A 23-kDa protein as a substrate for protein kinase C in bovine neutrophils. Purification and partial characterization

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Abstract
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, we 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 23kDa protein [.gamma.-32P]ATP in the presence of bovine neutophil 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 .mu.M. 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. IEF of the 32P-labeled 23-kDa protein followed by autoradiography revealed four discrete bands with distinct isoelectric points similar to those of the bands stained by Coomassie blue after IEF on nonlabeled 23-kDa protein; the more acidic bands were the more labeled. The bands of the 23-kDa protein resolved by IEF and transferred to nitrocellulose showed ability to bind [35]GTP-.gamma.-S. The immunoreactivity of antibodies raised in rabbits against the bovine neutrophil 23-kDa protein was demonstrated on immunoblots after SDS-PAGE. Binding of antibodies prevented the PKC-dependent phosphorylation of the protein. The antibodies reacted with a 23-kDa protein present in a bovine liver extract, possibly due to the macrographic Kupffer cells present in the liver tissue. No reaction occurred in extracts from bovine heart, skeletal muscle, platelets, and brain. Compared to human and rabbit neutrophils, the 23-kDa protein from bovine neutrophils appears to be overexpressed. A similarly sized protein from neutrophils, which is the regulatory light chain of myosin isolated from bovine neutrophil cytosol, was phosphorylated in the presence of [.gamma.32P]ATP by neutrophil PKC, like the 23-kDa protein. Yet the two proteins differed in their amino acid composition, the nature of their phosphorylated amino acids, and the absence of immunological cross-reactivity. The 23-kDa protein differed also from several other proteins of similar molecular mass that have been identified in neutrophils, namely calmodulin, the small subunit of the low-potential cytochrome b, and a low molecular weight protein which is ADP-ribosylated by the botulinium toxin.