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The Journal of Biological Chemistry logoLink to The Journal of Biological Chemistry
. 2013 Oct 15;288(47):34019–34029. doi: 10.1074/jbc.M113.480103

The Cdc42/Rac Nucleotide Exchange Factor Protein β1Pix (Pak-interacting Exchange Factor) Modulates β-Catenin Transcriptional Activity in Colon Cancer Cells

EVIDENCE FOR DIRECT INTERACTION OF β1PIX WITH β-CATENIN*

Ahmed Chahdi ‡, Jean-Pierre Raufman ‡,§,¶,1
PMCID: PMC3837141  PMID: 24129564

Background: Rac1 and β-catenin signaling are both dysregulated in colon cancer.

Results: Rac1-independent association of β-catenin with β1Pix, a Rac1/Cdc42 guanine nucleotide exchange factor, modulates β-catenin transcriptional activity and colon cancer cell proliferation.

Conclusion: The physical interaction between β1Pix and β-catenin is functional and independent of Rac1 activity.

Significance: β1Pix modulates β-catenin transcriptional activity independently of β1Pix guanine nucleotide exchange activity.

Keywords: β-Catenin, Cell Proliferation, Colon Cancer, Guanine Nucleotide Exchange Factor (GEF), Small GTPases

Abstract

Wnt/β-catenin signaling is highly regulated and critical for intestinal epithelial development and repair; aberrant β-catenin signaling is strongly associated with colon cancer. The small GTPase Rac1 regulates β-catenin nuclear translocation and signaling. Here we tested the hypothesis that β1Pix, a Cdc42/Rac guanine nucleotide exchange factor (GEF), regulates β-catenin-dependent transcriptional activity and cell function. We report the novel observations that β1Pix binds directly to β-catenin, an action requiring both the β1Pix DH and dimerization domains but not β1Pix GEF activity. In human colon cancer cells, activation of β-catenin signaling with LiCl decreased β1Pix/β-catenin association in the cytosol and increased nuclear binding of β-catenin to β1Pix. Nuclear association of β1Pix and β-catenin was independent of Rac1 expression and activation; down- and up-regulating Rac1 expression levels did not alter nuclear β1Pix/β-catenin association. Ectopic β1Pix expression enhanced LiCl-induced β-catenin transcriptional activity. Conversely, siRNA knockdown of β1Pix attenuated both LiCl-induced β-catenin transcriptional activity and colon cancer cell proliferation. Ectopic expression of β1Pix stimulated β-catenin transcriptional activity, whereas β1PixΔ(602–611), which is unable to bind β-catenin, had no effect. Altogether, these findings suggest that β1Pix functions as a transcriptional regulator of β-catenin signaling through direct interaction with β-catenin, an action that may be functionally relevant to colon cancer biology.

Introduction

β-Catenin plays a critical role in regulating key cellular processes. For example, β-catenin regulates cell adhesion and migration by linking E-cadherin to α-catenin and mediating E-cadherin binding to the actin cytoskeleton (1–3).

A prime example of β-catenin's critical biological role is its function as the central mediator of Wnt/β-catenin signaling, which regulates embryonic development, cellular proliferation, and maintenance of organs and tissues in adults (4, 5). In the absence of activation by Wnt ligands, cytoplasmic β-catenin associates with a multi-molecular destruction complex including glycogen synthase kinase-3β (GSK-3β),2 casein kinase 1α (CK1α), and the tumor suppressors axin and APC. GSK-3β- and CK1α-induced β-catenin phosphorylation promotes β-catenin ubiquitination and proteasomal degradation (6–8). Wnt ligands activate a signal transduction mechanism that inhibits both GSK-3β- and CK1α-induced β-catenin phosphorylation, thus stabilizing and freeing β-catenin from the cytoplasmic complex and allowing it to translocate to the nucleus (8, 9). Nuclear β-catenin interacts with TCF/LEF-1 family transcription factors, thereby inducing expression of key pro-proliferative genes (6, 7).

Nuclear accumulation of β-catenin, a critical event in initiation and promotion of many cancers, is arguably best studied in colon cancer. Mutations affecting the APC gene, and thus assembly of the β-catenin destruction complex, are observed in 80% of colon cancers (10). Phosphorylation of key amino acids in the β-catenin N-terminal region facilitates binding of the β-TrCP ubiquiting ligase as well as ubiquitination and proteasomal degradation of β-catenin (11, 12). Approximately 10% of colon cancers harbor N-terminal β-catenin mutations that thwart ubiquitination and proteasomal degradation, resulting in aggressive tumor growth and a worse outcome (10, 13–15).

Small GTPases of the Rho family control a wide range of cellular tasks ranging from the maintenance of cell polarity to control of cell-cell adhesion and cellular migration (16, 17). As molecular switches, GTPases shuttle between inactive GDP-bound and active GTP-bound states; they are activated by guanine nucleotide exchange factors (GEF) like β1Pix (Pak-interacting exchange factor), a GEF for Rac 1 and Cdc42 (18, 19). Several lines of evidence support the role of dysregulated Rac1 signaling in cancer - Rac1 expression is increased in colon neoplasia (20). Likewise, aberrant Rac1 activation, attributed to altered regulation and expression of upstream regulators, is reported in several human colon cancer cell lines (21). Rac1 is a critical regulator of β-catenin activation and nuclear translocation (22, 23).

