InteractiveFly: GeneBrief

Nedd4: Biological Overview | References

Gene name - Nedd4

Synonyms -

Cytological map position - 74D3-74D3

Function - E3 ubiquitin ligase

Keywords - Negatively regulates the Notch signaling pathway and the genes comm, Amph. contributes to neuromuscular synaptogenesis, transverse tubule formation in muscles, and muscle function, formation and actin-dependent patterning of the fly heart.

Symbol - Nedd4

FlyBase ID: FBgn0259174

Genetic map position - chr3L:17,530,081-17,546,376

NCBI classification - ubiquitin protein ligase

Cellular location - cytoplasmic

NCBI link: EntrezGene
Nedd4 orthologs: Biolitmine
Recent literature
Wasserman, S. S., Shteiman-Kotler, A., Harris, K., Iliadi, K. G., Persaud, A., Zhong, Y., Zhang, Y., Fang, X., Boulianne, G. L., Stewart, B. and Rotin, D. (2018). Regulation of SH3PX1 by dNedd4-long at the Drosophila Neuromuscular Junction. J Biol Chem. PubMed ID: 30518551
Drosophila Nedd4 (dNedd4) is a HECT E3 ubiquitin ligase present in two major isoforms: short (dNedd4S) and long (dNedd4Lo), with the latter containing two unique regions (N-terminus and Middle). While dNedd4S promotes neuromuscular synaptogenesis (NMS), dNedd4Lo inhibits it and impairs larval locomotion. To explain how dNedd4Lo inhibits NMS, mass spectrometry was performed to find its binding partners and identified SH3PX1, which binds dNedd4Lo unique Middle region. SH3PX1 contains SH3, PX and BAR domains and is present at neuromuscular junctions, where it regulates active zone ultrastructure and presynaptic neurotransmitter release. This study demonstrates direct binding of SH3PX1 to the dNedd4Lo Middle region (which contains a Pro rich sequence) in vitro and in cells, via the SH3PX1-SH3 domain. In Drosophila S2 cells, dNedd4Lo overexpression reduces SH3PX1 levels at the cell periphery. In vivo overexpression of dNedd4Lo post-synaptically, but not pre-synaptically, reduces SH3PX1 levels at the subsynaptic reticulum and impairs neurotransmitter release. Unexpectedly, larvae that overexpress dNedd4Lo post-synaptically and are heterozygous for a null mutation in SH3PX1 display increased neurotransmission compared to dNedd4Lo or SH3PX1 mutant larvae alone, suggesting a compensatory effect from the remaining SH3PX1 allele. These results suggest a postsynaptic - specific regulation of SH3PX1 by dNedd4Lo.
Zhang, T., Periz, G., Lu, Y. N. and Wang, J. (2020). USP7 regulates ALS-associated proteotoxicity and quality control through the NEDD4L-SMAD pathway. Proc Natl Acad Sci U S A 117(45): 28114-28125. PubMed ID: 33106424
An imbalance in cellular homeostasis occurring as a result of protein misfolding and aggregation contributes to the pathogeneses of neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). This study reports the identification of a ubiquitin-specific protease, USP7, as a regulatory switch in a protein quality-control system that defends against proteotoxicity. A genome-wide screen in a Caenorhabditis elegans model of SOD1-linked ALS identified the USP7 ortholog as a suppressor of proteotoxicity in the nervous system. The actions of USP7 orthologs on misfolded proteins were found to be conserved in Drosophila and mammalian cells. USP7 acts on protein quality control through the SMAD2 transcription modulator of the transforming growth factor β pathway, which activates autophagy and enhances the clearance of misfolded proteins. USP7 deubiquitinates the E3 ubiquitin ligase NEDD4L, which mediates the degradation of SMAD2. Inhibition of USP7 protected against proteotoxicity in mammalian neurons, and SMAD2 was found to be dysregulated in the nervous systems of ALS patients. These findings reveal a regulatory pathway of protein quality control that is implicated in the proteotoxicity-associated neurodegenerative diseases.
Zhang, T., Periz, G., Lu, Y. N. and Wang, J. (2020). USP7 regulates ALS-associated proteotoxicity and quality control through the NEDD4L-SMAD pathway. Proc Natl Acad Sci U S A 117(45): 28114-28125. PubMed ID: 33106424
An imbalance in cellular homeostasis occurring as a result of protein misfolding and aggregation contributes to the pathogeneses of neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). This study reports the identification of a ubiquitin-specific protease, USP7, as a regulatory switch in a protein quality-control system that defends against proteotoxicity. A genome-wide screen in a Caenorhabditis elegans model of SOD1-linked ALS identified the USP7 ortholog as a suppressor of proteotoxicity in the nervous system. The actions of USP7 orthologs on misfolded proteins were found to be conserved in Drosophila and mammalian cells. USP7 acts on protein quality control through the SMAD2 transcription modulator of the transforming growth factor β pathway, which activates autophagy and enhances the clearance of misfolded proteins. USP7 deubiquitinates the E3 ubiquitin ligase NEDD4L, which mediates the degradation of SMAD2. Inhibition of USP7 protected against proteotoxicity in mammalian neurons, and SMAD2 was found to be dysregulated in the nervous systems of ALS patients. These findings reveal a regulatory pathway of protein quality control that is implicated in the proteotoxicity-associated neurodegenerative diseases.

Drosophila Nedd4 (dNedd4) is a HECT ubiquitin ligase with two main splice isoforms: dNedd4 short (dNedd4S) and long (dNedd4Lo). DNedd4Lo has a unique N-terminus containing a Pro-rich region. While dNedd4S promotes neuromuscular synaptogenesis, dNedd4Lo inhibits it and impairs larval locomotion. To delineate the cause of the impaired locomotion, binding partners to the N-terminal unique region of dNedd4Lo were sought in larval lysates. Mass-spectrometry identified Amphiphysin (dAmph). dAmph is a postsynaptic protein containing SH3-BAR domains, which regulates muscle transverse tubule (T-tubule) formation in flies. The interaction was validated by coimmunoprecipitation, and direct binding between dAmph-SH3 domain and dNedd4Lo-N-terminus was demonstrated. Accordingly, dNedd4Lo was colocalized with dAmph postsynaptically and at muscle T-tubules. Moreover, expression of dNedd4Lo in muscle during embryonic development led to disappearance of dAmph and to impaired T-tubule formation, phenocopying amph null mutants. This effect was not seen in muscles expressing dNedd4S or a catalytically-inactive dNedd4Lo(C->A). It is proposed that dNedd4Lo destabilizes dAmph in muscles, leading to impaired T-tubule formation and muscle function (Safi, 2016).

The Drosophila melanogaster larval body wall muscles are established during embryogenesis beginning with the invagination of the mesoderm, which spreads along the ectoderm and then forms numerous mesodermal derivatives. Somatic mesodermal specification produces three different types of myoblasts. Fusion of muscle founder cells and fusion-competent myoblasts form the syncytial myotube, which develops into the embryonic and larval body wall muscles. After myoblast fusion, nuclei are positioned correctly throughout the myotube and form connections to surrounding tendon cells to establish the myotendinous junction, which is innervated by motorneurons in a process called neuromuscular (NM) synaptogenesis. The contractile apparatus is then assembled, and muscles begin to contract. During larval stages, the essential muscle pattern created in the embryo does not change, except that the muscles continue to expand along with the growth of the larva. In Drosophila larva, a repeated pattern of 30 unique muscle fibers is present in each abdominal hemisegment, which are innervated by 36 motor neurons. Each muscle fiber is distinguishable by size, shape, orientation, number of nuclei, innervation, and tendon attachment sites. Throughout development, internal and external cues guide muscles to adopt specific properties that allow them to perform particular functions (Safi, 2016).

