title: Essential role of the NH2-terminal WD/EPF motif in the phosphorylation-activated protective function of mammalian Hsp27.
abstract: Hsp27 is expressed at high levels after mild heat shock and contributes to making cells extremely resistant to subsequent treatments. The activity of the protein is regulated at the transcriptional level, but also by phosphorylation, which occurs rapidly during stress and is responsible for causing the dissociation of large 700-kDa Hsp27 oligomers into dimers. We investigated the mechanism by which phosphorylation and oligomerization modulate the protective activity of Chinese hamster Hsp27. In contrast to oligomer dissociation, which only required Ser90 phosphorylation, activation of Hsp27 thermoprotective activity required the phosphorylation of both Ser90 and Ser15. Replacement of Ser90 by Ala90, which prevented the dissociation of the oligomer upon stress, did cause a severe defect in the protective activity. Dissociation was, however, not a sufficient condition to activate the protein because replacement of Ser15 by Ala15, which caused little effect in the oligomeric organization of the protein, also yielded an inactive protein. Analyzes of mutants with short deletions in the NH2 terminus identified the Hsp27 WD/EPF or PF-rich domain as essential for protection, maintenance of the oligomeric structure, and in vitro chaperone activity of the protein. In light of a three-dimensional model of Hsp27 based on the crystallographic structure of wheat Hsp16.9, we propose that the conserved WD/EPF motif of mammalian Hsp27 mediates important intramolecular interactions with hydrophic surfaces of the alpha-crystallin domain of the protein. These interactions are destabilized by Ser90 phosphorylation, making the motif free to interact with heterologous molecular targets upon the additional phosphorylation of the nearby Ser15.
authors.affiliation: Centre de recherche en cancérologie de l'Université Laval, L'Hôtel-Dieu de Québec, Centre hospitalier universitaire de Québec, 9 rue McMahon, Québec G1R 2J6, Canada.
authors.forename: Jimmy R
authors.initials: JR
authors.lastname: Thériault
authors.forename: Herman
authors.initials: H
authors.lastname: Lambert
authors.forename: Aura T
authors.initials: AT
authors.lastname: Chávez-Zobel
authors.forename: Gabriel
authors.initials: G
authors.lastname: Charest
authors.forename: Pierre
authors.initials: P
authors.lastname: Lavigne
authors.forename: Jacques
authors.initials: J
authors.lastname: Landry
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D006224:Cricetinae; D006360:Heat-Shock Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010766:Phosphorylation; D016415:Sequence Alignment; D013329:Structure-Activity Relationship; D013696:Temperature
chemical_list: D006360:Heat-Shock Proteins