Regulation of the epithelial sodium channel by N4WBP5A,a novel Nedd4/Nedd4-2-interacting protein

The amiloride-sensitive epithelial sodium channel (ENaC) plays a critical role in fluid and electrolyte homeostasis and consists of alpha, beta, and gamma subunits. The carboxyl terminus of each ENaC subunit contains a PPXY motif that is believed to be important for interaction with the WW domains of the ubiquitin-protein ligases, Nedd4 and Nedd4-2. Disruption of this interaction, as in Liddle's syndrome where mutations delete or alter the PPXY motif of either the beta or gamma subunits, has been shown to result in increased ENaC activity and arterial hypertension. Here we present evidence that N4WBP5A, a novel Nedd4/Nedd4-2-binding protein, is a potential regulator of ENaC. In Xenopus laevis oocytes N4WBP5A increases surface expression of ENaC by reducing the rate of ENaC retrieval. We further demonstrate that N4WBP5A prevents sodium feedback inhibition of ENaC possibly by interfering with the xNedd4-2-mediated regulation of ENaC. As N4WBP5A binds Nedd4/Nedd4-2 via PPXY motif/WW domain interactions and appears to be associated with specific intracellular vesicles, we propose that N4WBP5A functions by regulating Nedd4/Nedd4-2 availability and trafficking. Because N4WBP5A is highly expressed in native renal collecting duct and other tissues that express ENaC, it is a likely candidate to modulate ENaC function in vivo.     

The Golgi-associated hook3 protein is a member of a novel family of microtubule-binding proteins

Microtubules are central to the spatial organization of diverse membrane-trafficking systems. Here, we report that Hook proteins constitute a novel family of cytosolic coiled coil proteins that bind to organelles and to microtubules. The conserved NH(2)-terminal domains of Hook proteins mediate attachment to microtubules, whereas the more divergent COOH-terminal domains mediate the binding to organelles. Human Hook3 bound to Golgi membranes in vitro and was enriched in the cis-Golgi in vivo. Unlike other cis-Golgi-associated proteins, however, a large fraction of Hook3 maintained its juxtanuclear localization after Brefeldin A treatment, indicating a Golgi-independent mechanism for Hook3 localization. Because overexpression of Hook3 caused fragmentation of the Golgi complex, we propose that Hook3 participates in defining the architecture and localization of the mammalian Golgi complex.     

Nedd4 family-interacting protein 1 (Ndfip1) is required for the exosomal secretion of Nedd4 family proteins

The ability to remove unwanted proteins is an important cellular feature. Classically, this involves the enzymatic addition of ubiquitin moieties followed by degradation in the proteasome. Nedd4 proteins are ubiquitin ligases important not only for protein degradation, but also for protein trafficking. Nedd4 proteins can bind to target proteins either by themselves or through adaptor protein Ndfip1 (Nedd4 family-interacting protein 1). An alternative mechanism for protein removal and trafficking is provided by exosomes, which are small vesicles (50-90-nm diameter) originating from late endosomes and multivesicular bodies (MVBs). Exosomes provide a rapid means of shedding obsolete proteins and also for cell to cell communication. In the present work, we show that Ndfip1 is detectable in exosomes secreted from transfected cells and also from primary neurons. Compared with control, Ndfip1 increases exosome secretion from transfected cells. Furthermore, while Nedd4, Nedd4-2, and Itch are normally absent from exosomes, expression of Ndfip1 results in recruitment of all three Nedd4 proteins into exosomes. Together, these results suggest that Ndfip1 is important for protein trafficking via exosomes, and provides a mechanism for cargoing passenger proteins such as Nedd4 family proteins. Given the positive roles of Ndfip1/Nedd4 in improving neuronal survival during brain injury, it is possible that exosome secretion provides a novel route for rapid sequestration and removal of proteins during stress.     

Alternative splicing determines the domain structure of WWP1, a Nedd4 family protein.

Nedd-4-like proteins are E3 ubiquitin-ligase molecules which regulate key trafficking decisions, including targeting of proteins to proteosomes or lysosomes. Here we show that a human Nedd4 family gene, WWP1, is localized on 8q21 and generates at least six isoforms through alternative splicing. We show that alternative splicing affects the domain structure of WWP1, with forms that contain or lack an N-terminal C2 domain. Interestingly, the relative ratio of these forms varies in a tissue-specific manner. Other splice forms were also identified which may disrupt the structure of the C2 domain by removing its predicted C-terminal beta-strands. One splice form generates, through the introduction of a reading frame shift, a C2 domain-only form of WWP1. We discuss the hypothesis that regulation of splice site usage may modulate the activity of WWP1 and possibly other Nedd4 family proteins.     

