Measurement of effector protein injection by type III and type IV secretion systems by using a 13-residue phosphorylatable glycogen synthase kinase tag
Measurement of effector protein injection by type III and type IV secretion systems by using a 13-residue phosphorylatable glycogen synthase kinase tag
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DOI：
10.1128/iai.00690-06
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发表时间：
2006-10-01
影响因子：
3.1
通讯作者：
Plano, Gregory V.
中科院分区：
文献类型：
--
作者：
Garcia, Julie Torruellas;Ferracci, Franco;Plano, Gregory V.
许多细菌病原体使用III型分泌系统（t3ss）或t4ss将毒力蛋白注射或转运到真核细胞中。已经开发了几种不同的报告系统来测量这些蛋白质的易位。本研究开发了一种基于肽标签的报告系统，用于监测T3S和T4S底物的注射情况。糖原合成酶激酶（GSK）标签是源自人GSK-3 β激酶的13个残基可磷酸化肽标签。将gsk标记的蛋白易位到真核细胞中会导致宿主细胞蛋白激酶依赖性磷酸化，这可以用磷酸化特异性GSK-3 β抗体检测到。构建了一系列编码Yop-GSK融合蛋白的表达质粒，评价GSK标签检测鼠疫耶尔森菌T3SS注射Yops的能力。gsk标记的YopE、YopH、LcrQ、YopK、YopN和YopJ在转运到HeLa细胞时被有效磷酸化。同样地，通过磷酸化GSK标签，通过幽门螺杆菌T4SS将GSK- caga注射到不同类型的细胞中。GSK标签提供了一种简单的方法来监测T3S和T4S底物的易位。
Numerous bacterial pathogens use type III secretion systems (T3SSs) or T4SSs to inject or translocate virulence proteins into eukaryotic cells. Several different reporter systems have been developed to measure the translocation of these proteins. In this study, a peptide tag-based reporter system was developed and used to monitor the injection of T3S and T4S substrates. The glycogen synthase kinase (GSK) tag is a 13-residue phosphorylatable peptide tag derived from the human GSK-3 beta kinase. Translocation of a GSK-tagged protein into a eukaryotic cell results in host cell protein kinase-dependent phosphorylation of the tag, which can be detected with phosphospecific GSK-3 beta antibodies. A series of expression plasmids encoding Yop-GSK fusion proteins were constructed to evaluate the ability of the GSK tag to measure the injection of Yops by the Yersinia pestis T3SS. GSK-tagged YopE, YopH, LcrQ, YopK, YopN, and YopJ were efficiently phosphorylated when translocated into HeLa cells. Similarly, the injection of GSK-CagA by the Helicobacter pylori T4SS into different cell types was measured via phosphorylation of the GSK tag. The GSK tag provides a simple method to monitor the translocation of T3S and T4S substrates.