The Ubiquitin Ligase Nedd4-1 Is Required for Heart Development and Is a Suppressor of Thrombospondin-1

Abstract

Nedd4 (Nedd4-1) is a Hect domain E3 ubiquitin ligase that also contains a C2 domain and three WW domains. Despite numerous in vitro studies, its biological function in vivo is not well understood. Here we show that disruption of Nedd4-1 in mice (leaving Nedd4-2 intact) caused embryonic lethality at mid gestation, with pronounced heart defects (double-outlet right ventricle and atrioventricular cushion defects) and vasculature abnormalities. Quantitative mass spectrometry and immunoblot analyses of lysates from the wild type and knock-out mouse embryonic fibroblasts to identify Nedd4-1 in vivo targets revealed dramatically increased amounts of thrombospondin-1 (Tsp-1) in the knock-out mouse embryonic fibroblasts and embryos. Tsp-1 is an inhibitor of angiogenesis, and its elevated level was mediated primarily by enhanced transcription. Interestingly, the administration of aspirin (an inhibitor of Tsp-1) to the pregnant heterozygote mothers led to a reduction in Tsp-1 levels and a substantial rescue of the embryonic lethality. These results suggest that Nedd4-1 is a suppressor of Tsp1 and that increased levels of Tsp-1 in the Nedd4-1 knock-out mice may have contributed to the developmental defect observed in the embryos.Ubiquitination regulates stability of many cellular proteins (mainly cytosolic) by tagging them for degradation by the 26 S proteasome (1). It also controls other cellular fates such as endocytosis and vesicular sorting of transmembrane proteins (2-4). The ubiquitination reaction is carried out by the sequential activation of E1, 4 E2, and E3 enzymes (1) , and HECW2) (6, 7). The WW domains of Nedd4 proteins usually bind their substrates by recognizing a short sequence, the PY motif ((L/P)PXY) (8 -10). Nedd4-2 was shown to regulate endocytosis of the epithelial Na ϩ channel by ubiquitination (11-15), an effect impaired in Liddle syndrome patients in whom the PY motif of the epithelial Na ϩ channel (binding site for Nedd4-2) is mutated. Other ion channels are also negatively regulated by . Although Nedd4-1 is more widely expressed than Nedd4-2, less is known about its function. Nedd4-1 was reported to bind to and regulate stability or vesicular sorting of several proteins, including CNrasGEF (21); LAPTM5 (22); the viral proteins HTLV-1 Gag (23), Marburg/Ebola (24 -26), and MLV Gag (27, 28); AP-1 (27); APOBEC3G (29); Grb10 (30, 31); and c-Cbl (32). Also, the Drosophila homolog of Nedd4-1, dNedd4, was recently shown to regulate neuromuscular synaptogenesis in flies by binding to commissureless and promoting its endocytosis (33). Nedd4-1 was also shown to indirectly regulate the function of the endocytic protein Eps15 (34). The proposed role of Nedd4-1 in regulating PTEN stability and nuclear translocation (35, 36) was recently challenged (37).Despite the numerous reports published on Nedd4-1 over the past decade, its in vivo function(s) in mammals are less clear (see "Discussion"). Here we describe the knock-out of Nedd4-1 in mice. We show that these mice exhibit severe cardiac and some vascular defects...

Nedd4-1 and β-Arrestin-1 Are Key Regulators of Na+/H+ Exchanger 1 Ubiquitylation, Endocytosis, and Function

“…4D). In a next step we aimed to validate these findings in the recently generated Nedd4-1 wild-type and knock-out mouse embryonic fibroblasts (MEFs) (17,36). As shown in Fig.…”

“…S1 and "Experimental Procedures"). Eluates were then denatured by boiling in 1% SDS to remove putative ubiquitylated NHE1-associated proteins (17,32). After this denaturation step, immunoprecipitation was performed with the indicated antibodies in Fig.…”

“…Generation and characterization of ␤-arrestin-1 knock-out and wild-type MEFs was described previously (16). Nedd4-1 wild-type and knock-out MEFs were a gift of H. Kawabe and N. Brose and were described in two recent publications (17,18). Generation and characterization of immortalized skin fibroblasts derived from NHE1 wild-type and knock-out mice was described (19).…”

Rac1 acts in conjunction with Nedd4 and Dishevelled-1 to promote maturation of cell-cell contacts

“…Deficiency of Nedd4 leads to elevated levels of thrombospondin, an inhibitor of angiogenesis, and consequent heart defects and embryonic lethality (Fouladkou et al, 2010). In addition, loss of Nedd4 results in impaired formation of the neuromuscular junction, a specialized type of heterotypic cell-cell contact (Liu et al, 2009).…”

The Rac1 hypervariable region in targeting and signaling

“…However, Rac1 is not ubiquitylated by Nedd4. 75,93 Nedd4 has been implicated in various physiological processes including heart development, 101 adaptive immunity 102 and development of the neuromuscular junction, a specialized form of cell-cell contact. 103 In line with this latter finding, our lab showed that Nedd4 is required for the formation of stable cell-cell contacts in epithelial HeLa cells, which is a Rac1-dependent process.…”