Phospho-substrate profiling of Epac-dependent protein kinase C activity
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Exchange protein directly activated by cAMP (Epac) and protein kinase A are effectors for cAMP with distinct actions and regulatory mechanisms. Epac is a Rap guanine nucleotide exchange factor that activates Rap1; protein kinase C (PKC) is a major downstream target of Epac-Rap1 signaling that has been implicated in a variety of pathophysiological processes, including cardiac hypertrophy, cancer, and nociceptor sensitization leading to chronic pain. Despite the implication of both Epac and PKC in these processes, few downstream targets of Epac-PKC signaling have been identified. This study characterized the regulation of PKC activity downstream of Epac activation. Using an antibody that recognizes phospho-serine residues within the consensus sequence phosphorylated by PKC, we analyzed the 1-dimensional banding profile of PKC substrate protein phosphorylation from the Neuro2A mouse neuroblastoma cell line. Activation of Epac either indirectly by prostaglandin PGE2, or directly by 8-pCPT-2-O-Me-cAMP-AM (8pCpt), produced distinct PKC phospho-substrate protein bands that were suppressed by co-administration of the Epac inhibitor ESI09. Different PKC isoforms contributed to the induction of individual phospho-substrate bands, as determined using isoform-selective PKC inhibitors. Moreover, the banding profile after Epac activation was altered by disruption of the cytoskeleton, suggesting that the orchestration of Epac-dependent PKC signaling is regulated in part by interactions with the cytoskeleton. The approach described here provides an effective means to characterize Epac-dependent PKC activity.
KeywordsSignal transduction Phosphorylation Prostaglandins Cytoskeleton
All work reported here was funded by the National Institutes of Health Institute of General Medical Sciences, Grants RO1102346 and P20GM103643.
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Conflict of interest
All authors certify that they have no conflicts of interest.
This article does not contain any studies with animals performed by any of the authors. This article does not contain any studies with human participants performed by any of the authors.
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