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Nedd4 and Nedd4-2: Ubiquitin ligases at work in the neuron
The International Journal of Biochemistry & Cell Biology 45 (2013) 706–710
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The International Journal of Biochemistry & Cell Biology journal homepage: www.elsevier.com/locate/biocel
Molecules in focus
Nedd4 and Nedd4-2: Ubiquitin ligases at work in the neuron Prudence Donovan a,∗ , Philip Poronnik b,c a
Swiss Institute for Experimental Cancer Research (ISREC), School of Life Sciences, Ecole Polytechnique Fédérale de Lausanne (EPFL), CH 1015 Lausanne, Switzerland Health Innovations Research Institute (HiRi), School of Medical Sciences, RMIT University, PO Box 71, Bundoora, Victoria, Australia c School of Medical Sciences, The University of Sydney, Sydney, New South Wales, Australia b
a r t i c l e
i n f o
Article history: Received 30 September 2012 Received in revised form 4 December 2012 Accepted 7 December 2012 Available online 20 December 2012 Keywords: Nedd4 Nedd4-2 Neurons Ubiquitination Trafficking
a b s t r a c t Ubiquitination of proteins by the Nedd4 family of ubiquitin ligases is a significant mechanism in protein trafficking and degradation and provides for tight spatiotemporal regulation. Ubiquitination is gaining increasing recognition as a central mechanism underpinning the regulation of neuronal development and homeostasis in the brain. This review will focus on the Nedd4 and Nedd4-2 E3 ubiquitin ligases that are implicated in an increasing number of neuronal protein–protein interactions. Understanding of the contribution of Nedd4 and Nedd4-2 in regulating key functions in the brain is shedding new light on the ubiquitination signal not only in orchestrating degradation events but also in protein trafficking. Furthermore, the description of several novel Nedd4/4-2 targets in neurons is changing the way we conceptualize how neurons maintain normal function and how this is altered in disease. © 2013 Elsevier Ltd. All rights reserved.
1. Introduction Nedd4 (Nedd4-1) and Nedd4-2 (Nedd4L, Nedd4.2) are structurally related proteins belonging to the family of HECT (Homologous to the E6-AP Carboxyl Terminus) E3 ubiquitin ligases. These proteins were initially identified in a screen for developmentally down regulated genes in the early embryonic mouse central nervous system (Kumar et al., 1992). Since their discovery, there has been significant interest in how Nedd4 and Nedd4-2 regulate neuronal function and plasticity in the developing and adult brain. Catalysis of ubiquitin to a substrate protein is dependent on a series of events between 3 enzymes, an ubiquitin activating enzyme (E1), multiple ubiquitin conjugating enzymes (E2), and several hundreds of ubiquitin-protein ligase (E3) (Yang and Kumar, 2010). The E3 ligases determine the substrate specificity of the reactions. Substrate recruitment by Nedd4 proteins is mediated via WW domains that recognize L/PPXY motifs in target proteins. The fate of the target protein is determined by the number of and manner by which ubiquitin moieties are conjugated. A protein can be monoubiquitinated, multi-monoubiquitinated, or polyubiquitinated. As a consequence of the various types of ubiquitination, Nedd4/4-2 can mediate degradation in one of three cellular compartments; the endoplasmic reticulum, lysosomes and classically, in the proteasome. It is important to note that not only can
∗ Corresponding author. Tel.: +41 21 69 30718. E-mail addresses: prudence.donovan@epfl.ch, [email protected] (P. Donovan). 1357-2725/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biocel.2012.12.006
ubiquitination target a protein for degradation, but it also acts as an important trafficking signal by causing a protein to be recycled through the endocytic machinery. Thus, Nedd4 and Nedd4-2 typically play a key role in maintaining levels of the target protein at the plasma membrane by balancing the steady state insertion and retrieval of proteins critical to the development and maintenance of neuronal function in the brain. 2. Structure The Nedd4 (Neuronally Expressed Developmentally Downregulated 4) family of HECT ubiquitin ligases comprises nine distinct members encoded by unique genes in humans, defined by a shared modular structure (Ingham et al., 2004). Human Nedd4 proteins include a Calcium-phospholipid-binding domain (C2), tryptophan (WW) modules and a catalytic HECT domain (Fig. 1). The protein structure is highly conserved across eukaryotes from yeast to mammals with the most striking divergence being an increase in the number of tryptophan substrate binding modules (Ingham et al., 2004). The largest enrichment of these modules is observed in the human proteins, Nedd4 and Nedd4-2 (Harvey et al., 2001). In vitro studies suggest that WW3 and WW4 of both proteins are the most utilized for substrate recruitment (Itani et al., 2003). Expression of these WW domains is maintained in all isoforms of NEDD4 and NEDD4-2. Two isoforms of the human NEDD4 genes are expressed. Isoform-1 of NEDD4 differs in the 5 untranslated region and contains alternate exons in the 5 coding region, which gives rise to a shorter gene and protein than isoform-2 (Kamynina et al., 2001). There are seven known isoforms of Nedd4-2 and notably, the
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Fig. 1. Schematic representation of the structure of Nedd4 and Nedd4-2 proteins. The Nedd4 family is defined by a modular structure, Nedd4 proteins are characterized by a Ca2+ /lipid-binding (C2) domain at the N-terminus, multiple WW domains located in the middle part of the protein, and a ubiquitin-protein ligase domain at the C-terminus, that is necessary for ligase function. Nedd4 proteins show a high degree of homology and the presence of WW domains and the HECT domain is evolutionarily conserved. Key divergences can be seen in mouse Nedd4-2 and in some variants of human Nedd4-2 that lack the C2 domain. While some splice variants of mouse Nedd4-2 lack the C2-domain, the predominatnly expressed forms of mouse Nedd4-2 in neurons contain a C2 domain. Higher-order organisms have an expansion in the number of WW domains. These domains regulate recruitment of substrates to be ubiquitinated and may represent an expansion in the flexibility and functionality of regulation by Nedd4/4-2.
