Guzman Javier Rivera, Fukuda Seiji, Pelus Louis M
Department of Microbiology & Immunology and the Walther Oncology Center, Indiana University School of Medicine, Indianapolis, IN, 46202 USA.
Gene Ther Mol Biol. 2009;13B:264-273.
The anti-apoptotic protein Survivin and the cyclin-dependent kinase p34Cdc2 regulate cell cycle progression and apoptosis. p34Cdc2 activation is required for its pro-apoptotic activity and phosphorylation of p34Cdc2 at Tyrosine-15 (Tyr15) maintains p34Cdc2 in an inactive state. In BaF3 IL-3-dependent murine hematopoietic cells, over-expression of wild-type (wt)-Survivin increased Tyrosine phosphorylation of p34Cdc2, while over-expression of dominant-negative (dn) T34A-Survivin decreased Tyr15 phosphorylation. The increased phospho-Tyr15 levels associated with ectopic wt-Survivin directly correlated with enhanced BaF3 cell survival upon growth factor withdrawal, while conversely, low phospho-Tyr15 levels and decreased survival were seen in BaF3 cells expressing ectopic dn-Survivin. Tyrosine-15 phosphorylation of p34Cdc2 is mediated by the Wee1 Kinase, a known target of caspase-3. In BaF3 cells over-expressing wt-Survivin, 2-fold higher levels of Wee1 protein were detected compared to cells expressing vector or dn-Survivin. Treatment of control vector-transduced BaF3 cells with the selective caspase-3 inhibitor Ac-DEVD-CHO increased p34Cdc2-Tyr15 phosphorylation and Wee1 protein levels. In a similar fashion, over-expression of wt-Survivin maintained high levels of phospho-Tyr15-p34Cdc2 and Wee1 protein. Since Survivin requires Hsp90 for stability, we treated cells with the Hsp90 inhibitors AICAR and 17-AAG to further link Survivin to blocking p34Cdc2 activation. Treatment of BaF3 cells expressing ectopic wt-Survivin with AICAR or 17-AAG significantly reduced p34Cdc2-Tyr15 phosphorylation compared to vehicle-treated controls. These results suggest that Survivin protects the p34Cdc2-Tyr15-targeting kinase Wee1 from degradation by blocking caspase-3 activation leading to inhibition of the pro-apoptotic function of p34Cdc2 and enhanced cell survival.
抗凋亡蛋白Survivin和细胞周期蛋白依赖性激酶p34Cdc2调节细胞周期进程和细胞凋亡。p34Cdc2的激活是其促凋亡活性所必需的，并且p34Cdc2在酪氨酸-15（Tyr15）位点的磷酸化使其维持在无活性状态。在依赖白细胞介素-3的BaF3小鼠造血细胞中，野生型（wt）-Survivin的过表达增加了p34Cdc2的酪氨酸磷酸化，而显性负性（dn）T34A-Survivin的过表达则降低了Tyr15磷酸化。与异位wt-Survivin相关的磷酸化Tyr15水平升高与生长因子撤除后BaF3细胞存活率提高直接相关，相反，在表达异位dn-Survivin的BaF3细胞中观察到低磷酸化Tyr15水平和存活率降低。p34Cdc2的酪氨酸-15磷酸化由已知的caspase-3靶点Wee1激酶介导。在过表达wt-Survivin的BaF3细胞中，与表达载体或dn-Survivin的细胞相比，检测到的Wee1蛋白水平高2倍。用选择性caspase-3抑制剂Ac-DEVD-CHO处理对照载体转导的BaF3细胞可增加p34Cdc2-Tyr15磷酸化和Wee1蛋白水平。以类似的方式，wt-Survivin的过表达维持了高水平的磷酸化Tyr15-p34Cdc2和Wee1蛋白。由于Survivin的稳定性需要热休克蛋白90（Hsp90），我们用Hsp90抑制剂AICAR和17-AAG处理细胞，以进一步将Survivin与阻断p34Cdc2激活联系起来。与载体处理的对照相比，用AICAR或17-AAG处理表达异位wt-Survivin的BaF3细胞可显著降低p34Cdc2-Tyr15磷酸化。这些结果表明，Survivin通过阻断caspase-3激活保护靶向p34Cdc2-Tyr15的激酶Wee1不被降解，从而导致p34Cdc2的促凋亡功能受到抑制并增强细胞存活。
