A single WW domain is the predominant mediator of the interaction between the human ubiquitin-protein ligase Nedd4 and the human epithelial sodium channel

2002 ◽  
Vol 361 (3) ◽  
pp. 481-488 ◽  
Author(s):  
J. Shaun LOTT ◽  
Sarah J. CODDINGTON-LAWSON ◽  
Paul H. TEESDALE-SPITTLE ◽  
Fiona J. MCDONALD
Keyword(s):  
The Body ◽  
Single Amino Acid ◽  
Na Channel ◽  
Α Subunit ◽  
Ww Domain ◽  
Ww Domains ◽  
Ubiquitin Protein Ligase ◽  
Single Amino Acid Change ◽  
Domain 3 ◽  
Neuronal Precursor Cell

The activity of the epithelial Na+ channel (ENaC) is required for the maintenance of salt and water balance in the body. Channel activity is regulated by the ubiquitin-protein ligase Nedd4 ['neuronal precursor cell-expressed developmentally down-regulated (gene 4)'] that interacts with the channel via its WW domains. Mutations in channel subunits that disrupt this interaction cause Liddle's syndrome, a severe inherited form of hypertension. In previous studies we showed that WW domains 2, 3 and 4 of human Nedd4 bound to the human ENaC (hENaC) subunits, whereas WW domain 1 did not. Here we extend this observation to determine the binding affinities of the human Nedd4 WW domains for hENaC C-terminal peptides. We show that WW domains 2, 3 and 4 bind with differing affinities to Na+ channel subunit peptides. WW domain 3 has the highest affinity and we predict that WW domain 3 contributes most of the binding because a construct containing the three WW domains bound no better than WW domain 3 alone. Further, a single amino acid change (Arg165 → Thr) in WW domain 1 enables binding to the α subunit of the channel to occur, with an affinity comparable with that of WW domain 4. Differential binding propensities between the various WW domains and Na+ channel subunit peptides are explained on the basis of quantitative structural modelling of the complexes and their isolated components.

scholarly journals Human Nedd4 interacts with the human epithelial Na+ channel: WW3 but not WW1 binds to Na+-channel subunits

2000 ◽  
Vol 345 (3) ◽  
pp. 503-509 ◽  
Author(s):  
Tracy J. FARR ◽  
Sarah J. CODDINGTON-LAWSON ◽  
Peter M. SNYDER ◽  
Fiona J. MCDONALD
Keyword(s):  
Binding Sites ◽  
Xenopus Oocytes ◽  
Na Channel ◽  
Channel Activity ◽  
Α Subunit ◽  
Ww Domains ◽  
Ubiquitin Protein Ligase ◽  
Human Lung Cells ◽  
Epithelial Na Channel ◽  
Proline Rich Region

The epithelial Na+ channel (ENaC) regulates Na+ absorption in epithelial tissues including the lung, colon and sweat gland, and in the distal nephrons of the kidney. When Na+-channel function is disrupted, salt and water homoeostasis is affected. The cytoplasmic regions of the Na+-channel subunits provide binding sites for other proteins to interact with and potentially regulate Na+-channel activity. Previously we showed that a proline-rich region of the α subunit of the Na+ channel bound to a protein of 116 kDa from human lung cells. Here we report the identification of this protein as human Nedd4, a ubiquitin-protein ligase that binds to the Na+-channel subunits via its WW domains. Further, we show that WW domains 2, 3 and 4 of human Nedd4 bind to the α, β and γ Na+-channel subunits but not to a mutated β subunit. In addition, when co-expressed in Xenopus oocytes, human Nedd4 down-regulates Na+-channel activity.

scholarly journals Identification of multiple proteins expressed in murine embryos as binding partners for the WW domains of the ubiquitin-protein ligase Nedd4

2000 ◽  
Vol 351 (3) ◽  
pp. 557-565 ◽  
Author(s):  
Corina N. JOLLIFFE ◽  
Kieran F. HARVEY ◽  
Bryan P. HAINES ◽  
Gayathri PARASIVAM ◽  
Sharad KUMAR
Keyword(s):  
Lipid Binding ◽  
Site Directed Mutagenesis ◽  
Mrna Levels ◽  
Developmentally Regulated ◽  
Ww Domain ◽  
Ww Domains ◽  
Ubiquitin Protein Ligase ◽  
Embryonic Tissues ◽  
Binding Partners ◽  
Protein Ligases

