Greenwood J A, Johnson G V
Department of Psychiatry and Behavioral Neurobiology, University of Alabama, Birmingham 35294, USA.
Exp Cell Res. 1995 Oct;220(2):332-7. doi: 10.1006/excr.1995.1323.
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 32Pi 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-5 nuclei were isolated, incubated with [gamma-32P]ATP, extracted, and tau was immunoprecipitated. Although numerous nuclear proteins were 32P-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.
研究了LA-N-5神经母细胞瘤细胞中tau蛋白的定位和磷酸化状态。我们的结果表明，LA-N-5细胞中有两种tau蛋白群体：胞质tau蛋白和核tau蛋白。间接免疫荧光显微镜显示，核tau蛋白特异性定位于核仁，而胞质tau蛋白则呈弥散分布。为了通过亚细胞分级分离来定位和定量LA-N-5细胞中的tau蛋白，我们开发了一种从细胞核中提取tau蛋白同时保持其内源状态的方法。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。通过用32Pi标记LA-N-5细胞并从不同部分免疫沉淀tau蛋白，检测了核tau蛋白和胞质tau蛋白的磷酸化状态。这些数据表明，核tau蛋白和胞质tau蛋白的磷酸化程度大致相同。为了确定核tau蛋白的磷酸化是否发生在细胞核中，分离了LA-N-5细胞核，与[γ-32P]ATP一起孵育，提取后免疫沉淀tau蛋白。尽管许多核蛋白被32P标记，但tau蛋白未被磷酸化。这些结果表明，核tau蛋白不是在细胞核中被磷酸化，而是在转运到细胞核之前在细胞质中被磷酸化。核tau蛋白的特异性定位强烈表明它在细胞核中具有功能作用。然而，需要进一步的研究来确定核tau蛋白的功能以及它如何被磷酸化调节。
