Epac and PKA: a tale of two intracellular cAMP receptors

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Acta Biochim Biophys

Sin 2008, 40: 651-662

doi:10.1111/j.1745-7270.2008.00438.x

Epac and PKA: a tale of two

intracellular cAMP receptors

Xiaodong Cheng*, Zhenyu Ji, Tamara Tsalkova, and Fang Mei

Department of Pharmacology and Toxicology,

Sealy Center for Cancer Cell Biology and Sealy Center for Structural Biology

and Molecular Biophysics, University of Texas Medical Branch, Galveston, Texas

77555-1031, USA

Received: May 6,

2008       

Accepted: May 21,

2008

This work was

supported by grants from the National Institutes of Health (No. GM061770) and the

American Heart Association (No. 0755049Y)

*corresponding author: Tel,

1-409-772-9656; Fax, 1-409-772-9642; E-mail, [email protected]

cAMP-mediated signaling pathways regulate a

multitude of important biological processes under both physiological and pathological

conditions, including diabetes, heart failure and cancer. In eukaryotic cells,

the effects of cAMP are mediated by two ubiquitously expressed intracellular

cAMP receptors, the classic protein kinase A (PKA)/cAMP-dependent protein

kinase and the recently discovered exchange protein directly activated by camp (Epac)/cAMP-regulated guanine

nucleotide exchange factors. Like PKA, Epac contains an evolutionally conserved

cAMP binding domain that acts as a molecular switch for sensing intracellular

second messenger cAMP levels to control diverse biological functions. The

existence of two families of cAMP effectors provides a mechanism for a more

precise and integrated control of the cAMP signaling pathways in a spatial and

temporal manner. Depending upon the specific cellular environments as well as

their relative abundance, distribution and localization, Epac and PKA may act

independently, converge synergistically or oppose each other in regulating a

specific cellular function.

Keywords    cAMP; exchange protein directly activated

by cAMP (Epac)/cAMP-regulated guanine exchange factor; protein kinase A

(PKA)/cAMP-dependent protein kinase; signal transduction.

Overview of the cAMP second messenger system

Eukaryotic cells respond to a wide range of extracellular signals,

including hormones, growth factors and neurotransmitters, by eliciting the

generation of intracellular second messengers. Second messengers in turn

trigger a myriad of cellular reactions by orchestrating a network of

intracellular signaling events. The discovery of cAMP 50 years ago marked the

birth of second messenger theory and the age of signal transduction. cAMP

regulates many physiological processes ranging from learning and memory in the

brain, as well as contractility and relaxation in the heart, to water uptake in

the gut and kidney. At the cellular level, cAMP plays an important role in

virtually every known function, such as metabolism, gene expression, cell

division and growth, cell differentiation, apoptosis, secretion and

neurotransmission. In addition to regulating many important cellular processes

directly, cAMP is also implicated in an array of cross-talks between

intracellular signaling pathways. For example, cAMP exerts its growth effects

through interactions with the Ras-mediated mitogen-activated protein kinase

pathways [1–5]. There is evidence that suggests that cAMP cross talks with the

Ca2+-dependent signaling pathway [6,7]. It has also been reported that

cAMP can potentially modulate cytokine signaling through inhibiting the

Jak/STAT pathway [8]. cAMP signaling is closely interwoven with the

phosphatidylinositol-3 kinase/protein kinase B (PKB) pathway [9,10]. For many years, the consensus was that the cAMP-mediated signaling

in eukaryotic cells, which involves the sequential activation of a series of

signaling molecules consisting of both plasma membrane and intracellular

components, existed as a linear pathway. Upon binding of ligand, the

G-protein-coupled receptor at the cell surface transduces the extracellular

signal across the cell membrane via stimulatory or inhibitory heterotrimeric

G-proteins that interact with the membrane-bound adenylate cyclase to regulate

cAMP production inside the cell. It was believed until recently that the major effects of cAMP in

mammalian cells, with the exception of cyclic nucleotide-gated ion channels in

photoreceptor cells, olfactory sensory neurons and cardiac sinoatrial node

cells [11], were mediated intracellularly by protein kinase A (PKA), also known

as cAMP-dependent protein kinase.

