Wall N R, Mohammad R M, Reddy K B, Al-Katib A M
Division of Hematology and Oncology, Department of Internal Medicine, Karmanos Cancer Institute at Wayne State University School of Medicine, Detroit, MI 48201, USA.
Int J Mol Med. 2000 Feb;5(2):165-71. doi: 10.3892/ijmm.5.2.165.
The ubiquitin-mediated proteolytic system has been implicated in the turnover of a number of intracellular proteins. In the present study, we investigated the novelty and potential role of bryostatin 1, a macrocyclic lactone isolated from the marine bryozoan, Bugula neritina, in inducing the ubiquitin-mediated proteolysis of the oncoprotein Bcl-2. Immunoprecipitation and immunoblotting analyses revealed that Bcl-2 is ubiquitinated following exposure of the acute lymphoblastic leukemia (ALL) cell line Reh to 1 nM bryostatin 1. Bcl-2 protein rapidly decreases to 50% of that recorded in the control after 24 h of bryostatin 1 treatment. In the subsequent 24 h, Bcl-2 protein again rapidly decreases to 6% of its pre-bryostatin 1 level at which time a plateau is reached and maintained for another 72 h. Furthermore, ubiquitin-Bcl-2 conjugates are detected in untreated as well as bryostatin 1 treated cells, indicating that ubiquitin-dependent proteolysis plays a role in the normal turnover of Bcl-2. However, ubiquitin-Bcl-2 conjugates increase in a time-dependent manner following bryostatin 1 treatment. Lactacystin, which inhibits the proteinase activities of the proteasome, inhibited the bryostatin 1-induced decrease of Bcl-2 protein. The effect of bryostatin 1 on the proteolytic efficiency of the 26S proteasome in Reh cell extracts was also investigated and shown to increase following 1 h of bryostatin 1 treatment. Proteolytic activity reached its highest point by 3 h, and subsequently returned to control levels by 12 h, post-bryostatin 1 treatment. In addition, bryostatin 1 treatment of the Reh cell line decreased expression of bcl-2 mRNA within 3 h. However, bcl-2 mRNA expression returned after 24 h. We speculate that this decrease in mRNA together with increased 26S proteolytic activity accounts for the initial rapid decrease recorded in Bcl-2 protein. These findings indicate that bryostatin 1 treatment of Reh ALL cells decreases Bcl-2 expression through two processes: a) enhanced Bcl-2 protein degradation through the activation of the ubiquitin-proteasome pathway and b) decreased bcl-2 mRNA expression.
泛素介导的蛋白水解系统与多种细胞内蛋白质的周转有关。在本研究中，我们调查了从海洋苔藓虫类动物红苔藓虫中分离出的大环内酯类化合物苔藓抑素1在诱导泛素介导的癌蛋白Bcl-2蛋白水解方面的新颖性和潜在作用。免疫沉淀和免疫印迹分析显示，急性淋巴细胞白血病（ALL）细胞系Reh暴露于1 nM苔藓抑素1后，Bcl-2会发生泛素化。苔藓抑素1处理24小时后，Bcl-2蛋白迅速降至对照组记录水平的50%。在随后的24小时内，Bcl-2蛋白再次迅速降至其苔藓抑素1处理前水平的6%，此时达到平台期并维持另外72小时。此外，在未处理以及经苔藓抑素1处理的细胞中均检测到泛素-Bcl-2缀合物，这表明泛素依赖性蛋白水解在Bcl-2的正常周转中起作用。然而，苔藓抑素1处理后，泛素-Bcl-2缀合物呈时间依赖性增加。抑制蛋白酶体蛋白酶活性的乳胞素抑制了苔藓抑素1诱导的Bcl-2蛋白减少。我们还研究了苔藓抑素1对Reh细胞提取物中26S蛋白酶体蛋白水解效率的影响，结果显示在苔藓抑素1处理1小时后其活性增加。蛋白水解活性在3小时时达到最高点，随后在苔藓抑素1处理后12小时恢复到对照水平。此外，用苔藓抑素1处理Reh细胞系3小时内bcl-2 mRNA表达下降。然而，24小时后bcl-2 mRNA表达恢复。我们推测，mRNA的这种下降以及26S蛋白水解活性的增加导致了Bcl-2蛋白最初记录到的快速下降。这些发现表明，用苔藓抑素1处理Reh ALL细胞通过两个过程降低Bcl-2表达：a）通过激活泛素-蛋白酶体途径增强Bcl-2蛋白降解，b）降低bcl-2 mRNA表达。
