cheap michael kors functional diversity within a common modular architecture

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functional diversity within a common modular architecture

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

Robert J Ingham1, Gerald Gish1 and Tony Pawson1,2Top of pageAbstractNeuronal precursor cell expressed developmentally downregulated 4 (Nedd4) is the prototypical protein in a family of E3 ubiquitin ligases that have a common domain architecture. They are comprised of a catalytic C terminal HECT domain and N terminal C2 domain and WW domains responsible for cellular localization and substrate recognition. These proteins are found throughout eukaryotes and regulate diverse biological processes through the targeted degradation of proteins that generally have a PPxY motif for WW domain recognition, and are found in the nucleus and at the plasma membrane. Whereas the yeast Saccharomyces cerevisiae uses a single protein, Rsp5p, to carry out these functions, evolution has provided higher eukaryotes with several related Nedd4 proteins that appear to have specialized roles. In this review we discuss how knowledge of individual domain function has provided insight into the physiological roles of the Nedd4 proteins and describe recent results that suggest discrete functions for individual family members.

Keywords: ubiquitination, Nedd4, E3 ubiquitin ligase, HECT, WW, C2

Top of pageIntroductionE3 protein ubiquitin ligases have the critical role of selecting specific proteins for conjugation to ubiquitin (Varshavsky, 1997). There are two main classes of such E3 ligases, proteins with a HECT (homologous to E6 AP carboxyl terminus) catalytic domain (Huibregtse et al., 1995) and proteins with a zinc binding RING finger (really interesting novel gene) (Joazeiro and Weissman, 2000) adaptor domain. Three additional E3 ligase domains, the U Box (Hatakeyama et al., 2001; Jiang et al., 2001), PHD (Boname and Stevenson, 2001; Coscoy et al., 2001; Lu et al., 2002) and HUL 1 (Van Sant et al., 2001) sequences have recently been described. Polypeptides of the Nedd4 family belong to the HECT class of E3 ligases (Harvey and Kumar, 1999; Rotin et al., 2000).

Nedd4 was originally identified in 1992, in a screen for genes developmentally downregulated in the early embryonic mouse central nervous system (Kumar et al., 1992), and resembles other regulatory proteins in containing multiple, distinct modular domains (Pawson and Nash, 2003). Subsequently, related proteins with similar structures have been characterized. All contain a catalytic HECT domain at the C terminus, and an N terminal region involved in substrate recognition, that includes a C2 domain and a series of WW domains. Although the common modular architecture of the Nedd4 family might suggest a redundant functional role for these proteins, recent work has begun to define specific cellular activities for individual members, and to suggest mechanisms that underlie this specificity. This review will provide an overview of the functional properties of the modular domains comprising the Nedd4 family, and highlight physiological processes regulated by these proteins.

Members of the Nedd4 protein familyNedd4 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 in Rotin et al., 2000), and transcription (Huibregtse et al., 1997). Furthermore, the function of this protein is critical, as disruption of the rsp5 gene is lethal (Hein et al., 1995). 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

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In Schizosaccharomyces pombe, genetic disruption of any one of the three family members, pub1, pub2, and pub3, results in a viable phenotype (Nefsky and Beach, 1996; Tamai and Shimoda, 2002). However, pub1 and pub3 double mutants are lethal, suggesting that Pub1p and Pub3p may have overlapping functions (Tamai and Shimoda, 2002). In contrast, a mutant deficient in both Pub1p and Pub2p is viable suggesting either that Pub2p is nonessential, or that Pub3p can compensate for essential functions of Pub2p.

Caenorhabditis elegans and Drosophila melanogaster each have three Nedd4 family members. Downregulation by RNA interference of mRNA encoding the C. elegans protein CeWWP1 causes defects in morphogenesis in the developing embryo (Huang et al., 2000a). In Drosophila, dNedd4 is involved in axon guidance by inhibiting signaling through the Robo receptor (Myat et al., 2002) while Suppressor of deltex (Su(dx)) negatively regulates signaling through the Notch receptor (Fostier et al., 1998; Cornell et al., 1999). dSmurf antagonizes the transforming growth factor (TGF ) homolog decapentaplegic (DPP) (Podos et al., 2001), which regulates dorsoventral patterning in the developing embryo.

The Nedd4 family has expanded further in mammals, and the existence of multiple spliced isoforms suggests additional complexity (Chen et al., 2001; Dunn et al., 2002; Flasza et al., 2002; Itani et al., 2003). There are only limited data regarding the functional similarities and differences of the various Nedd4 family proteins and their isoforms. However, mice homozygous for a loss of function mutation in the itch gene develop a variety of inflammatory problems, indicating that the Itch protein has nonredundant functions (Hustad et al., 1995; Perry et al., 1998).