Accumulating evidence indicates that β1Pix integrates signaling pathways that control cellular adhesion and cytoskeletal organization. Previous work showed that endothelin-1 induces β1Pix translocation to focal complexes by a protein kinase A-dependent mechanism (24) and that binding of 14-3-3β modulates β1Pix activity (25). β1Pix also mediates endothelin-1 signaling by interacting with Gαi3 and caveolin-1 (26, 27). Moreover, β1Pix down-regulates p27kip1 levels thereby increasing cell proliferation by a mechanism involving interaction with the adaptor protein p66Shc and the transcription factor FOXO3a (26, 28).

Based on these collective observations we tested the novel hypothesis that β1Pix plays a role in regulating β-catenin transcriptional activation. Using two human colon cancer cell lines we sought evidence for direct interaction between β-catenin and β1Pix, and determined whether this was dependent on β1Pix GEF and Rac1 activity. To test the functional significance of our findings we elucidated the actions of ectopic expression and depletion of β1Pix on β-catenin transcriptional activity and human colon cancer cell proliferation. Our findings suggest that β1Pix regulates β-catenin transcriptional activation by means of direct interaction with β-catenin; actions that are likely relevant to the critical role of β-catenin signaling in colon cancer biology.

EXPERIMENTAL PROCEDURES

Reagents and Antibodies

Cell culture media and supplements were obtained from Invitrogen (Carlsbad, CA). GST, GST-β-catenin, GST-axin(275–510), anti-β1Pix, and anti-Rac1 antibodies were from Millipore (Temecula, CA). GST-GSK-3β was from Proqinase (Freiburg, Germany). Antibodies against GSK-3β, axin1, β-catenin, β-actin, and histone 2A were purchased from Cell Signaling (Danvers, MA). Anti-Myc antibody was from Santa Cruz Biotechnologies (Santa Cruz, CA). Anti-HA antibody was purchased from Covance (Princeton, NJ). Anti-FLAG antibody was from Sigma-Aldrich.

Cell Lines and Transfection

HCT116 and Caco2 cell lines were purchased from American Type Culture Collection (ATCC). HEK293T and HEK293 cells were kindly provided by Dr. Andrey Sorokin (Medical College of Wisconsin, Milwaukee, WI) and Dr. Geoffrey Girnun (Marlene and Stewart Greenebaum Cancer Center, University of Maryland School of Medicine, Baltimore, MD), respectively. HCT116 cells were grown in McCoy's 5A medium supplemented with 10% fetal bovine serum. Caco2, HEK293, and HEK293T cells were cultured in DMEM (Invitrogen) supplemented with 10% fetal bovine serum, penicillin (100 units/ml), and streptomycin (100 μg/ml) in a 37 °C humidified incubator with 5% CO2. Transient transfection of cells with mammalian expression vectors was performed using Trans-It transfection 2020 reagent (Mirus, Madison, WI) according to the manufacturer's instructions.

Plasmids

FLAG-β1Pix, Myc-tagged-β1Pix, β1PixDHm(L238R, L239S), β1PixSH3m(W43K), β1PixΔDH, β1PixΔPH, and β1PixΔ(602–611) plasmids were described previously (24, 25). TOPFLASH/FOPFLASH, Renilla, and β-catenin plasmids were generously provided by Dr. Geoffrey Girnun (Marlene and Stewart Greenebaum Cancer Center, University of Maryland School of Medicine). HA-N17Rac1 and HA-V12Rac1 plasmids were obtained from the Missouri S&T cDNA Resource Center (www.cdna.org). Flag-β-catenin (plasmid 16828) and Flag-ΔN89 β-catenin (plasmid 19288) were obtained from Addgene (www.addgene.org). These plasmids were deposited by Dr. Eric Fearon (University of Michigan School of Medicine) who also generously contributed FlagΔC695 β-catenin.

Immunoprecipitation and Immunoblotting

Cells were transfected with appropriate constructs for 24 h, washed twice in phosphate-buffered saline (PBS), and lysed in a solution containing 20 mm Tris, pH 7.5, 100 mm NaCl, 5 mm MgCl2, 1 mm EDTA, 1% Triton X-100, 1 mm sodium fluoride, 1 mm sodium vanadate, 1 mm phenylmethylsulfonyl fluoride, 1 μg/ml pepstatin, and 1 μg/ml leupeptin. Equal amounts of proteins were separated using 7.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred onto polyvinylidene difluoride membranes (Millipore), immunoblotted with appropriate antibodies and visualized using enhanced chemiluminescence (ECL; Amersham Biosciences, Inc.). For immunoprecipitation, antibodies against β-catenin, axin, GSK-3β or β1Pix (500 μg) were added to cell lysates for 2 h, followed by addition of protein A- or protein G-agarose beads for an additional hour. Beads were washed three times in PBS. Immunoprecipitated proteins were released from beads by boiling in 1× sample buffer for 5 min and analyzed by immunoblotting. Total cell lysates (TCL) were analyzed to assess over-expression of constructs. Expression of recombinant and Myc-β1Pix mutants was verified by immunoblotting with anti-Myc antibody.

Purification of Recombinant β1Pix

β1Pix was expressed as N-terminal His6 tag fusion proteins in bacteria (BLD21DE3). Recombinant β1Pix was purified using the nickel-nitrilotriacetic acid purification system (Invitrogen) and eluted from the resin with 250 mm imidazole at room temperature.

