A Novel Cyclic AMP-Dependent Epac-Rit Signaling Pathway Contributes to PACAP38-Mediated Neuronal Differentiation

Abstract

Pituitary adenylate cyclase-activating polypeptide (PACAP38) stimulation results in the activation of G_sα protein-coupled receptors to regulate neuronal differentiation in a cyclic AMP (cAMP)-dependent manner. These pathways involve protein kinase A (PKA)-dependent processes, but a growing body of evidence indicates that cAMP also regulates cellular functions through PKA-independent signaling cascades. Here we show that the Rit small GTPase is regulated by PACAP38 in a cAMP-dependent but PKA-independent fashion. Rit activation results from stimulation of the cAMP-activated guanine nucleotide exchange factor Epac but does not appear to rely upon the activation of Rap GTPases, the accepted cellular Epac substrates. Although RNA interference studies demonstrated that Epac is required for PACAP38-mediated Rit activation, neither Epac1 nor Epac2 activates Rit directly, indicating that Epac signals to Rit through a novel mechanism in which Rap signaling is not essential. Loss-of-function analysis demonstrated that Rit makes an important contribution to PACAP38-mediated neuronal differentiation. Surprisingly, although Rit is required for sustained extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase signaling following nerve growth factor stimulation of pheochromocytoma 6 (PC6) cells, Rit silencing selectively suppressed PACAP38-elicited activation of p38, without obvious effects on ERK signaling in the same cells. Moreover, the ability of PACAP38 to stimulate CREB-dependent transcription and to promote neurite outgrowth was inhibited by Rit knockdown. Together, these studies identify an unsuspected connection between cAMP and Rit signaling pathways and imply that Rit can function downstream of G_sα/cAMP/Epac in a novel signal transduction pathway necessary for PACAP38-mediated neuronal differentiation and CREB signaling.

The cDNAs encoding G_sαQ227L (QL), Gβ1, and Gγ2 and the plasmids needed to analyze CREB transcription were kindly provided by J. Kehrl (National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD). The cDNAs encoding WT Epac1/2, constitutively active Epac2, and RapGAP were a gift from L. Quilliam (Indiana University School of Medicine, Indianapolis, IN).

This work was supported by Public Health Service grant NS045103 (to D.A.A.) from the National Institute of Neurological Disorders and Stroke and by grant P20RR20171 from the COBRE program of the National Center for Research Resources, a component of the National Institutes of Health (NIH). H.R. was supported by the Chemical Sciences of The Netherlands Organization for Scientific Research (NWO-CW).

The contents of this work are solely the responsibility of the authors and do not necessarily represent the official views of NCRR or NIH.