The Nedd4 Family of E3 Ubiquitin Ligases: Functional Diversity Within a Common Modular Architecture

The Nedd4 family of E3 ubiquitin ligases: functional diversity within a common modular architecture

Robert J Ingham*,1, Gerald Gish1 and Tony Pawson1,2

1Samuel Lunenfeld Research Institute, Mt. Sinai Hospital, Toronto, Ontario, Canada M5G 1X5; 2Department of Molecular and Medical Genetics, University of Toronto, Toronto, Ontario, Canada M5S 1A8

Neuronal precursor cell-expressed developmentally down- Nedd4 was originally identified in 1992, in a screen for regulated 4 (Nedd4)is the prototypical protein in a family genes developmentally downregulated in the early of E3 ubiquitin ligases that have a common domain embryonic mouse central nervous system (Kumar et al., architecture. They are comprised of a catalytic C-terminal 1992), and resembles other regulatory proteins in HECT domain and N-terminal C2 domain and WW containing multiple, distinct modular domains (Pawson domains responsible for cellular localization and substrate and Nash, 2003). Subsequently, related proteins with recognition. These proteins are found throughout eukar- similar structures have been characterized. All contain a yotes and regulate diverse biological processes through the catalytic HECT domain at the C-terminus, and an N- targeted degradation of proteins that generally have a terminal region involved in substrate recognition, that PPxY motif for WW domain recognition, and are found includes a C2 domain and a series of WW domains. in the nucleus and at the plasma membrane. Whereas the Although the common modular architecture of the yeast Saccharomyces cerevisiae uses a single protein, Nedd4 family might suggest a redundant functional role Rsp5p, to carry out these functions, evolution has provided for these proteins, recent work has begun to define higher eukaryotes with several related Nedd4 proteins that specific cellular activities for individual members, and to appear to have specialized roles. In this review we discuss suggest mechanisms that underlie this specificity. This how knowledge of individual domain function has provided review will provide an overview of the functional insight into the physiological roles of the Nedd4 proteins properties of the modular domains comprising the and describe recent results that suggest discrete functions Nedd4 family, and highlight physiological processes for individual family members. regulated by these proteins. Oncogene (2004) 23, 1972–1984. doi:10.1038/sj.onc.1207436 Members of the Nedd4 protein family Keywords: ubiquitination; Nedd4; E3 ubiquitin ligase; HECT; WW; C2 Nedd4-like proteins are found in eukaryotes from yeast to mammals and are defined by a similar domain organization (Figure 1). In Saccharomyces cerevisiae,a single member, Rsp5p, regulates a number of processes including mitochondrial inheritance (Fisk and Yaffe, 1999), internalization of cell surface receptors (reviewed Introduction in Rotin et al., 2000), and transcription (Huibregtse et al., 1997). Furthermore, the function of this protein is E3 protein-ubiquitin ligases have the critical role of critical, as disruption of the rsp5 gene is lethal (Hein selecting specific proteins for conjugation to ubiquitin et al., 1995). (Varshavsky, 1997). There are two main classes of such In Schizosaccharomyces pombe, genetic disruption of E3 ligases, proteins with a HECT (homologous to E6- any one of the three family members, pub1, pub2, and AP carboxyl terminus) catalytic domain (Huibregtse pub3, results in a viable phenotype (Nefsky and Beach, et al., 1995) and proteins with a zinc-binding RING 1996; Tamai and Shimoda, 2002). However, pub1 and finger (really interesting novel gene) (Joazeiro and pub3 double mutants are lethal, suggesting that Pub1p Weissman, 2000) adaptor domain. Three additional E3 and Pub3p may have overlapping functions (Tamai and ligase domains, the U Box (Hatakeyama et al., 2001; Shimoda, 2002). In contrast, a mutant deficient in both Jiang et al., 2001), PHD (Boname and Stevenson, 2001; Pub1p and Pub2p is viable suggesting either that Pub2p Coscoy et al., 2001; Lu et al., 2002) and HUL-1 (Van is nonessential, or that Pub3p can compensate for Sant et al., 2001) sequences have recently been essential functions of Pub2p. described. Polypeptides of the Nedd4 family belong to Caenorhabditis elegans and Drosophila melanogaster the HECT class of E3 ligases (Harvey and Kumar, 1999; each have three Nedd4 family members. Downregula- Rotin et al., 2000). tion by RNA interference of mRNA encoding the C. elegans protein CeWWP1 causes defects in morpho- *Correspondence: R Ingham; E-mail: [email protected] genesis in the developing embryo (Huang et al., 2000a). Nedd4 family of E3 ubiquitin ligases RJ Ingham et al 1973

Figure 1 The Nedd4 Family of E3 ubiquitin ligases. The domain architectures of all known Nedd4 proteins are illustrated and designated by accession number and common name. The relative position of the C2 (green), WW (red), and HECT (blue) domains were obtained by blasting the indicated sequences against the Conserved Domain Database (Marchler-Bauer et al., 2002, 2003). In cases where multiple protein products have been reported only the largest protein product is illustrated. Based on analysis from the Conserved Domain Database the C2 domain of murine NEDD4-1 may be truncated. The bar at the top represents 200 amino acids

