The complex of the subunits (RI␣, C␣) of cAMP-dependent protein kinase I (cA-PKI) was much more stable (K d ‫؍‬ 0.25 M) in the presence of excess cAMP than previously thought. The ternary complex of C subunit with cAMP-saturated RI␣ or RII␣ was devoid of catalytic activity against either peptide or physiological protein substrates. The ternary complex was destabilized by protein kinase substrate. Extrapolation from the in vitro data suggested about one-fourth of the C subunit to be in ternary complex in maximally cAMP-stimulated cells. Cells overexpressing either RI␣ or RII␣ showed decreased CRE-dependent gene induction in response to maximal cAMP stimulation. This could be explained by enhanced ternary complex formation. Modulation of ternary complex formation by the level of R subunit may represent a novel way of regulating the cAMP kinase activity in maximally cAMP-stimulated cells.The cAMP-dependent protein kinase (cA-PK) 1 differs from other kinases in having the catalytic site and the autoinhibitory site on two different subunits. The inactive cA-PK holoenzyme, when studied at nanomolar concentrations, dissociates into catalytic (C) and regulatory (R) subunits in the presence of cAMP (1). There is sparse evidence about the behavior of cA-PK at higher, more physiologically relevant, concentrations. Apparently, it is tacitly assumed that both isozymes (cA-PKI and cA-PKII) are completely dissociated by cAMP in the intact cell. The cAMP-induced decrease of fluorescent resonance transfer between microinjected C␣-FITC and RI␣-TRITC (2), and between genetically encoded fluorescent C␣ and RII␤ (3) has reinforced this notion, although such studies are not designed to tell whether the dissociation of cA-PK is complete or not (4). Recently, C/EBP␤ null mice were shown to have increased liver RI and RII, and attenuated cAMP-stimulated hepatic gene induction (5). Protein kinase inhibitor null mice, having 50% increased muscle RI␣, showed deficient cAMP-stimulated CREB phosphorylation and CRE-dependent gene expression in muscle (6). We have previously observed relatively more holoenzyme-associated kinase than expected from the tissue cAMP content during the pre-replicative cAMP surge in the regenerating liver, in which both RI and RII were up-regulated (7). These observations suggest the possibility that RI or RII subunits may have a negative effect on cA-PK dissociation even at high cAMP concentrations. We used the CRE-luciferase reporter gene to probe for dissociation of cA-PKI and cA-PKII in intact cells. Nuclear translocation of the C subunit requires cA-PK dissociation and is considered a prerequisite for phosphorylation of the CREB/CREM family of nuclear transcription factors, and hence for cAMP stimulation of CRE-governed reporter gene expression (8 -10). We show that cells overexpressing either hRI␣ or hRII␣, even when maximally cAMP challenged, had decreased cAMP responsive gene induction, suggesting that cAMP produced incomplete dissociation of either isozyme in intact cells. We will also present evidence that...

The IPC-81 cell line is derived from the transplantable BNML model of acute myelogenic leukemia (AML), known to be a reliable predictor of the clinical efficiency of antileukemic agents, like the first-line AML anthracycline drug daunorubicin (DNR). We show here that cAMP acted synergistically with DNR to induce IPC cell death. The DNR-induced death differed from that induced by cAMP by (1) not involving Bim induction, (2) being abrogated by GSK3β inhibitors, (3) by being promoted by the HSP90/p23 antagonist geldanamycin and truncated p23 and (4) by being insensitive to the CRE binding protein (CREB) antagonist ICER and to cyclin-dependent protein kinase (CDK) inhibitors. In contrast, the apoptosis induced by cAMP correlated tightly with Bim protein expression. It was abrogated by Bim (BCL2L11) downregulation, whether achieved by the CREB antagonist ICER, by CDK inhibitors, by Bim-directed RNAi, or by protein synthesis inhibitor. The forced expression of BimL killed IPC-81WT cells rapidly, Bcl2-overexpressing cells being partially resistant. The pivotal role of CREB and CDK activity for Bim transcription is unprecedented. It is also noteworthy that newly developed cAMP analogs specifically activating PKA isozyme I (PKA-I) were able to induce IPC cell apoptosis. Our findings support the notion that AML cells may possess targetable death pathways not exploited by common anti-cancer agents.