G1 specific increases in cyclic AMP levels and protein kinase activity in Chinese hamster ovary cells.

ABSTRACT

Chinese hamster ovary cells were synchronized by selective detachment of cells in mitosis. The adenosine 3'5'-cyclic monophosphate (cyclic AMP) intracellular concentrations and cyclic AMP-dependent protein kinase activities were measured as these cells traversed G1 phase and entered S phase. Protein kinase activity, assayed in the presence or absence of saturating exogenous cyclic AMP in the reaction mixture, was lowest in early G1 phase (2 h after mitosis), increased 2-fold (plus exogenous cyclic AMP in reaction mixture) or 3.5-fold (minus cyclic AMP in reaction mixture) to maximum values in mid to late G1 phase (4-5 h after mitosis), and then decreased as cells entered S phase. Intracellular cyclic AMP concentrations were minimal 1 h after mitosis, increased 5-fold to maximum levels at 4-6 after mitosis, and decreased as cells entered S phase. Similar to the fluctuations in intracellular cyclic AMP, the cyclic AMP-dependent protein kinase activity ratio increased more than 40% in late G1 or early S phase. Puromycin (either 10 mug/ml or 50 mug/ml) administered 1 h after mitosis inhibited cyclic AMP-dependent protein kinase activity up to 50% by 5 h after mitosis, while similar treatment (10 mug/ml) had no effect on the increase in cyclic AMP formation. These data demonstrate that (1) total protein kinase activity changed during G1 phase and this increase was dependent on new protein synthesis; (2) the increased intracellular concentrations of cyclic AMP were not dependent on new protein synthesis; and (3) the activation of cyclic AMP-dependent protein kinase was temporally coordinated with increased intracellular concentration of cycli AMP as Chinese hamster ovary cells traversed G1 phase and entered S phase. These results suggest that cyclic AMP acts during G1 phase to regulate the activation of cyclic AMP-dependent protein kinase.