Formation of Inactive cAMP-saturated Holoenzyme of cAMP-dependent Protein Kinase under Physiological Conditions

Abstract

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...

Differential Effects of Substrate on Type I and Type II PKA Holoenzyme Dissociation

“…On the other hand, it is known that increases in intracellular cAMP significantly increase concentrations of free C subunit in cells, especially in the nucleus (26), so some dissociation of holoenzyme by cAMP must occur. The recent studies by Døskelund and colleagues (11), as well as earlier fluorescence studies (9,10), suggest that the types I and II holoenzymes are much more stable in the presence of cAMP than previously thought. The results of the experiments presented herein confirm and extend this idea, using an equilibrium method that does not require any protein modifications.…”

“…Døskelund et al (11) reported that the cAMP-saturated type IR holoenzyme complex was inactive toward substrate. However, we observed that addition of both cAMP and substrate to the type IR holoenzyme causes full dissociation of R and C subunits, indicating that cAMP-saturated holoenzyme could be fully active toward substrate.…”

“…Data based on fluorescence resonance energy transfer (FRET) and fluorescence anisotropy measurements performed under equilibrium conditions (9,10) show that high levels of cAMP are insufficient to dissociate the type II holoenzyme. In addition, Døskelund and colleagues (11) have published the results of in vivo experiments that suggest undissociated type I holoenzyme can be present at high cAMP concentrations and that the cAMP-saturated holoenzyme is catalytically inactive toward its substrates. They also performed in vitro experiments and calculated the dissociation constant, K D , between R and C subunits in the presence of cAMP to be about 0.2 µM and showed that the substrate could further destabilize the complex.…”

Using Markov State Models to Develop a Mechanistic Understanding of Protein Kinase A Regulatory Subunit RIα Activation in Response to cAMP Binding

“…through binding to the C-subunit catalytic cleft and promoting activation (22,23). A simple model of kemptide buffering was created (Fig.…”

“…A simple model of kemptide buffering was created (Fig. 4A) and fit to experimental data with varying concentrations of kemptide (8,70, and 140 M) in the presence of excess ATP and Mg 2ϩ (23) to determine the buffering capabilities of kemptide on free C-subunit concentration (Fig. 4B).…”