Nuclear protein kinase activities during the cell cycle of HeLa S3 cells

To ascertain the activity and substrate specificity of nuclear protein kinases during various stages of the cell cycle of HeLa S3 cells, a nuclear phospho-protein-enriched sample was extracted from synchronised cells and assayed in vitro in the presence of homologous substrates. The nuclear protein kinases increased in activity during S and G2 phase to a level that was twice that of kinases from early S phase cells. The activity was reduced during mitosis but increased again in G1 phase. When the phosphoproteins were separated into five fractions by cellulose-phosphate chromatography each fraction, though not homogenous, exhibited differences in activity. Variations in the activity of the protein kinase fractions were observed during the cell cycle, similar to those observed for the unfractionated kinases. Sodium dodecyl sulfate polyacrylamide gel electrophoretic analysis of the proteins phosphorylated by each of the five kinase fractions demonstrated a substrate specificity. The fractions also exhibited some cell cycle stage-specific preference for substrates; kinases from G1 cells phosphorylated mainly high molecular weight polypeptides, whereas lower molecular weight species were phosphorylated by kinases from the S, G2 and mitotic stages of the cell cycle. Inhibition of DNA and histone synthesis by cytosine arabinoside had no effect on the activity or substrate specificity of S phase kinases. Some kinase fractions phosphorylated histones as well as non-histone chromosomal proteins and this phosphorylation was also cell cycle stage dependent. The presence of histones in the in vitro assay influenced the ability of some fractions to phosphorylate particular non-histone polypeptides; non-histone proteins also appeared to affect the in vitro phosphorylation of histones.     

tsJT16, a cell-cycle ts mutant defective in a function operating soon after growth stimulation, fails to induce a primarily activated labile nuclear protein

tsJT16 is a temperature-sensitive (ts) mutant of rat fibroblasts that has a ts defect in a function operating soon after the growth stimulation from the G0 phase. After the growth stimulation, the cells express several cell-cycle-dependent genes at both temperatures while they fail at the nonpermissive temperature to synthesize a protein p70 identified on two-dimensional gel electrophoresis. Here we report that 1) synthesis of p70 began within 1 h of stimulation, continued up to the 7th hour and then decreased; 2) the half-life of p70 was shortened after 6 h after the stimulation; 3) p70 was localized in the nuclear fraction; 4) p70 was likely to be a primarily induced protein; 5) mRNA of p70 was supposed to be synthesized exclusively within 2 h of growth stimulation. These and the previous results suggest that p70 is a nuclear protein responsible for the early stage of transition of cells from the G0 toward the S phase and is induced via a different signal transduction sequence from that for the c-fos gene.