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D Rotin

Publications and source records attributed to D Rotin.

At least 19 recordsLinked to original sources
PubMed2001

Nedd4 regulates ubiquitination and stability of the guanine-nucleotide exchange factor CNrasGEF.

Cyclic nucleotide ras GEF (CNrasGEF) is a guanine-nucleotide exchange factor previously isolated in a screen for Nedd4-WW domain interacting proteins (Pham, N., Cheglakov, I., Koch, C. A., de Hoog, C. L., Moran, M. F., and Rotin, D. (2000) Curr. Biol. 10, 555-558). It activates Ras in a cAMP-dependent manner and Rap-1 independent of cAMP. Here we show that CNrasGEF is a likely substrate of the ubiquitin protein ligase Nedd4. CNrasGEF possesses two PY motifs at its C terminus that are responsible for binding to Nedd4 in vitro. Moreover, Nedd4 and CNrasGEF co-immunoprecipitate from 293T cells expressing ectopic CNrasGEF and endogenous Nedd4, and this co-immunoprecipitation is abrogated in PY motif-mutated CNrasGEF (CNrasGEFDelta2PY). CNrasGEF is ubiquitinated in cells, and this ubiquitination is augmented upon overexpression of wt-Nedd4 but is inhibited in cells overexpressing a catalytically inactive Nedd4 (Nedd4(CS)) or in cells expressing CNrasGEFDelta2PY, which cannot bind Nedd4. Moreover, pulse-chase experiments have demonstrated that the half-life of CNrasGEF is reduced 5-fold (from approximately 10 to approximately 2 h) in cells co-expressing Nedd4 with CNrasGEF but not with CNrasGEFDelta2PY (t(0.5) approximately 14 h). CNrasGEF is also stabilized in cells co-expressing Nedd4(CS) or following treatment with lactacystin, indicating proteasomal degradation of this protein. Deletion/mutation of the CDC25 domain to abrogate Ras (or Rap-1) binding leads to impaired ubiquitination of CNrasGEF, suggesting that such binding is critical for ubiquitination. Treatment of cells with the cAMP analogue 8-bromo-cAMP does not affect the ability of CNrasGEF to bind Nedd4 nor its level of ubiquitination, suggesting that Ras binding per se and not its activation is the critical step in triggering ubiquitination of CNrasGEF. These results suggest that CNrasGEF is a substrate for Nedd4, which regulates its ubiquitination and stability in cells.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed2001

Solution structure of a Nedd4 WW domain-ENaC peptide complex.

Nedd4 is a ubiquitin protein ligase composed of a C2 domain, three (or four) WW domains and a ubiquitin ligase Hect domain. Nedd4 was demonstrated to bind the epithelial sodium channel (alphabetagammaENaC), by association of its WW domains with PY motifs (XPPXY) present in each ENaC subunit, and to regulate the cell surface stability of the channel. The PY motif of betaENaC is deleted or mutated in Liddle syndrome, a hereditary form of hypertension caused by elevated ENaC activity. Here we report the solution structure of the third WW domain of Nedd4 complexed to the PY motif-containing region of betaENaC (TLPIPGTPPPNYDSL, referred to as betaP2). A polyproline type II helical conformation is adopted by the PPPN sequence. Unexpectedly, the C-terminal sequence YDSL forms a helical turn and both the tyrosine and the C-terminal leucine contact the WW domain. This is unlike other proline-rich peptides complexed to WW domains, which bind in an extended conformation and lack molecular interactions with residues C-terminal to the tyrosine or the structurally equivalent residue in non-PY motif WW domain targets. The Nedd4 WW domain-ENaC betaP2 peptide structure expands our understanding of the mechanisms involved in WW domain-ligand recognition and the molecular basis of Liddle syndrome.

Amino Acid Motifs↗
PubMed2001

Trafficking and cell surface stability of ENaC.

The epithelial Na(+) channel (ENaC) plays a key role in the regulation of Na(+) and water absorption in several epithelia, including those of the distal nephron, distal colon, and lung. Accordingly, mutations in ENaC leading to reduced or increased channel activity cause human diseases such as pseudohypoaldosteronism type I or Liddle's syndrome, respectively. The gain of ENaC function in Liddle's syndrome is associated with increased activity and stability of the channel at the plasma membrane. Thus understanding the regulation of channel processing and trafficking to and stability at the cell surface is of fundamental importance. This review describes some of the recent advances in our understanding of ENaC trafficking, including the role of glycosylation, ENaC solubility in nonionic detergent, targeting signal(s) and hormones. It also describes the regulation of ENaC stability at the cell surface and the roles of the ubiquitin ligase Nedd4 (and ubiquitination) and clathrin-mediated endocytosis in that regulation.

