Optimizing photoswitchable MEK
Optimizing photoswitchable MEK
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DOI：
10.1073/pnas.1912320116
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发表时间：
2019-12
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影响因子：
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通讯作者：
Aleena L. Patel;E. Yeung;S. McGuire;Andrew Y Wu;Jared E. Toettcher;R. Burdine;S. Shvartsman
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文献类型：
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作者：
Aleena L. Patel;E. Yeung;S. McGuire;Andrew Y Wu;Jared E. Toettcher;R. Burdine;S. Shvartsman
使用光二聚化结构域控制对激酶活性位点的访问彻底改变了细胞信号传导的可能光遗传学扰动的类型。然而，这项技术只有在有强激酶活性可供利用时才有用。我们报告了一种策略，以系统地增强和调整功能的激酶激活细胞外信号调节激酶（ERK）途径，MEK，使用获得功能的点突变，不稳定的MEK。光开关突变体MEK活性通过可逆地笼化其活性位点控制体内ERK信号传导的大动态范围。我们优化和调节光激活蛋白激酶的方法并不限于MEK，并且可以用于系统地扩展光激活调节酶的能力。光遗传学方法正在改变细胞信号系统的定量研究。最近开发的一种光可转换的有丝分裂原活化蛋白激酶激酶1（MEK 1）酶（psMEK）在效应激酶激活的最接近步骤处短路高度保守的细胞外信号调节激酶（ERK）信号级联。然而，由于这种光遗传学工具依赖于MEK激活环中的磷酸化模拟取代，因此预测其催化活性显著低于在这些残基处磷酸化的野生型MEK的催化活性。在这里，我们提出的证据表明，psMEK确实有次优的功能在体内，并提出了一种策略，以规避这一限制，利用获得的功能，不稳定的突变MEK。具体而言，我们证明，结合磷酸模拟突变与MEK中的其他突变，选择其激活潜力，恢复最大的激酶活性在体外。我们确定，这种修饰可以通过选择去稳定化突变来调节，并且不会干扰果蝇和斑马鱼体内psMEK的可逆激活。为了说明优化的psMEK所能实现的扰动类型，我们用它来传递斑马鱼胚胎发生过程中ERK激活的脉冲，揭示ERK依赖的形态发生事件的变阻器样反应。
Significance Controlling access to the active sites of kinases using photo-dimerizable domains revolutionizes the types of possible optogenetic perturbations of cell signaling. This technology, however, is useful only when there is strong kinase activity to harness. We report a strategy to systematically enhance and tune functionality of a kinase activating the Extracellular Signal-Regulated Kinase (ERK) pathway, MEK, using gain-of-function point mutations that destabilize MEK. Photoswitching mutant MEK activity by reversibly caging its active site controls a large dynamic range of ERK signaling in vivo. Our approach for optimizing and tuning light-activated protein kinases is not limited to MEK and can be used to systematically extend the capabilities of light-activated regulatory enzymes. Optogenetic approaches are transforming quantitative studies of cell-signaling systems. A recently developed photoswitchable mitogen-activated protein kinase kinase 1 (MEK1) enzyme (psMEK) short-circuits the highly conserved Extracellular Signal-Regulated Kinase (ERK)-signaling cascade at the most proximal step of effector kinase activation. However, since this optogenetic tool relies on phosphorylation-mimicking substitutions in the activation loop of MEK, its catalytic activity is predicted to be substantially lower than that of wild-type MEK that has been phosphorylated at these residues. Here, we present evidence that psMEK indeed has suboptimal functionality in vivo and propose a strategy to circumvent this limitation by harnessing gain-of-function, destabilizing mutations in MEK. Specifically, we demonstrate that combining phosphomimetic mutations with additional mutations in MEK, chosen for their activating potential, restores maximal kinase activity in vitro. We establish that this modification can be tuned by the choice of the destabilizing mutation and does not interfere with reversible activation of psMEK in vivo in both Drosophila and zebrafish. To illustrate the types of perturbations enabled by optimized psMEK, we use it to deliver pulses of ERK activation during zebrafish embryogenesis, revealing rheostat-like responses of an ERK-dependent morphogenetic event.