Characterization of the WW Domain of Human Yes-associated Protein and Its Polyproline-containing Ligands*

(Received for publication, February 3, 1997, and in revised form, April 4, 1997)

Henry I. Chen Dagger , Aaron Einbond §, Sahng-June Kwak §, Hillary Linn §, Edward Koepf , Scott Peterson , Jeffery W. Kelly and Marius Sudol §par

From the Dagger  Laboratory of Molecular Oncology, The Rockefeller University, New York, New York 10021, the § Department of Biochemistry, Mount Sinai School of Medicine, New York, New York 10029, and the  Department of Chemistry, Texas A&M University, College Station, Texas 77843

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

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 domain-binding 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 site-directed 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.


INTRODUCTION

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 kinase-associated protein (YAP) that interacts in vitro with the SH3 domain of the Yes proto-oncogene product via a proline-rich region (13). In addition, the YAP sequence contains a semiconserved region of 38 amino acids which has since been found in a variety of unrelated proteins including dystrophin (implicated in Duchenne's and Becker's muscular dystrophy), Pub1 (a ubiquitin-ligase involved in cell cycle regulation in fission yeast), Rsp5/Nedd4 (transcriptional regulators with ubiquitin-ligase activity), and FE65 (an adaptor protein that binds Alzheimer's beta -amyloid precursor protein) (14-16). This protein repeat has been named the WW domain after the two highly conserved tryptophan residues spaced 20-22 residues apart within the consensus sequence. The list of WW domain-containing proteins continually grows, and these proteins seem to share the characteristic of being involved in regulatory or signaling processes (11).

More recently, we have shown that the WW domain of YAP is indeed a novel domain capable of mediating protein-protein interactions (17-19). By screening an expression library constructed from 16-day-old mouse embryo, we identified two putative ligands binding with high specificity and relatively high affinity (Kd = 1-50 mM; Refs. 17 and 20) to the WW domain of YAP, namely WBP-1 and WBP-2 (WW domain-binding protein). Using the technique of Western ligand blotting in performing in vitro binding assays, we further narrowed the binding region of WBP-1 to a proline-rich motif with the sequence Pro-Pro-Pro-Pro-Tyr (PPPPY), which we chose to call the PY motif, interestingly, the only region of perfect homology between WBP-1 and WBP-2. Binding assays using mutant forms of the PY motif established the preliminary minimal binding consensus as XPPXY (where X signifies any amino acid) required for interaction with the WW domain of YAP. Based on these results and the observation that the PY motif failed to bind arbitrarily to selected SH3 domains, we proposed that the PY motif differed from the binding consensus determined for SH3 ligands.

Although the WW and SH3 domains are functionally similar, their three-dimensional structures are different (for review, see Ref. 11). The WW domain has its NH2 and COOH termini in close juxtaposition, opposite from the ligand binding surface, allowing the modular unit to exist close to the protein surface exposed to the solvent (20). The NMR solution structure solved for the WW domain of YAP consists of a bent three-stranded antiparallel beta  sheet and a hydrophobic ligand binding pocket composed of leucine 190, tyrosine 188, and tryptophan 199 (the second conserved tryptophan). The two central proline residues of the consensus ligand (XPPXY) contact tryptophan 199 through van der Waals interactions, whereas the tyrosine residue of the PY ligand fits into a hydrophobic pocket of the WW domain formed by leucine 190 and histidine 192.

In this report, we describe the cloning and characterization of the complete cDNA clones for the murine and human orthologs of WBP-1 and WBP-2. Furthermore, the human genes encoding these two proteins were localized to chromosomes 2p12 and 17q25, respectively. To elucidate further the binding requirements of the PY motif, we replaced various positions of the motif with all possible amino acid residues and then assayed for in vitro binding to the WW domain of YAP using the "SPOTs" technique of multiple peptide synthesis on derivatized cellulose (28, 29). Finally, through site-directed mutagenesis of the conserved residues of the WW domain, we showed that the conservative substitution of the residues tyrosine 188, tryptophan 199, histidine 192, and proline 202 with related amino acids abolished binding, suggesting that these residues are important for binding.