GST Pulldown Assays

GST pulldown assays were performed using the Pierce® GST Protein Interaction Pulldown kit (Thermo Scientific, Rockford, IL) according to the manufacturer's instructions. Cell lysates from HEK293T cells overexpressing Myc-β1Pix (300 μg) were incubated with 5 μg of glutathione-agarose beads bound to GST, GST-β-catenin, GST-axin(275–510), or GST-GSK-3β. After overnight incubation at 4 °C, beads were washed five times and bound proteins were immunoblotted with anti-Myc antibody. For in vitro binding assays, 5 μg of GST, GST-β-catenin, GST-axin(275–510), and GST-GSK-3β, immobilized on glutathione-agarose, were incubated with (His)6-β1Pix (∼5 μg). After overnight incubation, resin beads were washed five times and boiled for 5 min in SDS-PAGE sample buffer. Reduced proteins were fractionated on 4–15% gradient gels, and bound proteins detected with anti-β1Pix antibody.

Nuclear and Cytosolic Fractionation

Cells were washed twice with ice-cold PBS, harvested by scraping with a rubber policeman, and lysed in a solution containing 20 mm HEPES, pH 7.0, 10 mm KCl, 2 mm MgCl2, 0.5% Nonidet P-40, 1 mm Na3VO4, 10 mm NaF, 1 mm phenylmethanesulfonyl fluoride, and 2 μg/ml aprotinin. After incubation on ice for 10 min, cells were homogenized by 20 strokes in a tightly fitting Dounce homogenizer. To sediment nuclei the homogenate was centrifuged at 1,500 × g for 5 min. The supernatant was then centrifuged at 16,000 × g for 20 min; the resulting supernatant was considered the non-nuclear fraction. The nuclear pellet was washed three times with lysis buffer to remove contamination from cytoplasmic membranes. To extract nuclear proteins, isolated nuclei were resuspended in a solution containing 150 mm NaCl, 1 mm EDTA, 20 mm Tris-Cl, pH 8.0, 0.5% Nonidet P-40, 1 mm Na3VO4, 10 mm NaF, 1 mm phenylmethanesulfonyl fluoride, and 2 μg/ml aprotinin, and the mixture was sonicated briefly to facilitate nuclear lysis. Nuclear lysates were collected after centrifugation (16,000 × g for 20 min at 4 °C). Lysate samples were subjected to electrophoresis on 7.5% SDS-polyacrylamide gels. Proteins were transferred to polyvinylidene difluoride membranes, immunoblotted with antibodies, and detected by electrochemiluminescence.

TCF/LEF Luciferase Reporter Assay

Cells grown on 24-well plates were transfected in triplicate with cDNAs for TCF-luciferase reporter (TOPFLASH) or the mutated control reporter (FOPFLASH) along with the Renilla reporter driven by the thymidine kinase promoter (tk-RL) and with other plasmids of interest. In all samples, total cDNA was equalized by using corresponding empty vectors. 48 h after transfection with increasing amounts of FLAG-β1Pix cDNA, cells were lysed, and luciferase activity measured and normalized to the corresponding Renilla activity using the dual-luciferase assay kit (Promega, Madison, WI). Normalized FOPFLASH values were subtracted from corresponding TOPFLASH values.

Cell Proliferation Assays

Caco2 and HCT116 cells were cultured in 6-well plates and transfected with control, β1Pix siRNA#1or β1Pix siRNA#2 using TransIT-siQUEST (Mirus) transfection reagent according to the manufacturer's instructions. 24 h later, siRNA-transfected cells were harvested and seeded into 96-well plates at a density of 7 × 103 cells per well. At the times indicated, cell proliferation was determined using the CellTiter-Glo assay kit (Promega).

Small Interfering (si) RNA

siRNAs targeting human β1Pix were as follows: siRNA#1, 5′-GAGCUCGAGAGACACAUGGTT-3′; siRNA#2, 5′-GGAUAUUAGUGUCGUGCAATT-3′. Silencer Negative Control siRNA#1 (Invitrogen) was used as control. Rac1 siGENOME SMART siRNA was from Thermo Scientific.

Statistical Analysis

Data are presented as means ± S.E. and analyzed by two-tailed unpaired Student's t-tests. A p value of <0.05 was considered statistically significant.

RESULTS

Physical Interaction of β1Pix with the β-Catenin Destruction Complex

To elucidate the potential role of β1Pix, a Rac1 GEF, as a modulator of β-catenin signaling, we first asked whether β1Pix binds to β-catenin, axin, or GSK-3β. Caco2 and HCT116 human colon cancer cells were transfected with Myc-tagged β1Pix and cell lysates were immunoprecipitated using anti-β-catenin, anti-axin, and anti-GSK-3β antibodies. Myc-β1Pix was not co-immunoprecipitated by control rabbit IgG (Fig. 1). The results shown in Fig. 1, A–C indicate that in both Caco2 and HCT116 cells Myc-β1Pix was co-immunoprecipitated with anti-β-catenin, anti-axin, and anti-GSK-3β antibodies. In both colon cancer cell lines, we were unable to detect a difference in the amount of Myc-β1Pix that co-immunoprecipitated with β-catenin, axin, and GSK-3β. This finding suggested that β1Pix interacts with these proteins as part of the β-catenin destruction complex.

FIGURE 1.

FIGURE 1.

