Protein Kinase A-α Directly Phosphorylates FoxO1 in Vascular Endothelial Cells to Regulate Expression of Vascular Cellular Adhesion Molecule-1 mRNA

FoxO1, a forkhead box O class transcription factor, is abundant in insulin-responsive tissues. Akt, downstream from phosphatidylinositol 3-kinase in insulin signaling, phosphorylates FoxO1 at Thr(24), Ser(256), and Ser(319), negatively regulating its function. We previously reported that dehydroepiandrosterone-stimulated phosphorylation of FoxO1 in endothelial cells requires cAMP-dependent protein kinase alpha (PKA-alpha). Therefore, we hypothesized that FoxO1 is a novel direct substrate for PKA-alpha. Using an immune complex kinase assay with [gamma-P-32] ATP, purified PKA-alpha directly phosphorylated wildtype FoxO1 but not FoxO1-AAA (mutant with alanine substitutions at known Akt phosphorylation sites). Phosphorylation of wild-type FoxO1 (but not FoxO1-AAA) was detectable using phospho-specific antibodies. Similar results were obtained using purified GST-FoxO1 protein as the substrate. Thus, FoxO1 is a direct substrate for PKA-alpha in vitro. In bovine aortic endothelial cells, interaction between endogenous PKA-alpha and endogenous FoxO1 was detected by co-immunoprecipitation. In human aortic endothelial cells (HAEC), pretreatment with H89 (PKA inhibitor) or siRNA knockdown of PKA-alpha decreased forskolin-or prostaglandin E-2-stimulated phosphorylation of FoxO1. In HAEC transfected with a FoxO-promoter luciferase reporter, co-expression of the catalytic domain of PKA-alpha, catalytically inactive mutant PKA-alpha, or siRNA against PKA-alpha caused corresponding increases or decreases in trans-activation of the FoxO promoter. Expression of vascular cellular adhesion molecule-1 mRNA, up-regulated by FoxO1 in endothelial cells, was enhanced by siRNA knockdown of PKA-alpha or treatment of HAEC with the PKA inhibitor H89. Adhesion of monocytes to endothelial cells was enhanced by H89 treatment or overexpression of FoxO1-AAA, similar to effects of TNF-alpha treatment. We conclude that FoxO1 is a novel physiological substrate for PKA-alpha in vascular endothelial cells.