NEDD4 (neural precursor cell expressed developmentally downregulated 4) was first identified in a screen for genes that are downregulated during development of the mouse brain (Kumar et al. 1992). Further characterization showed that mouse NEDD4 is a HECT type of ubiquitin ligase, comprising three WW domains (involved in protein-protein interactions) and a C2 calcium/phospholipid binding domain (Staub et al. 1996; Kumar et al. 1997). Discovery of the HECT domain gave the initial clues as to the function of NEDD4, as this domain was first identified in the papilloma virus oncoprotein E6-associated protein (E6-AP) and shown to be involved in the ubiquitination of p53 (Boase and Kumar 2015). The HECT family of ubiquitin ligases is found in all eukaryotic organisms, with NEDD4 the founding member of the NEDD4 family of HECT ligases, which contains nine human proteins (Scheffner and Kumar 2014).
NEDD4 Conservation, Structure and Expression
Regulation of Development and Growth Signaling
NEDD4 knockout in mouse results in embryonic lethality (Cao et al. 2008). Close analysis of the first NEDD4−/− embryos generated revealed delayed embryonic development, reduced growth and body weight, and decreased mitogenic activity. This was attributed to reduced insulin growth factor 1 (IGF1) and insulin signaling caused in part by upregulation of Grb10, a negative regulator of IGF1 signaling (Cao et al. 2008). Other studies suggest that NEDD4 also regulates IGF1 and insulin signaling via targeting AKT and PTEN (Shi et al. 2014; Boase and Kumar 2015). Recently, it was demonstrated that mice haploin–sufficient for NEDD4 display insulin resistance, enhanced lipolysis, and protection from high-fat diet-induced obesity (Li et al. 2015).
Other growth factor signaling pathways shown to be regulated by NEDD4 include the fibroblast growth factor receptor 1 (FGFR1) (Persaud et al. 2011), and the vascular endothelial growth factor receptor 2 (VEGF-R2) via NEDD4 regulation of Grb10 (Murdaca et al. 2004). Other potential growth signaling pathways, not always validated in vivo, have been summarized previously (Boase and Kumar 2015).
Another independently generated NEDD4−/− mouse line has been reported to display embryonic lethality with heart defects (double-outlet right ventricle and atrioventricular cushion defects) and vasculature abnormalities (Fouladkou et al. 2010). This was attributed to increased levels of thrombospondin-1 (Tsp1), which is an inhibitor of angiogenesis, and shows a role for NEDD4 in regulation of heart development.
NEDD4 in the Nervous System
Mouse knockout studies indicate that NEDD4 has a critical function in dendrite formation and arborization in hippocampal and cortical neurons through the regulation of Ras-related protein 2A (Rap2A) (Kawabe et al. 2010). NEDD4 also plays an important role in cranial neural crest cell survival and craniofacial development (Wiszniak et al. 2016) and in nerve regeneration (Christie et al. 2012), although the mechanisms for this remain unclear. Given its role in the nervous system, it is not surprising that aberrant NEDD4 is linked to multiple neurological disorders. For example, lower levels of NEDD4 in Parkinson’s disease (characterized by the abnormal accumulation of aggregates of α-synuclein protein in neurons) contribute to neuronal death by elevating levels of the proapoptotic protein RTP801 (Canal et al. 2016). In Alzheimer’s disease, accumulation of the neurotoxic ß-amyloid induces synaptic alterations which involve, in part, NEDD4-mediated ubiquitination of AMPARs (α-amino-3-hydroxy-5-methyl-4-isoxazolepro pionic acid receptors) to promote their internalization and weaken synaptic strength (Rodrigues et al. 2016). Additional evidence for the regulation of AMPAR trafficking by NEDD4 has been shown recently; for example, learning and memory are significantly impaired in NEDD4 heterozygous mice (Camera et al. 2016).
NEDD4 in the Neuromuscular Junction (NMJ) and Skeletal Muscle
NEDD4 is also implicated in regulating the formation and function of neuromuscular junctions (NMJ) (Liu et al. 2009). In a skeletal muscle specific NEDD4 knockout mouse, subjected to denervation induced skeletal muscle atrophy, heavier weights and larger fiber cross sectional area demonstrated a role for NEDD4 in the development of this atrophy (Nagpal et al. 2012). The mechanism for this is not known, but recent studies have identified NEDD4 targets including PDLIM7 (PDZ and LIM domain 7) that may contribute to the observed phenotype (D’Cruz et al. 2016). NEDD4 has also been shown to control skeletal muscle progenitor fate by regulating Pax7, leading to its degradation by the proteasome and promoting muscle differentiation (Bustos et al. 2015).