Domain architecture of the Nedd4 proteinsAlthough structural studies have not yet been reported for a full length Nedd4 protein, the individual domains have received considerable attention (Figure 2). The catalytic HECT domain was first revealed as a 40 kDa region of the protein E6 AP, a cellular enzyme recruited by the E6 oncoprotein of the human papillomavirus (HPV) associated with cervical cancers (Huibregtse et al., 1993a, 1993b). In these cancers, downregulation of the tumor suppressor protein p53 is an ubiquitin mediated process originating from the E6 induced association of p53 with E6 AP. Biochemical studies of the E6 AP HECT domain found that this region is necessary and sufficient for ubiquitin transfer and that a cysteine residue played a critical role (Scheffner et al., 1995). The mechanism involves initial binding of an ubiquitin conjugated E2 to the HECT domain with subsequent thiol ester exchange to transfer the ubiquitin moiety from the E2 to the catalytic cysteine in the E3. Transfer of ubiquitin then 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 amino group forming an isopeptide bond (Schwarz et al., 1998; Wang et al., 1999). (a) The two lobes that compose the HECT domain are shown in the E6 AP crystal structure (PDB 1D5F). The E2, UbcH7, binds to the N terminal lobe of the HECT domain in an orientation that would direct a conjugated ubiquitin molecule towards the catalytic cysteine within the E6AP C terminal lobe. (b) WW domains are formed by a three stranded anti parallel sheet as shown for the Nedd4 WW domain (PDB 1I5H). A ligand that contains a PPxY motif, forming a polyproline type II helix, binds primarily to one face of the WW domain sheet. (c) The loop regions of the C2 domain, found at one end of the sandwhich fold, form the binding site for calcium and phospholipids as shown in the Synaptotagmin 1 C2B domain (PDB 1K5W)

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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 UbcH7 with 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 region (Kishino et al., 1997; Jiang et al., 1999). However, the E6 AP/UbcH7 structure also presented a problem in that the position of the E3 catalytic cysteine is 40 distant from the E2 cysteine conjugate. Clearly, large conformational changes would be required to allow efficient thiol ester exchange to occur. The WWP1/AIP5 structure supported this notion, since the N and C terminal lobes were positioned such that the two cysteines would be closer together (16 Further, molecular modeling showed that this distance could be reduced to 5 if the relative orientation of the lobes were altered by rotation about the hinge loop (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 inhibited autoubiquitination. The inferred importance of the hinge has led to the proposal that the HECT domain functions as a ratchet to transfer ubiquitin from the E2 to the E3 catalytic cysteine. Motion about the hinge causes separation of the two lobes allowing the C terminal lobe to ubiquitinate the substrate while the N terminal lobe is recharged with an E2 ubiquitin conjugate.

Although these structural data have provided insight into the mechanism of action of the HECT domain, they have not definitively addressed how ubiquitin is transferred to the eventual substrate. It is unclear, for example, whether a polyubiquitin chain is formed by individual transfer of single ubiquitin molecules or whether a polyubiquitin chain is assembled on the E3 ligase and then directly added to the substrate. Recent data suggesting that some HECT domains may control the site of attachment on growing ubiquitin chains further complicates this issue. Proteins designated for proteasomal degradation are modified by attachment of a chain of ubiquitin molecules linked through lysine 48 of ubiquitin (Chan and Hill, 2001). For the HECT domain containing protein KIAA0010, assembly of ubiquitin chains linked through lysine 29 or 48 is possible (You and Pickart, 2001). Furthermore, Rsp5p can promote the formation of polyubiquitin chains linked through lysine 63 (Galan and Haguenauer Tsapis, 1997; Springael et al., 1999; Soetens et al., 2001). In addition, it is not known if the HECT domain plays a direct role in selecting substrates for mono or polyubiquitination.

The C2 domain and multiple WW domains N terminal to the HECT domain regulate cellular localization and substrate selection by the Nedd4 proteins. The WW domain, a small module consisting of approximately 35 amino acids and named for the presence of two conserved tryptophan residues spaced 20 amino acids apart, appears to mediate substrate recognition (reviewed in Zarrinpar and Lim (2000); Macias et al., 2002). Each family member has at least one WW domain with most possessing three or four modules located over a large middle region of the protein to form a substrate recognition scaffold.

WW domains are found in a variety of different proteins and have been shown to bind predominately proline rich motifs, including PPxY (amino acid single letter code; where x is any amino acid) (Chen and Sudol, 1995), PPLP (Bedford et al., 1997), PR (Bedford et al., 1998, 2000) and phosphoserine/threonine (pS/pT) residues that precede a proline residue (Yaffe et al., 1997; Lu et al., 1999). The Nedd4 family WW domains primarily recognize PPxY motifs but they have also been shown to bind to pS/pT residues (Lu et al., 1999) and may interact with other ligands (Qiu et al., 2000; Courbard et al., 2002; Murillas et al., 2002).

