Rap1-Mediated Activation of Extracellular Signal-Regulated Kinases by Cyclic AMP Is Dependent on the Mode of Rap1 Activation

Abstract

Like other small G proteins of the Ras superfamily, Rap1 is activated by distinct guanine nucleotide exchange factors (GEFs) in response to different signals to elicit cellular responses. Activation of Rap1 by cyclic AMP (cAMP) can occur via cAMP-dependent protein kinase A (PKA)-independent and PKA-dependent mechanisms. PKA-independent activation of Rap1 by cAMP is mediated by direct binding of cAMP to Rap1-guanine nucleotide exchange factors (Rap1-GEFs) Epac1 (exchange protein directly activated by cAMP 1) and Epac2 (Epac1 and Epac2 are also called cAMP-GEFI and -GEFII). The availability of cAMP analogues that selectively activate Epacs, but not PKA, provides a specific tool to activate Rap1. It has been argued that the inability of these analogues to regulate extracellular signal-regulated kinases (ERKs) signaling despite activating Rap1 provides evidence that Rap1 is incapable of regulating ERKs. We confirm that the PKAindependent activation of Rap1 by Epac1 activates a perinuclear pool of Rap1 and that this does not result in ERK activation. However, we demonstrate that this inability to regulate ERKs is not a property of Rap1 but is rather a property of Epacs themselves. The addition of a membrane-targeting motif to Epac1 (Epac-CAAX) relocalizes Epac1 from its normal perinuclear locale to the plasma membrane. In this new locale it is capable of activating ERKs in a Rap1-and cAMP-dependent manner. Rap1 activation by Epac-CAAX, but not wild-type Epac, triggers its association with B-Raf. Therefore, we propose that its intracellular localization prevents Epac1 from activating ERKs. C3G (Crk SH3 domain Guanine nucleotide exchanger) is a Rap1 exchanger that is targeted to the plasma membrane upon activation. We show that C3G can be localized to the plasma membrane by cAMP/PKA, as can Rap1 when activated by cAMP/PKA. Using a small interfering RNA approach, we demonstrate that C3G is required for the activation of ERKs and Rap1 by cAMP/PKA. This activation requires the GTP-dependent association of Rap1 with B-Raf. These data demonstrate that B-Raf is a physiological target of Rap1, but its utilization as a Rap1 effector is GEF specific. We propose a model that specific GEFs activate distinct pools of Rap1 that are differentially coupled to downstream effectors.

Regulation of PC12 Cell Differentiation by cAMP Signaling to ERK Independent of PKA: Do All the Connections Add Up?

“…Specifically, stimulation of ERK by forskolin is dependent on PKA, Rap1, and C3G and independent of EPAC, and these findings are recapitulated in COS cells, which lack endogenous EPAC (13). PC12 cells contain very low levels of EPAC, and even overexpression of EPAC in PC12 cells does not lead to PKA-independent forskolin-mediated ERK activation (13), which is consistent with previous observations (3). Instead, forskolin uses a PKA-dependent C3G pathway to activate ERK in PC12 cells (13).…”

“…This implies that EPAC is not upstream of ERK for PACAP-mediated neuritogenesis in PC12 cells. There are two Rap GEFs-EPAC and Crk SH3 domain guanine nucleotide exchanger (C3G)-and in PC12 cells ERK activation is dependent on which Rap GEF is used (13). Specifically, stimulation of ERK by forskolin is dependent on PKA, Rap1, and C3G and independent of EPAC, and these findings are recapitulated in COS cells, which lack endogenous EPAC (13).…”

“…Several groups have since used this compound or its derivatives to probe the involvement of EPAC in various physiological and signaling processes, including β-adrenergic receptor-mediated calcium release in cardiac myocytes (4), leukocyte adhesion and chemotaxis (5), anti-inflammatory effects of cytokines in endothelial cells (6), activation of R-Ras and H-Ras (7,8), modulation of Ca 2+ -regulated potassium channels through p38 MAPK (9), and activation of ERK (8,(10)(11)(12). However, it must be pointed out that EPAC remains only a candidate for noncanonical cAMP signaling to ERK in PC12 and neuronal cells: Direct application of EPAC-specific agonists fails to activate ERK in either PC12 cells (3,10,13) or primary locus ceruleus neurons (10). It might be involved in ERK activation indirectly, because EPAC agonist treatment in some cases lifts the inhibition of GPCR-stimulated ERK activation after treatment with adenylate cyclase inhibitors (10).…”

“…The critical link missing from these studies is the component responsible for cAMPdependent PKA-independent activation of ERK in PC12 cells (3,13,16,20 (24) (Fig. 2).…”

ERK5 Activity Is Required for Nerve Growth Factor-induced Neurite Outgrowth and Stabilization of Tyrosine Hydroxylase in PC12 Cells

“…Adenovirus encoding FLAG-Rap1GAP1 was constructed using FLAG-Rap1GAP1 (26). C3G siRNA (sense, GGACUUUGAUGUUGAAUGUtt; antisense, ACAUUCAACAUCAAAGUCCtg) was purchased from Ambion (Austin, TX) (27).…”