Animals↗
PubMed2000

Ubiquitination and endocytosis of plasma membrane proteins: role of Nedd4/Rsp5p family of ubiquitin-protein ligases.

In addition to its well-known role in recognition by the proteasome, ubiquitin-conjugation is also involved in downregulation of membrane receptors, transporters and channels. In most cases, ubiquitination of these plasma membrane proteins leads to their internalization followed by targeting to the lysosome/vacuole for degradation. A crucial role in ubiquitination of many plasma membrane proteins appears to be played by ubiquitin-protein ligases of the Nedd4/Rsp5p family. All family members carry an N-terminal Ca2+-dependent lipid/protein binding (C2) domain, two to four WW domains and a C-terminal catalytic Hect-domain. Nedd4 is involved in downregulation of the epithelial Na+ channel, by binding of its WW domains to specific PY motifs of the channel. Rsp5p, the unique family member in S. cerevisiae, is involved in ubiquitin-dependent endocytosis of a great number of yeast plasma membrane proteins. These proteins lack apparent PY motifs, but carry acidic sequences, and/or phosphorylated-based sequences that might be important, directly or indirectly, for their recognition by Rsp5p. In contrast to polyubiquitination leading to proteasomal recognition, a number of Rsp5p targets carry few ubiquitins per protein, and moreover with a different ubiquitin linkage. Accumulating evidence suggests that, at least in yeast, ubiquitin itself may constitute an internalization signal, recognized by a hypothetical receptor. Recent data also suggest that Nedd4/Rsp5p might play a role in the endocytic process possibly involving its C2 domain, in addition to its role in ubiquitinating endocytosed proteins.

Animals↗
PubMed2000

Apical membrane targeting of Nedd4 is mediated by an association of its C2 domain with annexin XIIIb.

Nedd4 is a ubiquitin protein ligase (E3) containing a C2 domain, three or four WW domains, and a ubiquitin ligase HECT domain. We have shown previously that the C2 domain of Nedd4 is responsible for its Ca(2+)-dependent targeting to the plasma membrane, particularly the apical region of epithelial MDCK cells. To investigate this apical preference, we searched for Nedd4-C2 domain-interacting proteins that might be involved in targeting Nedd4 to the apical surface. Using immobilized Nedd4-C2 domain to trap interacting proteins from MDCK cell lysate, we isolated, in the presence of Ca(2+), a approximately 35-40-kD protein that we identified as annexin XIII using mass spectrometry. Annexin XIII has two known isoforms, a and b, that are apically localized, although XIIIa is also found in the basolateral compartment. In vitro binding and coprecipitation experiments showed that the Nedd4-C2 domain interacts with both annexin XIIIa and b in the presence of Ca(2+), and the interaction is direct and optimal at 1 microM Ca(2+). Immunofluorescence and immunogold electron microscopy revealed colocalization of Nedd4 and annexin XIIIb in apical carriers and at the apical plasma membrane. Moreover, we show that Nedd4 associates with raft lipid microdomains in a Ca(2+)-dependent manner, as determined by detergent extraction and floatation assays. These results suggest that the apical membrane localization of Nedd4 is mediated by an association of its C2 domain with the apically targeted annexin XIIIb.

Amino Acid Sequence↗
PubMed2000

The guanine nucleotide exchange factor CNrasGEF activates ras in response to cAMP and cGMP.

Small GTPase proteins such as Ras are key regulators of cellular proliferation and are activated by guanine nucleotide exchange/releasing factors (GEFs/GRFs). Three classes of Ras GRFs have been identified to date, represented by Sos1/2, Ras-GRF1/2 and Ras-GRP. Here, we describe a novel candidate Ras activator, cyclic nucleotide rasGEF (CNrasGEF), which contains CDC25, Ras exchange motif (REM), Ras-association (RA), PDZ and cNMP (cAMP/cGMP) binding (cNMP-BD) domains, two PY motifs and a carboxy-terminal SxV sequence. CNrasGEF can activate Ras in vitro, and it binds cAMP directly via its cNMP-BD. In cells, CNrasGEF activates Ras in response to elevation of intracellular cAMP or cGMP, or treatment with their analogues 8-Br-cAMP or 8-Br-cGMP, independently of protein kinases A and G (PKA and PKG). This activation is prevented in CNrasGEF lacking its CDC25 domain or cNMP-BD. CNrasGEF can also activate the small GTPase Rap1 in cells, but this activation is constitutive and independent of cAMP. CNrasGEF is expressed mainly in the brain and is localized at the plasma membrane, a localization dependent on the presence of intact PDZ domain but not the SxV sequence. These results suggest that CNrasGEF may directly connect cAMP-generating pathways or cGMP-generating pathways to Ras.

