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）用山梨糖醇处理细胞以诱导高渗透导致PAK2和SYK转移到核周围的区域，并显着增强其关联。 PAK2和SYK的共转染导致在高渗条件下JNK的激活。 PAK2简短干扰RNA抑制了山梨糖醇介导的内源性SYK和JNK的激活，从而鉴定了通过Cdc42.2）识别出一种新的JNK激活途径）高亲和力IgE受体（FC Varepsilon RI）的聚集会诱导Basbophielic liukemia rikemia rbl-2h3 rbl-2h3。用蛋白酶体抑制剂治疗细胞会增强LYN的泛素化。共转染研究表明，C-CBL和CBL-B都可以诱导COS细胞中活化的LYN的泛素化。 C-CBL的膜锚定形式的过表达抑制了RBL-2H3细胞中Fc Varepsilon RI介导的脱粒和细胞因子基因的产生，通过通过增强的泛素化对LYN的激酶活性下调。此外，CBL-B是Lyn-Syk-LAT和GAB2的负调节剂，在Fcepsilonri介导的肥大细胞激活中介导的互补信号通路。3）使用COS-7细胞中的瞬态表达系统，3BP2在Y174，Y183，Y4446中主要磷酸化，主要磷酸化。一项体外结合研究表明，SYK对Y446的磷酸化可能会为RBL-2H3细胞中Lyn-SH2结构域创建一个结合位点。另外，与lyn-SH3结构域结合的3BP2的富含脯氨酸区域。 RBL-2H3细胞中3BP2的过表达导致LYN自磷酸化的增强。因此，衔接蛋白3BP2是Lyn的潜在调节剂，是其在肥大细胞中Ecepsilonri介导的信号传导中其SH3/SH2结构域的配体。4）作为SYK与吞噬性吞噬相关的作用，我们显示SYK对于在交叉胶片中对溶液的内体融合至关重要。