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A key phosphorylation site in AC8 mediates regulation of Ca2+-dependent cAMP dynamics by an AC8–AKAP79–PKA signalling complex

Summary Adenylyl cyclase (AC) isoforms can participate in multimolecular signalling complexes incorporating A-kinase anchoring proteins (AKAPs). We recently identified a direct interaction between Ca2+-sensitive AC8 and plasma membrane-targeted AKAP79/150 (in cultured pancreatic insulin-secreting cells and hippocampal neurons), which attenuated the stimulation of AC8 by Ca2+ 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 Ca2+ entry. We show that the actions of PKA are not mediated indirectly via PKA-dependent activation of protein phosphatase 2A (PP2A) B56&dgr; 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 Ca2+ 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 Ca2+ activity. This fine-tuning of Ca2+-dependent cAMP dynamics by targeted PKA could be highly significant for cellular events that depend on the interplay of Ca2+ and cAMP, such as pulsatile hormone secretion and memory formation.