title: Regulation of Hsp27 oligomerization, chaperone function, and protective activity against oxidative stress/tumor necrosis factor alpha by phosphorylation.
abstract: The small heat shock proteins (sHsps) from human (Hsp27) and mouse (Hsp25) form large oligomers which can act as molecular chaperones in vitro and protect cells from heat shock and oxidative stress when overexpressed. In addition, mammalian sHsps are rapidly phosphorylated by MAPKAP kinase 2/3 at two or three serine residues in response to various extracellular stresses. Here we analyze the effect of sHsp phosphorylation on its quaternary structure, chaperone function, and protection against oxidative stress. We show that in vitro phosphorylation of recombinant sHsp as well as molecular mimicry of Hsp27 phosphorylation lead to a significant decrease of the oligomeric size. We demonstrate that both phosphorylated sHsps and the triple mutant Hsp27-S15D,S78D,S82D show significantly decreased abilities to act as molecular chaperones suppressing thermal denaturation and facilitating refolding of citrate synthase in vitro. In parallel, Hsp27 and its mutants were analyzed for their ability to confer resistance against oxidative stress when overexpressed in L929 and 13.S.1.24 cells. While wild type Hsp27 confers resistance, the triple mutant S15D,S78D,S82D cannot protect against oxidative stress effectively. These data indicate that large oligomers of sHsps are necessary for chaperone action and resistance against oxidative stress whereas phosphorylation down-regulates these activities by dissociation of sHsp complexes to tetramers.
authors.affiliation: Max-Delbrück-Centrum für Molecule Medizin, Robert-Rössle-Str. 10, D-13122 Berlin, Germany.
authors.forename: T
authors.initials: T
authors.lastname: Rogalla
authors.forename: M
authors.initials: M
authors.lastname: Ehrnsperger
authors.forename: X
authors.initials: X
authors.lastname: Preville
authors.forename: A
authors.initials: A
authors.lastname: Kotlyarov
authors.forename: G
authors.initials: G
authors.lastname: Lutsch
authors.forename: C
authors.initials: C
authors.lastname: Ducasse
authors.forename: C
authors.initials: C
authors.lastname: Paul
authors.forename: M
authors.initials: M
authors.lastname: Wieske
authors.forename: A P
authors.initials: AP
authors.lastname: Arrigo
authors.forename: J
authors.initials: J
authors.lastname: Buchner
authors.forename: M
authors.initials: M
authors.lastname: Gaestel
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D002460:Cell Line; D002942:Circular Dichroism; D005347:Fibroblasts / Q000378:metabolism; D055551:HSP27 Heat-Shock Proteins; D006360:Heat-Shock Proteins / Q000502:physiology*; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D051379:Mice; D018832:Molecular Chaperones; D018716:Molecular Mimicry; D016297:Mutagenesis, Site-Directed; D009363:Neoplasm Proteins / Q000235:genetics / Q000502:physiology*; D018384:Oxidative Stress; D010766:Phosphorylation; D011108:Polymers / Q000378:metabolism; D011487:Protein Conformation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D051381:Rats; D012694:Serine / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*; D014812:Vitamin K / Q000494:pharmacology; D000099190:MAP-Kinase-Activated Kinase 2
chemical_list: D055551:HSP27 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D047908:Intracellular Signaling Peptides and Proteins; D018832:Molecular Chaperones; D009363:Neoplasm Proteins; D011108:Polymers; D017346:Protein Serine-Threonine Kinases; D012694:Serine; D014409:Tumor Necrosis Factor-alpha; D014812:Vitamin K; D000099190:MAP-Kinase-Activated Kinase 2; C057725:HSPB1 protein, human; C486113:Hsbp1 protein, mouse; C486114:Hspb1 protein, rat