cAMP-dependent protein kinase regulatory subunit typeII β

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 prop-erties of RII and two mutant RII subunits, engineered by converting a conserved Arg to Lys in each cAMP-binding domain thereby yielding a protein that contains one intact, high affinity cAMP-binding site and one de-fective site. Structure and function were characterized by circular dichroism, steady-state fluorescence, sur-face plasmon resonance and holoenzyme activation as-says. The Ka for RII is 610 nM, which is 10-fold greater than its Kd(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 Ka of the A domain mutant protein is 21-fold greater than that of wild-type and the Kd(cAMP) is increased 7-fold, confirming that cAMP must bind to the mutated site to initiate activa-tion. The domain B mutant Ka is 2-fold less than its Kd(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 Ka identical to the Kd(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 ac-tivate than the wild-type protein. Specificity in biochemical reactions is critical for proper cel-lular functioning, and one way to achieve specificity is through isoform diversity. cAMP-dependent protein kinase (cAPK),1 an enzyme involved in numerous physiological processes, is spa-tially and negatively regulated by four regulatory (R) subuni