Critical roles of SMYD2-mediated β-catenin methylation for nuclear translocation and activation of Wnt signaling

Abstract

Accumulation of β-catenin in the nucleus is a hallmark of activation of the Wnt/β-catenin signaling pathway, which drives development of a large proportion of human cancers. However, the mechanism of β-catenin nuclear translocation has not been well investigated. Here we report biological significance of SMYD2-mediated lysine 133 (K133) methylation of β-catenin on its nuclear translocation. Knockdown of SMYD2 attenuates the nuclear localization of β-catenin protein in human cancer cells. Consequently, transcriptional levels of well-known Wnt-signaling molecules, cMYC and CCND1, are significantly reduced. Substitution of lysine 133 to alanine in β-catenin almost completely abolishes its nuclear localization. We also demonstrate the K133 methylation is critical for the interaction of β-catenin with FOXM1. Furthermore, after treatment with a SMYD2 inhibitor, significant reduction of nuclear β-catenin and subsequent induction of cancer cell death are observed. Accordingly, our results imply that β-catenin methylation by SMYD2 promotes its nuclear translocation and activation of Wnt signaling.

Keywords: SMYD2; Wnt signaling pathway; lysine methylation; β-catenin.

Figure 1. SMYD2 methylates β-catenin in vitro and in vivo

A. Recombinant GST-WT-β-catenin protein (118 kDa) was methylated by His-SMYD2 (50 kDa) in a dose-dependent manner. Human recombinant GST-WT-β-catenin protein and S-adenosyl-L-methionine (SAM) were incubated in the absence or presence of recombinant His-SMYD2 protein. Methylated β-catenin was detected by fluorography, and amounts of loading proteins were evaluated by MemCode^TM Reversible Protein Stain. B. In vitro validation of SMYD2-mediated β-catenin methylation using anti-monomethylated K133 β-catenin antibody (meK133-β-catenin). Human recombinant GST-WT-β-catenin protein, FLAG-K133A-β-catenin protein and S-adenosyl-L-methionine (SAM) were incubated in the absence or presence of recombinant His-SMYD2. Samples were immunoblotted with the anti-meK133-β-catenin antibody. Methylated β-catenin was detected in the presence of His-SMYD2 protein, while no methylation band could be detected in FLAG-K133A-β-catenin. C. Detection of methylated β-catenin in 293T cells. 293T cells were transfected with a FLAG-WT-β-catenin vector or a FLAG-K133A-substituted β-catenin vector together with HA-Mock or HA-SMYD2 vector, followed by immunoprecipitation with anti-FLAG M2 agarose. Samples were immunoblotted with anti-meK133-β-catenin antibody after immunoprecipitation, and with anti-FLAG, anti-HA, anti-SMYD2 and anti-α-tubulin antibody before immunoprecipitation (input). D. Detection of methylated β-catenin in 293T cells. Cells were transfected with HA-Mock, HA-SMYD2 or enzyme-dead HA-SMYD2 (ΔNHSC/ ΔGEEV), followed by immunoblotting with anti-meK133-β-catenin, anti-β-catenin, anti-HA, anti-SMYD2 and anti-α -tubulin antibodies. E. Endogenous interaction of β-catenin and SMYD2 in HCT116 and SNU475 cell lines. Cell extracts of HCT116 and SNU475 were subjected to immunoprecipitation using anti-SMYD2 antibody or IgG, followed by immunoblotting with anti-β-catenin antibody (upper panels). Reciprocal immunoprecipitation was done using anti-β-catenin antibody or control IgG, followed by immunoblotting with anti-SMYD2 antibody (lower panels).

Figure 2. SMYD2-mediated methylation of β-catenin plays a critical role on nuclear translocation of β-catenin

(A, B) Effect of SMYD2 knockdown on the amount of nuclear β-catenin and that of K133-methylated endogenous β-catenin in cytoplasm and nucleus of SNU449 A. and SNU475 B. cells by western blot analysis. Cells were transfected with control siRNA (siNC) or either of two SMYD2-specific siRNAs (siSMYD2-#1 and -#2). 72 h after siRNA transfection, nuclear and cytoplasmic fractions were prepared and immunoblotted with anti-SMYD2, anti-β-catenin, anti-meK133-β-catenin, anti-α -tubulin and anti-histone H3 antibodies. (C-F) Disappearance or significant reduction of nuclear β-catenin by SMYD2 knockdown was observed in SNU449 C., SNU475 D., HCT116 E. and SW480 F. cells by ICC analysis. Cells were transfected with siNC (control) or siSMYD2 (SMYD2#1). After 48h-incubation with siRNA for SNU449 and SNU475 cells, or after 24h-incubation for HCT116 and SW480 cells, cells were fixed with 4% paraformaldehyde, and stained with an anti-α -tubulin antibody (Alexa Fluor^® 594, red), anti-β-catenin antibody (Alexa Fluor^® 488, green) and 4′,6′-diamidine-2′-phenylindole dihydrochloride (DAPI, blue). (G, H) Lack of nuclear β-catenin after introduction of methylation-deficient (K133A) β-catenin was observed by western blot analysis G. and ICC analysis H.. 293T cells were transfected with FLAG-WT-β-catenin or FLAG-K133A-substituted β-catenin, together with HA-SMYD2. For western blot analysis, nuclear and cytoplasmic fractions were prepared from the cells after 48h-incubation and immunoblotted with anti-FLAG, anti-HA, anti-α -tubulin and anti-histone H3 antibodies. For ICC analysis, after 48 h of incubation, the cells were fixed and stained with an anti-FLAG (K133A-substituted β-catenin) antibody (mouse, Alexa Fluor^®594, red), an anti-HA (SMYD2) antibody (rat, Alexa Fluor^® 488, green) and 4′,6′-diamidine-2′-phenylindole dihydrochloride (DAPI, blue).