Protein kinase A

PKA was one of the first protein kinases to be discovered [12].

Unlike most eukaryotic protein kinases, PKA is composed of two separate

subunits: the catalytic (C) and regulatory (R) subunits. The C subunit is

initially phosphorylated by phosphoinositide-dependent protein kinase at an

essential phosphorylation site Threonine 197 (T197) [13,14]. Phosphorylation of

T197 in the activation loop is necessary for the maturation and optimal

biological activity of PKA [15,16]. However, once phosphorylated, the C subunit

of PKA is fully active, and the T197 phosphate does not turn over readily [17].

The C subunit of PKA is then regulated via interaction with the inhibitory R

subunit, a major intracellular cAMP receptor that sequesters the C subunit in

an inactive heterotetrameric holoenzyme, R2C2. The activating ligand cAMP binds to the R subunit and induces

conformational changes that lead to the dissociation of the holoenzyme into its

constituent C and R subunits [18]. The free active C subunit can then affect a

range of diverse cellular events by phosphorylating an array of cytoplasmic and

nuclear protein substrates, including enzymes and transcriptional factors [19].

There are two general classes of PKA, designated as PKA(I) and

PKA(II), that are distinguished by differences in the R subunits, RI and RII,

which interact with an identical C subunit [18]. Four different R subunit

genes, RIa [20], RIb [21], RIIa [22], and RIIb [23] have been identified. Three C subunit genes, Ca, Cb, and Cg have also been discovered. However, preferential expression of any

of these C subunits with either RI or RII has not been found [24]. While both

RI and RII contain two tandem and highly conserved cAMP binding domains (CBD)

at the C-terminus [25], RI and RII differ significantly at their amino

terminus, especially at the proteolytically sensitive hinge region that binds

to the peptide recognition site of the C subunit. The hinge region of the RII

subunits contains a serine at the P site that can be auto-phosphorylated by the

C subunit [26], whereas RI contains a pseudo-phosphorylation site. R isoforms are differentially expressed in tissues [27–29], and their

subcellular distribution also appears to be distinct [30–33]. The

existence of a family of A-kinase anchoring proteins (AKAP) that tether RII

subunits to specific subcellular structures has been well documented [34], and

the majority of AKAPs preferentially bind RII subunits. However, AKAPs specific

to both RI and RII have also been identified recently [35]. These kinase

anchoring proteins interact exclusively with the dimerization domain of the R

subunits, and only the first 50 N-terminal amino acid residues of the R subunits

are required for binding of AKAPs [36]. The extensive sequence diversity at

this region between RI and RII may account for the difference in their AKAP

binding affinities. Large numbers of AKAPs have been identified.

Compartmentalization of PKA molecules to discrete intracellular locations

through association with anchoring proteins may ensure specificity in signal

transduction by placing the kinase close to its appropriate effectors or

substrates [34]. While the ratio of the total R subunits:C subunits in normal tissue

was found to be relatively constant at around 1:1, the relative amount of RI

and RII varies and depends highly on physiological conditions and the hormonal

status of the tissue [28,29,37,38]. One study showed that, in knockout mice

lacking the gene encoding RIIb, an

increased level of RIa compensates for the

loss of RIIb in brown fat cells. The

switching of PKA isoform from PKA(IIb) to

PK(Ia) results in an elevated basal

level of PKA activity and increased energy expenditure. The RIIb knockout mice are leaner and protected against diet-induced obesity

[39]. These results clearly demonstrate that RIa and RIIb are functionally

distinct. Although many of the physiologic effects of cAMP can be ascribed to

the action of one or more of the PKA isoforms, some of the cAMP-dependent

effects can not be explained based on the functions of PKA. For example, the

ability of cAMP to enhance the secretion of insulin from pancreatic beta cells

is not affected by specific inhibitors of PKA [40]. Many similar experimental

observations have hinted at the existence of “PKA-independent”

mechanisms of cAMP action.

