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

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.

[1]  H. Heng,et al.  High-resolution mapping of mammalian genes by in situ hybridization to free chromatin. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[2]  M. Sudol,et al.  WW domains and retrovirus budding , 1996, Nature.

[3]  B. Margolis,et al.  A region in Shc distinct from the SH2 domain can bind tyrosine-phosphorylated growth factor receptors. , 1994, The Journal of biological chemistry.

[4]  L. Schild,et al.  A de novo missense mutation of the beta subunit of the epithelial sodium channel causes hypertension and Liddle syndrome, identifying a proline-rich segment critical for regulation of channel activity. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[5]  M. Sudol,et al.  The WW domain of Yes-associated protein binds a proline-rich ligand that differs from the consensus established for Src homology 3-binding modules. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[6]  R. Frank,et al.  An efficient method for anchoring fmoc-anino acids to hydroxyl-functionalised solid supports , 1990 .

[7]  P. Kraulis.  A program to produce both detailed and schematic plots of protein structures , 1991 .

[8]  P. Bork,et al.  Characterization of a novel protein‐binding module — the WW domain , 1995, FEBS letters.

[9]  D. Beach,et al.  Pub1 acts as an E6‐AP‐like protein ubiquitiin ligase in the degradation of cdc25. , 1996, The EMBO journal.

[10]  H. Tilly,et al.  Translocations involving band 3q27 and Ig gene regions in non-Hodgkin's lymphoma. , 1992, Blood.

[11]  H. Omran,et al.  Mapping of a gene for familial juvenile nephronophthisis: refining the map and defining flanking markers on chromosome 2. APN Study Group. , 1993, American journal of human genetics.

[12]  T Pawson,et al.  Specific motifs recognized by the SH2 domains of Csk, 3BP2, fps/fes, GRB-2, HCP, SHC, Syk, and Vav , 1994, Molecular and cellular biology.

[13]  C. Cameron,et al.  An assembly domain of the Rous sarcoma virus Gag protein required late in budding , 1994, Journal of virology.

[14]  Andrea Musacchio,et al.  High-resolution crystal structures of tyrosine kinase SH3 domains complexed with proline-rich peptides , 1994, Nature Structural Biology.

[15]  M. Saraste,et al.  Structure of the WW domain of a kinase-associated protein complexed with a proline-rich peptide , 1996, Nature.

[16]  L. Williams,et al.  An alternative to SH2 domains for binding tyrosine-phosphorylated proteins. , 1994, Science.

[17]  O. Staub,et al.  WW domains of Nedd4 bind to the proline‐rich PY motifs in the epithelial Na+ channel deleted in Liddle's syndrome. , 1996, The EMBO journal.

[18]  T Pawson,et al.  SH2 and SH3 domains , 1993, Current Biology.

[19]  P. Bork,et al.  Characterization of the Mammalian YAP (Yes-associated Protein) Gene and Its Role in Defining a Novel Protein Module, the WW Domain (*) , 1995, The Journal of Biological Chemistry.

[20]  L. Schild,et al.  Liddle disease caused by a missense mutation of beta subunit of the epithelial sodium channel gene. , 1996, The Journal of clinical investigation.

[21]  M. Sudol,et al.  Structure and function of the WW domain. , 1996, Progress in biophysics and molecular biology.

[22]  G. Liddle.  A familial renal disorder simulating primary aldosteronism but with negligible aldosterone secretion , 1964 .

[23]  D. Baltimore,et al.  Modular binding domains in signal transduction proteins , 1995, Cell.

[24]  M. Sudol,et al.  Towards prediction of cognate complexes between the WW domain and proline‐rich ligands , 1996, FEBS letters.

[25]  V. Desmet,et al.  Alveolar soft‐part sarcoma: evidence for its myogenic origin and for the involvement of 17q25 , 1993, Histopathology.

[26]  D. Baltimore,et al.  Signalling through SH2 and SH3 domains. , 1993, Trends in cell biology.

[27]  M. Sudol,et al.  Yes-associated protein (YAP65) is a proline-rich phosphoprotein that binds to the SH3 domain of the Yes proto-oncogene product. , 1994, Oncogene.

[28]  Hongtao Yu,et al.  Structural basis for the binding of proline-rich peptides to SH3 domains , 1994, Cell.

[29]  M. Sudol,et al.  Identification and characterization of protein ligands to the WW domain by western ligand blotting , 1996 .

[30]  B. Hemmings,et al.  Pleckstrin domain homology , 1993, Nature.

[31]  L. Schild,et al.  Identification of a PY motif in the epithelial Na channel subunits as a target sequence for mutations causing channel activation found in Liddle syndrome. , 1996, The EMBO journal.

[32]  P. Bork,et al.  The WW domain: a signalling site in dystrophin? , 1994, Trends in biochemical sciences.

[33]  H. Heng,et al.  FISH detection on DAPI-banded chromosomes. , 1994, Methods in molecular biology.

[34]  J. Wills,et al.  Positionally independent and exchangeable late budding functions of the Rous sarcoma virus and human immunodeficiency virus Gag proteins , 1995, Journal of virology.

[35]  Peer Bork,et al.  A phosphotyrosine interaction domain , 1995, Cell.

[36]  S. Aaronson,et al.  An efficient directional cloning system to construct cDNA libraries containing full-length inserts at high frequency. , 1989, Gene.

[37]  T. Pawson,et al.  SH2 domains recognize specific phosphopeptide sequences , 1993, Cell.

[38]  R. Frank,et al.  Simultaneous multiple peptide synthesis under continuous flow conditions on cellulose paper discs as segmental solid supports , 1988 .

[39]  L. Schild,et al.  A mutation in the epithelial sodium channel causing Liddle disease increases channel activity in the Xenopus laevis oocyte expression system. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[40]  Peggy J. Farnham,et al.  Expression cloning of a cDNA encoding a retinoblastoma-binding protein with E2F-like properties , 1992, Cell.

[41]  F. Sanger,et al.  DNA sequencing with chain-terminating inhibitors. , 1977, Proceedings of the National Academy of Sciences of the United States of America.