Single-molecule studies reveal a hidden key step in the activation mechanism of membrane-bound protein kinase C-α.
Single-molecule studies reveal a hidden key step in the activation mechanism of membrane-bound protein kinase C-α.
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DOI：
10.1021/bi4016082
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发表时间：
2014-03-18
期刊：
影响因子：
2.9
通讯作者：
Falke, Joseph J.
中科院分区：
文献类型：
--
作者：
Ziemba, Brian P.;Li, Jianing;Landgraf, Kyle E.;Knight, Jefferson D.;Voth, Gregory A.;Falke, Joseph J.
蛋白激酶C-α (PKCα)是常规蛋白激酶C异构体（cPKCs）家族的一员，可调节多种细胞信号通路，具有共同的激活机制，并与多种病理相关。cPKC结构域是模块化的，由一个n端假底物肽、两个抑制结构域（C1A和C1B）、一个靶向结构域（C2）和一个激酶结构域组成。成熟的细胞质cPKCs是无活性的，直到它们被主要发生在质膜表面的多步骤激活反应打开。通常，这种激活始于细胞质Ca2+信号，触发C2结构域靶向到质膜，在那里它结合磷脂酰丝氨酸（PS）和磷脂酰肌醇4,5-二磷酸（PIP2）。随后，信号脂质二酰基甘油（DAG）的出现通过从激酶结构域招募抑制性假底物和一个或两个C1结构域来激活膜结合酶。为了进一步研究这一机制，本研究利用单分子全内反射荧光显微镜（TIRFM）定量分析了全长PKCα和缺失特定结构域片段在支持的脂质双层上的结合和横向扩散。脂质结合事件，以及在此过程中额外的蛋白质插入到双分子层中，通过它们对平衡结合粒子密度和二维扩散速率的影响来检测。除了先前提出的激活步骤外，研究结果还揭示了一个主要的，未描述的，激酶无活性的中间体。在含有PS或PS和PIP2的双层膜上，全长PKCα首先通过其C2结构域与膜对接，然后其C1A结构域在DAG出现之前嵌入双层膜中。由此产生的DAG前中间体具有膜结合的C1A和C2结构域，是PKCα在等待DAG信号时的主要状态。新检测到的这种前dag中间体的膜嵌入C1A结构域具有多种有用的特征，包括增强的膜亲和力和更长的结合态寿命。研究结果还确定了激酶激活的关键分子步骤：由于C1A已经在膜上嵌入激酶关闭状态，因此DAG或phorbol酯将C1B募集到双分子层是稳定激酶打开状态的关键调控事件。更广泛地说，这项研究说明了单分子方法在阐明膜结合信号蛋白的激活机制和隐藏调节状态方面的力量。
Protein kinase C-α (PKCα) is a member of the conventional family of protein kinase C isoforms (cPKCs) that regulate diverse cellular signaling pathways, share a common activation mechanism, and are linked to multiple pathologies. The cPKC domain structure is modular, consisting of an N-terminal pseudosubstrate peptide, two inhibitory domains (C1A and C1B), a targeting domain (C2), and a kinase domain. Mature, cytoplasmic cPKCs are inactive until they are switched on by a multistep activation reaction that occurs largely on the plasma membrane surface. Often, this activation begins with a cytoplasmic Ca2+ signal that triggers C2 domain targeting to the plasma membrane where it binds phosphatidylserine (PS) and phosphatidylinositol 4,5-bisphosphate (PIP2). Subsequently, the appearance of the signaling lipid diacylglycerol (DAG) activates the membrane-bound enzyme by recruiting the inhibitory pseudosubstrate and one or both C1 domains away from the kinase domain. To further investigate this mechanism, this study has utilized single-molecule total internal reflection fluorescence microscopy (TIRFM) to quantitate the binding and lateral diffusion of full-length PKCα and fragments missing specific domain(s) on supported lipid bilayers. Lipid binding events, and events during which additional protein is inserted into the bilayer, were detected by their effects on the equilibrium bound particle density and the two-dimensional diffusion rate. In addition to the previously proposed activation steps, the findings reveal a major, undescribed, kinase-inactive intermediate. On bilayers containing PS or PS and PIP2, full-length PKCα first docks to the membrane via its C2 domain, and then its C1A domain embeds itself in the bilayer even before DAG appears. The resulting pre-DAG intermediate with membrane-bound C1A and C2 domains is the predominant state of PKCα while it awaits the DAG signal. The newly detected, membrane-embedded C1A domain of this pre-DAG intermediate confers multiple useful features, including enhanced membrane affinity and longer bound state lifetime. The findings also identify the key molecular step in kinase activation: because C1A is already membrane-embedded in the kinase off state, recruitment of C1B to the bilayer by DAG or phorbol ester is the key regulatory event that stabilizes the kinase on state. More broadly, this study illustrates the power of single-molecule methods in elucidating the activation mechanisms and hidden regulatory states of membrane-bound signaling proteins.
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影响因子：
4.7
作者：
ARCOLEO, JP;WEINSTEIN, IB
通讯作者：
WEINSTEIN, IB
影响因子：
2.9
作者：
Edwards, AS;Newton, AC
通讯作者：
Newton, AC
影响因子：
5.8
作者：
Arnold, K;Bordoli, L;Schwede, T
通讯作者：
Schwede, T
DOI：
10.1152/ajplung.2000.279.3.l429
发表时间：
2000-09-01
影响因子：
4.9
作者：
Dempsey, EC;Newton, AC;Messing, RO
通讯作者：
Messing, RO
影响因子：
3.4
作者：
Chen, Chiu-Hao;Malkova, Sarka;Schlossman, Mark L.
通讯作者：
Schlossman, Mark L.