Ras Is Required for the Cyclic AMP-Dependent Activation of Rap1 via Epac2

Abstract

Exchange proteins activated by cAMP (cyclic AMP) 2 (Epac2) is a guanine nucleotide exchange factor for Rap1, a small G protein involved in many cellular functions, including cell adhesion, differentiation, and exocytosis. Epac2 interacts with Ras-GTP via a Ras association (RA) domain. Previous studies have suggested that the RA domain was dispensable for Epac2 function. Here we show for the first time that Ras and cAMP regulate Epac2 function in a parallel fashion and the Ras-Epac2 interaction is required for the cAMPdependent activation of endogenous Rap1 by Epac2. The mechanism for this requirement is not allosteric activation of Epac2 by Ras but the compartmentalization of Epac2 on the Ras-containing membranes. A computational modeling is consistent with this compartmentalization being a function of both the level of Ras activation and the affinity between Ras and Epac2. In PC12 cells, a well-established model for sympathetic neurons, the Epac2 signaling is coupled to activation of mitogen-activated protein kinases and contributes to neurite outgrowth. Taken together, the evidence shows that Epac2 is not only a cAMP sensor but also a bona fide Ras effector. Coincident detection of both cAMP and Ras signals is essential for Epac2 to activate Rap1 in a temporally and spatially controlled manner.Rap1 is a small GTPase involved in the regulation of multiple cellular functions such as adhesion, differentiation, and exocytosis. Like all small G proteins, Rap1 cycles between a GTP-loaded active state and a GDP-loaded inactive state, which is mediated via the opposing actions of G protein activation proteins that promote hydrolysis of bound GTP to GDP and guanine nucleotide exchange factors (GEFs) that catalyze the exchange of bound GDP for GTP.Exchange proteins activated by cAMP (Epac1 and Epac2) are unique Rap1 GEFs that link cAMP elevation to Rap1 activation (5). This is achieved via the direct binding of cAMP to the Epac protein itself, thereby defining a novel cAMP signaling pathway that is independent of protein kinase A (PKA). Both Epac1 and Epac2 contain a catalytic region and a regulatory region. The catalytic region consists of a CDC25 homology domain that catalyzes Rap1 activation, a REM domain, and a Ras association (RA) domain that lies in between. The regulatory region consists of one or two cAMP binding domains (cNBDs) and a DEP (disheveled, Egl-10, and pleckstrin homology) domain. Under resting conditions, Epac proteins are inactive due to the inhibitory interaction between the regulatory and catalytic regions. The binding of cAMP to the cNBD relieves the intramolecular inhibition by exposing the catalytic site to . Previous studies have been largely focused on the mechanism of cAMP regulation of Epacs. Whether other molecular interactions play a role in Epac regulation is unknown.A number of features of Epac2 distinguish it from Epac1, which suggests distinctive regulatory mechanisms for Epac2. While Epac1 is expressed ubiquitously, Epac2 is highly enriched in neuronal tissues (22). Moreover, Epac1 i...

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