title: cAMP-dependent protein kinase regulatory subunit type IIbeta: active site mutations define an isoform-specific network for allosteric signaling by cAMP.
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 RIIbeta and two mutant RIIbeta 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 defective site. Structure and function were characterized by circular dichroism, steady-state fluorescence, surface plasmon resonance and holoenzyme activation assays. The Ka for RIIbeta is 610 nM, which is 10-fold greater than its Kd(cAMP) and significantly higher than for RIalpha and RIIalpha. 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 activation. The domain B mutant Ka is 2-fold less than its Kd(cAMP), demonstrating that, unlike RIalpha, 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 RIIbeta, indicating that the B domain inhibits holoenzyme activation for RIIbeta. In RIalpha, removal of the B domain generates a protein that is more difficult to activate than the wild-type protein.
authors: Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla 92093-0654, USA.
authors: Kerri M
authors: KM
authors: Zawadzki
authors: Susan S
authors: SS
authors: Taylor
mesh_terms: D000495:Allosteric Site; D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000737:chemistry; D001665:Binding Sites; D001710:Biotin / Q000737:chemistry; D002942:Circular Dichroism; D000242:Cyclic AMP / Q000737:chemistry / Q000378:metabolism; D054757:Cyclic AMP-Dependent Protein Kinase RIIbeta Subunit; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry* / Q000235:genetics; D004305:Dose-Response Relationship, Drug; D007700:Kinetics; D008239:Lysine / Q000737:chemistry; D008301:Maleimides / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011485:Protein Binding; D017510:Protein Folding; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D051381:Rats; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D020349:Surface Plasmon Resonance; D013816:Thermodynamics; D013997:Time Factors; D014364:Tryptophan / Q000737:chemistry; D014466:Ultraviolet Rays; D014508:Urea / Q000494:pharmacology
chemical_list: D054757:Cyclic AMP-Dependent Protein Kinase RIIbeta Subunit; D008301:Maleimides; C517839:Prkar2b protein, rat; D020033:Protein Isoforms; C043592:maleimide; D001710:Biotin; D014364:Tryptophan; D014508:Urea; D001120:Arginine; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D008239:Lysine