second isoform, Isoform-2, lacks the C2 phospholipid-binding domain (Itani et al., 2003). 3. Expression and post-translational modification of Nedd4 proteins Expression of Nedd4 and Nedd4-2 is regulated both temporally and spatially. Nedd4 expression is detectable in the developing central nervous system of mouse embryos and peaks during neurogenesis and is decreased into adulthood. A subsequent study indicated that Nedd4-2 expression was greatest at E10 (Kumar et al., 1997). Nedd4 is expressed ubiquitously whereas Nedd4-2 expression is more restricted, with prominent expression in the kidney, brain and liver (Anan et al., 1998; Kumar et al., 1997). Despite the ubiquitous expression of Nedd4 and its close homology with Nedd4-2, it is becoming increasingly clear that the two proteins have different physiological substrates. For example, Nedd4-2 is the major physiological regulator of ENaC in the kidney (Fotia et al., 2004; Harvey et al., 2001) although Nedd4 can also interact with ENaC in vitro. More recent studies in knockout animals provide further evidence for a lack of functional redundancy between the two proteins. The subcellular localization of both Nedd4 and Nedd4-2 is diffusely cytoplasmic (Anan et al., 1998) and localization of Nedd4 proteins to the cytoplasm is dependent on the presence of the N-terminal portion of the protein. In cells overexpressing an N-terminal mutant of Nedd4, the distribution of the protein is primarily peri-nuclear (Anan et al., 1998). Interestingly, both Nedd4 and Nedd4-2 have also been detected in exosomes (Putz et al., 2008), this finding expands the role for Nedd4 proteins in exosome biogenesis and raises new questions regarding the functions Nedd4 and Nedd4-2 perform when excreted from the cells. The fidelity and activation of Nedd4-2 activity is also regulated at the post-translational level. Nedd4-2 plays a crucial role in sodium homeostasis by regulating the levels of the epithelial
sodium channel, ENaC. The binding of Nedd4-2 to ENaC is regulated by serum glucocorticoid kinase (SGK1) phosphorylation of Nedd4-2 (Faresse et al., 2012). SGK1 binds to Nedd4-2 and phosphorylates three residues (Ser-221, Thr-246, and Ser-327) to alter Nedd4-2 binding to ENaC (Debonneville et al., 2001). A novel selfinhibitory regulatory mechanism for Nedd4-2 was also identified in studies assessing Nedd4-2 protein stability. Pulse-chase experiments demonstrated that mutation of the HECT PY-motif decreased the stability of Nedd4-2, suggesting that the PY motif itself can stabilize Nedd4-2 levels. Furthermore, Bruce et al. found that Nedd4-2 itself was ubiquitinated following substrate interaction and subsequently degraded (Bruce et al., 2008). To date, mechanisms that regulate Nedd4 activation are unknown. However, it is clear that these tight regulatory mechanisms are critical for maintaining Nedd4-2 at the levels and location appropriate to maintain homeostatic mechanisms. 4. Biological function The first physiological role for Nedd4 and Nedd4-2-mediated ubiquitination was reported in the epithelial cells of the kidney tubules. It was shown that Nedd4 and Nedd4-2 could regulate membrane levels of ENaC (Yang and Kumar, 2010) through the binding of WW domains to the PY motifs in the cytoplasmic tails of the ENaC subunits. Of interest, the number of WW domains is enriched in humans, rats and mice compared to Drosophila and recently it was demonstrated that only WW3 and WW4 of Nedd4-2 are involved in the regulation of ENaC and substrate recruitment (Fotia et al., 2003). Subsequent to studies describing the regulation of ENaC, a growing list of proteins regulated by Nedd4 and Nedd4-2 has emerged (for review see (Persaud et al., 2009). The diversity of proteins targeted by Nedd4 and Nedd4-2 reveals a broad range of potential functions regulated by these ligases (Fig. 2). The absolute requirement for regulation of cellular function by Nedd4 proteins is demonstrated in studies of Nedd4 and
