RB1 methylation by SMYD2 enhances cell cycle progression through an increase of RB1 phosphorylation

Abstract

It is well known that RB functions are regulated by posttranslational modifications such as phosphorylation and acetylation, but the significance of lysine methylation on RB has not been fully elucidated. Our expression analysis of SMYD2 by quantitative real-time polymerase chain reaction showed that expression levels of SMYD2 are significantly elevated in human bladder carcinomas compared with nonneoplastic bladder tissues (P < .0001), and its expression levels in tumor tissues were much higher than those of any other normal tissues. SMYD2 knockdown resulted in the suppression of cancer cell growth, and cell cycle analysis indicated that SMYD2 might play a crucial role in the G(1)/S transition. According to an in vitro methyltransferase assay, we found that SMYD2 methylates RB1 protein, and liquid chromatography-tandem mass spectrometry analysis revealed lysine 810 of RB1 to be methylated by SMYD2. Importantly, this methylation enhanced Ser 807/811 phosphorylation of RB1 both in vitro and in vivo. Furthermore, we demonstrated that methylated RB1 accelerates E2F transcriptional activity and promotes cell cycle progression. SMYD2 is an important oncoprotein in various types of cancer, and SMYD2-dependent RB1 methylation at lysine 810 promotes cell cycle progression of cancer cells. Further study may explore SMYD2-dependent RB1 methylation as a potential therapeutic target in human cancer.

Figure 1

SMYD2 is overexpressed in cancer tissues and involved in the growth of cancer cells. (A) Expression analysis of SMYD2 at mRNA levels in 125 bladder cancer cases and 28 normal bladder cases by quantitative RT-PCR, and results are shown by box-whisker plot. GAPDH and SDH were used as housekeeping genes. Mann-Whitney U test was used for statistical analysis (P < .0001). (B) Comparison of mRNA levels of SMYD2 between bladder cancer samples and normal organ tissues. The normal organ tissues include brain, breast, colon, esophagus, eye, heart, liver, lung, pancreas, placenta, kidney, rectum, spleen, stomach, and testis. (C) Immunohistochemical analysis of bladder cancer and normal bladder tissues. All tissue samples were purchased from BioChain. Original magnification, x200. (D) Validation of SMYD2 knockdown at the protein level. Lysates from SW780 and RT4 cells 72 hours after siRNA treatment were immunoblotted with anti-SMYD2 and anti-ACTB (an internal control) antibodies. (E) Effects of SMYD2 knockdown on the proliferation of bladder cancer cell lines (SW780 and RT4) measured by Cell Counting Kit-8. Relative cell numbers are normalized to the number of siNC-treated cells (siNC = 1): results are the mean ± SD (error bars) of three independent experiments. P values were calculated using Student's t test (*, P < .05). (F) Methylation activity of SMYD2 is critical for its growth-promoting effect. COS-7 cells were transfected with FLAG-Mock, -SMYD2 (WT or ΔNHSC/ΔGEEV) and, 10 days after transfection, were stained with Giemsa. Expression of SMYD2 (WT or ΔNHSC/ΔGEEV) was confirmed by Western blot using anti-FLAG antibody. Expression of ACTB served as a control.

