Ubiquitin is double-phosphorylated by PINK1 for enhanced pH-sensitivity of conformational switch
Ubiquitin is double-phosphorylated by PINK1 for enhanced pH-sensitivity of conformational switch
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泛素被 PINK1 双磷酸化，增强构象转换的 pH 敏感性
DOI：
10.1007/s13238-019-0644-x
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发表时间：
2019-07
期刊：
影响因子：
21.1
通讯作者：
Tang Chun
中科院分区：
文献类型：
--
作者：
Ye Shang-Xiang;Gong Zhou;Yang Ju;An Yu-Xin;Liu Zhu;Zhao Qun;Lescop Ewen;Dong Xu;Tang Chun
在许多信号蛋白中发现了多位点磷酸化，这赋予了蛋白质新的功能。多位点磷酸化的蛋白质可以表现出对细胞刺激的开关样反应，其中通常成簇的磷酸化位点被相同的激酶未磷酸化或完全磷酸化（Kapuy等人，2009年）。还报道了多位点磷酸化促进内在无序蛋白质的折叠，并因此调节与其他蛋白质的结合亲和力（Bah等人，2015）。在这里，我们表明，多位点磷酸化使蛋白质成为一个更好的pH传感器，迅速切换替代构象状态之间的pH变化在生理条件下的响应。蛋白质组学研究表明，Ub中几乎所有丝氨酸/苏氨酸残基都可以磷酸化（Swatek和Komander，2016）。然而，迄今为止鉴定的唯一Ub激酶是PINK 1，其在残基S65处磷酸化Ub（Kane等人，2014; Koyano等人，2014; Gladkova等人，2017年）。Ub的PINK 1磷酸化可以激活PARKIN（Gladkova等人，2018年），E3连接酶，这反过来又导致细胞线粒体自噬和减缓帕金森病的进展。因此，PINK 1的突变与帕金森病的早期发作有关（Kane等人，2014; Koyano等人，2014年）。在这里，我们报告，PINK 1也可以磷酸化Ub在残基T66。我们首先从体外酶促测定中鉴定了对应于磷酸化的S65和T66两者的质量的肽（图1A）。在将UB 7 KR（将所有7个赖氨酸突变为赖氨酸，从而阻断UB链形成）和可溶性PINK 1（Gao et al. 2016）共转染至HEK 293细胞后，我们还鉴定了S65和T66双磷酸化肽（图1 B）。我们使用核磁共振（NMR）光谱来表征重组纯化的pS 65/pT 66 Ub蛋白的结构。双磷酸化Ub的二维1H-15 N杂原子单量子相关（HSQC）光谱显示出超过120个峰，几乎是预期的76个残基蛋白质的两倍。这些峰可以归属于两种不同的构象
Multisite phosphorylation is observed in many signaling proteins, which confers the proteins new functions. A multisite phosphorylated protein can exhibit switch-like response to cellular stimuli, with the often-clustered phosphorylation sites either unphosphorylated or fully phosphorylated by the same kinase (Kapuy et al., 2009). Multisite phosphorylation has also been reported to promote the folding of an intrinsically disordered protein, and consequently modulate the binding affinity to other proteins (Bah et al., 2015). Here we show that multisite phosphorylation makes a protein a better pH sensor, promptly switching between alternative conformational states in response to pH change under physiological conditions.Ubiquitin (Ub) is a 76-residue signaling protein in cells. Proteomics studies have shown that almost all serine/threonine residues in Ub can be phosphorylated (Swatek and Komander, 2016). However, the only Ub kinase identified to date is PINK1, which phosphorylates Ub at residue S65 (Kane et al., 2014; Koyano et al., 2014; Gladkova et al., 2017). PINK1 phosphorylation of Ub can activate PARKIN (Gladkova et al., 2018), an E3 ligase, which in turn leads to cell mitophagy and slows the progression of Parkinson’s disease. Thus, mutations of PINK1 are implicated in the early onset of Parkinson’s disease (Kane et al., 2014; Koyano et al., 2014). Here we report that PINK1 can also phosphorylate Ub at residue T66. We first identified a peptide from in vitro enzymatic assay that corresponds to the mass of both S65 and T66 phosphorylated (Fig. 1 A). Upon co-transfection of Ub 7KR (mutating all seven lysines to arginines thus blocking Ub chain formation) and soluble PINK1 (Gao et al. 2016) to HEK293 cells, we also identified S65 and T66 double-phosphorylated peptide (Fig. 1 B). We used nuclear magnetic resonance (NMR) spectroscopy to characterize the structure of the recombinantly purified pS65/pT66 Ub protein. The 2D 1H-15N heteronuclear single quantum correlation (HSQC) spectrum of the doublephosphorylated Ub shows more than 120 peaks, nearly twice as much as expected for a 76-residue protein. The peaks can be assigned to two alternative conformational
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影响因子：
44.1
作者：
Swatek KN;Komander D
通讯作者：
Komander D
影响因子：
64.8
作者：
Gladkova C;Maslen SL;Skehel JM;Komander D
通讯作者：
Komander D
影响因子：
4.6
作者：
Okatsu K;Sato Y;Yamano K;Matsuda N;Negishi L;Takahashi A;Yamagata A;Goto-Ito S;Mishima M;Ito Y;Oka T;Tanaka K;Fukai S
通讯作者：
Fukai S
影响因子：
4
作者：
White, Katharine A.;Grillo-Hill, Bree K.;Barber, Diane L.
通讯作者：
Barber, Diane L.
影响因子：
2.7
作者：
Bienkiewicz, EA;Lumb, KJ
通讯作者：
Lumb, KJ