EXPERIMENTAL PROCEDURES

cDNA Cloning and Sequencing

Mouse WBP-1 and WBP-2 partial cDNAs (17) were used as probes to screen a lambda  pCEV9 cDNA library derived from M426 human lung fibroblast cells (21) (a gift from Dr. Stuart Aaronson). The low stringency conditions of hybridization were as follows: 4 × SSPE, 10 × Denhardt's, 2% SDS, 0.2 mg/ml salmon sperm DNA, and 106 cpm/ml 32P-labeled cDNA at 65 °C overnight. The filters were washed twice at 60 °C for 20 min with 0.1 × SSC, 0.1% SDS. The lambda  pCEV9 cDNA library contained phages with a plasmid portion that carried the insert. The plasmids with inserts were easily rescued from the lambda  genome following the published protocol (21). The apparently complete sequence of human WBP-1 cDNA was contained in one recombinant plasmid pCEV9-hWBP1 (clone 43) with a BamHI-HindIII insert of 1.2 kilobases. Human WBP-2 cDNA was contained in one recombinant plasmid pCEV9-hWBP2 (clone 29) with a BamHI-HindIII insert of 1.8 kilobases. Both strands of the cDNA clones were subsequently sequenced directly using the Sanger method (22).

To isolate the remainder of the cDNA for murine WBP-1, a 16-day mouse embryo cDNA library was screened with the partial cDNA clone coding for murine WBP-1 that was isolated previously according to published protocols (17, 23). Plaques representing the library were lifted onto nitrocellulose filters, which were subsequently incubated in hybridization solution (6 × SSC, 0.01 M EDTA, 5 × Denhardt's solution, 0.5% SDS, 100 µg/ml denatured salmon sperm DNA) with random labeled cDNA probe overnight at 68 °C. The filters were then washed in 2 × SSC, 0.5% SDS, and 2 × SSC, 0.1% SDS at room temperature, and then 0.1 × SSC, 0.5% SDS at 65 °C before exposing to film. The longest clone obtained by this method was selected for direct DNA sequencing by the Sanger method (22).

The remainder of the murine WBP-2 cDNA clone was successfully isolated using the technique of 5' rapid amplification of cDNA ends (Life Technologies, Inc.). First, the cDNA coding for WBP-2 was derived from poly(A)+-purified mouse liver RNA with reverse transcriptase using the following primer to the coding region: 5' dTCCCTTAATGAAGTTCGC. A poly(C) tail was added to the 5' end of the cDNA following the protocol recommended by the manufacturer. The 5' regions of WBP-2 cDNA were amplified using polymerase chain reaction with primers annealing to the 5' poly(C) tail (5'-dCUACUACUACUAGGCCACGCGTCGACTAGTACGGGIIGGGIIGGGIIG, I denoting deoxyinosine) and to a region in the coding region of WBP-2 (5'- dAUGAUCAUAGGACAUUAG). The cDNA fragments were then isolated according to the manufacturer's recommendations.

Preparation of Chromosomal Slides

Lymphocytes isolated from human blood were cultured in a minimal essential medium supplemented with 10% fetal calf serum and phytohemagglutinin at 37 °C for 68-72 h. The lymphocytes cultures were treated with bromodeoxyuridin (0.18 mg/ml, Sigma) to synchronize the cell population. The synchronized cells were washed three times with serum-free medium to release the block and recultured at 37 °C for 6 h in minimal essential medium with thymidine (2.5 µg/ml, Sigma). Cells were harvested, and slides were made using standard procedures including hypotonic treatment, fixation, and air drying.

Chromosomal Localization by Fluorescence in Situ Hybridization (FISH)

The cDNA inserts for WBP-1 and WBP-2 were separately biotinylated with dATP using the Life Technologies, Inc. BioNick labeling kit at 15 °C for 1 h as described elsewhere (24). The procedure for FISH detection was performed as described previously (24, 25). Slides were baked at 55 °C for 1 h. After RNase treatment, the slides were denatured in 70% formamide in 2 × SSC for 2 min at 70 °C followed by dehydration with ethanol. The biotinylated probes were denatured at 75 °C for 5 min in a hybridization solution consisting of 50% formamide and 10% dextran sulfate. The probes were loaded on the denatured chromosomal slides. After an overnight hybridization, the slides were washed and detected as well as amplified. FISH signals and the DAPI banding pattern were recorded separately by photography, and assignment of the FISH mapping data with chromosomal bands was accomplished by superimposing FISH signals with DAPI-banded chromosomes (26).