The Nedd4-like protein KIAA0439 is a potential regulator of the epithelial sodium channel

The amiloride-sensitive epithelial sodium channel (ENaC) plays a critical role in fluid and electrolyte homeostasis and consists of alpha, beta, and gamma subunits. The carboxyl terminus of each ENaC subunit contains a PPxY, motif which is believed to be important for interaction with the WW domains of the ubiquitin-protein ligase, Nedd4. Disruption of this interaction, as in Liddle's syndrome, where mutations delete or alter the PPxY motif of either the beta or gamma subunits, has been proposed to result in increased ENaC activity. Here we present evidence that KIAA0439 protein, a close relative of Nedd4, is also a potential regulator of ENaC. We demonstrate that KIAA0439 WW domains bind all three ENaC subunits. We show that a recombinant KIAA0439 WW domain protein acts as a dominant negative mutant that can interfere with the Na(+)-dependent feedback inhibition of ENaC in whole-cell patch clamp experiments. We propose that KIAA0439 and Nedd4 proteins either play a redundant role in ENaC regulation or function in a tissue- and/or signal-specific manner to down-regulate ENaC.     

Mouse whey acidic protein is a novel member of the family of 'four-disulfide core' proteins.

Unlike in other mammalian species, the major whey protein in mouse is not alpha-lactalbumin, but a cysteine rich, acidic protein with a molecular weight of 14.0 kDa. We have deduced the amino acid sequence of this mouse acidic of whey protein from the nucleotide sequence of cloned cDNA. The positions of the half cysteines suggest that mouse whey acidic protein (WAP) is a two domain protein, very similar in structure to the plant lectin wheat germ agglutinin and the hypothalamic carrier protein neurophysin.     

Transport of LAPTM5 to lysosomes requires association with the ubiquitin ligase Nedd4, but not LAPTM5 ubiquitination

LAPTM5 is a lysosomal transmembrane protein expressed in immune cells. We show that LAPTM5 binds the ubiquitin-ligase Nedd4 and GGA3 to promote LAPTM5 sorting from the Golgi to the lysosome, an event that is independent of LAPTM5 ubiquitination. LAPTM5 contains three PY motifs (L/PPxY), which bind Nedd4-WW domains, and a ubiquitin-interacting motif (UIM) motif. The Nedd4-LAPTM5 complex recruits ubiquitinated GGA3, which binds the LAPTM5-UIM; this interaction does not require the GGA3-GAT domain. LAPTM5 mutated in its Nedd4-binding sites (PY motifs) or its UIM is retained in the Golgi, as is LAPTM5 expressed in cells in which Nedd4 or GGA3 is knocked-down with RNAi. However, ubiquitination-impaired LAPTM5 can still traffic to the lysosome, suggesting that Nedd4 binding to LAPTM5, not LAPTM5 ubiquitination, is required for targeting. Interestingly, Nedd4 is also able to ubiquitinate GGA3. These results demonstrate a novel mechanism by which the ubiquitin-ligase Nedd4, via interactions with GGA3 and cargo (LAPTM5), regulates cargo trafficking to the lysosome without requiring cargo ubiquitination.     

Bap31 is a novel target of the human papillomavirus E5 protein

The E5 proteins of human papillomaviruses (HPVs) are small hydrophobic proteins that are expressed in the early and late stages of the viral life cycle; however, their role in HPV pathogenesis is not clearly understood. In this study, a split-ubiquitin yeast (Saccharomyces cerevisiae) two-hybrid system was used to identify B-cell-associated protein 31 (Bap31) as a binding partner of HPV E5 proteins. The association of these proteins was confirmed by coimmunoprecipitation of complexes of Bap31 with either HPV type 16 (HPV16) or HPV31 E5. In addition, Bap31 and E5 were found to colocalize in perinuclear patterns consistent with localization to the endoplasmic reticulum. Mutational analysis of E5 identified amino acids in the extreme C terminus as important for stabilizing the interaction with Bap31. Deletion of these C-terminal amino acids of E5 in the context of complete HPV31 genomes resulted in impaired proliferative capacity of HPV-positive keratinocytes following differentiation. When small interfering RNAs were used to reduce the levels of Bap31, the proliferative ability of HPV-positive keratinocytes upon differentiation was also reduced, implicating Bap31 as a regulator of this process. These studies identify a novel binding partner of the high-risk HPV E5 proteins and provide insight into how the E5 proteins may modulate the life cycle in differentiating cells.     

