Herbst R, Zhang X, Qin J, Simon M A
Department of Biological Sciences, Stanford University, Stanford, CA 94305-5020, USA.
EMBO J. 1999 Dec 15;18(24):6950-61. doi: 10.1093/emboj/18.24.6950.
The pleckstrin homology (PH) domain-containing protein Daughter of Sevenless (DOS) is an essential component of the Sevenless receptor tyrosine kinase (SEV) signaling cascade, which specifies R7 photoreceptor development in the Drosophila eye. Previous results have suggested that DOS becomes tyrosine phosphorylated during SEV signaling and collaborates with the protein tyrosine phosphatase CSW. We have investigated this possibility by identifying tyrosine residues 801 and 854 of DOS as the phosphorylated binding sites for the CSW SH2 domains. We show that these sites become phosphorylated in response to SEV activation and that phosphorylation of both sites is required to allow CSW to bind DOS. Mutant DOS proteins in which either Y801 or Y854 of DOS has been changed to phenylalanine are unable to function during signaling by SEV and other receptor tyrosine kinases. In contrast, we find that a mutant DOS protein in which all tyrosine phosphorylation sites except Y801 and Y854 have been removed is able effectively to provide DOS function during SEV signaling and to rescue the lethality associated with dos loss-of-function mutations. These results indicate that a primary role for DOS during signaling by SEV and other receptor tyrosine kinases is to become phosphorylated at Y801 and Y854 and then recruit CSW.
含普列克底物蛋白同源（PH）结构域的“七无之女”（DOS）蛋白是七无受体酪氨酸激酶（SEV）信号级联反应的重要组成部分，该信号级联反应决定了果蝇眼睛中R7光感受器的发育。先前的研究结果表明，DOS在SEV信号传导过程中发生酪氨酸磷酸化，并与蛋白酪氨酸磷酸酶CSW协同作用。我们通过将DOS的酪氨酸残基801和854鉴定为CSW SH2结构域的磷酸化结合位点，对这一可能性进行了研究。我们发现，这些位点在SEV激活后发生磷酸化，并且两个位点的磷酸化都是CSW结合DOS所必需的。将DOS的Y801或Y854突变为苯丙氨酸的突变型DOS蛋白在SEV和其他受体酪氨酸激酶的信号传导过程中无法发挥作用。相比之下，我们发现一种突变型DOS蛋白，其中除Y801和Y854外的所有酪氨酸磷酸化位点均已去除，该蛋白在SEV信号传导过程中能够有效地发挥DOS功能，并挽救与dos功能丧失突变相关的致死性。这些结果表明，DOS在SEV和其他受体酪氨酸激酶信号传导过程中的主要作用是在Y801和Y854处发生磷酸化，然后招募CSW。