Figure 3. SMYD2-mediated β-catenin methylation is required for expression of Wnt downstream genes and β-catenin nuclear translocation by FOXM1

A. SMYD2 knockdown attenuated transcriptional levels of two pivotal Wnt pathway downstream genes, CCND1 and cMYC in two HCC cell lines, SNU449 (upper left panel ) and SNU475 (upper right panel ), and two colon cancer cell lines, HCT116 (lower left panel) and SW480 cells (lower right panel). Cells were transfected with siNC or siSMYD2 (siSMYD2#1). After 48h-incubation, RNAs were prepared from these cells and transcriptional levels of SMYD2, CCND1 and cMYC were measured by quantitative RT-PCR. Statistical analyses were performed using unpaired Student's t-test (two groups). The asterisks indicate statistical significance; *, **, and *** indicate p-value of < 0.05, < 0.01 and < 0.005, respectively, compared to the corresponding value of the siNC (control) group. Error bars indicate values of one standard deviation (n = 3). B. SMYD2-mediated methylation at K133 of β-catenin is required for its interaction with FOXM1, BCL9 and α -catenin. 293T cells were co-transfected with FLAG-WT-β-catenin or FLAG-K133A-substituted β-catenin, and HA-SMYD2. After 48-h incubation, cell lysates were immunoblotted with anti-FLAG (WT-β-catenin or K133A-substituted β-catenin), anti-FOXM1, anti-BCL9, anti-α -catenin, and anti-HA (SMYD2) antibodies before immunoprecipitation (input), and after immunoprecipitation with anti-FLAG M2 agarose. FOXM1 and BCL9 were co-immunoprecipitated with FLAG-WT-β-catenin, but not with FLAG-K133A-substituted β-catenin. α -catenin was co-immunoprecipitated more preferentially with FLAG-K133A-substituted β-catenin than FLAG-WT-β-catenin. However, HA-SMYD2 was co-immunoprecipitated almost equally with both WT- and K133A-substituted β-catenin proteins. C. Structural analysis of an α -catenin-binding region of β-catenin including lysine 133. Without methylation of lysine 133 of β-catenin, hydrogen bonds are made between lysine 133 (K133) and methionine 98 (M98), and between lysine 133 and glutamic acid 101 (Glu 101) in an α -catenin-binding region of β-catenin. When K133 is methylated, these hydrogen bonds might be affected and the affinity of β-catenin to α -catenin could be reduced. The crystal structure of residues 84-682 of β-catenin is obtained from PDB: 4ONS and 2Z6G.

Figure 4. SMYD2-mediated β-catenin methylation is critical for cancer cell growth

A. Significant decrease of nuclear β-catenin after treatment with SMYD2 inhibitor (LLY-507) was observed by ICC. SNU449 cells were treated with 5 μM of LLY-507 (a SMYD2-specific inhibitor) or DMSO, and incubated for 48 h. Cells were then fixed and stained with an anti-β-catenin antibody (rabbit, Alexa Fluor^® 488, green), an anti-α -tubulin antibody (mouse, Alexa Fluor^® 594, red) and 4′,6′-diamidine-2′-phenylindole dihydrochloride (DAPI, blue). B. The effect on the viability of SNU449 cells by LLY-507. LLY-507 was added to the culture medium of SNU449 cells at different concentrations (0, 3, 5, or 7 μM). C. Cell growth curves of four cancer cell lines under the treatment of LLY-507 at 0 and 3 μM for six days. Significant growth-suppressive effects on cancer cells were observed in the cells treated with inhibitors in comparison of the control group. D. Cell viability assay of four cancer cell lines treated with siRNA. Significant decrease of the number of viable cells was observed in cells treated with siSMYD2-#1 and #2, compared with those treated with siNC after 5-day (SNU449 and SNU475) or 7-day (HCT116 and SW480) of the treatment. The asterisks indicate p value of < 0.005 compared with the control using an unpaired Student's t-test (two groups). Error bars indicate values of one standard deviation (n = 3). (E, F) Schematic presentation of the proposed mechanism of the Wnt signaling pathway in the presence E. or absence F. of SMYD2.