Research for regulation system and new function of tyrosine kinase Syk

酪氨酸激酶Syk调控系统及新功能研究

基本信息

批准号：
14370044
负责人：
YAMAMURA Hirohei
金额：
$ 8.96万
依托单位：
Kobe University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2003
项目状态：
已结题

项目摘要

1) Treatment of cells with sorbitol to induce hyperosmolarity results in the translocation of Pak2 and Syk to the region surrounding the nucleus and in dramatic enhancement of their association. Cotransfection of Pak2 and Syk leads to the activation of JNK under hyperosmotic conditions. Pak2 short interfering RNA suppresses sorbitol-mediated activation of endogenous Syk and JNK, thus identifying a novel pathway for JNK activation by Cdc42.2) Aggregation of the high affinity IgE receptor (Fc varepsilon RI) induces the rapid ubiquitination of Lyn in rat basophilic leukaemia RBL-2H3 cells. Treatment of cells with a proteasome inhibitor enhances the ubiquitination of Lyn. Co-transfection study shows that both c-Cbl and Cbl-b could induce the ubiquitination of activated Lyn in COS cells. Over-expression of membrane-anchored form of c-Cbl inhibits the Fc varepsilon RI-mediated degranulation and cytokine gene production in RBL-2H3 cells by the down-regulation of the kinase activity of Lyn through the enhanced ubiquitination. Furthermore Cbl-b is a negative regulator of both Lyn-Syk-LAT and Gab2 mediated complementary signaling pathways in FcepsilonRI-mediated mast cell activation.3) Using the transient expression system in COS-7 cells, 3BP2 was predominantly phosphorylated on Y174, Y183, and Y446 when it was coexpressed with Syk. An in vitro binding study revealed that phosphorylation of Y446 by Syk was likely to create a binding site for the Lyn-SH2 domain in RBL-2H3 cells. In addition, proline-rich region of 3BP2 bound to the Lyn-SH3 domain. Overexpression of 3BP2 in RBL-2H3 cells resulted in an enhancement of Lyn autophosphorylation. Thus the adaptor protein 3BP2 is a potential regulator of Lyn as a ligand of its SH3/SH2 domains in EcepsilonRI-mediated signaling in mast cells.4) As the role of Syk related to phagocytosis, we showed that Syk is essential for the endosomal fusion to lysosomes in the crosslinking-induced-endocytosis.

1) 用山梨醇处理细胞以诱导高渗透压导致 Pak2 和 Syk 易位到细胞核周围的区域，并显着增强它们的关联。 Pak2 和 Syk 的共转染导致 JNK 在高渗条件下激活。 Pak2 短干扰 RNA 抑制山梨醇介导的内源性 Syk 和 JNK 激活，从而确定了 Cdc42 激活 JNK 的新途径。2) 高亲和力 IgE 受体 (Fc varepsilon RI) 的聚集诱导大鼠嗜碱性白血病中 Lyn 的快速泛素化RBL-2H3 细胞。用蛋白酶体抑制剂处理细胞可增强 Lyn 的泛素化。共转染研究表明，c-Cbl和Cbl-b均可诱导COS细胞中活化的Lyn泛素化。膜锚定形式的 c-Cbl 的过度表达通过增强泛素化下调 Lyn 激酶活性，抑制 RBL-2H3 细胞中 Fc varepsilon RI 介导的脱颗粒和细胞因子基因产生。此外，Cbl-b 是 FcepsilonRI 介导的肥大细胞激活中 Lyn-Syk-LAT 和 Gab2 介导的互补信号通路的负调节因子。3) 使用 COS-7 细胞中的瞬时表达系统，3BP2 主要在 Y174、Y183 上磷酸化，以及与 Syk 共表达时的 Y446。一项体外结合研究表明，Syk 对 Y446 的磷酸化可能会在 RBL-2H3 细胞中为 Lyn-SH2 结构域创建一个结合位点。此外，3BP2 富含脯氨酸的区域与 Lyn-SH3 结构域结合。 RBL-2H3 细胞中 3BP2 的过度表达导致 Lyn 自磷酸化增强。因此，接头蛋白3BP2是Lyn的潜在调节剂，作为EcepsilonRI介导的肥大细胞信号传导中其SH3/SH2结构域的配体。4)由于Syk的作用与吞噬作用相关，我们表明Syk对于内体融合至关重要交联诱导的内吞作用中的溶酶体。