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.

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 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 ◽  
Author(s):  
Corina N. JOLLIFFE ◽  
Kieran F. HARVEY ◽  
Bryan P. HAINES ◽  
Gayathri PARASIVAM ◽  
Sharad KUMAR
Keyword(s):  
Ww Domains ◽  
Ubiquitin Protein Ligase ◽  
Binding Partners ◽  
Murine Embryos

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.

scholarly journals Substrate proteolysis is inhibited by dominant-negative Nedd4 and Rsp5 mutants harboring alterations in WW domain 1

2002 ◽  
Vol 115 (5) ◽  
pp. 1041-1048 ◽  
Author(s):  
Natalia Shcherbik ◽  
Sharad Kumar ◽  
Dale S. Haines
Keyword(s):  
Mammalian Cells ◽  
Budding Yeast ◽  
Large Family ◽  
Lipid Binding ◽  
Dominant Negative ◽  
Growth Media ◽  
Ww Domain ◽  
Ww Domains ◽  
Multiple Protein ◽  
Dominant Negative Activity

Mammalian Nedd4 and its budding yeast orthologue Rsp5 are members of a large family of HECT-domain-containing ubiquitin ligases. Besides possessing a Ca2+/lipid-binding domain, both ligases have multiple protein-interacting modules termed WW domains. The C-terminal WW domains mediate interactions with substrates, but the function of the first WW domain remains unclear. We found that expression of a WW domain 1 Nedd4 mutant inhibits the growth of budding yeast by affecting the rsp5-ole1pathway. The WW domain 1 mutant-induced phenotype is suppressed by ole1 cDNA overexpression or oleic acid supplementation of growth media and ole1 RNA levels are reduced in cells expressing this Nedd4 mutant. Also, the WW domain 1 Nedd4 mutant associates via WW domains 2 and 3 with Spt23, a Rsp5 target and ole1 transactivator. The dominant-negative activity of this mutant is associated with promoting accumulation of unprocessed Spt23 and inhibiting generation of processed and presumably active protein. Also, Spt23 processing is inhibited by a Nedd4 mutant that lacks ubiquitin ligase activity and Spt23-binding-competent Rsp5 mutants harboring WW domain 1 or ligase domain mutations. Interestingly, in mammalian cells, wild-type Nedd4 promotes proteasome-mediated degradation of the precursor form of Spt23. WW domain 1 and ligase domain Nedd4 mutants block its degradation. These results indicate that WW domain 1 of these ligases interacts with cofactors that are required for ubiquitin/proteasome-dependent proteolysis of bound substrates.

scholarly journals An Internalization Signal in ClC-5, an Endosomal Cl−Channel Mutated in Dent's Disease

2000 ◽  
Vol 276 (15) ◽  
pp. 12049-12054 ◽  
Author(s):  
Michael Schwake ◽  
Thomas Friedrich ◽  
Thomas J. Jentsch
Keyword(s):  
Surface Expression ◽  
Dominant Negative ◽  
Dent’S Disease ◽  
Ww Domains ◽  
Ubiquitin Protein Ligase ◽  
Dent's Disease ◽  
Carboxyl Terminal ◽  
Py Motif ◽  
Protein Ligases ◽  
Inactivating Mutations

The ClC-5 chloride channel resides mainly in vesicles of the endocytotic pathway and contributes to their acidification. Its disruption in mice entails a broad defect in renal endocytosis and causes secondary changes in calciotropic hormone levels. Inactivating mutations in Dent's disease lead to proteinuria and kidney stones. Possibly by recycling, a small fraction of ClC-5 also reaches the plasma membrane. Here we identify a carboxyl-terminal internalization motif in ClC-5. It resembles the PY motif, which is crucial for the endocytosis and degradation of epithelial Na+channels. Mutating this motif increases surface expression and currents about 2-fold. This is probably because of interactions with WW domains, because dominant negative mutants of the ubiquitin-protein ligase WWP2 increased surface expression and currents of ClC-5 only when its PY motif was intact. Stimulating endocytosis by expressing rab5 or its GTPase-deficient Q79L mutant decreased WT ClC-5 currents but did not affect channels with mutated motifs. Similarly, decreasing endocytosis by expressing the inactive S34N mutant of rab5 increased ClC-5 currents only if its PY-like motif was intact. Thus, the endocytosis of ClC-5, which itself is crucial for the endocytosis of other proteins, depends on the interaction of a carboxyl-terminal internalization signal with ubiquitin-protein ligases containing WW domains.

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.

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.

Developmentally regulated expression of a human "finger"-containing gene encoded by the 5' half of the ret transforming gene

1988 ◽  
Vol 8 (4) ◽  
pp. 1853-1856
Author(s):  
M Takahashi ◽  
Y Inaguma ◽  
H Hiai ◽  
F Hirose
Keyword(s):  
Human Gene ◽  
Mrna Levels ◽  
Nucleic Acid Binding ◽  
Developmentally Regulated ◽  
Unique Transcript ◽  
Binding Domains ◽  
Regulated Expression ◽  
Pachytene Spermatocytes ◽  
Human Finger ◽  
Transforming Gene

We isolated and sequenced a cDNA clone of the human gene encoded by the 5' half of the ret transforming gene. The nucleotide sequence indicates that it encodes a protein with "finger" structures which represent putative metal- and nucleic acid-binding domains. Transcription of this gene was detected at high levels in a variety of human and rodent tumor cell lines, mouse testis, and embryos. In addition, a unique transcript was observed in testis RNA. When the expression of the unique transcript was examined at different stages of spermatogenesis, a striking increase in mRNA levels accompanied progression from meiotic prophase pachytene spermatocytes to postmeiotic round spermatids. This finger-containing gene may thus function in male germ cell development.

scholarly journals Physical and Functional Interaction of the HECT Ubiquitin-protein Ligases E6AP and HERC2

2011 ◽  
Vol 286 (22) ◽  
pp. 19410-19416 ◽  
Author(s):  
Simone Kühnle ◽  
Ulrike Kogel ◽  
Sandra Glockzin ◽  
Andreas Marquardt ◽  
Aaron Ciechanover ◽  
...  
Keyword(s):  
Amino Acid ◽  
Angelman Syndrome ◽  
Amino Acid Residues ◽  
Cervical Carcinogenesis ◽  
Tight Control ◽  
Functional Interaction ◽  
Ubiquitin Protein Ligase ◽  
Protein Ligases ◽  
Translational Level

Deregulation of the ubiquitin-protein ligase E6AP contributes to the development of the Angelman syndrome and to cervical carcinogenesis suggesting that the activity of E6AP needs to be under tight control. However, how E6AP activity is regulated at the post-translational level under non-pathologic conditions is poorly understood. In this study, we report that the giant protein HERC2, which is like E6AP a member of the HECT family of ubiquitin-protein ligases, binds to E6AP. The interaction is mediated by the RCC1-like domain 2 of HERC2 and a region spanning amino acid residues 150–200 of E6AP. Furthermore, we provide evidence that HERC2 stimulates the ubiquitin-protein ligase activity of E6AP in vitro and within cells and that this stimulatory effect does not depend on the ubiquitin-protein ligase activity of HERC2. Thus, the data obtained indicate that HERC2 acts as a regulator of E6AP.

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