Cloning and characterization of N4WBP5A, an inducible, cyclosporine-sensitive, Nedd4-binding protein in human T lymphocytes.
We have cloned and characterized a human cDNA, designated N4WBP5A, that belongs to the family of Nedd4-binding proteins. We originally identified N4WBP5A as an unknown expressed sequence tag (AA770150) represented in a cDNA microarray analysis that was up-regulated upon activation of T cells and inhibited by cell treatment with the calcineurin phosphatase inhibitors, cyclosporine (CsA) and tacrolimus (FK506). The predicted N4WBP5A amino acid sequence of 242 amino acid residues reveals an open reading frame of 729 nucleotides with a corresponding molecular mass of 27.1 kDa. Detection of N4WBP5A mRNA by reverse transcription-PCR was consistent with the induction of N4WBP5A following mitogenic stimulation of T lymphocytes and inhibition by CsA. Immunoblot analysis revealed endogenous N4WBP5A protein to be up-regulated following T cell activation and inhibited by CsA. This regulation of N4WBP5A mRNA expression differed from that of its homologue (51% identical; 65% similar) N4WBP5. Like N4WBP5, however, expression of epitope-tagged N4WBP5A indicated that the protein is localized predominantly to the Golgi network. Here we show by co-precipitation experiments that N4WBP5A interacts with the WW domains of Nedd4, an E3 ubiquitin ligase. Taken together, our data suggest that N4WBP5A may play a regulatory role in modulating Nedd4 activity at the level of the Golgi apparatus in T lymphocytes.
Cristillo
Anthony D
AD
Laboratory of Lymphocyte Biology, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.
Nie
Linghu
L
Macri
Mirtha J
MJ
Bierer
Barbara E
BE
D000595:Amino Acid Sequence; D000818:Animals; D015152:Blotting, Northern; D019556:COS Cells; D002135:Calcium-Binding Proteins / Q000737:chemistry*; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics*; D002478:Cells, Cultured; D003001:Cloning, Molecular; D016572:Cyclosporine / Q000494:pharmacology*; D018076:DNA, Complementary / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004721:Endoplasmic Reticulum / Q000378:metabolism; D056827:Endosomal Sorting Complexes Required for Transport; D000939:Epitopes; D020224:Expressed Sequence Tags; D006056:Golgi Apparatus / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting; D007166:Immunosuppressive Agents / Q000494:pharmacology*; D008025:Ligases / Q000737:chemistry*; D008213:Lymphocyte Activation; D008565:Membrane Proteins; D008856:Microscopy, Fluorescence; D008928:Mitochondria / Q000378:metabolism; D008957:Models, Genetic; D008969:Molecular Sequence Data; D000075702:Nedd4 Ubiquitin Protein Ligases; D020411:Oligonucleotide Array Sequence Analysis; D016366:Open Reading Frames; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D013601:T-Lymphocytes / Q000378:metabolism*; D016559:Tacrolimus / Q000494:pharmacology; D013997:Time Factors; D014018:Tissue Distribution; D014162:Transfection; D044767:Ubiquitin-Protein Ligases; D015854:Up-Regulation
D002135:Calcium-Binding Proteins; D002352:Carrier Proteins; D018076:DNA, Complementary; D056827:Endosomal Sorting Complexes Required for Transport; D000939:Epitopes; D007166:Immunosuppressive Agents; D008565:Membrane Proteins; C477747:NDFIP2 protein, human; D012333:RNA, Messenger; D016572:Cyclosporine; D000075702:Nedd4 Ubiquitin Protein Ligases; C000618483:Nedd4 protein, human; D044767:Ubiquitin-Protein Ligases; D008025:Ligases; D016559:Tacrolimus