Nedd4-like proteins: an emerging family of ubiquitin-protein ligases implicated in diverse cellular functions

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Nedd4-like proteins: an emerging family of ubiquitin-protein ligases implicated in diverse cellular functions Kieran F. Harvey and Sharad Kumar

The members of an emerging family of proteins similar to Nedd4 have a unique modular structure consisting of a Ca21/lipidbinding domain, multiple protein–protein interaction modules and a ubiquitin-protein ligase domain. Although little is known about the physiological roles of these proteins, studies in both mammals and yeast are providing evidence that members of this family might be involved in diverse cellular functions, such as regulation of membrane channels and permeases, the cell cycle and transcription. This article attempts to bring together what is currently known about these evolutionarily conserved ubiquitin-protein ligases.

The authors are at the Hanson Centre for Cancer Research, Institute of Medical and Veterinary Science, Frome Road, Adelaide, SA 5000, Australia. E-mail: sharad.kumar@ imvs.sa.gov.au

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Nedd4 was identified originally as a developmentally regulated mouse gene highly expressed in early embryonic central nervous system1. Further analysis revealed that the expression of Nedd4 is not restricted to the embryonic central nervous system and that it is expressed at varying levels in different tissues2,3. At the time that Nedd4 was first cloned in 1992, the only identifiable structure in the protein was a Ca21/lipid-binding domain (C2 domain) and three repeats of approximately 40 amino acids. These repeats, now known as WW domains, are protein–protein interaction modules found in a variety of proteins4. In 1993, the C-terminal region of Nedd4 was discovered to be similar to human papilloma virus (HPV) oncoprotein E6-associated protein (E6-AP). E6-AP is a ubiquitin-protein ligase involved in the E6-mediated ubiquitination of p535,6. The presence of these three domains in Nedd4 suggested that Nedd4 could be involved in Ca21-mediated ubiquitination of a membrane protein(s). Recently,

a number of proteins have been discovered that share the same modular structure as Nedd4 and appear to be part of a family of ubiquitin-protein ligases. Some of these proteins have been implicated in a variety of cellular functions (Table 1). Modular structure of the Nedd4 family of proteins The characteristic feature of the Nedd4 family of proteins is the organization of the C2, WW and ubiquitin-protein ligase domains. In all cases, the C2 domain is located towards the N-terminus (Fig. 1). The C2 domain was first identified in protein kinase C and is responsible for Ca21-dependent binding of membrane phospholipids7. It is found in a number of proteins, most of which are involved in signal transduction or membrane traffic. The C2 domain is believed to regulate the function of proteins by mediating their translocation to phospholipid membranes in response to increased cytosolic Ca21. The function of the C2 domain in the Nedd4 family of proteins is not well understood, but it might mediate redistribution of these proteins to intracellular membranes upon fluctuations in Ca21 concentration. All members of the Nedd4 family of proteins contain multiple WW domains, located between the C2 and ubiquitin-protein ligase domains. WW domains (also called WWP domains) derive their name from the presence of two highly conserved tryptophan residues and a conserved proline residue in a sequence of ~35 amino acids4. These domains consist of a hydrophobic core surrounded by beta sheets containing a number of charged residues and have a preference for binding small proline-rich sequences, called PY motifs, the most common of which is PPxY4. However, WW domains from some proteins can bind to alternative proline-rich motifs – for example, the WW domains of formin-binding proteins have a preference for PPLP8. Single WW domains are found in many proteins, but 2–4 copies are present in Nedd4-like proteins. Different WW domains from the same protein possess differential substrate specificity in vitro4. Therefore, it is plausible that each of the Nedd4-like proteins interacts with a number of different proteins through WW domains in vivo. The ubiquitin-protein ligase domain is situated at the C-termini of the Nedd4 family members. It is a large domain (approximately 350 residues) and was first characterized in the human ubiquitin-protein ligase E6-AP, and hence is often referred to as the HECT (homologous to E6-AP C-terminus) domain5,6. Ubiquitin-protein ligases (E3s) comprise the substrate-specificity arm of the ubiquitin pathway (reviewed in Ref. 9). Initially, ubiquitin is activated by a ubiquitin activating enzyme (E1), transferred to a ubiquitin conjugating enzyme (E2) and then linked to a substrate protein either directly or via a ubiquitin-protein ligase (E3)9. The HECT domain proteins are a major subclass of E3s and contain a conserved cysteine residue, located towards the carboxyl end of the HECT domain, that is capable of forming a thioester with ubiquitin5,6,10. Many classes of proteins are regulated by ubiquitination, including cellcycle proteins (e.g. cyclins), transcription factors

