Phosphorylation of alphaB-crystallin in response to various types of stress.
Phosphorylation of alphaB-crystallin, a member of the hsp27 family, in human glioma (U373 MG) cells was stimulated by exposure of the cells to various stimuli, which included heat, arsenite, phorbol 12-myristate 13-acetate (PMA), okadaic acid, H2O2, anisomycin, and high concentrations of NaCl or sorbitol, but not in response to agents that elevated intracellular levels of cyclic AMP. Cells exposed to PMA together with okadaic acid yielded three bands of 32P-labeled alphaB-crystallin when immunoprecipitated samples were subjected to electrophoresis on an isoelectric focusing gel. All of the phosphorylated residues were identified as serine, an indication that three different serine residues can act as sites of phosphorylation in alphaB-crystallin. Structural analysis by mass spectrometry revealed that phosphorylation of alphaB-crystallin occurred at serines 19, 45, and 59. Dithiothreitol and staurosporine selectively inhibited the phosphorylation induced by arsenite and the phorbol ester, respectively. SB202190, an inhibitor of p38 mitogen-activated protein (MAP) kinase, suppressed the phosphorylation induced by arsenite, anisomycin, H2O2, sorbitol, NaCl, and heat shock, but not that induced by PMA and okadaic acid. The PMA-induced phosphorylation was selectively suppressed by an inhibitor of p44 MAP kinase kinase, PD98059. Although PMA and arsenite preferentially stimulated the phosphorylation of Ser-45 and Ser-59, respectively, as determined with antibodies that recognized the respective phosphorylated forms of alphaB-crystallin, all three sites were phosphorylated in response to each stimulus. These results suggest that p38 MAP kinase or p44 MAP kinase might be involved in the signal transduction cascade that leads to the phosphorylation of alphaB-crystallin. The phosphorylation of alphaB-crystallin was also enhanced in the heart and diaphragm when rats were exposed to heat stress (42 degrees C for 20 min).
Ito
H
H
Department of Biochemistry, Institute for Developmental Research, Aichi Human Service Center, 713-8 Kamiya, Kasugai, Aichi 480-03, Japan.
Okamoto
K
K
Nakayama
H
H
Isobe
T
T
Kato
K
K
D000818:Animals; D018053:Arsenites / Q000378:metabolism; D001665:Binding Sites; D015153:Blotting, Western; D003459:Crystallins / Q000378:metabolism*; D004229:Dithiothreitol / Q000494:pharmacology; D006360:Heat-Shock Proteins / Q000378:metabolism*; D006358:Hot Temperature; D006801:Humans; D018832:Molecular Chaperones / Q000378:metabolism*; D010449:Peptide Mapping; D010766:Phosphorylation; D051381:Rats; D019311:Staurosporine / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D018053:Arsenites; D003459:Crystallins; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D019311:Staurosporine; C015001:arsenite; D004229:Dithiothreitol