title: Phosphorylation analysis of 90 kDa heat shock protein within the cytosolic arylhydrocarbon receptor complex.
abstract: The arylhydrocarbon receptor (AhR) functions as a ligand-activated transcription factor that regulates the transcription of genes encoding xenobiotic metabolizing enzymes and also mediates most of the toxic effects caused by dioxins and polycyclic aromatic hydrocarbons. The cytosolic AhR complex exists as a transcriptionally cryptic complex, consisting of the 90 kDa heat shock protein (HSP90) and the hepatitis B virus X-associated protein 2 (XAP2). The posttranslational modifications, especially phosphorylation, of the cytosolic AhR-HSP90-XAP2 complex are poorly understood, although the phosphorylation of a transcriptionally active heterodimer of AhR and an AhR nuclear translocator is critically involved in AhR function. To reveal the phosphorylation status involved in AhR function, we used mass spectrometry to determine the site-specific phosphorylation of the steady-state cytosolic AhR complex, prepared from Chinese hamster ovary cells stably expressing mouse AhR. We identified phosphorylations of the HSP90 subunits within the AhR complex at Ser225 and Ser254 of HSP90beta and Ser230 of HSP90alpha. By site-directed mutagenesis, these serine residues were substituted with alanine and glutamic acid to elucidate the role of the HSP90beta serine phosphorylations in the AhR function. Immunoprecipitation assays using COS7 transfectants showed that the replacement of Ser225 and Ser254 by Ala, S225/254A, increased the binding affinity for AhR, as compared with the Glu replacement. In a ligand-induced AhR transcription activity assay using Hepa1 transfectants, the S255/254A mutant exhibited more potent transcription activity than the S225/254E mutant, which had activity similar to that of wild-type HSP90beta. These results suggest that the phosphorylations in the charged linker region of the HSP90 molecule modulate the formation of the functional cytosolic AhR complex.
authors.affiliation: Protein Research Group, RIKEN Genomic Sciences Center, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama 230-0045, Japan.
authors.forename: Hideo
authors.initials: H
authors.lastname: Ogiso
authors.forename: Noriko
authors.initials: N
authors.lastname: Kagi
authors.forename: Eiko
authors.initials: E
authors.lastname: Matsumoto
authors.forename: Madoka
authors.initials: M
authors.lastname: Nishimoto
authors.forename: Ryoichi
authors.initials: R
authors.lastname: Arai
authors.forename: Mikako
authors.initials: M
authors.lastname: Shirouzu
authors.forename: Junsei
authors.initials: J
authors.lastname: Mimura
authors.forename: Yoshiaki
authors.initials: Y
authors.lastname: Fujii-Kuriyama
authors.forename: Shigeyuki
authors.initials: S
authors.lastname: Yokoyama
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D019556:COS Cells; D045744:Cell Line, Tumor; D002522:Chlorocebus aethiops; D006224:Cricetinae; D003600:Cytosol / Q000378:metabolism*; D018841:HSP90 Heat-Shock Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D020033:Protein Isoforms / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D011506:Proteins / Q000378:metabolism; D018336:Receptors, Aryl Hydrocarbon / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism* / Q000502:physiology; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012694:Serine / Q000235:genetics / Q000378:metabolism; D014162:Transfection
chemical_list: D018841:HSP90 Heat-Shock Proteins; C493165:HSP90AB1 protein, human; D047908:Intracellular Signaling Peptides and Proteins; D010750:Phosphoproteins; D020033:Protein Isoforms; D011506:Proteins; D018336:Receptors, Aryl Hydrocarbon; D011994:Recombinant Proteins; C103265:aryl hydrocarbon receptor-interacting protein; D012694:Serine