gamma2-Adaptin, a ubiquitin-interacting adaptor, is a substrate to coupled ubiquitination by the ubiquitin ligase Nedd4 and functions in the endosomal pathway

gamma2-Adaptin is a putative member of the clathrin adaptor protein family with unknown physiological function. We previously reported that gamma2-adaptin acts as a ubiquitin receptor by virtue of its ubiquitin-interacting motif. Here we demonstrate that this motif mediates a specific physical interaction with the ubiquitin ligase Nedd4 and promotes ubiquitination of gamma2-adaptin. By mapping regions of Nedd4 involved in binding to gamma2-adaptin, we identified its C2 domain to be essential, whereas the WW and HECT domains are dispensable. Consistent with this, we uncovered that the C2 domain of Nedd4 is ubiquitinated itself and as such is recruited by the ubiquitin-interacting motif of gamma2-adaptin for subsequent ubiquitin conjugation. Unlike known coupled ubiquitination reactions, this novel type of interaction leads to mono- and multi/polyubiquitinated gamma2-adaptin. In addition, we show that gamma2-adaptin functions in the endosomal/multivesicular body (MVB) pathway. Depletion of gamma2-adaptin impairs the degradation of internalized epidermal growth factor and results in defective MVB morphology characterized by significantly enlarged vesicles. These defects cannot be rescued by gamma1-adaptin, a closely related homolog of gamma2-adaptin, which is unable to bind ubiquitin. Together, these results indicate that gamma2-adaptin may operate within the MVB sorting system in a manner different from that of classic adaptins.     

Molecular determinants of voltage-gated sodium channel regulation by the Nedd4/Nedd4-like proteins

The voltage-gated Na⁺ channels (Na_v) form a family composed of 10 genes. The COOH termini of Na_v contain a cluster of amino acids that are nearly identical among 7 of the 10 members. This COOH-terminal sequence, PPSYDSV, is a PY motif known to bind to WW domains of E3 protein-ubiquitin ligases of the Nedd4 family. We recently reported that cardiac Na_v1.5 is regulated by Nedd4-2. In this study, we further investigated the molecular determinants of regulation of Na_v proteins. When expressed in HEK-293 cells and studied using whole cell voltage clamping, the neuronal Na_v1.2 and Na_v1.3 were also downregulated by Nedd4-2. Pull-down experiments using fusion proteins bearing the PY motif of Na_v1.2, Na_v1.3, and Na_v1.5 indicated that mouse brain Nedd4-2 binds to the Na_v PY motif. Using intrinsic tryptophan fluorescence imaging of WW domains, we found that Na_v1.5 PY motif binds preferentially to the fourth WW domain of Nedd4-2 with a K_d of 55 µM. We tested the binding properties and the ability to ubiquitinate and downregulate Na_v1.5 of three Nedd4-like E3s: Nedd4-1, Nedd4-2, and WWP2. Despite the fact that along with Nedd4-2, Nedd4-1 and WWP2 bind to Na_v1.5 PY motif, only Nedd4-2 robustly ubiquitinated and downregulated Na_v1.5. Interestingly, coexpression of WWP2 competed with the effect of Nedd4-2. Finally, using brefeldin A, we found that Nedd4-2 accelerated internalization of Na_v1.5 stably expressed in HEK-293 cells. This study shows that Nedd4-dependent ubiquitination of Na_v channels may represent a general mechanism regulating the excitability of neurons and myocytes via modulation of channel density at the plasma membrane. ubiquitin; Nedd4-2; PY motif; Na_v1.5; human ether-à-go-go-related gene     

STOP-like protein 21 is a novel member of the STOP family, revealing a Golgi localization of STOP proteins

