Title: The Axin1 scaffold protein promotes formation of a degradation complex for c-Myc
Abstract: Article8 January 2009free access The Axin1 scaffold protein promotes formation of a degradation complex for c-Myc Hugh K Arnold Hugh K Arnold Department of Molecular and Medical Genetics, Oregon Health and Science University, Portland, OR, USAPresent address: Department of Human Biology, D4-100, Fred Hutchinson Cancer Research Center, PO Box 19024, 1100 Fairview Avenue N, Seattle, WA 98109-1024, USA Search for more papers by this author Xiaoli Zhang Xiaoli Zhang Department of Molecular and Medical Genetics, Oregon Health and Science University, Portland, OR, USA Search for more papers by this author Colin J Daniel Colin J Daniel Department of Molecular and Medical Genetics, Oregon Health and Science University, Portland, OR, USA Search for more papers by this author Deanne Tibbitts Deanne Tibbitts Department of Molecular and Medical Genetics, Oregon Health and Science University, Portland, OR, USA Search for more papers by this author Julie Escamilla-Powers Julie Escamilla-Powers Department of Molecular and Medical Genetics, Oregon Health and Science University, Portland, OR, USA Search for more papers by this author Amy Farrell Amy Farrell Department of Molecular and Medical Genetics, Oregon Health and Science University, Portland, OR, USA Search for more papers by this author Sara Tokarz Sara Tokarz Department of Molecular and Medical Genetics, Oregon Health and Science University, Portland, OR, USA Search for more papers by this author Charlie Morgan Charlie Morgan Department of Molecular and Medical Genetics, Oregon Health and Science University, Portland, OR, USA Search for more papers by this author Rosalie C Sears Corresponding Author Rosalie C Sears Department of Molecular and Medical Genetics, Oregon Health and Science University, Portland, OR, USA Search for more papers by this author Hugh K Arnold Hugh K Arnold Department of Molecular and Medical Genetics, Oregon Health and Science University, Portland, OR, USAPresent address: Department of Human Biology, D4-100, Fred Hutchinson Cancer Research Center, PO Box 19024, 1100 Fairview Avenue N, Seattle, WA 98109-1024, USA Search for more papers by this author Xiaoli Zhang Xiaoli Zhang Department of Molecular and Medical Genetics, Oregon Health and Science University, Portland, OR, USA Search for more papers by this author Colin J Daniel Colin J Daniel Department of Molecular and Medical Genetics, Oregon Health and Science University, Portland, OR, USA Search for more papers by this author Deanne Tibbitts Deanne Tibbitts Department of Molecular and Medical Genetics, Oregon Health and Science University, Portland, OR, USA Search for more papers by this author Julie Escamilla-Powers Julie Escamilla-Powers Department of Molecular and Medical Genetics, Oregon Health and Science University, Portland, OR, USA Search for more papers by this author Amy Farrell Amy Farrell Department of Molecular and Medical Genetics, Oregon Health and Science University, Portland, OR, USA Search for more papers by this author Sara Tokarz Sara Tokarz Department of Molecular and Medical Genetics, Oregon Health and Science University, Portland, OR, USA Search for more papers by this author Charlie Morgan Charlie Morgan Department of Molecular and Medical Genetics, Oregon Health and Science University, Portland, OR, USA Search for more papers by this author Rosalie C Sears Corresponding Author Rosalie C Sears Department of Molecular and Medical Genetics, Oregon Health and Science University, Portland, OR, USA Search for more papers by this author Author Information Hugh K Arnold1, Xiaoli Zhang1, Colin J Daniel1, Deanne Tibbitts1, Julie Escamilla-Powers1, Amy Farrell1, Sara Tokarz1, Charlie Morgan1 and Rosalie C Sears 1 1Department of Molecular and Medical Genetics, Oregon Health and Science University, Portland, OR, USA *Corresponding author. Department of Molecular and Medical Genetics, Oregon Health and Science University, 3181 SW Sam Jackson Park Road, Portland, OR 97239, USA. Tel.: +1 503 494 6885; Fax: +1 503 494 4411; E-mail: [email protected] The EMBO Journal (2009)28:500-512https://doi.org/10.1038/emboj.2008.279 There is a Have you