ACTIVE-SITE MUTATIONS DEFINE THE PATHWAY FOR THE COOPERATIVE ACTIVATION OF CAMP-DEPENDENT PROTEIN-KINASE

cAMP-dependent protein kinase (cAPK) is,a heterotetramer containing tw o regulatory (R) and two catalytic (C) subunits. Each R-subunit contai ns two tandem cAMP-binding domains, and activation of cAPK is mediated by the cooperative, high affinity binding of cAMP to these two domain s, Mutant R-subunits containing one intact high affinity cAMP-binding site and one defective site were used to define the pathway for activa tion and to delineate the unique roles that each cAMP-binding domain p lays, 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 ac tivation 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 th e holoenzyme: the low affinity Site A must also be occupied, In rR(R33 3K), Site A retains its high affinity for cAMP, but Sire A cannot bind until the low affinity Site B is occupied, Thus, both mutants, for di fferent reasons? have similar K-a's for activation that are approximat ely 20-fold higher than that of the wild-type holoenzyme, The double m utant 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 presenc e 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 act ivation based on these and other mutants and from kinetic data, the fo llowing conclusions are reached: Site A provides the major contact sit e 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 Si te B is fully accessible. Access of cAMP to Site A is mediated by Site B, Thus Site B not only helps to shield Sire Al it also provides the specific signal that ''opens up'' Site A, Finally, a nonfunctional Sit e A in the holoenzyme prevents stable binding of cAMP to Site B in the absence of subunit dissociation.