Two distinct forms of MAPKAP kinase-2 in adult cardiac ventricular myocytes.
Hsp27 kinase activities were studied in adult rat ventricular myocytes following sequential chromatography on Mono Q and Mono S. A basal level of activity was present following cell isolation. FPLC on Mono Q revealed three peaks of activity, peaks 'a', 'b', and 'c'. A fourth peak, 'd', was detected upon subsequent chromatography of the Mono Q flow-through on Mono S. Immunoblotting revealed that peaks 'a', 'b', and 'c' contained predominantly a 49 kDa form of MAPKAP kinase-2. Peak 'd' contained a 43 kDa form. 'In-gel' kinase assays using hsp27 indicated both forms of MAPKAP kinase-2 were active. No other bands of hsp27 kinase activity were detected. Both forms of hsp27 kinase immunoprecipitated with a MAPKAP kinase-2 antibody and have therefore been named MAPKAP kinase-2alpha (p49) and MAPKAP kinase-2beta (p43). MAPKAP kinase-2beta chromatographed on Superose 12 as a 60.7 kDa monomer whereas the behavior of MAPKAP kinase-2alpha suggested both a 65.7 kDa monomer and higher molecular mass complexes. Both activities phosphorylated hsp27 on serine residues, and two-dimensional phosphopeptide mapping indicated the same sites were phosphorylated. A tumor-promoting phorbol ester, phorbol 12-myristate 13-acetate (PMA), stimulated both MAPKAP kinase-2alpha and MAPKAP kinase-2beta activity. Inhibition of MEK activation with PD 98059 or p38alpha/beta MAP kinase activity with SB203580 blocked activation by PMA. However, whereas PD 98059 inhibited only the PMA-stimulated activation, SB203580 inhibited both PMA-stimulated and basal hsp27 phosphorylation. These data demonstrate the presence of two forms of MAPKAP kinase-2 in adult ventricular myocytes. Both forms are activated indirectly by the ERK MAP kinase pathway and directly by p38 MAP kinase but independently regulated.
Chevalier
D
D
Institut de Cardiologie de Montréal, Centre de Recherche, 5000 rue Bélanger est, Montréal, PQ, Canada, H1T 1C8.
Allen
B G
BG
D000595:Amino Acid Sequence; D000818:Animals; D002852:Chromatography, Ion Exchange; D004285:Dogs; D055551:HSP27 Heat-Shock Proteins; D006367:HeLa Cells; D006360:Heat-Shock Proteins / Q000378:metabolism; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism; D008297:Male; D018832:Molecular Chaperones; D008969:Molecular Sequence Data; D009206:Myocardium / Q000166:cytology / Q000201:enzymology*; D009363:Neoplasm Proteins / Q000302:isolation & purification / Q000378:metabolism; D016716:PC12 Cells; D010766:Phosphorylation; D011233:Precipitin Tests; D017346:Protein Serine-Threonine Kinases / Q000302:isolation & purification / Q000378:metabolism*; D011817:Rabbits; D051381:Rats; D017207:Rats, Sprague-Dawley; D000099190:MAP-Kinase-Activated Kinase 2
D055551:HSP27 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D047908:Intracellular Signaling Peptides and Proteins; D007527:Isoenzymes; D018832:Molecular Chaperones; D009363:Neoplasm Proteins; D017346:Protein Serine-Threonine Kinases; D000099190:MAP-Kinase-Activated Kinase 2; C057725:HSPB1 protein, human; C486114:Hspb1 protein, rat