Identification of Golgi-localized acyl transferases that palmitoylate and regulate endothelial nitric oxide synthase.
Lipid modifications mediate the subcellular localization and biological activity of many proteins, including endothelial nitric oxide synthase (eNOS). This enzyme resides on the cytoplasmic aspect of the Golgi apparatus and in caveolae and is dually acylated by both N-myristoylation and S-palmitoylation. Palmitoylation-deficient mutants of eNOS release less nitric oxide (NO). We identify enzymes that palmitoylate eNOS in vivo. Transfection of human embryonic kidney 293 cells with the complementary DNA (cDNA) for eNOS and 23 cDNA clones encoding the Asp-His-His-Cys motif (DHHC) palmitoyl transferase family members showed that five clones (2, 3, 7, 8, and 21) enhanced incorporation of [3H]-palmitate into eNOS. Human endothelial cells express all five of these enzymes, which colocalize with eNOS in the Golgi and plasma membrane and interact with eNOS. Importantly, inhibition of DHHC-21 palmitoyl transferase, but not DHHC-3, in human endothelial cells reduces eNOS palmitoylation, eNOS targeting, and stimulated NO production. Collectively, our data describe five new Golgi-targeted DHHC enzymes in human endothelial cells and suggest a regulatory role of DHHC-21 in governing eNOS localization and function.
Fernández-Hernando
Carlos
C
Department of Pharmacology and Program in Vascular Cell Signaling and Therapeutics, Boyer Center for Molecular Medicine, Yale University School of Medicine, New Haven, CT 06536, USA.
Fukata
Masaki
M
Bernatchez
Pascal N
PN
Fukata
Yuko
Y
Lin
Michelle I
MI
Bredt
David S
DS
Sessa
William C
WC
D000215:Acylation; D000217:Acyltransferases / Q000378:metabolism*; D020816:Amino Acid Motifs; D000818:Animals; D019556:COS Cells; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D042783:Endothelial Cells / Q000166:cytology / Q000201:enzymology; D006056:Golgi Apparatus / Q000201:enzymology*; D006801:Humans; D047468:Immunoprecipitation; D009569:Nitric Oxide / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism*; D019308:Palmitic Acid / Q000378:metabolism*; D011485:Protein Binding; D021381:Protein Transport
D019308:Palmitic Acid; D009569:Nitric Oxide; D052250:Nitric Oxide Synthase Type III; D000217:Acyltransferases; C105599:HHAT protein, human