Bécsi Bálint, Dedinszki Dóra, Gyémánt Gyöngyi, Máthé Csaba, Vasas Gábor, Lontay Beáta, Erdődi Ferenc
MTA-DE Cell Biology and Signaling Research Group, University of Debrecen, Debrecen, Hungary; Department of Medical Chemistry, University of Debrecen, Debrecen, Hungary.
Department of Medical Chemistry, University of Debrecen, Debrecen, Hungary.
J Photochem Photobiol B. 2014 Sep 5;138:240-8. doi: 10.1016/j.jphotobiol.2014.06.004. Epub 2014 Jun 14.
Identification of the interacting proteins of protein phosphatases is crucial to understand the cellular roles of these enzymes. Microcystin-LR (MC-LR), a potent inhibitor of protein phosphatase-1 (PP1), -2A (PP2A), PP4, PP5 and PP6, was biotinylated, immobilized to streptavidin-coupled sensorchip surface and used in surface plasmon resonance (SPR) based binding experiments to isolate phosphatase binding proteins. Biotin-MC-LR captured PP1 catalytic subunit (PP1c) stably and the biotin-MC-LR-PP1c complex was able to further interact with the regulatory subunit (MYPT1) of myosin phosphatase. Increased biotin-MC-LR coated sensorchip surface in the Surface Prep unit of Biacore 3000 captured PP1c, PP2Ac and their regulatory proteins including MYPT1, MYPT family TIMAP, inhibitor-2 as well as PP2A-A and -Bα-subunits from normal and UVA-irradiated HaCaT cell lysates as revealed by dot blot analysis of the recovered proteins. Biotin-MC-LR was used for the subcellular localization of protein phosphatases in HaCaT cells by identification of phosphatase-bound biotin-MC-LR with fluorescent streptavidin conjugates. Partial colocalization of the biotin-MC-LR signals with those obtained using anti-PP1c and anti-PP2Ac antibodies was apparent as judged by confocal microscopy. Our results imply that biotin-MC-LR is a suitable capture molecule in SPR for isolation of protein phosphatase interacting proteins from cell lysates in sufficient amounts for immunological detection.
鉴定蛋白磷酸酶的相互作用蛋白对于理解这些酶在细胞中的作用至关重要。微囊藻毒素-LR（MC-LR）是一种强效的蛋白磷酸酶-1（PP1）、-2A（PP2A）、PP4、PP5和PP6抑制剂，将其生物素化后固定在链霉亲和素偶联的传感芯片表面，并用于基于表面等离子体共振（SPR）的结合实验，以分离磷酸酶结合蛋白。生物素-MC-LR能稳定捕获PP1催化亚基（PP1c），且生物素-MC-LR-PP1c复合物能够进一步与肌球蛋白磷酸酶的调节亚基（MYPT1）相互作用。通过对回收蛋白的斑点印迹分析发现，Biacore 3000表面预处理单元中增加的生物素-MC-LR包被传感芯片表面，能从正常和紫外线照射的HaCaT细胞裂解物中捕获PP1c、PP2Ac及其调节蛋白，包括MYPT1、MYPT家族的TIMAP、抑制剂-2以及PP2A-A和-Bα亚基。通过用荧光链霉亲和素偶联物鉴定与磷酸酶结合的生物素-MC-LR，生物素-MC-LR被用于HaCaT细胞中蛋白磷酸酶的亚细胞定位。通过共聚焦显微镜判断，生物素-MC-LR信号与使用抗PP1c和抗PP2Ac抗体获得的信号部分共定位明显。我们的结果表明，生物素-MC-LR是SPR中一种合适的捕获分子，可从细胞裂解物中分离出足够量的用于免疫检测的蛋白磷酸酶相互作用蛋白。
