P27 Regulates the Autophagy-Lysosomal Pathway Via the Control of Ragulator and Mtor
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bioRxiv preprint doi: https://doi.org/10.1101/2020.01.07.896860; this version posted January 7, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 1 p27 regulates the autophagy-lysosomal pathway via the control of Ragulator and mTOR 2 activity in amino acid deprived cells 3 4 Ada Nowosad1, Pauline Jeannot1, Caroline Callot1, Justine Creff1, Renaud T. Perchey1, Carine 5 Joffre2, Patrice Codogno3,4, Stephane Manenti2 and Arnaud Besson1,5 6 7 1LBCMCP, Centre de Biologie Intégrative, Université de Toulouse, CNRS, UPS, 31062 8 Toulouse Cedex, France. 9 2Cancer Research Center of Toulouse (CRCT), INSERM U1037, CNRS ERL5294, University of 10 Toulouse, Toulouse, France. 11 3Institut Necker-Enfants Malades (INEM), INSERM U1151-CNRS UMR 8253, F-75993 Paris, 12 France. 13 4Université Paris Descartes-Sorbonne Paris Cité, F-75005 Paris, France. 14 5Corresponding author 15 16 Correspondence to: 17 Arnaud Besson 18 LBCMCP UMR5088 CNRS - Université Paul Sabatier, 19 Batiment 4R3b1 20 118 route de Narbonne 21 31062 Toulouse cedex 9, France 22 Email: [email protected] 23 Tel: +33 (0)561558486 24 25 26 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.07.896860; this version posted January 7, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 2 27 Summary 28 Autophagy is a catabolic process whereby cytoplasmic components are degraded within 29 lysosomes, allowing cells to maintain energy homeostasis during nutrient depletion. Several 30 studies have shown that the CDK inhibitor p27Kip1 promotes starvation-induced autophagy. 31 However, the underlying mechanism remains unknown. Here, we report that in amino acid 32 deprived cells, p27 controls autophagy via an mTORC1-dependent mechanism. During 33 prolonged amino acid starvation, a fraction of p27 is recruited to lysosomes where it interacts 34 with LAMTOR1, a component of the Ragulator complex required for mTORC1 lysosomal 35 localization and activation. p27 binding to LAMTOR1 prevents Ragulator assembly and function 36 and subsequent mTORC1 activation, thereby promoting autophagy. Conversely, upon amino 37 acid withdrawal, p27-/- cells exhibit elevated mTORC1 signaling, impaired lysosomal activity 38 and autophagy, and resistance to apoptosis. This is associated with sequestration of TFEB in the 39 cytoplasm, preventing the induction of lysosomal genes required for lysosomal function. 40 Silencing of LAMTOR1 or mTOR inhibition restores autophagy and induces apoptosis in p27-/- 41 cells. Together, these results reveal a direct, coordinated regulation between the cell cycle and 42 cell growth machineries. 43 44 45 46 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.07.896860; this version posted January 7, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 3 47 INTRODUCTION 48 In all organisms, cell growth is coupled to cell division to allow normal development and 49 maintain homeostasis. How cells coordinate the machinery that regulates cell growth, at the heart 50 of which lies the mTOR kinase, with the machinery that controls cell division, driven by 51 cyclin/CDK complexes, has been the subject of considerable interest for several decades1. This 52 coordination has been mostly studied under normal metabolic conditions and except for notable 53 exceptions, such as early embryonic development, it appears that growth control drives the 54 activity of the cell cycle machinery. Here we investigated this question in conditions of 55 metabolic restriction to determine if the cell cycle machinery could in turn regulate the growth 56 control machinery. 57 p27Kip1 (p27) was initially identified as a cyclin/CDK inhibitor2, 3. Due to its ability to induce 58 cell cycle arrest, p27 acts as a tumor suppressor and p27-/- mice display multiple organ 59 hyperplasia and spontaneously develop pituitary tumors4. Nevertheless, in contrast to classic 60 tumor suppressors such as p53 or Rb, inactivating mutations of the p27 gene are extremely rare 61 and its inactivation in cancer is rather caused by enhanced degradation, attenuated transcription 62 or translation, or mislocalization in the cytoplasm2, 3, 5. The latter correlates with poor prognosis 63 in a variety of cancers, suggesting a direct contribution of cytoplasmic p27 to tumor progression2, 64 3. In fact, knock-in mice in which p27 is largely sequestered in the nucleus due to defective 65 export to the cytoplasm (p27S10A) are partially resistant to tumorigenesis6. Conversely, another 66 knock-in model in which p27 cannot bind to cyclin-CDKs (p27CK-) revealed that p27 can act as 67 an oncogene, as these mice display an increased susceptibility to both spontaneous and induced 68 tumorigenesis compared to p27-/- mice7, 8. How exactly p27 acts as an oncogene remains elusive, 69 but could be due to the regulation of several other cellular processes by p27. Indeed, p27 has 70 been involved in the control, sometimes in a CDK-independent manner, of cell migration and 71 invasion, differentiation, cytokinesis, transcriptional repression, apoptosis and autophagy2, 9-15. 