LOCALIZATION AND IN-SITU PHOSPHORYLATION STATE OF NUCLEAR-TAU
LOCALIZATION AND IN-SITU PHOSPHORYLATION STATE OF NUCLEAR-TAU
复制标题
DOI：
10.1006/excr.1995.1323
复制
发表时间：
1995-10-01
影响因子：
3.7
通讯作者：
JOHNSON, GVW
中科院分区：
文献类型：
--
作者：
GREENWOOD, JA;JOHNSON, GVW
检查了LA-N-5神经母细胞瘤细胞中tau的定位和磷酸化状态我们的结果表明LA-N-5细胞中有两个tau群体：胞质tau和核tau间接免疫荧光显微镜显示核tau特异性定位于核仁，而胞质tau弥散分布。为了通过亚细胞分级分离定位和定量LA-N-5细胞中的tau，开发了一种从细胞核中提取tau同时保持蛋白质内源状态的方法。这些研究表明，LA-N-5细胞中总tau蛋白的16%位于细胞核中，更具体地说，主要存在于含有DNA、染色质和相关蛋白的染色质部分中。通过用P-32（i）标记LA-N-5细胞并免疫沉淀来自不同级分的tau来检查细胞核和细胞溶质tau的磷酸化状态。这些数据表明，核tau蛋白和胞质tau蛋白磷酸化的程度大致相同。为了确定核tau的磷酸化是否发生在核中，分离LA-N-B核，与[γ-P-32]ATP孵育，提取，并免疫沉淀tau。尽管许多核蛋白被P-32标记，但tau蛋白未被磷酸化。这些结果表明，细胞核tau蛋白不是在细胞核中磷酸化，而是在细胞质中运输到细胞核之前。核tau蛋白的特异性定位强烈表明它在细胞核中具有功能性作用。然而，还需要进一步的研究来确定核tau蛋白的功能以及它如何通过磷酸化来调节。(C)出版社：Academic Press
The localization and phosphorylation state of tau in LA-N-5 neuroblastoma cells was examined Our results demonstrate that there are two populations of tau in LA-N-5 cells: cytosolic tau and nuclear tau Indirect immunofluorescent microscopy revealed that nuclear tau is specifically localized to the nucleolus while cytosolic tau is diffusely distributed, To localize and quantitate tau in LA-N-5 cells by subcellular fractionation, a method was developed to extract tau from the nucleus while preserving the endogenous state of the protein. These studies revealed that 16% of the total tau protein in LA-N-5 cells is located in the nucleus and more specifically was found predominantly in the chromatin fraction containing DNA, chromatin, and associated proteins. The phosphorylation state of nuclear and cytosolic tau was examined by labeling LA-N-5 cells with P-32(i) and immunoprecipitating tau from the different fractions. These data demonstrated that nuclear tau and cytosolic tau are phosphorylated approximately to the same extent. To determine if the phosphorylation of nuclear tau occurs in the nucleus, LA-N-B nuclei were isolated, incubated with [gamma-P-32]ATP, extracted, and tau was immunoprecipitated. Although numerous nuclear proteins were P-32-labeled, tau was not phosphorylated. These results suggest that nuclear tau is not phosphorylated in the nucleus but rather in the cytosol prior to transport into the nucleus. The specific localization of nuclear tau strongly suggests that it has a functional role in the nucleus. However, further studies are necessary to determine the function of nuclear tau and how it may be regulated by phosphorylation. (C) 1995 Academic Press, Inc.