Reversible phosphorylation of Rpn1 regulates 26S proteasome assembly and function
Reversible phosphorylation of Rpn1 regulates 26S proteasome assembly and function
复制标题
Rpn1 的可逆磷酸化调节 26S 蛋白酶体组装和功能
DOI：
10.1073/pnas.1912531117
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发表时间：
2020-01-07
影响因子：
11.1
通讯作者：
Guo, Xing
中科院分区：
文献类型：
--
作者：
Liu, Xiaoyan;Xiao, Weidi;Guo, Xing
26S蛋白酶体是真核生物中降解大部分细胞蛋白质的酶。在这项研究中，我们的特点是一个共同的和基本的机制，通过可逆的磷酸化的关键蛋白酶体亚基，Rpn 1，在Ser361蛋白酶体调节。这一磷酸化位点的突变会损害蛋白酶体复合物的形成并扰乱细胞内稳态。利用遗传密码扩增系统，我们获得了磷酸化的Rpn1蛋白，并提供了一个明确的生化解释Rpn1-S361磷酸化如何促进蛋白酶体组装。我们进一步确定了相应的激酶和磷酸酶的这个网站，这可能会导致新的方法操纵蛋白酶体活性的治疗目的。26S蛋白酶体在健康和疾病中的重要性表明，它的功能必须得到精细的控制，但我们对蛋白酶体调控的知识仍然有限。蛋白酶体亚基的翻译后修饰，特别是磷酸化，已被证明通过不同的机制影响蛋白酶体功能，尽管绝大多数蛋白酶体磷酸化事件尚未被研究。在这里，我们的特点是1最常见的检测到的蛋白酶体磷酸化位点，即Ser361的Rpn 1，19S调节颗粒的一个碱基亚基。使用多种方法，包括CRISPR/Cas9介导的基因编辑和定量质谱，我们发现Rpn1-S361磷酸化的丧失会降低蛋白酶体活性，损害细胞增殖，并导致氧化应激以及线粒体功能障碍。人类激酶组的筛选鉴定了几种激酶，包括催化S361磷酸化的PIM 1/2/3，而其水平由蛋白酶体驻留磷酸酶UBLCP 1可逆控制。从机制上讲，Rpn 1-S361磷酸化是26 S蛋白酶体正确组装所必需的，我们利用遗传密码扩展系统直接证明了S361磷酸化的Rpn 1更容易与Rpt 2形成前体复合物，这是19 S碱基组装的第一步。这些发现揭示了一个普遍的和生物学上重要的机制，蛋白酶体的形成和功能。
Significance The 26S proteasome is responsible for the degradation of the majority of cellular proteins in eukaryotes. In this study, we characterized a common and basic mechanism for proteasome regulation through reversible phosphorylation of a key proteasome subunit, Rpn1, at Ser361. Mutation of this single phosphosite impairs proteasome complex formation and perturbs cellular homeostasis. Using the genetic code expansion system, we obtained phosphorylated Rpn1 protein and provided a clear biochemical explanation of how Rpn1-S361 phosphorylation promotes proteasome assembly. We further identified the corresponding kinases and phosphatases of this site, which may lead to new approaches of manipulating proteasome activity for therapeutic purposes. The fundamental importance of the 26S proteasome in health and disease suggests that its function must be finely controlled, and yet our knowledge about proteasome regulation remains limited. Posttranslational modifications, especially phosphorylation, of proteasome subunits have been shown to impact proteasome function through different mechanisms, although the vast majority of proteasome phosphorylation events have not been studied. Here, we have characterized 1 of the most frequently detected proteasome phosphosites, namely Ser361 of Rpn1, a base subunit of the 19S regulatory particle. Using a variety of approaches including CRISPR/Cas9-mediated gene editing and quantitative mass spectrometry, we found that loss of Rpn1-S361 phosphorylation reduces proteasome activity, impairs cell proliferation, and causes oxidative stress as well as mitochondrial dysfunction. A screen of the human kinome identified several kinases including PIM1/2/3 that catalyze S361 phosphorylation, while its level is reversibly controlled by the proteasome-resident phosphatase, UBLCP1. Mechanistically, Rpn1-S361 phosphorylation is required for proper assembly of the 26S proteasome, and we have utilized a genetic code expansion system to directly demonstrate that S361-phosphorylated Rpn1 more readily forms a precursor complex with Rpt2, 1 of the first steps of 19S base assembly. These findings have revealed a prevalent and biologically important mechanism governing proteasome formation and function.