ADP enhances the allosteric activation of eukaryotic elongation factor 2 kinase by calmodulin.
ADP enhances the allosteric activation of eukaryotic elongation factor 2 kinase by calmodulin.
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DOI：
10.1073/pnas.2300902120
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发表时间：
2023-04-25
影响因子：
11.1
通讯作者：
Ghose, Ranajeet
中科院分区：
文献类型：
--
作者：
Piserchio, Andrea;Long, Kimberly J.;Browning, Luke S.;Bohanon, Amanda L.;Isiorho, Eta A.;Dalby, Kevin N.;Ghose, Ranajeet
核糖体翻译mRNA信息是真核细胞中最耗能的过程之一，需要在能量胁迫下下调其表达。抑制翻译延长的主要机制是通过特异性磷酸化降低GTP酶真核延长因子2(EEF-2)的核糖体亲和力。这种共价的EEF-2修饰是由钙调蛋白激活的EEF-2激酶(EEF-2K)唯一催化的。已有研究表明，EEF-2K通过能量储备耗尽时被激活的主感受器AMP激活的蛋白激酶间接感受细胞的能量状态。在这里，我们建议通过ADP介导的EEF-2K的直接能量传感作用，它与独特位置的激酶的结合导致对其变构激活剂钙调蛋白的敏感性增强。蛋白质翻译是真核细胞中最耗能的过程之一，需要强有力的调控，特别是在缺乏能量的条件下。这一调控的一个关键组成部分是通过减少GTP酶真核延长因子2(EEF-2)的核糖体结合来抑制翻译延长，这是由于钙调蛋白(CaM)激活的α激酶EEF-2激酶(EEF-2K)对其进行特异性磷酸化而导致的。有研究表明，EEF-2K对细胞能量水平降低的反应是间接的，并由通用能量传感器AMP激活的蛋白激酶(AMPK)通过直接刺激磷酸化和/或下调EEF-2K抑制的营养感知mTOR途径来介导。在这里，我们提供了结构、生化和细胞生物学证据，证明EEF-2K通过ADP的刺激发挥直接的能量传感作用。CaM与EEF-2K功能核心之间的核苷酸结合复合体的晶体结构显示，ADP结合在与激酶活性位点相对的面上的一个独特的口袋上。在这个基本口袋(BP)中，通过与两个相互作用伙伴的多次相互作用，ADP通过与两个相互作用伙伴的多次相互作用而稳定。利用野生型EEF-2K和特定的BP突变体进行的生化分析表明，ADP稳定了活性复合体中的CaM，增加了该激酶对CaM的敏感性。与表达野生型EEF-2K的细胞相比，通过糖酵解抑制诱导的能量应激导致表达ADP结合受损BP突变体的细胞中磷酸化EEF-2水平的显著降低。这些结果表明，EEF-2K通过与CaM和ADP的协同作用发挥直接的能量传感作用。
Translation of an mRNA message by the ribosome represents one of the most energy-consuming processes in a eukaryotic cell, necessitating its downregulation under energy stress. The primary mechanism of suppressing translational elongation is by a reduction in the ribosome affinity of the GTPase eukaryotic elongation factor 2 (eEF-2) through specific phosphorylation. This covalent eEF-2 modification is uniquely catalyzed by the calmodulin-activated eEF-2 kinase (eEF-2K). It has been suggested that eEF-2K indirectly senses the cellular energy state through the master sensor, AMP-activated protein kinase, activated upon depletion of energy reserves. Here, we suggest a direct energy-sensing role for eEF-2K mediated by ADP whose engagement with the kinase at a unique site leads to enhanced sensitivity toward its allosteric activator, calmodulin. Protein translation, one of the most energy-consumptive processes in a eukaryotic cell, requires robust regulation, especially under energy-deprived conditions. A critical component of this regulation is the suppression of translational elongation through reduced ribosome association of the GTPase eukaryotic elongation factor 2 (eEF-2) resulting from its specific phosphorylation by the calmodulin (CaM)-activated α–kinase eEF-2 kinase (eEF-2K). It has been suggested that the eEF-2K response to reduced cellular energy levels is indirect and mediated by the universal energy sensor AMP-activated protein kinase (AMPK) through direct stimulatory phosphorylation and/or downregulation of the eEF-2K-inhibitory nutrient-sensing mTOR pathway. Here, we provide structural, biochemical, and cell-biological evidence of a direct energy-sensing role of eEF-2K through its stimulation by ADP. A crystal structure of the nucleotide-bound complex between CaM and the functional core of eEF-2K phosphorylated at its primary stimulatory site (T348) reveals ADP bound at a unique pocket located on the face opposite that housing the kinase active site. Within this basic pocket (BP), created at the CaM/eEF-2K interface upon complex formation, ADP is stabilized through numerous interactions with both interacting partners. Biochemical analyses using wild-type eEF-2K and specific BP mutants indicate that ADP stabilizes CaM within the active complex, increasing the sensitivity of the kinase to CaM. Induction of energy stress through glycolysis inhibition results in significantly reduced enhancement of phosphorylated eEF-2 levels in cells expressing ADP-binding compromised BP mutants compared to cells expressing wild-type eEF-2K. These results suggest a direct energy-sensing role for eEF-2K through its cooperative interaction with CaM and ADP.
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EEF2激酶通过阻止翻译伸长来赋予对营养剥夺的耐药性。
影响因子：
64.5
作者：
Leprivier G;Remke M;Rotblat B;Dubuc A;Mateo AR;Kool M;Agnihotri S;El-Naggar A;Yu B;Somasekharan SP;Faubert B;Bridon G;Tognon CE;Mathers J;Thomas R;Li A;Barokas A;Kwok B;Bowden M;Smith S;Wu X;Korshunov A;Hielscher T;Northcott PA;Galpin JD;Ahern CA;Wang Y;McCabe MG;Collins VP;Jones RG;Pollak M;Delattre O;Gleave ME;Jan E;Pfister SM;Proud CG;Derry WB;Taylor MD;Sorensen PH
通讯作者：
Sorensen PH
影响因子：
--
作者：
Kenney, Justin W.;Moore, Claire E.;Proud, Christopher G.
通讯作者：
Proud, Christopher G.
影响因子：
14.9
作者：
Brzezinski D;Porebski PJ;Kowiel M;Macnar JM;Minor W
通讯作者：
Minor W
影响因子：
7.1
作者：
Jan A;Jansonius B;Delaidelli A;Bhanshali F;An YA;Ferreira N;Smits LM;Negri GL;Schwamborn JC;Jensen PH;Mackenzie IR;Taubert S;Sorensen PH
通讯作者：
Sorensen PH
影响因子：
4.8
作者：
Browne, GJ;Finn, SG;Proud, CG
通讯作者：
Proud, CG