seen ...? (March 2009) associated with this Article. PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Expression of the c-Myc proto-oncoprotein is tightly regulated in normal cells. Phosphorylation at two conserved residues, threonine58 (T58) and serine62 (S62), regulates c-Myc protein stability. In cancer cells, c-Myc can become aberrantly stabilized associated with altered T58 and S62 phosphorylation. A complex signalling cascade involving GSK3β kinase, the Pin1 prolyl isomerase, and the PP2A-B56α phosphatase controls phosphorylation at these sites. We report here a novel role for the tumour suppressor scaffold protein Axin1 in facilitating the formation of a degradation complex for c-Myc containing GSK3β, Pin1, and PP2A-B56α. Although knockdown of Axin1 decreases the association of c-Myc with these proteins, reduces T58 and enhances S62 phosphorylation, and increases c-Myc stability, acute expression of Axin1 reduces c-Myc levels and suppresses c-Myc transcriptional activity. Moreover, the regulation of c-Myc by Axin1 is impaired in several tested cancer cell lines with known stabilization of c-Myc or loss of Axin1. This study provides critical insight into the regulation of c-Myc expression, how this can be disrupted in three cancer types, and adds to our knowledge of the tumour suppressor activity of Axin1. Introduction c-Myc is a transcription factor that regulates the expression of numerous genes involved in the regulation of cellular proliferation, growth, apoptosis, and differentiation (Dang et al, 1999). Normal cell function requires proper regulation of c-Myc expression, which occurs at multiple levels through transcriptional, translational, and post-translational mechanisms. Recent research has identified a complex signalling pathway that controls c-Myc protein expression at the post-translational level through sequential and reversible phosphorylation at two highly conserved sites, threonine58 (T58) and serine62 (S62), that regulate ubiquitination and 26S proteasomal turnover of c-Myc (reviewed in Sears, 2004). Specifically, S62 phosphorylation increases c-Myc protein stability, whereas T58 phosphorylation stimulates its ubiquitination and degradation by the SCFFbw7 complex (Sears et al, 2000; Welcker et al, 2004; Yada et al, 2004). A number of reports have identified GSK3β, the prolyl isomerase Pin1, and protein phosphatase 2A (PP2A) with the associated B56α regulatory subunit as critical components in c-Myc protein turnover as GSK3β mediates T58 phosphorylation and Pin1 and PP2A-B56α cooperate to dephosphorylate S62 (Sears et al, 2000; Gregory et al, 2003; Yeh et al, 2004; Arnold and Sears, 2006). Axin1 is a multi-domain scaffold protein that coordinates several different protein complexes that are involved in regulating Wnt, TGFβ, SAPK/JNK, and p53 signalling (Zeng et al, 1997; Zhang et al, 1999; Rui et al, 2004; Liu et al, 2006). Thus far Axin1 has been characterized to be a tumour suppressor and numerous gene mutations have been identified throughout AXIN1 from a number of different cancers (Salahshor and Woodgett, 2005). These mutations most likely compromise the ability of Axin1 to form complexes with DVL, MEKK, GSK3α/β, PP2A, APC, β-catenin, and even Axin1 with itself (summarized in Salahshor and Woodgett, 2005). It has been shown in some cases of hepatocellular carcinomas (HCCs) harbouring mutations in AXIN1 that re-introduction of wild-type Axin1 expression increases apoptosis (Satoh et al, 2000), suggesting that Axin1 may be an important molecular target in HCC as well as other cancers with compromised Axin1 function. We now report an additional regulatory role for Axin1 in negatively controlling c-Myc protein levels at the post-translational level. Axin1 facilitates the interaction of c-Myc with GSK3β, PP2A, and Pin1, stimulates c-Myc ubiquitin-mediated degradation, and inhibits c-Myc transcriptional activity. Interestingly, several cancer cell lines with increased c-Myc stability have impaired formation of the Axin1–c-Myc degradation complex. Altogether, our results add to our understanding of the regulation of c-Myc expression and provide a new mechanism for the tumour suppressor activity of Axin1. Results Axin1 associates with