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Acta Biochim Biophys |
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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
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抯 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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