Animals↗
PubMed2000

Sequential assignment of proline-rich regions in proteins: application to modular binding domain complexes.

Many protein-protein interactions involve amino acid sequences containing proline-rich motifs and even polyproline stretches. The lack of amide protons in such regions complicates assignment, since 1HN-based triple-resonance assignment strategies cannot be employed. Two such systems that we are currently studying include an SH2 domain from the protein Crk with a region containing 9 prolines in a 14 amino acid sequence, as well as a WW domain that interacts with a proline-rich target. A modified version of the HACAN pulse scheme, originally described by Bax and co-workers [Wang et al. (1995) J. Biomol. NMR, 5, 376-382], and an experiment which correlates the intra-residue 1Halpha, 13Calpha/13Cbeta chemical shifts with the 15N shift of the subsequent residue are presented and applied to the two systems listed above, allowing sequential assignment of the molecules.

Amino Acid Sequence↗
PubMed2000

Regulation of the epithelial Na+ channel by Nedd4 and ubiquitination.

The epithelial Na+ channel (ENaC) is comprised of three subunits, alpha, beta and gamma, and plays an essential role in Na+ and fluid absorption in the kidney, colon and lung. We had identified proline-rich sequences at the C termini of alpha beta gamma ENaC, which include the sequence PPxY, the PY motif. This sequence in beta or gamma ENaC is deleted or mutated in Liddle's syndrome, a hereditary form of arterial hypertension. Our previous work demonstrated that these PY motifs bind to the WW domains of Nedd4, a ubiquitin protein ligase containing a C2 domain, three or four WW domains and a ubiquitin protein ligase Hect domain. Accordingly, we have recently demonstrated that Nedd4 regulates ENaC function by controlling the number of channels at the cell surface, that this regulation is impaired in ENaC bearing Liddle's syndrome mutations, and that ENaC stability and function are regulated by ubiquitination. The C2 domain is responsible for localizing Nedd4 to the plasma membrane in a Ca(2+)-dependent manner, and in polarized epithelial MDCK cells this localization is primarily apical. In accordance, electrophysiological characterization of ENaC expressed in MDCK cells revealed inhibition of channel activity by elevated intracellular Ca2+ levels. Thus, in response to Ca2+, Nedd4 may be mobilized to the apical membrane via its C2 domain, where it binds ENaC via Nedd4-WW:ENaC-PY motifs' interactions, leading to ubiquitination of the channel by the Nedd4-Hect domain and subsequent channel endocytosis and lysosomal degradation. This process may be at least partially impaired in Liddle's syndrome due to reduced Nedd4 binding, leading to increased retention of ENaC at the cell surface.

Animals↗
PubMed2000

Regulation of the epithelial sodium channel (ENaC) by accessory proteins.

The epithelial sodium channel (ENaC) plays a key role in the regulation of fluid absorption in the kidney, lung, colon and exocrine glands, and in the regulation of blood pressure. Abnormal functioning of ENaC is associated with several human diseases, including pseudohypoaldosteronism type I, Liddle's syndrome, pulmonary edema, and cystic fibrosis. ENaC is regulated by several hormones, ions and accessory proteins. This review focuses on the regulation of ENaC by recently described accessory proteins, mainly Nedd4, syntaxin 1A, CFTR, sgk, K-Ras2A and Cap-1.

Calcium-Binding Proteins↗
PubMed2000

Latent membrane protein 2A of Epstein-Barr virus binds WW domain E3 protein-ubiquitin ligases that ubiquitinate B-cell tyrosine kinases.

