Caveolin versus Calmodulin

Michel, Jeffrey B.; Féron, Olivier; Sase, Kazuhiro; Prabhakar, Prakash; Michel, Thomas

doi:10.1074/jbc.272.41.25907

Cited by 277 publications

(108 citation statements)

References 23 publications

(38 reference statements)

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“…5, the maximal activity (V max ) of eNOS in the presence of phosphorylated CaM is reduced In contrast, the affinity of phosphorylated CaM or CaM for eNOS is similar (K A of 13.1 vs. 16.2 nM, respectively, P ϭ not significant). This K A of CaM is in good agreement with our previously determined value of 20 nM for E. coli-expressed eNOS (23).…”

Section: Ck2 Treatment Of Cam Ser-81 Phosphorylation-site Mutants In supporting

confidence: 79%

“…Moreover, CK2 has been shown to catalyze in vitro phosphorylation of the scaffolding protein caveolin, the principal protein in caveolae (39). We have previously shown that eNOS undergoes a regulatory cycle of enzymatic activation and inhibition based on its reversible association with Ca 2ϩ -CaM and caveolin, respectively (23,43). It is possible that CK2 plays a role in regulating this cycle through catalyzing phosphorylation of CaM or caveolin and thereby altering allosteric regulation of eNOS.…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Calmodulin phosphorylation and modulation of endothelial nitric oxide synthase catalysis

Greif

Sacks²,

Michel³

2004

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

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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...

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Section: Ck2 Treatment Of Cam Ser-81 Phosphorylation-site Mutants In supporting

confidence: 79%

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.

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“…5C). In contrast, previous reports have indicated that the inhibition of eNOS activity by the same peptide was competitive and completely reversed by Ca 2ϩ /CaM (24). These results suggest that the inhibition mode of nNOS by caveolin is different from that of eNOS and that CaV1p1 does not directly interact with CaM and the CaM binding site (discussed below).…”

Section: Discussioncontrasting

confidence: 53%

“…Besides localization, the interaction of eNOS and nNOS with these structural proteins inhibits NO formation (19 -22). The inhibition of eNOS by caveolin is reversed by Ca 2ϩ /CaM binding (23,24), but neither this interaction nor its mechanism has previously been studied in nNOS.…”

mentioning

confidence: 99%

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

Sato

Sagami

Shimizu

2004

Journal of Biological Chemistry

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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...

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“…Previous reports have suggested the presence of a Cav-1 consensus binding motif in the eNOS oxygenase domain, which also consists of a series of hydrophobic residues (FSAAPF-SGW) (9, 16); however, based on the crystal structure of the eNOS oxygenase, this domain is buried in the protein, stabilizes the heme prosthetic group, and is unlikely to participate in protein-protein interactions (17). Based on biochemical studies, Cav-1 can interact with eNOS or neuronal NOS by means of binding to sites in both the oxygenase and reductase domains of the enzymes (9,(18)(19)(20). Mechanistically, Cav-1 binding to NOS inhibits the reductase function of the enzymes, thereby reducing NO synthesis (21).…”

Section: Discussionmentioning

confidence: 99%

Dissecting the molecular control of endothelial NO synthase by caveolin-1 using cell-permeable peptides

Bernatchez

Bauer

et al. 2005

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

174

199

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In endothelia, NO is synthesized by endothelial NO synthase (eNOS), which is negatively regulated by caveolin-1 (Cav-1), the primary coat protein of caveolae. We show that delivery of Cav-1 amino acids 82-101 (Cav) fused to an internalization sequence from Antennapedia (AP) blocks NO release in vitro and inflammation and tumor angiogenesis in vivo. To characterize the molecular mechanism by which the AP-Cav peptide and Cav-1 mediate eNOS inhibition, we subdivided the Cav portion of AP-Cav into three domains (Cav-A C aveolae are cholesterol-and sphingomyelin-rich invaginations of the plasma membrane found throughout the cardiovascular system (1). Numerous lines of evidence suggest that these macromolecular organelles have the capacity to segregate or concentrate spatially distinct signaling molecules that may modulate many cellular responses. Electron microscopy studies have revealed the presence of caveolae in several cell types, and vascular endothelial cells are a major source of caveolae. Caveolin-1 (Cav-1), the main coat protein of caveolae in blood vessels, is believed to be instrumental in the caveolae-dependent compartmentalization and modulation of signaling systems. Emerging evidence also suggests that Cav-1 is in part targeted to the cell membrane, likely through palmitoylation, where it can bind to other molecules through its scaffolding domain (residues 82-101 in Cav-1) or carboxyl tail (2).One well described molecular target of Cav-1 is endothelial nitric oxide (NO) synthase (eNOS), the NO synthase (NOS) isoform that produces low levels of NO in response to mechanical forces and diverse agonists in endothelial cells. eNOS in the plasma membrane is negatively regulated by its protein-protein interaction with Cav-1 (3, 4). This finding is supported by recent genetic data showing that Cav-1 knockout mice have increased endothelium-dependent relaxations and NOx levels in blood (5, 6). Additional evidence supporting a negative regulatory role for Cav-1 on eNOS functions stems from using a cell-permeable version of the inhibitory Cav-1 scaffolding domain that selectively blocks eNOS-mediated NO release, vasorelaxation, inflammation, and tumor angiogenesis in variety of settings (7,8). Hence, this peptide, called Antennapedia (AP)-Cav or cavtratin, is a valuable tool for mimicking and studying Cav-1 molecular interactions with eNOS.To gain insights into the molecular determinants of how Cav-1 and the peptide AP-Cav may mediate eNOS inhibition, we have designed several cell-permeable peptides containing truncated portions of Cav-1 and examined their effect on eNOS function in four distinct bioassays. Here, we show that truncated cellpermeable peptides that contain the hydrophobic sequence FT-TFTVT (Cav-1 amino acids 89-95) can inhibit recombinant eNOS activity, NO release from endothelial cells, microvessel permeability in vitro, and inflammation in vivo similarly to AP-Cav (residues 82-101). By performing alanine scanning of this seven amino acid sequence, residues Thr-90 and -91 (T90,91) and Phe-92 (F92) ...

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Caveolin versus Calmodulin

Cited by 277 publications

References 23 publications

Calmodulin phosphorylation and modulation of endothelial nitric oxide synthase catalysis

Calmodulin phosphorylation and modulation of endothelial nitric oxide synthase catalysis

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

Dissecting the molecular control of endothelial NO synthase by caveolin-1 using cell-permeable peptides

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