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Physical interaction between β1Pix, β-catenin, axin, and GSK-3β. Caco2 and HCT116 cells were transfected with Myc-β1Pix for 24 h, and cell lysates were immunoprecipitated with control IgG, anti-β-catenin (A), anti-axin (B), or anti-GSK-3β (C) antibodies. Immunoprecipitates were immunoblotted with anti-Myc antibody. Results are representative of three independent experiments. IP, immunoprecipitation. TCL, total cell lysate.

To characterize further the interaction between β1Pix, β-catenin, axin, and GSK-3β, we performed GST-pulldown assays. Lysates from HEK293T cells overexpressing Myc-β1Pix were incubated with GST, GST-β-catenin, GST-axin(275–510), and GST-GSK-3β bound to glutathione-agarose beads. As shown in Fig. 2A, β1Pix interaction with β-catenin, axin, and GSK-3β confirmed results obtained by co-immunoprecipitation (Fig. 1, A–C). To determine whether β1Pix binds directly to β-catenin, axin, or GSK-3β, an in vitro pulldown assay was performed wherein immobilized GST, GST-β-catenin, GST-axin(275–510), and GST-GSK-3β were incubated with bacterial-expressed (His)6-tagged β1Pix. Immunoblotting with anti-β1Pix antibody showed that β1Pix and β-catenin bound directly in vitro; β1Pix failed to bind either axin(275–510) or GSK-3β (Fig. 2B). Jointly, these data demonstrated that in human colon cancer cells β1Pix binds directly to β-catenin, both individually (Fig. 2B) and when β-catenin is a component of the destruction complex containing axin and GSK-3β (Fig. 1A).

FIGURE 2.

FIGURE 2.

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β1Pix binds directly to β-catenin. A, cell lysates from HEK293T cells overexpressing Myc-β1Pix were incubated with glutathione-agarose beads bound to GST, GST-β-catenin, GST-axin(275–510), and GST-GSK-3β. After overnight incubation at 4 °C, the beads were washed five times, and bound proteins were immunoblotted with anti-Myc antibody. B, bacterial-expressed (His)6-tagged-β1Pix was purified and incubated with immobilized GST, GST-β-catenin, GST-axin(275–510), and GST-GSK-3β. Proteins bound to GST-fusion proteins were immunoblotted with anti-β1Pix antibody. GST fusion proteins used for the pulldown assay were stained with Coomassie Blue (lower panels). CB, Coomassie Blue. C, endogenous interaction between β1Pix and β-catenin. Caco2 and HCT116 cells were lysed, immunoprecipitated with control IgG or anti-β1Pix antibody, and immunoblotted with anti-β-catenin antibody. Results are representative of three independent experiments.

Also, we explored interaction of endogenous β1Pix and β-catenin. Co-immunoprecipitation experiments in both Caco2 and HCT116 cells showed that anti-β1Pix antibody precipitated β-catenin (Fig. 2C). Immunoprecipitates obtained with control IgG did not contain β-catenin. These results provide strong evidence that β1Pix interacts specifically with β-catenin and that this interaction occurs at physiological levels of protein expression.

Binding to β-Catenin Requires β1Pix Dimerization and the Presence of the DH Domain but Does Not Require β1Pix GEF Activity

To gain mechanistic insight into the physical interaction between β1Pix and β-catenin we employed a series of mutated and truncated β1Pix constructs. Key structural domains of β1Pix are shown in Fig. 3A. In general, GEF proteins contain catalytic Dbl homology (DH) and pleckstrin homology (PH) domains. In addition, a β1Pix Src homology 3 (SH3) domain binds with high affinity to a polyproline stretch in Pak1 (p21-activated kinase) (18). β1Pix binds GIT1 through a GIT1-binding domain (29). Finally, the C terminus of β1Pix contains a leucine zipper domain required for β1Pix dimerization (30).

FIGURE 3.

FIGURE 3.

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β1Pix binding to β-catenin requires the β1Pix DH domain and β1Pix dimerization but not β1Pix-GEF activity. A, illustrations showing key structural domains of WT and mutant β1Pix. B, HEK293T cells were transfected with empty vector, Myc-tagged β1Pix, β1PixSH3m, β1PixΔ(602–611), β1PixΔPH, or β1PixΔDH for 24 h. Cell lysates were immunoprecipitated with anti-β-catenin antibody followed by immunoblotting with anti-Myc antibody. C, HEK293T cells were transfected with empty vector, Myc-β1Pix or Myc-β1PixDHm(L238R, L239S) for 24 h, and cell lysates were immunoprecipitated with anti-β-catenin antibody. To verify successful transfection, total cell lysates (TCL) were probed with anti-Myc antibody. Results are representative of three independent experiments.