Neuronal microtubules are stabilized by two calmodulin-regulated microtubule-associated proteins, E-STOP and N-STOP, which when suppressed in mice induce severe synaptic and behavioral deficits. Here we show that mature neurons also contain a 21-kDa STOP-like protein, SL21, which shares calmodulin-binding and microtubule-stabilizing homology domains with STOP proteins. Accordingly, in different biochemical or cellular assays, SL21 has calmodulin binding and microtubule stabilizing activity. However, in cultured hippocampal neurons, SL21 antibodies principally stain the somatic Golgi and punctate Golgi material in neurites. In cycling cells, transfected SL21 decorates microtubules when expressed at high levels but is otherwise principally visible at the Golgi. The Golgi targeting of SL21 depends on the presence of cysteine residues located within the SL21 N-terminal domain, suggesting that Golgi targeting may require SL21 palmitoylation. Accordingly we find that SL21 is palmitoylated in vivo. N-STOP and E-STOP, which contain the Golgi targeting sequences present in SL21, also display distinct Golgi staining when expressed at low level in cycling cells. Thus neuronal proteins of the STOP family have the capacity to associate with Golgi material, which could be important for STOP synaptic functions.     

The direct p53 target gene, FLJ11259/DRAM, is a member of a novel family of transmembrane proteins

The tumor suppressor p53 regulates diverse biological processes primarily via activation of downstream target genes. Even though many p53 target genes have been described, the precise mechanisms of p53 biological actions are uncertain. In previous work we identified by microarray analysis a candidate p53 target gene, FLJ11259/DRAM. In this report we have identified three uncharacterized human proteins with sequence homology to FLJ11259, suggesting that FLJ11259 is a member of a novel family of proteins with six transmembrane domains. Several lines of investigation confirm FLJ11259 is a direct p53 target gene. p53 siRNA prevented cisplatin-mediated up-regulation of FLJ11259 in NT2/D1 cells. Likewise in HCT116 p53+/+ cells and MCF10A cells, FLJ11259 is induced by cisplatin treatment but to a much lesser extent in isogenic p53-suppressed cells. A functional p53 response element was identified 22.3 kb upstream of the first coding exon of FLJ11259 and is shown to be active in reporter assays. In addition, chromatin immunoprecipitation assays indicate that p53 binds directly to this element in vivo and that binding is enhanced following cisplatin treatment. Confocal microscopy showed that an FLJ-GFP fusion protein localizes mainly in a punctate pattern in the cytoplasm. Overexpression studies in Cos-7, Saos2, and NT2/D1 cells suggest that FLJ11259 is associated with increased clonal survival. In summary, we have identified FLJ11259/DRAM as a p53-inducible member of a novel family of transmembrane proteins. FLJ11259/DRAM may be an important modulator of p53 responses in diverse tumor types.     

The direct p53 target gene, FLJ11259/DRAM, is a member of a novel family of transmembrane proteins

The tumor suppressor p53 regulates diverse biological processes primarily via activation of downstream target genes. Even though many p53 target genes have been described, the precise mechanisms of p53 biological actions are uncertain. In previous work we identified by microarray analysis a candidate p53 target gene, FLJ11259/DRAM. In this report we have identified three uncharacterized human proteins with sequence homology to FLJ11259, suggesting that FLJ11259 is a member of a novel family of proteins with six transmembrane domains. Several lines of investigation confirm FLJ11259 is a direct p53 target gene. p53 siRNA prevented cisplatin-mediated up-regulation of FLJ11259 in NT2/D1 cells. Likewise in HCT116 p53+/+ cells and MCF10A cells, FLJ11259 is induced by cisplatin treatment but to a much lesser extent in isogenic p53-suppressed cells. A functional p53 response element was identified 22.3 kb upstream of the first coding exon of FLJ11259 and is shown to be active in reporter assays. In addition, chromatin immunoprecipitation assays indicate that p53 binds directly to this element in vivo and that binding is enhanced following cisplatin treatment. Confocal microscopy showed that an FLJ-GFP fusion protein localizes mainly in a punctate pattern in the cytoplasm. Overexpression studies in Cos-7, Saos2, and NT2/D1 cells suggest that FLJ11259 is associated with increased clonal survival. In summary, we have identified FLJ11259/DRAM as a p53-inducible member of a novel family of transmembrane proteins. FLJ11259/DRAM may be an important modulator of p53 responses in diverse tumor types.     

