JBC Papers in Press. Published on December 29, 2014 as Manuscript M114.602532 The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.M114.602532 Cyclin E/Cdk2 Phosphorylates Plant Homeodomain Finger Protein 8 (PHF8) and Regulates Its Function in Cell Cycle Liping Sun1, 2, Yan Huang1, 2, Qian Wei1, 2, Xiaomei Tong1, Rong Cai1, 2，Grzegorz Nalepa3 and Xin Ye1 1CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences (CAS), Beijing 100101, P. R. China, 2University of Chinese Academy of Sciences, Beijing 100101, P. R. China. 3Department of Pediatrics and Division of Pediatric Hematology-Oncology, Herman B Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, Indiana, United States of America; Downloaded from Department of Medical and Molecular Genetics, Indiana University School of Medicine, Indianapolis, Indiana, United States of America. http://www.jbc.org/ Running title: Cyclin E/Cdk2 regulates PHF8 function To whom correspondence should be addressed: Xin Ye, CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences (CAS), 1 Beichen West Road, Chaoyang District, Beijing 100101, China. Phone: +86-10 at Indiana University School of Medicine on July 1, 2015 -64807508; Fax: +86-10-64807513; E-mail: [email protected] Keywords: cyclin E/Cdk2, PHF8, phosphorylation, cell cycle, rRNA transcription Background: Cyclin E/Cdk2 is a key finger protein 8). In this report, we protein kinase in cell cycle. confirmed that PHF8 is a novel cyclin Results: Phosphorylation of PHF8 by E/Cdk2 substrate. By taking the cyclin E/Cdk2 enhances its demethylase approach of mass spectrometry, we activity towards H3K9me2 and promotes identified that PHF8 Ser844 is rDNA transcription as well as S phase phosphorylated by cyclin E/Cdk2. progression. Immunoblotting analysis indicated that Conclusion: Cyclin E/Cdk2-dependent wt-PHF8 demethylates histone phosphorylation of PHF8 affects its H3K9me2 more efficiently than the function. cyclinE/Cdk2 phosphorylation-deficient Significance: We unveiled the mechanism PHF8-S844A mutant. Furthermore, of how cyclin E/Cdk2 regulates the flow cytometry analysis showed that function of PHF8 in cell cycle regulation. wt-PHF8 promotes S phase progression more robustly than PHF8-S844A. SUMMARY Real-time PCR results demonstrated Cyclin E/Cdk2 is a key regulator in that PHF8 increases transcription of G /S transition. Previously we identified 1 cyclin E, E2F3 and E2F7 to significantly a number of Cdk2 interacting proteins higher levels compared to PHF8-S844A. including PHF8 (Plant homeodomain Further analysis by ChIP assay 1 Copyright 2014 by The American Society for Biochemistry and Molecular Biology, Inc. indicated that PHF8 binds to cyclin E by the cyclin E/Cdk2 complex to increase promoter stronger than PHF8-S844A, histone transcription required for S-phase and reduces H3K9me2 level at the entry and progression (13). In addition to cyclin E promoter more efficiently than its function on promoting G1/S transition, PHF8-S844A. In addition, we found cyclin E/Cdk2 is also involved in the DNA that cyclinE/Cdk2-mediated damage checkpoint control phosphorylation of PHF8 Ser844 by phosphorylating DNA helicase promotes PHF8-dependent rRNA complex subunit MCM3 and MCM7 (14, transcription in luciferase reporter 15). assays and real-time PCR. Taken As cyclins/Cdks are orderly activated together, these results indicate that during cell cycle progression, their phosphorylation substrates orchestrate the cyclin E/Cdk2 phosphorylates PHF8 to cellular changes that ultimately lead to cell Downloaded from stimulate its demethylase activity in division. However, many of these order to promote rRNA transcription as substrates remain unknown. In order to well as cell cycle progression. understand the molecular mechanisms of cyclin E/Cdk2 and its associated proteins http://www.jbc.org/ Cyclins and cyclin-dependent kinases in cell cycle regulation, we employed TAP (CDKs) play important roles during cell (Tandem Affinity Purification) technique cycle progression (1). Different cyclin/Cdk to identify 14 novel proteins interacting complexes exert their function at different with Cdk2, including the histone at Indiana University School of Medicine on July 1, 2015 cell cycle phases. Among them, cyclin demethylase PHF8 (16). PHF8 belongs to E/Cdk2 plays critical role at the G1 /S a class of PHD domain-containing zinc transition (2, 3) via phosphorylating a finger proteins, which play an important number of downstream signaling proteins role in histone demethylation, gene (4-7). Multiple cyclin E/Cdk2 substrates transcription and chromatin remodeling. are cell division regulators. Cyclin E/Cdk2 PHF8 can demethylate H3K9me2/1, assists cyclin D/Cdk4 in phosphorylating H3K27me2 or H4K20me1, and relieve the retinoblastoma protein (Rb), which leads transcriptional repression and promote the to the release of E2F transcriptional transcription of related genes (17, 18, 19, factors from Rb-dependent inhibition in 20). It has been found that PHF8 can also order