To identify the β-catenin binding domain of β1Pix, HEK293T cells were transiently transfected with a series of mutated and truncated Myc-tagged β1Pix constructs (Fig. 3A). Cell lysates were immunoprecipitated with anti-β-catenin antibody and resulting precipitates analyzed by immunoblotting with anti-Myc antibody. Fig. 3B shows that β1Pix, β1PixSH3m(W43K) and β1PixΔPH associated with β-catenin. The β1PixSH3 domain is essential for β1Pix-induced Rac1 activation (31) and Pak1 binding (18); mutant β1PixSH3m (W43K) abolishes interaction of β1Pix with Pak1 and β1Pix-induced Rac1 activation (31). Thus, our finding that association of β1PixSH3m(W43K) with β-catenin was indistinguishable from that of wild-type β1Pix suggests that Pak1 binding is not essential for β1Pix association with β-catenin. In contrast, β1PixΔDH and a β1Pix dimerization-deficient mutant, β1PixΔ(602–611), failed to interact with β-catenin, thereby indicating that β1Pix interaction with β-catenin requires both β1Pix dimerization and the DH domain. To test whether β1Pix GEF activity within the DH domain is needed for β1Pix interaction with β-catenin, we transfected HEK293T cells with Myc-β1Pix and a Myc-β1PixDHm(L238R, L239S) mutant unable to catalyze GDP/GTP exchange on Rac (18). These experiments showed that both β1Pix and the β1PixDHm(L238R, L239S) mutant interacted alike with β-catenin (Fig. 3C), a finding that provides evidence that β1Pix interaction with β-catenin is independent of β1Pix GEF activity.

Both N and C Terminals Regions of β-Catenin Interact with β1Pix

β-Catenin has a central domain (residues 141–664) comprised of 12 armadillo repeats, an N-terminal domain that harbors the α-catenin binding site as well as GSK3 and CK1 phosphorylation sites (32, 33), and a C-terminal domain (34). The armadillo repeat sequences are critical for TCF-4 binding to β-catenin (35, 36). To identify β-catenin domains required for interaction with β1Pix and whether these overlapped with the TCF-4 binding site, HCT116 cells were transfected FLAG-tagged wild-type β-catenin, and N- (FLAG-ΔN89 β-catenin) and C-terminal (FLAG-ΔC695 β-catenin) truncations; both β-catenin truncations contained the TCF-4 binding region. Immunoprecipitation with anti-β1Pix antibody revealed lack of β1Pix interaction with either FLAG-ΔN89 or FLAG-ΔC695 β-catenin (Fig. 4A). These findings identified broadly distributed binding of β1Pix with β-catenin that could potentially overlap with the TCF-4 binding domain.

FIGURE 4.

FIGURE 4.

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The N- and C-terminal regions of β-catenin are required for its interaction with β1Pix. A, HCT116 cells were transfected with empty vector, FLAG-β-catenin, FLAG ΔN89 β-catenin, or FLAG ΔC695 β-catenin for 24 h. Cell lysates were immunoprecipitated using anti-β1Pix antibody followed by immunoblotting with anti-FLAG antibody. B, HCT116 cells were co-transfected with increasing amounts of FLAG-β1Pix along with constant amount of Myc-TCF-4 for 24 h. Cell lysates were immunoprecipitated with anti-β-catenin antibody and immunocomplexes immunoblotted with anti-FLAG, anti-Myc, and anti-β-catenin antibodies. Successful overexpression of different constructs was verified in total cell lysates (TCL).

Therefore, to exclude the possibility of competition between β1Pix and TCF-4 for β-catenin binding, we transfected HCT116 cells with increased amounts of FLAG-β1Pix while maintaining a constant amount of Myc-TCF4. Immunprecipitation experiments using anti-β-catenin antibody revealed that β1Pix binding to β-catenin increased in a dose-response manner (Fig. 4B). At the same time, binding of TCF-4 to β-catenin was unchanged (Fig. 4), thereby indicating that β-catenin can interact concurrently with β1Pix and TCF-4. Based on these experimental results, we concluded that β1Pix does not compete with TCF-4 binding to β-catenin.

Stimulation of β-Catenin Signaling Increases Nuclear Association of β1Pix with β-Catenin Independently of Rac1

To clarify the biological importance of molecular interaction between β1Pix and β-catenin, we examined effects on cytosolic and nuclear β1Pix/β-catenin association of activating β-catenin signaling with LiCl, a GSK-3β inhibitor. Caco2 and HCT116 cells were stimulated with 5 mm LiCl for 1 h, cytosolic and nuclear fractions were immunoprecipitated with anti-β1Pix antibody, and immunoprecipitates were immunoblotted with anti-β-catenin antibody. As shown in Fig. 5A, in both Caco2 and HCT116 cells, activation of β-catenin signaling with LiCl decreased cytosolic binding of β-catenin to β1Pix, whereas nuclear binding of β-catenin to β1Pix was increased. These findings suggested that β1Pix/β-catenin association is dynamically regulated by β-catenin signaling. Also, stimulation of Caco2 and HCT116 cells with LiCl resulted in increased cytosolic and nuclear β-catenin levels (Fig. 5A, third panel from the top) but did not alter levels of β1Pix (Fig. 5A, fourth panel from the top).

FIGURE 5.

FIGURE 5.

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Activation of β-catenin signaling induces nuclear association of β-catenin to β1Pix independently of Rac1. A, Caco2 and HCT116 cells were treated with vehicle or LiCl (5 mm) for 60 min and cytosolic (C) and nuclear (N) fractions were immunoprecipitated with anti-β1Pix antibody followed by immunoblotting with anti-β-catenin antibody. β-actin was used as a cytosolic marker and histone 2A as a nuclear marker. Total levels of cytosolic and nuclear β-catenin (third panel from the top) and β1Pix (fourth panel from the top) are shown. B, Caco2 and HCT116 cells were transfected with control or Rac1 siRNA for 48 h before adding LiCl (5 mm) for an additional 60 min. Nuclear fractions were immunoprecipitated with anti-β1Pix antibody and resulting immunoprecipitates subjected to immunoblotting analysis using anti-β-catenin antibody. C, Caco2 and HCT116 cells were transfected with empty vector, HA-N17Rac1 or HA-V12Rac1 for 24 h before adding LiCl (5 mm) for an additional 60 min. Nuclear fractions were immunoprecipitated with anti-β1Pix antibody followed by immunoblotting with anti-β-catenin antibody. To verify successful transfection, total cell lysates (TCL) were probed with anti-HA antibody. Results are representative of three independent experiments.