The latent membrane protein (LMP) 2A of Epstein-Barr virus (EBV) is implicated in the maintenance of viral latency and appears to function in part by inhibiting B-cell receptor (BCR) signaling. The N-terminal cytoplasmic region of LMP2A has multiple tyrosine residues that upon phosphorylation bind the SH2 domains of the Syk tyrosine kinase and the Src family kinase Lyn. The LMP2A N-terminal region also has two conserved PPPPY motifs. Here we show that the PPPPY motifs of LMP2A bind multiple WW domains of E3 protein-ubiquitin ligases of the Nedd4 family, including AIP4 and KIAA0439, and demonstrate that AIP4 and KIAA0439 form physiological complexes with LMP2A in EBV-positive B cells. In addition to a C2 domain and four WW domains, these proteins have a C-terminal Hect catalytic domain implicated in the ubiquitination of target proteins. LMP2A enhances Lyn and Syk ubiquitination in vivo in a fashion that depends on the activity of Nedd4 family members and correlates with destabilization of the Lyn tyrosine kinase. These results suggest that LMP2A serves as a molecular scaffold to recruit both B-cell tyrosine kinases and C2/WW/Hect domain E3 protein-ubiquitin ligases. This may promote Lyn and Syk ubiquitination in a fashion that contributes to a block in B-cell signaling. LMP2A may potentiate a normal mechanism by which Nedd4 family E3 enzymes regulate B-cell signaling.

Amino Acid Motifs↗
PubMed1999

mGrb10 interacts with Nedd4.

We have utilized the yeast two-hybrid system to identify proteins interacting with mouse Grb10, an adapter protein known to interact with both the insulin and the insulin-like growth factor-I receptors. We have isolated a mouse cDNA clone containing the C2 domain of mouse Nedd4, a ubiquitin protein ligase (E3) that also contains a hect (homologous to the E6-AP carboxyl-terminus) domain and three WW domains. The interaction with Grb10 in the two-hybrid system was confirmed using the full-length Nedd4, and it was abolished by deleting the last 148 amino acids of Grb10, a region that includes the SH2 domain and the newly identified BPS domain. The interaction between Grb10 and Nedd4 was also reproduced in vivo in mouse embryo fibroblasts, where endogenous Nedd4 co-immunoprecipitated constitutively with both the endogenous and an overexpressed Grb10. This interaction was Ca(2+)-independent. Grb10 interacting with Nedd4 was not ubiquitinated in vivo, raising the possibility that this interaction may be used to target other proteins, like tyrosine kinase receptors, for ubiquitination.

Animals↗
PubMed1999

Proline-rich motifs of the Na+/H+ exchanger 2 isoform. Binding of Src homology domain 3 and role in apical targeting in epithelia.

The NHE2 isoform of the Na+/H+ exchanger (NHE) displays two proline-rich sequences in its C-terminal region that resemble SH3 (Src homology 3)-binding domains. We investigated whether these regions (743PPSVTPAP750, termed Pro-1, and 786VPPKPPP792, termed Pro-2) can bind to SH3 domains and whether they are essential for NHE2 function and targeting. A fusion protein containing the Pro-1 region showed promiscuous binding to SH3 domains of several proteins in vitro, whereas a Pro-2 fusion bound preferentially to domains derived from kinases. In contrast, cytoplasmic regions of NHE1, NHE3, or NHE4 failed to interact. When expressed in antiporter-deficient cells, truncated NHE2 lacking both Pro-rich regions catalyzed Na+/H+ exchange, retained sensitivity to intracellular ATP, and was activated by hyperosmolarity, resembling full-length NHE2. The role of the Pro-rich regions in subcellular targeting was examined by transfection of epitope-tagged forms of NHE2 in porcine renal epithelial LLC-PK1 cells. Both full-length and Pro-2-truncated NHE2 localized almost exclusively to the apical membrane. By contrast, a mutant devoid of both Pro-1 and Pro-2 was preferentially sorted to the basolateral surface but also accumulated intracellularly. These observations indicate that the region encompassing Pro-1 is essential for appropriate subcellular targeting of NHE2.

Adenosine Triphosphate↗
PubMed1999

Neuronal defects and posterior pituitary hypoplasia in mice lacking the receptor tyrosine phosphatase PTPsigma.

The LAR-family protein tyrosine phosphatase sigma (PTPsigma, encoded by the gene Ptprs) consists of a cell adhesion-like extracellular domain composed of immunoglobulin and fibronectin type-III repeats, a single transmembrane domain and two intracellular catalytic domains. It was previously shown to be expressed in neuronal and lung epithelial tissues in a developmentally regulated manner. To study the role of PTPsigma in mouse development, we inactivated Ptprs by gene targeting. All Ptprs+/- mice developed normally, whereas 60% of Ptprs-/- mice died within 48 hours after birth. The surviving Ptprs-/- mice demonstrated stunted growth, developmental delays and severe neurological defects including spastic movements, tremor, ataxic gait, abnormal limb flexion and defective proprioception. Histopathology of brain sections revealed reduction and hypocellularity of the posterior pituitary of Ptprs-/- mice, as well as a reduction of approximately 50-75% in the number of choline acetyl transferase-positive cells in the forebrain. Moreover, peripheral nerve electrophysiological analysis revealed slower conduction velocity in Ptprs-/- mice relative to wild-type or heterozygous animals, associated with an increased proportion of slowly conducting, small-diameter myelinated fibres and relative hypomyelination. By approximately three weeks of age, most remaining Ptprs-/- mice died from a wasting syndrome with atrophic intestinal villi. These results suggest that PTPsigma has a role in neuronal and epithelial development in mice.

