Presence of cyclic-AMP-independent protein kinase activity in RNA-binding proteins of embryonic chicken muscle.

Abstract

A class of protein(s) with high affinity for RNA was isolated by affinity chromatography on poly(U)‐Sepharose from an extract of embryonic chicken muscle which had been freed of ribosomes and cytoplasmic messenger ribonucleoprotein complexes. The RNA‐binding protein comprised about 1% of the total protein of the post‐ribosomal extract. Analysis of the polypeptide composition of the RNA‐binding proteins by two‐dimensional gel electrophoresis, using separation according to charge in the first dimension and molecular weight in the second, showed the presence of at least 37 polypeptides. These polypeptides were non‐ribosomal in nature. The major class of polypeptides falls into three molecular weight groups with Mr of 34000–41000, 46000–54000 and 68000–78000. Nine of these polypeptides showed similar electrophoretic migration properties to that of polypeptides from nonpolysomal cytoplasmic messenger ribonucleoprotein. Furthermore, four polypeptides, including one acidic 38000‐Mr polypeptide, displayed electrophoretic migration properties similar to those of polypeptides present in crude initiation factor preparation.The RNA‐binding proteins can form a ribonucleoprotein complex(es) when incubated with either synthetic or natural RNAs. Furthermore, in competition binding studies, mRNAs were found to be more efficient in complexing with the RNA‐binding proteins than were non‐mRNA molecules.A cyclic‐AMP‐independent protein kinase activity was detected in the RNA‐binding proteins. This protein kinase phosphorylates several polypeptides of the RNA‐binding proteins. Among these, the 38000‐Mr acidic polypeptide was the best acceptor of phosphate group(s). In contrast, ribosomal proteins were found to be poor substrates for the protein kinase. The acidic 38000‐Mr polypeptide of RNA‐binding protein, which is most intensely phosphorylated by the endogenous protein kinase, also appeared to be the common polypeptide found in the crude initiation factor preparation. The similarity of this protein kinase with the cyclic‐AMP‐independent protein kinase activity, previously reported in the nonpolysomal cytoplasmic messenger ribonucleoprotein complexes of embryonic chicken muscle [Bag, J. and Sells, B. H. (1979) J. Biol. Chem. 254, 3137–3140] has been discussed.These RNA‐binding proteins interfered with translation of both homologous and heterologous mRNAs in the rabbit reticulocyte cell‐free system. The effects obtained, however, were concentration‐dependent in the range 7–30 μg/ml: the proteins inhibited polypeptide formation while at higher concentrations (70 μg/ml) they stimulated translation. The presence of both inhibitor and activator of mRNA translation in RNA‐binding protein has been discussed.