Several WW domain structures have been solved and the ligand binding determinants for the PPxY motif characterized (Figure 2b) (Kanelis et al., 1998: Huang et al., 2000b; Verdecia et al., 2000; Kanelis et al., 2001; Pires et al., 2001). WW domains possess a consensus three stranded anti parallel sheet fold that forms a hydrophobic ligand binding groove, together with a conserved binding pocket (termed the XP groove), which is generated by the juxtaposition of two large aromatic residues. The PPxY ligand yields a polyproline type II helix, and the two prolines are packed into the XP groove. The tyrosine makes several important interactions including a hydrogen bond with a conserved histidine residue as seen in the dystrophin WW domain (Huang et al., 2000b). This might explain the requirement for the tyrosine residue seen in biochemical studies (Chen and Sudol, 1995; Chen et al., 1997; Kasanov et al., 2001; Otte et al., 2003). Other interactions between the peptide and WW domain may also be important. For instance, the structure of the third WW domain of rat Nedd4 with a peptide from the ENaC channel shows an extended PPxY motif, PPxYxxL, binds the WW domain, with the leucine making important contacts. Binding studies support this result as a peptide with a leucine to alanine substitution displayed a six fold lower affinity than the wild type peptide (Henry et al., 2003).

The C2 domain was first identified in classical Protein Kinase C isoforms as a Ca2+ dependent phospholipid binding domain (reviewed in Rizo and Sudhof, 1998). It is approximately 120 amino acids and is composed of two four stranded sheets oriented to form an eight stranded sandwich structure (Figure 2c) (Sutton et al., 1995; Grobler et al., 1996; Essen et al., 1997; Perisic et al., 1998). Loop regions between the strands at the ‘top’ of the sandwich contain a number of conserved aspartate residues that are responsible for Ca2+ binding (Sutton et al., 1995; Essen et al., 1996; Shao et al., 1996). C2 domains have been shown to interact with a variety of phospholipids and proteins (Nalefski and Falke, 1996) and are thought to function as membrane recruitment domains involved in protein localization and trafficking. Indeed, the C2 domain is found in many proteins that require association with phospholipids at membrane surfaces for function, such as RasGAP, Synaptotagmin and several phospholipases (Ponting and Parker, 1996).

Physiological processes mediated by Nedd4 proteinsUbiquitin mediated processes are involved not only in the targeting of proteins for degradation by the 26S proteasome but also in the sorting of proteins at different steps in biosynthetic and endocytic pathways (Hicke, 2001; Bonifacino and Traub, 2003). Proteins at the plasma membrane are tagged with monoubiquitin to direct internalization. Within vesicles the ubiquitinated proteins are then transported to the late endosome and further sorted, in the multivesicular body, for recycling to the plasma membrane or destruction at the lysosome. Ubiquitin mediated protein sorting also occurs at the trans Golgi network where newly synthesized proteins, such as the amino acid permeases, are directed to the cell surface or targeted for destruction in the lysosome. The Nedd4 proteins participate in these processes through ubiquitination of target proteins.

While S. cerevisiae has only a single Nedd4 member, Rsp5p, other eukaryotes have several Nedd4 proteins that appear to have both redundant and specialized functions (Figure 3). Here, we describe one function of Rsp5p in the regulation of cell surface receptors in yeast. As examples of how the Nedd4 proteins function in mammals, we discuss how several Nedd4 proteins appear to operate in the downregulation of the epithelial sodium channel (ENaC) while Smurf1 and 2 have evolved specialized functions in the control of TGF signaling. Finally, we discuss how viruses have co opted the Nedd4 proteins to regulate various aspects of the viral life cycle. In particular, that Epstein virus (EBV) recruits Nedd4 proteins for the maintenance of viral latency in B cells and retroviruses utilize these proteins to bud from the cell. 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

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Regulation of yeast membrane proteinsRsp5p mediated regulation of cell surface receptors is one mechanism used by S. cerevisiae to adapt to environmental changes. For example, the Gap1p general amino acid permease is upregulated when it is necessary to use amino acids as a nitrogen source (reviewed in Magasanik and Kaiser, 2002). However, when yeast are grown on ammonia, Gap1p is degraded in an ubiquitin mediated manner (Springael and Andr 1998). Similarly, the factor receptor, Ste2p, the uracil permease, Fur4p, and several other cell surface receptors are regulated by ubiquitination in response to different stimuli (reviewed in Rotin et al., 2000).

The ubiquitination, internalization, and degradation of Ste2p, Gap1p, and Fur4p are all dependent on Rsp5p (Hein et al., 1995; Galan et al., 1996; Dunn and Hicke, 2001a). In the case of Ste2p and Fur4p, receptor degradation occurs not through the proteasome but via the lysosome like vacuole (Galan et al., 1996; Hicke and Riezman, 1996). In fact, in frame fusion of a single ubiquitin molecule to the C terminus of Ste2p is sufficient for internalization and degradation of the receptor (Terrell et al., 1998; Shih et al., 2000; Dunn and Hicke, 2001b), and monoubiquitination of Fur4p is also sufficient for internalization (Springael et al., 1999a). Fur4p and Gap1p are also polyubiquitinated (Galan and Haguenauer Tsapis, 1997; Springael et al., 1999a; Soetens et al., 2001). However, the polyubiquitin chains are not linked through lysine 48, which normally targets proteins to the proteasome, but are linked through lysine 63. This is consistent with polyubiquitinated Fur4p and Gap1p not being substrates of the proteasome. Furthermore, Rsp5p has functions in addition to ubiquitination of Ste2p, as the Ste2p/ubiquitin chimera still requires Rsp5p for internalization and degradation (Dunn and Hicke, 2001b).

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