c-Myc, GSK3, PP2A-B56, and Pin1 Owing to the high level of regulation and rapid turnover of c-Myc protein, we hypothesized that some of the proteins involved in regulating c-Myc protein turnover might be organized into a complex by associating with a scaffolding protein. We focused our attention on the scaffold protein Axin1, as it had been shown to associate with GSK3α/β and PP2A-B56α (Behrens et al, 1998; Fagotto et al, 1999; Li et al, 2001). To examine whether Axin1 associates with c-Myc, we transiently transfected 293 cells with c-Myc and Axin1 expression vectors and found that c-Myc co-immunoprecipitated with Axin1, and that endogenous GSK3β, B56α, PP2A-C (catalytic subunit), and Pin1 also co-immunoprecipitated with Axin1 (Figure 1A, lane 4). To further examine the association between c-Myc and Axin1, we performed the reverse co-immunoprecipitation and found that Axin1 co-immunoprecipitated with c-Myc (Supplementary Figure 1). Additionally, we found that endogenous Axin1, GSK3β, B56α, PP2A-C, and Pin1 co-immunoprecipitated with endogenous c-Myc, but not another endogenous transcription factor, Sp1 (Figure 1B, lane 3 versus 2). Lastly, immunoprecipitation of in vitro translated Axin1 co-precipitated in vitro translated c-Myc (Figure 1C, lane 2). Altogether, these findings demonstrate that Axin1 and c-Myc associate, which can be detected at endogenous levels along with endogenous GSK3β, PP2A-B56α, and Pin1. Figure 1.Axin1 associates with c-Myc along with GSK3β, PP2A-B56α, and Pin1. (A) V5–Axin1 co-immunoprecipitates c-Myc and proteins that stimulate c-Myc degradation. 293 cells were co-transfected with expression plasmids for V5–Axin1 and c-Myc as indicated. Cells were lysed in co-IP buffer and subject to α-V5 immunoprecipitation (IP). Input lysate and immunoprecipitated proteins were detected by western blotting as indicated. (B) Axin1 and c-Myc interact at endogenous levels. 293 cells were lysed in co-IP buffer and subject to IP with αc-Myc or α-Sp1 (control) as indicated. Input and immunoprecipitated proteins were detected by western blotting as indicated. (C) In vitro synthesized Axin1 and c-Myc interact. V5–Axin1 and c-Myc were produced by in vitro transcription/translation. Indicated reactions were suspended in co-IP buffer and subject to α-V5 IP, and input and immunoprecipitated proteins were detected by western blot as indicated. (D) APC associates with Axin1, but not with c-Myc. 293 cells were co-transfected with HA–c-Myc and V5–Axin1, cells were lysed in co-IP buffer, and the lysate was split into thirds. Protein G beads, α-HA, or α-V5 were used for immunoprecipitation, and input and IP proteins were visualized by western blot as indicated. (E) Wnt signalling does not affect c-Myc expression levels. 293T cells were transfected with expression plasmid for c-Myc and starved in 0.2% FBS for 48 h. Cells were then treated with 80 ng/ml Wnt3a for indicated times and c-Myc, endogenous activated β-catenin (anti-active β-catenin; Millipore), and GAPDH were detected by western blot. Fold-change relative to lane 1 and GAPDH is indicated. (F) Activation of the β-catenin/TCF-responsive luciferase reporter plasmid, TOPFLASH, upon stimulation with Wnt3a. 293T cells were co-transfected with TOPFLASH and CMV-β-gal. Cells were starved in 0.2% FBS for 48 h and then treated with 80 ng/ml Wnt3a for indicated times. Luciferase activity was measured and normalized to β-gal activity. Download figure Download PowerPoint The adenomatous polyposis coli (APC) gene product has a critical function in the recruitment and turnover of β-catenin on the Axin1 scaffold protein (Xing et al, 2003). We examined whether APC also has a function in the recruitment of c-Myc to Axin1. 