Thr497

Site Information
AGITRKktFKEVANA SwissProt Entrez-Gene
Blast this site against: NCBI SwissProt PDB
Site Group ID: 447885

In vivo Characterization
Methods used to characterize site in vivo:	[32P] bio-synthetic labeling ( 20 ) , immunoprecipitation ( 2 ) , mutation of modification site ( 6 , 9 , 12 , 18 , 20 , 23 ) , phospho-antibody ( 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 ) , phosphopeptide mapping ( 24 ) , western blotting ( 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 16 , 17 , 19 , 20 , 21 , 22 , 25 , 26 )
Relevant cell line - cell type - tissue:	293 (epithelial) ( 7 , 10 , 14 , 18 ) , BAEC (endothelial) ( 1 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 12 , 13 , 17 , 18 , 19 , 20 , 21 , 22 , 24 , 25 ) , BAE ( 11 , 23 ) , BLMVEC (endothelial) ( 16 ) , COS (fibroblast) ( 12 , 18 , 20 , 23 , 26 ) , endothelial-aorta ( 14 , 15 ) , PPAEC (endothelial) ( 2 ) , vascular smooth muscle cell ('muscle, smooth') [eNOS (cow)] ( 9 )

In vivo Characterization

Methods used to characterize site in vivo:

[32P] bio-synthetic labeling ( 20 ) , immunoprecipitation ( 2 ) , mutation of modification site ( 6 , 9 , 12 , 18 , 20 , 23 ) , phospho-antibody ( 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 ) , phosphopeptide mapping ( 24 ) , western blotting ( 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 16 , 17 , 19 , 20 , 21 , 22 , 25 , 26 )

Relevant cell line - cell type - tissue:

293 (epithelial) ( 7 , 10 , 14 , 18 ) , BAEC (endothelial) ( 1 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 12 , 13 , 17 , 18 , 19 , 20 , 21 , 22 , 24 , 25 ) , BAE ( 11 , 23 ) , BLMVEC (endothelial) ( 16 ) , COS (fibroblast) ( 12 , 18 , 20 , 23 , 26 ) , endothelial-aorta ( 14 , 15 ) , PPAEC (endothelial) ( 2 ) , vascular smooth muscle cell ('muscle, smooth') [eNOS (cow)] ( 9 )

Upstream Regulation
Regulatory protein:	eNOS (cow) ( 12 ) , PKCZ (human) ( 6 ) , PTEN (human) ( 26 ) , RAC1 (human) ( 8 ) , TIE2 (human) ( 6 )
Putative in vivo kinases:	PKCA (cow) ( 19 ) , PKCZ (human) ( 6 )
Kinases, in vitro:	PKCA (human) ( 19 )
Phosphatases, in vitro:	PPP1CA (human) ( 24 ) , PPP2CA (human) ( 20 )
Treatments:	17-beta-estradiol ( 2 ) , A23187 ( 4 ) , angiopoietin-1 ( 6 ) , angiotensin_2 ( 9 ) , aphidicolin ( 3 ) , aPL ( 5 ) , apoA-I ( 15 ) , atenolol ( 8 ) , ATP ( 1 ) , BAPTA-AM ( 4 ) , bisindolylmaleimide ( 4 , 6 ) , bradykinin ( 13 , 25 ) , butoxamine ( 8 ) , calyculin_A ( 24 ) , cAMP_analog ( 21 ) , ciclosporin ( 22 , 25 ) , colforsin ( 24 ) , fluid_shear_stress ( 21 ) , geldanamycin ( 14 ) , Go_6976 ( 6 ) , H2O2 ( 7 ) , HDL ( 15 ) , IBMX ( 24 ) , IgG ( 5 ) , insulin ( 4 ) , ionomycin ( 12 , 18 ) , lactacystin ( 10 ) , LDL ( 15 ) , LY333531 ( 1 ) , MG132 ( 10 ) , nebivolol ( 13 ) , okadaic_acid ( 24 , 25 ) , phorbol_ester ( 6 , 18 , 19 , 24 ) , PKCnPS ( 1 ) , PP2 ( 8 ) , pulsatile shear stress ( 1 ) , quercetin ( 11 ) , racepinefrine ( 8 ) , radicicol ( 14 ) , rottlerin ( 4 ) , SKI-1 ( 1 ) , SR59230A ( 8 ) , staurosporine ( 19 ) , thapsigargin ( 4 ) , troglitazone ( 17 ) , U0126 ( 8 ) , vanadate ( 16 ) , VEGF ( 4 , 5 , 6 , 18 , 21 , 22 , 23 , 25 ) , wortmannin ( 8 , 25 )

Downstream Regulation
Effects of modification on eNOS:	enzymatic activity, inhibited ( 1 , 18 , 19 , 24 ) , molecular association, regulation ( 2 )
Induce interaction with:	Calmodulin (cow) ( 2 )

