title: Cyclin E-CDK2 protein phosphorylates plant homeodomain finger protein 8 (PHF8) and regulates its function in the cell cycle.
abstract: Cyclin E-CDK2 is a key regulator in G1/S transition. Previously, we identified a number of CDK2-interacting proteins, including PHF8 (plant homeodomain finger protein 8). In this report, we confirmed that PHF8 is a novel cyclin E-CDK2 substrate. By taking the approach of mass spectrometry, we identified that PHF8 Ser-844 is phosphorylated by cyclin E-CDK2. Immunoblotting analysis indicated that WT PHF8 demethylates histone H3K9me2 more efficiently than the cyclin E-CDK2 phosphorylation-deficient PHF8-S844A mutant. Furthermore, flow cytometry analysis showed that WT PHF8 promotes S phase progression more robustly than PHF8-S844A. Real-time PCR results demonstrated that PHF8 increases transcription of cyclin E, E2F3, and E2F7 to significantly higher levels compared with PHF8-S844A. Further analysis by ChIP assay indicated that PHF8 binds to the cyclin E promoter stronger than PHF8-S844A and reduces the H3K9me2 level at the cyclin E promoter more efficiently than PHF8-S844A. In addition, we found that cyclin E-CDK2-mediated phosphorylation of PHF8 Ser-844 promotes PHF8-dependent rRNA transcription in luciferase reporter assays and real-time PCR. Taken together, these results indicate that cyclin E-CDK2 phosphorylates PHF8 to stimulate its demethylase activity to promote rRNA transcription and cell cycle progression.
authors.affiliation: From the Chinese Academy of Sciences Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China, the University of the Chinese Academy of Sciences, Beijing 100101, China, and.
authors.forename: Liping
authors.initials: L
authors.lastname: Sun
authors.affiliation: From the Chinese Academy of Sciences Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China, the University of the Chinese Academy of Sciences, Beijing 100101, China, and.
authors.forename: Yan
authors.initials: Y
authors.lastname: Huang
authors.affiliation: From the Chinese Academy of Sciences Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China, the University of the Chinese Academy of Sciences, Beijing 100101, China, and.
authors.forename: Qian
authors.initials: Q
authors.lastname: Wei
authors.affiliation: From the Chinese Academy of Sciences Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China.
authors.forename: Xiaomei
authors.initials: X
authors.lastname: Tong
authors.affiliation: From the Chinese Academy of Sciences Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China, the University of the Chinese Academy of Sciences, Beijing 100101, China, and.
authors.forename: Rong
authors.initials: R
authors.lastname: Cai
authors.affiliation: the Department of Pediatrics and Division of Pediatric Hematology-Oncology, Herman B. Wells Center for Pediatric Research, and Department of Medical and Molecular Genetics, Indiana University School of Medicine, Indianapolis, Indiana 46202.
authors.forename: Grzegorz
authors.initials: G
authors.lastname: Nalepa
authors.affiliation: From the Chinese Academy of Sciences Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China, yex@im.ac.cn.
authors.forename: Xin
authors.initials: X
authors.lastname: Ye
mesh_terms: D015153:Blotting, Western; D049109:Cell Proliferation; D002478:Cells, Cultured; D047369:Chromatin Immunoprecipitation; D019927:Cyclin E / Q000235:genetics / Q000378:metabolism*; D051357:Cyclin-Dependent Kinase 2 / Q000235:genetics / Q000378:metabolism*; D019175:DNA Methylation; D004275:DNA, Ribosomal / Q000235:genetics; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D005786:Gene Expression Regulation; D006367:HeLa Cells; D056466:Histone Demethylases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D006657:Histones / Q000378:metabolism*; D006801:Humans; D047468:Immunoprecipitation; D015513:Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016196:S Phase / Q000502:physiology*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014157:Transcription Factors / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*
chemical_list: C475820:CCNE1 protein, human; D019927:Cyclin E; D004275:DNA, Ribosomal; D006657:Histones; D015513:Oncogene Proteins; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D014157:Transcription Factors; D056466:Histone Demethylases; C511934:PHF8 protein, human; C495894:CDK2 protein, human; D051357:Cyclin-Dependent Kinase 2
keywords: Cell Cycle; Cell Signaling; Cyclin; Cyclin E-CDK2, PHF8, Phosphorylation, rRNA Transcription; Cyclin-dependent Kinase (CDK); Phosphorylation