Rac1 plays an important role in β-catenin nuclear localization and β-catenin-dependent transcriptional activation (22, 23, 37). To determine the role of Rac1 in nuclear interaction of β1Pix with β-catenin, we modulated endogenous expression of Rac1 by transfecting Caco2 and HCT116 cells with specific Rac1 and control siRNA. Aliquots of cell lysates were immunoblotted to confirm reduced expression of Rac1 in cells transfected with Rac1 but not control siRNA (Fig. 5B). Rac1 depletion in Caco2 and HCT116 cells did not alter LiCl-induced nuclear association between β1Pix and β-catenin (Fig. 5B). These findings provide evidence that Rac1 is not involved in regulating nuclear association of β1Pix with β-catenin.

To elucidate further the role of Rac1 in nuclear association of β1Pix and β-catenin we examined the effects of blocking or increasing endogenous Rac1 activity by transient transfection of a dominant-negative mutant, N17Rac1, and a dominant-positive mutant, V12Rac1. Caco2 and HCT116 cells were transiently transfected with empty vector, HA-N17Rac1 and HA-V12Rac1, and nuclear fractions were immunoprecipitated with anti-β1Pix antibody followed by immunoblotting with anti-β-catenin antibody. To confirm successful transfection of HA-N17Rac1 and HA-V12Rac1, aliquots of cell lysates were immunoblotted with anti-HA antibody. Fig. 5C shows that LiCl-induced nuclear association between β1Pix and β-catenin was not altered by either dominant-negative or -positive Rac1 mutants. Taken together, these results provide strong evidence that LiCl-induced nuclear association between β1Pix and β-catenin is independent of both Rac1 expression and activity.

β1Pix Modulates β-Catenin Signaling in Colon Cancer Cells

To assess the functional importance of nuclear association between β1Pix and β-catenin, we examined the effects of β1Pix on β-catenin-regulated transcriptional activation using a β-catenin/LEF/TCF luciferase reporter assay (TOPFLASH) (38). HEK293 cells which have tightly regulated β-catenin signaling were transfected with fixed amounts of Myc-β-catenin and increasing concentrations of FLAG-β1Pix. Our results revealed that β1Pix stimulated dose-dependent β-catenin-regulated transcriptional activation (TOPFLASH activity) (Fig. 6A). Likewise, in HCT116 cells with dysregulated β-catenin signaling (39) β1Pix overexpression dose-dependently increased TOPFLASH activity (Fig. 6B), indicating that β1Pix enhances β-catenin-dependent transcriptional activity.

FIGURE 6.

FIGURE 6.

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β1Pix stimulates β-catenin-mediated transcription in HEK293 and HCT116 human colon cancer cells. A, HEK293 cells were transfected for 48 h with β-catenin and increasing amounts of FLAG-β1Pix along with TOPFLASH/FOPFLASH reporter plasmids. B, HCT116 cells were transfected with increasing amounts of FLAG-β1Pix along with TOPFLASH/FOPFLASH reporter plasmids for 48 h. Caco2 (C) and HCT116 (D) cells were transfected with empty vector, Myc-β1Pix, Myc-β1PixDHm(L238R, L239S), or Myc-β1PixΔ(602–611) along with TOPFLASH/FOPFLASH reporter plasmids for 48 h, and cells were harvested and assayed for luciferase activity as described in “Experimental Procedures.” Data shown are representative of three independent experiments carried out in triplicate.

To determine the biological significance of β1Pix binding to β-catenin, we investigated the effects of transfecting cells with β1Pix mutants on β-catenin transcriptional activity. We transfected Caco2 and HCT116 cells with wild-type β1Pix, a β1Pix GEF-deficient mutant [β1PixDHm(L238R, L239S)] or a mutant unable to bind β-catenin [β1PixΔ(602–611)]. Results of these experiments confirmed that β1Pix overexpression stimulated β-catenin-dependent transcriptional activity, and that TOPFLASH activity levels were similar in cells ectopically overexpressing wild type β1Pix and the β1Pix-GEF deficient mutant, β1PixDHm(L238R, L239S) (Fig. 6, C and D). In contrast, in cells with ectopic expression of the β1Pix mutant unable to bind β-catenin, TOPFLASH activity was the same as control (Fig. 6, C and D). These data provide evidence that β1Pix binding to β-catenin plays an important role in modulating β-catenin transcriptional activity and added support for the conclusion that this action does not require GEF activity.

To examine further the role of β1Pix in regulating Wnt/β-catenin transcriptional activity, TOPFLASH/FOPFLASH plasmids and vector expressing FLAG-β1Pix were transfected into Caco2 and HCT116 cells for 48 h. The results of these experiments showed that LiCl or ectopic β1Pix expression stimulated comparable levels of TOPFLASH activation in both Caco2 (Fig. 7A) and HCT116 (Fig. 7B) colon cancer cells. Interestingly, ectopic β1Pix expression enhanced LiCl-induced TOPFLASH activity (Fig. 7, A and B), indicating that β1Pix enhances β-catenin signaling.