In Drosophila, dNedd4 is involved in axon guidance by and their isoforms. However, mice homozygous for a inhibiting signaling through the Robo receptor (Myat loss-of-function mutation in the itch gene develop a et al., 2002) while Suppressor of deltex (Su(dx)) variety of inﬂammatory problems, indicating that the negatively regulates signaling through the Notch recep- Itch protein has nonredundant functions (Hustad et al., tor (Fostier et al., 1998; Cornell et al., 1999). dSmurf 1995; Perry et al., 1998). antagonizes the transforming growth factor-b (TGF-b) et al homolog decapentaplegic (DPP) (Podos ., 2001), Domain architecture of the Nedd4 proteins which regulates dorsoventral patterning in the developing embryo. Although structural studies have not yet been reported The Nedd4 family has expanded further in mammals, for a full-length Nedd4 protein, the individual domains and the existence of multiple spliced isoforms suggests have received considerable attention (Figure 2). The additional complexity (Chen et al., 2001; Dunn et al., catalytic HECT domain was ﬁrst revealed as a 40 kDa 2002; Flasza et al., 2002; Itani et al., 2003). There are region of the protein E6-AP, a cellular enzyme recruited only limited data regarding the functional similarities by the E6 oncoprotein of the human papillomavirus and differences of the various Nedd4 family proteins (HPV) associated with cervical cancers (Huibregtse et al.,

Oncogene Nedd4 family of E3 ubiquitin ligases RJ Ingham et al 1974 occurs to either a target protein or to another ubiquitin molecule to promote a polyubiquitin chain. In both cases, the ubiquitin is transferred from the E3 catalytic cysteine to a lysine side-chain e-amino group forming an isopeptide bond (Schwarz et al., 1998; Wang et al., 1999). Two HECT domain structures, from E6-AP (Lan et al., 1999) and the Nedd4 family member WWP1/AIP5 (Verdecia et al., 2003), have been determined by X-ray crystallography. Significant differences exist between the two structures, providing insight into how the catalytic activity of the HECT domain is regulated. In both structures the domain is composed of two lobes linked by a flexible hinge loop (Figure 2a). As the E6-AP HECT domain was cocrystallized with the E2 UbcH7, the structure reveals the E2 binding site within the N- terminal lobe. This region is only moderately conserved between different HECT domains, which likely reflects differing specificities for one of the over 30 different E2s (Jentsch, 1992). In particular, E6-AP binds the E2 UbcH7with high affinity, while the yeast Nedd4 protein Rsp5p prefers UbcH5 (Kumar et al., 1997). All known catalytic residues are located in the C-terminal lobe of the HECT domain, including the catalytic cysteine that is the transient acceptor for ubiquitin prior to transfer to the final product. The two structures differ in the relative orientation of the N and C lobes. In the E6-AP structure, the two lobes display an L-shaped architecture, with conserved residues lining a broad catalytic cleft at the junction between the lobes. Consistent with the suggestion that this cleft forms part of the HECT active site, inactivating mutations in E6-AP that cause the neurological disorder Angelman Syndrome are localized to this Figure 2 Molecular structure of domains found in the Nedd4 region (Kishino et al., 1997; Jiang et al., 1999). family of E3 ubiquitin ligases illustrating the domain binding However, the E6-AP/UbcH7structure also presented a surfaces of associated ligands and proteins. (a) The two lobes that problem in that the position of the E3 catalytic cysteine compose the HECT domain are shown in the E6-AP crystal is 40 A˚ distant from the E2 cysteine–ubiquitin conjugate. structure (PDB 1D5F). The E2, UbcH7, binds to the N-terminal lobe of the HECT domain in an orientation that would direct a Clearly, large conformational changes would be re- conjugated ubiquitin molecule towards the catalytic cysteine within quired to allow efficient thiol-ester exchange to occur. the E6AP C-terminal lobe. (b) WW domains are formed by a three- The WWP1/AIP5 structure supported this notion, since stranded anti-parallel b-sheet as shown for the Nedd4 WW domain the N- and C-terminal lobes were positioned such that (PDB 1I5H). A ligand that contains a PPxY motif, forming a ˚ polyproline type II helix, binds primarily to one face of the WW the two cysteines would be closer together (16 A). domain b-sheet. (c) The loop regions of the C2 domain, found at Further, molecular modeling showed that this distance one end of the b-sandwhich fold, form the binding site for calcium could be reduced to 5 A˚ if the relative orientation of the and phospholipids as shown in the Synaptotagmin 1 C2B domain lobes were altered by rotation about the hinge loop (PDB 1K5W) (Verdecia et al., 2003). The importance of the flexible hinge linking the two lobes was further demonstrated by mutation and deletion analysis, predicted to restrict the relative movement of the lobes, which resulted in 1993a, b). In these cancers, downregulation of the tumor inhibited autoubiquitination. The inferred importance suppressor protein p53 is an ubiquitin-mediated process of the hinge has led to the proposal that the HECT originating from the E6-induced association of p53 with domain functions as a ratchet to transfer ubiquitin from E6-AP. Biochemical studies of the E6-AP HECT the E2 to the E3 catalytic cysteine. Motion about the domain found that this region is necessary and sufficient hinge causes separation of the two lobes allowing the for ubiquitin transfer and that a cysteine residue played C-terminal lobe to ubiquitinate the substrate while a critical role (Scheffner et al., 1995). The mechanism the N-terminal lobe is recharged with an E2-ubiquitin involves initial binding of an ubiquitin-conjugated E2 to conjugate. the HECT domain with subsequent thiol-ester exchange Although these structural data have provided insight to transfer the ubiquitin moiety from the E2 to the into the mechanism of action of the HECT domain, they catalytic cysteine in the E3. Transfer of ubiquitin then have not definitively addressed how ubiquitin is