293 cells were transfected with V5-tagged Axin1 and HA-tagged c-Myc. Immunoprecipitation of V5–Axin1 co-immunoprecipitated APC as expected (Figure 1D, lane 4). In contrast, immunoprecipitation of HA–Myc co-immunoprecipitated Axin1, but not APC (Figure 1D, lane 3). In addition, shRNA knockdown of APC does not affect c-Myc or P-T58-Myc levels in conditions in which Axin1 knockdown does, and increasing APC expression does not affect the ability of c-Myc to complex with Axin1 (Dr Bruno Amati, European Institute of Oncology, Italy, personal communication). Lastly, because Wnt signalling inhibits β-catenin turnover by the Axin–APC complex, we tested the effects of Wnt signalling on ectopic c-Myc expression levels. We did not observe a significant induction of c-Myc under conditions where we did observe an increase in activated β-catenin and in β-catenin/TCF-mediated transcription consistent with other reports in the literature (Staal et al, 2002) (Figure 1E and F). Taken together, these data suggest that the Axin1–c-Myc complex is most likely distinct from the Wnt-regulated Axin1–β-catenin complex involving APC. Additionally, c-Myc is primarily nuclear and as such would be expected to associate with a nuclear pool of Axin1. Axin1 knockdown increases c-Myc levels and decreases its association with GSK3, PP2A, and Pin1 We generated shRNA that efficiently knocks down Axin1 expression (Figure 2A). Knockdown of Axin1 increases endogenous c-Myc protein levels in low-serum growth conditions (Figure 2B). Endogenous c-myc mRNA levels are also increased with Axin1 knockdown (Figure 2C), presumably due to increased β-catenin activity with Axin1 knockdown as β-catenin/TCF has been shown to transcriptionally activate the c-myc gene (He et al, 1998). To focus our research on potential post-transcriptional regulation of c-Myc by Axin1, we examined ectopically expressed c-Myc, driven by the constitutively active CMV enhancer in the following experiments. We found that Axin1 knockdown significantly increased expression of CMV-driven c-Myc (Figure 2D, lower panel, lane 2 versus 1). Moreover, despite a significant increase in immunoprecipitated c-Myc with Axin1 knockdown (5.2-fold), approximately 40% less of both GSK3β and PP2A-C co-immunoprecipitated with c-Myc as compared with control (Figure 2D, compare lane 4 with 3). Quantitation of the amount of GSK3β and PP2A-C co-precipitated relative to the amount of c-Myc indicated an 88% decrease in the association of these proteins with c-Myc upon Axin1 knockdown. This result suggests that c-Myc, which accumulates in the absence of Axin1, does not efficiently associate with GSK3β or PP2A. To address whether this could be partially due to limiting GSK3β and/or PP2A, we tested the effects of ectopic Axin1 expression on the association of c-Myc with GSK3β, PP2A, and Pin1. As shown in Figure 2E, ectopic expression of Axin1 increased the amount of GSK3β, PP2A, and Pin1 that co-immunoprecipitated with c-Myc approximately two-fold (Figure 2E, lane 6 versus 5). Additionally, the association of GSK3β and PP2A with Axin1 is enhanced under low-serum culture conditions where c-Myc protein levels are reduced (Sears et al, 1999) (Supplementary Figure 2, lanes 4 and 6 versus 2). Figure 2.Axin1 facilitates the association of c-Myc with GSK3β and PP2A, and negatively regulates c-Myc protein stability. (A) shRNA-mediated knockdown of Axin1. 293 cells were co-transfected with V5–Axin1 and either vector expressing scramble or Axin1-targeted shRNA. Extracts were normalized for transfection efficiency and V5–Axin1 was detected by western blotting and quantified using the LI-COR imager software. (B) Knockdown of Axin1 increases endogenous c-Myc protein expression. Axin1 or scramble shRNA expression vectors were transfected into 293 cells. Cells were maintained in low serum for 48 h. Endogenous c-Myc and β-tubulin were detected by western blot. (C) Knockdown of Axin1 increases c-myc RNA levels. 293tr–shAxin1 cells were treated with or without Dox to induce the expression of Axin1 shRNA under low-serum conditions, and c-myc mRNA expression was analysed by qRT–PCR. (D) Axin1 knockdown reduces the interaction of c-Myc with GSK3β and PP2A. 293 cells were co-transfected with HA–c-Myc and either scramble or Axin1 shRNA. HA–c-Myc was immunoprecipitated and input and immunoprecipitated proteins were detected by western blotting. The change in expression between lane 4 versus 3 was quantified using the LI-COR software. (E) The interaction of c-Myc with GSK3β and PP2A is enhanced by increased Axin1 expression. V5–c-Myc was immunoprecipitated from 293 cells co-transfected with V5–c-Myc and/or HA–Axin1 as indicated. Input and immunoprecipitated proteins were detected by western blot and the fold increase in expression between lane 6 versus 5 was quantified using the LI-COR software. (F) Axin1 knockdown inhibits T58 phosphorylation and increases S62 phosphorylation. 