FIGURE 7.

FIGURE 7.

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β1Pix positively regulates β-catenin-mediated transcription in colon cancer cells. Caco2 (A) and HCT116 (B) cells were transfected with empty vector or FLAG-β1Pix along with TOPFLASH/FOPFLASH plasmids for 48 h before adding LiCl (5 mm) for 6 h. 30 nm control, β1Pix siRNA#1 or β1Pix siRNA#2 were transfected into Caco2 (C, E) and HCT116 (D, F) cells followed by transfection with TOPFLASH/FOPFLASH plasmids for 48 h. The cells were treated with vehicle or LiCl (5 mm) for 6 h, harvested, and luciferase activity measured as described in “Experimental Procedures”. Bar graphs show means ± S.E. of three independent experiments performed in triplicate. *, p < 0.05. Immunoblots shown are representative of three independent experiments.

To confirm that β1Pix was involved in LiCl-induced β-catenin-regulated transcriptional activation, we used two specific siRNAs to reduce endogenous β1Pix expression in Caco2 and HCT116 cells. β1Pix and control siRNA were transfected into cells with TOPFLASH and FOPFLASH plasmids. Immunoblotting of cell lysates confirmed reduced β1Pix expression (Fig. 7, C–F, lower panels). In both Caco2 and HCT116 cells siRNA knockdown of β1Pix significantly attenuated LiCl-induced TOPFLASH activity; control siRNA had no effect (Fig. 6, C–F, upper panels). In the absence of LiCl treatment, β1Pix siRNAs no. 1 and no. 2 did not alter TOPFLASH activity (not shown). Overall, these results confirm that β1Pix modulates β-catenin-dependent transcriptional activity.

Lastly we tested whether β1Pix modulated a functional end point of β-catenin-dependent transcriptional activation-colon cancer cell proliferation. We transfected two different specific β1Pix siRNAs and control siRNA into Caco2 and HCT116 cells. Analysis of cell lysates by immunoblotting with anti-β1Pix antibody confirmed depletion of β1Pix after treatment with both specific siRNAs, but not with control siRNA (Fig. 8, A and B, insets). As shown in Fig. 8, A and B, in both Caco2 and HCT116 cells β1Pix knockdown attenuated cell proliferation. Furthermore, in control experiments, β1Pix siRNA-attenuated cell proliferation was restored by ectopically expressing β1Pix (Fig. 8C). These findings suggest that β1Pix-induced enhancement of β-catenin signaling modulates a biologically meaningful outcome, human colon cancer cell proliferation.

FIGURE 8.

FIGURE 8.

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Effect of β1Pix knockdown on human colon cancer cell proliferation. In 6-well plates, Caco2 (A) and HCT116 (B) cells were transfected for 24 h with 30 nm control siRNA, β1Pix siRNA#1 or β1Pix siRNA#2. siRNA-transfected cells were harvested and seeded into 96-well plates (7 × 103 cells/well). At the times indicated, cell proliferation was measured using the CellTiter-Glo assay. At day 3 after transfection, both β1Pix siRNAs induced ∼70% reduction in expression levels of endogenous β1Pix (insets). C, HCT116 cells were transfected with control or β1Pix siRNA#1 and #2 for 48 h followed by transfection with FLAG-β1Pix for 36 h. Data shown are means ± S.E. of three independent experiments performed in triplicate. *, p < 0.05; **, p < 0.01.

DISCUSSION

By modulating gene transcription in both normal and neoplastic intestinal epithelial cells, canonical Wnt/β-catenin signaling regulates fundamental cell functions including proliferation, migration and invasion. Others showed that Rac1-mediated JNK activation stimulates β-catenin phosphorylation, thereby promoting β-catenin nuclear localization. Rac1 was also reported to associate with β-catenin/TCF complexes and facilitate transcriptional control of Wnt target genes (37) and regulate nuclear accumulation of an armadillo protein SmgGDS by a mechanism involving both the C-terminal polybasic region (PBR) and Rac activation (40). However, the Rac1 GEF involved in these actions was uncertain (23). Based on its function as a Cdc42/Rac1-GEF, we sought a role for β1Pix in modulating β-catenin signaling.

Herein, we demonstrate for the first time that, although β1Pix interacts directly with β-catenin and does indeed modulate its transcriptional activity, these actions are Rac1-independent. Using a series of truncated and mutated proteins to provide molecular detail, we show clearly that β-catenin interacts directly with β1Pix; an interaction that requires the β1PixDH and dimerization domains.

Our data show convincingly that stimulation of β-catenin signaling-induced nuclear association of β1Pix with β-catenin is independent of either Rac1 expression or activation; this association was not altered by Rac1 depletion or ectopic expression of dominant-negative and dominant-positive Rac1 mutants. In accord with these findings, we demonstrated that β-catenin binding to β1Pix via the β1PixDH domain does not require β1Pix-GEF activity. The SH3 domain mediates binding of β1Pix to Rac (31) and to a polyproline stretch in Pak1 (18, 41), and is required for β1Pix-induced Rac1 activation (31). Thus, our finding that association of β1PixSH3m(W43K) with β-catenin was indistinguishable from that of wild-type β1Pix indicate that Pak1 binding to β1Pix and β1Pix-GEF activity are not essential for β1Pix/β-catenin association. Altogether, our data provide strong evidence that Rac1 activation is not required for β1Pix/β-catenin molecular interaction or its effects on β-catenin transcriptional activation.