Epac, a new intracellular

cAMP receptor

Recently, a family of novel cAMP sensor proteins, named exchange

protein directly activated by camp

(Epac) or cAMP-regulated guanine exchange factor (cAMP-GEF), was identified

[41,42]. These proteins contain a CBD that is homologous to that of PKA R

subunits and the prokaryotic transcription regulator, cAMP receptor protein

(CRP) (Fig. 1). Epac proteins bind to cAMP with high affinity and

activate the Ras superfamily small GTPases Rap1 and Rap2. Rap1 was initially

identified as an antagonist for the transforming function of Ras [43]. It can

be activated in response to a variety of second messengers, including cAMP [45].

Although PKA can phosphorylate Rap1 at its C-terminus, PKA phosphorylation is

not required for cAMP-dependent activation of Rap1 [41]. There are two isoforms of Epac, Epac1 and Epac2, which are products

of independent genes in mammals. While Epac1 is ubiquitously expressed in all

tissues, Epac2 has a more limited distribution [41,42]. Epac1 and Epac2 share

extensive sequence homology, and both contain an N-terminal regulatory region

and a C-terminal catalytic region. The catalytic region of Epac1 consists of a

Ras exchange motif domain, Ras association domain and a classic CDC25-homology

domain responsible for nucleotide exchange activity. Whereas the regulatory

region of Epac1 and Epac2 shares a Dishevelled/Egl-10/pleckstrin (DEP) domain

followed by a CBD domain that is evolutionally conserved to the CBD of PKA and

the bacterial transcriptional factor CRP, an additional CBD N-terminal to the

DEP domain is presented in Epac2 (Fig. 1). The function of this extra

CBD domain is not clear, as it binds cAMP with low affinity and does not seem

to be essential for Epac2 regulation by cAMP [45].

Cellular functions regulated by Epac

Cellular functions regulated by Epac

The discovery of Epac proteins as a new family of intracellular cAMP

receptors suggests that the cAMP-mediated signaling mechanism is much more

complex than what was believed earlier. Many cAMP-mediated effects that were

previously thought to act through PKA alone may also be transduced by Epac.

Extensive studies have so far established that Epac proteins are involved in a

host of cAMP-related cellular functions, such as cell adhesion [46,47],

cell-cell junction [48,49], exocytosis/secretion [50–53], cell differentiation

[54] and proliferation, gene expression, apoptosis, cardiac hypertrophy and

phagocytosis. With the exception of a few preliminary reports of Epac knockout

in fly and worm models, so far no detailed in vivo genetic and function

analyses of either Epac isoform in an animal model system have been reported.

Our discussion of Epac? biological functions will mainly be based on ex vivo studies

in cell culture models.

Epac and cell adhesion

One of the first cellular functions attributed to Epac is its

ability to enhance cell adhesion. When Epac is ectopically overexpressed in

HEK293 cells, it induces flattened cell morphology and increases cell adhesion

[55]. This is not surprising since one of the major functions of Rap1, a

downstream effector of Epac, is control of cell morphology/adhesion [56,57]. A

study using an Epac-selective cAMP analog, 8-(4-chloro-phenylthio)-2‘-O-methy­lade­nosine-3‘,5‘-cyclic

monophosphate [58], suggests that activation of Epac induces Rap-dependent

integrin-mediated cell adhesion to fibronectin in Ovcar3, a human ovarian carcinoma cell line [46]. Subsequent analysis further

revealed that the cAMP-Epac-Rap1 pathway regulates cell spreading and cell

adhesion to laminin-5 through the a3b1

integrin but not through the a6b4

integrin [47]. Interaction between Epac1 and light chain 2 of the microtubule-associated protein 1A enhances Rap1-dependent cell

adhesion to laminin [59]. Activation of Epac1 increases the b2-integrin-dependent

adhesion of human endothelial progenitor cells to endothelial cell monolayers and to ICAM-1, as well as the b1-integrin-dependent adhesion of human endothelial progenitor cells and mesenchymal stem

