Abstract
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.
Highlights
The cyclin-dependent kinase inhibitor p27Kip[1] (p27) has been involved in promoting autophagy and survival in conditions of metabolic stress
Revealed that LAMTOR1 silencing increased the peripheral distribution of autophagosomes in p27+/+ cells, as previously reported[47,48], but had no effect on autophagosome localization in p27−/− cells (Fig. S3D, E). These results indicate that p27 regulates autophagosome positioning in a LAMTOR1-independent manner in glucosedeprived cells
While Trichostatin A (TSA) treatment did not affect autophagy rate in full medium (Fig. S5A, B, D, G), suggesting that microtubule acetylation is dispensable for basal autophagy, TSA rescued impaired autophagosome maturation and autophagy flux in p27−/− cells in glucose-deprivation conditions (Fig. S5A, C, E, H), without significant effect in p27+/+ Mouse embryo fibroblasts (MEFs) in which tubulin acetylation levels are already elevated
Summary
P27 promotes autophagy during glucose deprivation p27 was previously reported to promote autophagy and cell survival in response to glucose and/or serum deprivation[17]. While TSA treatment did not affect autophagy rate in full medium (Fig. S5A, B, D, G), suggesting that microtubule acetylation is dispensable for basal autophagy, TSA rescued impaired autophagosome maturation and autophagy flux in p27−/− cells in glucose-deprivation conditions (Fig. S5A, C, E, H), without significant effect in p27+/+ MEFs in which tubulin acetylation levels are already elevated. Consistent with the positive effect of TSA on autophagosome maturation, we observed a marked reduction in p62 levels in p27−/− MEFs, indicative of a restored autophagy flux (Fig. S6) Taken together, these data suggest that p27 regulates autophagy flux via a MT acetylation-dependent mechanism in glucosedeprived cells. These results indicate that in glucose-deprived cells, p27 promotes autophagy by controlling autophagosome positioning via ATAT1-dependent MT acetylation
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