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TABLE 1 – KNOWN FUNCTIONS OF SOME NEDD4 FAMILY MEMBERS Protein

Biochemical activity

Physiological function

Nedd4

Ubiquitin-dependent downregulation of epithelial Na1 channel Regulation of unknown protein Ubiquitination of permeases (uracil permease, general amino acid permease and maltose permease) and RNA polymerase II Ubiquitin-mediated turnover of Cdc25p phosphatase and membrane permeases

Regulation of intracellular Na1 concentration

Itch Rsp5p

Pub1p

(e.g. NF-kB) and membrane proteins such as the epithelial sodium channel (ENaC) and c-Kit9. Once ubiquitinated, nuclear and cytoplasmic substrate proteins are generally targeted for degradation by the 26S proteasome. By contrast, many membrane proteins are endocytosed following ubiquitination and are either recycled to the membrane or degraded by the lysosomal pathway (reviewed in Ref. 11). Pleiotropic functions of Nedd4/Rsp5 protein To date, Nedd4 orthologues have been found in yeast, mouse, rat and human1–3. While yeast, mouse and rat proteins have a similar structure, human Nedd4 has an additional WW domain that might allow it to interact with other proteins. The first known substrate for Nedd4 was the epithelial sodium channel (ENaC)3. The WW domains in Nedd4 interact with the PY motifs found in the C-termini of all three ENaC subunits3. Mutations that delete or alter the PY motif of either the b or g ENaC subunit cause Liddle’s Syndrome, an autosomal-dominant form of hypertension3,12. This led to the hypothesis that Nedd4 normally negatively regulates ENaC by ubiquitin-mediated protein turnover. This theory was confirmed recently with the demonstration that Nedd4 downregulates ENaC activity in a ubiquitin-dependent fashion13. Goulet et al. supported this finding by showing that the conserved cysteine residue that is required for the ubiquitin ligase activity of the HECT domain of Nedd4 was necessary for downregulation of ENaC14. There are Nedd4 homologues in both Saccharomyces cerevisiae and Schizosaccharomyces pombe. The S. cerevisiae protein Rsp5p/Npi1p was identified originally as a suppressor of mutations in the SPT3 gene, which encodes a transcription factor that interacts with a TATA-binding protein15. More recently, it has been identified in numerous screens and implicated in a plethora of apparently unrelated functions. It is required for the ubiquitin-mediated turnover of at least three membrane proteins – the general amino acid permease16, the uracil permease16–18 and the maltose transporter19. The regulation of membrane transporters by Rsp5p/Npi1p is similar to the regulation of ENaC by Nedd4 and is discussed more extensively in a recent article11. Rsp5p/Npi1p is also believed to play a role in minichromosome maintenance20, mitochondrial/ cytoplasmic protein distribution21 and is required for vegetative growth, sporulation and the stress response22. Rsp5p/Npi1p also interacts with and ubiquitinates trends in CELL BIOLOGY (Vol. 9) May 1999

Role in inflammatory response Control of import of metabolites and nutrients and regulation of transcription Regulation of cell cycle, membrane transport and pH tolerance

RNA polymerase II23. A recent report shows that WW domains 2 and 3 and the HECT domain are indispensable for the essential function of Rsp5p/Npi1p24. The C2 domain and WW domain 1 do not appear to be required for viability but might be required for a non-essential function of Rsp5p/Npi1p. Pub1p, the S. pombe homologue of Rsp5p/Npi1p, targets the mitosis-activating tyrosine phosphatase cdc25 for degradation by the ubiquitin pathway25. Like Rsp5p, Pub1p is also implicated in the regulation of membrane permeases26. Human Nedd4 and Rsp5p also potentiate hormone-dependent activation of transcription by progesterone and glucocorticoid receptors, in a manner apparently independent of ubiquitinprotein ligase function and interactions through the WW domains27. In addition, Nedd4 interacts C2 domain

