P-Tyr42 RhoA GTPase amplifies superoxide formation through p47phox, phosphorylated by ROCK
P-Tyr42 RhoA GTPase amplifies superoxide formation through p47phox, phosphorylated by ROCK
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DOI：
10.1016/j.bbrc.2020.01.001
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发表时间：
2020-03-19
影响因子：
3.1
通讯作者：
Park, Jae-Bong
中科院分区：
文献类型：
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作者：
Cap, Kim Cuong;Kim, Jae-Gyu;Park, Jae-Bong
活性氧（ROS）的最佳水平在细胞生理功能中起着至关重要的作用。NADPH氧化酶是产生细胞内超氧化物的来源之一。Rac 1/2和RhoA GTP酶参与NADPH氧化酶活性的调节，RhoA的Tyr42磷酸化（p-Tyr42 RhoA）在这方面似乎很重要，因为最近显示过氧化氢能够增加p-Tyr42 RhoA水平。佛波醇肉豆蔻酸酯乙酸酯（PMA），一种肿瘤促进剂，也诱导超氧化物的产生; PMA激活Src，一种酪氨酸激酶，并增加p-Tyr42 RhoA水平。在探索PMA作用的机制中，我们用si-RhoA降低测试细胞中的RhoA水平，然后恢复各种形式的RhoA以响应细胞对PMA的作用并产生超氧化物。与WT和Y42E RhoA相比，RhoA Y42F（脱磷酸模拟形式）的恢复仍然具有响应于PMA的减少的超氧化物形成。这与细胞迁移和细胞增殖的测定类似，细胞对PMA有反应。ROCK（Rho相关卷曲螺旋激酶）抑制剂Y27632也抑制超氧化物的产生，并且还降低p-Y416 Src和p-p47 phox水平。ROCK活性片段也能够在Ser345残基处磷酸化p47phox（p-Ser345 p47phox），这是NADPH氧化酶的组分。总之，我们证明PMA处理后p-Tyr42 RhoA水平增加，这是通过产生超氧化物和激活Src。这些反过来又通过p47phox的ROCK磷酸化放大超氧化物的产生，并维持超氧化物产生的正反馈回路，并有助于肿瘤进展。(C)2020爱思唯尔公司All rights reserved.
Optimal levels of reactive oxygen species (ROS) play a critical role in cellular physiological function. For production of intracellular superoxide, NADPH oxidase is one of the sources. Rac1/2 and RhoA GTPases are involved in regulation of NADPH oxidase activity and Tyr42 phosphorylation of RhoA (p-Tyr42 RhoA) seems significant in this regard as it was recently shown that hydrogen peroxide was able to increase p-Tyr42 RhoA levels. Phorbol myristate acetate (PMA), a tumor promoter, also induces production of superoxides; PMA activates Src, a tyrosine kinase, and increases p-Tyr42 RhoA levels. In exploring the mechanism of PMA effects, we reduced RhoA levels in test cells with si-RhoA and then restoration of various versions of RhoA for effect in response of the cells to PMA and producing superoxides. Restoration of RhoA Y42F (a dephospho-mimic form) still had reduced superoxide formation in response to PMA, compared with WT and Y42E RhoA. This was similarly seen with assays for cell migration and proliferation with cells responding to PMA. Y27632, a ROCK (Rho associated coiled coil kinase) inhibitor, also inhibited superoxide production, and also reduced p-Y416 Src and p-p47phox levels. A ROCK active fragment was also able to phosphorylate p47phox at Ser345 residue (p-Ser345 p47phox), a component of NADPH oxidase. Overall, we demonstrate that p-Tyr42 RhoA levels increase following PMA treatment and this is through production of superoxide and activation of Src. These in turn amplify superoxide production through ROCK phophorylation of p47phox and maintain a positive feedback loop for superoxide generation, and contribute to tumor progression. (C) 2020 Elsevier Inc. All rights reserved.