[en] The cyclin-dependent kinase inhibitor p27Kip1 (p27) has been involved in promoting autophagy and survival in conditions of metabolic stress. While the signaling cascade upstream of p27 leading to its cytoplasmic localization and autophagy induction has been extensively studied, how p27 stimulates the autophagic process remains unclear. Here, we investigated the mechanism by which p27 promotes autophagy upon glucose deprivation. Mouse embryo fibroblasts (MEFs) lacking p27 exhibit a decreased autophagy flux compared to wild-type cells and this is correlated with an abnormal distribution of autophagosomes. Indeed, while autophagosomes are mainly located in the perinuclear area in wild-type cells, they are distributed throughout the cytoplasm in p27-null MEFs. Autophagosome trafficking towards the perinuclear area, where most lysosomes reside, is critical for autophagosome-lysosome fusion and cargo degradation. Vesicle trafficking is mediated by motor proteins, themselves recruited preferentially to acetylated microtubules, and autophagy flux is directly correlated to microtubule acetylation levels. p27-/- MEFs exhibit a marked reduction in microtubule acetylation levels and restoring microtubule acetylation in these cells, either by re-expressing p27 or with deacetylase inhibitors, restores perinuclear positioning of autophagosomes and autophagy flux. Finally, we find that p27 promotes microtubule acetylation by binding to and stabilizing α-tubulin acetyltransferase (ATAT1) in glucose-deprived cells. ATAT1 knockdown results in random distribution of autophagosomes in p27+/+ MEFs and impaired autophagy flux, similar to that observed in p27-/- cells. Overall, in response to glucose starvation, p27 promotes autophagy by facilitating autophagosome trafficking along microtubule tracks by maintaining elevated microtubule acetylation via an ATAT1-dependent mechanism.
Disciplines :
Biochemistry, biophysics & molecular biology
Author, co-author :
Nowosad, Ada ; MCD, Centre de Biologie Intégrative, Université de Toulouse, CNRS, UPS, 31062, Toulouse, Cedex, France
Creff, Justine; MCD, Centre de Biologie Intégrative, Université de Toulouse, CNRS, UPS, 31062, Toulouse, Cedex, France
Jeannot, Pauline; MCD, Centre de Biologie Intégrative, Université de Toulouse, CNRS, UPS, 31062, Toulouse, Cedex, France
Culerrier, Raphael; MCD, Centre de Biologie Intégrative, Université de Toulouse, CNRS, UPS, 31062, Toulouse, Cedex, France
Codogno, Patrice ; Institut Necker-Enfants Malades (INEM), INSERM U1151-CNRS UMR 8253, F-75015, Paris, France ; Université Paris, F-75005, Paris, France
Manenti, Stephane; Cancer Research Center of Toulouse (CRCT), INSERM U1037, CNRS ERL5294, University of Toulouse, Toulouse, France
Fondation ARC pour la Recherche sur le Cancer [FR]
Funding text :
The authors thank Raphael Culerrier for technical assistance with cloning. We are grateful to Maxence Nachury (Stanford University) and Jayanta Debnath (University of California San Francisco) for providing reagents. A.N. was supported by a studentship from the Fondation ARC pour la Recherche sur le Cancer. S.M. is supported by a grant from the Ligue Nationale Contre le Cancer. This project was supported by funds from the Ligue Nationale Contre le Cancer (# 2FI13911UPQV) and Fondation ARC pour la Recherche sur le Cancer (PJA 20171206148) to A.B. A.B. is supported by funds from the Fondation pour la Recherche Médicale (FRM Equipe EQU202103012639).A.N. was supported by a studentship from the Fondation ARC pour la Recherche sur le Cancer. S.M. is supported by a grant from the Ligue Nationale Contre le Cancer. This project was supported by funds from the Ligue Nationale Contre le Cancer (# 2FI13911UPQV) and Fondation ARC pour la Recherche sur le Cancer (PJA 20171206148) to A.B. A.B. is supported by funds from the Fondation pour la Recherche Médicale (FRM Equipe EQU202103012639).
