Ubiquitin-protein ligase WWP2 binds to and  downregulates the epithelial Na<sup>+</sup> channel

McDonald, Fiona J.; Western, Andrea; McNeil, J.; Thomas, Brittany C.; Olson, Diane R.; Snyder, Peter M.

doi:10.1152/ajprenal.00080.2002

Cited by 51 publications

(38 citation statements)

References 31 publications

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“…We sequenced the S1P 1 -associated polypeptides from FTY720P-treated HEK293 cells by LC/MS/MS analysis. The NEDD4 family member E3 ubiquitin ligase WWP2 (25) was identified with high statistical confidence in the proteomic analysis ( Figure 3A and Table 2). To confirm the proteomic identification of WWP2, we used co-IP analysis to confirm that these 2 proteins are found in a complex.…”

Section: Phosphorylation Leads To Multisite Polyubiquitinylation Of Tmentioning

confidence: 89%

“…In addition, WWP2 was capable of associating with S1P 1 -S5A and S1P 1 -K4R mutants, which suggests that phosphorylation and ubiquitinylation of the receptor are not required. Indeed, WWP2 protein contains discrete protein interaction domains such as the WW domain and a C2 domain, which could be involved in targeting the E3 ligase to membrane domains containing S1P 1 (25). A similar NEDD4 family ubiquitin E3 ligase called AIP4 targets the chemokine receptor CXCR4 for degradation (36).…”

Section: Figurementioning

confidence: 99%

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Engagement of S1P1-degradative mechanisms leads to vascular leak in mice

Oo¹,

Chang²,

Thangada³

et al. 2011

J. Clin. Invest.

201

205

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GPCR inhibitors are highly prevalent in modern therapeutics. However, interference with complex GPCR regulatory mechanisms leads to both therapeutic efficacy and adverse effects. Recently, the sphingosine-1-phosphate (S1P) receptor inhibitor FTY720 (also known as Fingolimod), which induces lymphopenia and prevents neuroinflammation, was adopted as a disease-modifying therapeutic in multiple sclerosis. Although highly efficacious, dose-dependent increases in adverse events have tempered its utility. We show here that FTY720P induces phosphorylation of the C-terminal domain of S1P receptor 1 (S1P 1 ) at multiple sites, resulting in GPCR internalization, polyubiquitinylation, and degradation. We also identified the ubiquitin E3 ligase WWP2 in the GPCR complex and demonstrated its requirement in FTY720-induced receptor degradation. GPCR degradation was not essential for the induction of lymphopenia, but was critical for pulmonary vascular leak in vivo. Prevention of receptor phosphorylation, internalization, and degradation inhibited vascular leak, which suggests that discrete mechanisms of S1P receptor regulation are responsible for the efficacy and adverse events associated with this class of therapeutics.

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Section: Phosphorylation Leads To Multisite Polyubiquitinylation Of Tmentioning

confidence: 89%

Section: Figurementioning

confidence: 99%

Engagement of S1P1-degradative mechanisms leads to vascular leak in mice

Oo¹,

Chang²,

Thangada³

et al. 2011

J. Clin. Invest.

201

205

View full text Add to dashboard Cite

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“…Although the Nedd4-2-independent pathways of ENaC regulation remain unclear, it is possible that some of the genes regulated by aldosterone could act through pathways independent of Nedd4-2. Alternatively, it is possible that another WW domain-containing protein, perhaps Nedd4 or other members of Nedd4 family of HECT ubiquitin ligases, partially substitutes for the actions of Nedd4-2, albeit with less efficiency (18,24,30,31). Inactivation of mouse Nedd4 gene resulted in perinatal lethality (Cao et al, unpublished data) and therefore precluded us from assessing its role in Na ϩ homeostasis and BP regulation.…”

Section: Discussionmentioning

confidence: 99%

Salt-sensitive hypertension and cardiac hypertrophy in mice deficient in the ubiquitin ligase Nedd4-2