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Fig. 2. Schematic representation of Nedd4 and Nedd4-2 function through the ubiquitin axis. (A) The Nedd4 and Nedd4-2 ubiquitin axis. Studies of the regulation of ENaC demonstrated that Nedd4-2 WW domains binds to the PY motifs of ENaC, leading to channel ubiquitination and endocytosis. Since the initial characterization of Nedd4/4-2 function, it is now understood that Nedd4/4-2 mediated ubiquitination can target a protein to all proteolytic organelles and all destinations mediated by the ubiquitin axis. The ubiquitin axis describes organelles essential to the trafficking of ubiquitin tagged proteins to the relevant proteolytic compartment and includes, endosomes, lysosomes, endoplasmic reticulum associated degradation (ERAD) and classical degradation in the proteasome. Examples of Nedd4 and Nedd4-2 substrates and their cellular fate subsequent to ubiquitination are indicated next to each compartment of the ubiquitin axis. Some substrates such as Cav 1.2␣1 can be trafficked and degraded in multiple ubiquitin compartments. In these cases, this involves recognition, ubiquitination and dislocation of the substrate in the endoplasmic reticulum followed by degradation in the proteasome. (B) Ubiquitin in chains. Attachment of ubiquitin is catalyzed by Nedd4 and Nedd4-2 and occurs at lysine (K) residues on target proteins. Ubiquitination can occur on both transmembrane and intracellular proteins. Generally, ubiquitination of an intracellular protein signals protein degradation in proteasomes. The cellular fate of an ubiquitinated protein is determined by the number of ubiquitin moieties attached the substrate and at what lysine number within ubiquitin the chain is extended upon. The schematic above depicts the several species of ubiquitin that can be attached to target protein. The number of ubiquitin molecules attached to the substrate and how in the case of multiple ubiquitination these are linked directs the fate of the protein (left to right). Mono-ubiquitination and multiple mono-ubiquitination of a transmembrane protein generally result in internalization and recycling. Whereas, poly-ubiquitination and ubiquitin chain extension at K63 on a target protein initiates trafficking to the lysosome. For degradation in the proteasome, K48-linked ubiquitin chains are necessary.
Nedd4-2 transgenic mice. Both the Nedd4 and Nedd4-2 knockout mice are peri-natal lethal (Boase et al., 2011; Cao et al., 2008; Shi et al., 2008). Nedd4 knockout mice show significant developmental retardation and are much smaller, due to a defect associated with impaired insulin-like growth factor-1 signaling. In contrast, Nedd42 mice show no difference in size and die of an ENaC related lung phenotype. Given the number of potential target proteins associated with Nedd4 and Nedd4-2 and their essential roles, the great challenge in this area of research is ascribing physiological roles for the observed interactions. Importantly, Nedd4 proteins also modulate immune system function (Guo et al., 2012), viral budding, protein sorting, and cell division (Ingham et al., 2004). Recent studies have shown that Nedd4 and Nedd4-2 are critical to all stages of neuronal development such as neuronal cell fate determination, neurite outgrowth, axon guidance and neuronal cell survival. In zebrafish, Nedd4 is involved in the dorsoventral patterning of the neural ectoderm (Bakkers et al., 2005). The role of Nedd4 in determining cell fate is also conserved in Drosophila, and is achieved by the regulation of the transmembrane receptor, Notch, of which Nedd4 is a negative regulator (Dalton et al., 2011; Sakata et al., 2004). If Notch is expressed on cells early in development, the cell is prevented from differentiating into any cell type of the neuronal cell lineage. Recent studies have found that adaptor proteins can also mediate the effect of Nedd4 on Notch, Nedd4 binding protein, Ndfip, has been reported to anchor the proteolytic complex to the lumen of the lysosome, enhancing Nedd4 downregulation of Notch (Dalton et al., 2011).
4.1. Neurite outgrowth Common to all mechanisms of neurite outgrowth is the need for timely regulation of pathways that all converge onto the cytoskeleton. Two recent studies have explained a role for Nedd4
in the growth and branching of axons and dendrites. Analyses of glutamatergic neurons from conditional Nedd4 knockout mice show a reduction in the complexity of branching (Kawabe and Brose, 2010). Using a biochemical approach, Kawabe and Brose (2010) identified an important protein–protein interaction between Nedd4, Rap2A and Tink, resulting in the ubiquitination of Rap2A. Similarly, Drinjakovic et al. (2010) showed that downregulation of PTEN by Nedd4-mediated-ubiquitination controls branching of retinal ganglion cell axons. The outcome of Nedd4 regulation on axon branching appears to be influenced by the environment of the target neuron. For example, Liu et al. (2009) demonstrated that neuromuscular junctions of Nedd4 knockout mice had an increased number of pre-synaptic nerve terminal branches, indicating that Nedd4 is an important regulator of dendritic and axonal branching and that the effect of Nedd4 on branching complexity may differ between regions of the nervous system.