Besson, A., Dowdy, S. F. & Roberts, J. M. CDK Inhibitors: cell cycle regulators and beyond. Developmental Cell 14, 159–169 (2008). DOI: 10.1016/j.devcel.2008.01.013
Fero, M. L., Randel, E., Gurley, K. E., Roberts, J. M. & Kemp, C. J. The murine gene p27Kip1 is haplo-insufficient for tumour suppression. Nature 396, 177–180 (1998). DOI: 10.1038/24179
Fero, M. L. et al. A syndrome of multiorgan hyperplasia with features of gigantism, tumorigenesis, and female sterility in p27(Kip1)-deficient mice. Cell 85, 733–744 (1996). DOI: 10.1016/S0092-8674(00)81239-8
Min, Y. H. et al. Cytoplasmic mislocalization of p27Kip1 protein is associated with constitutive phosphorylation of Akt or protein kinase B and poor prognosis in acute myelogenous leukemia. Cancer Res. 64, 5225–5231 (2004). DOI: 10.1158/0008-5472.CAN-04-0174
Liang, J. et al. PKB/Akt phosphorylates p27, impairs nuclear import of p27 and opposes p27-mediated G1 arrest. Nat. Med. 8, 1153–1160 (2002). DOI: 10.1038/nm761
Fujita, N., Sato, S., Katayama, K. & Tsuruo, T. Akt-dependent phosphorylation of p27Kip1 promotes binding to 14-3-3 and cytoplasmic localization. J. Biol. Chem. 277, 28706–28713 (2002). DOI: 10.1074/jbc.M203668200
Wander, S. A., Zhao, D. & Slingerland, J. M. p27: a barometer of signaling deregulation and potential predictor of response to targeted therapies. Clin. Cancer Res. 17, 12–18 (2011). DOI: 10.1158/1078-0432.CCR-10-0752
Chu, I. M., Hengst, L. & Slingerland, J. M. The Cdk inhibitor p27 in human cancer: prognostic potential and relevance to anticancer therapy. Nat. Rev. Cancer 8, 253–267 (2008). DOI: 10.1038/nrc2347
Besson, A., Gurian-West, M., Schmidt, A., Hall, A. & Roberts, J. M. p27Kip1 modulates cell migration through the regulation of RhoA activation. Genes Dev. 18, 862–876 (2004). DOI: 10.1101/gad.1185504
Besson, A., Assoian, R. K. & Roberts, J. M. Regulation of the cytoskeleton: an oncogenic function for CDK inhibitors? Nat. Rev. Cancer 4, 948–955 (2004). DOI: 10.1038/nrc1501
Baldassarre, G. et al. p27Kip1-stathmin interaction influences sarcoma cell migration and invasion. Cancer cell 7, 51–63 (2005). DOI: 10.1016/j.ccr.2004.11.025
Jeannot P. et al. p27(Kip1) promotes invadopodia turnover and invasion through the regulation of the PAK1/Cortactin pathway. Elife 6 10.7554/eLife.22207 (2017)
Besson, A. et al. Discovery of an oncogenic activity in p27Kip1 that causes stem cell expansion and a multiple tumor phenotype. Genes Dev. 21, 1731–1746 (2007). DOI: 10.1101/gad.1556607
Choudhury, S. et al. Molecular profiling of human mammary gland links breast cancer risk to a p27(+) cell population with progenitor characteristics. Cell Stem Cell 13, 117–130 (2013). DOI: 10.1016/j.stem.2013.05.004
Sun, X. et al. p27 Protein protects metabolically stressed cardiomyocytes from apoptosis by promoting autophagy. J. Biol. Chem. 289, 16924–16935 (2014). DOI: 10.1074/jbc.M113.542795
Su, M. et al. MicroRNA-221 inhibits autophagy and promotes heart failure by modulating the p27/CDK2/mTOR axis. Cell death Differ. 22, 986–999 (2015). DOI: 10.1038/cdd.2014.187