Oncogene Nedd4 family of E3 ubiquitin ligases RJ Ingham et al 1975 transferred to the eventual substrate. It is unclear, for 2001; Otte et al., 2003). Other interactions between the example, whether a polyubiquitin chain is formed by peptide and WW domain may also be important. For individual transfer of single ubiquitin molecules or instance, the structure of the third WW domain of whether a polyubiquitin chain is assembled on the E3 rat Nedd4 with a peptide from the b ENaC channel ligase and then directly added to the substrate. Recent shows an extended PPxY motif, PPxYxxL, binds the data suggesting that some HECT domains may control WW domain, with the leucine making important the site of attachment on growing ubiquitin chains contacts. Binding studies support this result as a peptide further complicates this issue. Proteins designated for with a leucine to alanine substitution displayed a six- proteasomal degradation are modified by attachment of fold lower affinity than the wild-type peptide (Henry a chain of ubiquitin molecules linked through lysine 48 et al., 2003). of ubiquitin (Chan and Hill, 2001). For the HECT The C2 domain was first identified in classical Protein domain-containing protein KIAA0010, assembly of Kinase C isoforms as a Ca2 þ -dependent phospholipid ubiquitin chains linked through lysine 29 or 48 is binding domain (reviewed in Rizo and Sudhof, 1998). It possible (You and Pickart, 2001). Furthermore, Rsp5p is approximately 120 amino acids and is composed of can promote the formation of polyubiquitin chains two four-stranded b-sheets oriented to form an eight- linked through lysine 63 (Galan and Haguenauer- stranded b-sandwich structure (Figure 2c) (Sutton et al., Tsapis, 1997; Springael et al., 1999; Soetens et al., 1995; Grobler et al., 1996; Essen et al., 1997; Perisic 2001). In addition, it is not known if the HECT domain et al., 1998). Loop regions between the b-strands at the plays a direct role in selecting substrates for mono- or ‘top’ of the b-sandwich contain a number of conserved polyubiquitination. aspartate residues that are responsible for Ca2 þ binding The C2 domain and multiple WW domains N- (Sutton et al., 1995; Essen et al., 1996; Shao et al., 1996). terminal to the HECT domain regulate cellular localiza- C2 domains have been shown to interact with a variety tion and substrate selection by the Nedd4 proteins. The of phospholipids and proteins (Nalefski and Falke, WW domain, a small module consisting of approxi- 1996) and are thought to function as membrane mately 35 amino acids and named for the presence of recruitment domains involved in protein localization two conserved tryptophan residues spaced 20–22 amino and trafficking. Indeed, the C2 domain is found in many acids apart, appears to mediate substrate recognition proteins that require association with phospholipids at (reviewed in Zarrinpar and Lim (2000); Macias et al., membrane surfaces for function, such as RasGAP, 2002). Each family member has at least one WW Synaptotagmin and several phospholipases (Ponting domain with most possessing three or four modules and Parker, 1996). located over a large middle region of the protein to form a substrate recognition scaffold. Physiological processes mediated by Nedd4 proteins WW domains are found in a variety of different proteins and have been shown to bind predominately Ubiquitin-mediated processes are involved not only in proline-rich motifs, including PPxY (amino-acid single the targeting of proteins for degradation by the 26S letter code; where x is any amino acid) (Chen and Sudol, proteasome but also in the sorting of proteins at 1995), PPLP (Bedford et al., 1997), PR (Bedford et al., different steps in biosynthetic and endocytic pathways 1998, 2000) and phosphoserine/threonine (pS/pT) resi- (Hicke, 2001; Bonifacino and Traub, 2003). Proteins at dues that precede a proline residue (Yaffe et al., 1997; the plasma membrane are tagged with monoubiquitin to Lu et al., 1999). The Nedd4 family WW domains direct internalization. Within vesicles the ubiquitinated primarily recognize PPxY motifs but they have also been proteins are then transported to the late endosome and shown to bind to pS/pT residues (Lu et al., 1999) and further sorted, in the multivesicular body, for recycling may interact with other ligands (Qiu et al., 2000; to the plasma membrane or destruction at the lysosome. Courbard et al., 2002; Murillas et al., 2002). Ubiquitin-mediated protein sorting also occurs at the Several WW domain structures have been solved and trans-Golgi network where newly synthesized proteins, the ligand binding determinants for the PPxY motif such as the amino-acid permeases, are directed to the characterized (Figure 2b) (Kanelis et al., 1998: Huang cell surface or targeted for destruction in the lysosome. et al., 2000b; Verdecia et al., 2000; Kanelis et al., 2001; The Nedd4 proteins participate in these processes Pires et al., 2001). WW domains possess a consensus through ubiquitination of target proteins. three-stranded anti-parallel b-sheet fold that forms a While S. cerevisiae has only a single Nedd4 member, hydrophobic ligand-binding groove, together with a Rsp5p, other eukaryotes have several Nedd4 proteins conserved binding pocket (termed the XP groove), that appear to have both redundant and specialized which is generated by the juxtaposition of two large functions (Figure 3). Here, we describe one function of aromatic residues. The PPxY ligand yields a polyproline Rsp5p in the regulation of cell surface receptors in yeast. type II helix, and the two prolines are packed into the As examples of how the Nedd4 proteins function in XP groove. The tyrosine makes several important mammals, we discuss how several Nedd4 proteins interactions including a hydrogen bond with a conserved appear to operate in the downregulation of the epithelial histidine residue as seen in the dystrophin WW domain sodium channel (ENaC) while Smurf1 and 2 have (Huang et al., 2000b). This might explain the require- evolved specialized functions in the control of TGF-b ment for the tyrosine residue seen in biochemical studies signaling. Finally, we discuss how viruses have co-opted (Chen and Sudol, 1995; Chen et al., 1997; Kasanov et al., the Nedd4 proteins to regulate various aspects of the

