Galdiero M, D'Isanto M, Vitiello M, Finamore E, Peluso L, Galdiero M
Dipartimento di Medicina, Sperimentale, Sezione di Microbiologia e Microbiologia Clinica, facoltà di Medicina e Chiruga, Seconda Università degli Studi di Napoli, Italy.
J Infect. 2003 Feb;46(2):111-9. doi: 10.1053/jinf.2002.1068.
In the present study a monocytic cell line, U937, was used to investigate the possible involvement of protein tyrosine kinases (NT-PTKs), protein kinase A (PKA) and protein kinase C (PKC) in cell signaling pathways following Salmonella enterica serovar Typhimurium porin stimulation.
Different concentrations of porins and lipopolysaccharide (LPS) were analysed to evaluate changes in PTK activity by a non radioactive tyrosine kinase assay and in PKA and PKC phosphorylation by Western blotting analysis. The inhibitors of PTK, PKA and PKC activation used, were: 3,4-dihydroxybenzylidene-malononitrile (tyrphostin 23), inhibitor of epidermal growth factor (EGF) receptor tyrosine kinase activity; dihychloride (H-89), a selective inhibitor of PKA which is useful to discriminate between the effects of PKC and PKA; diacylglycerol kinase inhibitor II (R59949), which is useful for elucidating roles of PKC; calphostin C, a specific inhibitor of PKC.
Porins of the outer membrane of the ST were isolated to be used as a stimulus in the performed experiments. Following porin treatment, a dose-dependent increase in PTK, PKA and PKC activation was observed. U937 monocytes pretreated with inhibitors induced an evident decrease in PTK activity and PKA and PKC phosphorylation pattern in porin stimulated monocytes.
Our data support the important role played by NT-PTK, PKA and PKC in transducing the activating signal in macrophages stimulated with porins through the activation of the mitogen-activated protein kinase (MAPK) pathway that participate in the regulation of gene expression.
在本研究中，使用单核细胞系U937来研究蛋白酪氨酸激酶（非受体蛋白酪氨酸激酶，NT-PTKs）、蛋白激酶A（PKA）和蛋白激酶C（PKC）在鼠伤寒沙门氏菌孔蛋白刺激后的细胞信号通路中可能发挥的作用。
分析不同浓度的孔蛋白和脂多糖（LPS），通过非放射性酪氨酸激酶测定评估PTK活性的变化，并通过蛋白质印迹分析评估PKA和PKC的磷酸化变化。所使用的PTK、PKA和PKC激活抑制剂分别为：3,4-二羟基亚苄基丙二腈（ tyrphostin 23），表皮生长因子（EGF）受体酪氨酸激酶活性抑制剂；二盐酸盐（H-89），一种PKA的选择性抑制剂，可用于区分PKC和PKA的作用；二酰基甘油激酶抑制剂II（R59949），可用于阐明PKC的作用；钙磷蛋白C，一种PKC的特异性抑制剂。
分离出鼠伤寒沙门氏菌外膜的孔蛋白，用作所进行实验的刺激物。孔蛋白处理后，观察到PTK、PKA和PKC激活呈剂量依赖性增加。用抑制剂预处理的U937单核细胞导致孔蛋白刺激的单核细胞中PTK活性以及PKA和PKC磷酸化模式明显降低。
我们的数据支持NT-PTK、PKA和PKC在通过激活参与基因表达调控的丝裂原活化蛋白激酶（MAPK）途径转导孔蛋白刺激的巨噬细胞中的激活信号方面发挥的重要作用。