Liang, J. et al. The energy sensing LKB1-AMPK pathway regulates p27(kip1) phosphorylation mediating the decision to enter autophagy or apoptosis. Nat. Cell Biol. 9, 218–224 (2007). DOI: 10.1038/ncb1537
Campos, T. et al. Rheb promotes cancer cell survival through p27Kip1-dependent activation of autophagy. Mol. Carcinog. 55, 220–229 (2016). DOI: 10.1002/mc.22272
Campos, T. et al. Rapamycin requires AMPK activity and p27 expression for promoting autophagy-dependent Tsc2-null cell survival. Biochim. Biophys. Acta 1863, 1200–1207 (2016). DOI: 10.1016/j.bbamcr.2016.03.009
White, J. P. et al. The AMPK/p27(Kip1) axis regulates autophagy/apoptosis decisions in aged skeletal muscle stem cells. Stem Cell Rep. 11, 425–39 (2018). DOI: 10.1016/j.stemcr.2018.06.014
Nowosad, A. et al. p27 controls Ragulator and mTOR activity in amino acid-deprived cells to regulate the autophagy-lysosomal pathway and coordinate cell cycle and cell growth. Nat. Cell Biol. 22, 1076–90 (2020). DOI: 10.1038/s41556-020-0554-4
Levine, B. & Kroemer, G. Autophagy in the pathogenesis of disease. Cell 132, 27–42 (2008). DOI: 10.1016/j.cell.2007.12.018
White, E. The role for autophagy in cancer. J. Clin. Invest. 125, 42–46 (2015). DOI: 10.1172/JCI73941
Botti-Millet, J., Nascimbeni, A. C., Dupont, N., Morel, E. & Codogno, P. Fine-tuning autophagy: from transcriptional to posttranslational regulation. Am. J. Physiol. Cell Physiol. 311, C351–C362 (2016). DOI: 10.1152/ajpcell.00129.2016
Mackeh, R., Perdiz, D., Lorin, S., Codogno, P. & Pous, C. Autophagy and microtubules—new story, old players. J. Cell Sci. 126, 1071–1080 (2013). DOI: 10.1242/jcs.115626
Kast, D. J. & Dominguez, R. The cytoskeleton-autophagy connection. Curr. Biol. 27, R318–R26 (2017). DOI: 10.1016/j.cub.2017.02.061
Kimura, S., Noda, T. & Yoshimori, T. Dynein-dependent movement of autophagosomes mediates efficient encounters with lysosomes. Cell Struct. Funct. 33, 109–122 (2008). DOI: 10.1247/csf.08005
Ravikumar, B. et al. Dynein mutations impair autophagic clearance of aggregate-prone proteins. Nat. Genet. 37, 771–776 (2005). DOI: 10.1038/ng1591
Cardoso, C. M. et al. Depletion of kinesin 5B affects lysosomal distribution and stability and induces peri-nuclear accumulation of autophagosomes in cancer cells. PloS ONE 4, e4424 (2009). DOI: 10.1371/journal.pone.0004424
Jahreiss, L., Menzies, F. M. & Rubinsztein, D. C. The itinerary of autophagosomes: from peripheral formation to kiss-and-run fusion with lysosomes. Traffic 9, 574–587 (2008). DOI: 10.1111/j.1600-0854.2008.00701.x
Reed, N. A. et al. Microtubule acetylation promotes kinesin-1 binding and transport. Curr. Biol. 16, 2166–2172 (2006). DOI: 10.1016/j.cub.2006.09.014
Dompierre, J. P. et al. Histone deacetylase 6 inhibition compensates for the transport deficit in Huntington’s disease by increasing tubulin acetylation. J. Neurosci. 27, 3571–3583 (2007). DOI: 10.1523/JNEUROSCI.0037-07.2007
Kochl, R., Hu, X. W., Chan, E. Y. & Tooze, S. A. Microtubules facilitate autophagosome formation and fusion of autophagosomes with endosomes. Traffic 7, 129–145 (2006). DOI: 10.1111/j.1600-0854.2005.00368.x
