Abstract
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.
Highlights
Cyclic adenosine monophosphate is a ubiquitous second messenger that is involved in regulating many aspects of cell function, including cell differentiation [1], secretion [2], cell morphology [3], inflammatory pathways [4], contractility [5] and synapse formation [6]
This suggests that EPAC1 activation alone is not sufficient to promote maximal levels of cell spreading or cortical actin bundling in human umbilical vein endothelial cells (HUVEC), and that there is an additional requirement for protein kinase A (PKA)
Cooperativity must exist between exchange protein directly activated by Cyclic adenosine monophosphate (cAMP) (EPAC) and PKA signalling pathways in HUVECs that underlies the cytoskeletal reorganisation required for maximal cell spreading
Summary
Cyclic adenosine monophosphate (cAMP) is a ubiquitous second messenger that is involved in regulating many aspects of cell function, including cell differentiation [1], secretion [2], cell morphology [3], inflammatory pathways [4], contractility [5] and synapse formation [6]. Elevations in intracellular cAMP occur in response to activation of Gscoupled G-protein coupled receptors (GPCRs). Adenylate cyclase (AC), which catalyses the conversion of ATP into cAMP. This process is terminated through the action of the cAMP phosphodiesterase (PDE) family, which hydrolyses cAMP to 5′-AMP [7]. E. Parnell et al / Biochimica et Biophysica Acta 1853 (2015) 1749–1758 guanine nucleotide exchange factor (GEF) towards the small GTPases, Rap and Rap2 [14], as opposed to the kinase activity of PKA [10]
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