cells to the matrix protein fibronectin [60]. These

results demonstrate Epac’s therapeutic potential via enhancing

integrin-dependent homing functions of progenitor cells. Interestingly, cAMP-Epac1-Rap1 signaling also stimulates sickle red

blood cells adhesion to lammin. However, the adhesion of sickle red blood cells

to lammin promoted by Epac-Rap1 is not dependent on integrin, but

it is mediated by the cell adhesion molecule/Lutheran

receptor, a member of the Ig superfamily of receptors [61]. Consistent with the

stimulatory effect of Epac1-Rap1 on cell adhesion, activation of Epac1 inhibits

epithelial cell migration, which requires the disruption of cell-cell adhesion,

in response to both hepatocyte growth factor and transforming growth factor b (TGFb) [62]. Direct interaction between Epac1 and type I TGFb receptor has been reported and may be responsible for the observed

inhibitory effect of Epac1 on TGFb-mediated

cell migration [63]. While the effects of cAMP on cell adhesion are reported to be PKA

independent, cAMP-regulated integrin-dependent adhesions of vascular

endothelial cells to extracellular matrix proteins are coordinated by both PKA

and Epac [64]. In human primary monocytes and in monocytic U937 cells, Epac1-Rap1 has been shown to regulate b1-integrin-dependent

cell adhesion, cell polarization and chemotaxis [65]. However,

a similar study showed that, although Epac1 is expressed in human peripheral monocytes and activates Rap1, cAMP modulates most monocyte immune functions through PKA and not Epac1-Rap1 [66].

Therefore, it appears that the roles of Epac1 and PKA in monocytes also remain

unsettled.

Epac and cell junctions

In addition to its effects on integrin-mediated adhesion, Epac1/Rap1

signaling has also shown to contribute to E-cadherin-mediated adhesion [67].

This is consistent with the fact that Rap1 plays an important role in the

formation of cell-cell junctions [68]. Stable cell-cell contacts are critical

for the barrier function of epithelial and endothelial cells. Endothelial cell

junctions are of central importance for regulating vascular permeability. It is

well established that cAMP enhances the formation of cell junctions and

endothelial barrier function. cAMP decreases basal permeability

and reverse vascular leakage induced by inflammatory mediators.

Previously, it was believed that cAMP exerted its effects through activation of PKA. However, inhibition of

PKA activity does not block cAMP-enhanced endothelial

cell barrier function, suggesting the existence of

PKA-independent pathways.  Several studies in human umbilical vein endothelial

cells now show that Epac1 induces junction formation and actin remodeling, and

reduces endothelial permeability through activating Rap1, which is enriched at

endothelial cell-cell contacts [48,49,69]. Activation of Epac leads to enhanced

basal endothelial barrier function by increasing cortical actin and

redistributing adherens and tight junctional molecules to cell-cell contacts.

Moreover, activation of Epac offsets thrombin-induced hyperpermeability through

down-regulation of Rho GTPase activation [48]. Using VE-cadherin null mouse

cells immortalized with polyoma mT, Kooistra et al demonstrated that

regulation of endothelial permeability by Epac1 requires VE-cadherin and that

Epac-specific cAMP analog-induced actin rearrangements are independent of cell

junction formation [49]. Recently, it was shown that Epac1 can directly promote

microtubule (MT) growth independent of Rap1 activation [70] and that Epac

activation reverses MT-dependent increases in vascular

permeability induced by tumor necrosis factor-a and TGFb. Therefore, it appears that Epac1 promotes endothelial barrier

function through a two-leg strategy: a Rap1-depedent increase in cortical actin and a Rap-independent regulation of MTs [71].Studies using human pulmonary artery endothelial cells show that

barrier-protective effects of cAMP, downstream of Prostaglandin E2, prostacyclin and atrial natriuretic peptide, on pulmonary

endothelial cells are mediated by both PKA and Epac pathways. Activation of PKA

and Epac/Rap1 converges on Rac activation via stimulation of Rac-specific GEFs

Tiam1 and Vav2, leading to the enhancement of peripheral actin cytoskeleton and

adherens junctions [72,73]. In rat venular microvessels, activation of the Epac/Rap1 pathway significantly

attenuates the platelet-activating factor-induced increase in microvessel

permeability, as measured by hydraulic conductivity, and completely

prevents the platelet-activating factor-induced rearrangement of VE-cadherin

[74]. Collectively, these results suggest that Epac/Rap1 signaling plays

an important role in maintaining endothelial barrier function and vascular

integrity.