Shi¹,

Cao²,

Sweezer³

et al. 2008

American Journal of Physiology-Renal Physiology

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139

134

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. Salt-sensitive hypertension and cardiac hypertrophy in mice deficient in the ubiquitin ligase Nedd4-2. Am J Physiol Renal Physiol 295: F462-F470, 2008. First published June 4, 2008 doi:10.1152 doi:10. /ajprenal.90300.2008 has been proposed to play a critical role in regulating epithelial Na ϩ channel (ENaC) activity. Biochemical and overexpression experiments suggest that Nedd4-2 binds to the PY motifs of ENaC subunits via its WW domains, ubiquitinates them, and decreases their expression on the apical membrane. Phosphorylation of Nedd4-2 (for example by Sgk1) may regulate its binding to ENaC, and thus ENaC ubiquitination. These results suggest that the interaction between Nedd4-2 and ENaC may play a crucial role in Na ϩ homeostasis and blood pressure (BP) regulation. To test these predictions in vivo, we generated Nedd4-2 null mice. The knockout mice had higher BP on a normal diet and a further increase in BP when on a high-salt diet. The hypertension was probably mediated by ENaC overactivity because 1) Nedd4-2 null mice had higher expression levels of all three ENaC subunits in kidney, but not of other Na ϩ transporters; 2) the downregulation of ENaC function in colon was impaired; and 3) NaClsensitive hypertension was substantially reduced in the presence of amiloride, a specific inhibitor of ENaC. Nedd4-2 null mice on a chronic high-salt diet showed cardiac hypertrophy and markedly depressed cardiac function. Overall, our results demonstrate that in vivo Nedd4-2 is a critical regulator of ENaC activity and BP. The absence of this gene is sufficient to produce salt-sensitive hypertension. This model provides an opportunity to further investigate mechanisms and consequences of this common disorder. ion channels; kidney; sodium channels HYPERTENSION AFFECTS MORE than 25% of the adult population in most Westernized countries and is a major cause of coronary artery disease and cerebrovascular disease (9, 10). Despite its prevalence, the etiology of more than 90% of hypertension cases is unknown. Numerous studies revealed that interactions between genetic and environmental factors, especially the generous intake of dietary salt, play a critical role in its pathogenesis and a large body of evidence implicates the inappropriate retention of Na(Cl) by the kidney in the pathophysiology of hypertension (25). One of the most important molecular determinants of Na ϩ excretion is the activity of the epithelial Na ϩ channel (ENaC), a highly regulated, threesubunit ion channel complex that is active in the distal nephron (15,29). The activity of ENaC is regulated by many first and second messengers, the most widely studied of which is aldosterone (13, 22). The importance of ENaC in Na ϩ homeostasis and hypertension is underscored by the discovery that patients with Liddle syndrome, an autosomal dominant form of hypertension, have gain-of-function mutations in ENaC (28).The mutations in the ␤-and ␥-ENaC subunits leading to Liddle syndrome have focused attention on the specific regions of the COOH termini whose modificati...

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“…We and others have shown that the PY motifs interact with WW domains of a HECT (homologous to E6-AP-carboxy terminal [29]) domain-containing subfamily of E3 enzymes, the Nedd4/Nedd4-like family of ubiquitin-protein ligases (58). Specifically, binding to the WW domains of Nedd4-2 (6,11,21,25,34), Nedd4-1 (5,12,14,16,22,25,36,37,44,62,65,69), WWP1 (54), and WWP2 (48,54) was demonstrated by various approaches, suggesting that ENaC is regulated by ubiquitination. Indeed, it could be confirmed that ENaC subunits become ubiquitinated (70) and exhibit rapid turnover (47,70), a hallmark of ubiquitinated proteins.…”

mentioning

confidence: 99%

Participation of the Ubiquitin-Conjugating Enzyme UBE2E3 in Nedd4-2-Dependent Regulation of the Epithelial Na⁺ Channel

