Dynamic regulation of endothelial NOS mediated by competitive interaction with alpha-actinin-4 and calmodulin.
Alpha-actinins are critical components of the actin cytoskeleton. Here we show that alpha-actinins serve another important biological function by binding to and competitively inhibiting calcium-dependent activation of endothelial NOS (eNOS). Alpha-actinin-2 was found to associate with eNOS in a yeast two-hybrid screen. In vascular endothelial cells, which only express alpha-actinin-1 and -4, alpha-actinin-4 interacted and colocalized with eNOS. Addition of alpha-actinin-4 directly inhibited eNOS recombinant protein, and overexpression of alpha-actinin-4 inhibited eNOS activity in eNOS-transfected COS-7 cells and bovine aortic endothelial cells (BAECs). In contrast, knockdown of alpha-actinin-4 by siRNA increased eNOS activity in BAECs. The alpha-actinin-4-binding site on eNOS was mapped to a central region comprising the calmodulin-binding domain, and the eNOS-binding site on alpha-actinin-4 was mapped to the fourth spectrin-like rod domain, R4. Treatment of endothelial cells with a calcium ionophore, A23187, decreased alpha-actinin-4-eNOS interaction, leading to translocation of alpha-actinin-4 from plasma membrane to cytoplasm. Indeed, addition of calmodulin displaced alpha-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 alpha-actinin-4 and calmodulin.
Hiroi
Yukio
Y
Vascular Medicine Research, Brigham & Women's Hospital, 65 Landsdowne Street, Boston, MA 02139, USA.
Guo
Zhongmin
Z
Li
Yuxin
Y
Beggs
Alan H
AH
Liao
James K
JK
D000185:Actinin / Q000502:physiology*; D000818:Animals; D002147:Calmodulin / Q000502:physiology*; D002417:Cattle; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000187:drug effects / Q000201:enzymology*; D006801:Humans; D008840:Microfilament Proteins / Q000502:physiology*; D052250:Nitric Oxide Synthase Type III / Q000037:antagonists & inhibitors / Q000378:metabolism*; D020798:Two-Hybrid System Techniques
C111453:ACTN4 protein, human; D002147:Calmodulin; D008840:Microfilament Proteins; D000185:Actinin; D052250:Nitric Oxide Synthase Type III