Caged catalytic subunit of cAMP-dependent protein kinase
Caged catalytic subunit of cAMP-dependent protein kinase
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DOI：
10.1021/ja981649v
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发表时间：
1998-08-05
影响因子：
15
通讯作者：
Bayley, H
中科院分区：
文献类型：
--
作者：
Chang, CY;Fernandez, T;Bayley, H
光介导的活性生物分子从“笼”试剂中的释放已被广泛应用于生物问题。1笼状试剂特别有用，因为可以控制细胞中光释放的程度、时间和位置。笼状小分子包括核苷酸（ATP、GTP等），神经递质，第二信使（cAMP，Ca 2+，IP 3，一氧化氮等），最近又研究了缩氨酸2光介导的蛋白质活性调节也是非常需要的。已经采取了两种主要的方法;一种是可逆控制（开关）的活性与光异构化分子修饰的蛋白质。3在这种情况下，尚未实现异或非切换，可能是因为通常对蛋白质上的官能团进行随机而不是靶向修饰。第二种方法是灭活蛋白质的不可逆光活化（触发），即制备笼蛋白。后者包括蛋白酶、4种T4溶菌酶、5种免疫毒素、6种G-肌动蛋白、7种牛血清白蛋白、8种R-溶血素、9种重酶解肌球蛋白、10种β-半乳糖苷酶、11种抗体。[12]用光敏保护基团笼住蛋白质的化学修饰也常常是随机的，这种方法缺乏控制是一个严重的缺点。有针对性的修饰是更可取的，我们提出了这一想法，通过使用在单一的半胱氨酸残基引入诱变修饰。9，13对参与细胞信号传导的蛋白质（如蛋白激酶、G蛋白和转录因子）进行笼化是一个重要的挑战。cAMP依赖性蛋白激酶（PKA）的催化亚基是广泛信号传导事件的关键参与者，包括参与激素作用、14发育、15和神经元可塑性的那些。[16]四聚体PKA全酶含有两个调节亚基（R）和两个催化亚基（C），在cAMP与R结合之前是无活性的。全酶然后解离形成R2二聚体和两个单体活性C亚基。游离C亚基可以磷酸化细胞质或膜蛋白底物或扩散到细胞核中，并通过作用于转录因子改变基因表达。17在这里，我们描述了PKA的C亚基在活性位点口的Cys-199处被捕获（图1）。该酶在光活化后活性增加20至30倍，因此适合作为活细胞显微注射的试剂。含有两个半胱氨酸残基Cys-199和Cys-343的C亚基（图1）在很大程度上被各种巯基特异性试剂灭活。活性的丧失是由于Cys-199的衍生化，因为仅在Cys-343处修饰的C亚基仍然是活性的，并且用二硫苏糖醇解封闭用5，5′-二硫代双（2-硝基苯甲酸）修饰的Cys-199恢复酶活性。因此，我们选择通过在Cys-199处连接2-硝基苄基来笼化C亚基，Cys-199在Cys-199中。
The light-mediated release of active biological molecules from “caged” reagents has been widely applied to biological problems. 1 Caged reagents are especially useful as the extent, timing, and location of photorelease in cells can be controlled. Caged small molecules include nucleotides (ATP, GTP &c.), neurotransmitters, second messengers (cAMP, Ca2+, IP3, nitric oxide &c.), and, recently, peptides. 2 Light-mediated regulation of protein activity is also highly desirable. Two major approaches have been taken; one is reversible control (switching) of the activity of proteins modified with photoisomerizable molecules. 3 In this case, allor-none switching has not yet been achieved, possibly because random rather than targeted modification of functional groups on proteins has generally been applied. The second approach is irreversible photoactivation (triggering) of inactivated proteins, ie, the preparation of caged proteins. The latter have included proteinases, 4 T4 lysozyme, 5 an immunotoxin, 6 G-actin, 7 bovine serum albumin, 8 R-hemolysin, 9 heavy meromyosin, 10 β-galactosidase, 11 and antibodies. 12 The chemical modifications used to cage proteins with light-sensitive protecting groups have also often been random, and the lack of control in this approach is a serious drawback. Targeted modification is more desirable and we advanced this idea by using modification at single cysteine residues introduced by mutagenesis. 9, 13The caging of proteins involved in cell signaling such as protein kinases, G proteins, and transcription factors is an important challenge. The catalytic subunit of cAMP-dependent protein kinase (PKA) is a crucial participant in a wide range of signaling events including those involved in hormone action, 14 development, 15 and neuronal plasticity. 16 The tetrameric PKA holoenzyme, which contains two regulatory (R) and two catalytic (C) subunits, is inactive until cAMP binds to R. The holoenzyme then dissociates to form an R2 dimer and two monomeric, active C subunits. The free C subunit can phosphorylate cytoplasmic or membrane protein substrates or diffuse into the nucleus and alter gene expression by acting on transcription factors. 17 Here, we describe a C subunit of PKA caged at Cys-199 at the mouth of the active site (Figure 1). The enzyme undergoes a 20-to 30-fold increase in activity upon photoactivation, and it is therefore suitable as a reagent for microinjection into living cells. The C subunit, which contains two cysteine residues, Cys-199 and Cys-343 (Figure 1), is largely inactivated by a variety of sulfhydryl-specific reagents. The loss of activity is due to derivatization of Cys-199 as C subunit modified exclusively at Cys-343 is still active and unblocking of Cys-199 modified with 5, 5′-dithiobis (2-nitrobenzoic acid) with dithiothreitol restores enzymatic activity. 20 Therefore, we chose to cage the C subunit by attaching a 2-nitrobenzyl group at Cys-199, which is in the