Epac and secretion

While regulated exocytoses are mainly triggered by the elevation of

intracellular Ca2+, second messenger cAMP also plays a role in

modulating exocytosis in a variety of secretory cells. Epac has been implicated

in stimulating numerous secretory pathways, including insulin secretion in

pancreatic b cells [75,76], the release of the non-amyloidogenic soluble form of

amyloid precursor protein [53,77,78], progesterone secretion by luteinizing

human granulosa cells [79], secretory activity in mouse melanotrophs [80] and

rat chromaffin cells [81,82], neurotensin secretion in human endocrine cells

[51], acrosomal exocytosis in sperm [83], and apical exocytotic insertion of

aquaporin-2 in the inner medullary collecting duct [84]. As cAMP-regulated

exocytosis has been reviewed in detail [52], we will focus on some of the most

recent advances in the area of Epac-mediated insulin secretion. A recent study investigated the effect of PKA and Epac on two types

of secretory vesicles in mouse pancreatic -cells: large dense-core vesicles

(LVs) and small vesicles (SVs). By directly visualizing Ca2+-dependent exocytosis of both LVs and SVs with two-photon imaging,

it was revealed that Epac and PKA selectively regulate exocytosis of SVs and

LVs, respectively [85]. In a similar study, FM1-43 epifluorescence imaging was

used to dissect the distinct contributions of Epac and PKA in regulating the

number of plasma membrane (PM) exocytic sites and insulin secretory granule

(SG)-to-granule fusions in these exocytic events. Again, Epac and PKA modulate

both distinct and common exocytic steps to potentiate insulin exocytosis.

Whereas Epac activation mobilizes SGs to fuse at the PM and thereby increase

the number of PM exocytic sites, PKA and Epac activation synergistically

increases both the number of exocytic sites at the PM and SG-SG fusions [86].

Lastly, a study using primary cultured pancreatic b-cells isolated from

wild-type and mutant mice lacking Epac2 suggests that, although activation of

cAMP signaling alone does not cause either significant docking or fusion events

of insulin granules, it substantially potentiates both the first phase

(a prompt, marked and transient increase) and the second phase (a moderate and

sustained increase) of glucose-induced fusion events. Moreover,

cAMP-potentiated fusion events in the first phase of glucose-induced exocytosis

are markedly reduced in b-cells isolated from Epac2 null mice. The data indicates that Epac2

signaling is important in cAMP-regulated insulin secretion because it controls

insulin granule density near the PM [87].

Epac and differentiation

cAMP has been implicated in regulating differentiation in a variety

of cell systems, such as neurite outgrowth in the neuroendocrine model cell line PC12 [88] and adipocyte formation from mouse 3T3-L1 fibroblasts

[89]. The role that PKA plays in these processes is controversial, and it has

been speculated that a PKA-independent cAMP signaling component may be

involved. Indeed, several studies have revealed that Epac plays an important

role in mediating the effects of pituitary adenylate cyclase-activating

polypeptide in inducing neurite outgrowths in PC12 cells [54,90] and human

neuroblastoma SH-SY5Y cells [91]. However, as summarized in a recent Science

STKE perspective [92], the detailed signal transduction pathways that

mediate the neurotrophic effects of cAMP are not clear, and the involvement of

PKA remains contentious [93].Intracellular second messenger cAMP is essential for the induction

of adipocyte differentiation in the mouse 3T3-L1 preadipocyte cell line. Again, it is generally believed that cAMP exerts its

effects through activation of PKA. However, our recent

studies suggest that PKA catalytic activity is not required for cAMP-mediated adipocyte

