The cAMP sensors, EPAC1 and EPAC2, display distinct subcellular distributions despite sharing a common nuclear pore localisation signal

We have identified a conserved nuclear pore localisation signal (NPLS; amino acids 764–838 of EPAC1) in the catalytic domains of the cAMP-sensors, EPAC1 and EPAC2A. Consequently, EPAC1 is mainly localised to the nuclear pore complex in HEK293T cells where it becomes activated following stimulation with cAMP. In contrast, structural models indicate that the cAMP-binding domain of EPAC2A (CNBD1) blocks access to the conserved NPLS in EPAC2A, reducing its ability to interact with nuclear binding sites. Consequently, a naturally occurring EPAC2 isoform, EPAC2B, which lacks CNBD1 is enriched in nuclear fractions, similar to EPAC1. Structural differences in EPAC isoforms may therefore determine their intracellular location and their response to elevations in intracellular cAMP

EPAC isoform specificity: drug development, subcellular targeting and relevance to cell morphology

Cyclic adenosine monophosphate (cAMP) is a second messenger signalling molecule that has been reported to exert beneficial effects within the vasculature and other physiological systems. cAMP produces its effects within the cell through two key downstream effector molecules: exchange protein activated by cAMP (EPAC) and protein kinase A (PKA). Many of the effects of cAMP have been attributed to PKA, however there is a growing appreciation of the potential of EPAC, particularly isoform 1 (EPAC1), based therapies for the regulation of inflammatory responses within the vasculature, thereby promoting cardiovascular health. Furthermore, side effects associated with global cAMP elevating agents may be avoided by isoform selective EPAC regulation. To date no small molecule agonists have been discovered to effectively or selectively promote EPAC1 activity. In order to address this, we have developed a fluorescence based competition assay able to identify compounds which interact with the cyclic nucleotide binding domains (CNBs) of both EPAC1 and EPAC2. Rigorous testing of the assay has confirmed that it is able to reliably and reproducibly identify EPAC interacting compounds within high throughput screening (HTS) of small molecule libraries. Furthermore, dual screening of EPAC1 and EPAC2 has allowed isoform selective compounds to be identified from a small compound library, confirming the suitability of this assay for HTS. This HTS assay is likely to facilitate the discovery of EPAC1-selective interacting molecules with the potential to be effective, small molecule regulators of EPAC1. In order to classify small molecules isolated by HTS as either agonists or antagonists of EPAC1, we developed a secondary screen that is able to detect EPAC1 activation in vivo. This assay is based on the ability of EPAC1 to produce a rapid, cell spreading response in HEK293T cells stably transfected with EPAC1. However, the precise signalling pathways which produce these changes in cell shape are unknown. Therefore, we have attempted to identify pathways involved in EPAC1-mediated morphological change by assessing the effects of various inhibitors on cell spreading. Interestingly, we found that EPAC1 and PKA synergise to produce maximal cell spreading in HEK293T cells. Recent reports suggest that the cortical actin-membrane linker protein ezrin is required for the cell spreading effects of EPAC1. Here, we demonstrate that ezrin responds to elevations in intracellular cAMP in HEK293T cells in a PKA-dependent manner. Indeed, PKA activation promotes the post translational modification of ezrin and alters the response of EPAC1-expressing cells to cAMP. These results suggests that the PKA pathway is able to regulate ezrin by post translational modification and that this is required for PKA and EPAC1 to synergise and produce maximal cell spreading. In addition to agents which directly activate the catalytic activity of EPAC1, there is a body of evidence that supports the idea that compartmentalisation of cAMP effectors is an important mechanism for the determination of downstream signalling events leading to cellular responses, such as cell spreading. As such, we have attempted to identify the regions within EPAC proteins that determine their subcellular distribution. This was done through a combination of subcellular fractionation and the immunofluorescent detection of the localisation of EPAC isoforms. In particular, mutational analysis of EPAC1 revealed a carboxy terminal (C-terminal) nuclear localisation domain that is required for the perinuclear distribution of EPAC1 alongside the nuclear pore protein, RANBP2. Structural analyses suggest that this domain appears to be conserved within EPAC2 despite EPAC2 adopting a distinct cytoplasmic distribution. One explanation for this observation is steric interference within EPAC2 which blocks access to the conserved nuclear localisation domain. We have observed that the additional amino-terminal (N-terminal) CNB of EPAC2 appears to disrupt nuclear localisation and promote a cytoplasmic distribution within the cell. Indeed, the absence of the CNB1 promotes nuclear accumulation of EPAC2, with a pattern similar to that of EPAC1. The presence of this domain within EPAC2, absent in EPAC1, may represent a mechanism which regulates the subcellular distribution, and therefore function, of EPACs within the cellular environment. In summary, we have developed a screening cascade to identify small molecules which may form the basis of therapeutic agents able to selectively target EPAC1 to promote the beneficial effects of EPAC1. In addition, a secondary screen involving EPAC1 induced morphological change was developed and characterised as an effective assay in which to test the agonist properties of compounds identified by primary HTS screening. We have confirmed that HEK293T cell spreading in response to cAMP elevation requires the expression of EPAC1, but that a secondary pathway involving interactions between PKA and ezrin is able to supplement the primary cell spreading effects of EPAC1. Finally, we have identified a potential mechanism for the different subcellular localisation of EPAC1 and EPAC2: EPAC1 is targeted to the perinuclear compartment via a previously undiscovered C-terminal nuclear localisation domain

