Pseudomonas aeruginosa exoenzyme S ADP-ribosylates Ras at multiple sites
Pseudomonas aeruginosa exoenzyme S ADP-ribosylates Ras at multiple sites
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DOI：
10.1074/jbc.273.13.7332
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发表时间：
1998-03-27
影响因子：
4.8
通讯作者：
Barbieri, JT
中科院分区：
文献类型：
--
作者：
Ganesan, AK;Frank, DW;Barbieri, JT
铜绿假单胞菌外切酶S（ExoS）ADP核糖基化Ras后，每分子Ras可掺入2分子ADP核糖，表明ExoS可在多个精氨酸残基上对Ras进行ADP核糖基化。SDS-聚丙烯酰胺凝胶电泳分析表明ADP核糖基化Ras的迁移率低于非ADP核糖基化Ras，通过ExoS对体外转录/翻译的Ras的ADP-核糖基化的分析鉴定了Ras的两种精氨酸转移形式，这与Ras在两个不同的精氨酸残基处的ADP-核糖基化一致，对具有单独Arg-至-Ala取代的ADP-核糖基化的体外转录/翻译Ras突变体的分析表明，Arg-41是ADP-核糖基化的优选位点，并且第二ADP-核糖基化事件以比ADP-41慢的速率发生。核糖基化在Arg-41，但没有发生在特定的精氨酸残基。对细菌表达的野生型Ras Delta CAAX和Ras Delta CAAXR 41 K的分析支持Arg-41是ADP-核糖基化的优选位点的结论。Arg-41位于Ras的开关1区域附近，其参与效应器相互作用。将ExoS引入真核细胞中抑制Ras介导的真核信号转导，因为用铜绿假单胞菌的ExoS产生菌株感染PC-12细胞抑制神经生长因子刺激的神经突形成。这是ExoS破坏Ras介导的信号转导途径的第一个证明。
Pseudomonas aeruginosa exoenzyme S (ExoS) ADP-ribosylated Ras to a stoichiometry of similar to 2 molecules of ADP-ribose incorporated per molecule of Ras, which suggested that ExoS could ADP-ribosylate Ras at more than one arginine residue, SDS-polyacrylamide gel electrophoresis analysis showed that ADP-ribosylated Ras possessed a slower mobility than non-ADP-ribosylated Ras, Analysis of the ADP-ribosylation of in vitro transcribed/translated Ras by ExoS identified two electrophoretically shifted forms of Ras, which was consistent with the ADP-ribosylation of Ras at two distinct arginine residues, Analysis of ADP-ribosylated in vitro transcribed/translated Ras mutants possessing individual Arg-to-Ala substitutions showed that Arg-41 was the preferred site of ADP-ribosylation and that the second ADP-ribosylation event occurred at a slower rate than the ADP-ribosylation at Arg-41, but did not occur at a specific arginine residue. Analysis of bacterially expressed wild-type Ras Delta CAAX and Ras Delta CAAXR41K supported the conclusion that Arg-41 was the preferred site of ADP-ribosylation. Arg-41 is located adjacent to the switch 1 region of Ras, which is involved in effector interactions. Introduction of ExoS into eukaryotic cells inhibited Ras-mediated eukaryotic signal transduction since infection of PC-12 cells with an ExoS-producing strain of P. aeruginosa inhibited nerve growth factor-stimulated neurite formation, This is the first demonstration that ExoS disrupts a Ras-mediated signal transduction pathway.