title: A key phosphorylation site in AC8 mediates regulation of Ca(2+)-dependent cAMP dynamics by an AC8-AKAP79-PKA signalling complex.
abstract: Adenylyl cyclase (AC) isoforms can participate in multimolecular signalling complexes incorporating A-kinase anchoring proteins (AKAPs). We recently identified a direct interaction between Ca(2+)-sensitive AC8 and plasma membrane-targeted AKAP79/150 (in cultured pancreatic insulin-secreting cells and hippocampal neurons), which attenuated the stimulation of AC8 by Ca(2+) entry (Willoughby et al., 2010). Here, we reveal that AKAP79 recruits cAMP-dependent protein kinase (PKA) to mediate the regulatory effects of AKAP79 on AC8 activity. Modulation by PKA is a novel means of AC8 regulation, which may modulate or apply negative feedback to the stimulation of AC8 by Ca(2+) entry. We show that the actions of PKA are not mediated indirectly via PKA-dependent activation of protein phosphatase 2A (PP2A) B56δ subunits that associate with the N-terminus of AC8. By site-directed mutagenesis we identify Ser-112 as an essential residue for direct PKA phosphorylation of AC8 (Ser-112 lies within the N-terminus of AC8, close to the site of AKAP79 association). During a series of experimentally imposed Ca(2+) oscillations, AKAP79-targeted PKA reduced the on-rate of cAMP production in wild-type but not non-phosphorylatable mutants of AC8, which suggests that the protein-protein interaction may provide a feedback mechanism to dampen the downstream consequences of AC8 activation evoked by bursts of Ca(2+) activity. This fine-tuning of Ca(2+)-dependent cAMP dynamics by targeted PKA could be highly significant for cellular events that depend on the interplay of Ca(2+) and cAMP, such as pulsatile hormone secretion and memory formation.
authors.affiliation: Department of Pharmacology, University of Cambridge, Cambridge CB2 1PD,  UK.
authors.forename: Debbie
authors.initials: D
authors.lastname: Willoughby
authors.forename: Michelle L
authors.initials: ML
authors.lastname: Halls
authors.forename: Katy L
authors.initials: KL
authors.lastname: Everett
authors.forename: Antonio
authors.initials: A
authors.lastname: Ciruela
authors.forename: Philipp
authors.initials: P
authors.lastname: Skroblin
authors.forename: Enno
authors.initials: E
authors.lastname: Klussmann
authors.forename: Dermot M F
authors.initials: DM
authors.lastname: Cooper
mesh_terms: D054758:A Kinase Anchor Proteins / Q000378:metabolism*; D000262:Adenylyl Cyclases / Q000378:metabolism*; D015153:Blotting, Western; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D006801:Humans; D047468:Immunoprecipitation; D010766:Phosphorylation
chemical_list: D054758:A Kinase Anchor Proteins; C487775:AKAP5 protein, human; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D000262:Adenylyl Cyclases