In Vitro Site-directed Mutagenesis

The cDNA encoding the WW domain of human YAP was subcloned previously in-frame into the vector pGEX-2TK, allowing it to be expressed as a fusion protein with glutathione S-transferase (GST) (Pharmacia Biotech Inc.) (17). This vector in addition allowed the purified fusion protein to be directly labeled by 32Pi at a protein kinase A phosphorylation site in the region between the GST and WW portions, as described elsewhere (17, 18, 27). Substitution of selected amino acids in the WW domain was achieved using a double-stranded site-directed mutagenesis kit (Pharmacia). The selection primer changed the unique ScaI site in the vector to an MluI site, the introduced mutation in boldface (5'-CTGTGACTGGTGACGCGTCAACCAAGTC-3'). The target primer was used simultaneously to effect the desired substitutions within the WW domain, the introduced mutation in boldface (W177F: 5'-GTCTTTGCCATCTCGAAACCTGCTG-3'; Y188F: 5'-GTGATTTAAGAAAAATCTCTGACCA-3'; F189Y: 5'-GATGTGATTTAAATAGTATCTCTGA-3'; H192F: 5'-TGTCTGATCGATGAAATTTAAGAAG-3'; W199F: CCTGGGGTCCTGGAATGTTGTTGTC-3'; P202A: 5'-ATGGCCTTCCTGAGCTCCTGCCATG-3'). The mutagenesis reactions were performed according to the manufacturer's instructions. Plasmid DNA isolated from individual colonies was purified and sequenced by the Sanger method to confirm each mutation (22).

Coprecipitation and Binding Assays

HeLa cells were grown to confluence in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum in 10-cm plates and lysed in RIPA buffer (10 mM Tris-HCl, pH 7.4, 5 mM EDTA, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 1% Trasylol, 1 µM leupeptin, 1 µM antipain, 1 mM sodium vanadate). GST or GST-WW-YAP (100 µg) bound to glutathione-agarose was incubated with 200 mg of the cell lysate which was diluted 10-fold in Tris/Tween buffer (50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 1 mM EDTA, 0.1% Tween 20, 1% ovalbumin, 1 mM dithiothreitol) at 4 °C overnight. The complexes were then analyzed by Western ligand blotting using 32P-GST-WW-YAP protein as a probe as described elsewhere (17). Binding assays with the mutant WW-YAP protein was performed by western ligand blotting using 32P-GST-GTPPPPYTVG protein as a probe (17).

SPOTs Method of Peptide Synthesis on Derivatized Cellulose

The peptide synthesis was accomplished following the method described by Frank and co-workers (28, 29). Fmoc (N-(9-fluorenyl)methoxycarbonyl) L-amino acids, derivatized cellulose membranes, instruction manual and computer software SPOTs, Release 1.0, for generating various schedules of amino acid applications were all purchased from Genosys Biotechnologies, Inc. Phosphorylated serine, threonine, and tyrosine were obtained from Chem Impex International. The membranes were incubated with the 32P-labeled GST-WW domain protein of human YAP under the conditions described previously (17), except the 5% milk in the blocking solution was replaced with 1 × blocking buffer supplied (as 10 × solution) by the manufacturer. After probing the membranes with 32P-labeled WW proteins, the membranes were regenerated with urea, mercaptoethanol, and acetic acid-containing buffers (as described by the manufacturer), and stained with Coomassie Blue for 10 min and destained using standard protocol for visualizing proteins on SDS-polyacrylamide gels. Uniform light blue staining was observed for the peptides with the exception of those containing Lys, Arg, or His, which were always stained more intensely with Coomassie Blue.


RESULTS

Cloning of WBP-1 and WBP-2

We described previously the isolation of partial clones for murine WBP-1 and WBP-2 which were each missing part of the 5'-coding regions (17). In this paper, the remainder of the murine cDNA encoding these proteins was determined. Additionally, the human orthologs of WBP-1 and WBP-2 were isolated by screening a human cDNA library (M426 lung fibroblasts) under low stringency conditions with the corresponding murine cDNA probes. Human and murine WBP-1 share an 84% sequence identity (Fig. 1A), whereas human and murine WBP-2 are 94% identical (Fig. 1B), not considering those residues that are biochemically similar. The WBP-1 and WBP-2 sequences are dissimilar except for the conserved PY motif. Data-base searches using the FASTA program again did not reveal any known proteins with significant homology to WBP-1. However, human WBP-2 shared 26 and 29% homology with two proteins in Caenorhabditis elegans (unnamed, with GenBank accession numbers [GenBank] and [GenBank]) respectively, as predicted from the open reading frames. These two C. elegans proteins have yet to be characterized. Interestingly, the most extensive region of homology between WBP-2 and the C. elegans proteins contains the PY motif (marked by an asterisk). The PY motif in the C. elegans proteins lacks a proline in the first position but still conforms to the XPPXY consensus that has been established as the minimal sequence that can bind to the WW domain of YAP. Additionally, another region (marked by two asterisks) contains a sequence that has the XPPXY consensus. The above sequence comparison implies that these regions have been relatively well conserved through evolution because of their functional significance, presumably in mediating protein-protein interactions. A third region containing PPPPY sequence (indicated with arrows, Fig. 1B) is conserved between human and mouse WBP-2 sequences, but it is not present in the C. elegans protein.