4.2. Neuronal survival A role for Nedd4-2 in development and cell survival was recently observed by the regulation of cell surface levels of the nerve growth factor (NGF) receptor, neurotrophic tyrosine kinase receptor type 1 (TrkA), in dorsal root ganglia (Arévalo et al., 2006). More recently, Georgieva et al. (2011) showed that Nedd4-2 mediates multiple mono-ubiquitination of TrkA, and that this leads to receptor internalization and lysosomal trafficking of the receptor, and not receptor recycling or degradation in the proteasome. The role of Nedd4-2 in the trafficking of TrkA is further validated by the observation that, in Nedd4-2-depleted sensory neurons, TrkA is recycled back to the membrane in early endosomes, instead of trafficking into late endosomal and lysosomal compartments (Yu et al., 2011). The ability of Nedd4-2 to regulate receptor tyrosine kinase
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receptor trafficking is also demonstrated in studies of the epidermal growth factor and fibroblast growth factor receptor, where Nedd42-mediated ubiquitination, in contrast to the TrkA receptor, causes endosomal recycling (Persaud et al., 2009). These studies highlight that regulation by Nedd4 and Nedd4-2 is tissue specific and provides insights into the role of Nedd4 proteins in regulating protein traffic and endosomal cargo. 4.3. Regulation of neuronal ion channels by Nedd4 proteins The epithelial paradigm of ENaC regulation has also been shown to be of relevance in the brain. ENaC is also present in the brain and increased expression of ENaC has been detected in the brain of Nedd4-2 null mice, resulting in an increase of Na+ levels in the cerebrospinal fluid and highlighting a possible role in salt-induced hypertension (Van Huysse et al., 2012). In addition, ion channels responsible for excitability have also been shown to be targets for Nedd4 and Nedd4-2. These included the voltage-gated Na+ channels (Rougier et al., 2005), KCNQ channels (Schuetz et al., 2008; Ekberg et al., 2007) and more recently, voltage-dependent calcium channels (Rougier et al., 2011). Unlike the voltage-gated Na+ channels that appear to be regulated primarily by Nedd4-2, Rougier et al. showed that only Nedd4 was able to modulate whole cell Cav currents, and down-regulate surface expression of Cav 1.2␣1. Ubiquitination by Nedd4 resulted in the channels trafficking to the ER-Golgi network endosomes (Rougier et al., 2011). There is very strong evidence to suggest complex roles for Nedd4 and Nedd4-2 in the regulation of the fundamental electrical properties of neurons and elucidating the physiological relevance of these interactions is of great importance. 4.4. Nedd4 proteins: a role in the regulation of neurotransmitter signaling Nedd4-2 regulates the abundance of the glutamate transporters, EAAT, EAAT2 and EAAT3/4 (Sopjani et al., 2010) and is therefore a key regulator in maintaining the appropriate balance of glutamate at the synapse. In addition, protein kinase C (PKC) promotes the phosphorylation of Nedd4-2, increasing its association with EAAT2, resulting in ubiquitination of the transporter and its endocytosis (Garcia-Tardon et al., 2012). PKC activation is also necessary for the regulation of dopamine transporters by Nedd42 (Vina-Vilaseca and Sorkin, 2010). These studies highlight how finely tuned and responsive regulation of Nedd4-2 mediated ubiquitin is. This specificity and impact of the neuronal environment in dictating the Nedd4/4-2 response is elegantly realized in the ability of Nedd4 to not only regulate glutamate but also target of glutamate, Amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA) receptors. In hippocampal neurons, ubiquitination of GluA1-containing AMPA receptors by Nedd4 results in receptor internalization and trafficking to the lysosome (Lin et al., 2011). Nedd4-mediated ubiquitination of AMPA receptors is also agonist dependent, and ubiquitination of NMDA type receptors is not affected by knockdown of Nedd4 (Schwarz et al., 2010). In addition, it is also suggested that Nedd4-2 may regulate choline uptake by neurons through regulation of the choline transporter, CHT-1 (Yamada et al., 2012). These data show that Nedd4-2 is a key regulator of excitatory and inhibitory neurotransmission. The mechanisms that drive activation of Nedd4 and Nedd4-2 at the appropriate synapse are less well understood and represent an important field for further study. 5. Possible medical and industrial applications It appears that Nedd4-family proteins potentially regulate almost every facet of neuronal function, from development through
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The International Journal of Biochemistry & Cell Biology journal homepage: www.elsevier.com/locate/biocel
Molecules in focus
Nedd4 and Nedd4-2: Ubiquitin ligases at work in the neuron Prudence Donovan a,∗ , Philip Poronnik b,c a
Swiss Institute for Experimental Cancer Research (ISREC), School of Life Sciences, Ecole Polytechnique Fédérale de Lausanne (EPFL), CH 1015 Lausanne, Switzerland Health Innovations Research Institute (HiRi), School of Medical Sciences, RMIT University, PO Box 71, Bundoora, Victoria, Australia c School of Medical Sciences, The University of Sydney, Sydney, New South Wales, Australia b
a r t i c l e
i n f o