Oncogene Nedd4 family of E3 ubiquitin ligases RJ Ingham et al 1976

Figure 3 The NEDD4 family of proteins regulate diverse cellular processes. Selected cellular processes regulated by the NEDD4 family of proteins from yeast to higher eukaryotes are shown. Details regarding the speciﬁc roles of the NEDD4 family proteins in each of the processes are provided in the text. Current understanding of the role played by individual family members in the regulation of these processes is illustrated, however the involvement of other family members not yet investigated cannot be discounted

viral life cycle. In particular, that Epstein–Barr virus are not linked through lysine 48, which normally targets (EBV) recruits Nedd4 proteins for the maintenance of proteins to the proteasome, but are linked through viral latency in B cells and retroviruses utilize these lysine 63. This is consistent with polyubiquitinated proteins to bud from the cell. Fur4p and Gap1p not being substrates of the proteasome. Furthermore, Rsp5p has functions in addition to ubiquitination of Ste2p, as the Ste2p/ubiquitin chimera Regulation of yeast membrane proteins still requires Rsp5p for internalization and degradation Rsp5p-mediated regulation of cell surface receptors is (Dunn and Hicke, 2001b). one mechanism used by S. cerevisiae to adapt to The HECT and C2 domains play distinct functional environmental changes. For example, the Gap1p general roles in the function of Rsp5p. A temperature-sensitive amino-acid permease is upregulated when it is necessary allele containing a mutation in the HECT domain, rsp5- to use amino acids as a nitrogen source (reviewed in 1, is defective in the internalization of Ste2p at Magasanik and Kaiser, 2002). However, when yeast are nonpermissive temperatures (Dunn and Hicke, grown on ammonia, Gap1p is degraded in an ubiquitin- 2001a). The catalytic cysteine of Rsp5p is required as mediated manner (Springael and Andre´ , 1998). Simi- well for ubiquitination and internalization of Gap1p larly, the a factor receptor, Ste2p, the uracil permease, (Springael et al., 1999b). It appears that yeast do not Fur4p, and several other cell surface receptors are necessarily require the Rsp5p C2 domain, as a C2- regulated by ubiquitination in response to different deleted protein rescues rsp5-null cells (Wang et al., 1999; stimuli (reviewed in Rotin et al., 2000). Dunn and Hicke, 2001a). However, while the C2 The ubiquitination, internalization, and degradation domain is not required for ubiquitination of Ste2p, of Ste2p, Gap1p, and Fur4p are all dependent on Rsp5p Fur4p, or Gap1p (Springael et al., 1999b; Dunn and (Hein et al., 1995; Galan et al., 1996; Dunn and Hicke, Hicke, 2001a; Wang et al., 2001), nor for Ste2p 2001a). In the case of Ste2p and Fur4p, receptor internalization, it is important for internalization of degradation occurs not through the proteasome but Fur4p and Gap1p. via the lysosome-like vacuole (Galan et al., 1996; Hicke The WW domains of Rsp5p are also important for and Riezman, 1996). In fact, in-frame fusion of a single regulating these cell surface receptors. Mutation of any ubiquitin molecule to the C-terminus of Ste2p is single WW domain strongly reduces both the ubiquiti- sufﬁcient for internalization and degradation of the nation and internalization of Ste2p, with mutation of receptor (Terrell et al., 1998; Shih et al., 2000; Dunn and the third WW domain showing the greatest effect (Dunn Hicke, 2001b), and monoubiquitination of Fur4p is also and Hicke, 2001a). In contrast, ubiquitination and sufﬁcient for internalization (Springael et al., 1999a). internalization of Fur4p is dependent on both the Fur4p and Gap1p are also polyubiquitinated (Galan second and third WW domains (Gajewska et al., 2001). and Haguenauer-Tsapis, 1997; Springael et al., 1999a; It is interesting to note that Ste2p, Fur4p, and Gap1p Soetens et al., 2001). However, the polyubiquitin chains do not possess a PPxY motif. The C-terminal tail of