Figure 2

SMYD2 methylates RB1 and makes a complex through its C-terminal domain. (A) RB1 is methylated by SMYD2. In vitro methyltransferase reaction was performed using purified N-RAS, H-RAS, K-RAS, RB1, p53, Aurora B, and AKT1 recombinant proteins. Methylated proteins were visualized with fluorography. (B and C) Coimmunoprecipitation assays of SMYD2 and RB1 proteins. 293T cells were cotransfected with a SMYD2 expression vector and a RB1 expression vector or a mock control vector. The interaction of FLAG-SMYD2 and HA-RB1 (B) or FLAG-RB1 and HA-SMYD2 (C) was examined by immunoprecipitation using anti-FLAG M2 agarose and immunoblotted with anti-FLAG and anti-HA antibodies. (D) The C-terminal region of SMYD2 is essential for the interaction with RB1. 293T cells were cotransfected with a FLAG-RB1 expression vector and three different regions of HA-SMYD2 vectors (amino acids 1–250, 250–330, and 320–433 in SMYD2 protein). Immunoprecipitation was performed using HA-Agarose, and samples were immunoblotted with anti-FLAG and -HA antibodies. (E) Colocalization of SMYD2 and RB1 proteins in SBC5 cells. SBC5 cells were stained with anti-RB1 (Alexa Fluor 488 [green]) and anti-SMYD2 (Alexa Fluor 594 [red]) antibodies, and DAPI [blue]). Scale bar, 30 µm.

Figure 3

SMYD2 methylates RB1 at K810. (A) The C-terminal region of RB1 is methylated by SMYD2. In vitro methyltransferase assay was performed using purified RB1 recombinant proteins (RB1 (Full), RB1 (1–378), RB1 (379–928), and RB1 (773–928)), and methylated proteins were visualized with fluorography. (B) MS/MS spectrum of monomethyl peptide of RB1. RB1 protein (773–928) was treated with SMYD2 and then the mixture was subjected to SDS-PAGE. After CBB staining, a protein band of ∼25 kDa was digested with API and subjected to LC-MS/MS. A spectrum for the monomethylated RB1 is shown. The *K indicates monomethyl lysine. (C) K810A-RB1 is not methylated by SMYD2. In vitro methyltransferase assay was performed using RB1 (773–928, 773–813), K810A-RB1 (773–813). (D and E) Validation of the anti-K810me RB1 antibody. In vitro methyltransferase assay was conducted with RB1 (Full) and RB1 (773–928) (D) or RB1 (773–813) and K810A-RB1 (773–813) (E). The samples were immunoblotted with anti-RB1K810me and anti-His (internal control) antibodies. (F) 293T cells were cotransfected with a FLAG-WT-RB1 vector or a FLAG-K810A-RB1 vector and an HA-WT-SMYD2 vector or an HA-SMYD2 (ΔNHSC/GEEV) vector. Immunoprecipitation was performed using anti-FLAG M2 agarose, and the samples were immunoblotted with anti-RB1K810me, anti-FLAG, and anti-HA antibodies.

Figure 4

SMYD2 enhances RB1 phosphorylation. (A) Expression levels of SMYD2 correlate with phosphorylation levels of RB1 (Ser 807/811). Lysates from normal cell lines (CCD18Co and HFL1) and cancer cell lines (HeLa, ACC-LC-319, A549, SW480, SW780, HCT116, and SBC5) were immunoblotted with anti-phospho-RB1 (Ser 807/811), anti-SMYD2, and anti-ACTB (internal control) antibodies. (B) 293T cells were transfected with a FLAG-SMYD2 vector and a mock vector (negative control). Cells were lysed with RIPA-like buffer containing complete protease inhibitor cocktail, and samples were immunoblotted with anti-FLAG, anti-phospho-RB1 (Ser 807/811), and anti-RB1 (internal control) antibodies. (C) After transfection with an HA-SMYD2 vector into HeLa cells, cells were fixed with 4% paraformaldehyde (PFA) and permeabilized with 0.5% Triton X-100. The fixed cells were stained with anti-phospho-RB1 (Ser 807/811) (Alexa Fluor 488 [green]) and anti-HA (Alexa Fluor 594 [red]) antibodies, and DAPI (blue). (D) Knockdown of SMYD2 diminishes phosphorylation levels of RB1 (Ser 807/811). After knockdown of SMYD2 using SMYD2-specific siRNAs, cells were lysed with RIPA-like buffer containing complete protease inhibitor cocktail. Immunoblot was performed with anti-SMYD2, anti-phospho-RB1 (Ser 807/811), and anti-RB1 (internal control) antibodies. (E) 293T cells were transfected with a FLAG-RB1 (773–813) vector and an HA-WT-SMYD2 vector and an HA-SMYD2 (ΔNHSC/GEEV) vector. Immunoprecipitation was conducted with anti-FLAG M2 agarose. Anti-FLAG, anti-phospho-RB1 (Ser 807/811), and anti-phospho-RB1 (Ser 780) antibodies were used for immunoblot analysis.

