Yaich L, Ooi J, Park M, Borg J P, Landry C, Bodmer R, Margolis B
Department of Biology, University of Michigan, Ann Arbor, Michigan 48109, USA.
J Biol Chem. 1998 Apr 24;273(17):10381-8. doi: 10.1074/jbc.273.17.10381.
The Numb protein is involved in cell fate determination during Drosophila neural development. Numb has a protein domain homologous to the phosphotyrosine-binding domain (PTB) in the adaptor protein Shc. In Shc, 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 Shc PTB domain. Several amino acid residues in Shc have been implicated by site-directed mutagenesis to be critical for Shc binding to receptor tyrosine kinases. We have generated homologous mutations in Numb to test whether, in vivo, these changes affect Numb function during Drosophila sensory organ development. Two independent amino acid changes that interfere with Shc binding to phosphotyrosine residues do not affect Numb activity in vivo. In contrast, a mutation shown to abrogate the ability of the Shc PTB domain to bind residues upstream of the phosphotyrosine virtually eliminates Numb function. Similar results were observed in vitro by examining the binding of the Numb PTB domain to proteins from Schneider S2 cells. Our data confirm the importance of the PTB domain for Numb function but strongly suggest that the Numb PTB domain is not involved in phosphotyrosine-dependent interactions.
Numb蛋白参与果蝇神经发育过程中的细胞命运决定。Numb具有一个与衔接蛋白Shc中的磷酸酪氨酸结合结构域（PTB）同源的蛋白结构域。在Shc中，该结构域与受体酪氨酸激酶和其他信号分子上特定的含磷酸酪氨酸基序相互作用。磷酸酪氨酸N端的残基对于磷酸肽与Shc PTB结构域的结合也至关重要。通过定点诱变表明，Shc中的几个氨基酸残基对于Shc与受体酪氨酸激酶的结合至关重要。我们在Numb中产生了同源突变，以测试在果蝇感觉器官发育过程中，这些变化在体内是否会影响Numb功能。两个干扰Shc与磷酸酪氨酸残基结合的独立氨基酸变化在体内不影响Numb活性。相反，一个显示能消除Shc PTB结构域结合磷酸酪氨酸上游残基能力的突变几乎完全消除了Numb功能。通过检测Numb PTB结构域与施耐德S2细胞蛋白的结合，在体外也观察到了类似的结果。我们的数据证实了PTB结构域对Numb功能的重要性，但强烈表明Numb PTB结构域不参与磷酸酪氨酸依赖性相互作用。