Age Factors↗
PubMed1999

Defective regulation of the epithelial Na+ channel by Nedd4 in Liddle's syndrome.

Liddle's syndrome is an inherited form of hypertension linked to mutations in the epithelial Na+ channel (ENaC). ENaC is composed of three subunits (alpha, beta, gamma), each containing a COOH-terminal PY motif (xPPxY). Mutations causing Liddle's syndrome alter or delete the PY motifs of beta- or gamma-ENaC. We recently demonstrated that the ubiquitin-protein ligase Nedd4 binds these PY motifs and that ENaC is regulated by ubiquitination. Here, we investigate, using the Xenopus oocyte system, whether Nedd4 affects ENaC function. Overexpression of wild-type Nedd4, together with ENaC, inhibited channel activity, whereas a catalytically inactive Nedd4 stimulated it, likely by acting as a competitive antagonist to endogenous Nedd4. These effects were dependant on the PY motifs, because no Nedd4-mediated changes in channel activity were observed in ENaC lacking them. The effect of Nedd4 on ENaC missing only one PY motif (of beta-ENaC), as originally described in patients with Liddle's syndrome, was intermediate. Changes were due entirely to alterations in ENaC numbers at the plasma membrane, as determined by surface binding and immunofluorescence. Our results demonstrate that Nedd4 is a negative regulator of ENaC and suggest that the loss of Nedd4 binding sites in ENaC observed in Liddle's syndrome may explain the increase in channel number at the cell surface, increased Na+ reabsorption by the distal nephron, and hence the hypertension.

Animals↗
PubMed1998

Electrophysiological characterization of the rat epithelial Na+ channel (rENaC) expressed in MDCK cells. Effects of Na+ and Ca2+.

The epithelial Na+ channel (ENaC), composed of three subunits (alpha, beta, and gamma), is expressed in several epithelia and plays a critical role in salt and water balance and in the regulation of blood pressure. Little is known, however, about the electrophysiological properties of this cloned channel when expressed in epithelial cells. Using whole-cell and single channel current recording techniques, we have now characterized the rat alpha beta gamma ENaC (rENaC) stably transfected and expressed in Madin-Darby canine kidney (MDCK) cells. Under whole-cell patch-clamp configuration, the alpha beta gamma rENaC-expressing MDCK cells exhibited greater whole cell Na+ current at -143 mV (-1,466.2 +/- 297.5 pA) than did untransfected cells (-47.6 +/- 10.7 pA). This conductance was completely and reversibly inhibited by 10 microM amiloride, with a Ki of 20 nM at a membrane potential of -103 mV; the amiloride inhibition was slightly voltage dependent. Amiloride-sensitive whole-cell current of MDCK cells expressing alpha beta or alpha gamma subunits alone was -115.2 +/- 41.4 pA and -52.1 +/- 24.5 pA at -143 mV, respectively, similar to the whole-cell Na+ current of untransfected cells. Relaxation analysis of the amiloride-sensitive current after voltage steps suggested that the channels were activated by membrane hyperpolarization. Ion selectivity sequence of the Na+ conductance was Li+ > Na+ >> K+ = N-methyl-D-glucamine+ (NMDG+). Using excised outside-out patches, amiloride-sensitive single channel conductance, likely responsible for the macroscopic Na+ channel current, was found to be approximately 5 and 8 pS when Na+ and Li+ were used as a charge carrier, respectively. K+ conductance through the channel was undetectable. The channel activity, defined as a product of the number of active channel (n) and open probability (Po), was increased by membrane hyperpolarization. Both whole-cell Na+ current and conductance were saturated with increased extracellular Na+ concentrations, which likely resulted from saturation of the single channel conductance. The channel activity (nPo) was significantly decreased when cytosolic Na+ concentration was increased from 0 to 50 mM in inside-out patches. Whole-cell Na+ conductance (with Li+ as a charge carrier) was inhibited by the addition of ionomycin (microM) and Ca2+ (1 mM) to the bath. Dialysis of the cells with a pipette solution containing 1 microM Ca2+ caused a biphasic inhibition, with time constants of 1.7 +/- 0.3 min (n = 3) and 128.4 +/- 33.4 min (n = 3). An increase in cytosolic Ca2+ concentration from <1 nM to 1 microM was accompanied by a decrease in channel activity. Increasing cytosolic Ca2+ to 10 microM exhibited a pronounced inhibitory effect. Single channel conductance, however, was unchanged by increasing free Ca2+ concentrations from <1 nM to 10 microM. Collectively, these results provide the first characterization of rENaC heterologously expressed in a mammalian epithelial cell line, and provide evidence for channel regulation by cytosolic Na+ and Ca2+.

