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The WW Domain of Yes-Associated Protein Binds a Proline-Rich

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The WW Domain of Yes-Associated Protein Binds a Proline-Rich Proc. Natl. Acad. Sci. USA Vol. 92, pp. 7819-7823, August 1995 Biochemistry The WW domain of Yes-associated protein binds a proline-rich ligand that differs from the consensus established for Src homology 3-binding modules (protein-protein interactions/polyproline/modular domains) HENRY I. CHEN AND MARIUS SUDOL* Laboratory of Molecular Oncology, The Rockefeller University, 1230 York Avenue, New York, NY 10021 Communicated by Hidesaburo Hanafusa, The Rockefeller University, New York; NY, May 18, 1995 (received for review March 28, 1995) ABSTRACT The WW domain has previously been de- Various studies have shown that SH3 domains may mediate scribed as a motif of 38 semiconserved residues found in protein localization to the plasma membrane or the cytoskel- seemingly unrelated proteins, such as dystrophin, Yes- eton, as exemplified by the interaction between Grb2 and Sos associated protein (YAP), and two transcriptional regulators, that transduces signals from cell surface receptors to the Ras Rsp-5 and FE65. The molecular function of the WW domain pathway (13-15). Furthermore, similar to SH2 domains, SH3 has been unknown until this time. Using a functional screen domains may in fact mediate the targeting of proteins to their of a cDNA expression library, we have identified two putative substrates (16-21). ligands of the WW domain of YAP, which we named WBP-1 Recently we have characterized a Yes-associated protein and WBP-2. Peptide sequence comparison between the two (YAP) that binds to the SH3 domain of the Yes protoonco- partial clones revealed a homologous region consisting of a gene product via a proline-rich domain (22). Within the YAP proline-rich domain followed by a tyrosine residue (with the sequence, we have identified a protein motif of 38 amino acids shared sequence PPPPY), which we shall call the PY motif. first recognized to be shared by YAP, dystrophin, and a variety Binding assays and site-specific mutagenesis have shown that of other proteins (23, 24). This domain contains four well- the PY motif binds with relatively high affinity and specificity conserved aromatic amino acids among which are two tryp- to the WW domain of YAP, with the preliminary consensus tophan residues, hence the name WW domain. Computer- XPPXY being critical for binding. Herein, we have implicated aided structure analysis of the WW domain predicted a the WWV domain with a role in mediating protein-protein hydrophobic core consisting of these four aromatic residues interactions, as a variant of the paradigm set by Src homology flanked by 13 strands containing charged residues, reminiscent 3 domains and their proline-rich ligands. of the determined structure of the SH3 domain (25-27). The newly discovered domain has also been found in other proteins The progressive elucidation of the function of Src homology including utrophin, murine Nedd-4, yeast Rsp5, ORF-1 (a Ras (SH) 2 and SH3 domains, in addition to the identification of GTPase activator-related protein), and FE65 (a mammalian their ligands, has in the recent years spawned an era of protein implicated in the regulation of transcription) (23). remarkable developments in the fields of signal transduction These diverse proteins share the intriguing characteristic of and molecular oncology (1-3). As a result of this trend, two being involved in signaling or regulatory functions. As a result, new domains, the pleckstrin homology domain and the phos- it has been hypothesized that the WW domain may be a photyrosine interaction domain, have been identified and are putative protein-binding site important in the intracellular extensively studied in the context of signaling pathways (4-8). signaling network. The recognition of the existence of modular protein-binding Herein, we report the cloning and characterization of pro- domains has proven to be critical in understanding the intri- tein ligands for the WW domain of YAP-namely, WBP-1 and cacies of signal transduction where a variety of events occur. WBP-2 (WW domain bindingprotein). Furthermore, we have For example, proteins transiently interact with their upstream demonstrated that WBP-1 binds with high specificity to the and downstream partners, substrates are brought into prox- WW domain through a proline-rich domain with a sequence of imity of their catalytic centers, or proteins are localized to their PPPPY, which we have named the PY motif. Through site- subcellular compartments through the interaction of these directed mutagenesis, we have shown that the second and third domains with their ligands (9). proline residues, along with the tyrosine residue of the PY The SH2 domain was first described as a conserved sequence motif, are essential for binding to the domain of YAP (WW- of 100 amino acids in the noncatalytic domain of cytoplasmic YAP). In addition, the PY motif of WBP-1 does not bind with tyrosine kinases such as Src and