Ubiquitination is the process of conjugating ubiquitin onto proteins, and it plays an important role in controlling protein degradation/stability, as well as in trafficking, sorting, and endocytosis of transmembrane proteins. The ubiquitination cascade involves three enzymes: E1, E2, and E3, with the last responsible for substrate recognition and ubiquitin transfer, either indirectly (e.g., RING E3 ligases) or directly (Safi, 2016).

Although many proteins are involved in the regulation of NM synaptogenesis in flies, the role of the ubiquitin system in this process is less well characterized. Studies have shown that the RING-family ubiquitin ligase complex Highwire inhibits synapse formation and function by inhibiting the kinase Wallenda/DLK1, the activator of JNK , whereas the deubiquitinating enzyme Fat Facet targets Liquid Facet/Epsin and promotes synaptic growth (Safi, 2016 and references therein).

Neuronal precursor cell expressed developmentally down-regulated 4 (Nedd4) family members belong to the HECT family of E3 ligases and contain a common C2-WW(n)-HECT domain architecture. Drosophila contains a single dNedd4 gene, which undergoes alternative splicing to produce several splice isoforms, including two prominent ones: dNedd4-short (dNedd4S) and dNedd4-long (dNedd4Lo). Differences between the two isoforms of dNedd4 include an alternate start codon site, resulting in a longer N-terminal region in dNedd4Lo, and an extra exon inserted between those encoding WW1 and WW2 domains (Zhong, 2011; Safi, 2016 and references therein).

Previous studies have shown that dNedd4S promotes NM synaptogenesis in flies (Ing, 2007) by interacting and ubiquitinating Commissureless (Comm), which leads to endocytosis of Comm from the muscle surface, a step required for NM synaptogenesis. Whereas dNedd4S is essential for proper NM synaptogenesis (Ing, 2007), dNedd4Lo inhibits it (Zhong, 2011). Of importance, dNedd4Lo also inhibited normal larval locomotion (Zhong, 2011). These adverse effects of dNedd4Lo were caused by unique N-terminal and Middle regions found in dNedd4Lo (and absent from dNedd4S) and required a functional HECT domain (Zhong, 2011). Of interest, during embryonic muscle development, dNedd4Lo expression is dramatically decreased, whereas that of dNedd4S remains relatively high (Zhong, 2011; Safi, 2016 and references therein).

Because it was observed that the muscle and synaptogenesis defects of dNedd4Lo larvae were not caused by altered phosphorylation of dNedd4Lo, dNedd4Lo-mediated inhibition of catalytic activity of dNedd4S, or diminished effects of dNedd4Lo on Comm endocytosis (Zhong, 2011), it was suspected that dNedd4Lo might inhibit muscle development and/or function by interacting with other proteins via its unique regions (Safi, 2016).

This study identified, using mass spectrometry, Drosophila Amphiphysin (dAmph) as a binding partner of the unique N-terminal region of dNedd4Lo. Amphiphysins are members of the BAR-SH3 domain-containing family of proteins. Mammalian amphiphysin Amph I is involved in endocytosis and synaptic vesicle recycling during neurotransmission by interacting with clathrin/dynamin. In contrast, both mammalian Amph IIb (Bin1) and its fly orthologue, dAmph, are postsynaptic, lack binding sites for clathrin/dynamin, and are not involved in endocytosis. Instead, Bin1 and dAmph regulate transverse tubule (T-tubule) biogenesis in muscles (Safi, 2016).

This study demonstrates that the N-terminus of dNedd4Lo directly binds to dAmph-SH3 domain. In accord, dNedd4Lo and dAmph are colocalized postsynaptically at neuromuscular junctions (NMJs) and muscle transverse tubules (T-tubules). The data show that dNedd4Lo regulates the levels of dAmph postsynaptically and in muscles. Moreover, expression of dNedd4Lo in muscles results in impaired T-tubule formation, phenocopying amph-null mutants. These results demonstrate an important role of dNedd4Lo in regulating T-tubule organization of Drosophila muscles (Safi, 2016).

Previous work has shown that unlike dNedd4 (dNedd4S), muscle-specific overexpression of the dNedd4Lo isoform inhibits NM synaptogenesis and leads to impaired larval locomotion and lethality (Zhong, 2011). This effect required the catalytic activity of dNedd4Lo, since a mutant dNedd4Lo(C->A) with an inactivating mutation in the HECT domain (Cys -> Ala) was not inhibitory. This suggested that the same gene (dNedd4) can encode isoforms with opposite functions. In accord with this, it was observed that during stages of embryonic development when synaptogenesis takes place (14-24 h), dNedd4S expression remains relatively high, whereas that of the inhibitory dNedd4Lo is strongly reduced (Zhong, 2011), suggesting tight regulation of expression of these isoforms to promote muscle development at a very precise time. The inhibitory effect of dNedd4Lo is mediated by two regions unique to dNedd4Lo (N-terminus and Middle region), as deletion of these unique regions alleviated the synaptogenesis defects and increased viability. Therefore it is postulated that the unique regions of dNedd4Lo might negatively regulate NM synaptogenesis and muscle development by targeting specific substrate(s) (Safi, 2016).

This study identified dAmph as a binding partner for the unique N-terminal region of dNedd4Lo by mass spectrometry and validated the interaction in vitro and in vivo in flies. Transiently expressed dAmph coimmunoprecipitates with dNedd4Lo but not with dNedd4S in Drosophila S2 cells and demonstrated direct binding between dAmph-SH3 domain and the dNedd4Lo N-terminus. In vivo results showed that dAmph colocalizes with dNedd4Lo postsynaptically at neuromuscular junctions and muscle T-tubules, where their expression overlaps with the postsynaptic/T-tubule marker, Dlg. Of importance, it was demonstrated that dNedd4Lo expression significantly reduced the levels of dAmph in the postsynaptic region and muscles, an effect not observed in larvae expressing dNedd4S or dNedd4Lo(C->A). As expected, due to the disappearance of endogenous dAmph in larvae expressing dNedd4Lo (and the inability to 'treat' live larvae with proteasome inhibitors), it was not possible to detect ubiquitination of dAmph in these larvae. In addition to biochemical interactions, genetic interactions were also shown between dNedd4Lo and dAmph (Safi, 2016).

The reduction in dAmph levels in the dNedd4Lo-expressing muscles correlated with impaired T-tubule formation, mimicking the phenotype of the amph-null flies. These results could help explain (along with previously described NM synaptogenesis defects; Zhong, 2011) the observed locomotion defects in the dNedd4Lo-overexpressing larvae. At present, it is not possible to quantify the contribution of the T-tubule defects versus the NM synaptogenesis defects to the impaired muscle locomotion/function (Safi, 2016).

Interestingly, it was found that flies overexpressing the dAmph(ΔSH3) mutant in muscles showed reduced localization at the postsynaptic region and T-tubules and no longer colocalized with dNedd4. It is known that the SH3 domain of some membrane-associated proteins is important for their targeting to specific subcellular locations. Similarly, it was found that the SH3 domain of dAmph is also important for its location in the postsynaptic region and the muscle, since the ΔSH3 dAmph protein mislocalized to a region near the muscle plasma membrane. It is not known whether this SH3-dependent localization is related to the ability of this domain to bind dNedd4Lo or due to its interaction(s) with other molecules (Safi, 2016).

