Identification of multiple proteins expressed in murine embryos as binding partners for the WW domains of the ubiquitin-protein ligase Nedd4.
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.
Jolliffe
C N
CN
Hanson Centre for Cancer Research, Institute of Medical and Veterinary Science, P.O. Box 14, Rundle Mall, Adelaide, SA 5000, Australia.
Harvey
K F
KF
Haines
B P
BP
Parasivam
G
G
Kumar
S
S
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004622:Embryo, Mammalian / Q000378:metabolism*; D056827:Endosomal Sorting Complexes Required for Transport; D018507:Gene Expression Regulation, Developmental; D006801:Humans; D008025:Ligases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D000075702:Nedd4 Ubiquitin Protein Ligases; D011485:Protein Binding; D012333:RNA, Messenger / Q000235:genetics; D044767:Ubiquitin-Protein Ligases
D002135:Calcium-Binding Proteins; D056827:Endosomal Sorting Complexes Required for Transport; D008565:Membrane Proteins; D012333:RNA, Messenger; D000075702:Nedd4 Ubiquitin Protein Ligases; C000618483:Nedd4 protein, human; C000618461:Nedd4l protein, mouse; D044767:Ubiquitin-Protein Ligases; D008025:Ligases