to promote expression of genes bind to the promoter region of rDNA and (such as cyclin E, myc and DNA regulate rDNA transcription (21, 22). As a polymerase) that drive cells to enter the S histone demethylase, PHF8 regulates phase (8, 9). Cyclin E/Cdk2 also retinoic acid response in APL (acute phosphorylates Cdk inhibitor p27 to promyelocytic leukemia) (23). The promote its degradation (10). Smad3 is a germline F279S mutation in PHF8 that transcriptional factor that regulates disrupts the demethylase activity causes TGF-signal pathway. The hereditary XLMR (X-linked mental phosphorylation of Smad3 by cyclin retardation) (24, 25). E/Cdk2 inhibits its transcriptional activity PHF8 is involved in the regulation of (11). Cyclin E/Cdk2 phosphorylates cell cycle. PHF8 can interact with E2F1, CBP/p300 to stimulate its histone HCF-1 and SET1A, and promote cell acetyltransferase activity during cell cycle cycle G /S transition (26), while loss of progression (12). NPAT is phosphorylated 1 2 PHF8 leads to prolonged G2 phase and were purchased from Santa Cruz defective mitosis (27). However, the Biotechnology. Phosphor-Ser antibody mechanism of PHF8 regulation is not clear (2324S) was purchased from Cell and it remains to be elucidated whether Signaling. Histone H3 (ab1791) and phosphorylation influences PHF8 function. H3K9me2 (ab1220) antibodies were In this report, we demonstrate that cyclin purchased from Abcam. E/Cdk2 phosphorylates Ser-844 of the Co-immunoprecipitation and GST PHF8 demethylase and regulates its Pulldown- For immunoprecipitation, cellular distribution. We found that 293T cells were transfected with indicated mutation of the cyclinE/Cdk target site plasmids. The cell lysates were incubated (Ser-844) negatively affects the ability of with indicated antibodies at 4℃ for 2 h PHF8 to promote the S phase progression. followed by the addition of protein Further analysis revealed that A-agarose beads. After 4 h of incubation at Downloaded from overexpression of PHF8 upregulates the 4℃, the beads were washed with lysis cyclin E by affecting PHF8 and H3K9me2 buffer. The immune complexes were levels at the cyclin E promoter. separated by SDS-PAGE followed by Additionally, we found that wt-PHF8 immunoblotting analyses. http://www.jbc.org/ promotes rDNA transcription more For the GST pull down assay, 1 g of effectively than the PHF8-S844A mutant. GST-PHF8, GST-PHF8 (1-316), We conclude that phosphorylation of GST-PHF8 (317-680), GST-PHF8 PHF8 by cyclin E/Cdk2 plays an (681-1024) or GST as a negative control at Indiana University School of Medicine on July 1, 2015 important role in regulating gene were incubated with cell lysates from transcription and and cell cycle 293T cells overexpressing myc-tagged progression. cyclin E and Cdk2, or with His-Cdk2 purified from BL21. The glutathione beads EXPERIMENTAL PROCEDURES were then added and incubated for 2 h. Plasmids and Antibodies- The bound proteins were eluted with pCMV-SPORT6-PHF8 was purchased sample loading buffer and analyzed by from Openbiosystems. PHF8 and its immunoblotting with anti-myc or Cdk2 mutant PHF8-S844A were cloned into antibodies. pCMV FLAG. The truncated PHF8 (PHF8 Cell Culture and Synchronization- 293T (1-316), PHF8 (317-680), PHF8 and HeLa cells were cultured in (681-1024) and PHF8 mutants PHF8 Dulbecco’s modified Eagle’s medium (317-680) 381AGA, PHF8 (317-680) (DMEM) supplemented with 10% fetal 641AKA, PHF8 (681-1024) S844A) and bovine serum (PAA) and 100 μg/ml full-length Cdk2 were cloned into pET41b. streptomycin and 100 U/ml penicillin at The mammalian expression vectors for 37 ℃ with 5% CO2. HeLa cells were cyclin E and Cdk2 were generated as synchronized at early S phase by treatment described previously (16). with double thymidine in the same way as The following primary antibodies were for U2OS cells described in the previous used: FLAG antibody (M2) was purchased report (16). Briefly, cells were from Sigma. PHF8 (P-15), myc (9E10), synchronized at early S transition by His (H-3), Cdk2 (D-12), cyclin E (E-4), treatment with 2 mM thymidine for 16 h in β-actin (1-19), lamin B (M-20) antibodies complete medium, released in fresh 3 medium for 8 h and then incubated with 2 GFP-H2B plasmid for the selection of mM thymidine for another 16 h. Cells transfected cells. Cells were harvested at were synchronized to early S phase at this indicated time, fixed in ethanol, and point. stained with propidium iodide, and then In Vitro Kinase Assay- 1 g of subjected to flow cytometry analysis. GST-PHF8, GST-PHF8 (1-316), Dual-luciferase reporter activity assay- GST-PHF8 (317-680), and GST-PHF8 293T cells in a 24-well plate were (681-1024) or GST-PHF8 mutants were transfected with plasmids for expressing incubated with GST-cyclin E/Cdk2 in PHF8, PHF8-S844A, or siRNA targeting kinase buffer (50 mM Tris-Cl, pH7.5, 10 PHF8 or Cdk2, and rDNA luciferase mM MgCl2, 1 mM DTT) with 1 mM cold reporter pHrD-IRES-Luc (28) and ATP, 0.5 μCi of [γ-32P]ATP at 30℃ for 30 pRL-TK for 48 h.