Active Site Mutations Define the Pathway for the Cooperative Activation of cAMP-Dependent Protein Kinase

Abstract

cAMP-dependent protein kinase (cAPK) is a heterotetramer containing two regulatory (R) and two catalytic (C) subunits. Each R-subunit contains two tandem cAMP-binding domains, and activation of cAPK is mediated by the cooperative, high affinity binding of cAMP to these two domains. Mutant R-subunits containing one intact high affinity cAMP-binding site and one defective site were used to define the pathway for activation and to delineate the unique roles that each cAMP-binding domain plays. Two mutations were introduced by replacing the essential Arg in each cAMP-binding site with Lys (R209K in Site A and R333K in Site B). Also, the double mutant (R209/333K) was constructed. Analysis of cAMP binding and dissociation and the apparent constants for holoenzyme activation and R-and C-subunit interaction, measured by analytical gel filtration and surface plasmon resonance, established the following: (1) For rR(R209K), occupancy of Site B is not sufficient to activate the holoenzyme; the low affinity Site A must also be occupied. In rR(R333K), Site A retains its high affinity for cAMP, but Site A cannot bind until the low affinity Site B is occupied. Thus, both mutants, for different reasons, have similar K a 's for activation that are approximately 20-fold higher than that of the wild-type holoenzyme. The double mutant with two defective sites is no worse than either single mutant. (2) Kinetic analysis of cAMP binding showed that the mutation in Site A or B abolishes high affinity cAMP binding to that site and slightly weakens the affinity of the adjacent site for cAMP. (3) In the presence of MgATP, both mutants rapidly form a stable holoenzyme even in the presence of cAMP in contrast to the wild-type R where holoenzyme forms slowly in Vitro and requires dialysis. Regarding the mechanism of activation based on these and other mutants and from kinetic data, the following conclusions are reached: Site A provides the major contact site with the C-subunit; Site B is not essential for holoenzyme formation. Occupancy of Site A by cAMP mediates dissociation of the C-subunit. Site A is inaccessible to cAMP in the full length holoenzyme, while Site B is fully accessible. Access of cAMP to Site A is mediated by Site B. Thus Site B not only helps to shield Site A, it also provides the specific signal that "opens up" Site A. Finally, a nonfunctional Site A in the holoenzyme prevents stable binding of cAMP to Site B in the absence of subunit dissociation.The regulatory subunits of cAMP-dependent protein kinase (cAPK) 1 serve as negative regulators of cAPK and maintain an inactive holoenzyme complex in the absence of cAMP. All R-subunits contain an inhibitory site that resembles either a substrate or an inhibitor followed by two contiguous cAMP-binding domains in each protomer (Takio et al., 1984;Taylor et al., 1990;Titani et al., 1984). The cooperative activation of the holoenzyme is mediated by the sequential binding of cAMP to these two sites, which then leads to the dissociation of the active C-subunit (Øgreid & Døsk...

Communication between Tandem cAMP Binding Domains in the Regulatory Subunit of Protein Kinase A-Iα as Revealed by Domain-silencing Mutations

“…As phosphodiesterases start to catalyze the removal of cAMP from site A, CBD-A begins to undergo partial but global unfolding, which in turn promotes global unfolding of CBD-B, thus, accelerating the release of cAMP from site B as well. This conclusion is also corroborated by the observation that the residence time of cAMP in site B decreases by 60% in the R209K mutant relative to wild-type RI␣ (21).…”

“…K D from 30 nM to the ՆM range) as well as at the nonmutated site B by about 1 order of magnitude (i.e. K D from 15 to 130 nM) (21). A similar reciprocal "domain silencing" effect has been reported also for R333K (21).…”

“…K D from 15 to 130 nM) (21). A similar reciprocal "domain silencing" effect has been reported also for R333K (21). The ability of the R209K and R333K mutations to decrease the cAMP affinity for both PBCs indicates that these point substitutions cause long range perturbations and represent useful tools to start dissecting the pathways that mediate the interdomain communication in RI␣.…”

“…The ability of the R209K and R333K mutations to decrease the cAMP affinity for both PBCs indicates that these point substitutions cause long range perturbations and represent useful tools to start dissecting the pathways that mediate the interdomain communication in RI␣. Furthermore, despite the increased dissociation constants for cAMP, the inhibition of the C-subunit by either R209K or R333K RI␣ is still cAMP-dependent (21), indicating that both RI␣ mutants are functionally active. Overall, the three state (i.e.…”

Functional Roles of Loops 3 and 4 in the Cyclic Nucleotide Binding Domain of Cyclic AMP Receptor Protein from Escherichia coli

“…It has long been established that the binding affinity of site A is lower than site B, an observation parallel that of the deletion and wild-type CRP, respectively. It is not surprising that cooperativity of ligand binding is different between these two type of sites (35)(36)(37)(38)(39)(40)(41)(42).…”