Autoinhibition of Endothelial Nitric-oxide Synthase

Nishida, Clinton R.; Montellano, Paul R. Ortiz de

doi:10.1074/jbc.274.21.14692

Cited by 101 publications

(88 citation statements)

References 57 publications

(49 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In the absence of Ca 2ϩ /CaM, the autoinhibitory and C-terminal control elements repress electron transfers within and from the reductase domain in both nNOS and eNOS (11)(12)(13)(14)(15)(16)(17). The nNOS control element deletion mutants (⌬40 and ⌬C33) retained activity even in the absence of Ca 2ϩ /CaM (16,17) (Table II).…”

Section: Discussionmentioning

confidence: 99%

“…Deletion of either element affects the Ca 2ϩ /CaM sensitivity of nNOS and eNOS and allows electron transfers to proceed even in the absence of CaM (11)(12)(13)(14)(15)(16)(17). To investigate how caveolin binding regulates these CaM-independent electron transfers, we examined two deletion mutants of nNOS: ⌬40, in which 40 amino acids have been deleted from the FMN binding subdomain and ⌬C33, in which 33 amino acids have been truncated from the C-terminal end (Fig.…”

Section: Figmentioning

confidence: 99%

“…Peptides derived from this putative autoinhibitory domain in eNOS inhibited CaM binding and Ca 2ϩ activation of both nNOS and eNOS (11). Deletion of the autoinhibitory domains of eNOS and nNOS activated the reductase domains in the absence of CaM and affected Ca 2ϩ /CaM-dependent NO formation (12)(13)(14)(15)(16). A mutant nNOS in which this region was deleted (⌬40) was 10% as active as the wild-type enzyme in the absence of Ca 2ϩ /CaM (16).…”

mentioning

confidence: 99%

See 2 more Smart Citations

Identification of Caveolin-1-interacting Sites in Neuronal Nitric-oxide Synthase

Sato

Sagami

Shimizu

2004

Journal of Biological Chemistry

View full text Add to dashboard Cite

Caveolin is known to down-regulate both neuronal (nNOS) and endothelial nitric-oxide synthase (eNOS). In the present study, direct interactions of recombinant caveolin-1 with both the oxygenase and reductase domains of nNOS were demonstrated using in vitro binding assays. To elucidate the mechanism of nNOS regulation by caveolin, we examined the effects of a caveolin-1 scaffolding domain peptide (CaV1p1; residues (82-101)) on the catalytic activities of wild-type and mutant nNOSs. CaV1p1 inhibited NO formation activity and NADPH oxidation of wild-type nNOS in a dose-dependent manner with an IC 50 value of 1.8 M. Mutations of Phe 584 and Trp 587 within a caveolin binding consensus motif of the oxygenase domain did not result in the loss of CaV1p1 inhibition, indicating that an alternate region of nNOS mediates inhibition by caveolin. The addition of CaV1p1 also inhibited more than 90% of the cytochrome c reductase activity in the isolated reductase domain with or without the calmodulin (CaM) binding site, whereas CaV1p1 inhibited ferricyanide reductase activity by only 50%. These results suggest that there are significant differences in the mechanism of inhibition by caveolin for nNOS as compared with those previously reported for eNOS. Further analysis of the interaction through the use of several reductase domain deletion mutants revealed that the FMN domain was essential for successful interaction between caveolin-1 and nNOS reductase. We also examined the effects of CaV1p1 on an autoinhibitory domain deletion mutant (⌬40) and a C-terminal truncation mutant (⌬C33), both of which are able to form NO in the absence of CaM, unlike the wild-type enzyme. Interestingly, CaV1p1 inhibited CaM-dependent, but not CaMindependent, NO formation activities of both ⌬40 and ⌬C33, suggesting that CaV1p1 inhibits interdomain electron transfer induced by CaM from the reductase domain to the oxygenase domain.Nitric-oxide synthase (NOS) 1 generates the physiologically important nitric oxide molecule (NO) from L-arginine (L-Arg) (1-5).NOS consists of two functional domains: an N-terminal oxygenase domain containing a cytochrome P450 (P450)-like heme active site, and L-Arg and (6R)-5,6,7,8-tetrahydrobiopterin (H 4 B) binding sites (6), and a C-terminal reductase domain that contains the FAD, FMN, and NADPH binding sites (7,8). The two domains are connected by a calmodulin (CaM) binding site. Binding of CaM facilitates the interdomain electron transfers required for NO formation as well as intradomain electron transfers within the reductase domain. Two isoforms of NOS (neuronal NOS (nNOS) and endothelial NOS (eNOS)) are constitutively expressed and are regulated by intracellular Ca 2ϩ concentrations via the reversible binding of Ca 2ϩ /CaM (9). In contrast, the inducible NOS isoform (iNOS) is regulated at the transcriptional level, and binds CaM tightly even at basal Ca 2ϩ concentrations (10). CaM-dependent regulation of the three NOS isoforms is also controlled by isoform-specific sequences (i.e. insertions or extensions) and by pho...

