The WW domain has previously been described as a motif of 38 semiconserved residues found in seemingly unrelated proteins, such as dystrophin, Yesassociated protein (YAP), and two transcriptional regulators, Rsp-5 and FE65. The molecular function of the WW domain has been unknown until this time. Using a functional screen of a cDNA expression library, we have identified two putative ligands of the WW domain of YAP, which we named WBP-1 and WBP-2. Peptide sequence comparison between the two partial clones revealed a homologous region consisting of a proline-rich domain followed by a tyrosine residue (with the shared sequence PPPPY), which we shall call the PY motif. Binding assays and site-specific mutagenesis have shown that the PY motif binds with relatively high affinity and specificity to the WW domain of YAP, with the preliminary consensus XPPXY being critical for binding. Herein, we have implicated the WWV domain with a role in mediating protein-protein interactions, as a variant of the paradigm set by Src homology 3 domains and their proline-rich ligands.The progressive elucidation of the function of Src homology (SH) 2 and SH3 domains, in addition to the identification of their ligands, has in the recent years spawned an era of remarkable developments in the fields of signal transduction and molecular oncology (1-3). As a result of this trend, two new domains, the pleckstrin homology domain and the phosphotyrosine interaction domain, have been identified and are extensively studied in the context of signaling pathways (4-8).The recognition of the existence of modular protein-binding domains has proven to be critical in understanding the intricacies of signal transduction where a variety of events occur. For example, proteins transiently interact with their upstream and downstream partners, substrates are brought into proximity of their catalytic centers, or proteins are localized to their subcellular compartments through the interaction of these domains with their ligands (9).The SH2 domain was first described as a conserved sequence of 100 amino acids in the noncatalytic domain of cytoplasmic tyrosine kinases such as Src and Fps, but it rapidly became recognized in other molecules involved in signaling pathways and cellular transformation including Crk, Ras-GAP, and phospholipase C-,y. When the SH2 domain was later shown to bind with high affinity to phosphotyrosine residues, it was quickly realized that the SH2 domain represented a means by which many molecules sharing a common signaling pathway may associate to regulate signal propagation from the extracellular environment into the cell and its nucleus (10). In contrast, the SH3 domain was originally identified as a 50-amino-acid-long region of homology shared between the Crk and Src oncogene products and phospholipase C-,y (11, 12).Various studies have shown that SH3 domains may mediate protein localization to the plasma membrane or the cytoskeleton, as exemplified by the interaction between Grb2 and Sos that transduces signals from cell surfa...

We have identified, characterized and cloned human, mouse and chicken cDNA of a novel protein that binds to the Src homology domain 3 (SH3) of the Yes proto-oncogene product. We subsequently named it YAP for Yes-associated protein. Analysis of the YAP sequence revealed a protein module that was found in various structural, regulatory and signaling molecules. Because one of the prominent features of this sequence motif is the presence of two conserved tryptophans (W), we named it the WW domain. Using a functional screen of a cDNA expression library, we have identified two putative ligands of the WW domain of YAP which we named WBP-1 and WBP-2. Peptide sequence comparison between the two partial clones revealed a homologous proline-rich region. Binding assays and site-specific mutagenesis have shown that the proline-rich motif binds with relatively high affinity and specificity to the WW domain of YAP, with a preliminary consensus that is different from the SH3-binding PXXP motif. This suggests that the WW domain has a role in mediating protein-protein interactions via proline-rich regions, similar but distinct from Src homology 3 (SH3) domains. Based on this finding, we hypothesize that additional protein modules exist and that they could be isolated using proline-rich peptides as functional probes.Key words: Protein protein interaction; Protein module; Polyproline Background and rationale -Retrospective lookWhat started as a pilot project ended up being the main focus of an entire lab. Our general aim has been to decipher molecular steps of signaling by the Yes proto-oncogene product which represents a non-receptor type protein-tyrosine kinase of the Src family [1]. The specific goal was to isolate substrates and regulators of the Yes kinase in order to understand at least some aspects of the molecular role it plays in normal and the viral-Yes oncogene transformed cells [2]. Initially, our experimental approaches were descriptive of nature [3,4]. We argued that by characterizing the pattern of Yes expression in various tissues and cells, we would have been able to find a common denominator that would provide a clue regarding the physiological function of the Yes protein [5]. Localization of the Yes proto-oncogene transcript and protein in cerebellar Purkinje cells was an exciting finding which gave us hope for functional clues [6]. Unfortunately, that was immediately followed by the frustration of trying to study the Yes kinase in difficult experimental systems of isolated Purkinje neurons or cerebellar slices. The direction of our research activities shifted swiftly when Hirai and Varmus proposed that the amino termini of Src family kinases form complexes with cellular proteins and that these apparently transient and dynamic complexes constitute a part of the mechanism by which Src, Yes and other kinases signal [7]. The SH2 domain, residing at the amino terminal half of the Src kinases had already been delineated at that time and was a primary candidate for a signaling domain [8]. The proposal of Hirai...

We had previously identified the WW domain as a novel globular domain that is composed of 38 -40 semiconserved amino acids and is involved in mediating protein-protein interaction. The WW domain is shared by proteins of diverse functions including structural, regulatory, and signaling proteins in yeast, nematode, and mammals. Functionally it is similar to the Src homology 3 domain in that it binds polyproline ligands. By screening a 16-day mouse embryo expression library, we identified two putative ligands of the WW domain of Yes kinase-associated protein which we named WW domainbinding proteins 1 and 2. These proteins interacted with the WW domain via a short proline-rich motif with the consensus sequence of four consecutive prolines followed by a tyrosine. Herein, we report the cDNA cloning and characterization of the human orthologs of WW domain-binding proteins 1 and 2. The products encoded by these cDNA clones represent novel proteins with no known function. Furthermore, these proteins show no homology to each other except for a proline-rich motif. By fluorescence in situ hybridization on human metaphase chromosomes, we mapped the human genes for WW domain-binding proteins 1 and 2 to chromosomes 2p12 and 17q25, respectively. In addition, using sitedirected mutagenesis, we determined which residues in the WW domain of Yes kinase-associated protein are critical for binding. Finally, by synthesizing peptides in which the various positions of the four consecutive proline-tyrosine motif and the five surrounding residues were replaced by all possible amino acid residues, we further elucidated the binding requirements of this motif.The Src homology (SH) 1 2 and SH3 domains have assumed essential roles in furthering the understanding of how an extracellular signal is transmitted from the cellular membrane, through the cytoplasm, and finally into the nucleus where the signal is interpreted through the process of gene-specific transcription. The SH2 domain has been shown to interact specifically with sequences containing a phosphotyrosine residue, whereas the SH3 domain mediates binding to proline-rich sequences with the minimal consensus of PXXP (P represents proline, and X designates any amino acid) (1, 2). The SH2 and SH3 domains thus consist of a common binding core that recognizes phosphotyrosine-or proline-rich motifs, respectively, and which achieve binding specificity through unique flanking sequences (3-5). As a result, these domains determine which proteins can interact, and equally important, in what order the interaction occurs in the closely regulated pathways of signal transduction. Recently, two other important signaling modules were characterized: the pleckstrin homology domain and the protein interaction domain/phosphotyrosine binding domain (6 -10). These modular repeats represent true protein domains in that they constitute structurally distinct three-dimensional units that can properly fold and function in the context of other proteins or in isolation (11,12).We have previously identified a Yes ki...