Active site mutations define the pathway for the cooperative activation of cAMP-dependent protein kinase.
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 Ka'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.
Herberg
F W
FW
Department of Chemistry, University of California, San Diego, La Jolla, 92093-0654, USA.
Taylor
S S
SS
Dostmann
W R
WR
D000595:Amino Acid Sequence; D001665:Binding Sites; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis / Q000378:metabolism; D002850:Chromatography, Gel; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D004789:Enzyme Activation; D007700:Kinetics; D046911:Macromolecular Substances; D008961:Models, Structural; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D017384:Sequence Deletion; D013379:Substrate Specificity; D013997:Time Factors
D046911:Macromolecular Substances; D011994:Recombinant Proteins; D000242:Cyclic AMP; D015500:Chloramphenicol O-Acetyltransferase; D017868:Cyclic AMP-Dependent Protein Kinases