GSK-3 磷酸化并稳定Bim 的机制及其在神经元凋亡中的作用

Bim, a BH3-only protein, plays a critical role during neuronal apoptosis. We have reported the transcriptional mechanism of Bim during neuronal apoptosis（J Neurosci, 2011）. Recently, we also found that in addition to transcriptional up-regulation, the stability of Bim protein was also increased during neuronal apoptosis. Stability is an important decisive factor of Bim protein level. However, the mechanism underlying Bim stability regulation remains unkown. We had demonstrated GSK-3, a pro-apoptotic kinase, mediates the stability of Bim protein. The further data showed that GSK-3 phosphorylates Bim directly and PKA pre-treatment enhances Bim phosphorylation by GSK-3. Based on these observations, we propose the possible mechanism: taken PKA as a primed kinase, GSK-3 phosphorylates Bim directly, increases Bim stability and then mediates neuronal apoptosis. The purpose of this project is to identify the exact phosphorylated site of Bim by PKA/GSK-3, clarify the mechanism of phosphorylation-regulated the stability of Bim using the methods of phosphorylated site-directed mutation, kinase assay and transgenic mice. We propose to demonstrate the role the Bim phosphorylation by GSK-3 during neuronal apoptosis, providing scientific basis for establishment of therapeutic target for neurodegenerative diseases.

BH3-only家族蛋白Bim在神经元凋亡中起重要作用。我们已报道了bim的转录上调机制（J Neurosci，2011）。最近我们发现：除了转录上调，凋亡时Bim蛋白的稳定性显著增加，这是维持Bim上调的重要因素，但机制不清楚。文献提示并经我们证实，促凋亡激酶GSK-3介导了Bim蛋白的稳定性。进一步发现GSK-3可直接磷酸化Bim，且PKA预处理可有效增强GSK-3对Bim的磷酸化。由此，我们提出假设：以PKA作为预激酶，GSK-3进一步磷酸化Bim，从而促进Bim稳定并介导了神经元凋亡。本项目拟采用磷酸化位点突变（S82、S86、S90）、激酶分析和转基因鼠等手段，旨在鉴定PKA/GSK-3磷酸化Bim的位点，阐明磷酸化调控Bim稳定性的机制，评价GSK-3增加Bim蛋白稳定性这一事件在神经元凋亡中的作用，为寻找神经退行性疾病治疗新靶标提供科学依据。

中脑黑质多巴胺能神经元(Dopaminergic neuron, DAN)进行性死亡是帕金森病(Parkinson’s disease, PD)的主要病理特征。我们及其他实验室均证明糖原合成酶激酶-3(Glycogen synthase kinase-3, GSK-3)是介导DAN死亡的关键激酶，但其发挥促死效应的机制及底物不清。我们前期结果证明，GSK-3可以上调BH3-only促凋亡蛋白Bim。为了提升研究的意义，我们拟在MPTP诱导的PD动物模型中验证这个发现。在本项目资助下，我们引进了GSK-3两个亚型(α和β)的条件性敲除小鼠，在MPTP诱导的PD模型中首次证明，只有β亚型有促死作用。进一步实验结果显示，GSK-3β通过转录后上调c-Jun激活Bim表达，促进DAN死亡。本项目首次在PD模型中区分GSK-3两个亚型对DAN死亡的差异作用，并证实GSK-3β通过c-Jun上调Bim，进而促进DAN死亡，为揭示PD的发病机制及确立新治疗靶点提供科学依据。