Dynamic regulation of endothelial NOS mediated by competitive interaction with α‐actinin‐4 and calmodulin

Alpha‐actinins are critical components of the actin cytoskeleton. Here we show that α‐actinins serve another important biological function by binding to and competitively inhibiting calcium‐dependent activation of endothelial NOS (eNOS). α‐actinin‐2 was found to associate with eNOS in a yeast two‐hybrid screen. In vascular endothelial cells, which only express α‐actinin‐1 and ‐4, α‐actinin‐4 interacted and colocalized with eNOS. Addition of α‐actinin‐4 directly inhibited eNOS recombinant protein, and overexpression of a‐actinin‐4 inhibited eNOS activity in eNOS‐ transfected COS‐7 cells and bovine aortic endothelial cells (BAECs). In contrast, knockdown of α‐actinin‐4 by siRNA increased eNOS activity in BAECs. The α‐actinin‐4‐binding site on eNOS was mapped to a central region comprising the calmodulin‐binding domain, and the eNOS‐binding site on α‐actinin‐4 was mapped to the fourth spectrin‐like rod domain, R4. Treatment of endothelial cells with a calcium ionophore, A23187, decreased α‐actinin‐4‐eNOS interaction, leading to translocation of α‐actinin‐4 from plasma mem‐brane to cytoplasm. Indeed, addition of calmodulin dis‐placed α‐actinin‐4 binding to eNOS and increased eNOS activity. These findings indicate that eNOS activity in vascular endothelial cells is tonically and dynamically regulated by competitive interaction with α‐actinin‐4 and calmodulin.—Hiroi, Y., Guo, Z., Li, Y., Beggs, A. H., Liao, J. K. Dynamic regulation of endothelial NOS mediated by competitive interaction with a‐actinin‐4 and calmodulin. FASEB J . 22, 1450–1457 (2008)