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lps刺激的巨噬细胞中发现了一种65 kda的磷酸化蛋白，该蛋白被证明是人类l -活蛋白的小鼠同源物，l -活蛋白最初被发现是癌细胞中转化诱导的磷酸化蛋白。p65/L-plastin是plastin家族的一员，其特征是一系列Ca^<2+>，钙调蛋白和b-actin结合域。l -异构体仅在白细胞中表达，表明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LPS刺激巨噬细胞通过整合素的粘附显著增加时p65/L-plastin的动态变化。lps诱导的丝氨酸激酶磷酸化p65/L-plastin的抑制剂抑制p65/L-plastin重排和细胞粘附。在这种与p65/ l -质体相关的粘附中，可以清楚地观察到该蛋白密集地位于靠近基质的podosomes样结构中。沙门氏菌感染小鼠脾脏中积聚的b_2整合素阳性细胞黏附性增加，p65/ l -活质蛋白重排。利用肽底物进行的体外激酶测定显示，在体外LPS刺激的腹膜巨噬细胞和沙门氏菌感染的脾脏中浸润的cd11b阳性细胞中，磷酸化p65/ l - plasin -peptide的激酶活性均增强。此外，在lps刺激的巨噬细胞中，p65/ l -活蛋白易位的过氧化氢（一种杀菌物质）的生成比静止的巨噬细胞高得多。这些发现表明p65/L-plastin和b-actin在巨噬细胞中响应细菌刺激紧密耦合，组织含有整合素的粘附结构，这也可能作为细胞骨架支架有效组织巨噬细胞的抗菌活性。

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.