Algorithms↗
PubMed1998

The second catalytic domain of protein tyrosine phosphatase delta (PTP delta) binds to and inhibits the first catalytic domain of PTP sigma.

The LAR family protein tyrosine phosphatases (PTPs), including LAR, PTP delta, and PTP sigma, are transmembrane proteins composed of a cell adhesion molecule-like ectodomain and two cytoplasmic catalytic domains: active D1 and inactive D2. We performed a yeast two-hybrid screen with the first catalytic domain of PTP sigma (PTP sigma-D1) as bait to identify interacting regulatory proteins. Using this screen, we identified the second catalytic domain of PTP delta (PTP delta-D2) as an interactor of PTP sigma-D1. Both yeast two-hybrid binding assays and coprecipitation from mammalian cells revealed strong binding between PTP sigma-D1 and PTP delta-D2, an association which required the presence of the wedge sequence in PTP sigma-D1, a sequence recently shown to mediate D1-D1 homodimerization in the phosphatase RPTP alpha. This interaction was not reciprocal, as PTP delta-D1 did not bind PTP sigma-D2. Addition of a glutathione S-transferase (GST)-PTP delta-D2 fusion protein (but not GST alone) to GST-PTP sigma-D1 led to approximately 50% inhibition of the catalytic activity of PTP sigma-D1, as determined by an in vitro phosphatase assay against p-nitrophenylphosphate. A similar inhibition of PTP sigma-D1 activity was obtained with coimmunoprecipitated PTP delta-D2. Interestingly, the second catalytic domains of LAR (LAR-D2) and PTP sigma (PTP sigma-D2), very similar in sequence to PTP delta-D2, bound poorly to PTP sigma-D1. PTP delta-D1 and LAR-D1 were also able to bind PTP delta-D2, but more weakly than PTP sigma-D1, with a binding hierarchy of PTP sigma-D1 >> PTP delta-D1 > LAR-D1. These results suggest that association between PTP sigma-D1 and PTP delta-D2, possibly via receptor heterodimerization, provides a negative regulatory function and that the second catalytic domains of this and likely other receptor PTPs, which are often inactive, may function instead to regulate the activity of the first catalytic domains.

Amino Acid Sequence↗
PubMed1998

NMR studies of tandem WW domains of Nedd4 in complex with a PY motif-containing region of the epithelial sodium channel.

Nedd4 (neuronal precursor cell-expressed developmentally down-regulated 4) is a ubiquitin-protein ligase containing multiple WW domains. We have previously demonstrated the association between the WW domains of Nedd4 and PPxY (PY) motifs of the epithelial sodium channel (ENaC). In this paper, we report the assignment of backbone 1H alpha, 1HN, 15N, 13C', 13C alpha, and aliphatic 13C resonances of a fragment of rat Nedd4 (rNedd4) containing the two C-terminal WW domains, WW(II+III), complexed to a PY motif-containing peptide derived from the beta subunit of rat ENaC, the betaP2 peptide. The secondary structures of these two WW domains, determined from chemical shifts of 13C alpha and 13C beta resonances, are virtually identical to those of the WW domains of the Yes-associated protein YAP65 and the peptidyl-prolyl isomerase Pin1. Triple resonance experiments that detect the 1H alpha chemical shift were necessary to complete the chemical shift assignment, owing to the large number of proline residues in this fragment of rNedd4. A new experiment, which correlates sequential residues via their 15N nuclei and also detects 1H alpha chemical shifts, is introduced and its utility for the chemical shift assignment of sequential proline residues is discussed. Data collected on the WW(II+III)-betaP2 complex indicate that these WW domains have different affinities for the betaP2 peptide.

Amino Acid Sequence↗