Stephen A G, Trausch-Azar J S, Handley-Gearhart P M, Ciechanover A, Schwartz A L
Edward Mallinckrodt Department of Pediatrics, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
J Biol Chem. 1997 Apr 18;272(16):10895-903. doi: 10.1074/jbc.272.16.10895.
The ubiquitin-activating enzyme exists as two isoforms: E1a, localized predominantly in the nucleus, and E1b, localized in the cytoplasm. Previously we generated hemagglutinin (HA) epitope-tagged cDNA constructs, HA1-E1 (epitope tag placed after the first methionine) and HA2-E1 (epitope tag placed after the second methionine) (Handley-Gearhart, P. M., Stephen, A. G., Trausch-Azar, J. S., Ciechanover, A., and Schwartz, A. L. (1994) J. Biol. Chem. 269, 33171-33178), which represent the native isoforms. HA1-E1 is exclusively nuclear, whereas HA2-E1 is found predominantly in the cytoplasm. Using high resolution isoelectric focusing and SDS-polyacrylamide gel electrophoresis, we confirm that these epitope-tagged constructs HA1-E1 and HA2-E1 represent the two isoforms E1a and E1b. HA1-E1/E1a exists as one non-phosphorylated and four phosphorylated forms, and HA2-E1/E1b exists as one predominant non-phosphorylated form and two minor phosphorylated forms. We demonstrate that the first 11 amino acids are essential for phosphorylation and exclusive nuclear localization of HA1-E1. Within this region are four serine residues and a putative nuclear localization sequence (NLS; 5PLSKKRR). Removal of these four serine residues reduced phosphorylation levels by 60% but had no effect on nuclear localization of HA1-E1. Each serine residue was independently mutated to an alanine and analyzed by two-dimensional electrophoresis; only serine 4 was phosphorylated. Disruption of the basic amino acids within the NLS resulted in loss of exclusive nuclear localization and a 90-95% decrease in the phosphorylation of HA1-E1. This putative NLS was able to confer nuclear import on a non-nuclear protein in digitonin-permeabilized cells in a temperature- and ATP-dependent manner. Thus the predominant requirement for efficient phosphorylation of HA1-E1/E1a is a functional NLS, suggesting that E1a may be phosphorylated within the nucleus.
E1a，主要定位于细胞核；E1b，定位于细胞质。此前我们构建了带有血凝素（HA）表位标签的cDNA构建体，HA1-E1（表位标签位于第一个甲硫氨酸之后）和HA2-E1（表位标签位于第二个甲硫氨酸之后）（汉德利-吉尔哈特，P.M.，斯蒂芬，A.G.，特劳施-阿扎尔，J.S.，齐查诺弗，A.，以及施瓦茨，A.L.（1994年）《生物化学杂志》269，33171 - 33178），它们代表天然同工型。HA1-E1仅存在于细胞核中，而HA2-E1主要存在于细胞质中。通过高分辨率等电聚焦和SDS - 聚丙烯酰胺凝胶电泳，我们证实这些带有表位标签的构建体HA1-E1和HA2-E1分别代表两种同工型E1a和E1b。HA1-E1/E1a以一种非磷酸化形式和四种磷酸化形式存在，HA2-E1/E1b以一种主要的非磷酸化形式和两种次要的磷酸化形式存在。我们证明前11个氨基酸对于HA1-E1的磷酸化和排他性核定位至关重要。在该区域内有四个丝氨酸残基和一个假定的核定位序列（NLS；5PLSKKRR）。去除这四个丝氨酸残基使磷酸化水平降低了60%，但对HA1-E1的核定位没有影响。每个丝氨酸残基分别突变为丙氨酸并通过二维电泳分析；只有丝氨酸4被磷酸化。NLS内碱性氨基酸的破坏导致排他性核定位丧失，并且HA1-E1的磷酸化水平降低90 - 95%。这个假定的NLS能够以温度和ATP依赖的方式赋予非核蛋白进入洋地黄皂苷通透细胞的核转运能力。因此，HA1-E1/E1a高效磷酸化的主要要求是一个功能性的NLS，这表明E1a可能在细胞核内被磷酸化。