293 cells were co-transfected with V5–c-Myc and either scramble or Axin1 shRNA. Lysates were normalized for transfection efficiency and phosphorylated c-Myc was detected by phospho-specific antibodies. Total c-Myc on the same blots was detected by α-V5 and a representative blot is shown. The signal in lane 2 versus 1 was quantified using the LI-COR software and three separate experiments are graphed showing average change with standard deviation (s.d.) for P-T58/total c-Myc and P-S62/total c-Myc. (G) Axin1 knockdown increases c-Myc protein stability. 293 cells were co-transfected with V5–c-Myc and either scramble or Axin1 shRNA, maintained in low serum, and then treated with cycloheximide and collected at the indicated time points. c-Myc half-life was determined from western blots and quantified using the LI-COR software for V5–c-Myc protein levels using α-tubulin as a control. Download figure Download PowerPoint Knockdown of Axin1 decreases T58 phosphorylation, increases S62 phosphorylation, and increases c-Myc protein stability As our data demonstrate that Axin1 is important for c-Myc association with GSK3β and PP2A, and GSK3β phosphorylates T58, whereas PP2A-B56α dephosphorylates S62, we examined the effects of knockdown of Axin1 on c-Myc phosphorylation at T58 and S62. To accurately quantify the level of P-T58 and P-S62 relative to total c-Myc, we dual probed our western blots with α-V5 for total c-Myc and phospho-specific antibodies for P-T58 or P-S62 levels. Upon Axin1 knockdown, we observed an increase in the P-S62 signal, but a decrease in the P-T58 signal (Figure 2F, lane 2 versus 1). Quantifying the ratio of P-T58 to total c-Myc revealed a substantial decrease in the amount of T58 phosphorylated c-Myc with Axin1 knockdown (Figure 2F, graph). In contrast, the signal for P-S62 increased relative to total c-Myc, resulting in a modest increase in S62 phosphorylated c-Myc compared with control (Figure 2F, graph). We also examined whether Axin1 knockdown affects c-Myc protein stability. 293 cells were transfected with CMV-driven c-Myc and either shRNA to Axin1 or scramble control. Following cycloheximide treatment to inhibit protein synthesis, we found that knockdown of Axin1 substantially increased c-Myc protein stability to 117 min, as compared with scramble control shRNA with a 32-min half-life for c-Myc (Figure 2G). Thus, Axin1 post-translationally regulates c-Myc protein stability consistent with its effects on c-Myc T58 and S62 phosphorylation and its effects on the ability of c-Myc to associate with GSK3β, PP2A, and Pin1. Ectopic Axin1 expression increases the ubiquitination and degradation of c-Myc Given the effect of Axin1 expression on c-Myc protein stability, we tested whether Axin1 affects c-Myc ubiquitination. Cells were transfected as indicated in Figure 3A and treated with MG132/MG115 for 4 h prior to collection to prevent turnover of multi-ubiquitinated c-Myc. Ubiquitinated proteins were immunoprecipitated and c-Myc was detected by western blotting. We observed an approximate five-fold increase in ubiquitinated forms of c-Myc with ectopic expression of Axin1 (Figure 3A, lane 4 versus 3). Although our findings support a model where Axin1 coordinates the association of GSK3β, PP2A-B56α, and Pin1 with c-Myc and thus facilitates c-Myc degradation, c-Myc input levels in our co-immunoprecipitation experiments are often not substantially affected by co-expression of Axin1 (see Figure 2E, bottom panel, lanes 2 and 3). This is most likely due to altered stoichiometry of components for complex formation, as the overexpression of scaffold proteins, including Axin1 have been reported to result in dominant-negative effects (Lee et al, 2003). We therefore generated a stable 293tr cell line with tetracycline/doxycycline (Dox)-inducible V5–Axin1 expression to more carefully control the amount and time of Axin1 expression. As shown in Figure 3B, 4 h of Dox treatment induced expression of Axin1 in the 293tr–V5–Axin1 cells (lane 2 versus 1), and this expression of Axin1 decreased ectopic c-Myc protein levels consistently by 60% as compared with control (lane 2 versus 1 and see Figure 5D). Figure 3.Acute expression of Axin1 increases c-Myc ubiquitination and downregulates c-Myc protein expression. (A) Axin1 expression increases c-Myc ubiquitination. 