Article history: Received 30 September 2012 Received in revised form 4 December 2012 Accepted 7 December 2012 Available online 20 December 2012 Keywords: Nedd4 Nedd4-2 Neurons Ubiquitination Trafficking
a b s t r a c t Ubiquitination of proteins by the Nedd4 family of ubiquitin ligases is a significant mechanism in protein trafficking and degradation and provides for tight spatiotemporal regulation. Ubiquitination is gaining increasing recognition as a central mechanism underpinning the regulation of neuronal development and homeostasis in the brain. This review will focus on the Nedd4 and Nedd4-2 E3 ubiquitin ligases that are implicated in an increasing number of neuronal protein–protein interactions. Understanding of the contribution of Nedd4 and Nedd4-2 in regulating key functions in the brain is shedding new light on the ubiquitination signal not only in orchestrating degradation events but also in protein trafficking. Furthermore, the description of several novel Nedd4/4-2 targets in neurons is changing the way we conceptualize how neurons maintain normal function and how this is altered in disease. © 2013 Elsevier Ltd. All rights reserved.
1. Introduction Nedd4 (Nedd4-1) and Nedd4-2 (Nedd4L, Nedd4.2) are structurally related proteins belonging to the family of HECT (Homologous to the E6-AP Carboxyl Terminus) E3 ubiquitin ligases. These proteins were initially identified in a screen for developmentally down regulated genes in the early embryonic mouse central nervous system (Kumar et al., 1992). Since their discovery, there has been significant interest in how Nedd4 and Nedd4-2 regulate neuronal function and plasticity in the developing and adult brain. Catalysis of ubiquitin to a substrate protein is dependent on a series of events between 3 enzymes, an ubiquitin activating enzyme (E1), multiple ubiquitin conjugating enzymes (E2), and several hundreds of ubiquitin-protein ligase (E3) (Yang and Kumar, 2010). The E3 ligases determine the substrate specificity of the reactions. Substrate recruitment by Nedd4 proteins is mediated via WW domains that recognize L/PPXY motifs in target proteins. The fate of the target protein is determined by the number of and manner by which ubiquitin moieties are conjugated. A protein can be monoubiquitinated, multi-monoubiquitinated, or polyubiquitinated. As a consequence of the various types of ubiquitination, Nedd4/4-2 can mediate degradation in one of three cellular compartments; the endoplasmic reticulum, lysosomes and classically, in the proteasome. It is important to note that not only can
∗ Corresponding author. Tel.: +41 21 69 30718. E-mail addresses: prudence.donovan@epfl.ch, [email protected] (P. Donovan). 1357-2725/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biocel.2012.12.006
ubiquitination target a protein for degradation, but it also acts as an important trafficking signal by causing a protein to be recycled through the endocytic machinery. Thus, Nedd4 and Nedd4-2 typically play a key role in maintaining levels of the target protein at the plasma membrane by balancing the steady state insertion and retrieval of proteins critical to the development and maintenance of neuronal function in the brain. 2. Structure The Nedd4 (Neuronally Expressed Developmentally Downregulated 4) family of HECT ubiquitin ligases comprises nine distinct members encoded by unique genes in humans, defined by a shared modular structure (Ingham et al., 2004). Human Nedd4 proteins include a Calcium-phospholipid-binding domain (C2), tryptophan (WW) modules and a catalytic HECT domain (Fig. 1). The protein structure is highly conserved across eukaryotes from yeast to mammals with the most striking divergence being an increase in the number of tryptophan substrate binding modules (Ingham et al., 2004). The largest enrichment of these modules is observed in the human proteins, Nedd4 and Nedd4-2 (Harvey et al., 2001). In vitro studies suggest that WW3 and WW4 of both proteins are the most utilized for substrate recruitment (Itani et al., 2003). Expression of these WW domains is maintained in all isoforms of NEDD4 and NEDD4-2. Two isoforms of the human NEDD4 genes are expressed. Isoform-1 of NEDD4 differs in the 5 untranslated region and contains alternate exons in the 5 coding region, which gives rise to a shorter gene and protein than isoform-2 (Kamynina et al., 2001). There are seven known isoforms of Nedd4-2 and notably, the
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Fig. 1. Schematic representation of the structure of Nedd4 and Nedd4-2 proteins. The Nedd4 family is defined by a modular structure, Nedd4 proteins are characterized by a Ca2+ /lipid-binding (C2) domain at the N-terminus, multiple WW domains located in the middle part of the protein, and a ubiquitin-protein ligase domain at the C-terminus, that is necessary for ligase function. Nedd4 proteins show a high degree of homology and the presence of WW domains and the HECT domain is evolutionarily conserved. Key divergences can be seen in mouse Nedd4-2 and in some variants of human Nedd4-2 that lack the C2 domain. While some splice variants of mouse Nedd4-2 lack the C2-domain, the predominatnly expressed forms of mouse Nedd4-2 in neurons contain a C2 domain. Higher-order organisms have an expansion in the number of WW domains. These domains regulate recruitment of substrates to be ubiquitinated and may represent an expansion in the flexibility and functionality of regulation by Nedd4/4-2.
