Fig 1: SMYD2-mediated β-catenin methylation is required for expression of Wnt downstream genes and β-catenin nuclear translocation by FOXM1A. 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.