differentiation in 3T3-L1 preadipocyte cells, as IBMX- or

forskolin-induced 3T3-L1 adipocyte differentiation is not sensitive to two

mechanistically distinct PKA inhibitors, H89 and PKI. On the other hand,

selectively suppressing Epac1 expression using short hairpin RNAs substantially

reduces the efficiency of IBMX- or forskolin-induced 3T3-L1 adipocyte

differentiation.Interestingly, while Epac1 is required for cAMP-mediated 3T3-L1

adipocyte differentiation, Epac-selective cAMP analog, 8-CPT-2′-O-Me-cAMP, is

not sufficient to replace IBMX or forskolin to induce 3T3-L1 adipocyte

differentiation, nor are cAMP analogs selective for PKA RI or RII. 3T3-L1

adipocyte differentiation requires the combination treatment of cAMP analogs

selective for Epac, PKA RI and RII (Cheng et al, unpublished data). We

are currently investigating the signaling mechanism of cAMP/Epac-mediated

adipocyte differentiation.

Epac and cardiomyocyte

hypertrophy

cAMP is the main second messenger in cardiomyocytes, which can be

activated by the sympathetic and parasympathetic systems, cardioactive hormones

and drugs [94]. cAMP regulates many important processes, such as contractility

and relaxation, in both normal and failing hearts [95]. Traditionally, these

effects have been attributed to the classic intracellular cAMP receptor, PKA

[96]. For example, PKA has been shown to phosphorylate key Ca2+-handling proteins, such as voltage-gated L-type Ca2+ channel [97], ryanodine receptor [98], and phospholamban [99,100].

The net result in increase in the sarcoplasmic reticulum Ca2+ release via ryanodine receptor 2 and enhanced uptake by SR Ca2+ pump results in larger intracellular Ca2+

transients. Increased Ca2+ transients significantly

enhance contractility. However, emerging evidence suggests that Epac may also

play an important role in many cellular functions, particularly cardiac

hypertrophy, as a new mediator of cAMP signaling in the cardiovascular system

[101]. Recent studies have shown that the expression of both Epac1 and

Epac2 are developmentally increased in the heart from neonatal stages to

adulthood, and Epac levels are significantly up-regulated in mouse hearts with

myocardial hypertrophy induced by chronic isoproterenol infusion or with

pressure overload by transverse aortic banding [102]. In cardiomyocytes, Epac

is involved in the formation of gap junctions, which are essential for gating

ions and small molecules to coordinate cardiac contractions [103]. Epac also

enhances intracellular Ca2+ release during cardiac

excitation-contraction coupling in cardiac myocytes by activating

calcium-calmodulin-dependent protein kinase II [104] or activation of

phospholipase Ce [105], which is known to

associate with cardiac hypertrophy. Interestingly, activation of Epac leads to

induction of hypertrophic program based on morphological changes, cytoskeletal

reorganization, increase in protein synthesis and induction of cardiac

hypertrophic markers. This effect is mediated by a Ca2+-dependent

activation of Rac, calcineurin and its primary downstream effector, NFAT [106].

It has been reported that Epac1 is the major Epac isoform expressed in the

human heart, and its level increases during heart failure. Knockdown of Epac1

strongly suppresses beta-adrenergic receptor-induced hypertrophic program. Surprisingly,

Epac1’s hypertrophic effects are mediated by the small GTPase Ras, the

phosphatase calcineurin and Ca(2+)/calmodulin-dependent protein kinase II,

independent of Rap1, a canonical Epac effector [107].

Cross-talk

between Epac and PKA

The discovery of second intracellular cAMP receptor raises many

questions regarding the mechanism of cAMP-mediated signaling. The existence of

two highly coordinated cAMP effectors provides a mechanism for a more precise

and integrated control of the cAMP signaling pathways in a spatial and temporal

manner. Since both PKA and Epac are ubiquitously expressed in all tissues, an

increase in intracellular cAMP levels will lead to the activation of both PKA

and Epac, and possibly other potential cAMP effectors as well. Therefore, the

net cellular effects of cAMP are not just dictated by PKA or Epac alone, but by

the sum of all the relevant pathways. As such, it is critical to consider which

cAMP effects are mediated by Epac and which by PKA, as well as whether there is

cross-talk between Epac and PKA.Our earlier studies demonstrated that although PKA and Epac are

activated by the same second messenger cAMP, they can exert opposing effects on

the regulation of the PKB/AKT pathway. While PKA suppresses PKB phosphorylation

and activity, activation of Epac leads to increased PKB phosphorylation [108].