Fig. 1. Alignment of human and mouse WBP-1 and WBP-2 amino acid sequences. Panel A, comparison of human and murine WBP-1. The asterisk denotes the only region of homology between the two proteins, the PY motif. Panel B, alignment of human and murine WBP-2 and homologous proteins. The bar with one asterisk marks the PY motif identical to that found in WBP-1 and which is highly conserved among the proteins, including two proteins from C. elegans which share 26 and 29% homology to human WBP-2, respectively. The bar with two asterisks indicates a consensus PY motif (XPPXY) that is shared by the proteins. Two arrows mark the third PY motif in the WBP-2 which is not conserved in C. elegans. Note that WBP-1 and WBP-2 sequences are dissimilar except for the conserved PY motifs. Sequence alignments were done using the CLUSTALW and BOXSHADE programs.
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Chromosomal Localization of WBP-1 and WBP-2

Under the conditions described, the hybridization efficiency for the WBP-1 cDNA probe was approximately 81% (among 100 mitotic figures examined, 81 showed signals on one pair of chromosomes). Similarly, the efficiency for the WBP-2 cDNA probe was approximately 76%. The detailed positions of the loci were determined further based on the summary from 10 FISH signal photographs/probe; representative hybridization pictures are shown in Fig. 2, A and D. By superimposing the signals obtained from FISH onto a representation of DAPI-banded chromosomes, we localized the WBP-1 locus to chromosome 2p12 and the WBP-2 locus to chromosome 17q25 (Fig. 2, C and F, respectively). Some hybridization of the WBP-1 cDNA occurred at chromosome 2p11.2 as well, but this signal was considerably weaker compared with hybridization at region p12.


Fig. 2. Chromosomal localization of human WBP-1 and WBP-2 genes. Panel A, FISH of normal human metaphase chromosomes using WBP-1 cDNA as a probe. Arrow indicates positive staining on chromosome 2, region p12. Panel B, DAPI staining of chromosomes. C, schematic representation of the results of the FISH of WBP-1. Each filled circle indicates a fluorescent signal on chromosome 2p12. Panel D, FISH of normal human metaphase chromosomes using WBP-2 cDNA as a probe. Arrow indicates positive staining on chromosome 17, region q25. Panel E, DAPI staining of chromosomes. Arrow indicates chromosome 17. Panel F, schematic representation of the results of the FISH of WBP-2. Each filled circle indicates a fluorescent signal on chromosome 17q25.
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Filter Binding Assays with Mutant WW Domains

To determine the residues of the WW domain which are important for binding, we performed in vitro site-directed mutagenesis of selected residues in the WW domain fused to GST (Fig. 3). The most conserved residues in the domain were chosen, including Trp177, Tyr188, Phe189, His92, Trp199, and Pro202. Since we were interested in elucidating the residues directly involved in forming the binding surface and contacting the ligand, we replaced the above residues with amino acids similar in structure and size. Replacing with amino acids too dissimilar in size would have risked disturbing the natural tertiary structure of the WW fold. We conducted filter binding assays in which these GST-WW mutant proteins were transferred to a nitrocellulose membrane that was subsequently probed with 32P-labeled GST-PY protein. The WW mutants Y188F, H192F, P202A, and W199F failed to bind to the ligand probe (Fig. 4A). However, the constructs W177F and F189Y were still able to bind to the ligand, and the former construct may bind at a slightly higher affinity than even the wild type GST-WW protein (Fig. 4A). Thus, according to these results, the residues Tyr188, His192, Trp199, and Pro202 seem to be involved in establishing the binding surface of the WW domain. Alternatively, some of these residues may be critical for maintaining the three-dimensional structure of the binding site on the nitrocellulose membrane, should a renatured form of the GST-WW protein be required to retain binding function.


