Studies on the phosphorylation and synthesis of type I regulatory subunit of cyclic AMP-dependent protein kinase in intact S49 mouse lymphoma cells.

Phosphorylation and synthesis of type I regulatory subunit (RI) of cAMP-dependent protein kinase were studied using two-dimensional polyacrylamide gel electrophoresis of [35S]methionine-labeled proteins from intact S49 mouse lymphoma cells. [32P]Phosphate labeling, peptide mapping, and acid hydrolysis confirm that charge heterogeneity in RI results from phosphorylation of a single serine residue. In drug-free cells, phosphorylation proceeds to a steady state proportion of 90 to 95% of total RI with a half-time of about 25 min. The rate and steady state extent of RI phosphorylation are reduced by some, but not all, agents causing intracellular kinase activation. These results suggest that RI might assume different conformations in association with different amounts of cAMP or different analogs of cAMP. Endogenous kinase activation has no immediate effect on RI synthesis but leads to a moderate increase in RI synthesis after several hours; this induction occurs with all agents tested. Mutants of S49 cells lacking catalytic activity of cAMP-dependent protein kinase exhibit reduced phosphorylation and synthesis of RI. Comparative studies suggest that the phosphorylation of RI and its induction by kinase activation are fairly general phenomena; the extent of RI phosphorylation and the relative rate of RI synthesis are variable among cell types.