RB1 Methylation by SMYD2 Enhances Cell Cycle Progression through an Increase of RB1 Phosphorylation

Primary human fibroblasts undergoing oncogene-induced or replicative senescence are known to form senescence-associated heterochromatin foci (SAHF), which can stabilize the state of senescence. The retinoblastoma (RB) protein has an important role in SAHF; cells that lack active RB pathway fail to form SAHF. It has been known that the posttranslational modifications of RB, for example, phosphorylation, regulate its function. To date, whether methylation of RB impacts on the SAHF formation is unknown. Here we report that JMJD3, a histone demethylase catalyzing the tri-methylation of H3K27 (H3K27me3), can demethylate the nonhistone protein RB at the lysine810 residue (K810), which is a target of the methyltransferase Set7/9. We detected a significant upregulation of JMJD3 during cellular senescence and SAHF formation in WI38 cells induced by H-RasV 12 , and we found that ectopic expression of JMJD3 promoted cellular senescence and SAHF formation in WI38 cells. Furthermore, during the process of SAHF assembly, JMJD3 was transported to the cytoplasm and interacted with RB through its demethylase domain JmjC. Significantly, our data demonstrated that the JMJD3-mediated demethylation of RB at K810 impeded the interaction of RB with the protein kinase CDK4 and resulted in reduced level of phosphorylation of RB at Serine807/811 (S807/811), implicating an important role of the interplay between the demethylation and phosphorylation of RB in SAHF assembly. This study highlights the role of JMJD3 as a novel inducer of SAHF formation through demethylating RB and provides new insights into the mechanisms of cellular senescence and SAHF assembly. Cellular senescence is an irreversible process of cell cycle arrest. The senescent cells remain metabolically active but are unable to express genes required for cell proliferation. 1,2 The known causes of cellular senescence include telomere shortening, oxidative stress, DNA damage and hyperoncogenic signaling. 3 H-RasV 12 has been used as a model to induce senescence in normal cells. [4][5][6] Senescent cells are typically characterized by a large flat morphology and the expression of a senescence-associated β-galactosidase activity (SA-β-gal), and nuclei of senescence cells may remodel to form the heterochromatin structures termed the senescenceassociated heterochromatin foci (SAHF). 7 SAHF are condensed regions of DNA that correlate with transcriptionally inactive sites. 8 These heterochromatin foci are hallmarked by H3K9me3 and the incorporation of heterochromatin protein HP1, macroH2A, PML (promyelocy leukemia protein) and HMGA1 (high mobility group AT-hook 1). 7,9-11 Recently, it has been shown that repressive markers, such as H3K9me3, H3K9me2 and H3K27me3, are rearranged into the nonoverlapping structural layers in SAHF. 12-14 Changes of heterochromatin organization generate a repressive chromatin environment that prevents transcription of genes that promote growth, thereby stabilize the state of senescence. 7,15 The retinoblastoma (RB) tumor suppressor is an important senesc...

show abstract

“…29 Apparently, our results are in favor of that described by Cho et al 29 that methylation of RB K810 promotes cell cycle progression.…”

Section: Discussioncontrasting

confidence: 65%

“…28 Contrarily, the SMYD2-dependent methylation of RB at K810 promotes cell cycle progression in SW780 and RT4 cells. 29 The mechanistic details underlying these discrepancies remain unclear. Moreover, whether the methylation of RB impacts the SAHF formation has not been studied prior to this report.…”

mentioning

confidence: 99%

JMJD3 promotes SAHF formation in senescent WI38 cells by triggering an interplay between demethylation and phosphorylation of RB protein

et al. 2015

View full text Add to dashboard Cite

show abstract

“…As we recently reported (17), perturbation of SMYD2 had no significant quantitative effect on global levels of histone methylation, but rather affected the global levels of several non-histone Kme1 sites, as we demonstrated here, suggesting that SMYD2 primarily functions as a non-histone PKMT. In addition, there was no overlap between the Kme1 sites we identified in cells and the Kme1 sites (and tryptic Kme1 peptides) reported in biochemical methylation assays using recombinant proteins and artificial cellbased overexpression systems (11,42,43). Although we cannot confidently assert that these Kme1 sites do not occur in endogenous cellular contexts, we do emphasize the Kme1 sites we report here are of sufficient abundance for robust and reproducible monitoring and warrant attention in additional studies into the cellular activity of SMYD2.…”