Fig. 3. Mutant WW domains used in binding assays. Selected conserved residues of the WW-YAP domain were substituted with similar residues in a fusion protein with GST. The consensus sequence of the WW domain and the delineation of three beta  strands (B) are indicated. Capitals indicate conserved and semiconserved amino acids; h, hydrophobic; t, turnlike or polar amino acids; x, positions without clear t or h designations.
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Fig. 4. Binding activity of different WW mutants. Panel A, purified GST-WW proteins with various amino acid substitutions in the WW domain were separated by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membrane. Blots were then probed with 32P-labeled GST fused to the PY peptide, GTPPPPYTVG. Panel B, coprecipitation of endogenous ligand by incubating glutathione-agarose coupled to different GST-WW proteins (100 µg each) with HeLa cell lysates. Incubation occurred at 4 °C for 18 h, after which the beads were washed three times with phosphate-buffered saline, solubilized in sample running buffer, separated by SDS-polyacrylamide gel electrophoresis, and transferred to nitrocellulose membrane. The blots were probed with 32P-labeled GST-WW (wild type) protein. Arrow indicates endogenous ligand coprecipitated with the GST-WW proteins. Panel C, Coomassie stain of a duplicate gel from panel A, to confirm equal amounts of protein (2 µg) loaded per lane. Lane 1, GST; lane 2, GST-WW wild type; lane 3, GST-WW Y188F; lane 4, GST-WW W177F; lane 5, GST-WW P202A; lane 6, GST-WW H192F; lane 7, GST-WW F189Y; lane 8, GST-WW W199F. Molecular mass in kDa indicated on the right.
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Binding of the WW Mutants in Solution

The binding of the WW domain while in its natural tertiary shape in solution may more closely resemble conditions encountered in vivo. As a result, coprecipitation assays were conducted in which GST-WW proteins coupled to glutathione-agarose were incubated with cell lysates to isolate the endogenous ligand to the WW domain of YAP. Using this method, the residues His192, Pro202, and Trp199 were found to be important for the WW domain to bind its ligand in solution (Fig. 4B). Remarkably, residue Tyr188, previously found to be important in filter binding assays, does not seem to be implicated in the binding activity of the WW domain in its natural form since the construct Y188F still was able to coprecipitate the endogenous ligand (Fig. 4B).

Mutational Analysis of the PY Motif

Each position of the target ligand, Gly1-Thr2-Pro3-Pro4-Pro5-Pro6-Tyr7-Thr8-Val9-Gly10, corresponding to residues 170-179 of the mouse WBP-1 sequence, was substituted with all possible amino acids using SPOTs peptide synthesis to determine the binding requirements of the YAP WW domain ligand. Certain points can be inferred from the results of the SPOTs analysis: The replacement of the Pro4 residue with leucine, serine, valine, or alanine resulted in weak binders to the WW domain of YAP. However, substitution with other amino acids in this position abolished binding completely, in agreement with the consensus established earlier (Fig. 5). When the Pro5 position was replaced with a tyrosine (Fig. 6, spot 20), the peptide bound with moderate affinity. All other substitutions at Pro5 were negative. Tyr7 could not be replaced with phenylalanine, tryptophan, histidine, or with any other amino acid without disrupting binding activity. Also, a target peptide containing a phosphorylated tyrosine failed to interact with the WW domain, suggesting a method of regulating binding activity by tyrosine phosphorylation. The Tyr7 residue seems to be the least tolerant of any substitution. Moreover, the target peptide with the reverse sequence (GVTYPPPPTG) does not bind to the WW domain of YAP (Fig. 5).


