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

Autor: Gemma L. Baillie, Alison Porter, Stuart P. McElroy, Ryan T. Cameron, George S. Baillie, Andreas Koschinski, Euan Parnell, Manuela Zaccolo, Stephen J. Yarwood
Rok vydání: 2015
Předmět:
GEF
guanine exchange factor
F-Actin
filamentous actin
F/R
forskolin/rolipram
Cell morphology
environment and public health
Cell membrane
0302 clinical medicine
Ezrin
Cell Movement
Chlorocebus aethiops
Cyclic AMP
Guanine Nucleotide Exchange Factors
Phosphorylation
Cytoskeleton
007
8-pCPT-2′-O-Me-cAMP
Genes
Dominant
0303 health sciences
ROCK
RhoA activated Protein Kinase
biology
Microfilament Proteins
3. Good health
Cell biology
medicine.anatomical_structure
Phosphothreonine
COS Cells
EPAC
exchange protein directly activated by cAMP
Intracellular
EPAC1
macromolecular substances
Article
03 medical and health sciences
medicine
Human Umbilical Vein Endothelial Cells
HUVEC
human umbilical vein endothelial cells
Animals
Humans
ERM
ezrin–radixin–moesin family
Actin-binding protein
Protein kinase A
Protein Kinase Inhibitors
Molecular Biology
030304 developmental biology
Cell Membrane
Cell Biology
Actin cytoskeleton
Cyclic AMP-Dependent Protein Kinases
Cytoskeletal Proteins
FERM
4.1 ezrin–radixin–moesin
HEK293 Cells
biology.protein
PKA
protein kinase A
030217 neurology & neurosurgery
Zdroj: Biochimica et Biophysica Acta
ISSN: 0167-4889
DOI: 10.1016/j.bbamcr.2015.04.009
Popis: 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.
Graphical abstract
Highlights • EPAC1 is absolutely required for cAMP-induced cell spreading in HEK293T cells. • EPAC1 facilitates further PKA-dependent cell spreading in HEK293T cells. • PKA-dependent cell spreading is associated with cortical actin bundling. • Ezrin is required for EPAC1- and PKA-dependent cell spreading. • Phosphorylated ezrin (pThr567) stabilises cortical actin and prevents membrane projections.
Databáze: OpenAIRE
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