Oncogene Nedd4 family of E3 ubiquitin ligases RJ Ingham et al 1977 Ste2p contains the sequence SINNDAKSS, which is such as aldosterone, vasopressin and insulin (Verry et al., phosphorylated on serine residues in response to a 2000). factor binding (Hicke and Riezman, 1996). This appears The ENaC complex is composed of three homologous to be a prerequisite for ubiquitination and internaliza- subunits (a, b, and g), each containing short intracellular tion of the receptor since mutation of the serine residues N- and C-termini and two transmembrane domains to alanine blocks ubiquitination of the receptor and linked by an extracellular loop (Canessa et al., 1993; stabilizes it at the surface (Hicke and Riezman, 1996; Canessa et al., 1994a, b). Although controversial, the Hicke et al., 1998). It has been suggested, but not shown stoichiometry of the subunit assembly appears to be a2 b experimentally, that these phosphorylated residues g (Firsov et al., 1998; Kosari et al., 1998). The C- could provide binding sites for the WW domains of terminal tail of each subunit contains proline-rich Rsp5p. This is plausible, since the WW domains of sequences including a PPxY motif known to bind the Nedd4 have been shown to bind serine/threonine WW domains of several Nedd4 family members includ- phosphorylated ligands (Lu et al., 1999). Alternatively, ing Nedd4-1 (Staub et al., 1996), WWP2 (McDonald the interaction between the receptors and Rsp5p could et al., 2002) and the likely in vivo binding partner be indirect. Nedd4-2 (Debonneville et al., 2001; Harvey et al., 2001). In support of the idea that Rsp5p may indirectly Individual WW domains of the Nedd4 proteins provide interact with substrates, Rsp5p associates with two specificity for ENaC binding. For example, of the four PPxY-containing proteins, Bul1 and 2 (Yashiroda et al., WW domains found in Human Nedd4-1 and Nedd4-2, 1996, 1998), that regulate Gap1p function (Helliwell the third domain appears to be most important for et al., 2001; Soetens et al., 2001). While an interaction ENaC binding while the first domain appears dispen- between Gap1p and Bul1 or 2 has not been demon- sable (Lott et al., 2002; Asher et al., 2003; Fotia et al., strated, it is clear that Bul1 and 2 regulate Gap1p 2003; Henry et al., 2003). Of the three WW domains of stability. When overexpressed, Bul1 or 2 inhibit Gap1p mouse and rat Nedd4-1, the second and third WW function (Helliwell et al., 2001) while, conversely, domains seem to be the most important (Harvey et al., disruption of bul1/bul2 results in an increase in Gap1p 1999; Asher et al., 2001). at the cell surface (Helliwell et al., 2001; Soetens et al., Functionally, these proteins bind and ubiquitinate 2001). Interestingly, the regulation of Gap1p may not lysine residues within the ENaC C-terminal region, occur at the cell surface but rather intracellularly. When resulting in downregulation of the sodium channel, grown on urea, a nonpreferred nitrogen source, Gap1p presumably through internalization and degradation via relocalizes to the cell surface from the golgi (Roberg a lysosomal pathway (Staub et al., 1997). Consistent et al., 1997; Helliwell et al., 2001). Bul1 and 2 play an with this result, the conserved cysteine residue in the important role in this sorting process. In cells over- Nedd4 HECT domain required for ubiquitin ligase expressing either Bul1 or Bul2, Gap1p is targeted activity is also necessary for ENaC downregulation. The directly from the golgi to the vacuole for degradation importance of this downregulation is highlighted by regardless of nitrogen source (Helliwell et al., 2001). natural mutations in the C-terminal tail in the b or g However, in bul1/bul2-deficient cells Gap1p is stabilized subunits that result in Liddle’s Syndrome, an auto- and concentrated at the cell surface (Helliwell et al., somal-dominant hypertensive disorder (Liddle et al., 2001; Soetens et al., 2001). Although it is clear that Bul1 1963; Botero-Velez et al., 1994). These mutations, which and Bul2 regulate the ubiquitination of Gap1p, it is still result in alteration or complete loss of the PPxY motif, controversial if Bul1 and 2 are absolutely required for impair binding by the WW domains of the Nedd4 Gap1p ubiquitination (Soetens et al., 2001) or rather proteins. This leads to increased surface stability of function to promote polyubiquitin chain assembly ENaC and consequently increased sodium ion uptake (Helliwell et al., 2001). (Snyder et al., 1995; Schild et al., 1996; Goulet et al., 1998; Abriel et al., 1999). Regulation of epithelial sodium channels The Nedd4 C2 domain has been shown to mediate localization of the protein to the plasma membrane. In Downregulation of ENaC by the Nedd4 family is an Madin-Darby canine kidney (MDCK) cells, a well important component in the physiological processes defined polarized epithelial cell line with distinct apical regulating fluid and electrolyte homeostasis (Garty and and basolateral surfaces, which contains ENaC com- Palmer, 1997; Rossier et al., 2002). In the kidney, lung, plexes localized to the apical surface, Nedd4 redistri- and distal colon, specialized absorptive epithelia express butes from the cytosol to apical and lateral membranes ENaC to establish a unidirectional flow of sodium ions when treated with Ca2 þ and ionomycin (Plant et al., within the organ. In the kidney, ENaC is localized to the 1997). A construct lacking the C2 domain failed to apical surface of epithelia composing the distal nephron undergo membrane localization, remaining cytosolic and drives the transport of sodium ions into the cell. At even in the presence of Ca2 þ . Although Ca2 þ -dependent the basolateral side of these cells a Na þ ,Kþ -ATPase binding of the C2 domain to phosphatidylcholine, acts to remove sodium ions in exchange for potassium phosphatidylserine, and phosphatidylinositol liposomes ions. The rate of this unidirectional sodium flow is was observed, and might account for the localization, a dependent on ENaC activity and therefore on the direct Ca2 þ -dependent protein–protein association with abundance of the channel at the apical surface. annexin XIIIb has also been implicated (Plant et al., Similarly, ENaC activity is modulated by hormones 2000). The annexins are a large family of proteins

