Ca2+/calmodulin-dependent protein kinase phosphatase (CaMKP/PPM1F) interacts with neurofilament L and inhibits its filament association.
Ca2+/calmodulin-dependent protein kinase phosphatase (CaMKP/PPM1F) interacts with neurofilament L and inhibits its filament association.
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Ca2 /钙调蛋白依赖性蛋白激酶磷酸酶 (CaMKP/PPM1F) 与神经丝 L 相互作用并抑制其丝缔合。
DOI：
10.1016/j.bbrc.2016.06.141
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发表时间：
2016
期刊：
影响因子：
--
通讯作者：
Ishida A
中科院分区：
文献类型：
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作者：
Ozaki H;Katoh T;Nakagawa R;Ishihara Y;Sueyoshi N;Kameshita I;Taniguchi T;Hirano T;Yamazaki T;Ishida A
Ca 2 +/钙调素依赖性蛋白激酶磷酸酶（CaMKP/PPM 1F）是一种丝氨酸/苏氨酸磷酸酶，属于PPM家族。越来越多的证据表明，PPM磷酸酶，包括CaMKP作为一个与其他蛋白质的复合物，以调节细胞功能。在这项研究中，使用二维远蛋白质印迹技术与地高辛标记的CaMKP作为探针，结合肽质量指纹分析，我们确定了神经丝L（NFL）作为一个钙MKP结合蛋白在Triton不溶性部分的大鼠脑。我们通过荧光偏振证实了荧光素标记的CaMKP（F-CaMKP）与溶液中的NFL的结合。分析表明，F-CaMKP对NFL的解离常数为73 ± 17 nM（n= 3）。使用NGF分化的PC 12细胞的胞质级分的免疫共沉淀测定表明，内源性CaMKP和NFL在细胞中形成复合物。此外，研究了CaMKP对NFL自组装的影响。电子显微镜显示，CaMKP显着防止NFL形成大的丝状聚集体，这表明CaMKP结合到NFL抑制其细丝协会。这些发现可能为神经元分化过程中调节神经丝网络形成的新机制提供新的见解。
Ca2+/calmodulin-dependent protein kinase phosphatase (CaMKP/PPM1F) is a Ser/Thr phosphatase that belongs to the PPM family. Growing evidence suggests that PPM phosphatases including CaMKP act as a complex with other proteins to regulate cellular functions. In this study, using the two-dimensional far-western blotting technique with digoxigenin-labeled CaMKP as a probe, in conjunction with peptide mass fingerprinting analysis, we identified neurofilament L (NFL) as a CaMKP-binding protein in a Triton-insoluble fraction of rat brain. We confirmed binding of fluorescein-labeled CaMKP (F-CaMKP) to NFL in solution by fluorescence polarization. The analysis showed that the dissociation constant of F-CaMKP for NFL is 73 ± 17 nM (n= 3). Co-immunoprecipitation assay using a cytosolic fraction of NGF-differentiated PC12 cells showed that endogenous CaMKP and NFL form a complex in cells. Furthermore, the effect of CaMKP on self-assembly of NFL was examined. Electron microscopy revealed that CaMKP markedly prevented NFL from forming large filamentous aggregates, suggesting that CaMKP-binding to NFL inhibits its filament association. These findings may provide new insights into a novel mechanism for regulating network formation of neurofilaments during neuronal differentiation.