title: The dynamics of histone H2A ubiquitination in HeLa cells exposed to rapamycin, ethanol, hydroxyurea, ER stress, heat shock and DNA damage.
abstract: Polyubiquitination plays key roles in proteasome-dependent and independent cellular events, whereas monoubiquitination is involved in gene expression, DNA repair, protein-protein interaction, and protein trafficking. We previously developed an FK2 antibody, which specifically recognizes poly-Ub moieties but not free Ub. To elucidate the role of Ub conjugation in response to cellular stress, we used FK2 to investigate whether chemical stress (rapamycin, ethanol, or hydroxyurea), ER stress (thapsigargin or tunicamycin), heat shock or DNA damage (H2O2 or methyl methanesulfonate) affect the formation of Ub conjugates including histone H2A (hH2A) ubiquitination. First, we found that all forms of stress tested increased poly-ubiquitinated proteins in HeLa cells. Furthermore, rapamycin and hydroxyurea treatment, and ER stress increased ubiquitination of hH2A, while methyl methanesulfonate (MMS) treatment induced deubiquitination of hH2A. The ethanol and H2O2 treatments, and heat shock transiently induced hH2A de-ubiquitination, although deubiquitinated hH2A were ubiquitinated again by subsequent cultivation. We also revealed that FK2 reacts with not only polyubiquitinated proteins but also mono-ubiquitinated hH2A. With the exception of MMS, all forms of stress tested increased the acetylation of K5-hH2A, K9-hH3 and K8-hH4 in addition to ubiquitination. K118 and K119 of hH2A were ubiquitinated in cells under normal conditions, and K119 was the major ubiquitination site. The MMS-treatment and heat shock induced the deubiquitination of both K118 and K119-histone H2A. Interestingly, MMS treatment did not affect cell HeLa cell viability expressing double-mutant hH2A (KK118,119AA-hH2A), while heat shock slightly but significantly decreased viability of double-mutant hH2A expressing cells, indicating that ubiquitination of both sites associates with recovery from heat shock but not MMS treatment. Thus, we characterized FK2 reactivity and demonstrated that various stresses alter the ubiquitination status, particularly ubiquitinated hH2A and with histone acetylation, and highlight the physiological importance of hH2A ubiquitination after exposure to stress stimuli.
authors.affiliation: Department of Cell Biology, Kyoto Pharmaceutical University, Misasagi-Shichonocho 1, Yamashinaku, Kyoto, 607-8412, Japan.
authors.forename: Shiori
authors.initials: S
authors.lastname: Nakata
authors.affiliation: Department of Cell Biology, Kyoto Pharmaceutical University, Misasagi-Shichonocho 1, Yamashinaku, Kyoto, 607-8412, Japan.
authors.forename: Tadashi
authors.initials: T
authors.lastname: Watanabe
authors.affiliation: Department of Pathophysiology and Therapeutics, Faculty of Pharmaceutical Sciences, Hokkaido University, Kita-12 Nishi-6, Kitaku, Sapporo, 060-0812, Japan.
authors.forename: Koji
authors.initials: K
authors.lastname: Nakagawa
authors.affiliation: Department of Pathophysiology and Therapeutics, Faculty of Pharmaceutical Sciences, Hokkaido University, Kita-12 Nishi-6, Kitaku, Sapporo, 060-0812, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Takeda
authors.affiliation: Chemical Genetics Laboratory, RIKEN, 2-1 Hirosawa, Wako, Saitama, 351-0198, Japan.
authors.forename: Akihiro
authors.initials: A
authors.lastname: Ito
authors.affiliation: Department of Cell Biology, Kyoto Pharmaceutical University, Misasagi-Shichonocho 1, Yamashinaku, Kyoto, 607-8412, Japan. Electronic address: fuji2@mb.kyoto-phu.ac.jp.
authors.forename: Masahiro
authors.initials: M
authors.lastname: Fujimuro
mesh_terms: D000107:Acetylation / Q000187:drug effects; D004249:DNA Damage; D059865:Endoplasmic Reticulum Stress; D000431:Ethanol / Q000633:toxicity; D006367:HeLa Cells; D018869:Heat-Shock Response; D006657:Histones / Q000378:metabolism*; D006801:Humans; D006861:Hydrogen Peroxide / Q000633:toxicity; D006918:Hydroxyurea / Q000633:toxicity; D008741:Methyl Methanesulfonate / Q000633:toxicity; D020123:Sirolimus / Q000633:toxicity; D013347:Subcellular Fractions / Q000187:drug effects / Q000378:metabolism; D057149:Ubiquitinated Proteins / Q000378:metabolism; D054875:Ubiquitination / Q000187:drug effects*
chemical_list: D006657:Histones; D057149:Ubiquitinated Proteins; D000431:Ethanol; D008741:Methyl Methanesulfonate; D006861:Hydrogen Peroxide; D020123:Sirolimus; D006918:Hydroxyurea
keywords: DNA damage; ER stress; Heat shock; Histone H2A; Stress response; Ubiquitin