Oncogene Nedd4 family of E3 ubiquitin ligases RJ Ingham et al 1978 involved in vesicle trafficking, endocytosis, and exocy- proteins expressed in oocytes revealed that only gThr623 tosis (reviewed in Gerke and Moss, 2002). Annexin was phosphorylated to detectable levels. Casein Kinase- XIIIb, specifically localizes to the apical membrane in 2-like kinase has also been shown to phosphorylate the MDCK cells, is myristoylated, and is found in lipid rafts bS631 as well as gT599 but the functional consequence (Fiedler et al., 1995; Lafont et al., 1998; Lecat et al., of this phosphorylation has yet to be determined (Shi 2000). Functionally, Annexin XIIIb plays a role in et al., 2002b). directing proteins from the trans-Golgi network to the apical face via apical sorting vesicles (Fiedler et al., 1995; Regulation of TGF-b signaling Lafont et al., 1998; Lecat et al., 2000). Thus, the binding of the C2 domain of Nedd4 to Annexin XIIIb provides a Smad ubiquitin regulatory factor (Smurf) 1 (Zhu et al., means for Nedd4 not only to be recruited to the 1999) and 2 (Kavsak et al., 2000; Lin et al., 2000; Zhang membrane but also to localize to specific microdomains et al., 2001) are Nedd4 family members that carry out within the membrane. However, the C2 domain is not distinct functions in the regulation of signaling path- absolutely required for downregulation of ENaC, as a ways triggered by the TGF-b superfamily. These path- C2-deleted construct of human Nedd4-1 could effi- ways control cellular responses leading to growth, ciently inhibit the ENaC channel (Kamynina et al., differentiation, morphogenesis and apoptosis (Roberts 2001; Snyder et al., 2001), and indeed the C2 domain and Sporn, 1990). Signaling is mediated through may actually be inhibitory to the process (Kamynina heteromeric complexes of types I and II transmembrane et al., 2001). S/T kinases which, upon activation, phosphorylate the The serum- and glucocorticoid-regulated kinase (Sgk) cellular receptor-regulated Smads (R-Smads) 1, 2, 3, 5, has also been implicated in regulating the interaction and 8 (Massague, 1998). Association of an individual R- between the Nedd4 proteins and ENaC. Induction of Smad with the common Smad, Smad4, leads to nuclear Sgk expression by aldosterone occurs in the cortical translocation where transcription is modulated by collecting duct of the nephron (Webster et al., 1993) interaction with DNA binding partners, including and, when tested through coexpression in Xenopus members of the Ski/SnoN family (Akiyoshi et al., oocytes, leads to an increase of ENaC expression and 1999; Luo et al., 1999; Stroschein et al., 1999; Sun activity (Alvarez de la Rosa et al., 1999; Chen et al., et al., 1999a, b; Xu et al., 2000), 50-TG-30-interaction 1999; Naray-Fejes-Toth et al., 1999). The Sgk kinase is a factor (Wotton et al., 1999) and histone acetyltransfer- member of the PKB/Akt family of serine/threonine ase CBP/p300 (Attisano and Wrana, 2000; Massague kinases and contains a PPFY motif that binds the WW and Chen, 2000; Miyazono, 2000). The inhibitory domains of both Nedd4-1 and Nedd4-2 (Debonneville Smads (I-Smad) 6 and 7disrupt the pathway through et al., 2001; Snyder et al., 2002). Curiously, only Nedd4- interaction with the activated receptor (Hayashi et al., 2 is phosphorylated by the kinase as it contains two 1997; Imamura et al., 1997; Nakao et al., 1997); Smad6 consensus phosphorylation sites [RxRCC(S/T)] on also appears to inhibit Smad1/Smad4 association (Hata either side of the second WW domain. Phosphorylation et al., 1998). Smurf1 and 2 regulate these pathways by of Nedd4-2 at these sites, Ser444 and to a lesser extent inducing ubiquitination and degradation of specific Ser338, inhibits its binding to ENaC. The Nedd4-2 pathway components. S444A/S338A double mutant also decouples ENaC Smurf1 is an antagonist of signals initiated by the upregulation by Sgk. The mechanism is potentially bone morphogenetic protein (BMP). It was originally more complicated as the interaction between Sgk and identified by its ability to ubiquitinate and degrade the the Nedd4 proteins also leads to Sgk ubiquitination and BMP-specific R-Smads 1 and 5 (Zhu et al., 1999). The degradation. It appears that other membrane proteins, interaction occurs through a PPxY motif common to such as the glutamine transporter SN1, are also both Smad1 and 5. Smad7also contains a PPPY motif; regulated by similar Sgk/Nedd4-2 interactions (Boehmer however, formation of the resulting complex with et al., 2003). Smurf1 does not lead to degradation of the Smad, but Phosphorylation of serine and threonine residues results in enhanced turnover of the TGF-b type I adjacent to the PPxY motifs found in the ENaC b and receptor (Ebisawa et al., 2001). The mechanism is g subunits has also been suggested as a mechanism for complex as Smurf1, normally localized in the cytoplasm, modulating sodium channel downregulation. The extra- is first induced to translocate to the nucleus where an cellular regulated kinases (ERK1 and 2) are reported to association with Smad7occurs. This Smurf1/Smad7 phosphorylate the b-subunit Thr613 and Thr623 in the complex then moves to the cytoplasm and targets the g-subunit yielding a pTPPPxY motif (Shi et al., 2002a). TGF-b type I receptor at the plasma membrane. This modification was observed, by surface plasmon Although Smad7has a critical role in TGF- b type I resonance experiments, to increase the affinity of Nedd4 receptor recognition, the Smurf1 C2 domain is also WW domains to synthetic peptides that mimic these important. A truncated Smurf1, with the C2 domain ligands. Consistent with the idea that phosphorylation removed, can translocate Smad7to the cytoplasm, but increases the interaction between ENaC and Nedd4 the complex fails to associate with the plasma membrane proteins; functional expression studies of wild type and resulting in the inhibition of TGF-b type I receptor bT613A/gT623A mutants in Xenopus oocytes displayed degradation (Suzuki et al., 2002). an increase in channel activity for the mutants. Smurf2, like Smurf1, can interact with Smads 1 and 5. However, monitoring the phosphorylation levels of the It also complexes with Smad7to regulate the TGF- b

