Two distinct forms of MAPKAP kinase-2 in adult cardiac ventricular myocytes

Biochemistry. 2000 May 23;39(20):6145-56. doi: 10.1021/bi9928389.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Chromatography, Ion Exchange
  • Dogs
  • HSP27 Heat-Shock Proteins
  • HeLa Cells
  • Heat-Shock Proteins / metabolism
  • Humans
  • Intracellular Signaling Peptides and Proteins
  • Isoenzymes / isolation & purification
  • Isoenzymes / metabolism
  • Male
  • Molecular Sequence Data
  • Myocardium / cytology
  • Myocardium / enzymology*
  • Neoplasm Proteins / isolation & purification
  • Neoplasm Proteins / metabolism
  • PC12 Cells
  • Phosphorylation
  • Precipitin Tests
  • Protein-Serine-Threonine Kinases / isolation & purification
  • Protein-Serine-Threonine Kinases / metabolism*
  • Rabbits
  • Rats
  • Rats, Sprague-Dawley

Substances

  • HSP27 Heat-Shock Proteins
  • HSPB1 protein, human
  • Heat-Shock Proteins
  • Hspb1 protein, rat
  • Intracellular Signaling Peptides and Proteins
  • Isoenzymes
  • Neoplasm Proteins
  • MAP-kinase-activated kinase 2
  • Protein-Serine-Threonine Kinases