Amphiphysin has been implicated in T-tubule biogenesis due to its N-terminal amphipathic helix and BAR domain (N-BAR), which promotes membrane curvature. The BAR domain of the isoform of mammalian amphiphysin 2 (Bin1) is known to be associated with T-tubule formation in skeletal and cardiac muscles, where it induces tubular plasma membrane invaginations. Similar to Bin1, dAmph was shown to participate in plasma membrane remodeling during cleavage furrow ingression, which is required for de novo formation of cells in the Drosophila embryo; the BAR domain of dAmph is required for the formation of endocytic tubules that form at the cleavage furrow tips. This study shows that dNedd4Lo expression reduces the levels of dAmph in the muscle and significantly inhibits T-tubule formation. The degradation of dAmph by dNedd4Lo could impair T-tubule biogenesis by the BAR domain of dAmph, which could help to explain the larval locomotion defects that were observed (Safi, 2016).

Cardiac Bin1 has been implicated in calcium channel trafficking and formation of the inner membrane folds of the cardiac T-tubules. Bin1 localizes to cardiac T-tubules with the L-type calcium channel, Cav1.2, by tethering dynamic microtubules to membrane scaffolds, allowing targeted delivery of Cav1.2 to cardiac T-tubules. Knockdown of Bin1 reduces surface Cav1.2 and delays development of the calcium transient. In cardiomyopathy, decrease in Bin1 alters T-tubule morphology and can cause arrhythmia. Mice with cardiac Bin1 deletion show decreased T-tubule folding, which leads to free diffusion of local extracellular ions, prolonging action-potential duration and increasing susceptibility to arrhythmias. Bin1 is also important for maintenance of intact T-tubule structure and Ca2+ homeostasis in adult skeletal muscle. Adult mouse skeletal muscles with Bin1 knockdown display swollen T-tubule structures, alterations to intracellular Ca2+ release, and compromised coupling between the voltage-gated calcium channel, dihydropyridine receptor (DHPR), and the intracellular calcium channel, ryanodine receptor 1 (Safi, 2016).

Similar to Bin1, dAmph is also required for the organization of the excitation-contraction coupling machinery of muscle. Accordingly, dAmph mutant larvae and flies show defects in T-tubule formation, severe locomotor defects, and flight impairments, indicative of defects in muscle function. Therefore degradation of dAmph in the muscle and inhibition of T-tubule biogenesis by dNedd4Lo could have adverse effects on the localization of T-tubule-associated calcium channels and coupling between the DHPR and ryanodine receptor, as a result altering calcium signaling in muscles. Efficient intracellular Ca2+ homeostasis in skeletal muscle requires intact triad junctional complexes comprising T-tubule invaginations of plasma membrane and terminal cisternae of sarcoplasmic reticulum. Because dNedd4Lo expression significantly reduced T-tubule projections, this would likely impair intracellular Ca2+ homeostasis and result in locomotor defects (Safi, 2016).

Although there is no direct homologue of dNedd4Lo in species other than Drosophila, the mammalian Nedd4 relative Itch has a proline-rich N-terminal region that binds the SH3 domain of Sorting Nexin 9 (Baumann, 2010). In yeast, Rsp5 (the yeast orthologue of Nedd4 proteins) regulates the Amphiphysin homologue Rvs167 by monoubiquitination of lysine in the SH3 domain of Amphiphysin, demonstrating that Nedd4 family members can interact with SH3 domains, including that of amphiphysin, in other species in addition to flies (Safi, 2016).

In addition to dAmph, this study identified several other interacting partners of the N-terminal and Middle regions of dNedd4Lo that could potentially be targeted by dNedd4Lo. Similar to dAmph, two of these proteins, Syndapin (which, like dAmph, bound the unique N-terminus region of dNedd4Lo), and Sorting Nexin 9 (SH3PX1, which bound the unique Middle region of dNedd4Lo), contain BAR and SH3 domains. The SH3 domain-containing protein Cindr/CG31012 (orthologue of the mammalian Cd2ap and Cin85) was also identified as a binding partner to the unique N-terminus of dNedd4Lo. It is not known whether these proteins are bone fide substrates of dNedd4Lo or contribute to the NMJ and T-tubule defects caused by overexpression of dNedd4Lo in the muscle during development (Safi, 2016).

In conclusion, the severely reduced locomotion activity of larvae overexpressing dNedd4Lo in the muscle may be explained by both impaired neuromuscular synaptogenesis, which has been demonstrated previously (Zhong, 2011), and by impaired T-tubule formation as a result of dAmph degradation by dNedd4Lo, which is shown in this study. The defective T-tubule branching would likely impair coupling between the DHPR and ryanodine receptor, possibly by affecting the localization of calcium channels to the T-tubule network. Reduced surface calcium channels on the T-tubule network would alter calcium homeostasis and compromise excitation and contraction coupling, causing larval locomotor defects (Safi, 2016).

The E3 ubiquitin ligase Nedd4/Nedd4L is directly regulated by microRNA 1

miR-1 is a small noncoding RNA molecule that modulates gene expression in heart and skeletal muscle. Loss of Drosophila miR-1 produces defects in somatic muscle and embryonic heart development, which have been partly attributed to miR-1 directly targeting Delta to decrease Notch signaling. This study shows that overexpression of miR-1 in the fly wing can paradoxically increase Notch activity independently of its effects on Delta. Analyses of potential miR-1 targets revealed that miR-1 directly regulates the 3'UTR of the E3 ubiquitin ligase Nedd4. Analysis of embryonic and adult fly heart revealed that the Nedd4 protein regulates heart development in Drosophila. Larval fly hearts overexpressing miR-1 have profound defects in actin filament organization that are partially rescued by concurrent overexpression of Nedd4. These results indicate that miR-1 and Nedd4 act together in the formation and actin-dependent patterning of the fly heart. Importantly, it was found that the biochemical and genetic relationship between miR-1 and the mammalian ortholog Nedd4-like (Nedd4l) is evolutionarily conserved in the mammalian heart, potentially indicating a role for Nedd4L in mammalian postnatal maturation. Thus, miR-1-mediated regulation of Nedd4/Nedd4L expression may serve to broadly modulate the trafficking or degradation of Nedd4/Nedd4L substrates in the heart (Zhu, 2017).

Unexpectedly, overexpression of miR-1 in the anterior-posterior (AP) organizer of the wing disc results in a dose-dependent loss of L3 vein structures, consistent with de-repression of Notch or weakening of a regulatory mechanism that dampens the Notch signal. Using genetic techniques, it was determined that the loss of the distal aspect of L3 could be phenocopied by reducing the gene dose of Notch co-repressors or Nedd4; in the case of Nedd4, the regulation by miR-1 was direct. An expanded model is proposed in which miR-1 expression in the AP organizer has complex effects on Notch signaling owing to its regulation of ligand availability and receptor trafficking. As lower levels of miR-1 expression (18°C) caused wing-vein thickening and tortuosity, and higher levels (22°C) caused vein loss, Delta and Nedd4 may be differentially sensitive to miR-1 regulation, although these studies were not designed to address this issue. It is also possible that indirect effects, such as reductions in Nedd4-mediated ubiquitylation of positive effectors of the Notch receptor (e.g. Deltex) or perturbations in Delta-mediated cis-inhibition, contributed to the de-repression of Notch in the wing-based assay system (Zhu, 2017).

