Department of Biochemistry, School of Life Science, Chungbuk National University, Cheongju 28644, Republic of Korea.
bHLBIO, Cheongju 28119, Republic of Korea.
Int J Mol Sci. 2024 Oct 1;25(19):10587. doi: 10.3390/ijms251910587.
Conventional biochemical methods for studying cellular signaling cascades have relied on destructive cell disruption. In contrast, the live cell imaging of fluorescent-tagged transfected proteins offers a non-invasive approach to understanding signal transduction events. One strategy involves monitoring the phosphorylation-dependent shuttling of a fluorescent-labeled kinase between the nucleus and cytoplasm using nuclear localization, export signals, or both. In this paper, we introduce a simple method to visualize intracellular signal transduction in live cells by exploring the translocation properties of PKC from the cytoplasm to the membrane. We fused bait protein to PKC, allowing the bait (RFP-labeled) and target (GFP-labeled) proteins to co-translocate from the cytoplasm to the membrane. However, in non-interacting protein pairs, only the bait protein was translocated to the plasma membrane. To verify our approach, we examined the Raf-MEK-ERK signaling cascade (ERK pathway). We successfully visualized direct Raf1/MEK2 interaction and the KSR1-containing ternary complex (Raf1/MEK2/KSR1). However, the interaction between MEK and ERK was dependent on the presence of the KSR1 scaffold protein under our experimental conditions.
传统的生化方法研究细胞信号级联反应依赖于细胞的破坏。相比之下，荧光标记转染蛋白的活细胞成像提供了一种非侵入性的方法来理解信号转导事件。一种策略是通过监测核定位、输出信号或两者兼用的荧光标记激酶在核质和细胞质之间的磷酸化依赖性穿梭来监测信号转导事件。在本文中，我们通过探索 PKC 从细胞质到膜的易位特性，介绍了一种在活细胞中可视化细胞内信号转导的简单方法。我们将诱饵蛋白与 PKC 融合，使诱饵（RFP 标记）和靶标（GFP 标记）蛋白从细胞质共易位到膜。然而，在非相互作用的蛋白对中，只有诱饵蛋白被易位到质膜。为了验证我们的方法，我们检查了 Raf-MEK-ERK 信号级联（ERK 途径）。我们成功地可视化了 Raf1/MEK2 的直接相互作用和包含 KSR1 的三元复合物（Raf1/MEK2/KSR1）。然而，在我们的实验条件下，ERK 与 MEK 之间的相互作用依赖于 KSR1 支架蛋白的存在。
