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 AC8 stimulation 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 negatively feedback on the stimulation of AC8 by Ca2+-entry. We show that the actions of PKA are not mediated indirectly via PKAdependent activation of PP2A B56I 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 Ca2+ and cAMP interplay, such as pulsatile hormone secretion and memory formation.
Willoughby, D., Halls, M. L., Everett, K. L., Ciruela, A., Skroblin, P., Klussmann, E., & Cooper, D. M. F. (2012). A key phosphorylation site in AC8 mediates regulation of Ca2+-dependent cAMP dynamics by an AC8-AKAP79-PKA signalling complex. Journal of Cell Science, 125(23), 5850 - 5859. https://doi.org/10.1242/jcs.111427