The findings in the mammalian heart indicate that the genetic and biochemical interaction between miR-1 and Nedd4l is physiologically relevant and may provide developmental or tissue-specific regulation of Nedd4l in the myocardium. It is speculated that the additional bands observed on western blots of heart lysates using an anti-Nedd4L antibody might result from post-translational modifications, because Nedd4L can autoregulate its stability through ubiquitylation of its HECT domain. Alternatively, they might represent heart-specific splice variants, because tissue-specific isoforms of Nedd4L have been found in the heart and the liver (Zhu, 2017).

Importantly, although miR-1-mediated reductions in Nedd4 activity caused wing-vein phenotypes induced by Notch, miR-1-mediated dysregulation of Nedd4L in the heart likely affects proteins outside the Notch pathway. Indeed, protein microarrays comparing human Nedd4 with human Nedd4L, suggest that Nedd4L (also known as Nedd4-2) preferentially targets ion channels, whereas Nedd4 targets are enriched for signaling pathways. Thus, in the heart, where miR-1 and murine Nedd4L are both expressed, their genetic and biochemical interaction might influence the excitability and connectivity of cardiomyocytes. Indeed, susceptibility to cardiac arrhythmias and sudden death in humans is associated with six genes that encode ion channels (SCN5A, KCNQ1, KCNH2, KCNE1, KCNE2 and RYR2). Murine Nedd4L regulates the cell-surface densities of the sodium channel, the voltage-gated type V alpha subunit (Scn5a), the potassium voltage-gated channel, KQT-like subfamily member 1 (Kcnq1) and the human Ether-a-go-go-related (KCNH2, previously hERG) channel. Furthermore, miR-1 directly regulates human KCNJ2, a channel that maintains cardiac resting potential. These findings suggest that the regulation of murine Nedd4l by miR-1 contributes to some of the electrophysiological abnormalities seen in miR-1 null mice. It would be interesting to determine whether Nedd4L is dysregulated in the heart after an infarction or under ischemic conditions, when miR-1 is upregulated and fatal cardiac dysrhythmias are common (Zhu, 2017).

Control of the dynamics and homeostasis of the Drosophila Hedgehog receptor Patched by two C2-WW-HECT-E3 Ubiquitin ligases

The conserved Hedgehog (HH) signals control animal development, adult stem cell maintenance and oncogenesis. In Drosophila, the HH co-receptor Patched (PTC) controls both HH gradient formation and signalling. PTC is post-translationally downregulated by HH, which promotes its endocytosis and destabilization, but the mechanisms of PTC trafficking and its importance in the control of PTC remain to be understood. PTC interacts with E3 Ubiquitin (UB)-ligases of the C2-WW-HECT family; two of them-SMURF and NEDD4-are known to regulate its levels. Mutation of the PTC PY motif, which mediates binding of C2-WW-HECT family members, inhibits its internalization but not its autonomous and non-autonomous signalling activities. In addition, the two related UB-C2-WW-HECT ligases NEDD4 and SU(DX) regulate PTC trafficking and finely tune its accumulation through partially redundant but distinct functions. While both NEDD4 and SU(DX) promote PTC endocytosis, only SU(DX) is able to induce its lysosomal targeting and degradation. In conclusion, PTC trafficking and homeostasis are tightly regulated by a family of UB-ligases (Brigui, 2015).

A splice isoform of DNedd4, DNedd4-long, negatively regulates neuromuscular synaptogenesis and viability in Drosophila

Neuromuscular (NM) synaptogenesis is a tightly regulated process. Previously work has shown that in flies, Drosophila Nedd4 (dNedd4/dNedd4S) is required for proper NM synaptogenesis by promoting endocytosis of Commissureless from the muscle surface, a pre-requisite step for muscle innervation. DNedd4 is an E3 ubiquitin ligase comprised of a C2-WW(x3)-Hect domain architecture, which includes several splice isoforms, the most prominent ones are dNedd4-short (dNedd4S) and dNedd4-long (dNedd4Lo). This study shows that while dNedd4S is essential for NM synaptogenesis, the dNedd4Lo isoform inhibits this process and causes lethality. The results reveal that unlike dNedd4S, dNedd4Lo cannot rescue the lethality of dNedd4 null (DNedd4(T121FS)) flies. Moreover, overexpression of UAS-dNedd4Lo specifically in wildtype muscles leads to NM synaptogenesis defects, impaired locomotion and larval lethality. These negative effects of dNedd4Lo are ameliorated by deletion of two regions (N-terminus and Middle region) unique to this isoform, and by inactivating the catalytic activity of dNedd4Lo, suggesting that these unique regions, as well as catalytic activity, are responsible for the inhibitory effects of dNedd4Lo on synaptogenesis. In accord with these findings, an increase was demonstrated in dNedd4S expression relative to the expression of dNedd4Lo during embryonic stages when synaptogenesis takes place. These studies demonstrate that splice isoforms of the same dNedd4 gene can lead to opposite effects on NM synaptogenesis (Zhong, 2011).

dNedd4S has been shown to be involved in NM synaptogenesis in Drosophila, most likely by promoting internalization of Comm from the muscle cell surface (a step necessary for initiation of NM synaptogenesis. Consistent with this model, knock down of dNedd4 during early muscle development or overexpression of Comm mutants that cannot bind dNedd4 yielded the same defects in NM synaptogenesis. This study provides genetic evidence that, in contrast to dNedd4S, the splice isoform dNedd4Lo has a negative role in NM synaptogenesis and embryo development in flies. In accord, expression of dNedd4Lo is reduced (and that of dNedd4S is increased) during synaptogenesis, to permit synaptogenesis to proceed. This negative role of dNedd4Lo does not involve Comm or phosphorylation of dNedd4Lo by Akt, nor does it involve an adverse effect of the unique regions of dNedd4Lo on the catalytic activity of the Hect domain of dNedd4S. Instead, it is likely that the unique Nterm and Mid regions of dNedd4Lo contribute to inhibition of NM synaptogenesis by interacting with other cellular factors or complexes, which are not yet known. Studies to analyze differences in general pattern of ubiquitylation upon overexpression of dNedd4Lo vs. dNedd4S in S2 cells did not reveal overt differences, most likely due to insufficient sensitivity of the system to detect changes in ubiquitylation of specific substrates among the many ubiquitylated cellular proteins (Zhong, 2011).

These studies demonstrate that muscle-specific overexpression of dNedd4Lo causes abnormal motor neuron innervation along the SNb branch on body wall muscles 13-->12. The types of defects found include inappropriate backward innervation from muscles 12-->13 and increased number of nerve branches on muscle 12. The backward innervation defect was previously observed for overexpression of Comm 2PY-->A (that cannot bind dNedd4) and Comm 10K-->R mutants (that cannot become ubiquitylated), as well as dNedd4 RNAi mutants. The other common defect observed was increased motor nerve branching on muscle 12 (Zhong, 2011).

It is known that disruption of genes involved in cell adhesion processes cause nerve branching defects, such as position specific (PS) β-integrin, fasciclinII (FasII), Calcium/Calmodulin dependent Kinase II (CaMKII), and DLG (a PDZ-domain Scaffold protein). They form a post-synaptic complex in the muscle to co-ordinately regulate defasiculation of the nerve terminal endings and fine-tune the interaction between motor neurons and their muscle targets. It has been proposed that β-integrin regulates recruitment of the cell adhesion molecule FasII on the muscle surface. Down-regulation of β-integrin and up-regulation of FasII on the muscle surface lead to nerve defasiculation. Whether or not the adverse effects of dNedd4Lo on NM synaptogenesis involve these (or other) proteins is currently unknown (Zhong, 2011).

