title: Human HSP27 is phosphorylated at serines 78 and 82 by heat shock and mitogen-activated kinases that recognize the same amino acid motif as S6 kinase II.
abstract: The intracellular concentration of the 27-kDa mammalian heat shock protein, HSP27, increases several-fold after heat and other metabolic stresses and is closely associated with the acquisition of thermotolerance. Posttranslational modifications may also affect the function of HSP27. Heat shock of HeLa cell cultures, or treatment with arsenite, phorbol ester, or tumor necrosis factor, caused a rapid phosphorylation of preexisting HSP27 and the appearance of three phosphorylated isoforms, HSP27 B, C, and D. Digestion with trypsin and fractionation of the peptides by reverse phase high performance liquid chromatography revealed three 32P-labeled phosphopeptides. Microsequence analysis identified peak I as Ala76-Leu77-Ser78-Arg79 and peak II as Gln80-Leu81-Ser82-Ser83-Gly84-Val85- Ser86-Glu87-Ile88-Arg89; peak III contained the undigested peptide pair Ala76-Arg89. Ser82 was the major site and Ser78 the minor site of phosphorylation. Mutant proteins with Ser78 or Ser82 altered to glycine or Ser78-Ser82 double mutants were phosphorylated to reduced extents in vivo after heat or arsenite treatment. Ser78 and Ser82 (and Ser15) occur in the sequence motif RXXS, which is recognized by ribosomal protein S6 kinase II. Mitogenic stimulation of serum-deprived, Go-arrested Chinese hamster cells with serum, thrombin, or fibroblast growth factor also stimulated phosphorylation of HSP27 Ser78 and Ser82, and mitogenic stimulation and heat shock activated protein kinase activities that phosphorylated HSP27 and protein S6 in vitro. These results suggest that HSP27 may exert phosphorylation-activated functions linked with growth signaling pathways in unstressed cells. A homeostatic function at this level could protect cells from adverse effects of signal transduction systems which may be activated inappropriately during stress.
authors.affiliation: Centre de Recherche en Cancérologie, Université Laval, Québec, Canada.
authors.forename: J
authors.initials: J
authors.lastname: Landry
authors.forename: H
authors.initials: H
authors.lastname: Lambert
authors.forename: M
authors.initials: M
authors.lastname: Zhou
authors.forename: J N
authors.initials: JN
authors.lastname: Lavoie
authors.forename: E
authors.initials: E
authors.lastname: Hickey
authors.forename: L A
authors.initials: LA
authors.lastname: Weber
authors.forename: C W
authors.initials: CW
authors.lastname: Anderson
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001151:Arsenic / Q000494:pharmacology; D018053:Arsenites; D015153:Blotting, Western; D002851:Chromatography, High Pressure Liquid; D006224:Cricetinae; D003412:Cricetulus; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D005346:Fibroblast Growth Factors / Q000378:metabolism; D006367:HeLa Cells; D006360:Heat-Shock Proteins / Q000378:metabolism*; D006801:Humans; D008934:Mitogens; D008969:Molecular Sequence Data; D010766:Phosphorylation / Q000187:drug effects; D011494:Protein Kinases / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D019893:Ribosomal Protein S6 Kinases; D012694:Serine / Q000378:metabolism*; D017670:Sodium Compounds; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013917:Thrombin / Q000378:metabolism; D014357:Trypsin / Q000737:chemistry; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
chemical_list: D018053:Arsenites; D006360:Heat-Shock Proteins; D008934:Mitogens; D017670:Sodium Compounds; D014409:Tumor Necrosis Factor-alpha; D012694:Serine; C017947:sodium arsenite; D005346:Fibroblast Growth Factors; D011494:Protein Kinases; D019893:Ribosomal Protein S6 Kinases; D014357:Trypsin; D013917:Thrombin; D001151:Arsenic; D013755:Tetradecanoylphorbol Acetate