高通量识别非经典蛋白激酶-M2型丙酮酸激酶的磷酸化底物并系统阐明其在促肿瘤发生发展中的机制

PKM2 (M2 pyruvate kinase) is a critical factor that contributes to aerobic glycolysis (Warburg effect). Increasing evidence demonstrates that aberrated expression of PKM2 is involved in reprogramming metabolic process and promoting tumor cell proliferation. PKM2 undergoes conformational conversion between tetramer and dimer. The tetrameric form possesses high pyruvate kinase activity and plays a crucial metabolic role in shifting glucose metabolism in favor of growth of cancer cells. In contrast, the dimeric form of PKM2 contributes to tumorigenesis in a "non-metabolic" way. It acts as a transcriptional co-factor to regulate gene transcription, or functions as a protein kinase to directly phosphorylate substrates involved in different signaling pathways. Recently, using protein-protein interaction proteomics to study the role of PKM2 in response to DNA damage, we found that PKM2 forms complex with some proteins related to DNA damage response (DDR). Moreover, we showed that PKM2 acts as a protein kinase to promote DNA damage signaling by directly binding and phohsphorylating histone H2AX at serine 139 in the nucleus. These findings reveal the new role of PKM2 in DDR. Based on these original findings, we plan to systematically identify substrates phosphorylated by non-canonical protein kinase-M2 pyruvate kinase in response to different stimuli using quantitative phosphoproteomics. These studies will shed new insights for the mechanism by which PKM2 modulates tumorigenicity and malignancy.

M2型丙酮酸激酶（M2 pyruvate kinase，PKM2）是导致肿瘤细胞发生Warburg效应的关键因子。越来越多的证据表明高表达的PKM2通过重新编译代谢进程及调节信号转导过程促进肿瘤细胞的增殖。高丙酮酸激酶活性的四聚体PKM2具有代谢调节作用；而二聚体PKM2作为转录共因子，或蛋白激酶发挥非代谢调节作用。最近，本课题组利用相互作用蛋白质组学技术，发现在DNA损伤的过程中，PKM2可以和一系列DNA损伤相关蛋白形成复合体。进一步研究发现，核内PKM2可以直接磷酸化组蛋白2的139位丝氨酸位点，从而提示了PKM2作为蛋白激酶在应对DNA损伤中的新功能。基于上述重要发现，本项目试图利用定量磷酸化蛋白质组学技术，高通量的识别在不同刺激因素作用下非经典蛋白激酶-PKM2的特异磷酸化底物，以期从一个全新的角度为深入研究PKM2在肿瘤发生发展的作用机制提供重要线索，推动新的学科生长点的形成。

M2型丙酮酸激酶作为糖酵解过程中一步重要的限速步骤，通过重新编译代谢进程及调节信号转导过程促进肿瘤细胞的增殖。然而，PKM2在肿瘤发生、发展中的作用尚未了解清楚。本课题组利用磷酸化蛋白质组学技术，选到了一个在DNA损伤过程中的新的PKM2蛋白激酶的底物，H2AX，并利用质谱解析出其磷酸化位点，生物学实验则证实了PKM2通过磷酸化H2AX促进了肿瘤细胞基因组的不稳定性；利用亚细胞-相互作用蛋白质组学技术，发现核内PKM2可以直接与P53、AML1-ETO相互作用，并最终证实了PKM2-P53-P21的信号通路在DNA受损的细胞发生周期非阻滞效应的作用和强化了PKM2作为促癌基因的理论依据，也提示了PKM2可能作为肿瘤两次打击学说的第二次打击导致的肿瘤发生。利用化学蛋白质组学技术，发现了新的药物靶标。综上所述，利用本课题的资助，我们深入阐明了PKM2代谢酶在肿瘤生长过程中发挥的非经典激酶功能，PKM2可以赋予肿瘤细胞生长优势从而避免凋亡, 这些结果为深入研究PKM2在肿瘤发生发展的作用机制提供重要线索，同时为确证PKM2成为肿瘤细胞的一个重要的药物靶点，夯实了细胞生物、分子生物等理论基础。