Fps, but it rapidly became high affinity to the SH3 domains of arbitrarily chosen proteins. recognized in other molecules involved in signaling pathways Therefore, the PY motif seems to diverge from the classical and cellular transformation including Crk, Ras-GAP, and PXXP consensus of SH3-binding proline-rich domains and phospholipase C-,y. When the SH2 domain was later shown to may in fact represent another type of modular protein-binding bind with high affinity to phosphotyrosine residues, it was sequence, pending the discovery of additional WW domain quickly realized that the SH2 domain represented a means by ligands. which many molecules sharing a common signaling pathway may associate to regulate signal propagation from the extra- MATERIALS AND METHODS cellular environment into the cell and its nucleus (10). In Construction of Fusion Proteins. Primers corresponding to contrast, the SH3 domain was originally identified as a 50- the 5' and 3' of the cDNA for human amino-acid-long region of homology shared between the Crk ends, respectively, and Src oncogene products and phospholipase C-,y (11, 12). Abbreviations: GST, glutathione S-transferase; SH, Src homology; WBP, WW domain binding protein; YAP, Yes-associated protein. The publication costs of this article were defrayed in part by page charge *To whom reprint requests should be addressed at: Laboratory of payment. This article must therefore be hereby marked "advertisement" in Molecular Oncology, The Rockefeller University, Box 169, 1230 accordance with 18 U.S.C. §1734 solely to indicate this fact. York Avenue, New York, NY 10021. Downloaded by guest on September 30, 2021 7819 7820 Biochemistry: Chen and Sudol Proc. Natl. Acad. Sci. USA 92 (1995) WW-YAP flanked by a 5' BamHI or a 3' EcoRI site (under- irradiation. The hybridization conditions were as described lined) are as follows: 5'-dCTATACGGATCCCAGTCT- (24, 32). The blots were washed for 30 min at room temper- TCTTTTGAGATACCT-3' and 5 '-dTACGACGAATIC- ature in 2x SSC/0.05% SDS and for 1 hr at 50°C in 0.1X GACTGGTGGGGGCTGTGACGTTCA-3'. The primers SSC/0.1% SDS. Removal of the WBP cDNA probes from the were subsequently used to amplify the WW-YAP gene frag- blot for subsequent hybridization with the human ,3-actin ment by PCR (Perkin-Elmer). The amplified fragments were probe was achieved by incubating the blot for 10 min in sterile subcloned in-frame into the pGEX-2TK vector between the H20 containing 0.5% SDS that was heated to 90°C. BamHI and EcoRI sites (Pharmacia). Expression of a gluta- thione S-transferase (GST) fusion protein migrating at the predicted molecular mass was verified by SDS/PAGE, and the RESULTS nucleic acid sequence of the construct was confirmed by direct Precipitation of the WW Domain Ligand from Cell Lysates. sequence analysis using the Sanger method (28). Fusion pro- To show the possible existence of specific protein ligands to teins of the cloned WBP-1 and fragments therein were con- WW-YAP, we conducted coprecipitation studies by incubating structed similarly. PCR-amplified fragments (using Pfu poly- GST-WW-YAP fusion proteins with lysates from various rat merase; Stratagene) of the full-length WBP-1 (5'-dAG- organs and established cell lines. Previous studies have shown TATCGGATCCAGCCACGGGGCTGGCCCTGTT-3' and that YAP is present at high levels in lung, ovary, cerebellum, 5 '-dGCATCCGAATTCGGTTCATGTCTCTT- and skeletal muscle, thus increasing the likelihood of detecting TAATGAG-3') and residues 1-74 of the N-terminal fragment the cognate ligand(s) in those organs as well (24). Western (BamHI primer, as above; EcoRI primer: 5'-dGCTATC- ligand blot analysis revealed a band of -38 kDa in size in lung, GAATTCAAACACCTTCTACATTTGTCC-3') were sub- ovary, and cerebellum, and an additional 34-kDa band present cloned into pGEX-2TK. An oligonucleotide and its comple- only in cerebellum when probed with 32P-labeled fusion pro- mentary strand coding for residues 34-43 (including the PY tein (Fig. 1B) but not with labeled GST (Fig. 1A). In addition, motif and flanking BamHI and EcoRI sites) of WBP-1 were precipitation with lysates of HeLa, A431, 3Y1, and v-src- annealed and then subcloned in-frame into the vector between transformed 3Y1 cell lines yielded a 38-kDa band as well (data the same two restriction sites (5'-dTACGTCGGATCCGG- not shown). CACACCGCCACCTCCTTACACTGTGGGCCGAAITCG- Cloning of Ligands for the WW Domain. We screened a TCTGC-3' and its complementary strand). Mutagenized PY in mouse embryo library with 32P-labeled GST-WW-YAP fusion GST fusion constructs was similarly constructed with a pair (sense protein and isolated three partial clones that were positive and antisense) of the above PY motif oligonucleotides, but each after four rounds of screening but that did not bind 32P-labeled of the respective codons for the residues PPPPY was alternately GST probe. Two of the clones proved to be identical. The replaced with the codon (GCA) for an alanine residue. predicted gene products of the two different clones, 700 bp and Purification and Labeling ofFusion Proteins.
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