Previously, DOCK4, a Rac1-GEF, was shown to mediate Rac1 activation in response to Wnt stimulation through GSK-3β-mediated phosphorylation and interaction with the β-catenin degradation complex but not with β-catenin itself (42). In this work we demonstrate that β1Pix interacts directly with β-catenin and enhances its transcriptional activity. Both the C- and N-terminal regions of β-catenin are essential for its interaction with β1Pix, suggesting that β1Pix and TCF-4 could bind concurrently to β-catenin. Numerous β-catenin binding partners have overlapping binding sites in the groove of the armadillo repeat domain. Biochemical studies confirmed that many of these partners (TCF-4, cadherin, ICAT, and APC) cannot bind to β-catenin simultaneously (43–45). β1Pix interacts with both β-catenin and TCF-4 and increased binding of β1Pix to β-catenin does not compete with the binding of TCF-4, indicating that the binding of β1Pix and TCF-4 to β-catenin is not mutually exclusive.

The precise molecular mechanism whereby β-catenin enters the nucleus and whether β1Pix/β-catenin association plays a role in this process remains uncertain and will require additional investigation. The coiled-coil C terminus region of β1Pix is critical for its dimerization and subcellular localization. β1Pix is localized in the cytosol and cell periphery, whereas β2Pix, which does not contain the coiled-coil region, is localized in the cytosol and the nucleus (46). β-Catenin itself contains neither nuclear localization signal (NLS) nor nuclear export signal (NES) sequences. Current evidence implicates other proteins, including components of the β-catenin destruction complex, as potential β-catenin chaperones. APC harbors a functional NES sequence and facilitates nuclear export of β-catenin (47). Axin was also identified as a nucleo-cytoplasmic shuttling factor that enhances cytoplasmic localization of β-catenin (48, 49). Alternatively, the adaptor protein 14-3-3 may function as a chaperone to mediate β-catenin nuclear transport since 14-3-3 proteins were shown to participate in dynamic nucleo-cytoplasmic transport (50). 14-3-3 proteins were found to interact with β-catenin and facilitate its activation (51). Interestingly, we recently showed that 14-3-3β binds to β1Pix and regulates its GEF activity (25). Hence, pursuing a role for β1Pix in β-catenin nuclear translocation appears worthy of further investigation.

Here, we show that ectopic expression of β1Pix alone increased β-catenin-dependent transcriptional activity in the absence of other stimulants of Wnt/β-catenin signaling. Converse experiments showing that β1Pix depletion attenuates β-catenin-dependent transcriptional activity suggested likely functional consequences of the β1Pix/β-catenin interaction. Indeed, β1PixΔ(602–611) that is unable to bind to β-catenin failed to stimulate β-catenin TOPFLASH activity, indicating that β1Pix modulates β-catenin transcriptional activity through direct interaction. In this context, our finding that β1Pix overexpression restored β1Pix siRNA-mediated inhibition of cell proliferation is not surprising and potentially important.

Regarding a regulatory role for β1Pix in neoplasia, over-expression of Rho-GEFs, including β1Pix, was reported for cervical, liver, and renal cancer (52). With particular regard to colon cancer a recent report indicates that the β1Pix gene, ARHGEF7, is highly down-regulated in PPARγ ligand-treated human colon cancer cells, an action correlated with inhibition of cell migration (53). The results of PPARγ ligand treatment were abrogated by ectopic expression of ARHGEF7 (53).

Although a novel role for β1Pix as a positive regulator of β-catenin signaling in cancer is attractive, future studies must provide additional mechanistic details. For example, whether ARHGEF7 is a target gene downstream of Wnt signaling is unknown. This is certainly conceivable since the gene for Tiam1, another Rac1 exchange factor, is Wnt-responsive. Tiam1 expression is up-regulated in mouse intestinal tumors and human colon adenomas, and promotes intestinal tumor formation and progression (54, 55). Tiam1 was also shown to associate with Wnt-responsive promoters and enhance Wnt signaling in colon cancer cells (37). Finding that β1Pix is a Wnt-responsive gene would suggest the existence of positive feedback whereby aberrant activation of β-catenin signaling induces transcription of target genes such as ARHGEF7, thus augmenting β1Pix expression and, consequently, increasing nuclear β1Pix/β-catenin association.

Our novel observations provide evidence that β1Pix modulates β-catenin signaling by a direct molecular interaction. These findings identify β1Pix as a potential therapeutic target to suppress aberrant β-catenin signaling in cancer, an action worthy of further investigation.

Acknowledgments

We thank Dr. Eric Fearon (University of Michigan School of Medicine) for providing FLAG-ΔC695 β-catenin and Dr. Geoffrey Girnun (Marlene and Stewart Greenebaum Cancer Center, University of Maryland School of Medicine) for assistance with TOPFLASH/FOPFLASH assays.

*

This work was supported, in whole or in part, by Public Health Service Grants CA-107345 and CA-120407 from the NCI and Grant DK-093406 from the NIDDK, National Institutes of Health.

2
The abbreviations used are:
GSK
glycogen synthase kinase
GEF
guanine nucleotide exchange factor
CK
casein kinase
Pix
Pak-interacting exchange factor.

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