second isoform, Isoform-2, lacks the C2 phospholipid-binding domain (Itani et al., 2003). 3. Expression and post-translational modification of Nedd4 proteins Expression of Nedd4 and Nedd4-2 is regulated both temporally and spatially. Nedd4 expression is detectable in the developing central nervous system of mouse embryos and peaks during neurogenesis and is decreased into adulthood. A subsequent study indicated that Nedd4-2 expression was greatest at E10 (Kumar et al., 1997). Nedd4 is expressed ubiquitously whereas Nedd4-2 expression is more restricted, with prominent expression in the kidney, brain and liver (Anan et al., 1998; Kumar et al., 1997). Despite the ubiquitous expression of Nedd4 and its close homology with Nedd4-2, it is becoming increasingly clear that the two proteins have different physiological substrates. For example, Nedd4-2 is the major physiological regulator of ENaC in the kidney (Fotia et al., 2004; Harvey et al., 2001) although Nedd4 can also interact with ENaC in vitro. More recent studies in knockout animals provide further evidence for a lack of functional redundancy between the two proteins. The subcellular localization of both Nedd4 and Nedd4-2 is diffusely cytoplasmic (Anan et al., 1998) and localization of Nedd4 proteins to the cytoplasm is dependent on the presence of the N-terminal portion of the protein. In cells overexpressing an N-terminal mutant of Nedd4, the distribution of the protein is primarily peri-nuclear (Anan et al., 1998). Interestingly, both Nedd4 and Nedd4-2 have also been detected in exosomes (Putz et al., 2008), this finding expands the role for Nedd4 proteins in exosome biogenesis and raises new questions regarding the functions Nedd4 and Nedd4-2 perform when excreted from the cells. The fidelity and activation of Nedd4-2 activity is also regulated at the post-translational level. Nedd4-2 plays a crucial role in sodium homeostasis by regulating the levels of the epithelial
sodium channel, ENaC. The binding of Nedd4-2 to ENaC is regulated by serum glucocorticoid kinase (SGK1) phosphorylation of Nedd4-2 (Faresse et al., 2012). SGK1 binds to Nedd4-2 and phosphorylates three residues (Ser-221, Thr-246, and Ser-327) to alter Nedd4-2 binding to ENaC (Debonneville et al., 2001). A novel selfinhibitory regulatory mechanism for Nedd4-2 was also identified in studies assessing Nedd4-2 protein stability. Pulse-chase experiments demonstrated that mutation of the HECT PY-motif decreased the stability of Nedd4-2, suggesting that the PY motif itself can stabilize Nedd4-2 levels. Furthermore, Bruce et al. found that Nedd4-2 itself was ubiquitinated following substrate interaction and subsequently degraded (Bruce et al., 2008). To date, mechanisms that regulate Nedd4 activation are unknown. However, it is clear that these tight regulatory mechanisms are critical for maintaining Nedd4-2 at the levels and location appropriate to maintain homeostatic mechanisms. 4. Biological function The first physiological role for Nedd4 and Nedd4-2-mediated ubiquitination was reported in the epithelial cells of the kidney tubules. It was shown that Nedd4 and Nedd4-2 could regulate membrane levels of ENaC (Yang and Kumar, 2010) through the binding of WW domains to the PY motifs in the cytoplasmic tails of the ENaC subunits. Of interest, the number of WW domains is enriched in humans, rats and mice compared to Drosophila and recently it was demonstrated that only WW3 and WW4 of Nedd4-2 are involved in the regulation of ENaC and substrate recruitment (Fotia et al., 2003). Subsequent to studies describing the regulation of ENaC, a growing list of proteins regulated by Nedd4 and Nedd4-2 has emerged (for review see (Persaud et al., 2009). The diversity of proteins targeted by Nedd4 and Nedd4-2 reveals a broad range of potential functions regulated by these ligases (Fig. 2). The absolute requirement for regulation of cellular function by Nedd4 proteins is demonstrated in studies of Nedd4 and