Role of NEDD4 in PTEN Regulation and Cancer
NEDD4 is frequently overexpressed in tumors and cancer cell lines (Zou et al. 2015). The tumor suppressor phosphatase PTEN has been reported to be a target of NEDD4-mediated ubiquitination and proteasomal degradation (Wang et al. 2007). Other findings suggest that NEDD4 modulates PTEN function both positively and negatively, depending on the level and localization of NEDD4. Importantly, PTEN levels and ubiquitination does not appear affected in NEDD4−/− mice, suggesting that this may be specific to certain conditions such as oncogenic stress (Cao et al. 2008). NEDD4 has also been associated with cancer independently of PTEN. For example, it has been shown to stabilize Mdm2, a ubiquitin ligase that exerts oncogenic activity primarily by suppressing p53, another prominent tumor suppressor (Zou et al. 2015). Furthermore, the elevated levels of NEDD4 may provide an additional mechanism to drive tumorigenesis in endometrial tumors, through regulation of PI3K/AKT signaling (Zhang et al. 2015). Conversely, NEDD4 ubiquitinates the oncoproteins N-Myc and c-Myc to target them for degradation (Liu et al. 2013). This effect is mediated by the direct binding of the histone deacetylase SIRT2 to NEDD4 to repress NEDD4 gene expression, thereby enhancing N-Myc and c-Myc expression. Hence, in this context decreased levels of NEDD4 contribute to cancer.
NEDD4 in the Immune Response
NEDD4 has been implicated in the regulation of T cell function. Due to the embryonic lethality of NEDD4−/− mice, in order to study this, fetal liver chimeras have been generated where only cells of hematopoetic origin are deficient in NEDD4 expression. T cells generated from these NEDD4−/− fetal liver chimeras developed normally, but proliferated less and produced less IL2, demonstrating that NEDD4 promotes the conversion of naïve to activated T cells (Yang et al. 2008). Mechanistically, NEDD4 has been shown to ubiquitinate Cbl-b (another E3 ubiquitin ligase), which plays a critical role in T cell activation (Guo et al. 2012). In the NEDD4 lacking T cells, higher levels of Cbl-b inhibited T cell activation by impeding the association of NEDD4 with PTEN, thereby suppressing PTEN activation. In addition, NEDD4−/− T cells were unable to provide support for B-cells to undergo immunoglobulin class switching (Yang et al. 2008).
Endocytosis and Viral Budding
Multiple proteins containing a ubiquitin interacting motif (UIM), such as γ2-adaptin, have been shown to interact with NEDD4, resulting in the targeting of proteins for ubiquitination to induce endocytosis (Boase and Kumar 2015). In addition, lysosomal proteins important for golgi to lysosome sorting including LAPTM5 (lysosomal associated protein transmembrane 5) have also been found to interact with NEDD4. Given its link to endocytosis, it is not surprising that many viruses also contain PY motifs within their matrix proteins to facilitate interaction with NEDD4 and viral budding. Some examples include Human Immunodeficiency Virus (HIV), Influenza, Ebola, and the Epstein-Bar virus (Boase and Kumar 2015; Chesarino et al. 2015). In addition, components of the ESCRT machinery which are required for viral budding have also been shown to interact with NEDD4 (Boase and Kumar 2015).
Regulation of NEDD4
NEDD4 is regulated in a number of ways, including by accessory proteins, post–translational modifications (such as phosphorylation), oxidative stress, and auto-ubiquitination (Boase and Kumar 2015). Ndfip1 and Ndfip2 are adaptor proteins that contain three PY motifs which interact with NEDD4 to facilitate its binding to substrate proteins that do not contain their own PY motifs (Foot et al. 2011). Post–translationally, NEDD4 can be phosphorylated by multiple proteins, including casein kinase 1δ (which then allows its regulation by the SCFβ-TRCP complex), and by c-Src to enhance its catalytic activity (Boase and Kumar 2015). Upregulation of NEDD4 has been reported by the Ras signaling pathway, and by the transcription factor FOXM1B (Forkhead box protein M1B) in response to oxidative stress (Boase and Kumar 2015).
Auto-ubiquitination also plays a major role in the regulation of NEDD4. In the absence of calcium, the enzymatic activity of NEDD4 is suppressed by its C2 domain binding to the HECT domain (Wang et al. 2007). Many factors have been shown to disrupt this autoinhibition, allowing NEDD4 to conduct its ubiquitin ligase activity. These include the presence of calcium, phosphorylation by c-Src, or when the adaptors proteins Ndfip1 and Ndfip2 bind the WW domains of NEDD4. Additional NEDD4 binding proteins such as p34 and Cbl-b also inhibit this autoubiquitination (Boase and Kumar 2015). Negative regulation of NEDD4 has been reported by the interferon inducible ISG15 and heclin (HECT ligase inhibitor) by preventing ubiquitin transfer or formation of thioester bonds. Other proteins shown to inhibit NEDD4 include the proto-oncogene ΔNp63α and PTEN (Boase and Kumar 2015).
NEDD4 is a member of the HECT E3 ubiquitin ligase family. It is highly conserved during evolution and is likely to be the most ancient member of the NEDD4 family. In vivo studies in mouse reveal the importance of this gene in the regulation of growth signaling and the immune response as well as development and function of the nervous system, muscle, and heart. In addition, there is much evidence implicating NEDD4 as a critical regulator of components and pathways leading to tumor initiation and progression. Given the pathological conditions involving misregulation of this protein, or its substrates, the opportunities for therapeutic interventions utilizing NEDD4 are extensive.