A caged phosphopeptide-based approach for photochemical activation of kinases in living cells
A caged phosphopeptide-based approach for photochemical activation of kinases in living cells
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DOI：
10.1002/cbic.200800116
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发表时间：
2008-07-02
期刊：
影响因子：
3.2
通讯作者：
Nagamune, Teruyuki
中科院分区：
文献类型：
--
作者：
Kawakami, Takashi;Cheng, Huan;Nagamune, Teruyuki
在所有真核生物中，激酶介导的蛋白质和脂质的磷酸化在细胞内信号和细胞功能的调节中起着核心作用。[1]许多激酶在静止的细胞中保持非活性状态，并在细胞刺激下被激活，通过磷酸化它们的底物来触发下游通路。重要的是，特定的细胞决策，如细胞周期检查点、分化和细胞迁移，取决于激活蛋白在细胞内的精确时间控制和相对空间分布。类似的时空定义，即特定于细胞和阶段的信号通路的激活发生在发育的早期。因此，在细胞、组织或整个动物中在所需的时间和/或位置人工激活感兴趣的激酶的能力将为研究生物系统以及最终为工程活的对象提供重要的工具。[2]可光激活的“笼子”蛋白质是这一目的的理想之选，因为这种激活可以由具有高时间和空间分辨率的光束触发。[3]沿着这条线，几个研究小组以前曾报道过光囚禁的蛋白激酶[4]和激酶调节蛋白的衍生物[5]，其中一些已经在细胞中发现了实际应用。虽然蛋白质笼化是一种直接的方法来赋予感兴趣的蛋白质光敏感性，但该策略受到与体外制备半合成笼状蛋白质相关的困难的限制。在这里，我们描述了一种新的基于多肽配体的活细胞中激酶的光激活方法。许多细胞内的激酶，包括Src家族酪氨酸激酶和磷脂酰肌醇3-激酶(PI3K)，具有模块结构域、Src同源2(SH2)和3(SH3)结构域，与含有共同序列的短肽基序结合。[6]这些激酶的催化活性受到模块结构域的严格调控，多肽结合以变构的方式诱导激酶的激活。[7]因此，我们认为这种模块结构域结合肽的笼形类似物应该提供一种通用的手段，无需化学修饰就可以用光激活这些激酶。为了测试这一策略，我们在这项工作中将PI3K作为光激活的目标。PI3K是一种脂酶，催化肌醇环D3位磷脂酰肌醇-4，5-二磷酸的磷酸化，产生脂质第二信使磷脂酰肌醇-3，4，5-三磷酸(PtdInsP3)。[8]PtdInsP3激活许多下游信号蛋白，调节不同的细胞功能，如细胞存活、代谢、运动、囊泡运输和轴突生长。尽管目前已有多种第二信使的笼状衍生物可用，但目前还没有在细胞内以光化学方式生产PtdInsP3的策略。因此，光激活PI3K的能力对于在(多)细胞环境中时空控制依赖于PtdInsP3的生物事件将是非常有价值的。以前的研究表明，含有pYXXM(Py，磷酸酪氨酸；X，任何氨基酸)序列的多肽可以通过与P85调节亚基中的SH2结构域结合来激活PI3K。[10]因此，我们决定制备一种SH2结构域靶向肽，其激活的酪氨酰磷酸部分用光保护基团保护。最近已经描述了笼状磷肽的化学合成。[11，12]然而，这些多肽包括经典的基于2-硝基苯基的笼状基团，因此对细胞毒性较小的紫外光(理想情况下超过350 nm)的光敏性可能不足以满足许多应用。因此，我们首先介绍了…
Kinase-mediated phosphorylation of proteins and lipids plays a central role in the regulation of intracellular signaling and cell functions in all eukaryotes.[1] Many kinases are maintained in an inactive state in quiescent cells and activated in response to cell stimulation, triggering the downstream pathways by phosphorylating their substrates. It is important that specific cellular decisions, such as cell-cycle checkpoints, differentiation, and cell migration, depend upon the precise temporal control and relative spatial distribution of activated proteins within cells. Analogous spatiotemporally defined, that is, cell-and stage-specific, activation of signaling pathways occurs during early development. Thus, the ability to artificially activate a kinase of interest at a desired time and/or location in cells as well as tissues or whole animals would provide important tools for investigating biological systems and ultimately for engineering living subjects.[2] Light-activatable,“caged” proteins are ideal for this purpose, as the activation can be triggered by a beam of light with high temporal and spatial resolution.[3] Along this line, several groups have previously reported photocaged derivatives of protein kinases [4] and kinase-regulated proteins,[5] and a few of them have found practical application in cells. Although protein caging is a straightforward approach to impart light sensitivity to proteins of interest, the strategy is limited by the difficulties associated with the preparation of semisynthetic caged proteins in vitro. Herein we describe a new peptide-ligand-based method for photoactivation of kinases in living cells. Many intracellular kinases, which include Src family tyrosine kinases and phosphatidylinositol 3-kinase (PI3K), possess modular domains, Src homology 2 (SH2), and 3 (SH3) domains, that bind short peptide motifs containing consensus sequences.[6] The catalytic activity of these kinases is tightly regulated by the modular domain, and the peptide-binding induces the activation of the kinase in an allosteric manner.[7] We therefore reasoned that caged analogues of such modular domain-binding peptides should provide a general means to activate these kinases by light without need of chemical modification. To test this strategy, we targeted PI3K for photoactivation in this work. PI3K is a lipid kinase that catalyzes the phosphorylation of phosphatidylinositide-4, 5-bisphosphate at the D3 position of the inositol ring, producing a lipid second messenger, phosphatidylinositide-3, 4, 5-trisphosphate (PtdInsP3).[8] PtdInsP3 activates a number of downstream signaling proteins and regulates diverse cellular functions, such as cell survival, metabolism, motility, vesicle trafficking, and neurite outgrowth. Although caged derivatives of various second messengers are currently available,[9] there is no strategy for photochemically producing PtdInsP3 inside cells. Therefore, the ability to activate PI3K by light will be highly valuable for spatiotemporally controlling PtdInsP3-dependent biological events in (multi) cellular contexts. Previous studies have revealed that peptides containing a pYXXM (pY, phosphotyrosine; X, any amino acid) sequence can activate PI3K through binding to the SH2 domains within the p85 regulatory subunit.[10] Accordingly, we decided to prepare a SH2 domain-targeting peptide of which the activating tyrosyl phosphate moiety is protected with a photolabile group. Chemical synthesis of caged phosphopeptides have been recently described.[11, 12] However, the peptides include classical 2-nitrobenzyl-based caging groups and thus the photosensitivity to less cell-toxic UV light (ideally over 350 nm) may not be sufficient for many applications. We thus first …