Nedd4 is a member of a growing family of ubiquitin-protein ligases which consist of a lipid-binding domain, two to four WW domains and a C-terminal ubiquitin-protein ligase domain. The Nedd4 mRNA levels are developmentally regulated and Nedd4 protein is highly expressed in many mouse embryonic tissues. In this study we have used a far-Western screen to identify embryonic proteins that interact with the WW domains in mouse Nedd4. We report here identification of eight Nedd4 WW-domain-interacting proteins from mouse embryonic cDNA expression libraries. Two of the proteins are novel, while two have been identified previously as ligands for a WW domain. All of these proteins contain one or more PY motifs. In seven of the eight proteins, these PY motifs are necessary for their interaction with the WW domains of Nedd4. Using site-directed mutagenesis, and by using individual WW domains of Nedd4 as probes for far-Western analysis, we show that the three WW domains in Nedd4 interact with varying affinities with the PY motifs present in various Nedd4-binding proteins. These results provide evidence that Nedd4 can potentially interact with multiple proteins, possibly simultaneously, through its WW domains.

Ubiquitin-protein ligase WWP2 binds to and downregulates the epithelial Na+ channel

2002 ◽  
Vol 283 (3) ◽  
pp. F431-F436 ◽  
Author(s):  
Fiona J. McDonald ◽  
Andrea H. Western ◽  
John D. McNeil ◽  
Brittany C. Thomas ◽  
Diane R. Olson ◽  
...  
Keyword(s):  
Functional Data ◽  
Direct Interaction ◽  
Na Channel ◽  
Critical Component ◽  
Ww Domains ◽  
Ubiquitin Protein Ligase ◽  
Salt And Water Balance ◽  
Protein Ligases ◽  
Epithelial Na Channel ◽  
Genetic Form

The epithelial Na+ channel (ENaC) is a critical component of the pathway maintaining salt and water balance. The channel is regulated by members of the Nedd4 family of ubiquitin-protein ligases, which bind to channel subunits and catalyze channel internalization and degradation. ENaC mutations that abolish this interaction cause Liddle's syndrome, a genetic form of hypertension. Here, we test the hypothesis that WW domain-containing protein 2 (WWP2), a member of the Nedd4 family of ubiquitin-protein ligases, is a candidate to regulate ENaC. Consistent with this hypothesis, we found that WWP2 is expressed in epithelial tissues that express ENaC, as well as in a wide variety of other tissues. WWP2 contains four WW domains, three of which bound differentially to ENaC subunits. In contrast, all four human Nedd4–2 WW domains bound to ENaC. WWP2 inhibited ENaC when coexpressed in epithelia, requiring a direct interaction between the proteins; mutation of the ENaC PY motifs abolished inhibition. Thus expression, binding, and functional data all suggest that WWP2 is a candidate to regulate ENaC-mediated Na+ transport in epithelia.

Distinct characteristics of two human Nedd4 proteins with respect to epithelial Na+ channel regulation

2001 ◽  
Vol 281 (3) ◽  
pp. F469-F477 ◽  
Author(s):  
Elena Kamynina ◽  
Caroline Tauxe ◽  
Olivier Staub
Keyword(s):  
Close Relative ◽  
Na Channel ◽  
Domain Interaction ◽  
Hect Domain ◽  
Ww Domains ◽  
Ubiquitin Protein Ligase ◽  
Activity Structure ◽  
Py Motif ◽  
Epithelial Na Channel ◽  
Salt Homeostasis

The epithelial Na+ channel (ENaC) is regulated via PY motif-WW domain interaction by the mouse (m) ubiquitin-protein ligase mNedd4-2 but not by its close relative mNedd4-1. Whereas mNedd4-1 is composed of one C2, three WW, and one HECT domain, mNedd4-2 comprises four WW domains and one HECT domain. Both proteins have human (h) homologs, hNedd4-1 and hNedd4-2; however, both of them include four WW domains. Therefore, we characterized hNedd4-1 and hNedd4-2 in Xenopus laevisoocytes with respect to ENaC binding and interaction. We found that hNedd4-2 binds to and abrogates ENaC activity, whereas hNedd4-1 does not coimmunoprecipitate with ENaC and has only modest effects on ENaC activity. Structure-function studies revealed that the C2 domain of hNedd4-1 prevents this protein from downregulating ENaC and that WW domains 3 and 4, involved in interaction with ENaC, do not by themselves provide specificity for ENaC recognition. Taken together, our data demonstrate that hNedd4-2 inhibits ENaC, implying that this protein is a modulator of salt homeostasis, whereas hNedd4-1 is not primarily involved in ENaC regulation.

