Phosphorylation of ubiquitin-activating enzyme in cultured cells.

Ubiquitin-activating enzyme, E1, is the first enzyme in the pathway leading to formation of ubiquitin-protein conjugates. E1 exists as two isoforms in human cells which are separable by electrophoresis. These isoforms migrate with apparent molecular sizes of 110 kDa and 117 kDa in SDS/polyacrylamide gels. Immunoprecipitation of E1 from lysates of HeLa cells metabolically labeled with [32P]phosphate indicated the presence of a phosphorylated form of E1 which migrates at 117 kDa. Phospho amino acid analysis identified serine as the phosphorylated residue in E1. Phosphorylated E1 was also detected in normal and transformed cells from another human cell line. Phosphatase-catalyzed dephosphorylation of E1 in vitro did not eliminate the 117-kDa E1 isoform detected by Coomassie staining after SDS/polyacrylamide gel electrophoresis, thereby demonstrating that phosphorylation is not the sole structural feature differentiating the isoforms of E1. These observations suggest new hypotheses concerning mechanisms of metabolic regulation of the ubiquitin conjugation pathway. IMAGES:

泛素激活酶E1（Ubiquitin-activating enzyme, E1）是介导泛素-蛋白质结合物形成通路的首个酶。人类细胞中的E1存在两种同工型，可通过电泳技术分离。这两种同工型在SDS-聚丙烯酰胺凝胶中电泳迁移时的表观分子量分别为110 kDa和117 kDa。对经[32P]磷酸代谢标记的海拉（HeLa）细胞裂解液进行E1免疫沉淀实验，结果显示存在一种迁移速率对应117 kDa的磷酸化E1形式。磷酸氨基酸分析鉴定出E1的磷酸化位点为丝氨酸残基。在另一株人类细胞系的正常细胞与转化细胞中，同样检测到了磷酸化E1。体外经磷酸酶催化去磷酸化的E1，在SDS-聚丙烯酰胺凝胶电泳后经考马斯亮蓝染色检测时，并未消除117 kDa的E1同工型，这表明磷酸化并非区分E1两种同工型的唯一结构特征。上述研究结果为泛素结合通路的代谢调控机制提出了新的研究假说。IMAGES: