AKT1 is a cytosolic serine/threonine kinase that is overexpressed in various

AKT1 is a cytosolic serine/threonine kinase that is overexpressed in various types of cancer and has a central role in human tumorigenesis. leiomyosarcoma and renal Wilms’ tumor (Supplementary Figure S2). In addition, the Cancer Genome Atlas (TCGA) database indicates frequent amplification of SMYD3 in various types of cancer 174484-41-4 supplier (Supplementary Figure S3). To further explore the biological functions of SMYD3 in human cancer, we performed liquid chromatography tandem-mass spectrometry (LC-MS/MS) analysis of AKT1 protein and identified that lysines 14, 30 and 39 (Lys 14, Lys 30 and Lys 39) in the PH domain of AKT1 were possibly methylated by SMYD3 (Figure 1B-1C and Supplementary Figures S4, S5 and S6). To verify the methylation in this region, we prepared the peptide that included methylation sites, and conducted an methyltransferase assay. As shown in Figure ?Figure1D,1D, the AKT1 peptide including three candidate lysines was methylated by SMYD3. Alignment of the PH domain of AKT1 protein showed that these methylation sites are conserved among various species, and in particular, Lys 14 is well conserved from to (Figure ?(Figure1E),1E), implying the importance of Lys 14 for AKT1 functions. Furthermore, glutamic acid 17 (Glu 17), which occupies the phosphoinositide-binding pocket and has a pivotal role in AKT1 activation [22, 23], is also well conserved among species, and basic Lys 14 is considered to form an ionic 174484-41-4 supplier interaction with acidic Glu 17 in this pocket. Figure 1 SMYD3 methylates AKT1 methylation of Lys 14 on AKT1 by SMYD3 To confirm SMYD3-mediated Lys 14 methylation on AKT1 (Supplementary Figure S8). To further validate methylation of AKT1 at Lys 14, we generated a specific antibody that recognizes Lys 14-monomethylated AKT1. Enzyme-linked immunosorbent assays (ELISAs) (Figure 3A, 3B) as well as an methyltransferase assay and subsequent western blot analysis (Figure ?(Figure3C)3C) revealed high specificity of anti-K14 monomethylated AKT1 antibody. To further investigate methylation of AKT1, we expressed FLAG-tagged wild-type AKT1 (AKT1-WT), or K14A- or K14R-substituted AKT1 proteins with a wild-type SMYD3 expression vector (SMYD3-WT) or an enzyme-inactive SMYD3 mutant (SMYD3EEL) expression vector, followed by immunoprecipitation using anti-FLAG? M2 affinity gel. Subsequent western blot analysis showed that the both Lys 14 monomethylation and Thr 308 phosphorylation signals of AKT1 were significantly attenuated in both K14A- and K14R-substituted AKT1 (Figure ?(Figure3D),3D), and that the enzyme-inactive SMYD3 mutant remarkably diminished Thr 308 phosphorylation signals of AKT1 (Figure ?(Figure3D).3D). These results suggest that SMYD3-mediated Lys 14 methylation of AKT1 is clearly observed and pivotal for phosphorylation of Thr 308 on AKT1. Figure 3 Validation of methylation on AKT1 at lysine 14 by specific antibody SMYD3-mediated Lys 14 methylation activates the AKT pathway in cancer cells A large body of literature and databases have documented frequent overexpression of SMYD3 (Supplementary Figures S2 and S3) and hyperactivation of the AKT pathway in a variety of malignancies [1, 11, 26]. Therefore, we examined the biological significance of SMYD3 on the AKT pathway in cancer cells. We knocked down SMYD3 in cancer cells by specific siRNAs and examined phosphorylation status of AKT1. As shown in Figure 4A and 4B, the phosphorylation level of AKT1 at Thr 308 was significantly diminished after knockdown of SMYD3 in the human colon cancer SW480 cells. Consistently, phosphorylation levels of mTOR, which is a major physiological substrate of AKT1, were also decreased (Figure 4A, 4B). 174484-41-4 supplier The similar results were obtained when we used the human breast cancer MDA-MB-231 cells (Figure 4C, 4D). We also examined the effect of BCI-121, a SMYD3 inhibitor [19], on the AKT activity. SW480 cells were treated with BCI-121 for 72 hours, followed by western blot analysis using anti-monomethyl AKT1 (K14) and anti-phospho AKT1 (Thr 308) antibodies (Figure 4E, 4F). As Cav2.3 we expected, BCI-121 treatment significantly attenuated Lys 14 monomethylation and Thr 308 phosphorylation of AKT1 in a dose-dependent manner, further implying the importance of SMYD3-mediated methylation on AKT1 activation. This inhibitor effect was also validated in MDA-MB-231 cells (Figure 4G, 4H). Figure 4 Knockdown and enzyme inhibitory of SMYD3 attenuate AKT1 activity Subsequently, to verify gain-of-function of SMYD3, we transfected wild-type FLAG-AKT1, and SMYD3 or Mock reflection vector into 293T cells, and performed traditional western mark evaluation 48 hours after the transfection (Amount ?(Figure5A).5A). Quantification of traditional western mark outcomes uncovered that SMYD3 overexpression enhances Lys 14 methylation and Thr 308 phopsphorylation of AKT1 as well as Ser 2448 phosphorylation of mTOR (Amount ?(Figure5B).5B). A very similar result was noticed when we utilized HeLa cells (Amount 5C, 5D), suggesting that SMYD3-mediated methylation can enhance phosphorylation of Thr 308 on AKT1 and power up the AKT1 path previously reported that glutamic acidity 17 (Glu 17) replacement to.