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. 2016 Mar;15(3):892-905.
doi: 10.1074/mcp.M115.053280. Epub 2016 Jan 10.

Quantitative Profiling of the Activity of Protein Lysine Methyltransferase SMYD2 Using SILAC-Based Proteomics

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Free PMC article

Quantitative Profiling of the Activity of Protein Lysine Methyltransferase SMYD2 Using SILAC-Based Proteomics

Jonathan B Olsen et al. Mol Cell Proteomics. .
Free PMC article

Abstract

The significance of non-histone lysine methylation in cell biology and human disease is an emerging area of research exploration. The development of small molecule inhibitors that selectively and potently target enzymes that catalyze the addition of methyl-groups to lysine residues, such as the protein lysine mono-methyltransferase SMYD2, is an active area of drug discovery. Critical to the accurate assessment of biological function is the ability to identify target enzyme substrates and to define enzyme substrate specificity within the context of the cell. Here, using stable isotopic labeling with amino acids in cell culture (SILAC) coupled with immunoaffinity enrichment of mono-methyl-lysine (Kme1) peptides and mass spectrometry, we report a comprehensive, large-scale proteomic study of lysine mono-methylation, comprising a total of 1032 Kme1 sites in esophageal squamous cell carcinoma (ESCC) cells and 1861 Kme1 sites in ESCC cells overexpressing SMYD2. Among these Kme1 sites is a subset of 35 found to be potently down-regulated by both shRNA-mediated knockdown of SMYD2 and LLY-507, a selective small molecule inhibitor of SMYD2. In addition, we report specific protein sequence motifs enriched in Kme1 sites that are directly regulated by endogenous SMYD2 activity, revealing that SMYD2 substrate specificity is more diverse than expected. We further show direct activity of SMYD2 toward BTF3-K2, PDAP1-K126 as well as numerous sites within the repetitive units of two unique and exceptionally large proteins, AHNAK and AHNAK2. Collectively, our findings provide quantitative insights into the cellular activity and substrate recognition of SMYD2 as well as the global landscape and regulation of protein mono-methylation.

Figures

Fig. 1.
Fig. 1.
Global identification of Kme1 sites using SILAC-based proteomics and Kme1 immunoaffinity enrichment. A, Western blot analysis of SMYD2 or β-actin in parental KYSE-150 cells infected with lentivirus expressing either control shRNA (shControl) or shRNA targeted against SMYD2 (shSMYD2). B, Western blot analysis of p53-K370me1, total p53, SMYD2, and β-actin following treatment of KYSE-150 cells overexpressing SMYD2 with either DMSO or LLY-507. C, Overview of the experimental approach used for SILAC-based peptide labeling and enrichment of lysine mono-methylated peptides in this study. D, Comparison of current and previously published datasets in terms of the number of Kme1 sites (black) and proteins with reported Kme1 sites (red). E, Venn diagram illustrating the number of reproducible Kme1 sites in parental cells and in SMYD2-overexpressing cells.
Fig. 2.
Fig. 2.
Kme1 sites down-regulated following knockdown or inhibition of SMYD2. A, Volcano plot illustrating the normalized peptide ratio (shSMYD2/shControl) and p value distribution of Kme1 sites identified in control or SMYD2 knockdown cells. B, Volcano plot illustrating the (LLY-507/DMSO) and p value distribution of Kme1 sites identified in SMYD2-overexpressing KYSE-150 cells following treatment with LLY-507 or DMSO. C, Normalized peptide ratios of the SMYD2-dependent Kme1 site in HSP90AA1 (K615) upon SMYD2 knockdown as well as Kme1 sites known to be regulated by other PKMTs. D, Venn diagram illustrating the overlap in Kme1 sites (identified in at least two of three replicate experiments) identified in parental KYSE-150 and SMYD2-overexpressing cell lines. E, Venn diagram illustrating the number of Kme1 sites down-regulated by SMYD2 knockdown in parental KYSE-150 cells and by LLY-507 treatment in SMYD2-overexpressing cells. F, Normalized peptide ratios for Kme1 sites down-regulated in response to SMYD2 knockdown and to LLY-507 treatment.
Fig. 3.
Fig. 3.
SMYD2 knockdown down-regulates the abundance of Kme1 sites commonly identified in proteomic experiments and across a variety of cancer cell lines, including BTF3-K2 and PDAP1-K126. A, Number of Kme1 sites (red) and mono-methylated proteins (black) identified in the indicated cell lines. Only the 273 reproducible Kme1 sites in the parental KYSE-150 cell line are represented. B, The number of overlapping Kme1 sites identified in the indicated proteomic datasets among with the cell lines that were profiled in the respective studies. Specific Kme1 sites are listed in Supplemental Table S3. C, Kme1 sites (and their sequences) that were identified in at least four of the seven cell lines profiles to date and their normalized SILAC peptide ratio in response to SMYD2 knockdown in parental KYSE-150 cells. * = p value ≤ 0.05 relative to AKAP13-K1670me1. D, Bioluminescence assays monitoring the production of SAH following incubation of 100 nm of protein substrate with the indicated concentrations of SMYD2. E, MS1 peak quantification of the indicated Kme1 peptide following biochemical methylation reactions using full-length recombinant BTF3 or PDAP1 with the indicated concentrations of SMYD2. 1, Cao et al., 2013; 2, Wu et al., 2014.
Fig. 4.
Fig. 4.
Enrichment of known and novel specificity sequence motifs in SMYD2-targeted Kme1 sites. A, Sequence motifs enriched in the Kme1 sites identified in parental KYSE-150 cells or in SMYD2-overexpressing cells. B, Fold-increase in the occurrence of each enriched sequence motif compared with the background proteome based on motif-X analysis. See Supplemental Table S4 for complete motif-X results. C, Normalized peptide ratios for Kme1 sites that contained an enriched sequence motif (black) and Kme1 sites that did not (blue). p value calculation is based on the normalized peptide ratios of Kme1 sites in each group using Student's t test. D, Normalized peptide ratios for Kme1 sites containing the indicated enriched sequence motif. p value calculations are based on comparison of the normalized peptide ratios of Kme1 sites in “No motif” using Student's t test. n.s., not significant; *p value ≤ 0.0005; **p value ≤ 0.00005, ***p value ≤ 0.000005. 1, Lanouette et al., 2015; 2, Rathert et al., 2008; 3, Dhayalan et al., 2011.
Fig. 5.
Fig. 5.
AHNAK and AHNAK2 are mono-methylated at multiple sites by SMYD2. A, Changes in the number of Kme1 sites per protein identified in parental KYSE-150 cells and in SMYD2-overexpressing cells. Only reproducible Kme1 sites (i.e. identified in two of three experimental replicates) are included. AHNAK and AHNAK2 are highlighted in red. B, Protein sequence alignment of select Kme1 sites in AHNAK and AHNAK2 that were down-regulated by SMYD2 knockdown. C, Protein domain architecture of AHNAK and AHNAK2 and the positioning of the identified Kme1 sites within a representative consensus central repeat unit (CRU) of each protein. The asterisk indicates that this site ambiguously maps to AHNAK as well. E, MS1 peak quantification of mono-methyl peptide products from biochemical methylation reactions of AHNAK2–4CRU with the indicated concentration of recombinant SMYD2. Abbreviations: me0, unmodified peptide; me1, mono-methylated peptide; 1Me, single mono-methylation site; 2Me, double mono-methylation site.

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