Oncogene Nedd4 family of E3 ubiquitin ligases RJ Ingham et al 1979 receptor (Kavsak et al., 2000), but it also has distinct 2000; Winberg et al., 2000; Ikeda et al., 2001, 2002). properties. Unlike Smurf1, it is localized to the nucleus These functions are dependent on the PPPY motifs of and binds R-Smads 2 and 3 (Lin et al., 2000; Zhang LMP2A and can be blocked by dominant negative AIP4 et al., 2001). Surprisingly, despite a 92% sequence and WWP2 mutants where the catalytic cysteine has similarity between Smad2 and Smad3, Smurf2 binds and been mutated (Winberg et al., 2000; Ikeda et al., 2002). degrades Smad2 preferentially over Smad3. Through the This suggests that LMP2A may maintain viral latency Smurf2/Smad2 interaction the transcriptional corepres- not only by sequestering kinases away from the BCR sor SnoN is also targeted for ubiquitination (Bonni et al., but by degrading them as well. However, an LMP2A 2001). Smad2 acts as an adaptor in SnoN degradation, molecule with mutated PPPY motifs was still able to having a PPGY binding site for Smurf2 and an MH2 block the induction of viral lytic genes (Ikeda et al., domain that associates with the N-terminal region of 2001) questioning whether ubiquitination is essential for SnoN. maintenance of viral latency. The ubiquitination of Lyn, Syk, and perhaps LMP2A may be more important in Maintenance of EBV latency LMP2A’s ability to mimic BCR signaling. Signals through the BCR required for B cell development and While it is clear that Nedd4 family proteins are for survival of mature B cells are thought to be antigen- important for many cellular processes, it has become independent and relatively weak in intensity. Therefore, evident that viruses also utilize this family of proteins. Nedd4 family proteins may titer LMP2A-mediated EBV is the causative agent of infectious mononucleosis signaling through the degradation of Lyn and Syk to and is associated with a number of cancers including provide a sufficient signal for survival yet prevent viral Burkitt’s Lymphoma. EBV infection can persist reactivation. throughout life, as the virus maintains a latent infection within the body’s memory B cells. The viral protein Nedd4 proteins are co-opted by retroviruses for viral latent membrane protein 2A (LMP2A) is required to budding maintain this latency (Miller et al., 1994). LMP2A is predicted to span the membrane 12 times, resulting in The puzzling early observation that free ubiquitin is both the N and C-terminal tails being located in the found in the virions of avian leukosis virus (Putterman cytoplasm. The N-terminus possesses a number of et al., 1990) has been resolved by recent work showing protein–protein interaction motifs including phospho- that these retroviruses co-opt cellular endocytic path- tyrosine-dependent binding sites for the SH2 domain of ways for viral assembly and budding from the plasma the Src family tyrosine kinase Lyn as well an ITAM membrane (Hicke, 2001). Indeed, the viral budding motif that binds the dual SH2 domains of the Syk process appears to be the topological reverse of tyrosine kinase (Burkhardt et al., 1992; Miller et al., endocytosis (Hicke, 2001). Consequently, current data 1995; Fruehling and Longnecker, 1997; Fruehling et al., suggest that retroviruses have rewired selected cellular 1998). LMP2A also has two PPPY motifs that have been processes, including those regulated by the Nedd4 shown to recruit the Nedd4 family proteins Nedd4-1, proteins, to carry out their life cycle. Nedd4-2, AIP4, and WWP2 (Ikeda et al., 2000; Winberg The late assembly domains found in the Gag et al., 2000). polyproteins of retroviruses, such as the Rous sarcoma LMP2A function in B cells is complex. In order to virus (Strack et al., 2000; Kikonyogo et al., 2001), maintain viral latency, LMP2A binds and sequesters the Mason-Pfizer monkey virus (M-PMV) (Yasuda and Lyn and Syk tyrosine kinases away from the B cell Hunter, 1998), and Moloney murine leukemia virus receptor (BCR), thereby potentially interfering with (Yuan et al., 1999), contain a PPxY motif that can bind BCR signaling (Miller et al., 1994, 1995; Fruehling and Nedd4 proteins. The importance of this PPxY motif has Longnecker, 1997; Fruehling et al., 1998). This is critical been demonstrated in the M-PMV Gag polyprotein, for maintenance of latency, as signaling pathways where it is critical for virion budding and release initiated by the BCR activate the viral BZLF1 gene (Yasuda and Hunter, 1998). Similar properties have which in turn activates viral lytic genes (Takada 1984; been observed for the VP40 protein of the Ebola virus Miller et al., 1994). In the same way, and somewhat and the M protein of rhabdoviruses, including the paradoxically, LMP2A can also mimic signaling vesicular stomatitis virus and rabies virus, which all through the BCR. Signals through the BCR are contain a PPxY sequence (Craven et al., 1999; Harty important for both B cell development and survival of et al., 1999, 2000, 2001; Jayakar et al., 2000). Beyond mature B cells in the periphery (reviewed in Gold, 2002; demonstrating the association of Nedd4 protein with Kurosaki, 2002). LMP2A has been shown to mimic viral structural proteins, few experiments have char- these signals by driving the maturation of rag1-deficient acterized the nature of these interactions further. A B cells and acting as a survival signal for mature B cells notable exception involves the Ebola virus matrix that have lost the expression of surface immunoglobulin protein VP40. This protein not only binds Nedd4-1 (Caldwell et al., 1998; Caldwell et al., 2000). LMP2A but contains a PTAP motif to recruit a protein involved therefore appears to have a dual function in maintaining in the vesicular protein sorting machinery, tumor B-cell survival while blocking BCR signaling. susceptibility gene 101 (Tsg101) (Martin-Serrano et al., Nedd4 proteins can ubiquitinate LMP2A and the 2001) which has also been implicated in viral budding of LMP2A-associated kinases Lyn and Syk (Ikeda et al., HIV (VerPlank et al., 2001). Curiously, both motifs