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Fig. 2. Schematic representation of Nedd4 and Nedd4-2 function through the ubiquitin axis. (A) The Nedd4 and Nedd4-2 ubiquitin axis. Studies of the regulation of ENaC demonstrated that Nedd4-2 WW domains binds to the PY motifs of ENaC, leading to channel ubiquitination and endocytosis. Since the initial characterization of Nedd4/4-2 function, it is now understood that Nedd4/4-2 mediated ubiquitination can target a protein to all proteolytic organelles and all destinations mediated by the ubiquitin axis. The ubiquitin axis describes organelles essential to the trafficking of ubiquitin tagged proteins to the relevant proteolytic compartment and includes, endosomes, lysosomes, endoplasmic reticulum associated degradation (ERAD) and classical degradation in the proteasome. Examples of Nedd4 and Nedd4-2 substrates and their cellular fate subsequent to ubiquitination are indicated next to each compartment of the ubiquitin axis. Some substrates such as Cav 1.2␣1 can be trafficked and degraded in multiple ubiquitin compartments. In these cases, this involves recognition, ubiquitination and dislocation of the substrate in the endoplasmic reticulum followed by degradation in the proteasome. (B) Ubiquitin in chains. Attachment of ubiquitin is catalyzed by Nedd4 and Nedd4-2 and occurs at lysine (K) residues on target proteins. Ubiquitination can occur on both transmembrane and intracellular proteins. Generally, ubiquitination of an intracellular protein signals protein degradation in proteasomes. The cellular fate of an ubiquitinated protein is determined by the number of ubiquitin moieties attached the substrate and at what lysine number within ubiquitin the chain is extended upon. The schematic above depicts the several species of ubiquitin that can be attached to target protein. The number of ubiquitin molecules attached to the substrate and how in the case of multiple ubiquitination these are linked directs the fate of the protein (left to right). Mono-ubiquitination and multiple mono-ubiquitination of a transmembrane protein generally result in internalization and recycling. Whereas, poly-ubiquitination and ubiquitin chain extension at K63 on a target protein initiates trafficking to the lysosome. For degradation in the proteasome, K48-linked ubiquitin chains are necessary.
Nedd4-2 transgenic mice. Both the Nedd4 and Nedd4-2 knockout mice are peri-natal lethal (Boase et al., 2011; Cao et al., 2008; Shi et al., 2008). Nedd4 knockout mice show significant developmental retardation and are much smaller, due to a defect associated with impaired insulin-like growth factor-1 signaling. In contrast, Nedd42 mice show no difference in size and die of an ENaC related lung phenotype. Given the number of potential target proteins associated with Nedd4 and Nedd4-2 and their essential roles, the great challenge in this area of research is ascribing physiological roles for the observed interactions. Importantly, Nedd4 proteins also modulate immune system function (Guo et al., 2012), viral budding, protein sorting, and cell division (Ingham et al., 2004). Recent studies have shown that Nedd4 and Nedd4-2 are critical to all stages of neuronal development such as neuronal cell fate determination, neurite outgrowth, axon guidance and neuronal cell survival. In zebrafish, Nedd4 is involved in the dorsoventral patterning of the neural ectoderm (Bakkers et al., 2005). The role of Nedd4 in determining cell fate is also conserved in Drosophila, and is achieved by the regulation of the transmembrane receptor, Notch, of which Nedd4 is a negative regulator (Dalton et al., 2011; Sakata et al., 2004). If Notch is expressed on cells early in development, the cell is prevented from differentiating into any cell type of the neuronal cell lineage. Recent studies have found that adaptor proteins can also mediate the effect of Nedd4 on Notch, Nedd4 binding protein, Ndfip, has been reported to anchor the proteolytic complex to the lumen of the lysosome, enhancing Nedd4 downregulation of Notch (Dalton et al., 2011).
4.1. Neurite outgrowth Common to all mechanisms of neurite outgrowth is the need for timely regulation of pathways that all converge onto the cytoskeleton. Two recent studies have explained a role for Nedd4
in the growth and branching of axons and dendrites. Analyses of glutamatergic neurons from conditional Nedd4 knockout mice show a reduction in the complexity of branching (Kawabe and Brose, 2010). Using a biochemical approach, Kawabe and Brose (2010) identified an important protein–protein interaction between Nedd4, Rap2A and Tink, resulting in the ubiquitination of Rap2A. Similarly, Drinjakovic et al. (2010) showed that downregulation of PTEN by Nedd4-mediated-ubiquitination controls branching of retinal ganglion cell axons. The outcome of Nedd4 regulation on axon branching appears to be influenced by the environment of the target neuron. For example, Liu et al. (2009) demonstrated that neuromuscular junctions of Nedd4 knockout mice had an increased number of pre-synaptic nerve terminal branches, indicating that Nedd4 is an important regulator of dendritic and axonal branching and that the effect of Nedd4 on branching complexity may differ between regions of the nervous system.
