Detailed analysis of the phosphorylation of the human La (SS-B) autoantigen. (De)phosphorylation does not affect its subcellular distribution
Detailed analysis of the phosphorylation of the human La (SS-B) autoantigen. (De)phosphorylation does not affect its subcellular distribution
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DOI：
10.1021/bi992308c
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发表时间：
2000-03-21
期刊：
影响因子：
2.9
通讯作者：
Pruijn, GJM
中科院分区：
文献类型：
--
作者：
Broekhuis, CHD;Neubauer, G;Pruijn, GJM
La (SS-B) 自身抗原是一种进化上保守的磷蛋白，在各种过程中发挥着重要作用，最有可能作为 RNA 伴侣，例如细胞核中 RNA 聚合酶 III 转录物的生物合成和成熟以及细胞质中翻译的（内部）起始。在本研究中，通过高分辨率二维 IEF/SDS-PAGE 分析来表征来自人 HeLa 和 HEp-2 细胞的该蛋白的磷酸化状态，并通过纳电喷雾质谱法绘制磷酸化位点。此外，通过定点诱变研究了所识别位点的磷酸化对蛋白质亚细胞分布的影响。在来自两种类型细胞的La蛋白的二维凝胶上至少识别出14种等电亚型。代谢标记与碱性磷酸酶处理相结合表明，这些亚型中只有有限数量可归因于磷酸化。通过对 HeLa 细胞中分离的 La 蛋白或该蛋白的羧基末端一半进行质谱分析，绘制了四个磷酸化位点：Thr-302、Ser-325、Thr-362 和 Ser-366。 La 突变体的分析，其中相应的磷酸化残基被中性（丙氨酸）或酸性（天冬氨酸）残基取代，一方面通过显微注射到非洲爪蟾卵母细胞中，另一方面转染 HEp-2 细胞，结果表明该蛋白质的亚细胞分布不受这些氨基酸取代的影响。这些结果强烈表明，决定该蛋白质亚细胞分布的信号不受所检查的靶残基的（去）磷酸化的调节。
The La (SS-B) autoantigen is an evolutionarily conserved phosphoprotein which plays an important role, most likely as an RNA chaperone, in various processes, such as the biosynthesis and maturation of RNA polymerase III transcripts in the cell nucleus and (internal) initiation of translation in the cytoplasm. In this study, the phosphorylation state of this protein from human HeLa and HEp-2 cells was characterized by high-resolution two-dimensional IEF/SDS-PAGE analysis, and phosphorylation sites were mapped by nanoelectrospray mass spectrometry. Furthermore, the effect of phosphorylation at the sites identified on the subcellular distribution of the protein was studied by site-directed mutagenesis, At least 14 isoelectric isoforms were discerned on 2-D gels with La protein from both types of cells. Metabolic labeling in combination with alkaline phosphatase treatment revealed that only a limited number of these isoforms could be attributed to phosphorylation. Four phosphorylation sites, Thr-302, Ser-325, Thr-362, and Ser-366, were mapped by mass spectrometric analysis of the isolated La protein from HeLa cells or the carboxy-terminal half of this protein. The analysis of mutants of La, in which the respective phosphorylated residues were replaced by either a neutral (alanine) or an acidic (aspartate) residue, by microinjection into Xenopus laevis oocytes on the one hand and transfection of HEp-2 cells on the other hand revealed that the subcellular distribution of this protein was not affected by these amino acid substitutions. These results strongly suggest that the signals that determine the subcellular distribution of this protein are not regulated by (de)phosphorylation of the target residues examined.