A 23-kDa protein as a substrate for protein kinase C in bovine neutrophils. Purification and partial characterization.
In 32Pi-loaded bovine neutrophils stimulated with phorbol myristate acetate (PMA), radioactivity was preferentially incorporated into a protein of low molecular mass, suggesting a PKC-dependent phosphorylation. This protein, termed 23-kDa protein, was predominantly localized in the cytosol. It was purified from bovine neutrophil cytosol by a series of chromatographic steps, including ion exchange on DE-52 cellulose and Mono Q, and filtration on Bio-Gel P60 in the presence of mercaptoethanol and urea. The apparent molecular mass of the purified protein, assessed by SDS-PAGE and mercaptoethanol by reference to protein markers, ranged between 20 and 23 kDa, depending on the percentage of polyacrylamide and conditions of migration. In the absence of mercaptoethanol, a dimer accumulated. Homogeneity of the 23-kDa protein was verified by 2D-PAGE analysis. Some properties of the 23-kDa protein, including its amino acid composition, were determined. Gel isoelectric focusing (IEF) of the purified 23-kDa protein followed by Coomassie blue staining allowed the visualization of four discrete protein bands with isoelectric points ranging between pH 6.3 and 6.7. Phosphorylation of the 23-kDa protein by [gamma-32P]ATP in the presence of bovine neutrophil PKC supplemented with Ca2+, phosphatidylserine, and diacylglycerol or with PMA occurred on serine and required the presence of mercaptoethanol. The apparent KM of ATP was 9 microM. The 23-kDa protein was also phosphorylated by PKM, the catalytic fragment of PKC obtained after removal of the regulatory domain, but not by cAMP-dependent protein kinase.(ABSTRACT TRUNCATED AT 250 WORDS)
Stasia
M J
MJ
Département de Recherche Fondamentale, Centre d'Etudes Nucléaires, Genoble, France.
Dianoux
A C
AC
Vignais
P V
PV
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002417:Cattle; D004586:Electrophoresis; D019204:GTP-Binding Proteins / Q000493:pharmacokinetics; D008970:Molecular Weight; D009218:Myosins / Q000302:isolation & purification; D009504:Neutrophils / Q000187:drug effects / Q000201:enzymology*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D013379:Substrate Specificity / Q000187:drug effects; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D000596:Amino Acids; D011493:Protein Kinase C; D019204:GTP-Binding Proteins; D009218:Myosins; D013755:Tetradecanoylphorbol Acetate