Phosphorylation of ezrin on Thr567 is required for the synergistic activation of cell spreading by EPAC1 and protein kinase A in HEK293T cells

Recent studies have demonstrated that the actin binding protein, ezrin, and the cAMP-sensor, EPAC1, cooperate to induce cell spreading in response to elevations in intracellular cAMP. To investigate the mechanisms underlying these effects we generated a model of EPAC1-dependent cell spreading based on the stable transfection of EPAC1 into HEK293T (HEK293T–EPAC1) cells. We found that direct activation of EPAC1 with the EPAC-selective analogue, 8-pCPT-2′-O-Me-cAMP (007), promoted cell spreading in these cells. In addition, co-activation of EPAC1 and PKA, with a combination of the adenylate cyclase activator, forskolin, and the cAMP phosphodiesterase inhibitor, rolipram, was found to synergistically enhance cell spreading, in association with cortical actin bundling and mobilisation of ezrin to the plasma membrane. PKA activation was also associated with phosphorylation of ezrin on Thr567, as detected by an electrophoretic band mobility shift during SDS-PAGE. Inhibition of PKA activity blocked ezrin phosphorylation and reduced the cell spreading response to cAMP elevation to levels induced by EPAC1-activation alone. Transfection of HEK293T–EPAC1 cells with inhibitory ezrin mutants lacking the key PKA phosphorylation site, ezrin-Thr567Ala, or the ability to associate with actin, ezrin-Arg579Ala, promoted cell arborisation and blocked the ability of EPAC1 and PKA to further promote cell spreading. The PKA phospho-mimetic mutants of ezrin, ezrin-Thr567Asp had no effect on EPAC1-driven cell spreading. Our results indicate that association of ezrin with the actin cytoskeleton and phosphorylation on Thr567 are required, but not sufficient, for PKA and EPAC1 to synergistically promote cell spreading following elevations in intracellular cAMP

The Potential of a Novel Class of EPAC-Selective Agonists to Combat Cardiovascular Inflammation

The cyclic 3′,5′-adenosine monophosphate (cAMP) sensor enzyme, EPAC1, is a candidate drug target in vascular endothelial cells (VECs) due to its ability to attenuate proinflammatory cytokine signalling normally associated with cardiovascular diseases (CVDs), including atherosclerosis. This is through the EPAC1-dependent induction of the suppressor of cytokine signalling gene, SOCS3, which targets inflammatory signalling proteins for ubiquitinylation and destruction by the proteosome. Given this important role for the EPAC1/SOCS3 signalling axis, we have used high throughput screening (HTS) to identify small molecule EPAC1 regulators and have recently isolated the first known non-cyclic nucleotide (NCN) EPAC1 agonist, I942. I942 therefore represents the first in class, isoform selective EPAC1 activator, with the potential to suppress pro-inflammatory cytokine signalling with a reduced risk of side effects associated with general cAMP-elevating agents that activate multiple response pathways. The development of augmented I942 analogues may therefore provide improved research tools to validate EPAC1 as a potential therapeutic target for the treatment of chronic inflammation associated with deadly CVDs