WW domains

Hect domain

Human Nedd4 Human KIAA0439 Human WWP2/AIP2 Human WWP1/AIP5 Human AIP-4 Human AC004893 Human KIAA0322 Mouse Nedd4 Mouse Itch Rat Nedd4 Xenopus AJ000085 S. cerevisiae Rsp5p/Npi1p S. pombe Pub1p S. pombe Z99759 FIGURE 1 The Nedd4 family of proteins. Members of this family are characterized by a Ca21/lipid-binding (C2) domain (black boxes) located at the N-terminus, multiple WW domains located in the middle part of the protein, and a ubiquitin-protein ligase domain (grey boxes) at the C-terminus. Some family members are predicted proteins derived from DNA databases (accession nos are indicated). The sequence of WWP1/AIP5 is incomplete at both ends, while the sequence of AIP4 is incomplete at the N-terminus.

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Note added in proof: It has recently been shown that the WW domains of Nedd4 can bind to phosphoserine and phosphothreonine residues34. Thus it is plausible that Nedd4-like proteins regulate a much larger repertoire of substrate proteins than first thought, by binding to proteins that might lack PY motifs but contain phosphorylated serine and threonine residues.

Acknowledgements We thank members of our laboratory for useful comments. Work in our laboratory is supported by the Wellcome Trust, the National Health and Medical Research Council and the National Heart Foundation. Owing to space restrictions, we were unable to include a comprehensive list of all primary papers pertaining to the work discussed in this article.

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in vitro through its WW domains with the PY motifs present in the haematopoietic transcription factor p45/NF-E2 and RNA polymerase II28. It is not clear, however, whether Nedd4 mediates ubiquitination of either p45 or RNA polymerase II. Mouse and human Nedd4 are cleaved rapidly by caspases during apoptosis, a process that releases the C2 domain from the rest of the protein29. The significance of Nedd4 cleavage during apoptosis is obscure at present, but it might be an energy-conserving mechanism. Alternatively, Nedd4 might normally be required to mediate the turnover of a protein(s) that is required for the apoptotic function. Other Nedd4 family members The protein most closely related to human Nedd4 is encoded by a human gene of unknown function (accession no. KIAA0439). This putative protein shares approximately 78% similarity with human Nedd4 and has a Xenopus homologue (accession no. AJ000085). Three human Nedd4-like proteins – WWP2/AIP2, WWP1/AIP5 and AIP4 – have been cloned recently and share a high degree of homology with each other30,31. WWP2/AIP2 and WWP1/AIP5 were identified based on their ability to bind to a PY motif peptide bait30. WWP2/AIP2, WWP1/AIP5 and an additional member, AIP4, were also cloned as molecules that interact with atrophin-1, a protein containing five PY motifs31. Certain WW domains from WWP2/AIP2 and WWP1/AIP5 possess binding specificity for peptides representing the PY motifs of several proteins, including RasGAP, ENaC and b-dystroglycan30. Whether these proteins or atrophin-1 are physiological targets of any of these Nedd4-like proteins is yet to be demonstrated. The likely murine homologue of the AIP-4 gene, the sequence of which is incomplete at the N-terminus, is Itch. Itch is disrupted in mice with the genetic disease non-agoutilethal 18H, which is characterized by a number of inflammatory disorders32. The protein target(s) of Itch is not yet known, but, since many cytokine receptors are involved in the inflammatory response and are regulated by the ubiquitin pathway, it is possible that its substrates include one or more cytokine receptors. Other members of the Nedd4 family include two predicted human proteins (accession nos AC004893 and KIAA0322), neither of which have been characterized as yet. Role of multiple WW domains in defining target specificity The WW domains of Nedd4-like proteins appear to mediate interactions with target proteins. Although in vitro data suggest that WW domains from different Nedd4-like proteins can bind to the same PY motif, the validity of these observations in vivo and the physiological significance of the in vitro interactions remain unknown. A likely scenario is that each WW domain of a Nedd4-like protein binds to a specific set of proteins. Support for this comes from recent studies with Nedd4–ENaC interactions. Only WW domains 2 and 3 of mouse Nedd4 interact in vitro with the three ENaC