Fig. 5. Mutational analysis of the Gly1-Thr2-Pro3-Pro4-Pro5-Pro6-Tyr7-Thr8-Val9-Gly10 target peptide that binds to the WW domain of human YAP. The SPOTs technique was used to generate a repertoire of 10-mer peptides (28, 29). Pro3, Pro4, and Pro6 were replaced consecutively with the remaining 19 amino acids. Peptide 1 is the parent sequence. Peptides 2-20 correspond to all of the Pro3 substitutions, peptides 21-39 correspond to Pro6 substitutions, and peptides 40-58 correspond to Pro4 substitutions. Tyr7 was replaced by phosphotyrosine, serine, phosphoserine, threonine, and phosphothreonine (a-f), and we also assayed these peptides with reverse sequence (g-l). For blotting 32P-labeled GST-WW domain of human YAP was used. Panel A, autoradiogram of the membrane exposed for 5 min; panel B, for 15 min; panel C, orientation of the derivatized spots on which peptides were synthesized. Panel D, individual sequences of the peptides corresponding to numbered spots. Controls indicated derivatized spots onto which amino acids were not applied. There was no binding detected when the blot was probed with 32P-labeled GST protein and exposed for 5 or 15 min.
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Fig. 6. Mutational analysis of the Gly1-Thr2-Pro3-Pro4-Pro5-Pro6-Tyr7-Thr8-Val9-Gly10 target peptide that binds to the WW domain of YAP. The SPOTs technique was used to synthesize a repertoire of mutated peptides. Pro5, Tyr7, and Gly10 were replaced consecutively with the remaining 19 amino acids. Peptide 1 is the parent sequence. Peptides 2-20 correspond to all of the Pro5 substitutions, peptides 21-37 correspond to Tyr7 substitutions, and peptides 38-56 correspond to Gly10 substitutions. For blotting 32P-labeled GST-WW domain of human YAP was used. Panel A, autoradiogram of the membrane exposed for 5 min; panel B, for 15 min; panel C, orientation of the derivatized spots on which peptides were synthesized. Panel D, individual sequences of the peptides corresponding to numbered spots. Controls indicated derivatized spots onto which amino acids were not applied.
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Although the lack of perfect normalization of the SPOTs membrane because of difficulties in precise measurements of the amount of peptide in each individual spot precludes a rigorous quantitation of the binding affinities, certain conclusions can be drawn. Based on the Coomassie Blue staining of the membranes, we assume that there is no significant difference in the amount of peptide between different spots. From the data shown in Figs. 5, 6, 7 we can observe that the replacement of Pro3, Pro6, Val9, and Gly10 with lysine, and Thr8 with proline increase the interaction with the GST-WW-YAP domain. Cysteine at position Gly1 and a cysteine or proline at position Thr2 seems to increase the binding slightly. In contrast, an isoleucine at position Pro3 and acidic residues at position Pro6, Thr8, Val9, or Gly10 reduce the interaction with the WW domain. To prove these conclusions, however, selected peptides would have to be synthesized, and the Kd value of their interaction with the WW domain, in solution and on solid supports, would have to be determined. Preliminary results obtained with an isothermal titration microcalorimeter indicate that peptide analogs containing lysine and proline substitutions at the carboxyl-terminal end of the parent peptide show at least 5-fold lower binding constants compared with the Kd of the parent peptide binding to the WW domain of YAP.2


Fig. 7. Mutational analysis of the Gly1-Thr2-Pro3-Pro4-Pro5-Pro6-Tyr7-Thr8-Val9-Gly10 target peptide that binds to the WW domain of YAP. The SPOTs technique was used to synthesize a repertoire of mutated peptides. Gly1, Thr2, Thr8, and Val9 were replaced consecutively with the remaining 19 amino acids. Peptide 1 is the parent peptide. Peptides 2-20 correspond to all of the Gly1 substitutions, peptides 21-39 correspond to Thr2 substitutions, peptides 40-58 correspond to Thr8 substitutions, and peptides 59-77 correspond to Val9 substitutions. For blotting 32P-labeled GST-WW domain of human YAP was used. Panel A, autoradiogram of the membrane exposed for 5 min; panel B, for 15 min; panel C, orientation of the derivatized spots on which peptides were synthesized. Panel D, individual sequences of the peptides corresponding to numbered spots. Controls indicated derivatized spots onto which amino acids were not applied.
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DISCUSSION

We have cloned the human homologs of the ligands to the WW domain of YAP, WBP-1 and WBP-2. These proteins allowed us to categorize the WW motif as a novel polyproline-binding module (17). The amino acid sequences of WBP-1 and WBP-2 have been well conserved through evolution, considering the high degree of homology between mouse and human orthologs. The degree of homology shared by the orthologs, especially in the region of the PY motif, suggests that the function of these proteins has been conserved between species. The PY motif, a region mediating protein interaction, is perfectly conserved between the mouse and human forms of WBP-1 and WBP-2 and represents the most conserved region shared between WBP-2 and two C. elegans proteins. This further supports the hypothesis that the biological function of these proteins depends on their ability to participate in protein interactions.

Using the cDNA of these clones, we localized the genes encoding human WBP-1 and WBP-2 to chromosomes 2p12 and 17q25, respectively. The chromosome region 2p12 has been shown to be potentially involved in several disorders. Chromosomal abnormalities are found in the majority of cases of non-Hodgkin's lymphoma, a subset of which shows a translocation involving the regions 3q27 and 2p12 (30). In addition, the gene for familial juvenile nephronophthisis, a leading genetic cause of juvenile end-stage renal failure, has been narrowed to the region 2p12 based on human linkage analysis (31). However, more work must be done to determine if WBP-1 can be implicated in these human conditions.

