Mosser E A, Kasanov J D, Forsberg E C, Kay B K, Ney P A, Bresnick E H
Department of Pharmacology, University of Wisconsin Medical School, Madison 53706, USA.
Biochemistry. 1998 Sep 29;37(39):13686-95. doi: 10.1021/bi981310l.
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.
β-珠蛋白基因座控制区中造血转录因子NF-E2的串联结合位点可激活转基因小鼠中高水平的β-珠蛋白基因表达。NF-E2是一种异二聚体，由造血亚基p45和普遍存在的亚基p18组成。加瓦等人[加瓦，N.R.，加瓦，R.，埃尔梅科娃，K.，苏多尔，M.，和沈，J.C.（1997年）《生物化学杂志》272卷，24105 - 24108页]报道，人p45含有一个与WW结构域结合的PPXY基序。我们发现，鼠类NF-E2在其反式激活结构域内含有两个PPXY基序（PPXY-1和-2），它与九种GST-WW结构域融合蛋白有不同的相互作用。定量分析显示，p45与在造血组织中表达的一种新型人泛素连接酶同源物（WWP1）的WW结构域有高亲和力结合（KD = 5.7 nM）。WWP1的氨基末端WW结构域与结合DNA的NF-E2形成多聚体复合物。通过噬菌体展示分离得到的一种WWP1配体肽和一个跨越PPXY-1的肽抑制了p45的结合，而一个与SH3结构域相互作用的肽和一个跨越PPXY-2的肽则没有。PPXY-1的突变而非PPXY-2的突变抑制了NF-E2的反式激活功能，这为WW结构域相互作用对NF-E2介导的反式激活很重要这一假说提供了支持。
