Physical and functional interactions between the transactivation domain of the hematopoietic transcription factor NF-E2 and WW domains.
Tandem binding sites for the hematopoietic transcription factor NF-E2 in the beta-globin locus control region activate high-level beta-globin gene expression in transgenic mice. NF-E2 is a heterodimer consisting of a hematopoietic subunit p45 and a ubiquitous subunit p18. Gavva et al. [Gavva, N. R., Gavva, R., Ermekova, K., Sudol, M., and Shen, J. C. (1997) J. Biol. Chem. 272, 24105-24108] reported that human p45 contains a PPXY motif that binds WW domains. We show that murine NF-E2, which contains two PPXY motifs (PPXY-1 and -2) within its transactivation domain, differentially interacted with nine GST-WW domain fusion proteins. Quantitative analysis revealed high-affinity binding (KD = 5.7 nM) of p45 to a WW domain from a novel human ubiquitin ligase homologue (WWP1) expressed in hematopoietic tissues. The amino-terminal WW domain of WWP1 formed a multimeric complex with DNA-bound NF-E2. A WWP1 ligand peptide, isolated by phage display, and a peptide spanning PPXY-1 inhibited p45 binding, whereas an SH3 domain-interacting peptide and a peptide spanning PPXY-2 did not. Mutation of PPXY-1, but not PPXY-2, inhibited the transactivation function of NF-E2, providing support for the hypothesis that WW domain interactions are important for NF-E2-mediated transactivation.
Mosser
E A
EA
Department of Pharmacology, University of Wisconsin Medical School, Madison 53706, USA.
Kasanov
J D
JD
Forsberg
E C
EC
Kay
B K
BK
Ney
P A
PA
Bresnick
E H
EH
D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism* / Q000502:physiology; D019281:Dimerization; D050981:Erythroid-Specific DNA-Binding Factors; D004920:Erythropoiesis / Q000235:genetics; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008025:Ligases / Q000378:metabolism; D046911:Macromolecular Substances; D050985:MafK Transcription Factor; D051379:Mice; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D050983:NF-E2 Transcription Factor; D050984:NF-E2 Transcription Factor, p45 Subunit; D009928:Organ Specificity / Q000235:genetics; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism* / Q000502:physiology; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000096:biosynthesis; D015534:Trans-Activators; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism* / Q000502:physiology; D015533:Transcriptional Activation / Q000502:physiology; D044767:Ubiquitin-Protein Ligases
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D050981:Erythroid-Specific DNA-Binding Factors; D047908:Intracellular Signaling Peptides and Proteins; D046911:Macromolecular Substances; D050985:MafK Transcription Factor; C494696:Mafk protein, mouse; D050983:NF-E2 Transcription Factor; D050984:NF-E2 Transcription Factor, p45 Subunit; C494692:NFE2 protein, human; C494693:Nfe2 protein, mouse; D010446:Peptide Fragments; D012333:RNA, Messenger; D015534:Trans-Activators; D014157:Transcription Factors; C106907:WBP1 protein, human; C106908:WBP2 protein, human; C484574:Wbp1 protein, mouse; C484576:Wbp2 protein, mouse; D004247:DNA; D044767:Ubiquitin-Protein Ligases; D008025:Ligases