Dynamic Ubiquitination of the Mitogen-activated Protein Kinase Kinase (MAPKK) Ste7 Determines Mitogen-activated Protein Kinase (MAPK) Specificity
Dynamic Ubiquitination of the Mitogen-activated Protein Kinase Kinase (MAPKK) Ste7 Determines Mitogen-activated Protein Kinase (MAPK) Specificity
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DOI：
10.1074/jbc.m113.475707
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发表时间：
2013-06-28
影响因子：
4.8
通讯作者：
Dohlman, Henrik G.
中科院分区：
文献类型：
--
作者：
Hurst, Jillian H.;Dohlman, Henrik G.
泛素化是一种翻译后修饰，标记蛋白质以供蛋白酶体降解。此外，人们越来越认识到泛素化可以影响蛋白质活性和定位。 Ste7 是酵母中的 MAPKK 原型，参与信息素信号传导和营养剥夺/侵入性生长途径。我们之前已经证明 Ste7 在信息素刺激下被泛素化。在这里，我们证明 Skp1/Cullin/F-box 泛素连接酶 SCFCdc4 和泛素蛋白酶 Ubp3 调节 Ste7 泛素化和信号特异性。使用纯化的成分，我们证明 SCFCdc4 直接泛素化 Ste7。使用基因缺失突变体，我们发现 SCFCdc4 和 Ubp3 对 Ste7 泛素化具有相反的作用。虽然 SCFCdc4 对于信息素 MAPK Fus3 的正确激活是必需的，但需要 Ubp3 来限制侵入性生长 MAPK Kss1 的激活。最后，我们证明 Fus3 直接磷酸化 Ubp3，并且 Ubp3 的磷酸化对于限制 Kss1 激活是必要的。这些结果揭示了一个反馈环路，其中一个 MAPK 限制上游 MAPKK 的泛素化，从而防止第二个竞争性 MAPK 的虚假激活。
Ubiquitination is a post-translational modification that tags proteins for proteasomal degradation. In addition, there is a growing appreciation that ubiquitination can influence protein activity and localization. Ste7 is a prototype MAPKK in yeast that participates in both the pheromone signaling and nutrient deprivation/invasive growth pathways. We have shown previously that Ste7 is ubiquitinated upon pheromone stimulation. Here, we show that the Skp1/Cullin/F-box ubiquitin ligase SCFCdc4 and the ubiquitin protease Ubp3 regulate Ste7 ubiquitination and signal specificity. Using purified components, we demonstrate that SCFCdc4 ubiquitinates Ste7 directly. Using gene deletion mutants, we show that SCFCdc4 and Ubp3 have opposing effects on Ste7 ubiquitination. Although SCFCdc4 is necessary for proper activation of the pheromone MAPK Fus3, Ubp3 is needed to limit activation of the invasive growth MAPK Kss1. Finally, we show that Fus3 phosphorylates Ubp3 directly and that phosphorylation of Ubp3 is necessary to limit Kss1 activation. These results reveal a feedback loop wherein one MAPK limits the ubiquitination of an upstream MAPKK and thereby prevents spurious activation of a second competing MAPK.