scholarly journals Single amino acid changes in the mumps virus haemagglutinin–neuraminidase and polymerase proteins are associated with neuroattenuation

2009 ◽  
Vol 90 (7) ◽  
pp. 1741-1747 ◽  
Author(s):  
Tahir H. Malik ◽  
Candie Wolbert ◽  
Laura Nerret ◽  
Christian Sauder ◽  
Steven Rubin
Keyword(s):  
Amino Acid ◽  
Clinical Isolate ◽  
Amino Acid Change ◽  
Mumps Virus ◽  
Site Directed Mutagenesis ◽  
Single Amino Acid ◽  
Supporting Evidence ◽  
L Protein ◽  
Single Amino Acid Change

It has previously been shown that three amino acid changes, one each in the fusion (F; Ala/Thr-91→Thr), haemagglutinin–neuraminidase (HN; Ser-466→Asn) and polymerase (L; Ile-736→Val) proteins, are associated with attenuation of a neurovirulent clinical isolate of mumps virus (88-1961) following serial passage in vitro. Here, using full-length cDNA plasmid clones and site-directed mutagenesis, it was shown that the single amino acid change in the HN protein and to a lesser extent, the change in the L protein, resulted in neuroattenuation, as assessed in rats. The combination of both amino acid changes caused neuroattenuation of the virus to levels previously reported for the clinical isolate following attenuation in vitro. The amino acid change in the F protein, despite having a dramatic effect on protein function in vitro, was previously shown to not be involved in the observed neuroattenuation, highlighting the importance of conducting confirmatory in vivo studies. This report provides additional supporting evidence for the role of the HN protein as a virulence factor and, as far as is known, is the first report to associate an amino acid change in the L protein with mumps virus neuroattenuation.

Genetic Analysis of Caenorhabditis elegans glp-1 Mutants Suggests Receptor Interaction or Competition

Genetics ◽  
2003 ◽  
Vol 163 (1) ◽  
pp. 115-132 ◽  
Author(s):  
Anita S-R Pepper ◽  
Darrell J Killian ◽  
E Jane Albert Hubbard
Keyword(s):  
Transmembrane Domain ◽  
Limiting Factors ◽  
Single Amino Acid ◽  
Receptor Interaction ◽  
Gain Of Function ◽  
Ligand Interaction ◽  
Receptor Proteins ◽  
C Elegans ◽  
Single Amino Acid Change ◽  
Functional Defect

Abstract glp-1 encodes a member of the highly conserved LIN-12/Notch family of receptors that mediates the mitosis/meiosis decision in the C. elegans germline. We have characterized three mutations that represent a new genetic and phenotypic class of glp-1 mutants, glp-1(Pro). The glp-1(Pro) mutants display gain-of-function germline pattern defects, most notably a proximal proliferation (Pro) phenotype. Each of three glp-1(Pro) alleles encodes a single amino acid change in the extracellular part of the receptor: two in the LIN-12/Notch repeats (LNRs) and one between the LNRs and the transmembrane domain. Unlike other previously described gain-of-function mutations that affect this region of LIN-12/Notch family receptors, the genetic behavior of glp-1(Pro) alleles is not consistent with simple hypermorphic activity. Instead, the mutant phenotype is suppressed by wild-type doses of glp-1. Moreover, a trans-heterozygous combination of two highly penetrant glp-1(Pro) mutations is mutually suppressing. These results lend support to a model for a higher-order receptor complex and/or competition among receptor proteins for limiting factors that are required for proper regulation of receptor activity. Double-mutant analysis with suppressors and enhancers of lin-12 and glp-1 further suggests that the functional defect in glp-1(Pro) mutants occurs prior to or at the level of ligand interaction.