Since our initial report, many studies have shown that Epac and PKA can act

antagonistically in controlling various cellular functions, such as

insulin-stimulated PKB phosphorylation [109], proliferation and differentiation

[54], myelin phagocytosis [110], regulation of hedgehog signaling and

glucocorticoid sensitivity in acute lymphoblastic leukemia cells [111], and

expression of high affinity choline transporter and the cholinergic locus

[112]. In contrast, we and others have shown that Epac and PKA, depending upon

the specific cellular context, can exert synergistic effects on downstream

signaling, such as stimulation of neurotensin secretion [51], promotion of PC12

cell neurite extension [93], regulation of sodium-proton exchanger isoform 3

[113], and attenuation of cAMP signaling through phosphodiesterases [114].

While a model of synergistic activation of Rap1 by Epac and PKA has been

proposed by Stork et al [44], the origin and causes of antagonism

between Epac and PKA is not understood. It is very likely that antagonism

between Epac and PKA involves complex mechanisms, and understanding the basis

of Epac and PKA cross-talk may represent a major research interest for future cAMP-mediated

signaling studies.

Mechanism of

cAMP-mediated Activation

Both Epac and PKA are regulated by a CBD, which is a compact and

evolutionally conserved structural/signaling motif that controls a set of

diverse functionalities when linked to other structural domains [115,116]. CBD,

the only common structural module between PKA and Epac, acts as a molecular

switch for sensing intracellular second messenger cAMP levels. X-ray crystal

structures and in-depth biochemical/biophysical analyses of PKA holoenzyme

complex and individual subunits reveal a molecular mechanism for cAMP-mediated

activation of PKA [117–120]. The R and C subunits form a large interface in the PKA

holoenzyme complex with several key residues (Y247 and W196) of the C subunit

binding directly to the phosphate binding cassette of the first CBD in the R

subunit [119]. cAMP not only competes directly with the C subunit for these

interactions, but it also induces major conformational changes in the R subunit,

particularly the helical subdomain of CBD, the inhibitor sequence and the

linker region [118–120]. Binding of the cAMP results in the retraction of the phosphate

binding cassette in the direction of cAMP-binding pocket and global

reorientation of the subhelical domain of CBD. The pivot motion around the

hydrophobic hinge dislodges the single extended B/C helix and, subsequently,

the inhibitor sequence from the docking site on the C subunit. In the absence

of the C subunit’s stabilizing/anchoring effects, the B/C helix bends in the

middle to form two individual helices, with the C helix portion folded back

onto the b barrel to form the “lid” of the cAMP-binding pocket.

These extensive cAMP-induced conformation changes eventually lead to the

activation of PKA.The CBD in Epac is covalently connected to the catalytic GEF domain

as a single polypeptide chain, and the intramolecular interaction between the

CBD and GEF domains sterically blocks the access of downstream effector Rap.

Recently, the crystal structure of Epac2 was solved in the absence of cAMP

[121]. In this autoinhibited Epac2 structure, the second CBD of Epac2, which is

common in both Epac1 and Epac2, is anchored to the catalytic core indirectly by

the Ras exchange motif domain through the so called “switchboard”.

One major structural difference between the CBD in Epac and PKA is located in

the lid region. The lid in CBD of PKA is a helix that covers the cAMP-binding

pocket, whereas the corresponding region in Epac points away from the cAMP binding-packet

in a two-strand b-sheet that forms the first part of the five-strand -sheet-like

“switchboard” structure. In addition, unlike the extensive interface

between the R and C subunits of PKA holoenzyme, the intramolecular interaction

between the regulatory and catalytic regions in Epac2 is surprisingly brief.

