Fig. 2: SMYD2 methylates BCAR3 in breast cancer cells.

a SILAC-based quantitative proteomic identification of methylome changes in SMYD2 activity proficient and deficient cell extracts. Scatter plot of proteins identified by MS in pan-methyl-protein pulldowns (3×MBT). The x-axis shows the Log₂ ratio of methylated proteins in the presence of active vs inactive SMYD2 (Forward). The y-axis shows the Log₂ ratio of a label-swap replicate experiment (Reverse). Candidates previously identified²⁶ are labeled in blue. b BCAR3 expression significantly correlates with SMYD2 expression in metastatic but not in non-metastatic breast cancer samples. Spearman correlation coefficients calculated for expression of genes encoding identified putative SMYD2 substrates and SMYD2 in indicated patient groups are shown. c Tandem MS spectrum identifying monomethylated K334 present on BCAR3 after in vitro SMYD2 methylation. Deuterated S-adenosyl-l-methionine was used as a methyl donor. d Autoradiogram of a radiolabeled methylation assay using recombinant SMYD2 and full-length recombinant WT or point mutants K5R and K334R BCAR3 (Top panel). Bottom panel, Coomassie stain of proteins in the reaction. e Immunodetection of endogenous BCAR3 K334me1 levels and upon genetic or pharmacologic SMYD2 repression in MDA-MB-231 cell line. Tubulin is shown as a loading control. f Immunoblot analysis with the indicated antibodies of whole cell lysate and immunoprecipitated (IP) proteins from MDA-MB-231 cells with doxycycline-induced (Dox) shRNA depletion of BCAR3 and rescue complementation with GFP-tagged WT or K334A (K > A) BCAR3, in combination with SMYD2 depletion (shSMYD2) or pharmacologic repression (SMYD2i). Tubulin is shown as a loading control. g, h Representative images (g) and signal quantification (h) of proximity ligation assay (PLA) detecting methylated BCAR3 by coupling antibodies against HA-tagged total BCAR3 and BCAR3 K334me1 in MDA-MB-231 cells carrying indicated modifications. Dotted red lines represent cells periphery and dotted white square represents the enlarged area depicted in Supplementary Fig. S2k. P-values were calculated by ANOVA with Tukey’s testing for multiple comparisons. Scale bars, 10 μm.