Section: Discussionmentioning

confidence: 56%

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

Olsen

Cao

Han

et al. 2016

Molecular & Cellular Proteomics

View full text Add to dashboard Cite

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. Protein lysine methyltransferases (PKMTs) 1 catalyze the sequence-specific transfer of one, two, or three methyl groups to the side chains of lysine residues (1-4). In addition to the extensively studied lysine methylation on histones, PKMTs can modify non-histone proteins (3,(5)(6)(7)(8)). An increasing number of non-histone proteins have been reported as PKMT substrates, and, as a result, potential roles for the dysregulation of non-histone lysine methylation in cancer development and progression have been proposed (9). The majority of PKMT substrates have been identified based on biochemical methylation assays using recombinant enzyme and substrate, followed by cell-based assays typically requiring overexpression of enzyme and/or substrate to maximize signal detection (10 -13). However, it is difficult to discern whether these substrates are bona fide physiological substrates of endogenous PKMT activity. With the development of PKMT-targeted drugs emerging as a key area of drug discovery (14 -18), unbiased and quantitative methods enabling the comprehensive identification of histone and non-histone substrates in cells are critical to clarifying the cell-relevant substrates of these enzymes and to more accurately understand the functions of non-histone methylation.Progressing from targeted biochemical...

show abstract

“…7). So far SMYD2 has been identified as a KMT catalyzing specific lysine monomethylation in diverse proteins (28,29), including the famous by guest on http://www.jbc.org/ Downloaded from SMYD2 regulates BMP but not TGFβ signaling transduction 8 tumor suppressor proteins p53 (30), RB (31,32) and PTEN (33). A recent comprehensive, large-scale proteomic study of lysine mono-methylation has identified several hundreds of potential SMYD2 methylation sites, and a subset of 35 sites were confirmed by SMYD2 knockdown (34).…”

Section: Discussionmentioning

confidence: 99%

The lysine methyltransferase SMYD2 methylates the kinase domain of type II receptor BMPR2 and stimulates bone morphogenetic protein signaling

Gao

Wang

et al. 2017

Journal of Biological Chemistry

View full text Add to dashboard Cite

Lysine methylation of chromosomal and nuclear proteins is a well-known mechanism of epigenetic regulation, but relatively little is known about the role of this protein modification in signal transduction. Using an RNAi-based functional screening of the SMYD family of lysine methyltransferases (KMTs), we identified SMYD2 as a KMT essential for robust bone morphogenic protein (BMP)-but not TGFβ-induced target gene expression in HaCaT keratinocyte cells. A role for SMYD2 in BMP-induced gene expression was confirmed by shRNA knockdown and CRISPR/Cas9-mediated knockout of SMYD2. We further demonstrate that SMYD2 knockdown or knockout impairs BMPinduced phosphorylation of the signaltransducing protein SMAD1/5 and SMAD1/5 nuclear localization and interaction with SMAD4. The SMYD2 KMT activity was required to facilitate BMP-mediated signal transduction, as treatment with the SMYD2 inhibitor AZ505 suppressed BMP2-induced SMAD1/5 phosphorylation. Furthermore, we present evidence that SMYD2 likely modulates the BMP response through its function in the cytosol. We show that, although SMYD2 interacted with multiple components in the BMP pathway, it specifically methylated the kinase domain of BMP type II receptor BMPR2. Taken together, our findings suggest that SMYD2 may promote BMP signaling by directly methylating BMPR2, which, in turn, stimulates BMPR2 kinase activity and activation of the BMP pathway.Lysine methylation has been extensively studied in the context of histone proteins as a mechanism of epigenetic regulation (1-3). With the presence of large number of lysine methyltransferases (KMTs) in mammalian genomes (4,5), it is not surprising that an increasingly larger number of non-histone chromosomal, nuclear and cytoplasmic proteins have been found to be lysine methylated (6-9). Lysine methylation can be dynamically removed by the action of demethylases (10,11), making it a feasible mechanism for signal transduction. However, up to now lysine methylation has been shown to regulate signaling proteins only in a limited cases. For instances, methylation of MAP3K2 by SMYD3 has been shown to increase MAP kinase signaling and promotes the formation of Ras-driven carcinomas (12), whereas methylation of VEGFR1 by SMYD3 activates its kinase activity (13), indicating that lysine methylation can play important roles in regulation of signal transduction.TGF-β/BMP superfamily of cytokines play pleiotropic roles in embryonic development, differentiation, organ morphogenesis and tissue homeostasis (14,15). These cytokines bind the cell surface type I and type II receptors that are also serine-threonine kinases (16,17). Upon ligand binding, the type II receptor activates the type I receptor by phosphorylating the GS motif of type I receptor (18). The activated type I receptor in turn phosphorylates regulatory SMADs (R-SMADs) at the C-terminal SSXS motif. This phosphorylation disrupts inhibitory intramolecular interaction between MH2 and MH1 domains of R-SMADs and stimulates R-SMADs to form a heteromeric complex with the C...

show abstract

RB1 Methylation by SMYD2 Enhances Cell Cycle Progression through an Increase of RB1 Phosphorylation

Cited by 180 publications

References 43 publications

JMJD3 promotes SAHF formation in senescent WI38 cells by triggering an interplay between demethylation and phosphorylation of RB protein

JMJD3 promotes SAHF formation in senescent WI38 cells by triggering an interplay between demethylation and phosphorylation of RB protein

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

The lysine methyltransferase SMYD2 methylates the kinase domain of type II receptor BMPR2 and stimulates bone morphogenetic protein signaling

Contact Info

Product

Resources

About