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

自噬，自食的过程，是从酵母到哺乳动物进化保守。该过程导致细胞溶质组分如细胞器、蛋白质或核酸的溶酶体降解。自噬开始于双膜囊的形成，该双膜囊扩张并形成可以吞噬胞质组分的自噬体。然后自噬体与溶酶体融合，溶酶体释放其消化酶以促进货物降解。自噬调节亚细胞区室的动态重构，并参与控制多种生理过程，如胚胎发生、饥饿反应、抗肿瘤和抗衰老。因此，理解这一重要过程对于基础研究和生物医学研究都至关重要。 我们对控制磷酸肌醇循环的候选基因进行了细胞内果蝇筛选，我们发现肌醇5-磷酸酶dOCRL控制PI（4，5）P2稳态（Ben El Kadhi等人，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去磷酸化以限制质膜上的磷酸肌醇。当通过RNAi敲低dOCRL时，细胞在巨大溶酶体表面异常积累PI（4，5）P2。此外，我们最近报道了PTEN激活通过激活内膜上的磷脂酶C（PLC）酶来促进PI（4，5）P2（其自身的酶产物）的水解。我们表明该功能可以挽救dOCRL损失（Ben El Kadhi等人，准备中）。 在我们未发表的观察中，我们表明dOCRL的耗尽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激活可以恢复dOCRL缺失细胞中的自噬通量。我们还发现，这种功能是独立的PTEN酶活性，但支持的最小嵌合体，包括两个非酶保守结构域。此外，我们发现PTEN缺失促进Pi（4，5）P2在内膜上的积累。 除了通过抑制PI 3 K/Akt/mTORC 1通路在自噬激活中的典型作用外，我们的结果表明PTEN在控制自噬通量中的新作用，其独立于其酶活性并通过调节溶酶体上的Pi（4，5）P2水平。 为了检验这一假设，我们将表征PTEN-PLC信号传导途径（目的1），并且我们将表征该途径对P1（4，5）P2稳态和自噬的作用（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).