Two groups of mutant clones were isolated from Y1 adrenocortical tumor cells. One group, Y1(Kin), exhibited altered cytosolic cyclic AMP-dependent protein kinase activity; the second group, Y1(Cyc), exhibited diminished corticotropin-responsive adenylate cyclase activity. Steroidogenic responses to corticotropin and cyclic nucleotides closely paralleled cyclic AMP-dependent protein kinase activity in the YI(Kin) mutants. In Y1(Cyc) mutants, corticotropin had little effect on steroidogenesis, whereas cyclic nucleotides were fully active. These data imply that adenylate cyclase and cyclic

A somatic cell genetic approach has been used to evaluate the role of cyclic AMP-dependent protein kinase in ACTH action on adrenal steroidogenesis. A mutant clone, 8BrcAMPr-1, previously was isolated from a n ACTH-sensitive adrenocortical tumor cell line (clone Y1) following mutagenesis and selective growth in 8-bromoadenosine 3', 5'-monophosphate. This study demonstrates that the 8BrcAMPr-1 cells have an altered cyclic AMPdependent protein kinase. The protein kinase in the cytosol of the mutant characteristically requires, for half-maximal activity, concentrations of cyclic AMP 7-fold higher than those required by the enzyme in preparations from the parent. The cytosolic cyclic AMP-dependent protein kinases of Y1 and 8BrcAMP'-1 cells chromatograph similarly on columns of DEAE-cellulose. From each cell line, a major peak of activity (2 70% of recovered activity), designated a s Peak I, elutes with 0.04-0.06 M NaCl; a second peak of activity, designated as Peak 11, elutes with 0.12-0.14 M NaCl. Protein kinase activity in the Peak I fraction of mutant cells has a decreased apparent affinity (4-fold) for cyclic AMP relative to the corresponding fraction of parental Y1 cells. The protein kinase activities present in Peak I1 fractions from Y1 and mutant cells are indistinguishable. The protein kinase mutant exhibits poor steroidogenic responses to added ACTH and cyclic AMP; and as shown previously does not display the growth arrest and morphological changes produced in Y 1 by these agents. These results suggest that cyclic AMP-dependent protein kinase is important in the regulation of adrenal steroidogenesis, morphology and growth by ACTH.Cyclic AMP 3-dependent protein kinase is regarded as a n important mediator of ACTH action in the adrenal (Garren et al., '71). Current evidence, however, does not offer unequivocal support for this concept. Protein kinase activity in the adrenal is increased by ACTH, but this increase is not detected a t concentrations of ACTH which stimulate steroidogenesis submaximally (Richardson and Schulster, '73; Moyle et al., '76); a putative substrate of the cyclic AMP-dependent protein kinase, controlling the action of ACTH on steroidogenesis, has not been identified; cyclic GMP-dependent protein kinase, not the cyclic AMP-dependent enzyme, has been implicated as the regulator of steroidogenesis at low levels of ACTH (Sharma e t al., '74, '76).In this study, we have used a n experimental approach of somatic cell genetics to examine the role of the cyclic AMP-dependent protein kinase in the steroidogenic pathway stimulated by ACTH. An ACTH-responsive, mouse adrenocortical tumor cell line (Clone Y1; Yasumura e t al., '66) behaves in many respects like cells from normal adrenal cortex, and provides a useful model for investigating the mechanism of ACTH action. A mutant subclone of the Y1 population, (8BrcAMPr-1), has a genetic defect in ACTH-stimulated steroidogenesis at an early step beyond cyclic AMP formation (Schimmer e t al., '77). We now dem-

In Y1 adrenocortical tumor cells, corticotropin (ACTH), cyclic AMP, and 8-bromoadenosine 3',5'-monophosphate (8BrcAMP) stimulated ornithine decarboxylase activity (L-ornithine carboxy-lyase, EC 4.1.1.17) and steroidogenesis. The concentrations required for half-maximal activation of ornithine decarboxylase were 60 pM for ACTH and 1 mM for 8BrcAMP; the concentrations required for half-maximal activation of steroidogenesis were 50 pM for ACTH and 0.2 mM for 8BrcAMP. Ornithine decarboxylase activity increased 1.5 hr after the addition of these agents, reached a maximum between 4 and 6 hr, and then declined. Mutant clones with impaired ACTH-responsive adenylate cyclase systems [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] did not respond to ACTH with increased ornithine decarboxylase activity, but they responded normally to added cyclic AMP. These results indicate that adenylate cyclase and cyclic AMP are necessary for the stimulation of ornithine decarboxylase activity by ACTH. In a series of Y1(Kin) mutants with altered cyclic AMP-dependent protein kinase activities (ATP:protein phosphotransferase, EC 2.7.1.37), the effects of ACTH on ornithine decarboxylase also were attenuated. These findings suggest that cyclic AMP-dependent protein kinase also plays a necessary role in the stimulation of ornithine decarboxylase activity by ACTH. The effects of ACTH on ornithine decarboxylase in the Kin mutants, however, were quantitatively different from the effects on steroidogenesis and did not closely reflect the degree of defect in cyclic AMP-dependent protein kinase activity. These differences suggest that the pathways of ACTH action leading to stimulation of steroidogenesis and ornithine decarboxylase activity diverge subsequent to activation of the protein kinase.