scholarly journals Viruses go modular

2020 ◽  
Vol 295 (14) ◽  
pp. 4604-4616 ◽  
Author(s):  
Ariel Shepley-McTaggart ◽  
Hao Fan ◽  
Marius Sudol ◽  
Ronald N. Harty
Keyword(s):  
Hippo Pathway ◽  
Host Proteins ◽  
Dna Viruses ◽  
Ww Domain ◽  
Ww Domains ◽  
Host Interactions ◽  
New Class ◽  
Domain Interactions ◽  
Molecular Aspects ◽  
The Hippo Pathway

The WW domain is a modular protein structure that recognizes the proline-rich Pro-Pro-x-Tyr (PPxY) motif contained in specific target proteins. The compact modular nature of the WW domain makes it ideal for mediating interactions between proteins in complex networks and signaling pathways of the cell (e.g. the Hippo pathway). As a result, WW domains play key roles in a plethora of both normal and disease processes. Intriguingly, RNA and DNA viruses have evolved strategies to hijack cellular WW domain–containing proteins and thereby exploit the modular functions of these host proteins for various steps of the virus life cycle, including entry, replication, and egress. In this review, we summarize key findings in this rapidly expanding field, in which new virus-host interactions continue to be identified. Further unraveling of the molecular aspects of these crucial virus-host interactions will continue to enhance our fundamental understanding of the biology and pathogenesis of these viruses. We anticipate that additional insights into these interactions will help support strategies to develop a new class of small-molecule inhibitors of viral PPxY-host WW-domain interactions that could be used as antiviral therapeutics.

A single amino acid change makes a rat neuronal sodium channel highly sensitive to pyrethroid insecticides

FEBS Letters ◽  
2000 ◽  
Vol 470 (2) ◽  
pp. 135-138 ◽  
Author(s):  
H. Vais ◽  
S. Atkinson ◽  
N. Eldursi ◽  
A.L. Devonshire ◽  
M.S. Williamson ◽  
...  
Keyword(s):  
Amino Acid ◽  
Sodium Channel ◽  
Amino Acid Change ◽  
Single Amino Acid ◽  
Pyrethroid Insecticides ◽  
Highly Sensitive ◽  
Single Amino Acid Change

scholarly journals Rsp5p, a New Link between the Actin Cytoskeleton and Endocytosis in the Yeast Saccharomyces cerevisiae

2002 ◽  
Vol 22 (20) ◽  
pp. 6946-6948 ◽  
Author(s):  
Joanna Kamińska ◽  
Beata Gajewska ◽  
Anita K. Hopper ◽  
Teresa ˙Zołądek
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Actin Cytoskeleton ◽  
Cytoskeletal Proteins ◽  
Wild Type ◽  
Yeast Saccharomyces Cerevisiae ◽  
Ww Domains ◽  
Ubiquitin Protein Ligase ◽  
Growth Defects ◽  
Genes Encoding ◽  
Cytoskeleton Dynamics

ABSTRACT Rsp5p is an ubiquitin-protein ligase of Saccharomyces cerevisiae that has been implicated in numerous processes including transcription, mitochondrial inheritance, and endocytosis. Rsp5p functions at multiple steps of endocytosis, including ubiquitination of substrates and other undefined steps. We propose that one of the roles of Rsp5p in endocytosis involves maintenance and remodeling of the actin cytoskeleton. We report the following. (i) There are genetic interactions between rsp5 and several mutant genes encoding actin cytoskeletal proteins. rsp5 arp2, rsp5 end3, and rsp5 sla2 double mutants all show synthetic growth defects. Overexpressed wild-type RSP5 or mutant rsp5 genes with lesions of some WW domains suppress growth defects of arp2 and end3 cells. The defects in endocytosis, actin cytoskeleton, and morphology of arp2 are also suppressed. (ii) Rsp5p and Sla2p colocalize in abnormal F-actin-containing clumps in arp2 and pan1 mutants. Immunoprecipitation experiments confirmed that Rsp5p and Act1p colocalize in pan1 mutants. (iii) Rsp5p and Sla2p coimmunoprecipitate and partially colocalize to punctate structures in wild-type cells. These studies provide the first evidence for an interaction of an actin cytoskeleton protein with Rsp5p. (iv) rsp5-w1 mutants are resistant to latrunculin A, a drug that sequesters actin monomers and depolymerizes actin filaments, consistent with the fact that Rsp5p is involved in actin cytoskeleton dynamics.

Sign in / Sign up

Export Citation Format

Share Document