The chromosome region 17q25 has been shown to be involved in certain forms of human carcinogenesis. Some cases of alveolar soft part sarcoma show a consistent abnormality of 17q25 (32). However, the region of 17q25, also occasionally translocated in chronic myelogenous leukemia, has not yet been associated with any known oncogenes (33). Additional genetic and biochemical studies would need to be performed to demonstrate the importance of WBP-2 as a potential oncogene in the etiology of these human cancers.

Using site-directed mutagenesis we have implicated specific residues in the WW domain of YAP which are important to maintain its ability to bind to the PY motif. Our results complement the recent data on the NMR solution structure of the WW domain of YAP in complex with the proline-rich ligand (20). The three-dimensional structure consists of a three-stranded, antiparallel beta  sheet with residues Tyr188 and Trp199 (the second conserved tryptophan) on the concave side and residues Phe189 and Trp177 on the convex side. The concave aspect contains an almost flat hydrophobic binding surface represented by the residues Tyr188, Trp199, His192, and Leu190. Moreover, the NH2 and COOH termini of the domain appose each other on the convex side in the manner of a hydrophobic buckle which presumably helps maintain the stable folded structure of the domain. This buckle forms when Pro174 and Pro202 pack against Trp177, the interaction of which is further stabilized by the presence of Ile167. These results from Macias and co-workers (20) support the binding consensus of the PY ligand established earlier as XPPXY (17). Moreover, the mutagenesis results presented here are for the most part in agreement with the NMR structure. Conservative substitution of the residues Tyr188, His192, Trp199, and Pro202 abrogated the binding function of the WW domain in our filter binding assays, implying in light of the NMR data that the hydrophobic binding surface is disrupted by the first three mutations or that the fold of the WW domain is severely compromised by the Pro202 substitution. Nuclear Overhauser effects between His192 and ligand residues Tyr7 and Val9, Trp199, and Pro5, and between Tyr188 and the carbonyl group of Pro6 provide corroborating evidence for the importance of these residues. The replacement of the histidine residue at position 192 with a phenylalanine suggests the participation of a hydrogen bond located between the imidazole nitrogen acceptor of histidine and the hydroxyl hydrogen donor of Tyr7 (Fig. 8). Elimination of this hydrogen bond likely destabilizes this structure, as does the introduction of the bulkier phenylalanine residue into such a confined space. Lack of ligand binding by the W199F and P202A mutants are not manifested through variations in charges or hydrogen bonds but likely reflect alteration in the architecture of the surface topology and changes in side chain packing. The removal of tryptophan at position 199 and replacement with a phenylalanine may eliminate enough of the surface recognition site to inhibit ligand binding. Alternatively, the newly introduced phenylalanine residue may not pack efficiently into the cavity designated by Pro4 and Pro5 (Fig. 8). Likewise, molecular van der Waals packing interactions are central to the P202A mutation. The incorporation of an alanine diminishes this tight hydrophobic packing which may ultimately destroy the molecular "fastener" thought to be responsible for maintaining the integrity of the core domain beta  sheet (Fig. 8). One should point out that the stability of the WW domain could be considered marginal (20) because the domain is only one beta sheet layer thick and thus may not have a well defined hydrophobic core.


Fig. 8. Models of the WW domain/target peptide complexes depicting changes introduced by selected point mutations. Panels A and B, Y188F and the wild type reference; panels C and D, H192F and the wild type reference; panels E and F, W199F and the wild type reference; panels G and H, P202A and the wild type reference. Graphical depictions of the WW domain-target peptide complexes were generated using MOLSCRIPT program (44). INSIGHTII (Biosym Technologies) was used to generate point mutations from the wild type WW domain protein data bank file (20). Torsion angles and molecular packing influences of each amino acid were monitored carefully to avoid the introduction of steric strain. RASMOL (Roger Sayle) was used to rotate and orient the protein data bank files and generate MOLSCRIPT script files. For details, see "Discussion."
[View Larger Version of this Image (38K GIF file)]

In contrast, mutations of Trp177 or Phe189 had no observable effect in the binding assays, suggesting that structural changes possibly imparted by these substitutions were insignificant to the overall function of the domain. The replacement of Tyr188 with phenylalanine failed to attenuate the binding ability of the domain in solution, as illustrated by the coprecipitation assays (Fig. 4B). Based upon NMR data, the hydroxyl proton of this tyrosine residue is within hydrogen bonding distance of the carbonyl oxygen of ligand residue Pro6 (Fig. 8, A and B). The ligand binding studies presented within suggest that such an interaction is not required to manifest ligand binding in solution. Comparison of the mutant Y188F structure with that of the wild type does not reveal any significant structural distinction. We also cannot rule out the possibility that another portion of the domain may compensate for or "mask" this mutation, depending on the conformation of the domain while bound to nitrocellulose membrane compared with its state in solution.