Figure 5

SMYD2-dependent monomethylation of RB1 at Lys 810 increases the phosphorylation of RB1 at Ser 807/811 in vitro. (A) Research strategy of sequential in vitro methylation and kinase assays. (B) In vitro methyltransferase assay was performed using recombinant RB1 (773–813) protein as a substrate reacted with bovine serum albumin (negative control) or SMYD2 as an enzyme. After confirmation of RB1 methylation by Western blot with anti-RB1K810me antibody, in vitro kinase assay was conducted using CDK4/Cyclin D1 complex as an enzyme. The samples were immunoblotted with an anti-phospho-RB1 (Ser 807/811) antibody. Amounts of loading proteins were visualized by MemCode Reversible Protein Stain (Thermo Fisher Scientific). (C) Methylation of RB1 at Lys 810 enhances phosphorelation levels of RB1 (Ser 807/811). After in vitro methyltransfease assay of RB1 treated with several different doses of SMYD2, in vitro kinase assay was performed with CDK4/Cyclin D1 complex as an enzyme. The samples were immunoblotted with anti-phopspho-RB1 (Ser 807/811) and anti-RB1 K810me antibodies. Amounts of loading proteins were visualized by MemCode Reversible Protein Stain (Thermo Fisher Scientific). (D) In vitro methyltransferase and kinase assays with WT-RB1 (773–813) and K810A-RB1 (773–813). Anti-RB1 K810me, anti-phospho-RB1 (Ser 807/811), and anti-His (internal control) antibodies were used for the immunoblot analysis. (E) Sequences of methylated and unmethylated peptides of RB1. (F) In vitro kinase assay of K810 methylated or unmethylated RB1 peptides was performed with CDK4/Cyclin D1 as an enzyme source. Anti-RB1 K810me and anti-phospho-RB1 (Ser 807/811) antibodies were used for immunoblot analysis. Amounts of loading peptides were visualized by MemCode Reversible Protein Stain (Thermo Fisher Scientific). Mean ± SD (error bars) of two independent experiments. P values were calculated using Student's t test (**P < .01). (G) In vitro kinase assay of RB1 peptides treated with two different doses of CDK4/Cyclin D1. Amounts of loading peptides were visualized by MemCode Reversible Protein Stain (Thermo Fisher Scientific).

Figure 6

Lys 810 methylation of RB1 enhances the phosphorylation of RB1 and E2F luciferase activity in vivo. (A) 293T cells were transfected with a HA-WT-SMYD2 vector and a FLAG-WT-RB1 vector or a FLAG-K810A-RB1 vector. Immunoprecipitation was conducted with anti-FLAG M2 agarose. Anti-FLAG, anti-RB1 K810me, and anti-phospho-RB1 (Ser 807/811) antibodies were used for immunoblot analysis. (B) 293T cells were transfected with a FLAG-WT-RB1 (773–813) vector or a FLAG-K810A-RB1 (773–813) vector and a HA-WT-SMYD2 vector. Immunoprecipitation was conducted with anti-FLAG M2 agarose. Anti-FLAG, anti-RB1 K810me, and anti-phospho-RB1 (Ser 807/811) antibodies were used for immunoblot analysis. (C) E2F reporter assay after overexpression of WT-RB1 and K810A-RB1 in 293T cells. Mean ± SD (error bars) of three independent experiments. P values were calculated using Student's t test (***P < .001). (D) A schematic model for the dynamic regulation of RB1 phosphorylation through methylation of RB1 by SMYD2.