Identification of developmentally expressed proteins that functionally interact with Nedd4 ubiquitin ligase.
Nedd4 is a HECT domain-containing ubiquitin ligase that mediates ubiquitylation and proteasome degradation of target proteins. The molecular basis for the interaction of Nedd4 with substrates lies in its WW domains, which can bind proline-rich (PY) domains in target proteins. Nedd4 is a developmentally expressed protein and may have a fundamental role to play in embryonic processes. However, whether Nedd4 has such a function is currently unknown, in part because few developmentally regulated ubiquitylation substrates have been identified or characterized. We have carried out a yeast two-hybrid screen and identified four proteins expressed in the mid-gestation embryo that are able to interact with Nedd4. Characterization of their functional interaction with Nedd4 in vitro and in vivo demonstrated that three of the four are bona fide Nedd4 binding partners, and two have the capacity to be ubiquitylation substrates. One of these is the first identified nonviral substrate for Nedd4-mediated monoubiquitylation. Interestingly, neither of these two ubiquitylated proteins interacts with Nedd4 through PY-mediated mechanisms. For one of the three Nedd4 binding partners, there was no discernable evidence of ubiquitylation. However, this protein clearly associates with Nedd4 through its PY domains and can alter the location of Nedd4 in cells, suggesting a role other than as a ubiquitylation substrate.
Murillas
Rodolfo
R
Experimental Immunology Branch, NCI, National Institutes of Health, Bethesda, Maryland 20892-1360, USA.
Simms
Kimberly S
KS
Hatakeyama
Shigetsugu
S
Weissman
Allan M
AM
Kuehn
Michael R
MR
D015153:Blotting, Western; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D003546:Cysteine Endopeptidases; D018076:DNA, Complementary / Q000378:metabolism; D056827:Endosomal Sorting Complexes Required for Transport; D020224:Expressed Sequence Tags; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D008025:Ligases / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors; D016297:Mutagenesis, Site-Directed; D000075702:Nedd4 Ubiquitin Protein Ligases; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D046988:Proteasome Endopeptidase Complex; D011485:Protein Binding; D014176:Protein Biosynthesis; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014162:Transfection; D020798:Two-Hybrid System Techniques; D025801:Ubiquitin / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases
D002135:Calcium-Binding Proteins; D018076:DNA, Complementary; D056827:Endosomal Sorting Complexes Required for Transport; D009097:Multienzyme Complexes; D011993:Recombinant Fusion Proteins; D025801:Ubiquitin; D000075702:Nedd4 Ubiquitin Protein Ligases; C000618483:Nedd4 protein, human; D044767:Ubiquitin-Protein Ligases; D005982:Glutathione Transferase; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D008025:Ligases