AMP-Activated Protein Kinase and Sirtuin 1 Coregulation of Cortactin Contributes to Endothelial Function

Tzu-Pin Shentu; Ming He; Xiaoli Sun; Jianlin Zhang; Fan Zhang; Brendan Gongol; Traci L Marin; Jiao Zhang; Liang Wen; Yinsheng Wang; Gregory G Geary; Yi Zhu; David A Johnson; John Y-J Shyy

doi:10.1161/ATVBAHA.116.307871

AMP-Activated Protein Kinase and Sirtuin 1 Coregulation of Cortactin Contributes to Endothelial Function

Arterioscler Thromb Vasc Biol. 2016 Dec;36(12):2358-2368. doi: 10.1161/ATVBAHA.116.307871. Epub 2016 Oct 6.

Authors

Affiliations

¹ From the Division of Cardiology, Department of Medicine, University of California, San Diego, La Jolla (T.-P.S., M.H., J.Z., J.Z.; L.W., J.Y.-J.S.); Department of Physiology and Pathophysiology, Peking University Health Science Center, Beijing, China (X.S., Y.Z.); Department of Chemistry, University of California, Riverside (F.Z., Y.W.); Department of Cardiopulmonary Sciences, Schools of Allied Health, Loma Linda University, CA (B.G., T.L.M.); Department of Kinesiology and Health Sciences, California State University, San Bernardino (G.G.G.); and Division of Biomedical Sciences, University of California, Riverside (D.A.J.).
² From the Division of Cardiology, Department of Medicine, University of California, San Diego, La Jolla (T.-P.S., M.H., J.Z., J.Z.; L.W., J.Y.-J.S.); Department of Physiology and Pathophysiology, Peking University Health Science Center, Beijing, China (X.S., Y.Z.); Department of Chemistry, University of California, Riverside (F.Z., Y.W.); Department of Cardiopulmonary Sciences, Schools of Allied Health, Loma Linda University, CA (B.G., T.L.M.); Department of Kinesiology and Health Sciences, California State University, San Bernardino (G.G.G.); and Division of Biomedical Sciences, University of California, Riverside (D.A.J.). [email protected].

Abstract

Objective: Cortactin translocates to the cell periphery in vascular endothelial cells (ECs) on cortical-actin assembly in response to pulsatile shear stress. Because cortactin has putative sites for AMP-activated protein kinase (AMPK) phosphorylation and sirtuin 1 (SIRT1) deacetylation, we examined the hypothesis that AMPK and SIRT1 coregulate cortactin dynamics in response to shear stress.

Approach and results: Analysis of the ability of AMPK to phosphorylate recombinant cortactin and oligopeptides whose sequences matched AMPK consensus sequences in cortactin pointed to Thr-401 as the site of AMPK phosphorylation. Mass spectrometry confirmed Thr-401 as the site of AMPK phosphorylation. Immunoblot analysis with AMPK siRNA and SIRT1 siRNA in human umbilical vein ECs and EC-specific AMPKα2 knockout mice showed that AMPK phosphorylation of cortactin primes SIRT1 deacetylation in response to shear stress. Immunoblot analyses with cortactin siRNA in human umbilical vein ECs, phospho-deficient T401A and phospho-mimetic T401D mutant, or aceto-deficient (9K/R) and aceto-mimetic (9K/Q) showed that cortactin regulates endothelial nitric oxide synthase activity. Confocal imaging and sucrose-density gradient analyses revealed that the phosphorylated/deacetylated cortactin translocates to the EC periphery facilitating endothelial nitric oxide synthase translocation from lipid to nonlipid raft domains. Knockdown of cortactin in vitro or genetic reduction of cortactin expression in vivo in mice substantially decreased the endothelial nitric oxide synthase-derived NO bioavailability. In vivo, atherosclerotic lesions increase in ApoE^-^/-/cortactin^+/- mice, when compared with ApoE^-/-/cortactin^+/+ littermates.

Conclusions: AMPK phosphorylation of cortactin followed by SIRT1 deacetylation modulates the interaction of cortactin and cortical-actin in response to shear stress. Functionally, this AMPK/SIRT1 coregulated cortactin-F-actin dynamics is required for endothelial nitric oxide synthase subcellular translocation/activation and is atheroprotective.

Keywords: AMP-activated protein kinases; atherosclerosis; caveolin-1; cortactin; nitric oxide synthase type III.

MeSH terms

AMP-Activated Protein Kinases / deficiency
AMP-Activated Protein Kinases / genetics
AMP-Activated Protein Kinases / metabolism*
Acetylation
Actins / metabolism
Animals
Apolipoproteins E / deficiency
Apolipoproteins E / genetics
Atherosclerosis / genetics
Atherosclerosis / metabolism*
Atherosclerosis / prevention & control
Cells, Cultured
Cortactin / deficiency*
Cortactin / genetics
Cortactin / metabolism*
Disease Models, Animal
Endothelial Cells / enzymology*
Genotype
Humans
Male
Mice, Knockout
Nitric Oxide Synthase Type III / metabolism
Phenotype
Phosphorylation
Protein Transport
Pulsatile Flow
RNA Interference
Signal Transduction
Sirtuin 1 / genetics
Sirtuin 1 / metabolism*
Stress, Mechanical
Transfection

Substances

Actins
Apolipoproteins E
CTTN protein, human
Cortactin
Cttn protein, mouse
NOS3 protein, human
Nitric Oxide Synthase Type III
Nos3 protein, mouse
AMPK alpha2 subunit, mouse
PRKAA2 protein, human
AMP-Activated Protein Kinases
SIRT1 protein, human
Sirt1 protein, mouse
Sirtuin 1

Abstract

MeSH terms

Substances

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