4.2. Neuronal survival A role for Nedd4-2 in development and cell survival was recently observed by the regulation of cell surface levels of the nerve growth factor (NGF) receptor, neurotrophic tyrosine kinase receptor type 1 (TrkA), in dorsal root ganglia (Arévalo et al., 2006). More recently, Georgieva et al. (2011) showed that Nedd4-2 mediates multiple mono-ubiquitination of TrkA, and that this leads to receptor internalization and lysosomal trafficking of the receptor, and not receptor recycling or degradation in the proteasome. The role of Nedd4-2 in the trafficking of TrkA is further validated by the observation that, in Nedd4-2-depleted sensory neurons, TrkA is recycled back to the membrane in early endosomes, instead of trafficking into late endosomal and lysosomal compartments (Yu et al., 2011). The ability of Nedd4-2 to regulate receptor tyrosine kinase
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receptor trafficking is also demonstrated in studies of the epidermal growth factor and fibroblast growth factor receptor, where Nedd42-mediated ubiquitination, in contrast to the TrkA receptor, causes endosomal recycling (Persaud et al., 2009). These studies highlight that regulation by Nedd4 and Nedd4-2 is tissue specific and provides insights into the role of Nedd4 proteins in regulating protein traffic and endosomal cargo. 4.3. Regulation of neuronal ion channels by Nedd4 proteins The epithelial paradigm of ENaC regulation has also been shown to be of relevance in the brain. ENaC is also present in the brain and increased expression of ENaC has been detected in the brain of Nedd4-2 null mice, resulting in an increase of Na+ levels in the cerebrospinal fluid and highlighting a possible role in salt-induced hypertension (Van Huysse et al., 2012). In addition, ion channels responsible for excitability have also been shown to be targets for Nedd4 and Nedd4-2. These included the voltage-gated Na+ channels (Rougier et al., 2005), KCNQ channels (Schuetz et al., 2008; Ekberg et al., 2007) and more recently, voltage-dependent calcium channels (Rougier et al., 2011). Unlike the voltage-gated Na+ channels that appear to be regulated primarily by Nedd4-2, Rougier et al. showed that only Nedd4 was able to modulate whole cell Cav currents, and down-regulate surface expression of Cav 1.2␣1. Ubiquitination by Nedd4 resulted in the channels trafficking to the ER-Golgi network endosomes (Rougier et al., 2011). There is very strong evidence to suggest complex roles for Nedd4 and Nedd4-2 in the regulation of the fundamental electrical properties of neurons and elucidating the physiological relevance of these interactions is of great importance. 4.4. Nedd4 proteins: a role in the regulation of neurotransmitter signaling Nedd4-2 regulates the abundance of the glutamate transporters, EAAT, EAAT2 and EAAT3/4 (Sopjani et al., 2010) and is therefore a key regulator in maintaining the appropriate balance of glutamate at the synapse. In addition, protein kinase C (PKC) promotes the phosphorylation of Nedd4-2, increasing its association with EAAT2, resulting in ubiquitination of the transporter and its endocytosis (Garcia-Tardon et al., 2012). PKC activation is also necessary for the regulation of dopamine transporters by Nedd42 (Vina-Vilaseca and Sorkin, 2010). These studies highlight how finely tuned and responsive regulation of Nedd4-2 mediated ubiquitin is. This specificity and impact of the neuronal environment in dictating the Nedd4/4-2 response is elegantly realized in the ability of Nedd4 to not only regulate glutamate but also target of glutamate, Amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA) receptors. In hippocampal neurons, ubiquitination of GluA1-containing AMPA receptors by Nedd4 results in receptor internalization and trafficking to the lysosome (Lin et al., 2011). Nedd4-mediated ubiquitination of AMPA receptors is also agonist dependent, and ubiquitination of NMDA type receptors is not affected by knockdown of Nedd4 (Schwarz et al., 2010). In addition, it is also suggested that Nedd4-2 may regulate choline uptake by neurons through regulation of the choline transporter, CHT-1 (Yamada et al., 2012). These data show that Nedd4-2 is a key regulator of excitatory and inhibitory neurotransmission. The mechanisms that drive activation of Nedd4 and Nedd4-2 at the appropriate synapse are less well understood and represent an important field for further study. 5. Possible medical and industrial applications It appears that Nedd4-family proteins potentially regulate almost every facet of neuronal function, from development through
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