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

Abstract

SummaryAdenylyl 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) B56d 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 finetuning 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.

Spatially compartmentalized phase regulation of a Ca2+-cAMP-PKA oscillatory circuit

“…AKAPs can recruit PKA to regulate channel activities (Dell'Acqua et al 2006;Mo et al 2017;Torres-Quesada et al 2017), such as in the regulation of voltage-mediated Ca 2+ entry via PKAdependent phosphorylation of Ca V 1.2 (Murphy et al 2014) or the modulation of store-operated Ca 2+ entry by both PKA-dependent STIM1 and Orai1 phosphorylation (Thompson et al 2015;Zhang et al 2019). Additional levels of regulatory feedback within the Ca 2+ -cAMP-PKA oscillatory circuit have also been identified, such as a negative feedback loop involving PKA phosphorylation of AC8, thereby fine-tuning the circuit dynamics (Willoughby et al 2012).…”

Control of βAR- and N-methyl-D-aspartate (NMDA) Receptor-Dependent cAMP Dynamics in Hippocampal Neurons

“…There are comparatively few known PKA targets that lead to reduced cAMP signaling in CA1 pyramidal neurons. One such mechanism is PKA inhibition of AC8 [ 91 ]; however, the modest inhibition of AC8 by PKA observed in HEK293 cells (~30% reduction of FRET after 3 min forskolin stimulation) is likely too weak to reduce NMDA-induced cAMP under our conditions. This is compounded by the relatively small contribution of AC8 to the cAMP response in the first place, due to its relatively low affinity for Ca 2+ /calmodulin (~800 nM vs. ~150 nM for AC1).…”

New Insights into Beta-Cell GLP-1 Receptor and cAMP Signaling

“…AKAP79 also inhibits Ca 2+ -stimulated AC8 activity by recruiting PKA resulting in AC8 phosphorylation [155,156]. From a pathophysiological perspective it is also interesting that, in HEK-293 cells, palmitoylation promotes AKAP79 association with lipid rafts and its regulation of AC8 [157] whereas, in neuronal synapses, CaMKII regulates depalmitoylation of AKAP79 [158].…”