Debonneville

Staub

2004

Molecular and Cellular Biology

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The epithelial Na ؉ channel (ENaC) is a heteromeric protein complex playing a fundamental role in Na ؉ homeostasis and blood pressure regulation. Specific mutations inactivating PY motifs in ENaC C termini cause Liddle's syndrome, an inherited form of hypertension. Previously we showed that these PY motifs serve as binding sites for the E3 enzyme Nedd4-2, implying ubiquitination as a regulatory mechanism of ENaC. Ubiquitination involves the sequential action of E1, E2, and E3 enzymes. Here we identify the E2 enzyme UBE2E3, which acts in concert with Nedd4-2, and show by coimmunoprecipitation that UBE2E3 and Nedd4-2 interact together. In Xenopus laevis oocytes, UBE2E3 reduces ENaC activity marginally, consistent with Nedd4-2 being the rate-limiting factor in this process, whereas a catalytically inactive mutant of UBE2E3 (UBE2E3-CS) causes elevated ENaC activity by increasing cell surface expression. No additive effect is observed when UBE2E3-CS is coexpressed with an inactive Nedd4-2 mutant, and the stimulatory role of UBE2E3-CS depends on the integrity of the PY motifs (Nedd4-2 binding sites) and the ubiquitination sites on ENaC. In renal mpkCCD cl4 cells, displaying ENaC-dependent transepithelial Na ؉ transport, Nedd4-2 and UBE2E3 can be coimmunoprecipitated and overexpression of UBE2E3 affects Na ؉ transport, corroborating the concept of a concerted action of UBE2E3 and Nedd4-2 in ENaC regulation.Covalent attachment of ubiquitin to proteins is a posttranslational modification that targets membrane proteins for internalization and/or degradation (17). Ubiquitination involves the successive action of a ubiquitin-activating enzyme (E1), a ubiquitin-conjugating or ubiquitin carrier enzyme (E2), and a ubiquitin-protein ligase (E3) (26). Possible involvement of E4 enzymes may lead to the formation of polyubiquitin chains (19,24,41). The unique E1 enzyme activates free ubiquitin in an ATP-dependent manner by forming a thioester bond with its catalytic cysteine and then transfers it to the cysteine of an E2 enzyme. Approximately 30 E2 enzymes are encoded in the human genome, and they can be separated into four classes on the basis of their primary structure. Class I E2 enzymes consist of approximately 150 residues which bear a highly conserved catalytic domain named UBC domain (ubiquitin conjugating domain, including a catalytic cysteine). Class II and III enzymes possess additional C-and N-terminal extensions, respectively (46). The class IV E2s have both N-and C-terminal elongations (4, 33). E2s usually form a complex with an E3 enzyme, which recognizes the target protein and catalyzes the attachment of ubiquitin via the formation of an isopeptide bond on lysine ε-NH 2 groups. There are probably hundreds of E3 enzymes comprising different structures. They can be divided into several main groups on the basis of the presence of one of the following domains: (i) RING fingers; (ii) U-boxes; (iii) PHD domains; (iv) HECT (homologous to E6-AP-carboxy-terminal) domains.The epithelial Na ϩ channel (ENaC) is located at the a...

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Ubiquitin-protein ligase WWP2 binds to and downregulates the epithelial Na⁺ channel

Cited by 51 publications

References 31 publications

Engagement of S1P1-degradative mechanisms leads to vascular leak in mice

Engagement of S1P1-degradative mechanisms leads to vascular leak in mice

Salt-sensitive hypertension and cardiac hypertrophy in mice deficient in the ubiquitin ligase Nedd4-2

Participation of the Ubiquitin-Conjugating Enzyme UBE2E3 in Nedd4-2-Dependent Regulation of the Epithelial Na⁺ Channel

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Ubiquitin-protein ligase WWP2 binds to and downregulates the epithelial Na+ channel

Cited by 51 publications

References 31 publications

Engagement of S1P1-degradative mechanisms leads to vascular leak in mice

Engagement of S1P1-degradative mechanisms leads to vascular leak in mice

Salt-sensitive hypertension and cardiac hypertrophy in mice deficient in the ubiquitin ligase Nedd4-2

Participation of the Ubiquitin-Conjugating Enzyme UBE2E3 in Nedd4-2-Dependent Regulation of the Epithelial Na+ Channel

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Ubiquitin-protein ligase WWP2 binds to and downregulates the epithelial Na⁺ channel

Participation of the Ubiquitin-Conjugating Enzyme UBE2E3 in Nedd4-2-Dependent Regulation of the Epithelial Na⁺ Channel