Phosphoregulation of Mitochondrial Survivin in Cancer Cells

惠特利实验室十多年来一直致力于癌症相关蛋白survivin的研究。这种迷人的小蛋白质对有丝分裂和细胞分裂至关重要，但也能防止细胞死亡（1,2）。它在所有癌症中都过表达，其丰度与患者预后不良、对常规化疗和放疗的耐药性以及转移有关。磷脂酰乙醇胺（PE）是一种对膜结构至关重要的磷脂：它通过其小的头基团促进正曲率。因此，在卵裂沟的质膜（PM）处暴露PE对细胞分裂的完成至关重要，其在线粒体嵴中的高表达控制着线粒体的完整性，最终影响细胞的代谢需求和细胞对凋亡刺激的反应(3)。PE也是自噬的关键调节因子，自噬是细胞在应激条件下启动的循环途径(4)。一开始，自噬是一种促进生存的机制，但如果持续不减，最终会导致细胞死亡。在动物细胞中，80%的PE是在线粒体中通过磷脂酰丝氨酸（PS）的脱羧合成的，磷脂酰丝氨酸是从内质网输入的。PS向PE的转化是由线粒体内膜生物磷脂酰丝氨酸脱羧酶（PSD）介导的，如果PSD缺失，PS/PE比值升高，线粒体形态受到干扰，影响生长速度、代谢和凋亡(3)。最近我们发现survivin与癌细胞线粒体中的PSD直接相互作用(5)。我们的初步数据表明，它是PE产生的负调节因子，我们假设生存素通过限制PE的可用性来改变膜结构。通过这种方式，survivin的表达可以影响许多基本过程，包括细胞分裂、线粒体健康、代谢重编程，并且可能能够防止细胞因过度自噬而死亡。这一新颖的分子见解表明，survivin明显的多重和不同的作用可能在这个蛋白质-脂质界面上有根本的联系。这一假说为研究survivin的功能及其如何导致癌症和其他代谢紊乱提供了一个全新的视角。在本项目中，我们将研究磷酸化（cdk1/p38）对survivin-PSD相互作用的调节。在初步工作中，我们已经将survivin和PSD之间的相互作用定位到survivin的杆状病毒凋亡重复抑制剂（BIR）结构域和PSD的脱羧酶结构域(5)。在BIR结构域中有一个独特的cdk1位点（T34），我们实验室之前的研究表明，表达T34突变体的细胞生长缓慢，对凋亡具有高度抗性，并且对照射不敏感(6)。此外，通过电子显微镜，我们发现线粒体完整性在这些细胞中受到损害。因此，本项目的目标是研究cdk1/p38磷酸化T34对survivin-PSD相互作用、PE产生的影响，并记录其在细胞分裂、代谢、对凋亡刺激（包括辐射）的反应和过度自噬方面的下游后果。

The Wheatley lab has been working for over a decade on the cancer-associated protein survivin. This fascinating little protein is essential for mitosis and cell division, but also prevents cells from dying (1,2). It is overexpressed in all cancers and its abundance correlates with poor patient prognosis, resistance to conventional chemotherapy and radiation, and metastasis.Phosphotidylethanolamine (PE) is a phospholipid that is critical to membrane architecture: it facilitates positive curvature by virtue of its small head group. Consequently PE exposure at the plasma membrane (PM) of the cleavage furrow is critical for completion of cell division, and its high expression in mitochondrial cristae governs mitochondrial integrity, ultimately impacting on the metabolic needs of the cell, and cellular response to apoptotic stimuli (3). PE is also a critical regulator of autophagy, the recycling pathway initiated by cells during stressful conditions (4). At its outset autophagy is a pro-survival mechanism, but if it continues unabated, it ultimately causes cell death. In animal cells 80% of the de novo pool of PE is synthesised in the mitochondria by decarboxylating phosphatidylserine (PS), which is imported from the endoplasmic reticulum. Conversion of PS to PE is mediated by the inner mitochondrial membrane resident, phosphatidylserine decarboxylase (PSD), and if PSD is depleted, the PS/PE ratio rises, mitochondrial morphology becomes perturbed, with growth rate, metabolism and apoptosis affected (3).Recently we discovered that survivin interacts directly with PSD in the mitochondria of cancer cells (5). Our initial data suggest that it is a negative regulator of PE production, and we hypothesise that survivin alters membrane architecture by limiting PE availability. In this manner survivin expression can influence many fundamental processes, including cell divisio, mitochondrial health, metabolic reprogamming, and may be able to prevent cells from dying through excessive autophagy. This novel molecular insight suggests that the apparently mutliple and disparate roles of survivin, may be fundamentally linked at this protein-lipid interface. This hypothesis opens up a completely new perspective on survivin function and how it contributes to cancer and other metabolic disorders.In this project, the regulation of survivin-PSD interaction by phosphorylation (cdk1/p38) will be investigated. In preliminary work we have mapped the interaction between survivin and PSD to the baculovirus inhibitor of apoptosis repeat (BIR) domain of survivin and the decarboxylase domain of PSD (5). Within the BIR domain there is a unique cdk1 site (T34), and previous work from our lab has shown that cells expressing T34 mutants, grow slowly, are highly resistant to apoptosis and are insentive to irradiation (6). Moreover, by electron microscopy we have discovered that mitochondrial integrity is compromised in these cells. Thus the goal of this project is to investigate the impact cdk1/p38 phosphorylation of T34 on survivin-PSD interaction, PE production and to document the downstream consequences in terms of cell division, metabolism, response to apoptotic stimuli including radiation, and excessive autophagy.