From the Division of Hematology Oncology, Department of Internal Medicine, University of Cincinnati Cancer Institute, Department of Neurosurgery, University of Cincinnati Neuroscience Institute, Brain Tumor Center University of Cincinnati, College of Medicine, Cincinnati, Ohio 45267.
J Biol Chem. 2014 Feb 14;289(7):3950-9. doi: 10.1074/jbc.M113.531178. Epub 2013 Dec 13.
Mammalian cells encode three closely related Ras proteins, H-Ras, N-Ras, and K-Ras. Oncogenic K-Ras mutations frequently occur in human cancers, which lead to dysregulated cell proliferation and genomic instability. However, mechanistic role of the Ras isoform regulation have remained largely unknown. Furthermore, the dynamics and function of negative regulation of GTP-loaded K-Ras have not been fully investigated. Here, we demonstrate RasG, the Dictyostelium orthologue of K-Ras, is targeted for degradation by polyubiquitination. Both ubiquitination and degradation of RasG were strictly associated with RasG activity. High resolution tandem mass spectrometry (LC-MS/MS) analysis indicated that RasG ubiquitination occurs at C-terminal lysines equivalent to lysines found in human K-Ras but not in H-Ras and N-Ras homologues. Substitution of these lysine residues with arginines (4KR-RasG) diminished RasG ubiquitination and increased RasG protein stability. Cells expressing 4KR-RasG failed to undergo proper cytokinesis and resulted in multinucleated cells. Ectopically expressed human K-Ras undergoes polyubiquitin-mediated degradation in Dictyostelium, whereas human H-Ras and a Dictyostelium H-Ras homologue (RasC) are refractory to ubiquitination. Our results indicate the existence of GTP-loaded K-Ras orthologue-specific degradation system in Dictyostelium, and further identification of the responsible E3-ligase may provide a novel therapeutic approach against K-Ras-mutated cancers.
哺乳动物细胞编码三种密切相关的 Ras 蛋白，即 H-Ras、N-Ras 和 K-Ras。致癌性 K-Ras 突变经常发生在人类癌症中，导致细胞增殖失调和基因组不稳定。然而，Ras 同工型调节的机制作用在很大程度上仍然未知。此外，负载 GTP 的 K-Ras 的负调控的动力学和功能尚未得到充分研究。在这里，我们证明 RasG，即 K-Ras 的粘菌同源物，通过多泛素化被靶向降解。RasG 的泛素化和降解都与 RasG 的活性严格相关。高分辨率串联质谱（LC-MS/MS）分析表明，RasG 的泛素化发生在 C 末端赖氨酸上，与人类 K-Ras 中的赖氨酸等效，但不在 H-Ras 和 N-Ras 同源物中。用精氨酸取代这些赖氨酸残基（4KR-RasG）可减少 RasG 的泛素化并增加 RasG 蛋白的稳定性。表达 4KR-RasG 的细胞无法进行适当的胞质分裂，导致多核细胞。外源性表达的人 K-Ras 在粘菌中经历多泛素介导的降解，而人 H-Ras 和粘菌 H-Ras 同源物（RasC）对泛素化有抗性。我们的结果表明，粘菌中存在负载 GTP 的 K-Ras 同工型特异性降解系统，进一步鉴定负责的 E3 连接酶可能为治疗 K-Ras 突变型癌症提供新的治疗方法。
