eNOS at a glanceWilliam C. SessaDepartment of Pharmacology and Vascular CellSignaling Program, Boyer Center for MolecularMedicine, Yale University School of Medicine,New Haven, CT 06536, USA

(e-mail: william.sessa@yale.edu)

Journal of Cell Science 117, 2427-2429Published by The Company of Biologists 2004doi:10.1242/jcs.01165

Endothelium-derived nitric oxide (NO)is a critical regulator of cardiovascularhomeostasis. Endothelial nitric oxide

synthase (eNOS or NOS3)-derived NOis an endogenous vasodilatory gas thatcontinually regulates the diameterof blood vessels and maintains ananti-proliferative and anti-apoptoticenvironment in the vessel wall. Initiallythought to be a simple, calmodulin(CaM) regulated enzyme, it is clear thateNOS has evolved to be tightlycontrolled by co- and post-translationallipid modifications, phosphorylation onmultiple residues and regulated protein-protein interactions (Fulton et al.,2001).

Various extracellular signals canpromote NO release fromendothelial cellsPhysiologically, endothelial cells areexposed to the hemodynamic forces ofblood including laminar shear stress.Shear stress, via G proteins (Gs), canactivate several signal transductionpathways, including the phosphoinoside3-kinase (PI3K) and adenylate cyclase(AC) pathways, leading to eNOSactivation via phosphorylation of serineresidues (S617 and S1179 for Akt, andS635 and S1179 for PKA), whichpromote eNOS activation. Shear stressalso increases S116 phosphorylation;however, the kinase responsible for thisphosphorylation and the function ofS116 are not well understood (Boo andJo, 2003). Additional stimuli, such asvascular endothelial growth factor(VEGF), estrogen, sphingosine 1-phosphate (S-1-P) and bradykinin, canbind to their cognate receptors and alsostimulate PI3K/Akt. However, they alsoactivate phospholipase C-γ (PLC-γ)to increase cytoplasmic calcium anddiacylglycerol (DAG) levels. Theincrease in cytoplasmic calcium levelsactivates CaM, which binds to thecanonical CaM-binding domain in eNOSto promote the alignment of theoxygenase and reductase domains ofeNOS, leading to efficient NO synthesis.In addition, CaM can activate CaMkinase II, which may phosphorylateeNOS on S1179. Increases in DAGlevels can activate PKC to phosphorylateT497, which may negatively regulateeNOS or influence its coupling.Finally, metabolic stress triggering thebreakdown of ATP will stimulate AMPkinase (AMPK) to phosphorylate eNOSon S1179 (Chen et al., 1999; Dimmeleret al., 1998; Fleming et al., 2001; Fultonet al., 1999; Harris et al., 2001; Hayneset al., 2000; Igarashi and Michel, 2001;Lin et al., 2003; Michell et al., 2002;Morales-Ruiz et al., 2001; Simoncini etal., 2000).

Intrinsic control of eNOS functionCo-translational N-terminal myristoyl-ation on G2 and post-translationalcysteine palmitoylation on C15 and C26control the subcellular targeting ofeNOS to the cytoplasmic aspect of theGolgi complex and to plasmalemmal

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Journal of Cell Science 2004 (117, pp. 2427-2429)

eNOS at a GlanceWilliam C. Sessa

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caveolae (García-Cardeña et al., 1996;Liu et al., 1995; Liu et al., 1997; Liu andSessa, 1994). Similar to nNOS andiNOS, eNOS contains a C-terminalreductase domain, which binds NADPH,and transfers electrons from NADPH toFAD to FMN, and ultimately to the N-terminal oxygenase domain, whichcontains a heme, and binding sites forarginine, tetrahydrobiopterin and CaM.eNOS utilizes molecular oxygen andelectrons from NADPH to oxidizethe substrate L-arginine into theintermediate OH-L-arginine, which isthen oxidized into NO and L-citrulline(Griffith and Stuehr, 1995). Twoautoinhibitory control elements (ACE-Iand ACE-II) impede eNOS activationand influence the calcium/CaMsensitivity of the enzyme (Lane andGross, 2002; Salerno et al., 1997). Giventhe localization of T497, S617 and S635in ACE-1 and S1179 in ACE-II, it islikely that phosphorylation removes thesteric hindrance imparted by thesenon-catalytic inserts and permits betterfidelity of electron flux from thereductase domain to NO generation inthe oxygenase domain (McCabe et al.,2000).

Regulated protein-proteininteractionseNOS can interact with variousproteins in its ‘less active’ and ‘moreactive’ states. N-myristoylated andpalmitoylated membrane-bound eNOSassociates with the caveolae coat proteincaveolin-1 (Cav-1) and with heat shockprotein 90 (Hsp90). The C-terminalHsp70-interacting protein (CHIP)interacts with both Hsp70 and Hsp90,and negatively regulates eNOStrafficking into the Golgi complex. Bycontrast, the nitric oxide synthase-interacting protein (NOSIP) and thenitric oxide synthase traffic inducer(NOSTRIN) can negatively regulateeNOS localization in the plasmamembrane (Jiang et al., 2003; Nedvetskyet al., 2002; Zabel et al., 2002).Endothelial cell stimulation by variousstimuli (top) activates eNOS catalysis[i.e. the conversion of L-arginine (L-Arg) to NO] through its association withCaM. Whether CaM is always boundand small changes in calcium determinecalcium-CaM dependence, more CaMis recruited to eNOS by large fluxes

in cytoplasmic calcium or howphosphorylation influences the calcium/CaM requirements of the enzyme in situare not known. However, the actions ofCaM are thought to be facilitated in cellsby the recruitment of Hsp90 to eNOSand from the dissociation of eNOSfrom Cav-1. Both calcium-dependentand -independent stimuli have beenshown to induce phosphorylation ofS1179 on eNOS. Phosphorylation of thisresidue by Akt, PKA or AMPK isassociated with increased enzymeactivity. Other proteins have been showto be associated with increased eNOSactivity or NO release, such as dynamin(Dyn), porin and the NO effector solubleguanylyl cyclase (sGC).

Effectors of NOOnce NO is produced by theendothelium, it can regulate severalaspects of vascular function viaactivation of the primary NO receptor,sGC, or initiate nitrosation reactionswith iron-sulphur-centered proteins orproteins with reactive thiols (S-nitrosylation). In the vascular system,NO-dependent relaxation of vascularsmooth muscle is predominatly sGCand protein kinas G (PKG) dependent,whereas the anti-proliferative actionsand ion channel modulation of NO canbe via PKG or via nitrosation reactions(Feil et al., 2003; Miranda et al., 2003).Nitrosylation of caspase-3 and caspase-8 inactivates the proteins, thus leading toinhibition of apoptosis (Stamler et al.,2001).

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Cell Science at a Glance

Cell Science at a Glance is included as a poster in the paper copy of the journal andavailable in several downloadable formats in the online version, which we encouragereaders to download and use as slides. Future contributions to this section willinclude signalling pathways, phylogenetic trees, multiprotein complexes, usefulreagents . . . and much more.

The JAK/STAT signaling pathway (March 2004)

Cell adhesion receptors in C. elegans (April 2004)

Polarity establishment in yeast (May 2004)

nNOS signalling (June 2004)

The Rb network (July 2004)

The matrix metalloproteinase family (August 2004)

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