Weiser Douglas C, Shenolikar Shirish
Duke University Medical Center, Durham, North Carolina, USA.
Curr Protoc Protein Sci. 2003 May;Chapter 13:Unit 13.10. doi: 10.1002/0471140864.ps1310s31.
Reversible protein phosphorylation is recognized as a major mechanism regulating the physiology of plant and animal cells. Virtually every biochemical process within eukaryotic cells is controlled by the covalent modification of key regulatory proteins. This in turn dictates the cellular response to a variety of physiological and environmental stimuli; errors in signals transduced by phosphoproteins contribute to many human diseases. Thus, defining protein phosphorylation events, and specifically, the phosphoproteins involved, is crucial for obtaining a better understanding of the physiological events that distinguish normal and diseased states. Protein phosphatase inhibitors are useful when deciphering physiological events regulated by reversible protein phosphorylation but the hormonal stimuli or signaling pathways involved are not known. They are also useful in analyzing the impact of hormones and other physiological stimuli on the function of a specific phosphoprotein. This unit describes protocols for inhibiting the cellular PP1/PP2A activity with okadaic acid, microcystin-LR, and PP2B/calcineurin and a widely utilized strategy for inhibiting protein tyrosine phosphatases.
可逆性蛋白质磷酸化被认为是调节植物和动物细胞生理机能的主要机制。真核细胞内几乎每一个生化过程都受关键调节蛋白的共价修饰控制。这进而决定细胞对各种生理和环境刺激的反应；磷蛋白转导信号的错误会导致许多人类疾病。因此，确定蛋白质磷酸化事件，特别是所涉及的磷蛋白，对于更好地理解区分正常和患病状态的生理事件至关重要。当解读由可逆性蛋白质磷酸化调节的生理事件，但所涉及的激素刺激或信号通路未知时，蛋白质磷酸酶抑制剂很有用。它们在分析激素和其他生理刺激对特定磷蛋白功能的影响方面也很有用。本单元介绍了用冈田酸、微囊藻毒素-LR和PP2B/钙调磷酸酶抑制细胞PP1/PP2A活性的方案，以及一种广泛使用的抑制蛋白质酪氨酸磷酸酶的策略。