One consequence of muscle innervation defects could be abnormal locomotor activity. Indeed, a significant reduction was found in the locomotor activity of larvae that overexpress dNedd4Lo specifically in the muscle. Furthermore, muscle-specific overexpression of dNedd4Lo leads to lethality during development. However, the muscle drivers 24B-GAL4 and 5-GAL4, used in this experiment, drive expression in the entire mesoderm, which derives into somatic (body wall) muscles for movement, visceral (gut) muscles for digestion, and cardiac muscles. Thus, while the innervation defects on body wall muscles may contribute to the larval lethality, defects in heart and/or gut muscle functions might contribute as well, since heart activity and feeding are essential for larval survival (Zhong, 2011).

Similar muscle innervation defects were also observed for the mouse homologue of dNedd4, mNedd4 (mNedd4-1) (Liu, 2009). In mNedd4 mutant embryos, motor nerves defasciculate upon reaching their skeletal muscle targets and the pre-synaptic nerve terminal branches are increased in number. It was also demonstrated that mNedd4 mutants had increased spontaneous miniature endplate potential (mEPP) frequency, which is consistent with the ultra structural alternation. In addition, β-catenin, a subunit of the cadherin protein complex, was proposed to be a potential substrate for mNedd4 in NM synapse formation and function. β-catenin deficient muscles show similar defects of nerve defasiculation as mNedd4 mutant. Similarly, molecular manipulation of β-integrins, which is also involved in the cell adhesion process, in muscles of mice also lead to abnormal development of pre-synaptic nerve terminals (Zhong, 2011).

Phosphorylation is an important mechanism for the regulation of Nedd4 proteins and other E3 ubiquitin ligases. For example, Nedd4-2 is known to be regulated by Akt/Sgk – mediated phosphorylation, which inhibits its ability to interact with its substrate ENaC. However, mutating the dAkt phosphorylation sites (S-->A) in dNedd4Lo did not affect the abnormal muscle innervation. Since the dAkt phosphorylation site in the unique N-terminal region of dNedd4Lo did not explain its negative function, the whole unique N-terminal region or the middle region were removed to determine their role in the negative effect of dNedd4Lo on viability and NM synaptogenesis. It was demonstrated that removing either the N-terminal or the middle region rescued the lethality and alleviated the muscle innervation defects. Therefore, both regions are involved in the negative function of dNedd4Lo in this event. Thus two possible underlying mechanisms were investigated for the negative regulation of dNedd4Lo in NM synaptogenesis. First, inhibition of function of dNedd4S through the unique regions of dNedd4Lo was tested. It is known that the catalytic activity of Nedd4 proteins can be regulated through auto-inhibition mechanism. For example, the WW domains of Nedd4-2 and a close relative of Nedd4, Itch, as well as the C2 domain of Smurf 2, were shown to bind to their own Hect domains and inhibit their catalytic activity. However, the data suggest that the unique N-terminal or middle regions of dNedd4Lo do not bind nor inhibit the catalytic activity of dNedd4S in vitro. Second, the effect of dNedd4Lo overexpression on dNedd4S-mediated Comm endocytosis was investigated in Drosophila S2 cells and body wall muscles. It is hypothesized that if dNedd4Lo acts to inhibit the function of dNedd4S, it would interfere with Comm endocytosis. However, the results show that overexpression of dNedd4Lo did not affect internalization of Comm. Therefore, the unique regions of dNedd4Lo regulate NM synaptogenesis by as yet unknown mechanisms, possibly by targeting other substrates (Zhong, 2011).

Interestingly, differential regulation of substrates is known for isoforms of the E3 ligase Cbl, namely dCblL (long) and dCblS (short). While the long isoform down-regulates EGFR signaling, the short isoform preferentially controls Notch signaling through regulation of the Notch ligand Delta. DNedd4 might use a similar mechanism to regulate Drosophila embryo development, particularly NM synaptogenesis. In addition, temporal regulation of expression of dNedd4S and dNedd4Lo differs, allowing NM synaptogenesis to proceed at the appropriate time in development (Zhong, 2011).

Regulation of Commissureless by the ubiquitin ligase DNedd4 is required for neuromuscular synaptogenesis in Drosophila melanogaster

Muscle synaptogenesis in Drosophila melanogaster requires endocytosis of Commissureless (Comm), a binding partner for the ubiquitin ligase dNedd4. This study investigated whether dNedd4 and ubiquitination mediate this process. Comm was shown to be expressed in intracellular vesicles in the muscle, whereas Comm bearing mutations in the two PY motifs (L/PPXY) responsible for dNedd4 binding [Comm(2PY--->AY)], or bearing Lys--->Arg mutations in all Lys residues that serve as ubiquitin acceptor sites [Comm(10K--->R)], localize to the muscle surface, suggesting they cannot endocytose. Accordingly, aberrant muscle innervation is observed in the Comm(2PY--->AY) and Comm(10K--->R) mutants expressed early in muscle development. Similar muscle surface accumulation of Comm and innervation defects are observed when dNedd4 is knocked down by double-stranded RNA interference in the muscle, in dNedd4 heterozygote larvae, or in muscles overexpressing catalytically inactive dNedd4. Expression of the Comm mutants fused to a single ubiquitin that cannot be polyubiquitinated and mimics monoubiquitination [Comm(2PY--->AY)-monoUb or Comm(10K--->R)-monoUb] prevents the defects in both Comm endocytosis and synaptogenesis, suggesting that monoubiquitination is sufficient for Comm endocytosis in muscles. Expression of the Comm mutants later in muscle development, after synaptic innervation, has no effect. These results demonstrate that dNedd4 and ubiquitination are required for Commissureless endocytosis and proper neuromuscular synaptogenesis (Ing, 2007).

The C-terminal tail of the Hedgehog receptor Patched regulates both localization and turnover

Patched is a membrane protein whose function in Hedgehog (Hh) signal transduction has been conserved among metazoans and whose malfunction has been implicated in human cancers. Genetic analysis has shown that Ptc negatively regulates Hh signal transduction, but its activity and structure are not known. This study investigated the functional and structural properties of Drosophila Ptc and its C-terminal domain (CTD), 183 residues that are predicted to reside in the cytoplasm. The results show that Ptc, as well as truncated Ptc deleted of its CTD, forms a stable trimer. This observation is consistent with the proposal that Ptc is structurally similar to trimeric transporters. The CTD itself trimerizes and is required for both Ptc internalization and turnover. Two mutant forms of the CTD, one that disrupts trimerization and the other that mutates the target sequence of the Nedd4 ubiquitin ligase, stabilize Ptc but do not prevent internalization and sequestration of Hh. Ptc deleted of its CTD is stable and localizes to the plasma membrane. These data show that degradation of Ptc is regulated at a step subsequent to endocytosis, although endocytosis is a likely prerequisite. It was also shown that the CTD of mouse Ptc regulates turnover (Lu, 2006).