subunits, but all three WW domains are required for downregulation of ENaC activity in vivo33. This suggests that WW domain 1 binds to a protein other than ENaC and that this interaction is required for Nedd4-mediated regulation of ENaC activity. In S. cerevisiae, a single Nedd4-like protein appears to regulate the ubiquitination of a variety of different proteins. The presence of multiple Nedd4-like proteins in mammals (seven in human) suggests that many mammalian proteins are likely to be modified through ubiquitination by Nedd4 family members. Interaction with a large number of protein targets might be achieved by means of multiple WW domains in individual Nedd4 family members, each interacting specifically with a small subset of target proteins. Outside the C2, WW and HECT domains, Nedd4-like proteins do not share significant homology. In addition to WW domains, other, as-yet-undefined, regions of Nedd4-like proteins might be important for binding to factors that further regulate substrate specificity. Concluding remarks Nedd4-like proteins define a unique family of HECT-domain-containing ubiquitin-protein ligases. At present, clear functional evidence is only available for mammalian Nedd4 and its yeast homologues Rsp5p/Npi1p and Pub1p. Consistent with the modular structure of these proteins, the main function for Nedd4/Rsp5p/Pub1p appears to be the regulation of membrane channels, receptors and transporters through ubiquitination, although additional roles in regulating cytoplasmic and nuclear proteins have also been demonstrated. Many membrane-bound channels, receptors and transporters are regulated by ubiquitination, but the proteins responsible for this regulation are largely unknown11. We speculate that members of the Nedd4 family of proteins are key regulators of membrane proteins, each targeting a select set of proteins for ubiquitination. The interaction of a Nedd4-like protein with its membrane-bound substrate could be direct (e.g. the WW domains of Nedd4 contact the PY motifs of ENaC) or could occur indirectly through an adaptor protein containing a PY motif. Such adaptor proteins might be required for Nedd4-like protein function as the intracellular regions of many membrane proteins do not contain PY motifs. As Nedd4-like proteins are an emerging family, there are numerous gaps in our understanding of how these proteins function. Identification of substrates of Nedd4 family members will provide important insights into the regulation of protein function by ubiquitin modification. References 1 Kumar, S., Tomooka, Y. and Noda, M. (1992) Biochem. Biophys. Res. Commun. 185, 1155–1161 2 Kumar, S. et al. (1997) Genomics 40, 435–443 3 Staub, O. et al. (1996) EMBO J. 15, 2371–2380 4 Sudol, M. (1996) Prog. Biophys. Mol. Biol. 65, 113–132 5 Scheffner, M. et al. (1993) Cell 75, 495–505 6 Scheffner, M., Nuber, U. and Huibregtse, J. M. (1995) Nature 373, 81–83 trends in CELL BIOLOGY (Vol. 9) May 1999

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7 Knopf, J. L. et al. (1986) Cell 46, 491–502 8 Chan, D., Bedford, M. T. and Leder, P. (1996) EMBO J. 15, 1045–1054 9 Hershko, A. and Ciechanover, A. (1998) Annu. Rev. Biochem. 67, 425–479 10 Huibregtse, J. M. et al. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 2563–2567 11 Hicke, L. (1999) Trends Cell Biol. 9, 107–112 12 Shimkets, R. A. et al. (1994) Cell 79, 407–414 13 Dinudom, A. et al. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 7169–7173 14 Goulet, C. C. et al. (1998) J. Biol. Chem. 273, 30012–30017 15 Eisenmann, D. M. et al. (1992) Genes Dev. 6, 1319–1331 16 Hein, C. et al. (1995) Mol. Microbiol. 18, 77–87 17 Galan, J. M. et al. (1996) J. Biol. Chem. 271, 10946–10952 18 Galan, J. M. and Hageunauer-Tsapis, R. (1997) EMBO J. 16, 5847–5854 19 Lucero, P. and Lagunas, R. (1997) FEMS Microbiol. Lett. 147, 273–277

