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

doi:10.1074/jbc.272.27.17070

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

From the ^‡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

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.

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 GenBank™/EMBL Data Bank with accession number(s) U40825 (mouse WBP-1), U40826 (mouse WBP-2), U79457 (human WBP-1), and U79458 (human WBP-2).
↵‖ 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 situhybridization; DAPI, 4,6-diamidino-2-phenylindole; GST, glutathioneS-transferase.
↵2 M. Lemmon and M. Sudol, unpublished data.
- Received February 3, 1997.
- Revision received April 4, 1997.