title: S6 kinase 2 is bound to chromatin-nuclear matrix cellular fractions and is able to phosphorylate histone H3 at threonine 45 in vitro and in vivo.
abstract: The activity of S6 kinases (S6K) is highly induced in cancer cells highlighting an essential role in carcinogenesis. The S6K family has two members: S6K1 and S6K2 which bear common as well as distinct features. In an attempt to identify S6K2 unique sequence features compared to S6K1, we applied extensive bioinformatic analysis and motif search approaches. Interestingly, we identified 14 unique protein signatures which are present in proteins directly connected to chromatin and/or involved in transcription regulation. Using chromatin binding assay, we biochemically showed that S6K2 is bound to chromatin as well as nuclear matrix cellular fractions in HEK293 cells. The presence of S6K2 in chromatin fractions raised the possibility that it may be in close proximity to a number of chromatin substrates. For that, we then searched for S6K phosphorylation consensus sites RXRXXT/S in mammalian proteins using the SWISS-PROT database. Interestingly, we identified some potential phosphorylation sites in histone H3 (Thr45). Using in vitro kinase assays and siRNA-based knockdown strategy; we confirmed that S6K2 but not S6K1 or AKT is essential for histone H3-Thr45 phosphorylation in HEK293 cells. Furthermore, we show that the nuclear localisation sequence in the S6K2 C-terminus is essential for this modification. We have found that, H3-Thr45 phosphorylation correlates to S6K activation in response to mitogens and TPA-induced cell differentiation of leukaemic cell lines U937, HL60 and THP1. Overall, we demonstrate that S6K2 is a novel kinase that can phosphorylate histone H3 at position Thr45, which may play a role during cell proliferation and/or differentiation.
authors.affiliation: Institute of Structural and Molecular Biology, University College London, London, WC1E 6BT, United Kingdom; Cancer Biology Department, National Cancer Institute, Cairo University, Cairo, Egypt.
authors.forename: Heba M S
authors.initials: HM
authors.lastname: Ismail
authors.forename: Paul J
authors.initials: PJ
authors.lastname: Hurd
authors.forename: Mahmoud I M
authors.initials: MI
authors.lastname: Khalil
authors.forename: Tony
authors.initials: T
authors.lastname: Kouzarides
authors.forename: Andrew
authors.initials: A
authors.lastname: Bannister
authors.forename: Ivan
authors.initials: I
authors.lastname: Gout
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002454:Cell Differentiation / Q000187:drug effects; D002458:Cell Fractionation; D045744:Cell Line, Tumor; D002843:Chromatin / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D018922:HL-60 Cells; D006657:Histones / Q000378:metabolism*; D006801:Humans; D051379:Mice; D008875:Middle Aged; D008969:Molecular Sequence Data; D041681:NIH 3T3 Cells; D015530:Nuclear Matrix / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D051057:Proto-Oncogene Proteins c-akt / Q000235:genetics / Q000378:metabolism; D034622:RNA Interference; D038762:Ribosomal Protein S6 Kinases, 70-kDa / Q000235:genetics / Q000378:metabolism; D038744:Ribosomal Protein S6 Kinases, 90-kDa / Q000235:genetics / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013912:Threonine / Q000378:metabolism*; D020298:U937 Cells
chemical_list: D002843:Chromatin; D006657:Histones; D013912:Threonine; D051057:Proto-Oncogene Proteins c-akt; D038762:Ribosomal Protein S6 Kinases, 70-kDa; D038744:Ribosomal Protein S6 Kinases, 90-kDa; C448901:ribosomal protein S6 kinase, 70kD, polypeptide 1; C069330:ribosomal protein S6 kinase, 90kDa, polypeptide 3; D013755:Tetradecanoylphorbol Acetate
keywords: CHROMATIN; H3 PHOSPHORYLATION; S6K2