Amoui M, Dráberová L, Tolar P, Dráber P
Department of Mammalian Gene Expression, Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Prague.
Eur J Immunol. 1997 Jan;27(1):321-8. doi: 10.1002/eji.1830270146.
Activation of rat mast cells through the receptor with high affinity for IgE (Fc epsilonRI) requires a complex set of interactions involving transmembrane subunits of the Fc epsilonRI and two classes of nonreceptor protein tyrosine kinase (PTK). the Src family PTK p53/p56(lyn) (Lyn) and the Syk/ZAP-family PTK p72(syk) (Syk). Early activation events involve increased activity of Lyn and Syk kinases and their translocation into membrane domains containing aggregated Fc epsilonRI, but the molecular mechanisms responsible for these changes have remained largely unclear. To determine the role of Fc epsilonRI subunits in this process, we have analyzed Syk- and Lyn-associated proteins in activated rat basophilic leukemia (RBL) cells and their variants deficient in the expression of Fc epsilonRI beta or gamma subunits. Sepharose 4B gel chromatography of postnuclear supernatants from Nonidet-P40-solubilized antigen (Ag)- or pervanadate-activated RBL cells revealed extensive changes in the size of complexes formed by Lyn and Syk kinases and other cellular components. A fusion protein containing Src homology 2 (SH2) and SH3 domains of Lyn bound Syk from lysates of nonactivated RBL cells; an increased binding was observed when lysates from Ag- or pervanadate-activated cells were used. A similar amount of Syk was bound when lysates from pervanadate-activated variant cells deficient in the expression of Fc epsilonRI beta or gamma subunits were used, suggesting that Fc epsilonRI does not function as the only intermediate in the formation of the Syk-Lyn complexes. Further experiments have indicated that Syk-Lyn interactions occur in Ag-activated RBL cells under in vivo conditions and that these interactions could involve direct binding of the Lyn SH2 domain with phosphorylated tyrosine of Syk. The physical association of Lyn and Syk during mast-like cell activation supports the recently proposed functional cooperation of these two tyrosine kinases in Fc epsilonRI signaling.
通过对IgE具有高亲和力的受体（FcεRI）激活大鼠肥大细胞，需要一系列复杂的相互作用，涉及FcεRI的跨膜亚基和两类非受体蛋白酪氨酸激酶（PTK），即Src家族PTK p53/p56(lyn)（Lyn）和Syk/ZAP家族PTK p72(syk)（Syk）。早期激活事件包括Lyn和Syk激酶活性增加及其向含有聚集FcεRI的膜结构域的转位，但导致这些变化的分子机制在很大程度上仍不清楚。为了确定FcεRI亚基在此过程中的作用，我们分析了活化的大鼠嗜碱性白血病（RBL）细胞及其缺乏FcεRIβ或γ亚基表达的变体中与Syk和Lyn相关的蛋白。对经Nonidet-P40溶解的抗原（Ag）或过钒酸盐激活的RBL细胞核后上清液进行琼脂糖4B凝胶层析，结果显示Lyn和Syk激酶与其他细胞成分形成的复合物大小发生了广泛变化。一种包含Lyn的Src同源2（SH2）和SH3结构域的融合蛋白能与未活化RBL细胞裂解物中的Syk结合；当使用Ag或过钒酸盐激活细胞的裂解物时，观察到结合增加。当使用缺乏FcεRIβ或γ亚基表达的过钒酸盐激活变体细胞的裂解物时，结合的Syk量相似，这表明FcεRI并非Syk-Lyn复合物形成的唯一中间体。进一步的实验表明，Syk-Lyn相互作用在体内条件下发生于Ag激活的RBL细胞中，且这些相互作用可能涉及Lyn的SH2结构域与Syk磷酸化酪氨酸的直接结合。肥大样细胞激活过程中Lyn和Syk的物理结合支持了最近提出的这两种酪氨酸激酶在FcεRI信号传导中的功能协同作用。
