title: Rearrangements in a hydrophobic core region mediate cAMP action in the regulatory subunit of PKA.
abstract: cAMP-dependent protein kinase (PKA) forms an inactive heterotetramer of two regulatory (R; with two cAMP-binding domains A and B each) and two catalytic (C) subunits. Upon the binding of four cAMP molecules to the R dimer, the monomeric C subunits dissociate. Based on sequence analysis of cyclic nucleotide-binding domains in prokaryotes and eukaryotes and on crystal structures of cAMP-bound R subunit and cyclic nucleotide-free Epac (exchange protein directly activated by cAMP), four amino acids were identified (Leu203, Tyr229, Arg239 and Arg241) and probed for cAMP binding to the R subunits and for R/C interaction. Arg239 and Arg241 (mutated to Ala and Glu) displayed no differences in the parameters investigated. In contrast, Leu203 (mutated to Ala and Trp) and Tyr229 (mutated to Ala and Thr) exhibited up to 30-fold reduced binding affinity for the C subunit and up to 120-fold reduced binding affinity for cAMP. Tyr229Asp showed the most severe effects, with 350-fold reduced affinity for cAMP and no detectable binding to the C subunit. Based on these results and structural data in the cAMP-binding domain, a switch mechanism via a hydrophobic core region is postulated that is comparable to an activation model proposed for Epac.
authors.affiliation: Department of Biochemistry, University of Kassel, D-34132 Kassel, Germany.
authors.forename: Claudia
authors.initials: C
authors.lastname: Hahnefeld
authors.forename: Daniela
authors.initials: D
authors.lastname: Moll
authors.forename: Maik
authors.initials: M
authors.lastname: Goette
authors.forename: Friedrich W
authors.initials: FW
authors.lastname: Herberg
mesh_terms: D000595:Amino Acid Sequence; D002942:Circular Dichroism; D000242:Cyclic AMP / Q000502:physiology*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry / Q000502:physiology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017386:Sequence Homology, Amino Acid; D020349:Surface Plasmon Resonance
chemical_list: D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases