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.
Institute of Molecular, Cellular and Systems Biology, College of Medical Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8QQ, UK.
Euan
E
Parnell
Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford OX1 3QX, UK.
Andreas
A
Koschinski
Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford OX1 3QX, UK.
Manuela
M
Zaccolo
Institute of Cardiovascular and Medical Sciences, College of Medical Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8QQ, UK.
Ryan T
RT
Cameron
Institute of Cardiovascular and Medical Sciences, College of Medical Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8QQ, UK.
George S
GS
Baillie
European Screening Centre, BioCity Scotland, Newhouse ML1 5UH, UK.
Gemma L
GL
Baillie
European Screening Centre, BioCity Scotland, Newhouse ML1 5UH, UK.
Alison
A
Porter
European Screening Centre, BioCity Scotland, Newhouse ML1 5UH, UK.
Stuart P
SP
McElroy
Institute of Molecular, Cellular and Systems Biology, College of Medical Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8QQ, UK. Electronic address: stephen.yarwood@glasgow.ac.uk.
Stephen J
SJ
Yarwood
D000818:Animals; D019556:COS Cells; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002465:Cell Movement / Q000187:drug effects; D002522:Chlorocebus aethiops; D000242:Cyclic AMP / Q000378:metabolism / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D003598:Cytoskeletal Proteins / Q000378:metabolism*; D003599:Cytoskeleton / Q000378:metabolism; D005799:Genes, Dominant; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism*; D057809:HEK293 Cells; D061307:Human Umbilical Vein Endothelial Cells; D006801:Humans; D008840:Microfilament Proteins / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D010769:Phosphothreonine / Q000378:metabolism*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D000099149:Ezrin
D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D003598:Cytoskeletal Proteins; D020662:Guanine Nucleotide Exchange Factors; D008840:Microfilament Proteins; D010769:Phosphothreonine; D047428:Protein Kinase Inhibitors; D000099149:Ezrin; C116122:RAPGEF3 protein, human
Cell morphology; Cyclic AMP; Cytoskeleton; EPAC1