A methylation and phosphorylation switch between an adjacent lysine and serine determines human DNMT1 stability

Abstract

The protein lysine methyltransferase SET7 regulates DNA methyltransferase-1 (DNMT1) activity in mammalian cells by promoting degradation of DNMT1 and thus allows epigenetic changes via DNA demethylation. Here we reveal an interplay between monomethylation of DNMT1 Lys142 by SET7 and phosphorylation of DNMT1 Ser143 by AKT1 kinase. These two modifications are mutually exclusive, and structural analysis suggests that Ser143 phosphorylation interferes with Lys142 monomethylation. AKT1 kinase colocalizes and directly interacts with DNMT1 and phosphorylates Ser143. Phosphorylated DNMT1 peaks during DNA synthesis, before DNMT1 methylation. Depletion of AKT1 or overexpression of dominant-negative AKT1 increases methylated DNMT1, resulting in a decrease in DNMT1 abundance. In mammalian cells, phosphorylated DNMT1 is more stable than methylated DNMT1. These results reveal cross-talk on DNMT1, through modifications mediated by AKT1 and SET7, that affects cellular DNMT1 levels.Multiple interdependent post-translational modifications of cellular proteins allow for combinatorial repertoires of interactions. Some of these modifications, including acetylation, phosphorylation, methylation and sumoylation, might participate in cross-talk for dynamic control of cellular signaling under various physiological conditions 1 . Protein phosphorylation is involved in various cellular processes, including cell growth, development and apoptosis, and regulates essential physiological systems, such as the period of the circadian rhythm within mammalian cells, via cellular signaling pathways 2 . Protein methylation, especially on lysines, is another important reversible post-translational modification of cellular proteins; for example, histone methylation is involved in the © 2011 Nature America, Inc. All rights reserved.Correspondence should be addressed to S.P. (pradhan@neb.com). Accession codes. Protein Data Bank: The coordinates and structure factors of the SET7-DNMT1 peptide complex have been deposited with accession code 3OS5.Note: Supplementary information is available on the Nature Structural & Molecular Biology website.AUTHOR CONT RIBUTIONS P.-O.E. performed cell biology and biochemistry experiments. M.S. made constructs, tested kinetics and performed the pull-down assay. G.R.F. performed quantitative PCR. Y.C. performed SET7 purifications, MS-based methylation assays on DNMT1 peptide, mutagenesis of K142R and crystallization of SET7-DNMT1 peptide. A.K.U. purified the DNMT1 N-terminal domain and performed MS-based methylation assays on this fragment. J.R.H. performed crystallographic experiments. X.C. and S.P. organized and analyzed data and wrote the manuscript. COMPETING FINANCIAL INT ERESTSThe authors declare no competing financial interests.Reprints and permissions information is available online at http://npg.nature.com/reprintsandpermissions/. Previously, we have demonstrated that methylation of Lys142 on DNMT1 leads to DNMT1 degradation15. Adjacent to Lys142 is Ser143, a known phosphorylation site 16 . Therefore, we s...

Blocking of JB6 Cell Transformation by Tanshinone IIA: Epigenetic Reactivation of Nrf2 Antioxidative Stress Pathway

“…7) [this enzymatic assay of DNMTs included all of the subtypes of DNMTs], the results suggested a specific DNMT rather than the total DNMTs might be crucial for Nrf2 promoter methylation. Furthermore, it has been reported that cellular DNMT level can be affected at both transcription and posttranslation levels (25)(26)(27), which could lead to changes of gene methylation profile. Thus, it is possible that TIIA alters Nrf2 promoter methylation pattern when total DNMTs activity remains unchanged.…”

Promoter methylation status of tumor suppressor genes and inhibition of expression of DNA methyltransferase 1 in non-small cell lung cancer

“…Several previous studies have demonstrated that SET7 protein affects DNMT protein enzymic activity via promoting degradation of the DNMT1 gene. 28 A recent study has confirmed that the histone methyltransferase SET7 can methylate the histone and the lysine142 residues of DNMT1, which can reduce the expression of the DNMT1 gene by the proteasome-mediated pathway. 28 It can, therefore, be assumed that methylation inhibitors can also inhibit the function of DNMT1 and also activate the histone methyltransferase, SET7.…”

Foxp3+ T-regulatory cells require DNA methyltransferase 1 expression to prevent development of lethal autoimmunity

“…[5][6][7] Dnmt1 stability and function are regulated by several posttranslational modifications, including phosphorylation, acetylation, ubiquitylation, methylation, and sumoylation. 8,9 Foxp31 T-regulatory (Treg) cells are key to immune regulation. Foxp3 is located on the X-chromosome, and Foxp3-deficient mice or male Scurfy mice, with a frameshift mutation that results in disruption in Foxp3 DNA-binding, lack functional Tregs and succumb to fatal autoimmunity within a month after birth, 10,11 unless adoptively transferred with normal CD41CD251 Tregs.…”