Gap junctions: more roles and new structural data Alexander M. Simon Characterization of the diverse functions of gap junctions is an ongoing effort, and there have been some interesting developments since the recent trends in CELL BIOLOGY review on which I was a coauthor1. The list of human diseases associated with gap junction abnormalities continues to grow, and two additional ones have been linked to mutations in connexins, the subunits of gap junction intercellular channels2,3. Surprisingly, the same connexin gene was fingered as the likely culprit in both cases. Mutations in the GJB3 gene, encoding Cx31, were found in two families with an autosomal–dominant hearing impairment3 and independently in four families with a dominantly transmitted skin disorder, erythrokeratodermia variabilis2. These disorders were associated with mutations affecting different amino acid residues in the Cx31 protein, suggesting that the distinct phenotypes might result from different effects on channel function. Hearing impairment was associated with a nonsense or a missense mutation in the second extracellular loop, while the skin disorder was linked to missense mutations in either the N-terminal domain or the second transmembrane domain. Two other recent papers examined the role of gap junctions, and particularly connexin 43, in migrating cells – in one case in neural crest cells4 and, in the other, in migrating keratinocytes5. Previous studies had shown that ablation of the Cx43 gene in mice (Cx43KO mice) resulted in conotruncal defects in trends in CELL BIOLOGY (Vol. 9) May 1999

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Yashiroda, Y. et al. (1996) Mol. Cell. Biol. 16, 3255–3263 Zoladek, T. et al. (1997) Genetics 145, 595–603 Kanda, T. (1996) Genes Genet. Syst. 71, 75–83 Huibregtse, J. M., Yang, J. C. and Beaudenon, S. L. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 3656–3661 Wang, G., Yang, J. and Huibregtse, J. M. (1999) Mol. Cell. Biol. 19, 342–352 Nefsky, B. and Beach, D. (1996) EMBO J. 15, 1301–1312 Saleki, R. et al. (1997) Mol. Gen. Genet. 20, 520–528 Imhof, M. O. and McDonnell, D. P. (1996) Mol. Cell. Biol. 16, 2594–2605 Gavva, N. R. et al. (1997) J. Biol. Chem. 272, 24105–24108 Harvey, K. F. et al. (1998) J. Biol. Chem. 273, 13524–13530 Pirozzi, G. et al. (1997) J. Biol. Chem. 272, 14611–14616 Wood, J. D. et al. (1998) Mol. Cell. Neurosci. 11, 149–160 Perry, W. L. et al. (1998) Nat. Genet. 18, 143–146 Harvey, K. F. et al. J. Biol. Chem. (in press) Lu, P-J. et al. (1999) Science 283, 1325–1328

the pulmonary outflow tract of the heart6, and transgenic mice expressing a dominant–inhibitory gap junction protein under the control of the EF-1a promoter (FC mice) displayed a similar phenotype7. As neural crest cells contribute to the development of the outflow tract, one model was that Cx43 expression might be important for cardiac neural crest cell migration or function. This idea was bolstered by the observation that neural crest cells do in fact express Cx43 and communicate extensively among themselves8. However, there was no direct evidence that Cx43 is crucial for neural crest cell function. Complicating the situation further, overexpression of Cx43 in neural crest cells using the CMV promotor (CMV43 mice) also resulted in conotruncal defects9. Now, Huang et al. have provided direct evidence that the migration efficiency of neural crest cells is particularly sensitive to Cx43 levels4. Using a neural tube explant culture system, they compared the ability of neural crest cells to migrate out of explants derived from wild-type, Cx43KO, FC and CMV43 mice. Neural crest cells overexpressing Cx43 migrated further than wild-type, while cells with Cx43 ablated or dominantly inhibited migrated less-thannormal distances. Furthermore, by following neural crest cells labelled with b-galactosidase in developing hearts, the number of neural crest cells present in the outflow tract in vivo could be correlated to Cx43 status. There were more neural crest cells in the outflow tract when Cx43 was overexpressed, and fewer when it was absent. How Cx43 status changes the migration efficiency of neural crest cells and how neural crest cells affect the development of the myocardium are still being worked out. Intriguingly, the CMV43 mice showed increased cellular proliferation in the myocardium, while myocardial proliferation was decreased in Cx43KO hearts, suggesting that neural crest cells influence myocardialization in some regions of the heart. Keratinocyte migration is important during wound healing of the epidermis. Lampe et al. found that

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The author is in the Physiology Dept, University of Arizona, Tucson, AZ 85724, USA. E-mail: amsimon@ u.arizona.edu

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