show abstract

Section: Discussionmentioning

confidence: 99%

Section: Figmentioning

confidence: 99%

mentioning

confidence: 99%

See 1 more Smart Citation

Identification of Caveolin-1-interacting Sites in Neuronal Nitric-oxide Synthase

Sato

Sagami

Shimizu

2004

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…Although eNOS and nNOS have similar features, their amino acid sequences are only 60% identical. Important differences exist in terms of the biological function and catalysis of these NOS isoforms and in the nature of their interaction with CaM (28)(29)(30)(31)(32)(33). Given these disparities, it is perhaps not surprising that the phosphorylation of CaM affects the kinetics of eNOS and nNOS differently.…”

Section: Discussionmentioning

confidence: 99%

Calmodulin phosphorylation and modulation of endothelial nitric oxide synthase catalysis

Greif

Sacks²,

Michel³

2004

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

The endothelial NO synthase (eNOS) is regulated by diverse protein kinase pathways, yet eNOS activity ultimately depends on the ubiquitous calcium regulatory protein calmodulin (CaM). In these studies, we establish that CaM itself undergoes phosphorylation in endothelial cells and that CaM phosphorylation attenuates eNOS activation. Using [ 32 P]orthophosphoric acid biosynthetic labeling, we found that CaM is a phosphoprotein in bovine aortic endothelial cells (BAEC) and that the kinase CK2 promotes CaM phosphorylation in BAEC. Phosphorylation of CaM by purified CK2 in vitro reduces the V max of immunopurified eNOS by a factor of 2 but has no effect on the K A for CaM or calcium. Additionally, [ 32 P]orthophosphoric acid biosynthetic labeling of mutant CaM-transfected BAEC revealed that phosphorylation of Ser-81 to alanine mutant CaM (''phosphonull'' S81A mutant) is dramatically reduced relative to WT, whereas phosphorylation of the ''phosphomimetic'' Ser-81 to aspartate (S81D) mutant is unchanged. Further studies using Escherichia coli-expressed and phenylSepharose-purified CaM mutants revealed that the S81A mutation abrogates in vitro CK2-mediated phosphorylation of CaM, whereas phosphorylation of the S81D CaM mutant by CK2 is preserved. Additionally, we found that the phosphomimetic S101D CaM mutant is impaired in its ability to activate eNOS. Taken together, these results suggest that phosphorylation of CaM inhibits eNOS catalysis and proceeds in a hierarchical manner, initially requiring phosphorylation of the CaM Ser-81 residue. We conclude that CaM phosphorylation may represent a unique pathway in the regulation of eNOS signaling and thereby may play a role in modulating NO-dependent vascular responses.T he vascular system uses a network of cell signaling pathways to maintain a delicate homeostatic balance. The endothelial NO synthase (eNOS) is a calmodulin-dependent enzyme that plays a key role in many of these signaling cascades by catalyzing the conversion of L-arginine and oxygen to L-citrulline and the labile gas NO (1). Because NO has fundamental effects on many aspects of vascular physiology (2), it is not surprising that eNOS is tightly regulated by a number of mechanisms. Subcellular localization, acylation, phosphorylation, and direct interaction with other proteins, including calmodulin (CaM), caveolin, and heat shock protein 90, have been shown to modulate eNOS (3). Recently, the phosphorylation of eNOS in endothelial cells has been extensively characterized (4-9); however, the role of phosphorylation pathways in modulating the protein partners of eNOS, such as CaM, is much less well understood.CaM is a ubiquitously expressed, highly conserved acidic protein that mediates a broad range of intracellular calcium-regulated enzymes (10, 11), including eNOS (12). CaM is comprised of 148 aa and has a ''dumbbell'' structure: a long flexible central helix joining two pairs of calcium-binding helix-loop-helix motifs, known as EF hands (13). Upon binding calcium, CaM exposes hydrophobic regions on its s...