293 cells were co-transfected with expression plasmids for c-Myc, HA–ubiquitin, and V5–Axin as indicated. Cells were placed in low-serum media and treated with proteasome inhibitors. Ubiquitinated proteins were immunoprecipitated with α-HA. Input proteins and immunoprecipitated c-Myc were detected by western blotting as indicated. Ubi-Myc (vertical arrow) was quantified by the LI-COR software and the fold change between lane 4 versus 3 is shown. (B) Acute expression of Axin1 suppresses c-Myc expression. Stable 293tr–V5–Axin cells were transfected with HA–c-Myc and then treated with Dox for 4 h as indicated. V5–Axin1 and HA–c-Myc were detected by western blot. (C) Endogenous c-Myc expression is suppressed by acute Axin1 expression. 293tr–V5–Axin cells were treated with or without Dox for the indicated times and cells were collected for western blot analysis as indicated. (D) Acute Axin1 expression suppresses endogenous c-Myc protein prior to affecting c-myc mRNA levels. c-Myc protein levels from (C) were quantified and adjusted to actin. Cells from (C) were also subject to quantitative RT–PCR for c-myc mRNA and 18S rRNA (control). Average fold change for c-Myc protein/actin and c-myc mRNA/18S relative to paired control are graphed as indicated from three independent experiments±s.d. Download figure Download PowerPoint Acute expression of Axin1 reduces endogenous c-Myc protein levels independent of its effects on c-myc transcription Our results have demonstrated that Axin1 can negatively regulate ectopic c-Myc protein levels driven from a constitutive enhancer. However, we also examined the effect of Axin1 overexpression on endogenous c-Myc protein levels and found that acute expression of Axin1 in the 293tr–V5–Axin1 cells consistently reduced endogenous c-Myc protein levels by approximately 50% (Figure 3C and D, lane 2 versus 1). We saw no effect of Dox treatment on endogenous c-Myc levels in parental 293tr cells that do not ectopically express Axin1 (Supplementary Figure 3). As a positive control for Axin1 post-translational function on a known target protein, we assessed endogenous β-catenin protein levels and found that β-catenin was also consistently reduced by approximately 50% under the same conditions (Figure 3C, lane 2 versus 1). We also examined c-myc mRNA levels in the above experiments by quantitative RT–PCR analysis as β-catenin/TCF can transcriptionally activate the c-myc gene (He et al, 1998). We found no effect on c-myc mRNA expression levels with activation of Axin1 out to 12 h (Figure 3D, grey bars). However, after 24 h of ectopic Axin1 expression, c-myc mRNA levels were reduced. Although short-term ectopic Axin1 expression reduced endogenous c-Myc protein expression without affecting its mRNA levels, longer term expression resulted in increased c-Myc protein levels, and β-catenin levels were also no longer reduced (Figure 3C, lanes 4, 6, 8, and 10). Endogenous c-Myc was consistently more sensitive to these 'dominant-negative' effects of increased Axin1 expression compared with ectopic c-Myc, which was degraded by higher Axin 1 expression with 4 h of Dox treatment (compare Figure 3B and C). The discrepancy in c-Myc protein versus c-myc mRNA expression suggests that long-term Axin1 overexpression in 293 cells stoichiometrically reduces productive degradation complexes, preventing efficient c-Myc and β-catenin degradation. Altogether, these results demonstrate that Axin1 can negatively regulate endogenous c-Myc protein expression independent of its effects on the β-catenin/TCF transcriptional regulation of c-myc mRNA expression. Axin1 expression reduces c-Myc-dependent transcription from the E2F2 promoter We assessed the effect of Axin1 expression on c-Myc-dependent activation of the E2F2 promoter. As shown in Figure 4A, ectopically expressed c-Myc increased E2F2-driven luciferase activity by two-fold from a luciferase reporter plasmid containing consensus c-Myc-binding E-box elements (Sears et