Oncogene Nedd4 family of E3 ubiquitin ligases RJ Ingham et al 1980 overlap within the sequence PTAPPEY. Reports that 1 receptor (IGFR-1) (Morrione et al., 1999; Vecchione Tsg101 binds to monomeric VP40 while Nedd4-1 is et al., 2003), and in the EGFR pathway to mono- preferentially bound to an oligomeric form of VP40 ubiquitinate Eps15 (Polo et al., 2002; Di Fiore et al., (Timmins et al., 2003) suggest that Tsg101 and Nedd4-1 2003) and Hgs (Katz et al., 2002). Of note, it appears might interact with VP40 at two different steps in virion that an ubiquitin-interacting motif (UIM) within Eps15 assembly and budding. A phosphorylation event may promotes monoubiquitination by Nedd4-1 (Polo et al., trigger the switch between Tsg101 and Nedd4-1 directed 2002; Katz et al., 2002) through interacting with the processes. Both VP40 and ENaC share a common ubiquitin and blocking polyubiquitin chain assembly. TPPPxY sequence that in ENaC undergoes ERK- AIP4/Itch is suggested to play a role in maintaining tight mediated threonine phosphorylation (Shi et al., 2002a), junctions through an interaction with occludin (Trawe- resulting in enhanced binding of Nedd4 proteins. A ger et al., 2002). Also, a connection to the Notch further complication, however, is that Tsg101 contains a pathway has been inferred from a report of Itch binding UEV domain that is structurally similar to the E2 Ubc to Notch (Qiu et al., 2000) that parallels the Suppressor proteins (Pornillos et al., 2002) and therefore may have of Deltex regulation of Notch seen in Drosophila some affinity for the E2 binding site on the Nedd4 (Fostier et al., 1998; Cornell et al., 1999). AIP4 has proteins. been found to associate with the RING finger protein Recent work by Yasuda et al. (2002) suggested that CBLC in a WW domain-dependent interaction not specific Nedd4 family members are recruited by retro- involving a PPxY motif (Courbard et al., 2002). It has viruses. Working with the M-PMV they identified also been proposed that this association between AIP4 budding-associated ubiquitin ligase 1 (BUL1), also and CBLC plays a role in the downregulation of the known as KIAA0322, as being functionally involved in EGFR. viral budding. The M-PMV viral capsid protein p27 Future work to characterize the function of individual bound with higher affinity to BUL1 than Nedd4 or the Nedd4 family members will require a detailed analysis of poorly characterized Nedd4 protein hNEDL2 the expression profile of each protein. The role that (KIAA1301). As expected, the BUL1 interaction is phosphorylation has on the activity of individual WW domain-mediated as an inactivating W791G members requires evaluation. In addition to the pS/pT mutation in the first WW domain abolished viral modification discussed above it has been reported that budding, as did a BUL1 fragment containing only the Nedd4 and AIP4 undergo tyrosine phosphorylation WW domains, presumably acting in a dominant upon stimulation of IGFR-1 (Morrione et al., 1999) and negative manner. EGFR (Courbard et al., 2002), respectively. Evaluation of nuclear import/export signals could also help clarify Summary and future prospects issues regarding cellular localization. Indeed, functional nuclear import/export signals have been identified in The modular nature of the Nedd4 family of proteins has Nedd4-1 and Smurf1 (Hamilton et al., 2001; Tajima provided guidance in understanding the function of this et al., 2003). Therefore, each family member needs to be class of E3 ubiquitin ligases. Indeed, techniques invol- examined for the presence of additional motifs that may ving far-western probing (Jolliffe et al., 2000), two- target these proteins to distinct locations within the cell hybrid analysis (Murillas et al., 2002), and mass or confer specific functionality. Finally, evaluation of spectrometry (Plant et al., 2000), using the WW and targeted mouse knockouts and the use of RNA C2 domains of individual members, continue to identify interference may provide further insight into this associated proteins. While further biochemical charac- important family of proteins. terization is required for many of these interactions, recent reports have further expanded the scope of cellular functions regulated by the Nedd4 proteins. Acknowledgements Nedd4-1 has been reported to regulate the guanine- Robert J Ingham is a Fellow of the Leukemia & Lymphoma nucleotide exchange factor CNrasGEF (Pham et al., Society. Tony Pawson is a Distinguished Scientist of the 2000; Pham and Rotin, 2001), to act in association with Canadian Institutes of Health Research (CIHR). We thank Grb10 in downregulating the insulin-like growth factor Julinor Bacani for critically reading the manuscript.

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