Gene activation during muscle differentiation and the role of nonhistone chromosomal protein phosphorylation

Abstract

The phosphorylation of nuclear proteins, in a standard assay with nuclei isolated from chick muscle cell cultures, increases steadily with time in culture. This increase is shown to reflect increased nuclear protein kinase activity. Most of the phosphorylated protein is chromatin bound and so are the enzymes responsible for the phosphorylation. Analysis of chromosomal proteins on hydroxylapatite columns shows that 32P is incorporated almost entirely into nonhistone proteins, while the specific radioactivity of the histones is low and does not increase during cell culture. Experiments in which cells were grown at different initial densities and, consequently, underwent cell fusion at different times show that phosphorylation is dependent neither on cell density nor on the degree of cell fusion. Chick thigh fibroblasts, prepared by repeated subculture of fibroblasts from muscle cell preparations to eliminate myogenic cells, do not show the increase in phosphorylation with culture time, suggesting that the changes may be specific for primary myoblast cultures. These changes are not shown by thigh muscle nuclei isolated during development in the whole embryo. In fact, nuclear protein phosphorylation activity decreases rapidly in ovo with increasing embryonic age. Possible roles for phosphorylation of nonhistone chromosomal proteins in gene activation during myogenesis are discussed in the light of these observations.