CHARACTERIZATION OF THE WW DOMAIN OF HUMAN YES-ASSOCIATED PROTEIN ANDITS POLYPROLINE-CONTAINING LIGANDS

We had previously identified the WW domain as a novel globular domain that is composed of 38-40 semi-conserved amino acids and is involved i n mediating protein-protein interaction, The WW domain is shared by pr oteins of diverse functions including structural, regulatory, and sign aling proteins in yeast, nematode, and mammals, Functionally it is sim ilar 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 protei n which we named WW domain-binding proteins 1 and 2, These proteins in teracted with the WW domain via a short proline-rich motif with She co nsensus sequence of four consecutive prolines followed by a tyrosine, Herein, we report the cDNA cloning and characterization of the human o rthologs of WW domain-binding proteins I and 2, The products encoded b y these cDNA clones represent novel proteins with no known function, F urthermore, these proteins show no homology to each other except for a proline-rich motif, By fluorescence in situ hybridization oil human m etaphase chromosomes, we mapped the human genes for WW domain-binding proteins 1 and 2 to chromosomes 2p12 and 17q25, respectively. In addit ion, using site-directed mutagenesis, we determined which residues in the WW domain of Yes kinase-associated protein are critical for bindin g. 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.