No abstract

cAMP-dependent protein kinase (cAPK) contains a regulatory (R) subunit dimer bound to two catalytic (C) subunits. Each R monomer contains two cAMP-binding domains, designated A and B. The sequential binding of two cAMPs releases active C. We describe here the properties of RII␤ and two mutant RII␤ subunits, engineered by converting a conserved Arg to Lys in each cAMPbinding domain thereby yielding a protein that contains one intact, high affinity cAMP-binding site and one defective site. Structure and function were characterized by circular dichroism, steady-state fluorescence, surface plasmon resonance and holoenzyme activation assays. The K a for RII␤ is 610 nM, which is 10-fold greater than its K d (cAMP) and significantly higher than for RI␣ and RII␣. The Arg mutant proteins demonstrate that the conserved Arg is important for both cAMP binding and organization of each domain and that binding to domain A is required for activation. The K a of the A domain mutant protein is 21-fold greater than that of wild-type and the K d (cAMP) is increased 7-fold, confirming that cAMP must bind to the mutated site to initiate activation. The domain B mutant K a is 2-fold less than its K d (cAMP), demonstrating that, unlike RI␣, cAMP can access the A site even when the B site is empty. Removal of the B domain yields a K a identical to the K d (cAMP) of full-length RII␤, indicating that the B domain inhibits holoenzyme activation for RII␤. In RI␣, removal of the B domain generates a protein that is more difficult to activate than the wild-type protein.

AbstractcAMP-dependent protein kinase (cAPK) is a heterotetramer containing a regulatory (R) subunit dimer bound to two catalytic (C) subunits and is involved in numerous cell signaling pathways. The C-subunit is activated allosterically when two cAMP molecules bind sequentially to the cAMP-binding domains, designated A and B (cAB-A and cAB-B, respectively). Each cAMP-binding domain contains a conserved Arg residue that is critical for high-affinity cAMP binding. Replacement of this Arg with Lys affects cAMP affinity, the structural integrity of the cAMP-binding domains, and cAPK activation. To better understand the local and long-range effects that the Arg-to-Lys mutation has on the dynamic properties of the R-subunit, the amide hydrogen/deuterium exchange in the RII␤ subunit was probed by electrospray mass spectrometry. Mutant proteins containing the Arg-to-Lys substitution in either cAMP-binding domain were deuterated for various times and then, prior to mass spectrometry analysis, subjected to pepsin digestion to localize the deuterium incorporation. Mutation of this Arg in cAB-A (Arg230) causes an increase in amide hydrogen exchange throughout the mutated domain that is beyond the modest and localized effects of cAMP removal and is indicative of the importance of this Arg in domain organization. Mutation of Arg359 (cAB-B) leads to increased exchange in the adjacent cAB-A domain, particularly in the cAB-A domain C-helix that lies on top of the cAB-B domain and is believed to be functionally linked to the cAB-B domain. This interdomain communication appears to be a unidirectional pathway, as mutation of Arg230 in cAB-A does not effect dynamics of the cAB-B domain.

The amino terminal dimerization/ docking domain and the two-tandem, carboxyterminal cAMP-binding domains (A and B) of cAMPdependent protein kinase regulatory (R) subunits are connected by a variable linker region. In addition to providing a docking site for the catalytic subunit, the linker region is a major source of sequence diversity between the R-subunit isoforms. The RII␤ isoform uniquely contains two endogenous tryptophan residues, one at position 58 in the linker region and the other at position 243 in cAMPbinding domain A, which can act as intrinsic reporter groups of their dynamics and microenvironment. Two single-point mutations, W58F and W243F, allowed the local environment of each Trp to be probed using steady-state and time-resolved fluorescence techniques. We report that: (a) the tryptophan fluorescence of the wild-type protein largely reflects Trp243 emission; (2) cAMP selectively quenches Trp243 and thus acts as a cAMP sensor; (3) Trp58 resides in a highly solvated, unstructured, and mobile region of the protein; and (4) Trp243 resides in a stable, folded domain and is relatively buried and rigid within the domain. The use of endogenous Trp residues presents a non-perturbing method for studying R-subunit subdomain characteristics in addition to providing the first biophysical data on the RII␤ linker region. Proteins 2003;51:552-561.