Analysis of CTD mutants revealed that the CTD controls Ptc localization and half-life. Both functions mapped to the CTD's 106 C-terminal residues. Deletion of this CTD reduced the levels of internal Ptc and stabilized the protein. Note that assays of Ptc localization measured its steady-state distribution and did not distinguish between effects on the rate of internalization or on recycling of internalized protein to the cell surface. Therefore, it is not known whether Hh directly affects the removal of Ptc from the cell surface, or if it affects a process that sorts internalized protein. Since internalized PtcWT has been observed to colocalize with Hh in multivesicular bodies, which are late endosomes that ferry cargo to lysosomes, it seems reasonable to propose that internalized Hh-bound Ptc is programmed for degradation, and that internalization is a requisite step in the pathway toward that fate. The observation that the instability conferred by the CTD is sensitive to NH4Cl, an inhibitor of lysosomal proteolysis, is consistent with this model. These experiments do not reveal whether unbound Ptc cycles between early endosomes and the cell surface in the absence of Hh, and so these experiments do not implicate Hh in the regulation of Ptc endocytosis per se (Lu, 2006).

The phenotypes of the ptc3P and ptcPPAA missense mutants add to the understanding of the Ptc degradation pathway. Both the Ptc3P and the PtcPPAA mutant proteins are processed by the degradation pathway less efficiently than PtcWT. Yet, both Ptc3P and PtcPPAA internalize in the presence of Hh. Since PtcPPAA mutates a PPXY motif in the CTD that is a recognition site for Nedd4, these results suggest that mono-ubiquitination in the CTD is a signal that targets Ptc to lysosomes, but mono-ubiquitination is not required for movement to early endosomes. Ptc3P retains the PPXY motif, but in contrast to CTDWT and CTDPPAA, its CTD cannot multimerize. This behavior suggests that the process that marks Ptc for sorting to late endosomes may require both the PPXY motif and a conformation that is generated by the trimerized CTD. Since both Ptc3P and the PtcPPAA proteins can sequester Hh, and both internalize and colocalize with Hh, these functions are apparently required for sorting, not for Hh binding or internalization. The inability of PtcΔ1/2C to internalize indicates that the 106 C-terminal residues also include a domain that targets Ptc to early endosomes (Lu, 2006).

The importance of regulated turnover to the proper function of signaling pathways has recently been illuminated by the isolation and analysis of the Drosophila vps25 and erupted genes. Both genes encode proteins that function in endosomal sorting, and mutants have impressive phenotypes characterized by unregulated growth and defective patterning. Endocytic defects in mutant clones result in accumulation of signaling receptors such as Notch and Thickveins as well as other signaling components, highlighting the critical role that endocytic sorting plays in regulating signaling. The multiple functions of the Ptc CTD that are necessary for proper trafficking and turnover testify to the many steps in this complex (Lu, 2006).

Cells that express a mouse Ptc CTD deletion (PtcΔCTD) have more than five times the number of binding sites for Shh as do cells expressing wild-type Ptc. The mouse PtcΔCTD mutant protein, like the Drosophila PtcΔCTD, has an increased half-life. It is noted that both mouse and human Ptc have a PPXY motif in their respective CTDs at a location that is comparable to that of the Drosophila PPAY sequence. Although the role of the PPXY motif in mouse Ptc was not investiated, it seems reasonable to propose that the functions of the CTD are generally conserved in the vertebrate and invertebrate proteins, that the increased stability of mouse PtcΔCTD derives in part from the absence of the PPXY sequence, that mouse PtcΔCTD is not internalized efficiently, and that these properties contribute to the increased binding of Shh to PtcΔCTD-expressing cells (Lu, 2006).

Drosophila Nedd4, a ubiquitin ligase, is recruited by Commissureless to control cell surface levels of the roundabout receptor

Crossing the midline produces changes in axons such that they are no longer attracted to the midline. In Drosophila, Roundabout reaches high levels on axons once they have crossed the midline, and this prohibits recrossing. Roundabout protein levels are regulated by Commissureless. Commissureless binds to and is regulated by the ubiquitin ligase DNedd4. The ability of Commissureless to regulate Roundabout protein levels requires an intact DNedd4 binding site and ubiquitin acceptor sites within the Commissureless protein. The ability of Commissureless to regulate Robo in the embryo also requires a Commissureless/DNedd4 interaction. These results show that changes in axonal sensitivity to external cues during pathfinding across the midline makes use of ubiquitin-dependent mechanisms to regulate transmembrane protein levels (Myat, 2002).

Within the embryo, Comm protein is located both at the cell surface and within intracellular vesicles in midline cells and commissural axons. This distribution is suggestive of a protein that can move between different locations in the cell. Robo, however, is expressed on the surface of longitudinal axons. Comm can regulate Robo protein levels, and the proteins are occasionally coexpressed in the same cell in the embryo when the Robo protein is found within intracellular vesicles with Comm. Thus, Comm may internalize Robo as part of its regulation of Robo. When Comm is expressed in Drosophila S2 cells, the protein displays a similar distribution to that seen in the embryos with the majority of the protein within intracellular vesicles. Robo, as expected, is expressed on the cell surface when expressed alone in S2 cells. However, when Comm and Robo are expressed together in S2 cells, the Robo protein is no longer found at the cell surface but is now colocalized with Comm within intracellular vesicles within the cell. Thus, Comm is able to change the site of Robo localization within the cell. This ability correlates with the observation in the embryo that overexpression of Comm results in the reduction of Robo protein at the cell surface. This study shows that the normal intracellular distribution of Comm requires an interaction with DNedd4. Removal or disruption of either the DNedd4 binding sites or the intracellular lysines in Comm or the reduction of DNedd4 levels in S2 cells results in Comm accumulating at the cell surface. Comm is no longer brought into the cell and is unable to remove coexpressed Robo from the cell surface. Thus, DNedd4 is a key cofactor that allows Comm to harness the ubiquitination pathway to target its removal from the cell surface together with other membrane receptors it may bind (Myat, 2002).

To test whether DNedd4 has an important role in Comm function, neural overexpression of Comm was used as a sensitive assay. Overexpression of a single copy of comm within all CNS neurons results in the downregulation of Robo in these cells and the production of a robo phenocopy where axons recross the midline. When the level of overexpressed Comm is increased, the phenotype becomes more severe and many axons remain at the CNS midline. Overexpression of DNedd4 alongside one copy of comm produces a phenotype similar to that seen when greater levels of comm are overexpressed. The presence of additional DNedd4 makes the overexpressed Comm more effective at downregulating Robo activity, suggesting it does indeed act with Comm to regulate Robo levels in the embryo. This is supported by the observation that the overexpression of a catalytically inactive form of DNedd4 partially suppresses the ability of overexpressed comm to cause a robo phenocopy. Thus, one activity of DNedd4 in Drosophila is to function with Comm to regulate Robo protein levels. Extrapolating from S2 cell observations, it is assumed that a similar process is taking place in the embryo whereby Comm acts with DNedd4 to internalize Robo into the cell. This suggests that normally Comm and DNedd4 function together in commissural neurons to reduce Robo activity and allow axons to cross the midline. Comm accumulates within commissural axons, and recent experiments have revealed that comm is expressed within these axons with Comm protein only reaching high levels at the midline (Myat, 2002).

