Department of Biochemistry, The University of Mississippi Medical Center, Jackson, Mississippi, United States of America.
PLoS One. 2011;6(10):e25743. doi: 10.1371/journal.pone.0025743. Epub 2011 Oct 3.
Coilin is a nuclear phosphoprotein that accumulates in Cajal bodies (CBs). CBs participate in ribonucleoprotein and telomerase biogenesis, and are often found in cells with high transcriptional demands such as neuronal and cancer cells, but can also be observed less frequently in other cell types such as fibroblasts. Many proteins enriched within the CB are phosphorylated, but it is not clear what role this modification has on the activity of these proteins in the CB. Coilin is considered to be the CB marker protein and is essential for proper CB formation and composition in mammalian cells. In order to characterize the role of coilin phosphorylation on CB formation, we evaluated various coilin phosphomutants using transient expression. Additionally, we generated inducible coilin phosphomutant cell lines that, when used in combination with endogenous coilin knockdown, allow for the expression of the phosphomutants at physiological levels. Transient expression of all coilin phosphomutants except the phosphonull mutant (OFF) significantly reduces proliferation. Interestingly, a stable cell line induced to express the coilin S489D phosphomutant displays nucleolar accumulation of the mutant and generates a N-terminal degradation product; neither of which is observed upon transient expression. A N-terminal degradation product and nucleolar localization are also observed in a stable cell line induced to express a coilin phosphonull mutant (OFF). The nucleolar localization of the S489D and OFF coilin mutants observed in the stable cell lines is decreased when endogenous coilin is reduced. Furthermore, all the phosphomutant cells lines show a significant reduction in CB formation when compared to wild-type after endogenous coilin knockdown. Cell proliferation studies on these lines reveal that only wild-type coilin and the OFF mutant are sufficient to rescue the reduction in proliferation associated with endogenous coilin depletion. These results emphasize the role of coilin phosphorylation in the formation and activity of CBs.
科尔林是一种核磷蛋白，在 Cajal 体（CB）中积累。CB 参与核糖核蛋白和端粒酶的生物发生，通常存在于转录需求高的细胞中，如神经元和癌细胞，但也可以在其他细胞类型中观察到较少，如成纤维细胞。许多在 CB 中富集的蛋白质被磷酸化，但不清楚这种修饰对这些蛋白质在 CB 中的活性有什么作用。科尔林被认为是 CB 的标记蛋白，对于哺乳动物细胞中 CB 的正确形成和组成是必不可少的。为了表征科尔林磷酸化在 CB 形成中的作用，我们使用瞬时表达评估了各种科尔林磷酸突变体。此外，我们生成了可诱导的科尔林磷酸突变细胞系，当与内源性科尔林敲低结合使用时，允许在生理水平上表达磷酸突变体。除了磷酸缺失突变体（OFF）之外，所有科尔林磷酸突变体的瞬时表达都显著降低了增殖。有趣的是，一个诱导表达科尔林 S489D 磷酸突变体的稳定细胞系显示突变体的核仁积累，并产生 N 端降解产物；这两种情况在瞬时表达时都没有观察到。在诱导表达科尔林磷酸缺失突变体（OFF）的稳定细胞系中也观察到 N 端降解产物和核仁定位。当内源性科尔林减少时，在稳定细胞系中观察到的 S489D 和 OFF 科尔林突变体的核仁定位减少。此外，与野生型相比，在内源性科尔林敲低后，所有磷酸突变细胞系的 CB 形成都显著减少。对这些细胞系的细胞增殖研究表明，只有野生型科尔林和 OFF 突变体足以挽救与内源性科尔林耗竭相关的增殖减少。这些结果强调了科尔林磷酸化在 CB 的形成和活性中的作用。
