title: Substrate proteolysis is inhibited by dominant-negative Nedd4 and Rsp5 mutants harboring alterations in WW domain 1.
abstract: Mammalian Nedd4 and its budding yeast orthologue Rsp5 are members of a large family of HECT-domain-containing ubiquitin ligases. Besides possessing a Ca(2+)/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-ole1 pathway. 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.
authors.affiliation: Fels Institute for Cancer Research and Molecular Biology, Temple University School of Medicine, 3307 N. Broad Street, Philadelphia, PA 19140, USA.
authors.forename: Natalia
authors.initials: N
authors.lastname: Shcherbik
authors.forename: Sharad
authors.initials: S
authors.lastname: Kumar
authors.forename: Dale S
authors.initials: DS
authors.lastname: Haines
mesh_terms: D002135:Calcium-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D002478:Cells, Cultured; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D056827:Endosomal Sorting Complexes Required for Transport; D004791:Enzyme Inhibitors / Q000494:pharmacology; D044943:Fatty Acid Desaturases / Q000235:genetics / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008025:Ligases / Q000235:genetics* / Q000378:metabolism; D008565:Membrane Proteins; D009097:Multienzyme Complexes / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D009154:Mutation / Q000235:genetics*; D000075702:Nedd4 Ubiquitin Protein Ligases; D010447:Peptide Hydrolases / Q000235:genetics* / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex; D017434:Protein Structure, Tertiary / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D013230:Stearoyl-CoA Desaturase; D015534:Trans-Activators; D014157:Transcription Factors; D043743:Ubiquitin-Protein Ligase Complexes; D044767:Ubiquitin-Protein Ligases; D014452:Ubiquitins / Q000235:genetics / Q000378:metabolism
chemical_list: D002135:Calcium-Binding Proteins; D056827:Endosomal Sorting Complexes Required for Transport; D004791:Enzyme Inhibitors; D005656:Fungal Proteins; D008565:Membrane Proteins; D009097:Multienzyme Complexes; C087570:SPT23 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D014452:Ubiquitins; D044943:Fatty Acid Desaturases; D013230:Stearoyl-CoA Desaturase; C029627:delta-9 fatty acid desaturase; D043743:Ubiquitin-Protein Ligase Complexes; D000075702:Nedd4 Ubiquitin Protein Ligases; C000618483:Nedd4 protein, human; D044767:Ubiquitin-Protein Ligases; D010447:Peptide Hydrolases; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D008025:Ligases; C469287:RSP5 protein, S cerevisiae; D003545:Cysteine