References
1	Muzorewa TT, Buerk DG, Jaron D, Barbee KA (2021) Coordinated regulation of endothelial calcium signaling and shear stress-induced nitric oxide production by PKCβ and PKCη. Cell Signal 87, 110125 34474112 Curated Info
2	Tran QK, et al. (2016) Estrogen Enhances Linkage in the Vascular Endothelial Calmodulin Network via a Feedforward Mechanism at the G Protein-coupled Estrogen Receptor 1. J Biol Chem 291, 10805-23 26987903 Curated Info
3	Park JH, et al. (2015) B56δ subunit of protein phosphatase 2A decreases phosphorylation of endothelial nitric oxide synthase at serine 116: Mechanism underlying aphidicolin-stimulated NO production. Nitric Oxide 50, 46-51 26255574 Curated Info
4	Haines RJ, Corbin KD, Pendleton LC, Eichler DC (2012) Protein Kinase Cα Phosphorylates a Novel Argininosuccinate Synthase Site at Serine 328 during Calcium-dependent Stimulation of Endothelial Nitric-oxide Synthase in Vascular Endothelial Cells. J Biol Chem 287, 26168-76 22696221 Curated Info
5	Ramesh S, et al. (2011) Antiphospholipid antibodies promote leukocyte-endothelial cell adhesion and thrombosis in mice by antagonizing eNOS via β2GPI and apoER2. J Clin Invest 121, 120-31 21123944 Curated Info
6	Oubaha M, Gratton JP (2009) Phosphorylation of endothelial nitric oxide synthase by atypical PKC zeta contributes to angiopoietin-1-dependent inhibition of VEGF-induced endothelial permeability in vitro. Blood 114, 3343-51 19564638 Curated Info
7	Hu Z, Chen J, Wei Q, Xia Y (2008) Bidirectional actions of hydrogen peroxide on endothelial nitric-oxide synthase phosphorylation and function: co-commitment and interplay of Akt and AMPK. J Biol Chem 283, 25256-63 18617528 Curated Info
8	Kou R, Michel T (2007) Epinephrine regulation of the endothelial nitric-oxide synthase: roles of RAC1 and beta3-adrenergic receptors in endothelial NO signaling. J Biol Chem 282, 32719-29 17855349 Curated Info
9	Suzuki H, et al. (2006) Activation of endothelial nitric oxide synthase by the angiotensin II type 1 receptor. Endocrinology 147, 5914-20 16980435 Curated Info
10	Wei Q, Xia Y (2006) Proteasome inhibition down-regulates endothelial nitric-oxide synthase phosphorylation and function. J Biol Chem 281, 21652-9 16737962 Curated Info
11	Jackson SJ, Venema RC (2006) Quercetin inhibits eNOS, microtubule polymerization, and mitotic progression in bovine aortic endothelial cells. J Nutr 136, 1178-84 16614401 Curated Info
12	Church JE, Fulton D (2006) Differences in eNOS activity because of subcellular localization are dictated by phosphorylation state rather than the local calcium environment. J Biol Chem 281, 1477-88 16257964 Curated Info
13	Dessy C, et al. (2005) Endothelial beta3-adrenoreceptors mediate nitric oxide-dependent vasorelaxation of coronary microvessels in response to the third-generation beta-blocker nebivolol. Circulation 112, 1198-205 16116070 Curated Info
14	Wei Q, Xia Y (2005) Roles of 3-phosphoinositide-dependent kinase 1 in the regulation of endothelial nitric-oxide synthase phosphorylation and function by heat shock protein 90. J Biol Chem 280, 18081-6 15737995 Curated Info
15	Drew BG, et al. (2004) High-density lipoprotein and apolipoprotein AI increase endothelial NO synthase activity by protein association and multisite phosphorylation. Proc Natl Acad Sci U S A 101, 6999-7004 15107497 Curated Info
16	Papapetropoulos A, et al. (2004) Vanadate is a potent activator of endothelial nitric-oxide synthase: evidence for the role of the serine/threonine kinase Akt and the 90-kDa heat shock protein. Mol Pharmacol 65, 407-15 14742683 Curated Info
17	Cho DH, Choi YJ, Jo SA, Jo I (2004) Nitric oxide production and regulation of endothelial nitric-oxide synthase phosphorylation by prolonged treatment with troglitazone: evidence for involvement of peroxisome proliferator-activated receptor (PPAR) gamma-dependent and PPARgamma-independent signaling pathways. J Biol Chem 279, 2499-506 14593122 Curated Info
18	Lin MI, et al. (2003) Phosphorylation of threonine 497 in endothelial nitric-oxide synthase coordinates the coupling of L-arginine metabolism to efficient nitric oxide production. J Biol Chem 278, 44719-26 12952971 Curated Info
19	Matsubara M, Hayashi N, Jing T, Titani K (2003) Regulation of endothelial nitric oxide synthase by protein kinase C. J Biochem (Tokyo) 133, 773-81 12869534 Curated Info
20	Greif DM, Kou R, Michel T (2002) Site-specific dephosphorylation of endothelial nitric oxide synthase by protein phosphatase 2A: evidence for crosstalk between phosphorylation sites. Biochemistry 41, 15845-53 12501214 Curated Info
21	Boo YC, et al. (2002) Shear stress stimulates phosphorylation of eNOS at Ser(635) by a protein kinase A-dependent mechanism. Am J Physiol Heart Circ Physiol 283, H1819-28 12384459 Curated Info
22	Kou R, Greif D, Michel T (2002) Dephosphorylation of endothelial nitric-oxide synthase by vascular endothelial growth factor. Implications for the vascular responses to cyclosporin A. J Biol Chem 277, 29669-73 12050171 Curated Info
23	Fulton D, et al. (2002) Localization of endothelial nitric-oxide synthase phosphorylated on serine 1179 and nitric oxide in Golgi and plasma membrane defines the existence of two pools of active enzyme. J Biol Chem 277, 4277-84 11729179 Curated Info
24	Michell BJ, et al. (2001) Coordinated control of endothelial nitric-oxide synthase phosphorylation by protein kinase C and the cAMP-dependent protein kinase. J Biol Chem 276, 17625-8 11292821 Curated Info
25	Harris MB, et al. (2001) Reciprocal phosphorylation and regulation of endothelial nitric-oxide synthase in response to bradykinin stimulation. J Biol Chem 276, 16587-91 11340086 Curated Info
26	Church JE, et al. Inhibition of endothelial nitric oxide synthase by the lipid phosphatase PTEN. Vascul Pharmacol 52, 191-8 19962452 Curated Info