (a) and C1199 Tiam1 717-727 (lacking the sequence between amino acids 17 and 727; b) were constructed according to the strategy described in Materials and Methods. (C) Anti-HA–mediated immunoblot of SP-1 cells transiently transfected with vector alone (lane 1), or HA-tagged C1199 Tiam1 cDNA (lane 2), or HA-tagged C1199 Tiam1 717-727 cDNA (lane 3). (D) The amount of 125I-ankyrin binding to anti-HA–mediated immunoprecipitates isolated from SP-1 cells transfected with vector alone (a), or HA-tagged C1199 Tiam1 cDNA (b), or HA-tagged C1199 Tiam1 717-727 cDNA (c).
Figure 8
Figure 8
Stimulation of Tiam1-catalyzed GDP/GTP exchange activity by ankyrin. Purified E. coli–derived GST-tagged GTPases (e.g., Rac1, Cdc42, or RhoA) was preloaded with GDP. 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., intact ankyrin or ARD; 1 μg/ml), 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-γ–35S (∼1,250 Ci/mmol). Subsequently, 2.5 pmol of GDP-loaded GST-tagged Rho GTPases (e.g., Rac1, Rac1, or Cdc42) was mixed with the reaction buffer containing Tiam1 and GTP-γ–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 in Materials and Methods. The termination reactions were filtered immediately through nitrocellulose filters, and the radioactivity associated with the filters was measured by scintillation fluid. The amount of GTP-γ–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%. (A–C) Kinetics of GTP-γ–35S bound to GDP-loaded GST-Rac1 by Tiam1 (isolated from SP-1 cells) in the absence (C) or in the presence of ankyrin, e.g., intact erythrocyte ankyrin (ANK1; A) or ARD fragment (B).
Figure 9
Figure 9
Transfection of SP1 cells with HA-tagged C1199 Tiam1 cDNA (A) or GFP-tagged Tiam1 fragment cDNA (B) or cotransfection of HA-tagged C1199 Tiam1 cDNA and GFP-tagged Tiam1 fragment cDNA (C). Detection of C1199 Tiam1 expression by anti-HA–mediated immunoblot in HA-tagged C1199 Tiam1 cDNA–transfected cells (B, a) or in vector-transfected cells (A, a). Detection of Tiam1 fragment expression by anti-GFP–mediated immunoblot in GFP-tagged Tiam1 fragment cDNA–transfected cells (C, b) or vector-transfected cells (A, b). Detection of coexpression of C1199 Tiam1 and Tiam1 fragment by immunoblotting of cells (cotransfected with HA-tagged C1199 Tiam1 cDNA and GFP-tagged Tiam1 fragment cDNA) with anti-HA antibody (D, a) and anti-GFP antibody (D, b), respectively. In controls, no signal was detected in HA-tagged C1199 Tiam1 cDNA–transfected cells or GFP-tagged Tiam1 fragment cDNA–transfected cells using anti-GFP (B, b) or anti-HA (C, a)–mediated immunoblotting, respectively.
Figure 10
Figure 10
Double immunofluorescence staining of ankyrin and Tiam1 in untransfected SP1 cells or SP1 transfectants. SP1 cells (untransfected or transfected with HA-tagged C1199 Tiam1 cDNA or GFP-tagged Tiam1 fragment cDNA or cotransfected with HA-tagged C1199 Tiam1 cDNA plus GFP-tagged Tiam1 fragment cDNA) were fixed by 2% paraformaldehyde. Subsequently, cells were rendered permeable by ethanol treatment and stained with various immunoreagents as described in Materials and Methods. (A–C) Rh-labeled anti-ANK3 staining (A) FITC-anti-Tiam1 staining (B) and colocalization of ankyrin and Tiam1 (C) in untransfected SP1 cells. (D–F) Rh-labeled anti-ANK3 staining (D) FITC-anti-HA–labeled C1199 Tiam1 staining (E), and colocalization of ankyrin and C1199 Tiam1 (F) in HA-tagged C1199 Tiam1 cDNA–transfected SP1 cells. (G–I) Rh-labeled anti-ANK3 staining (G), GFP-tagged Tiam1 fragment (H), and colocalization of ankyrin and Tiam1 fragment (I) in GFP-tagged Tiam1 fragment cDNA–transfected SP1 cells. (a–c) Rh-labeled normal mouse IgG staining (a), GFP-tagged Tiam1 fragment (b), and colocalization of normal mouse IgG and Tiam1 fragment (c) in GFP-tagged Tiam1 fragment cDNA–transfected SP1 cells. (J–L) Rh-labeled anti-HA staining of C1199 Tiam1 (J), GFP-tagged Tiam1 fragment (K), and colocalization of C1199 and Tiam1 fragment (L) in SP1 cells cotransfected with HA-tagged C1199 cDNA and GFP-tagged Tiam1 fragment cDNA. (d–f) Rh-labeled anti-ANK3 staining (d), GFP-tagged Tiam1 fragment (e), and colocalization of ankyrin and Tiam1 fragment (f) in SP1 cells cotransfected with HA-tagged C1199 cDNA and GFP-tagged Tiam1 fragment cDNA.
Figure 11
Figure 11
Kinetics of GTP-γ–35S bound to GDP-loaded GST-Rac1 in the presence of ankyrin-associated Tiam1 isolated from SP-1 cells: transfected with HA-tagged C1199 Tiam1 cDNA (a) or GFP-tagged Tiam1 fragment cDNA (d); or cotransfected with HA-tagged C1199 Tiam1 cDNA plus GFP-tagged Tiam1 fragment cDNA (c) or vector alone (b). Purified E. coli–derived GST-tagged GTPases (e.g., Rac1, Cdc42, or RhoA) were preloaded with GDP. First, 2 pmol ankyrin-associated Tiam1 isolated from various SP1 transfectants was added 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-γ–35S (∼1,250, Ci/mmol). Subsequently, 2.5 pmol GDP-loaded GST-tagged Rho GTPases (e.g., Rac1, RhoA, Cdc42 or GST alone) were mixed with the reaction buffer containing ankyrin-associated Tiam1 and GTP-γ–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 as described in Materials and Methods. The termination reactions were filtered immediately through nitrocellulose filters, and the radioactivity associated with the filters were measured by scintillation fluid. The amount of GTP-γ–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%.
See this image and copyright information in PMC

References

    1. Bagrodia S., Cerione R.A. PAK to the future. Trends Cell Biol. 1999;9:350–355. - PubMed
    1. Bennett V. Ankyrins. J. Biol. Chem. 1992;267:8703–8706. - PubMed
    1. Bennett V., Gilligan D.M. The spectrin-based membrane skeleton and micro-scale organization of the plasma membrane. Annu. Rev. Cell Biol. 1993;9:27–66. - PubMed
    1. Bourguignon L.Y.W. Interaction between membrane-cytoskeleton and CD44 during lymphocyte signal transduction and cell adhesion. Curr. Top. Membr. 1996;43:293–312.
    1. Bourguignon L.Y.W., Jin H. Identification of the ankyrin-binding domain of the mouse T-lymphoma cell inositol 1,4,5-triphosphate (IP3) receptor and its role in the regulation of IP3-mediated internal Ca2+ release. J. Biol. Chem. 1995;270:7257–7260. - PubMed

Publication types

MeSH terms