Identification of A Novel Class of Benzofuran Oxoacetic Acid-Derived Ligands that Selectively Activate Cellular EPAC1

Cyclic AMP promotes EPAC1 and EPAC2 activation through direct binding to a specific cyclic nucleotide-binding domain (CNBD) within each protein, leading to activation of Rap GTPases, which control multiple cell responses, including cell proliferation, adhesion, morphology, exocytosis, and gene expression. As a result, it has become apparent that directed activation of EPAC1 and EPAC2 with synthetic agonists may also be useful for the future treatment of diabetes and cardiovascular diseases. To identify new EPAC agonists we have developed a fluorescent-based, ultra-high-throughput screening (uHTS) assay that measures the displacement of binding of the fluorescent cAMP analogue, 8-NBD-cAMP to the EPAC1 CNBD. Triage of the output of an approximately 350,000 compound screens using this assay identified a benzofuran oxaloacetic acid EPAC1 binder (SY000) that displayed moderate potency using orthogonal assays (competition binding and microscale thermophoresis). We next generated a limited library of 91 analogues of SY000 and identified SY009, with modifications to the benzofuran ring associated with a 10-fold increase in potency towards EPAC1 over SY000 in binding assays. In vitro EPAC1 activity assays confirmed the agonist potential of these molecules in comparison with the known EPAC1 non-cyclic nucleotide (NCN) partial agonist, I942. Rap1 GTPase activation assays further demonstrated that SY009 selectively activates EPAC1 over EPAC2 in cells. SY009 therefore represents a novel class of NCN EPAC1 activators that selectively activate EPAC1 in cellulae

The future of EPAC-targeted therapies: agonism versus antagonism

yesPharmaceutical manipulation of cAMP levels exerts beneficial effects through the regulation of the exchange protein activated by cAMP (EPAC) and protein kinase A (PKA) signalling routes. Recent attention has turned to the specific regulation of EPAC isoforms (EPAC1 and EPAC2) as a more targeted approach to cAMP-based therapies. For example, EPAC2-selective agonists could promote insulin secretion from pancreatic β cells, whereas EPAC1-selective agonists may be useful in the treatment of vascular inflammation. By contrast, EPAC1 and EPAC2 antagonists could both be useful in the treatment of heart failure. Here we discuss whether the best way forward is to design EPAC-selective agonists or antagonists and the current strategies being used to develop isoform-selective, small-molecule regulators of EPAC1 and EPAC2 activity

Usp9X Controls Ankyrin-Repeat Domain Protein Homeostasis during Dendritic Spine Development

Variants in the ANK3 gene encoding ankyrin-G are\ua0associated with neurodevelopmental disorders, including intellectual disability, autism, schizophrenia, and bipolar disorder. However, no upstream regulators of ankyrin-G at synapses are known. Here, we\ua0show that ankyrin-G interacts with Usp9X, a neurodevelopmental-disorder-associated deubiquitinase (DUB). Usp9X phosphorylation enhances their interaction, decreases ankyrin-G polyubiquitination, and stabilizes ankyrin-G to maintain dendritic spine development. In forebrain-specific Usp9X knockout mice (Usp9X), ankyrin-G as well as multiple ankyrin-repeat domain (ANKRD)-containing proteins are transiently reduced at 2 but recovered at 12\ua0weeks postnatally. However, reduced cortical spine density in knockouts persists into adulthood. Usp9X mice display increase of ankyrin-G ubiquitination and aggregation and hyperactivity. USP9X mutations in patients with intellectual disability and autism ablate its catalytic activity or ankyrin-G interaction. Our data reveal a DUB-dependent mechanism of ANKRD protein homeostasis, the impairment of which only transiently\ua0affects ANKRD protein levels but leads to\ua0persistent neuronal, behavioral, and clinical abnormalities