Evaluating the Specificity and Potency of Activators and Inhibitors of the cAMP Sensors PKA, Epac2/Rapgef4, and NCS/Rapgef2 Using Cell‐Based Assays for Activation of CREB, p38 MAPK, and ERK

Abstract

Cyclic AMP elevation is well known to cause dividing neuroendocrine (e.g. PC12) cells to differentiate through a process that includes growth arrest, neurite extension, and induction of neuronal genes. Using the PC12 subclone NS‐1, we have previously shown that downstream of cAMP, signaling for differentiation is mediated by three insulated signaling pathways, initiated by cAMP activation of Epac2/Rapgef4, NCS/Rapgef2, and PKA, which regulate the activation of p38 MAP kinase, ERK, and CREB, respectively. These, in turn, are individually necessary for growth arrest (Epac2/Rapgef4), neurite extension (NCS/Rapgef2), and neuron‐specific gene expression (PKA) (Emery et al., 2014, J Biol Chem, 289:10126–39). Specific inhibitors and activators of PKA, Epac, and NCS/Rapgef2 would be useful to further explore the unique physiological functions of these three cAMP sensors in the CNS. Several compounds that show differential activity with respect to PKA and Epac exist, however, the selectivity of these agents across all three neuroendocrine cAMP sensors is not yet known. The goal of the work reported here was to identify cAMP analogs that provide specific activation and inhibition of each of these cAMP sensors. To this end, we have used a medium‐throughput assay platform that allows for quantitative measurement of the phosphorylation status of ERK, CREB, and p38 MAPK, which serve as read‐outs for activation of NCS/Rapgef2, PKA, and Epac2, respectively. 8‐CPT‐cAMP was found to be a full agonist with a similar potency at each sensor, while 8‐CPT‐2′‐O‐Me‐cAMP was highly specific for Epac relative to PKA (as reported by Enserink et al., 2002, Nat Cell Biol, 4:901–6) and also NCS/Rapgef2. We examined previously characterized PKA site‐specific agonists, i.e. compounds that stimulate PKA holoenzyme activity only when applied in combination, to determine if any of these provide SAR entree to further development of Epac or NCS/Rapgef2‐specific agonists. Our preliminary evidence suggests that several B site‐specific compounds, when administered singly, show significant activity at NCS/Rapgef2 without stimulating either Epac or the PKA holoenzyme. None of the cyclic nucleotide antagonists (Rp diastereomeric cAMP analogs) that we tested effectively inhibited cAMP‐dependent NCS/Rapgef2 signaling. Based on our previous work indicating that the nucleoside adenylate cyclase inhibitor SQ22,536 inhibits NCS/Rapgef2 (Emery et al., 2013, Mol Pharmacol, 83:95–105), along with new insights into structure‐activity relationships for NCS/Rapgef2 gleaned from our survey of B‐site‐specific activators, we propose that the development of a cell‐permeant, highly specific inhibitor of NCS/Rapgef2 is feasible.Support or Funding InformationSupported by NIMH‐IRP project ZO1‐MH002386