Mutational analysis of the target peptide (GTPPPPYTVG) for the WW domain of human YAP led us to the following conclusions. The data confirm our preliminary consensus Xaa-Pro4-Pro5-Xaa-Tyr7 generated by the "alanine scan" mutagenesis (17). In addition, these results are in agreement with the structure of the WW domain of human YAP in complex with the target peptide (20). The PY motif ligand (peptide GTPPPPYTVG) fits into the binding surface in the form of a polyproline type II helix. The Pro4 and Pro5 of the ligand are involved in the binding interface by forming van der Waals contacts with the second conserved Trp199. Moreover, the Tyr7 of the ligand peptide is accommodated by a hydrophobic surface containing conserved residues including Leu190 and His192 in the WW domain. Interestingly, mutations P5L and Y7H in the PY motif are analogous to those found in the amiloride-sensitive sodium channel beta  subunit of two Liddle's syndrome patients (34, 35). The proline-rich motif of amiloride-sensitive sodium channel is a target of deletions and point mutations resulting in Liddle's syndrome of hypertension. It is likely that the two mutations P616L and Y618H (Refs. 34 and 35), which are analogous to P5L and Y7H, result in the complete lack of binding to the WW domain of Nedd4. In addition, the fact that the WW domain does not bind to a target peptide with the reverse sequence provides suggestive evidence that the YPPPP motif in the WBP-2 protein (amino acids 179-183 of human WBP-2) may be nonfunctional in terms of binding to the WW domain of YAP.

The WW domain appears to be involved in various physiological and pathophysiological processes. The disruption of the WW-PY interaction may in fact be the molecular deficiency present in a form of inherited hypertension known as Liddle's syndrome (36-39). Furthermore, dystrophin, a protein implicated in Duchenne's and Becker's muscular dystrophy, forms a stable complex via its WW domain to a proline-rich motif in beta -dystroglycan in vitro (40). In addition, the Gag protein of retroviruses contains a PY motif that when mutated severely curtails the ability of the virus to bud from the host cell membrane (40-43).

Because of the relatively small size of the PY motif, low molecular weight mimotopes may be designed to fit into the binding pocket of the WW domain, to interfere with the viral life cycle, for example. Additional work on the WW domain will provide answers to fundamental biological questions and will ultimately lead to the development of therapeutic applications.


FOOTNOTES

*   This work was supported by Grant 5T32GM07739-16 from the National Institutes of Health (to H. I. C.), Grants CA45757 and CAO1605 from NCI, National Institutes of Health, Grant 3035 from the Council for Tobacco Research U. S. A. Inc., by a Human Frontier Science Program grant, and a Muscular Dystrophy Association grant (to M. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s)  U40825[GenBank] (mouse WBP-1), U40826[GenBank] (mouse WBP-2), U79457[GenBank] (human WBP-1), and U79458[GenBank] (human WBP-2).


par    To whom correspondence should be addressed: Dept. of Biochemistry, The Mount Sinai School of Medicine, One Gustave Levy Place, New York, NY 10029-6547. Tel.: 212-241-9431; Fax: 212-426-1483; E-mail: M_Sudol{at}smtplink.mssm.edu.
1   The abbreviations used are: SH, Src homology; YAP, Yes kinase-associated protein; WBP, WW domain-binding protein; PY, motif containing PPPPY sequence; FISH, fluorescence in situ hybridization; DAPI, 4,6-diamidino-2-phenylindole; GST, glutathione S-transferase.
2   M. Lemmon and M. Sudol, unpublished data.

ACKNOWLEDGEMENTS

We thank Dr. Hidesaburo Hanafusa for valuable comments and advice and the members of the Laboratory of Molecular Oncology at the Rockefeller University as well as the Department of Biochemistry at the Mount Sinai School of Medicine for stimulating discussions. We owe special thanks to Dr. Mark Lemmon for performing microcalorimetric measurements of WW domain-ligand interactions, to Drs. Matti Saraste and Hartmut Oschkinat for constructive discussions regarding the structure of the WW domain of human YAP, to Dr. Peer Bork for help in sequence analysis, and to Jason Paragas for critical comments on the manuscript.


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