Regulation of the voltage-gated K(+) channels KCNQ2/3 and KCNQ3/5 by ubiquitination. Novel role for Nedd4-2

The muscarine-sensitive K(+) current (M-current) stabilizes the resting membrane potential in neurons, thus limiting neuronal excitability. The M-current is mediated by heteromeric channels consisting of KCNQ3 subunits in association with either KCNQ2 or KCNQ5 subunits. The role of KCNQ2/3/5 in the regulation of neuronal excitability is well established; however, little is known about the mechanisms that regulate the cell surface expression of these channels. Ubiquitination by the Nedd4/Nedd4-2 ubiquitin ligases is known to regulate a number of membrane ion channels and transporters. In this study, we investigated whether Nedd4/Nedd4-2 could regulate KCNQ2/3/5 channels. We found that the amplitude of the K(+) currents mediated by KCNQ2/3 and KCNQ3/5 were reduced by Nedd4-2 (but not Nedd4) in a Xenopus oocyte expression system. Deletion experiments showed that the C-terminal region of the KCNQ3 subunit is required for the Nedd4-2-mediated regulation of the heteromeric channels. Glutathione S-transferase fusion pulldowns and co-immunoprecipitations demonstrated a direct interaction between KCNQ2/3 and Nedd4-2. Furthermore, Nedd4-2 could ubiquitinate KCNQ2/3 in transfected cells. Taken together, these data suggest that Nedd4-2 is potentially an important regulator of M-current activity in the nervous system.     

A novel mouse Nedd4 protein suppresses the activity of the epithelial Na+ channel.

Liddle's syndrome is a form of inherited hypertension linked to mutations in the genes encoding the epithelial Na+ channel (ENaC). These mutations alter or delete PY motifs involved in protein-protein interactions with a ubiquitin-protein ligase, Nedd4. Here we show that Na+ transporting cells, derived from mouse cortical collecting duct, express two Nedd4 proteins with different structural organization and characteristics of ENaC regulation: 1) the classical Nedd4 (herein referred to as Nedd4-1) containing one amino-terminal C2, three WW, and one HECT-ubiquitin protein ligase domain and 2) a novel Nedd4 protein (Nedd4-2), homologous to Xenopus Nedd4 and comprising four WW, one HECT, yet lacking a C2 domain. Nedd4-2, but not Nedd4-1, inhibits ENaC activity when coexpressed in Xenopus oocytes and this property correlates with the ability to bind to ENaC, as only Nedd4-2 coimmunoprecipitates with ENaC. Furthermore, this interaction depends on the presence of at least one PY motif in the ENaC complex and on WW domains 3 and 4 in Nedd4-2. Thus, these results suggest that the novel suppressor protein Nedd4-2 is the regulator of ENaC and hence a potential susceptibility gene for arterial hypertension.     

Cloning and characterization of DIP1, a novel protein that is related to the Id family of proteins

Using human cyclin D1 as the "bait" in a yeast two-hybrid system, together with a HL60 cDNA library, we identified a novel human nuclear protein designated DIP1. This protein is expressed in a variety of cell types, and in fibroblasts its level remains constant throughout the cell cycle. However, the level of this protein increases severalfold during the differentiation of HL60 cells. The DIP1 protein can be phosphorylated in vitro by a cellular kinase and this activity reaches its maximum in extracts obtained from cells in the G1 phase of the cell cycle. DIP1 contains a helix-loop-helix motif but lacks an adjacent basic DNA-binding domain, thus resembling the Id family of proteins. The dip1 gene is located on human chromosome 16p11.2-12, a locus that is amplified in several types of human cancer. These results suggest that DIP1 may be involved in the control of gene expression and differentiation, but its precise function remains to be determined.     

Target-assisted iterative screening reveals novel interactors for PSD95, Nedd4, Src,Abl and Crk proteins

A new in vitro screening method has been developed and applied to a commercial phage-displayed cDNA library to search for novel protein-protein interactions. PDZ, WW and SH3 domains from PSD95, Nedd4, Src, Abl and Crk proteins were used as targets. 12 novel putative and 2 previously reported interactions were identified in test screens. The novel screening format, dubbed TAIS (target-assisted iterative screening), is discussed as an alternative platform to existing technologies for a pair-wise characterization of protein-protein interactions.