FUNCTIONAL-ANALYSIS OF THE NUMB PHOSPHOTYROSINE-BINDING DOMAIN USING SITE-DIRECTED MUTAGENESIS

The Numb protein is involved in cell fate determination during Drosoph ila neural development. Numb has a protein domain homologous to the ph osphotyrosine-binding domain (PTB) in the adaptor protein She. In She, this domain interacts with specific phosphotyrosine containing motifs on receptor tyrosine kinases and other signaling molecules. Residues N-terminal to the phosphotyrosine are also crucial for phosphopeptide binding to the She PTB domain. Several amino acid residues in She have been implicated by site-directed mutagenesis to be critical for She b inding to receptor tyrosine kinases, We have generated homologous muta tions in Numb to test whether, in vivo, these changes affect Numb func tion during Drosophila sensory organ development, Two independent amin o acid changes that interfere with She binding to phosphotyrosine resi dues do not affect Numb activity in vivo, In contrast, a mutation show n to abrogate the ability of the She PTB domain to bind residues upstr eam of the phosphotyrosine virtually eliminates Numb function. Similar results were observed in vitro by examining the binding of the Numb P TB domain to proteins from Schneider S2 cells. Our data confirm the im portance of the PTB domain for Numb function but strongly suggest that the Numb PTB domain is not involved in phosphotyrosine-dependent inte ractions.