DataSheet2_Features and mechanisms of propofol-induced protein kinase C (PKC) translocation and activation in living cells.docx

Background and purpose: In this study, we aimed to elucidate the action mechanisms of propofol, particularly those underlying propofol-induced protein kinase C (PKC) translocation.Experimental approach: Various PKCs fused with green fluorescent protein (PKC-GFP) or other GFP-fused proteins were expressed in HeLa cells, and their propofol-induced dynamics were observed using confocal laser scanning microscopy. Propofol-induced PKC activation in cells was estimated using the C kinase activity receptor (CKAR), an indicator of intracellular PKC activation. We also examined PKC translocation using isomers and derivatives of propofol to identify the crucial structural motifs involved in this process.Key results: Propofol persistently translocated PKCα conventional PKCs and PKCδ from novel PKCs (nPKCs) to the plasma membrane (PM). Propofol translocated PKCδ and PKCη of nPKCs to the Golgi apparatus and endoplasmic reticulum, respectively. Propofol also induced the nuclear translocation of PKCζ of atypical PKCs or proteins other than PKCs, such that the protein concentration inside and outside the nucleus became uniform. CKAR analysis revealed that propofol activated PKC in the PM and Golgi apparatus. Moreover, tests using isomers and derivatives of propofol predicted that the structural motifs important for the induction of PKC and nuclear translocation are different.Conclusion and implications: Propofol induced the subtype-specific intracellular translocation of PKCs and activated PKCs. Additionally, propofol induced the nuclear translocation of PKCs and other proteins, probably by altering the permeability of the nuclear envelope. Interestingly, propofol-induced PKC and nuclear translocation may occur via different mechanisms. Our findings provide insights into the action mechanisms of propofol.

背景与目的：本研究旨在阐明丙泊酚的作用机制，特别是丙泊酚诱导的蛋白激酶C（PKC）转位的作用机制。实验方法：通过在HeLa细胞中表达融合有绿色荧光蛋白（PKC-GFP）或其他融合有GFP的蛋白的PKC，并利用共聚焦激光扫描显微镜观察其丙泊酚诱导的动态变化。使用C激酶活性受体（CKAR），作为细胞内PKC激活的指标，来估算细胞中丙泊酚诱导的PKC激活。我们还通过丙泊酚的同分异构体和衍生物来检查PKC转位，以识别参与此过程的至关重要的结构基序。主要结果：丙泊酚持续地将PKCα传统PKC和PKCδ从新型PKC（nPKCs）转位至质膜（PM）。丙泊酚将nPKCs中的PKCδ和PKCη转位至高尔基体和内质网。丙泊酚还诱导了非典型PKC或非PKC蛋白的PKCζ的核转位，使得核内外蛋白质浓度趋于一致。CKAR分析显示，丙泊酚在质膜和高尔基体中激活了PKC。此外，使用丙泊酚的同分异构体和衍生物的测试预测，诱导PKC和核转位的重要结构基序是不同的。结论与启示：丙泊酚诱导了PKC亚型特异性的细胞内转位并激活了PKC。此外，丙泊酚还诱导了PKC和其他蛋白的核转位，可能是通过改变核膜的通透性实现的。有趣的是，丙泊酚诱导的PKC和核转位可能通过不同的机制发生。我们的研究为丙泊酚的作用机制提供了洞见。