Xie, R., Nguyen, S., McKeehan, W. L. & Liu, L. Acetylated microtubules are required for fusion of autophagosomes with lysosomes. BMC Cell Biol. 11, 89 (2010). DOI: 10.1186/1471-2121-11-89
McLendon, P. M. et al. Tubulin hyperacetylation is adaptive in cardiac proteotoxicity by promoting autophagy. Proc. Natl Acad. Sci. USA 111, E5178–E5186 (2014). DOI: 10.1073/pnas.1415589111
Esteves, A. R. et al. Acetylation as a major determinant to microtubule-dependent autophagy: relevance to Alzheimer’s and Parkinson disease pathology. Biochim. Biophys. Acta Mol. Basis Dis. 1865, 2008–23 (2019). DOI: 10.1016/j.bbadis.2018.11.014
Short, J. D. et al. AMP-activated protein kinase signaling results in cytoplasmic sequestration of p27. Cancer Res. 68, 6496–6506 (2008). DOI: 10.1158/0008-5472.CAN-07-5756
Mizushima, N., Yoshimori, T. & Levine, B. Methods in mammalian autophagy research. Cell 140, 313–326 (2010). DOI: 10.1016/j.cell.2010.01.028
Pampliega, O. et al. Functional interaction between autophagy and ciliogenesis. Nature 502, 194–200 (2013). DOI: 10.1038/nature12639
Tian, W. et al. An antibody for analysis of autophagy induction. Nat. Methods 17, 232–239 (2020). DOI: 10.1038/s41592-019-0661-y
Kishi-Itakura, C., Koyama-Honda, I., Itakura, E. & Mizushima, N. Ultrastructural analysis of autophagosome organization using mammalian autophagy-deficient cells. J. Cell Sci. 127, 4089–4102 (2014). DOI: 10.1242/jcs.156034
Noda, N. N., Fujioka, Y., Hanada, T., Ohsumi, Y. & Inagaki, F. Structure of the Atg12-Atg5 conjugate reveals a platform for stimulating Atg8-PE conjugation. EMBO Rep. 14, 206–211 (2013). DOI: 10.1038/embor.2012.208
Kimura, S., Noda, T. & Yoshimori, T. Dissection of the autophagosome maturation process by a novel reporter protein, tandem fluorescent-tagged LC3. Autophagy 3, 452–460 (2007). DOI: 10.4161/auto.4451
Short, J. D. et al. AMPK-mediated phosphorylation of murine p27 at T197 promotes binding of 14-3-3 proteins and increases p27 stability. Mol. Carcinog. 49, 429–439 (2010). DOI: 10.1002/mc.20613
Korolchuk, V. I. et al. Lysosomal positioning coordinates cellular nutrient responses. Nat. Cell Biol. 13, 453–460 (2011). DOI: 10.1038/ncb2204
Jia, R., Guardia, C. M., Pu, J., Chen, Y. & Bonifacino, J. S. BORC coordinates encounter and fusion of lysosomes with autophagosomes. Autophagy 13, 1648–63 (2017). DOI: 10.1080/15548627.2017.1343768
Filipek, P. A. et al. LAMTOR/Ragulator is a negative regulator of Arl8b- and BORC-dependent late endosomal positioning. J. Cell Biol. 216, 4199–215 (2017). DOI: 10.1083/jcb.201703061
Pu, J., Keren-Kaplan, T. & Bonifacino, J. S. A Ragulator-BORC interaction controls lysosome positioning in response to amino acid availability. J. Cell Biol. 216, 4183–97 (2017). DOI: 10.1083/jcb.201703094
Yu, L. et al. Termination of autophagy and reformation of lysosomes regulated by mTOR. Nature 465, 942–946 (2010). DOI: 10.1038/nature09076
Morelli, G. et al. p27(Kip1) modulates axonal transport by regulating alpha-tubulin acetyltransferase 1 stability. Cell Rep. 23, 2429–42 (2018). DOI: 10.1016/j.celrep.2018.04.083