72 Autophagy is a catabolic process by which intracellular components are degraded and 73 recycled by the lysosomal machinery16, 17. Depending on the pathway implicated in the delivery 74 of cargo for degradation, there are three major types of autophagy: macroautophagy (hereafter 75 referred to as autophagy), microautophagy and chaperone-mediated autophagy17. Autophagic 76 degradation begins with formation of double-membrane autophagosomes that engulf the 77 cytoplasmic material destined for elimination. Autophagosomes then fuse with the endocytic bioRxiv preprint doi: https://doi.org/10.1101/2020.01.07.896860; this version posted January 7, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 4 78 compartment and eventually deliver their cargo to lysosomes for degradation by lysosomal 79 enzymes17, 18. Following proteolysis, the resulting molecules are released back to the cytosol 80 through lysosomal permeases for reuse19. Although autophagy occurs constantly in cells and is 81 required to control the quality of cytoplasm, its levels dramatically increase in stress situations, 82 such as nutrient withdrawal, and particularly under amino acid (aa) deprivation20. In these 83 conditions, autophagy recycles cellular components and the products of proteolysis are delivered 84 back to the cytoplasm, where they are used as energy source or as building blocks for protein 85 synthesis17. 86 Autophagy is negatively modulated by mTORC1, a master regulator of cell growth21, 22. 87 mTORC1 is a multi-protein complex consisting of mTOR and its regulatory binding partners 88 Raptor, MLST8, PRAS40 and Deptor22. The main function of mTORC1 is to orchestrate the 89 balance between anabolic and catabolic metabolism. Via a complex network of nutrient sensors, 90 in presence of aa, mTORC1 is recruited to lysosomal membranes by Rag GTPases, themselves 91 anchored by the Ragulator complex, where the kinase activity of mTORC1 can be stimulated by 92 the GTPase Rheb that also resides on lysosomes23-26. When nutrients are abundant, mTORC1 93 promotes protein synthesis via phosphorylation of substrates implicated in the regulation of 94 translation, such as S6 kinases and 4E-binding proteins. At the same time, mTORC1 inhibits 95 autophagy at multiple levels by phosphorylating and inactivating proteins involved in 96 autophagosome formation (such as ULK1, AMBRA1, Atg13 and Atg14) and maturation 97 (UVRAG)21, 22, 27, 28. mTORC1 also phosphorylates TFEB, a transcription factor that drives the 98 expression of many pro-autophagic and lysosomal genes, on S142 and S211, resulting in TFEB 99 cytoplasmic retention, thereby inhibiting its activity29-31. Conversely, under nutrient shortage, 100 mTOR is inactivated, allowing the autophagic machinery to form autophagosomes. During 101 prolonged aa starvation, partial mTORC1 reactivation triggers the reformation of lysosomes 102 from autolysosomes via a process called autophagic lysosome reformation (ALR)32. Thus, the 103 role of mTORC1 in autophagy is complex and context dependent: while mTORC1 inhibition is 104 required for autophagy initiation, its cyclic reactivation by autophagy-generated nutrients allows 105 maintaining autophagy during prolonged starvation by restoring the lysosomal population. 106 Cytoplasmic p27 was recently described as a positive regulator of basal and starvation- 107 induced autophagy and to protect cells in conditions of metabolic stress from apoptosis by 108 promoting autophagy14, 33-37. Glucose or serum deprivation activates the energy/nutrient sensing bioRxiv preprint doi: https://doi.org/10.1101/2020.01.07.896860; this version posted January 7, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 5 109 kinases LKB1 and AMPK, in turn AMPK phosphorylates p27 on S83, T170 and T198, causing 110 its stabilization and cytoplasmic retention14, 36, 38. Loss of Tsc2, a subunit of Tuberous Sclerosis 111 Complex that acts as a GTPase activating protein (GAP) for Rheb, also