Role of PTEN during Pi(4,5)P2 homeostasis and autophagy

自噬，即自食的过程，从酵母到哺乳动物在进化上都是保守的。这一过程导致溶酶体降解细胞器、蛋白质或核酸等胞质成分。自噬开始于双膜囊的形成，双膜囊扩展并形成自噬体，可以吞噬细胞质成分。自噬体随后与释放消化酶的溶酶体融合以促进货物降解。自噬调节亚细胞区室的动态重塑，参与控制胚胎发生、饥饿反应、抗肿瘤发生和抗衰老等生理过程。因此，了解这一重要过程对于基础研究和生物医学研究都是至关重要的。****我们对控制磷酸肌肽循环的候选基因进行了果蝇细胞筛选，发现肌醇5-磷酸酶dOCRL控制PI(4,5)P2稳态（Ben El Kadhi et al., Current biology 2011）。大部分PI(4,5)P2集中在质膜上，在那里它参与几乎所有涉及细胞表面的事件。我们证明了dOCRL与核内体和溶酶体相关，并且它使溶酶体上的PI(4,5)P2去磷酸化，从而在质膜上限制该磷酸肌肽。当dOCRL被RNAi敲低时，细胞异常地在巨溶酶体表面积累PI(4,5)P2。此外，我们最近报道了PTEN激活通过激活膜上的磷脂酶C （PLC）酶来促进其酶产物PI(4,5)P2的水解。我们证明该函数可以挽救dOCRL丢失（Ben El Kadhi et al., in prep）。****在我们未发表的观察中，我们发现dOCRL的消耗会导致自噬通量的缺陷。溶酶体不能再与自噬体融合。重要的是，我们发现激活PTEN可以恢复dOCRL缺失细胞的自噬通量。我们还发现该功能独立于PTEN酶活性，但由包含其两个非酶保守结构域的最小嵌合体支持。此外，我们发现PTEN的缺失促进了Pi(4,5)P2在细胞膜上的积累。***除了通过抑制PI3K/Akt/mTORC1通路在自噬激活中发挥典型作用外，我们的研究结果表明PTEN在控制自噬通量方面具有新的作用，不依赖于其酶活性，并通过调节溶酶体上的Pi(4,5)P2水平。****为了验证这一假设，我们将描述PTEN- PLC信号通路（目的1），并描述该通路在Pi(4,5)P2稳态和自噬中的作用（目的2）。***

Autophagy, the process of self-eating, is evolutionarily conserved from yeast to mammals. This process leads to the lysosomal degradation of cytosolic components such as organelles, proteins or nucleic acids. Autophagy starts with the formation of a double-membrane sac that expends and forms the autophagosome that can engulf cytosolic components. The autophagosome then fuse with lysosomes that discharge their digestive enzymes to promote cargo degradation. Autophagy regulates the dynamic remodeling of subcellular compartments and participates to the control of several physiological processes such as embryogenesis, response to starvation, anti-tumorigenesis and anti-senescence. Therefore, the understanding of this important process is essential for both fundamental and biomedical research.****We conducted an in cellulo Drosophila screen on candidate genes controlling the phosphoinositide cycle and we found that the inositol 5-phosphatase dOCRL controls PI(4,5)P2 homeostasis (Ben El Kadhi et al., Current biology 2011). The majority of the PI(4,5)P2 is concentrated at the plasma membrane where it participates in nearly all events that involve the cell surface. We demonstrated that dOCRL is associated with endosomes and lysosomes and that it dephosphorylates PI(4,5)P2 on lysosomes to restrict this phosphoinositide at the plasma membrane. When dOCRL is knocked-down by RNAi, cells abnormally accumulate PI(4,5)P2 at the surface of giant lysosomes. In addition, we recently reported that PTEN activation promotes the hydrolysis of PI(4,5)P2, its own enzymatic product, by activating Phospholipase C (PLC) enzymes on endomembranes. We showed that this function can rescue dOCRL loss (Ben El Kadhi et al., in prep).****In our unpublished observations we showed that depletion of dOCRL leads to a defect in the autophagic flux. Lysosomes cannot longer fuse with autophagosome. Importantly we showed that activation PTEN activation can restore the autophagic flux in dOCRL depleted cells. We also discovered that this function was independent of PTEN enzymatic activity but was supported by a minimal chimera encompassing two of its non-enzymatic conserved domains. In addition we found that PTEN depletion promotes accumulation of Pi(4,5)P2 on endomembranes. ***Besides its canonical role in autophagy activation by inhibiting the PI3K/Akt/mTORC1 pathway, our results suggest a novel role of PTEN in controlling the autophagic flux, independently of its enzymatic activity and through regulation of Pi(4,5)P2 levels on lysosomes.****To test this hypothesis we we will characterize the PTEN- PLC signaling pathway (aim 1) and we will characterize the role of this pathway on Pi(4,5)P2 homeostasis and autophagy (aim 2).***