Fass, E., Shvets, E., Degani, I., Hirschberg, K. & Elazar, Z. Microtubules support production of starvation-induced autophagosomes but not their targeting and fusion with lysosomes. J. Biol. Chem. 281, 36303–36316 (2006). DOI: 10.1074/jbc.M607031200
Godin Juliette, D. et al. p27Kip1 is a microtubule-associated protein that promotes microtubule polymerization during neuron migration. Dev. Cell 23, 729–744 (2012). DOI: 10.1016/j.devcel.2012.08.006
Perchey, R. T. et al. p27(Kip1) regulates the microtubule bundling activity of PRC1. Biochim. Biophys. Acta Mol. Cell Res. 1865, 1630–1639 (2018). DOI: 10.1016/j.bbamcr.2018.08.010
Hubbert, C. et al. HDAC6 is a microtubule-associated deacetylase. Nature 417, 455–458 (2002). DOI: 10.1038/417455a
Nagano, Y. et al. Siah1/SIP regulates p27(kip1) stability and cell migration under metabolic stress. Cell Cycle 10, 2592–2602 (2011). DOI: 10.4161/cc.10.15.16912
Dalvai, M., Schubart, K., Besson, A. & Matthias, P. Oct1 is required for mTOR-induced G1 cell cycle arrest via the control of p27(Kip1) expression. Cell Cycle 9, 3933–3944 (2010). DOI: 10.4161/cc.9.19.13154
Wang, J. et al. Phosphorylation at Ser10 triggered p27 degradation and promoted gallbladder carcinoma cell migration and invasion by regulating stathmin1 under glucose deficiency. Cell Signal 80, 109923 (2021). DOI: 10.1016/j.cellsig.2021.109923
Coats, S., Flanagan, W. M., Nourse, J. & Roberts, J. M. Requirement of p27Kip1 for restriction point control of the fibroblast cell cycle. Science 272, 877–880 (1996). DOI: 10.1126/science.272.5263.877
Fujimaki, K. et al. Graded regulation of cellular quiescence depth between proliferation and senescence by a lysosomal dimmer switch. Proc. Natl Acad. Sci. USA 116, 22624–34 (2019). DOI: 10.1073/pnas.1915905116
Geeraert, C. et al. Starvation-induced hyperacetylation of tubulin is required for the stimulation of autophagy by nutrient deprivation. J. Biol. Chem. 285, 24184–24194 (2010). DOI: 10.1074/jbc.M109.091553
Palazzo, A., Ackerman, B. & Gundersen, G. G. Cell biology: tubulin acetylation and cell motility. Nature 421, 230 (2003). DOI: 10.1038/421230a
Iwata, A., Riley, B. E., Johnston, J. A. & Kopito, R. R. HDAC6 and microtubules are required for autophagic degradation of aggregated huntingtin. J. Biol. Chem. 280, 40282–40292 (2005). DOI: 10.1074/jbc.M508786200
Guedes-Dias, P. et al. HDAC6 inhibition induces mitochondrial fusion, autophagic flux and reduces diffuse mutant huntingtin in striatal neurons. Biochim. Biophys. Acta 1852, 2484–2493 (2015). DOI: 10.1016/j.bbadis.2015.08.012
Gilardini Montani, M. S. et al. Histone deacetylase inhibitors VPA and TSA induce apoptosis and autophagy in pancreatic cancer cells. Cell Oncol. (Dordr.) 40, 167–80 (2017). DOI: 10.1007/s13402-017-0314-z
Lee, J.-Y. et al. HDAC6 controls autophagosome maturation essential for ubiquitin-selective quality-control autophagy. EMBO J. 29, 969–980 (2010). DOI: 10.1038/emboj.2009.405
Zhang, Y. et al. Mice lacking histone deacetylase 6 have hyperacetylated tubulin but are viable and develop normally. Mol. Cell. Biol. 28, 1688–1701 (2008). DOI: 10.1128/MCB.01154-06
Kuma, A., Komatsu, M. & Mizushima, N. Autophagy-monitoring and autophagy-deficient mice. Autophagy 13, 1619–28 (2017). DOI: 10.1080/15548627.2017.1343770
