Epac2 Mediates Cyclic AMP‐dependent Growth Arrest in NS‐1 cells Independently of Rap1

Abstract

We have investigated the upstream activators, and downstream effectors, of Epac2 signaling in the NS‐1 pheochromocytoma cell line in which three cyclic AMP sensors, Epac, PKA and NCS/Rapgef2 mediate distinct cellular outputs: p38‐dependent growth arrest; CREB‐dependent cell survival; and ERK‐dependent neuritogenesis, respectively (Emery et al., Sci. Sig. 6: ra51, 2013; Emery et al., JBC 289: 10126, 2014). Based on the growth arrest assay in NS‐1 cells, the Gs‐coupled GPCRs ADCYAP1R1, ADRB1, ADRB2 and ADORA2 all couple efficiently to Epac2 in these cells. We have previously shown that cAMP‐dependent growth arrest in these cells a) requires Epac2 (using siRNA and appropriate controls (Emery et al., JBC 289: 10126, 2014) and b) does not require Rap1 activation (ibid). Alternative candidates for mediation of growth arrest by the GPCR‐Gs‐cAMP‐Epac2 pathway include Rap2, Rit1, Rin, Rac and perhaps other small GTPase effector proteins for which Epac2 could conceivably provide catalysis of activation (GDP release or GDP/GTP exchange). Despite the observation that neither Epac1 nor Epac2 can function as a Rit‐GEF in vitro (Shi et al., MCB 26: 9136, 2006), quantitative expression analysis is consistent with Rit1 as the major candidate for mediating cAMP‐ and Epac2‐dependent activation of p38 leading to growth arrest in these cells, perhaps requiring an additional partner besides Epac2 for Rit1 activation in the pathway GPCR‐>Gs‐>AC‐>cAMP‐>Epac2‐>Rit1‐>MEKK‐>MEK‐>p38‐growth arrest. This pathway is distinctly parcellated from the GPCR‐>Gs‐>AC‐>cAMP‐>PKA‐>CREB and GPCR‐>Gs‐>AC‐>cAMP‐>NCS/Rapgef2‐>B‐Raf‐>MEK‐>ERK pathways which mediate cell survival and neuritogenesis, respectively, in NS‐1 cells.