Kihara H, Siraganian R P
Laboratory of Immunology, National Institute of Dental Research, National Institutes of Health, Bethesda, Maryland 20892.
J Biol Chem. 1994 Sep 2;269(35):22427-32.
Aggregation of the high affinity IgE receptor (Fc epsilon RI) on rat basophilic leukemia RBL-2H3 cells results in activation of the protein tyrosine kinases Syk and Lyn. Here, we report that fusion proteins containing the Src homology 2 (SH2) domains of Syk and Lyn precipitated tyrosine-phosphorylated proteins from RBL-2H3 cell lysates. There was no detectable precipitation with the N-terminal Syk SH2 domain, minimal with the C-terminal Syk SH2, and strong when the two SH2 domains were expressed in tandem. The Syk SH2 domains showed pronounced selectivity in the binding of tyrosine-phosphorylated proteins. Thus, Syk SH2 precipitated only three phosphoproteins, two of which were the beta and gamma subunits of Fc epsilon RI. In contrast, the fusion protein with Lyn SH2, in addition to precipitating the same Fc epsilon RI components, bound to many other phosphoproteins. Tyrosine phosphorylation of the beta and gamma subunits of Fc epsilon RI was essential for their precipitation with the SH2 fusion proteins. By direct binding studies, there was more binding of Syk SH2 to Fc epsilon RI gamma than to Fc epsilon RI beta, whereas Lyn SH2 bound only to Fc epsilon RI beta. The Syk SH2 fusion protein competitively inhibited the association of gamma and beta with Syk in lysates of activated cells. The SH2-mediated association of these two protein tyrosine kinases with Fc epsilon RI could play an important role in receptor signaling.
大鼠嗜碱性粒细胞白血病RBL-2H3细胞上高亲和力IgE受体（FcεRI）的聚集会导致蛋白酪氨酸激酶Syk和Lyn的激活。在此，我们报道含有Syk和Lyn的Src同源2（SH2）结构域的融合蛋白能从RBL-2H3细胞裂解物中沉淀出酪氨酸磷酸化蛋白。N端Syk SH2结构域未检测到沉淀，C端Syk SH2结构域沉淀极少，而当两个SH2结构域串联表达时沉淀强烈。Syk SH2结构域在结合酪氨酸磷酸化蛋白时表现出明显的选择性。因此，Syk SH2仅沉淀出三种磷酸化蛋白，其中两种是FcεRI的β和γ亚基。相比之下，含有Lyn SH2的融合蛋白除了沉淀相同的FcεRI组分外，还与许多其他磷酸化蛋白结合。FcεRI的β和γ亚基的酪氨酸磷酸化对于它们与SH2融合蛋白的沉淀至关重要。通过直接结合研究，Syk SH2与FcεRIγ的结合比与FcεRIβ的结合更多，而Lyn SH2仅与FcεRIβ结合。Syk SH2融合蛋白竞争性抑制活化细胞裂解物中γ和β与Syk的结合。这两种蛋白酪氨酸激酶通过SH2介导与FcεRI的结合可能在受体信号传导中起重要作用。