Sebti, S. et al. BAT3 modulates p300-dependent acetylation of p53 and autophagy-related protein 7 (ATG7) during autophagy. Proc. Natl Acad. Sci. USA 111, 4115–4120 (2014). DOI: 10.1073/pnas.1313618111
Huang, R. et al. Deacetylation of nuclear LC3 drives autophagy initiation under starvation. Mol. Cell 57, 456–466 (2015). DOI: 10.1016/j.molcel.2014.12.013
Lin, S. Y. et al. GSK3-TIP60-ULK1 signaling pathway links growth factor deprivation to autophagy. Science 336, 477–481 (2012). DOI: 10.1126/science.1217032
Ng, F. & Tang, B. L. Sirtuins’ modulation of autophagy. J. Cell Physiol. 228, 2262–2270 (2013). DOI: 10.1002/jcp.24399
Stankov, M. V. et al. Histone deacetylase inhibitors induce apoptosis in myeloid leukemia by suppressing autophagy. Leukemia 28, 577–588 (2014). DOI: 10.1038/leu.2013.264
Fullgrabe, J. et al. The histone H4 lysine 16 acetyltransferase hMOF regulates the outcome of autophagy. Nature 500, 468–471 (2013). DOI: 10.1038/nature12313
He, M. et al. MoSnt2-dependent deacetylation of histone H3 mediates MoTor-dependent autophagy and plant infection by the rice blast fungus Magnaporthe oryzae. Autophagy 14, 1543–61 (2018). DOI: 10.1080/15548627.2018.1458171
Akella, J. S. et al. MEC-17 is an alpha-tubulin acetyltransferase. Nature 467, 218–222 (2010). DOI: 10.1038/nature09324
Shida, T., Cueva, J. G., Xu, Z., Goodman, M. B. & Nachury, M. V. The major alpha-tubulin K40 acetyltransferase alphaTAT1 promotes rapid ciliogenesis and efficient mechanosensation. Proc. Natl Acad. Sci. USA 107, 21517–21522 (2010). DOI: 10.1073/pnas.1013728107
Vicinanza, M. et al. PI(5)P regulates autophagosome biogenesis. Mol. Cell 57, 219–234 (2015). DOI: 10.1016/j.molcel.2014.12.007
Cheong, H., Lindsten, T., Wu, J., Lu, C. & Thompson, C. B. Ammonia-induced autophagy is independent of ULK1/ULK2 kinases. Proc. Natl Acad. Sci. USA 108, 11121–11126 (2011). DOI: 10.1073/pnas.1107969108
Pena-Llopis, S. et al. Regulation of TFEB and V-ATPases by mTORC1. EMBO J. 30, 3242–3258 (2011). DOI: 10.1038/emboj.2011.257
Li, L. et al. A TFEB nuclear export signal integrates amino acid supply and glucose availability. Nat. Commun. 9, 2685 (2018). DOI: 10.1038/s41467-018-04849-7
Musiwaro, P., Smith, M., Manifava, M., Walker, S. A. & Ktistakis, N. T. Characteristics and requirements of basal autophagy in HEK 293 cells. Autophagy 9, 1407–1417 (2013). DOI: 10.4161/auto.25455
Wei, Y. et al. The stress-responsive kinases MAPKAPK2/MAPKAPK3 activate starvation-induced autophagy through Beclin 1 phosphorylation. Elife 4, e05289 (2015). DOI: 10.7554/eLife.05289
Nguyen, L. et al. p27kip1 independently promotes neuronal differentiation and migration in the cerebral cortex. Genes Dev. 20, 1511–1524 (2006). DOI: 10.1101/gad.377106
Serres, M. P. et al. p27(Kip1) controls cytokinesis via the regulation of citron kinase activation. J. Clin. Invest. 122, 844–858 (2012). DOI: 10.1172/JCI60376
N’Diaye, E. N. et al. PLIC proteins or ubiquilins regulate autophagy-dependent cell survival during nutrient starvation. EMBO Rep. 10, 173–179 (2009). DOI: 10.1038/embor.2008.238
