Park D S, Morris E J, Bremner R, Keramaris E, Padmanabhan J, Rosenbaum M, Shelanski M L, Geller H M, Greene L A
Neuroscience Research Institute, University of Ottawa, Ottawa, Ontario, K1H 8M5, Canada.
J Neurosci. 2000 May 1;20(9):3104-14. doi: 10.1523/JNEUROSCI.20-09-03104.2000.
Neuronal death evoked by DNA damage requires cyclin-dependent kinase 4 (Cdk4) and 6 activity and is accompanied by elevation of cyclin D1-associated kinase activity. Because Cdk4/6 phosphorylates retinoblastoma protein (pRb) family members that then modulate the transcriptional activity of E2F/DP1 complexes, we examined the involvement of these components in DNA damage-evoked neuronal death. Camptothecin induced rapid pRb and p107 phosphorylation at a Cdk4/6 phosphorylation site followed by selective loss of Rb and p107. The CDK inhibitor flavopiridol suppressed pRb and p107 phosphorylation and loss, implicating CDK activity in these events. Moreover, the loss of pRb and p107 appeared to be mediated by caspases because it was blocked by general caspase inhibitors. The role of phosphorylation and pRb and p107 loss in the death pathway was indicated by observations that virally mediated expression of pRb mutated at sites of phosphorylation, including the Cdk4/6 site, inhibited death. Finally, expression of dominant-negative versions of DP1, known to compromise E2F transcriptional activity, protects cortical neurons from death induced by camptothecin and sympathetic neurons from death evoked by UV treatment. Taken together, these results implicate the CDK-pRb/E2F/DP pathway as a required element in the neuronal death evoked by DNA damage.
DNA损伤引发的神经元死亡需要细胞周期蛋白依赖性激酶4（Cdk4）和6的活性，并伴有细胞周期蛋白D1相关激酶活性的升高。由于Cdk4/6使视网膜母细胞瘤蛋白（pRb）家族成员磷酸化，进而调节E2F/DP1复合物的转录活性，我们研究了这些成分在DNA损伤引发的神经元死亡中的作用。喜树碱诱导pRb和p107在Cdk4/6磷酸化位点快速磷酸化，随后Rb和p107选择性缺失。CDK抑制剂黄酮哌啶醇抑制pRb和p107的磷酸化及缺失，提示CDK活性参与了这些事件。此外，pRb和p107的缺失似乎是由半胱天冬酶介导的，因为它被通用的半胱天冬酶抑制剂所阻断。磷酸化以及pRb和p107缺失在死亡途径中的作用通过以下观察得以体现：病毒介导的在包括Cdk4/6位点在内的磷酸化位点发生突变的pRb的表达抑制了细胞死亡。最后，已知会损害E2F转录活性的DP1显性负性变体的表达，可保护皮质神经元免受喜树碱诱导的死亡，并保护交感神经元免受紫外线处理引发的死亡。综上所述，这些结果表明CDK-pRb/E2F/DP途径是DNA损伤引发的神经元死亡中必需的一个要素。