Nedd4 family proteins regulate the internalization of a number of cell surface proteins. Nedd4 regulates levels of the epithelial Na+ channel, while the yeast homolog Rsp5 catalyzes the internalization of a number of membrane transporters. Although Nedd4 was identified in a screen for transcripts expressed in the mouse nervous system during embryonic development, no targets for this molecule during neural development have yet been identified. DNedd4 can regulate Comm and consequently, Robo. Yet, removal of DNedd4 function in the embryo does not give rise to the same phenotype as a loss of comm. If DNedd4 was acting purely within the Comm pathway to regulate Robo protein levels, then one might expect that a loss of DNedd4 function would give rise to a comm-like phenotype, since Robo protein levels may stay high. However, inhibition of DNedd4 could result in the stabilization of Comm on neuronal membranes where it can bind Robo and possibly interfere with Robo function to produce a partial robo-like phenotype. RNA interference with DNedd4 gives rise to a phenotype where axons stall at the junction of the longitudinal and commissural axon tracts, resulting in thinner longitudinal and commissural axon tracts (i.e., neither a comm nor robo phenocopy). This phenotype suggests that DNedd4 may also regulate the cell surface levels of other axon guidance molecules. Additionally, DNedd4 may also affect neuronal fate decisions since a close homolog, Su(dx), acts as a regulator of Notch signaling. The isolation of DNedd4 loss-of-function mutations will aid full evaluation of the exact roles of DNedd4 in the embryo (Myat, 2002).

Functions of Nedd4 orthologs in other species Nedd4

NEDD4 controls intestinal stem cell homeostasis by regulating the Hippo signalling pathway

The Hippo pathway plays crucial roles in regulating organ size and stem cell homeostasis. Although the signalling cascade of the core Hippo kinases is relatively well understood, little is known about the mechanisms that modulate the activity of the Hippo pathway. This study reports the identification of human NEDD4, a HECT-type E3 ubiquitin ligase, as a regulatory component of the Hippo pathway. NEDD4 ubiquitylated and destabilized WW45 (see Drosophila Salvador) and LATS kinase (see Drosophila Warts, both of which are required for active Hippo signalling. Interestingly, MST1 protected WW45, but not LATS2, against NEDD4. The study also provided evidence indicating that NEDD4 inactivation at high cell density was a prerequisite for the elevated Hippo activity linked to contact inhibition. Moreover, NEDD4 promoted intestinal stem cell renewal in Drosophila by suppressing Hippo signalling. Collectively, the study presents a regulatory mechanism by which NEDD4 controls the Hippo pathway leading to coordinated cell proliferation and apoptosis (Bae, 2015).

NEDD4L regulates convergent extension movements in Xenopus embryos via Disheveled-mediated non-canonical Wnt signaling

During the early vertebrate body plan formation, convergent extension (CE) of dorsal mesoderm and neurectoderm is coordinated by the evolutionarily conserved non-canonical Wnt/PCP signaling. Disheveled (Dvl; see Drosophila Disheveled), a key mediator of Wnt/PCP signaling, is essential for the medial-lateral polarity formation in the cells undergoing convergent extension movements. NEDD4L, a highly conserved HECT type E3 ligase, has been reported to regulate the stability of multiple substrates including Dvl2. This study demonstrates that NEDD4L is required for the cellular polarity formation and convergent extension in the early Xenopus embryos. Depletion of NEDD4L in early Xenopus embryos results in the loss of mediolateral polarity of the convergent-extending mesoderm cells and the shortened body axis, resembling those defects caused by the disruption of non-canonical Wnt signaling. Depletion of xNEDD4L also blocks the elongation of the animal explants in response to endogenous mesoderm inducing signals and partially compromises the expression of Brachyury. Importantly, reducing Dvl2 expression can largely rescue the cellular polarity and convergent extension defects in NEDD4L-depleted embryos and explants. Together with the data that NEDD4L reduces Dvl2 protein expression in the frog embryos, these findings suggest that regulation of Dvl protein levels by NEDD4L is essential for convergent extension during early Xenopus embryogenesis (Zhang, 2014).

Ubiquitin ligase Nedd4L targets activated Smad2/3 to limit TGF-β signaling

TGF-beta induces phosphorylation of the transcription factors Smad2 and Smad3 at the C terminus as well as at an interdomain linker region. TGF-beta-induced linker phosphorylation marks the activated Smad proteins for proteasome-mediated destruction. This study identified Nedd4L as the ubiquitin ligase responsible for this step. Through its WW domain, Nedd4L specifically recognizes a TGF-beta-induced phosphoThr-ProTyr motif in the linker region, resulting in Smad2/3 polyubiquitination and degradation. Nedd4L is not interchangeable with Smurf1, a ubiquitin ligase that targets BMP-activated, linker-phosphorylated Smad1. Nedd4L limits the half-life of TGF-beta-activated Smads and restricts the amplitude and duration of TGF-beta gene responses, and in mouse embryonic stem cells, it limits the induction of mesoendodermal fates by Smad2/3-activating factors. Hierarchical regulation is provided by SGK1, which phosphorylates Nedd4L to prevent binding of Smad2/3. Previously identified as a regulator of renal sodium channels, Nedd4L is shown here to play a broader role as a general modulator of Smad turnover during TGF-beta signal transduction (Gao, 2009).

Abnormal development of the neuromuscular junction in Nedd4-deficient mice

Nedd4 (neural precursor cell expressed developmentally down-regulated gene 4) is an E3 ubiquitin ligase highly conserved from yeast to humans. The expression of Nedd4 is developmentally down-regulated in the mammalian nervous system, but the role of Nedd4 in mammalian neural development remains poorly understood. This study shows that a null mutation of Nedd4 in mice leads to perinatal lethality: mutant mice were stillborn and many of them died in utero before birth (between E15.5-E18.5). In Nedd4 mutant embryos, skeletal muscle fiber sizes and motoneuron numbers are significantly reduced. Surviving motoneurons project axons to their target muscles on schedule, but motor nerves defasciculate upon reaching the muscle surface, suggesting that Nedd4 plays a critical role in fine-tuning the interaction between the nerve and the muscle. Electrophysiological analyses of the neuromuscular junction (NMJ) demonstrate an increased spontaneous miniature endplate potential (mEPP) frequency in Nedd4 mutants. However, the mutant neuromuscular synapses are less responsive to membrane depolarization, compared to the wildtypes. Ultrastructural analyses further reveal that the pre-synaptic nerve terminal branches at the NMJs of Nedd4 mutants are increased in number, but decreased in diameter compared to the wildtypes. These ultrastructural changes are consistent with functional alternation of the NMJs in Nedd4 mutants. Unexpectedly, Nedd4 is not expressed in motoneurons, but is highly expressed in skeletal muscles and Schwann cells. Together, these results demonstrate that Nedd4 is involved in regulating the formation and function of the NMJs through non-cell autonomous mechanisms (Liu, 2009).

A Smad action turnover switch operated by WW domain readers of a phosphoserine code

When directed to the nucleus by TGF-β or BMP signals, Smad proteins undergo cyclin-dependent kinase 8/9 (CDK8/9) and glycogen synthase kinase-3 (GSK3) phosphorylations that mediate the binding of YAP and Pin1 for transcriptional action, and of ubiquitin ligases Smurf1 and Nedd4L for Smad destruction. This study demonstrates that there is an order of events-Smad activation first and destruction later-and that it is controlled by a switch in the recognition of Smad phosphoserines by WW domains in their binding partners. In the BMP pathway, Smad1 phosphorylation by CDK8/9 creates binding sites for the WW domains of YAP, and subsequent phosphorylation by GSK3 switches off YAP binding and adds binding sites for Smurf1 WW domains. Similarly, in the TGF-β pathway, Smad3 phosphorylation by CDK8/9 creates binding sites for Pin1 and GSK3, then adds sites to enhance Nedd4L binding. Thus, a Smad phosphoserine code and a set of WW domain code readers (see A Smad action turnover switch operated by WW domain readers of a phosphoserine code) provide an efficient solution to the problem of coupling TGF-β signal delivery to turnover of the Smad signal transducers (Aragón, 2011).

