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. Author manuscript; available in PMC: 2014 Dec 12.
Published in final edited form as: Nature. 2014 May 21;510(7504):283–287. doi: 10.1038/nature13320

Figure 3. SMYD3 methylates MAP3K2 in cancer cells.

Figure 3

a, Analysis of lung cancer development in Kras;Smyd3 mutant mice following infection with a lentivirus expressing Cre only or simultaneously Cre and WT SMYD3 or inactive SMYD3F183A. Histological analysis (HE staining) and IHC for pERK1/2 were performed 24 weeks post lentiviral infection. IHC confirms lentiviral-mediated expression of SMYD3. Scale bars, 50 μm. b, c, Quantification of total tumor area per lung and pERK1/2 positive area per lung, respectively (n=4 for each experimental group). Data are represented as mean +/− SEM. *: p-value<0.05 (two-tailed unpaired Student's t-test). d, SMYD3 directly methylates MAP3K2. In vitro methylation assay on full-length recombinant MAP3K2 with recombinant wild-type SMYD3, catalytic-dead SMYD3F183A, or GST control. Top panel: autoradiogram of methylation assay. Bottom panel: Coomassie stain of proteins in the reaction. e, SMYD3 methylates MAP3K2 at K260. In vitro methylation assay as in (d) with the indicated proteins on MAP3K2 aa1-350, MAP3K2 aa1-350 with a K260A substitution, and MAP3K2 aa351-619. Arrow indicates GST, which is a stable breakdown product of recombinant proteins. f-g, MAP3K2 is a specific substrate of SMYD3. In vitro methylation assays as in (d) on MAP3K2 using the indicated KMTs (positive controls for the known KMTs shown in Extended Data Fig. 6f). h, SMYD3 specifically methylates MAP3K2. In vitro SMYD3 methylation assay as in (d) on the indicated MAP Kinase pathway proteins. Asterisks indicates MAP3K2 breakdown product. Arrow indicates GST, which is a stable breakdown product for many of the screened substrates. i, Immunoblots with the indicated antibodies from input (cytoplasmic extract) or the indicated IPs (immunoprecipitations) from LKR10 cells stably expressing control or Smyd3 shRNA. j, Immunoblots with the indicated antibodies and samples as in (i) of lung tumor biopsy lysates isolated from Kras and Kras;Smyd3 mutant mice. Asterisks represent detection of IgG. For experiments e-k representative data based on three or more independent biological replica is shown.