There is only one direct contact point between the CBD and catalytic core of

Epac, described as the “ionic latch”. These major differences suggest

that although it is likely that Epac and PKA activations share the same

underlying principal, the detailed mechanisms of PKA and Epac activation by

cAMP will most likely be different at the structural level.Since the crystal structure of cAMP-bound Epac in its active state

is not currently available, the mechanism of Epac activation is not clear.

Extensive biochemical and structural studies by the Bos and Wittinghofer groups

suggest that the lid region of the C-terminus of CBD in Epac plays an important

role in communicating between the regulatory and catalytic domains and is

pivotal for the activation of Epac by cAMP [45,122–125]. To further probe the

mechanism of Epac activation, we used amide H/D exchange coupled with Fourier

transform infrared spectroscopy (FT-IR) and mass spectrometry to examine the

conformation and structural dynamics of Epac1 in the presence and absence of

cAMP. Our studies show that binding of cAMP to Epac1 does not induce

significant changes in overall secondary structure and structural dynamics,

suggesting that conformational changes induced by cAMP in Epac1 are most likely

local motion, such as hinge movements [126,127]. Hinge prediction based on

Gaussian Network Model first normal model displacement analysis revealed a

major hinge in Epac1 between residues 310 and 345 [127]. Indeed, our

amide H/D exchange mass spectrometry study reveals that the solvent

accessibility of this hinge region decreases upon cAMP binding, indicating

conformational changes [128]. Based on the cAMP-free Epac2 structure and our

in-depth H/D exchange and comparative sequence/structure analyses of Epac and

PKA, we propose a model of Epac activation (Fig. 2). In this model,

binding of cAMP induces an allosteric switch manifested by a hinge motion that

bends the extended C helix lid toward the b-barrel of the CBD. This

hinge movement pulls the b-strands S1 and S2 away from the five-strand b-sheet-like

switchboard to form the base of the cAMP-binding pocket. The conformational

changes induced upon cAMP binding result in a closed CBD conformation and

reorientation of the CBD/DEP domains relative to the rest of the molecule,

which releases the catalytic core from the inhibitory contact imposed by the

CBD. This structural transition allows Epac, albeit with a completely different

lid conformation in the inactive Epac structure, to utilize the same underlying

principal to bind cAMP in almost exactly the same manner as PKA and other

CBD-containing proteins [118,119,129,130]. Although final validation of the

model requires the three-dimensional structure of an Epac-cAMP complex, our

earlier studies using Epac-based fluorescence resonance energy transfer

indicators suggest that binding of cAMP leads to a more extended Epac

conformation [131], an observation in agreement with our model.

Conclusion

Since the discovery of Epac proteins a decade ago, the cAMP research

area has undergone a renaissance. It is now well recognized that eukaryotic

cAMP signaling is much more complex than it was initially believed and that the

classic PKA pathway is only part of the story. The net physiological effects of

cAMP necessitate the integration of Epac- and PKA-dependent pathways in a

spatial and temporal manner, which dramatically increases the complexity and,

consequently, the possible readouts of cAMP signaling. Depending upon the precise

cellular environment as well as their relative abundance, distribution and

localization, the two intracellular cAMP receptors may act independently,

converge synergistically or oppose each other in regulating a specific cellular

function. Therefore, careful dissection of the individual role and relative

contribution of Epac and PKA within the overall cAMP signaling in various model

systems will continue to be an important part of future research activity. In

addition, although we have learned a great deal about the structure and

functions of Epac, much remains to be discovered. Important future research in

the area includes but is not limited to understanding the physiological roles

of Epac isoforms using animal models, elucidating the mechanism of cross-talk

between Epac and PKA, and mapping the conformational cAMP-induced changes

during Epac activation.

Acknowledgements

The authors wish to apologize to the

investigators whose outstanding work was not cited here because of space limitations.

The authors would also like to thank Ms. Betty Redd and Mr. John Helms for

assisting in manuscript preparation.

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