NEDD4-1 is a proto-oncogenic ubiquitin ligase for PTEN

The tumor suppressor PTEN, a critical regulator for multiple cellular processes, is mutated or deleted frequently in various human cancers. Subtle reductions in PTEN expression levels have profound impacts on carcinogenesis. This study shows that PTEN level is regulated by ubiquitin-mediated proteasomal degradation, and its ubiquitin ligase has been purified as HECT-domain protein NEDD4-1. In cells NEDD4-1 negatively regulates PTEN stability by catalyzing PTEN polyubiquitination. Consistent with the tumor-suppressive role of PTEN, overexpression of NEDD4-1 potentiates cellular transformation. Strikingly, in a mouse cancer model and multiple human cancer samples where the genetic background of PTEN was normal but its protein levels were low, NEDD4-1 is highly expressed, suggesting that aberrant upregulation of NEDD4-1 can posttranslationally suppress PTEN in cancers. Elimination of NEDD4-1 expression inhibits xenotransplanted tumor growth in a PTEN-dependent manner. Therefore, NEDD4-1 is a potential proto-oncogene that negatively regulates PTEN via ubiquitination, a paradigm analogous to that of Mdm2 and p53 (Wang, 2007).

The functionally exchangeable L domains in RSV and HIV-1 Gag direct particle release through pathways linked by Tsg101

The functionally exchangeable L domains of HIV-1 and Rous sarcoma virus (RSV) Gag, bind Tsg101 and Nedd4, respectively. Tsg101 and Nedd4 function in endocytic trafficking, and studies show that expression of Tsg101 or Nedd4 fragments interferes with release of HIV-1 or RSV Gag, respectively, as virus-like particles (VLPs). To determine whether functional exchangeability reflects use of the same trafficking pathway, the effect on RSV Gag release of co-expression was tested with mutated forms of Vps4, Nedd4 and Tsg101. A dominant-negative mutant of Vps4A, an AAA ATPase required for utilization of endosomal sorting proteins that interferes with HIV-1 budding, also inhibits RSV Gag release, indicating that RSV uses the endocytic trafficking machinery, as does HIV. Nedd4 and Tsg101 interacts in the presence or absence of Gag and, through its binding of Nedd4, RSV Gag interacts with Tsg101. Deletion of the N-terminal region of Tsg101 or the HECT domain of Nedd4 does not prevent interaction; however, three-dimensional spatial imaging suggests that the interaction of RSV Gag with full-length Tsg101 and N-terminally truncated Tsg101 is not the same. Co-expression of RSV Gag with the Tsg101 C-terminal fragment interferes with VLP release minimally; however, a significant fraction of the released VLPs is tethered to Tsg101. The results suggest that, while Tsg101 is not required for RSV VLP release, alterations in the protein interfere with VLP budding/fission events. It is concluded that RSV and HIV-1 Gag direct particle release through independent ESCRT-mediated pathways that are linked through Tsg101-Nedd4 interaction (Medina, 2005).


Search PubMed for articles about Drosophila Nedd4

Aragon, E., Goerner, N., Zaromytidou, A. I., Xi, Q., Escobedo, A., Massague, J. and Macias, M. J. (2011). A Smad action turnover switch operated by WW domain readers of a phosphoserine code. Genes Dev 25(12): 1275-1288. PubMed ID: 21685363

Bae, S.J., Kim, M., Kim, S.H., Kwon, Y.E.,Lee, J.H., Kim, J., Chung, C.H., Lee, W.J. and Seol, J.H. (2015). NEDD4 controls intestinal stem cell homeostasis by regulating the Hippo signalling pathway. Nat Commun 6: 6314. PubMed ID: 25692647

Baumann, C., Lindholm, C. K., Rimoldi, D. and Levy, F. (2010). The E3 ubiquitin ligase Itch regulates sorting nexin 9 through an unconventional substrate recognition domain. FEBS J 277(13): 2803-2814. PubMed ID: 20491914

Brigui, A., Hofmann, L., Arguelles, C., Sanial, M., Holmgren, R. A. and Plessis, A. (2015). Control of the dynamics and homeostasis of the Drosophila Hedgehog receptor Patched by two C2-WW-HECT-E3 Ubiquitin ligases. Open Biol 5. PubMed ID: 26446620

Gao, S., et al. (2009). Ubiquitin ligase Nedd4L targets activated Smad2/3 to limit TGF-β signaling. Mol. Cell 36: 457-468. PubMed Citation: 19917253

Ing, B., Shteiman-Kotler, A., Castelli, M., Henry, P., Pak, Y., Stewart, B., Boulianne, G. L. and Rotin, D. (2007). Regulation of Commissureless by the ubiquitin ligase DNedd4 is required for neuromuscular synaptogenesis in Drosophila melanogaster. Mol Cell Biol 27(2): 481-496. PubMed ID: 17074801

Liu, Y., Oppenheim, R. W., Sugiura, Y. and Lin, W. (2009). Abnormal development of the neuromuscular junction in Nedd4-deficient mice. Dev Biol 330(1): 153-166. PubMed ID: 19345204

Lu, X., Liu, S. and Kornberg, T. B. (2006). The C-terminal tail of the Hedgehog receptor Patched regulates both localization and turnover. Genes Dev 20(18): 2539-2551. PubMed ID: 16980583

Medina, G., Zhang, Y., Tang, Y., Gottwein, E., Vana, M. L., Bouamr, F., Leis, J. and Carter, C. A. (2005). The functionally exchangeable L domains in RSV and HIV-1 Gag direct particle release through pathways linked by Tsg101. Traffic 6(10): 880-894. PubMed ID: 16138902

Myat, A., Henry, P., McCabe, V., Flintoft, L., Rotin, D. and Tear, G. (2002). Drosophila Nedd4, a ubiquitin ligase, is recruited by Commissureless to control cell surface levels of the roundabout receptor. Neuron 35(3): 447-459. PubMed ID: 12165468

Safi, F., Shteiman-Kotler, A., Zhong, Y., Iliadi, K. G., Boulianne, G. L. and Rotin, D. (2016). Drosophila Nedd4-long reduces Amphiphysin levels in muscles and leads to impaired T-tubule formation. Mol Biol Cell 27(6):907-18. PubMed ID: 26823013

Wang, X., et al. (2007). NEDD4-1 is a proto-oncogenic ubiquitin ligase for PTEN. Cell 128(1): 129-39. Medline abstract: 17218260

Zhang, Y., Ding, Y., Chen, Y. G. and Tao, Q. (2014). NEDD4L regulates convergent extension movements in Xenopus embryos via Disheveled-mediated non-canonical Wnt signaling. Dev Biol 392(1):15-25. PubMed ID: 24833518

Zhong, Y., Shtineman-Kotler, A., Nguyen, L., Iliadi, K. G., Boulianne, G. L. and Rotin, D. (2011). A splice isoform of DNedd4, DNedd4-long, negatively regulates neuromuscular synaptogenesis and viability in Drosophila. PLoS One 6(11): e27007. PubMed ID: 22110599

Zhu, J. Y., Heidersbach, A., Kathiriya, I. S., Garay, B. I., Ivey, K. N., Srivastava, D., Han, Z. and King, I. N. (2017). The E3 ubiquitin ligase Nedd4/Nedd4L is directly regulated by microRNA 1. Development 144(5): 866-875. PubMed ID: 28246214

Biological Overview

date revised: 2 November 2017

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