Intact S49 mouse lymphoma cells were used as a model system to study the effects of cyclic AMP (CAMP) and its analogs on the phosphorylation of regulatory (R) subunit of type I CAMP-dependent protein kinase. Phosphorylation of R subunit was negligible in mutants deficient in adenylate cyclase; low levels of cAMP analogs, however, stimulated R subunit phosphorylation in these cells to rates comparable to those in wild-type cells. In both wildtype and adenylate cyclase-deficient cells, R subunit phosphorylation was inhibited by a variety of N6-substituted derivatives of CAMP; C-8-substituted derivatives were generally poor inhibitors. Two derivatives that were inactive as kinase activators (N6-carbamoylmethyl-5'-AMP and 2'-deoxy-N6-monobutyryl-CAMP) were also ineffective as inhibitors of R subunit phosphorylation. Preferential inhibition by Nbmodified cAMP analogs could not be ascribed simply to selectivity for the more aminoterminal (site I) of the two CAMPbinding sites in R subunit: Analog concentrations required for inhibition of R subunit phosphorylation were always higher than those required for activat ion of endogenous ki nase; 8-pi per id i no-CAMP, a C-8-su bst i t u ted derivative that is selective for CAMP-binding site I, was relatively ineffective as an inhibitor; and, although thresholds for activation of endogenous kinase by site Iselective analogs could be reduced markedly by coincubation with low levels of site Il-selective analogs, no such synergism was observed for the inhibitory effect. The uncoupling of cyclic nucleotide effects on R subunit phosphorylation from activation of endogenous protein kinase suggests that, in intact cells, activation of CAMP-dependent protein kinase requires more than one and fewer than four molecules of cyclic nucleotide.
'Site I corresponds to site "2" (Rannels and Corbin, 1980), site "A" (Qgreid and Dgkkeland, 1980), or the "labile" site (de Wit and Hoppe, 1981); site I1 corresponds to site "1" (Rannels and Corbin, 1980), site "B" (ggreid and Dbskeland, 1980), or the "stable" site (de Wit and Hoppe, 1981).