show abstract

“…This has been confirmed by a number of groups (e.g. 24,25).Recently the C-terminal region has been shown to be necessary for control (26); C-terminal truncated enzymes are uncoupled in the absence of CaM, and the region contains a regulatory phosphorylation site (27). Mutations at this site (Ser-1179 in eNOS) (28) and the nearby FAD stacking residue (Phe-1164) (29) have an altered calcium response.…”

mentioning

confidence: 69%

The Function of the Small Insertion in the Hinge Subdomain in the Control of Constitutive Mammalian Nitric-oxide Synthases

Jones

Smith

Gao

et al. 2004

Journal of Biological Chemistry

View full text Add to dashboard Cite

Control of nitric oxide (NO) synthesis in the constitutive nitric-oxide synthases (NOS) by calcium/calmodulin is exerted through the regulation of electron transfer from NADPH through the reductase domains. This process has been shown previously to involve the calmodulin binding site, the autoinhibitory insertion in the FMN binding domain, and the C-terminal tail. Smaller sequence elements also appear to correlate with control. Although some of these elements appear well positioned to function in control, they are poorly conserved; their role in control is neither well established nor defined by available information. In this study mutations have been induced in the small insertion of the hinge subdomain, which has been shown recently to form a ␤ hairpin in structural studies of the neuronal NOS reductase domains adjacent to the calmodulin site and the autoinhibitory element. Modification of the small insertion in neuronal NOS tends to increase cytochrome c reduction but not NO synthetic activity; some modifications or deletions in the corresponding region in endothelial NOS modestly increase activity under some conditions. Unexpectedly, some minor changes in the sequence introduce a loss in the content of heme relative to flavin cofactors. Taken together, these results suggest that the small insertion protects the calmodulin binding site and that it may be a modulator of NOS activity.Nitric-oxide synthases (NOS) 1 are a growing family of modular enzymes, including three mammalian isoforms, as well as a number of related eukaryotic and more distantly related prokaryotic enzymes. Because of the discovery of the function of nitric oxide (NO) as a molecular messenger in a variety of signal transduction pathways (1-4), the endothelial and neuronal isoforms (eNOS and nNOS) have been shown to function as signal generators controlled by calcium/calmodulin (Ca 2ϩ / CaM) (5). The other mammalian isoform, iNOS, is induced during immune response (6 -9); it is constitutively active and binds CaM at basal Ca 2ϩ levels.NO synthesis from arginine and molecular oxygen requires 3 mol of electrons supplied by NADPH/mol of NO formed (10). The reductase domains in eukaryotic NOS are homologous to NADPH P450 reductase (11). These domains contain one flavin mononucleotide (FMN) and one flavin adenine dinucleotide (FAD) cofactor (11-18), which function in electron transfer from NADPH to the heme-containing active site (13,17,19,20).Ca 2ϩ /CaM control in NOS is exerted through the regulation of electron transfer between NADPH and heme (5). This regulation is primarily a function of the reductase domains (21). The canonical CaM binding site was recognized in the sequence region connecting the oxygenase and reductase regions (11). A major insertion in the FMN binding region, correlating with Ca 2ϩ /CaM control, was identified as a control element with autoinhibitory character (autoinhibitory insertion, or AI) (22,23). This has been confirmed by a number of groups (e.g. 24,25).Recently the C-terminal region has been shown to be necessary ...

show abstract

Autoinhibition of Endothelial Nitric-oxide Synthase

Cited by 101 publications

References 57 publications

Identification of Caveolin-1-interacting Sites in Neuronal Nitric-oxide Synthase

Identification of Caveolin-1-interacting Sites in Neuronal Nitric-oxide Synthase

Calmodulin phosphorylation and modulation of endothelial nitric oxide synthase catalysis

The Function of the Small Insertion in the Hinge Subdomain in the Control of Constitutive Mammalian Nitric-oxide Synthases

Contact Info

Product

Resources

About