The host defense mechanism by protein complexes assembling on the p65/L-plastin scaffold.

在我们之前的研究中，我们在细菌内毒素刺激的巨噬细胞中发现了一个65 kDa的磷酸化蛋白，并且被证明是人L-纤溶酶原蛋白的小鼠同源物，人纤溶酶原蛋白最初被鉴定为癌细胞中的一种转化诱导的磷蛋白。P65/L-纤溶酶原蛋白是纤溶酶原蛋白家族的成员，由一系列钙调素、钙调素和b-肌动蛋白结合区组成。L-异构体仅在白细胞中表达，提示它在白细胞特有的细胞功能中起着至关重要的作用。在本研究中，我们研究了在内毒素刺激下巨噬细胞通过整合素的黏附显著增加时p65/L-纤溶酶的动态变化。脂多糖诱导的丝氨酸激酶抑制剂使p65/L-纤溶酶原蛋白磷酸化，从而抑制p65/L-纤溶酶原蛋白重排和细胞黏附。在这种p65/L-纤溶酶参与的黏附中，可以清楚地观察到蛋白质密集地定位在靠近底物的足体样结构中。感染沙门氏菌的小鼠脾中b2整合素阳性细胞的黏附增加和p65/L-纤溶酶的重排也是其特点。采用多肽底物的体外激酶活性测定表明，p65/L-纤溶酶多肽在脂多糖刺激的体外巨噬细胞和感染沙门氏菌的脾组织中CD11b阳性细胞中的磷酸化活性均增强。此外，p65/L-纤溶酶转位的巨噬细胞产生的杀菌物质过氧化氢远高于静息状态下的巨噬细胞。这些发现表明，P65/L-纤溶酶原蛋白和b-肌动蛋白在巨噬细胞中紧密偶联，以响应细菌的刺激，组织含有整合素的黏附结构，这也可能是有效组织巨噬细胞抗菌活性的细胞骨架。

A 65-kDa protein was identified as a phosphorylated protein in bacterial LPS-stimulated macrophages in our previous study and has turned out to be a murine homologue of human L-plastin, a protein originally identified as a transformation-induced phosphoprotein in cancer cells. The p65/L-plastin is a member of the plastin family characterized by a series of Ca^<2+>, calmodulin, and b-actin binding domains. The L-isoform is exclusively expressed in leukocytes, suggesting it plays a vital role in cellular functions unique to leukocytes. In the present study, we have investigated the dynamics of p65/L-plastin when macrophage adhesion via integrins was remarkably increased by stimulation with LPS. An inhibitor of an LPS-inducible serine kinase phosphorylating p65/L-plastin inhibited both p65/L-plastin rearrangement and cellular adhesion. In this p65/L-plastin-involved adhesion, the protein was clearly observed to locate densely in podosome-like structures close to the substratum. The increased adhesion and the rearrangement of p65/L-plastin were also characteristic for b_2-integrin-positive cells accumulated in the spleen of mice infected with Salmonella organisms. in vitro kinase assay using peptide substrates revealed that the kinase activity phosphorylating the p65/L-plastin-peptides was augmented both in peritoneal macrophages stimulated in vitro with LPS and in CD11b-positive cells infiltrated in the Salmonella-infected spleen. Furthermore, generation of hydrogen peroxide, a bactericidal substance, was much higher in LPS-stimulated macrophages where p65/L-plastin was translocated than in resting macrophages. These findings suggest that p65/L-plastin and b-actin are closely coupled dynamically in macrophages in response to bacterial stimulation to organize adhesion structures containing integrins, and that this may also function as cytoskeletal scaffolds that effectively organize the anti-bacterial activities of macrophages.