Bonvini P, Nguyen P, Trepel J, Neckers L M
Department of Cell and Cancer Biology, Medicine Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.
Oncogene. 1998 Mar 5;16(9):1131-9. doi: 10.1038/sj.onc.1201625.
N-myc is a short-lived transcription factor, frequently amplified in human neuroblastomas. The ubiquitin-proteasome system is involved in the degradation of many short-lived cellular proteins and previous studies have shown that ubiquitin-dependent proteolysis is implicated in the turn-over of N-myc in vitro. However, calpain has also been implicated in N-myc degradation in vitro. Here we report that, in vivo, N-myc is a sensitive substrate for the 26S proteasome in N-myc amplified neuroblastoma cells. We observed that inhibition of the 26S proteasome with two inhibitors, ALLnL and lactacystin, led to an elevation of the N-myc protein steady-state and increased N-myc protein polyubiquitination, as revealed by ubiquitin Western blotting. Pulse-chase experiments have shown that the increased N-myc levels resulted from stabilization of the protein. In contrast treatment with several calpain and cathepsin inhibitors failed to block N-myc degradation in vivo. Furthermore, fluorescence microscopy of ALLnL-treated cells localized N-myc exclusively to the nuclear compartment, suggesting the absence of a requirement for transport to the cytoplasm prior to degradation.
N - myc是一种寿命短暂的转录因子，在人类神经母细胞瘤中经常发生扩增。泛素 - 蛋白酶体系统参与许多寿命短暂的细胞蛋白的降解，先前的研究表明，泛素依赖性蛋白水解在体外与N - myc的周转有关。然而，钙蛋白酶在体外也与N - myc的降解有关。在此我们报告，在体内，N - myc是N - myc扩增的神经母细胞瘤细胞中26S蛋白酶体的敏感底物。我们观察到，用两种抑制剂ALLnL和乳胞素抑制26S蛋白酶体，导致N - myc蛋白稳态升高，并增加了N - myc蛋白的多聚泛素化，这通过泛素蛋白质印迹法得以揭示。脉冲追踪实验表明，N - myc水平的增加是由于蛋白质的稳定化。相反，用几种钙蛋白酶和组织蛋白酶抑制剂处理未能在体内阻断N - myc的降解。此外，对ALLnL处理细胞的荧光显微镜观察显示，N - myc仅定位于核区室，这表明在降解之前不需要转运到细胞质中。
