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

QH301 Biology;QH345 Biochemistry;RM Therapeutics. Pharmacology

Parnell, Euan ; Yarwood, Stephen, J,

University:University of Glasgow

Department:Institute of Molecular Cell and Systems Biology

关键词: cAMP, EPAC, PKA, targetting, cell, high throughput screening, cell morphology, signalling;   

Others  :  http://theses.gla.ac.uk/6258/7/2015parnellphd.pdf

来源: University of Glasgow

【 摘 要 】

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.