白细胞介素-1受体相关激酶-2的共价修饰与激酶活性的研究

Study on the Modifications and Kinase Activity of IRAK2

白细胞介素-1受体相关激酶-2(IRAK2)是调节IL1和Toll样受体信号通路的一个关键性分子,目前对于共价修饰如何调节IRAK2的活性还所知甚少。当与IRAK1共转染时,IRAK2能被共价修饰,在SDS-PAGE分离中呈现出迁移率变慢的多条电泳带。在小鼠骨髓干细胞分化的巨噬细胞(BMMs)中,内源表达的IRAK2在TLR配体刺激下也呈现出类似的共价修饰。而且IRAK2的共价修饰具有磷酸酯酶敏感性,提示大部分为磷酸化修饰。通过体外磷酸激酶活性分析,发现巨噬细胞中表达的IRAK2能在LPS诱导下被激活,成为一个具有激酶活性的调节蛋白。进一步研究发现激酶灭活的IRAK2突变体不能重建IRAK2基因敲除巨噬细胞的功能。通过Western杂交和定量PCR分析,发现IRAK2的激酶活性是介导LPS诱导的信号通路和炎症因子表达所必须的。因此,在LPS诱导下,IRAK2可能被IRAK1进行磷酸化修饰而活化,从而介导下游的信号转导通路、诱导炎症因子的表达。

IRAK2 plays a critical role in IL1 and TLR-mediated signaling,however,the mechanisms by which IRAK2 regulates IL1/TLR signaling remain unclear.It is found that IRAK2 became modified by co-transfected IRAK1 in 293 cells.The modified IRAK2 were recognized by anti-IRAK2 Western blotting as slower mobility shift bands on SDS-PAGE.In bone-marrow derived macrophages,endogenous IRAK2 was also modified upon LPS induction.Moreover,the modified IRAK2 bands collapsed upon pretreatment by calf intestinal phosphatase,indicating that IRAK2 modification is mainly attributed to phosphorylation.By in vitro kinase assay,immunoprecipitated IRAK2 was demonstrated to be able to phosphorylate MBP in vitro,suggesting that LPS signaling induces the kinase activity of IRAK2.Furthermore,a kinase-inactive mutant of IRAK2 is made,which is unable to reconstitute the LPS-induced signaling and inflammatory gene expression in IRAK2-deficient macrophages.Taken together,it demonstrates that the kinase activity of IRAK2 is critical for LPS-induced downstream signaling and proinflammatory gene expression,in addition,phosphorylation of IRAK2,likely regulated by IRAK1 might have a key role in activating IRAK2 kinase.