al, 1997). This modest increase in c-Myc-dependent transcription is consistent with numerous previous reports and is dependent upon intact c-Myc-binding sites as the E2F2(-E-box)-Luc showed no change in luciferase activity (Figure 4A). Ectopic Axin1 expression alone had no observable effect on E2F2 promoter activity in 293 cells (Figure 4A). However, co-expression of Axin1 with c-Myc significantly reduced c-Myc-dependent E2F2 promoter activation in these cells (Figure 4A). We observed similar results in U2OS cells except that c-Myc expression alone caused a more robust activation of the E2F2 promoter (Figure 4B). This is most likely due to the deletion of ARF in these cells, as ARF has been shown to inhibit c-Myc transcriptional activity (Qi et al, 2004; Amente et al, 2006). We observed a consistent reduction in basal E2F2 promoter activity in U2OS cells with Axin1 expression alone and a dramatic reduction with co-expression of c-Myc (Figure 4B, third and fourth column sets). As Axin1 expression did not affect the Myc-binding site mutant (-E-box), it is most likely that Axin1 is affecting the activity of endogenous c-Myc as well as ectopic c-Myc on the E2F2 promoter in U2OS cells. Next, we assessed whether Axin1 could be found at the endogenous E2F2 promoter. 293tr–V5–Axin1 cells were stimulated as indicated in Figure 4C with Dox to induce V5–Axin1 expression. Chromatin immunoprecipitation (ChIP) with anti-V5 antibody revealed that Axin1 was present on the E2F2 promoter detected with primers spanning the Myc-binding sites (Figure 4C, lane 4). This finding suggests that Axin1 may suppress c-Myc-dependent transcription at c-Myc target gene promoters. Figure 4.Axin1 expression suppresses c-Myc-dependent transcription. (A) c-Myc-dependent activation of the E2F2 promoter is inhibited by Axin1 expression in 293 cells. 293 cells were co-transfected with expression plasmids for β-gal, c-Myc, Axin1, and either E2F2-Luc (containing wild-type E-boxes) or E2F2(-E-box)-Luc (containing mutant E-boxes) as indicated. Luciferase activity was measured and adjusted for transfection efficiency by β-gal assay and three separate experiments were graphed with averages±s.d. Statistical significance is indicated by P-values between c-Myc and control (*), and c-Myc+Axin and c-Myc (**). (B) Axin1 potently inhibits c-Myc activation of the E2F2 promoter in U2OS cells. The same as (A) except U2OS cells were used. (C) Axin1 is present at the c-Myc transcriptional target E2F2 gene promoter. 293tr–V5–Axin1 cells were treated with Dox as indicated and α-V5 conjugated to protein A beads was used for ChIP (see Materials and methods) followed by PCR for the E2F2 promoter region and GAPDH control. Download figure Download PowerPoint Figure 5.Axin1 associates with the transactivation domain of c-Myc dependent on S62 phosphorylation. (A) The transactivation domain of c-Myc is required for its interaction with Axin1. 293 cells were co-transfected with expression plasmids for Axin1 and either V5–empty, V5–c-MycWT, or V5–c-MycΔTAD and then subjected to immunoprecipitation with αV5. Input and immunoprecipitated proteins were detected by western blot as indicated. (B) Constitutive S62 phosphorylation increases the association of c-Myc with Axin1. 293 cells were co-transfected with Axin1 and either V5–empty, V5–c-MycWT, V5–c-MycT58A, or V5–c-MycS62D. α-V5 co-IPs were washed stringently with a buffer containing 300 mM NaCl. Input and immunoprecipitated proteins were detected by western blot as indicated. (C) Phosphorylation at S62 is required for c-Myc to associate with Axin1. 293 cells were co-transfected with Axin1 and either HA–empty, HA–c-MycWT, or HA–c-MycS62A. α-HA co-IPs were washed stringently as above. Input and immunoprecipitated proteins were detected by western blot. (D) T58 phosphorylation is required for Axin1-mediated c-Myc turnover. 293tr–V5–Axin cells were transfected with HA–c-MycWT, HA–c-MycT58A, or HA–c-MycS62A and then treated with Dox for 4 h as indicated. V5–Axin1 and HA–c-Myc were detected by western blot (representative shown with nonspecific band loading control (NS)), quantified using the LI-COR software, and averages±s.d. from three separate experiments were graphed. Download figure