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The WW domain is a globular protein domain that is involved in mediating protein-protein interaction and that ultimately participates in various intracellular signaling events. The domain binds to polyproline ligands containing the xPPxY consensus (where x signifies any amino acid, P is proline and Y is tyrosine). One of the first WW domain-ligand links that was characterized in vitro was the WW domain of Yes-Associated Protein (YAP) and its WBP-1 ligand. To further characterize this molecular interaction, we used two independent approaches, both of which focused on the mutational analysis of the WBP-1 ligand. We screened repertoires of synthetic decamer peptides containing the xPPxY core of WBP-1 in which all ten positions were sequentially replaced with the remaining amino acids. In addition, we screened decamer repertoires with all permutations of the amino acids which individually increased the binding to the WW domain of YAP, as compared to the wild type. In a parallel approach, we used a phage-displayed combinatorial peptide library biased for the presence of two consecutive prolines to study ligand preferences for the WW domain of YAP. Interestingly, these two lines of investigation converged and yielded the core sequence PPPPYP, which is preferred by the YAP-WW domain. This sequence was found within the p53 (tumor suppressor) binding protein-2, a probable cognate or alternative ligand interacting with YAP.

NF-E2 is an erythroid-specific transcription factor required for expression of several erythroid-specific genes. By Far-Western blotting and yeast two-hybrid assay, we demonstrate that p45, the large subunit of NF-E2, is capable of binding to a specific set of WW domain-containing proteins, including the ubiquitin ligase hRPF1. This binding is mediated through the interaction between the WW domains and a PY motif located within the amino-terminal region of p45. Interestingly, the carboxyl-terminal domain of mammalian RNA polymerase II binds a similar set of WW domains to which p45 interacts with. We discuss the data in terms of possible new pathways through which the processes of transcriptional regulation by NF-E2 could be regulated in erythroid and megakaryote cells.NF-E2 is an obligate heterodimer of basic leucine zipper polypeptides consisting of a larger p45 polypeptide and a smaller subunit belonging to the p18/Maf family (1-5). Of the two subunits of NF-E2, expression of p18/Maf is ubiquitous, while that of p45 is restricted to the erythroid and megakaryocytic lineages (1, 6). Indeed, intact p45 gene and its expression are required for transcriptional regulation of globin genes (Refs. 1 and 7-9 and references therein) as well as for normal differentiation of the megakaryocytes (10).As a transcriptional activator, NF-E2 exerts its function by binding to enhancers or promoters at a consensus sequence that are also binding sites of AP1 (Refs. 1 and 11-13 and references therein). The activation domain of p45 is located between amino acids 1-206 (9). Most likely, NF-E2 functions through DNA binding as well as interactions with other cell type-specific and ubiquitous co-activators. For example, binding of p45 with hormone receptor(s) and CREB-binding protein (14) potentiates transcriptional activation mediated by hormones. NF-E2 binding to its cognate sequence also induces local nucleosome disruption (15,16).The NH 2 -terminal region of p45 has been implicated as the transcriptional activation domain of NF-E2 (9), and it contains 18% of proline residues. Interestingly, during data base search and visual comparison of the p45 sequence, we have noticed the sequence PPPPY located at amino acids 79 -83 of human p45. In mouse, it is PPPSY. This sequence fits the consensus of the so-called "PY" motifs, XPPXY, in which P is a proline, Y is a tyrosine, and Xs are any amino acid (17, 18; Fig. 1). The PY motifs are ligands capable of binding to the WW domains, which in turn were first identified in the proto-oncogene Yesassociated protein (YAP), 1 dystrophin, transcriptional regulator FE65, and others (19). These domains are of the length 38 amino acids, and they contain ␤ strands grouped around four aromatic positions (17,20). Two of these positions are most frequently occupied by tryptophans, hence the name "WW" domain was given. The WW domains are found in a number of unrelated proteins, including human and mouse YAP (hYAP, mYAP), human dystrophin (hDys), human ORF1, yeast Rsp5, yeast Ess1 (yEss1), fission yeast Pub1,...

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