Autophagy regulates the localization and degradation of p16^INK4a

Abstract

The tumor suppressor protein p16 INK4a (p16) is a well-established hallmark of aging that induces cellular senescence in response to stress. Previous studies have focused primarily on p16 regulation at the transcriptional level; comparatively little is known about the protein's intracellular localization and degradation. The autophagy-lysosomal pathway has been implicated in the subcellular trafficking and turnover of various stressresponse proteins, but it is unclear whether p16 is involved in these pathways. Here, we investigate the role of autophagy, vesicular trafficking, and lysosomal degradation on p16 expression and localization in human epithelial cells. Time-lapse fluorescence microscopy using an endogenous p16-mCherry reporter revealed that autophagy induced by genotoxic stress stimulates rapid p16 recruitment to acidic cytoplasmic vesicles. When vesicular acidification was inhibited by NH4Cl, nuclear p16 levels increased. Single-cell imaging revealed that p16 localizes to lysosomes upon stress, implicating the autophagy pathway as a regulator of p16 localization. Blocking autophagy with bafilomycin, chloroquine, or NH4Cl resulted in elevated p16 protein levels without increased transcription. Increased p16 coincided with accumulation of autophagosome chaperone p62/SQSTM1 (p62) and decreased levels of phosphorylated-Rb. Furthermore, chloroquine caused p16 aggregation within stalled vesicles containing autophagosome marker LC3, demonstrating that p16 is transported and degraded by the autophagylysosomal pathway. Knockdown of p62 resulted in delocalization of p16 aggregates to autophagosomes, suggesting that p16 is targeted to these vesicles by p62. Taken together, these results implicate the autophagy pathway as a novel regulator of p16 degradation and localization, which could play a role in the etiology of cancer and agerelated diseases.

Cross-talk between redox signalling and protein aggregation

“…A third mechanism by which cells can clear and recycle cellular components is by autophagy. Many types of protein aggregates are cleared by autophagy, including τ, Aβ, α-synuclein, huntingtin, SOD1 and p16 INK4A [38][39][40][41][42][43], with autophagy defects leading to neurodegenerative disease [44,45].…”

CDKN2A/p16INK4a suppresses hepatic fatty acid oxidation through the AMPKα2-SIRT1-PPARα signaling pathway

“…Interestingly, p16 protein levels were increased in IHH cells in the refeeding experiment ( Figure 4D-E), suggesting that p16 is regulated post-translationally. A recent report demonstrated that p16 is degraded by autophagy (17). Accordingly, rapamycin treatment for 8h in IHH cells led to a strong decrease of p16 protein ( Figure 4F), suggesting that autophagy-mediated p16 degradation likely links nutritional status to p16 activity in hepatocytes.…”

“…We observed that p16 protein level is increased in IHH cells during refeeding conditions, without modulation of p16 mRNA expression by fasting. In line, p16 protein was recently demonstrated to be degraded by autophagy (17), a process induced by fasting and inhibited by refeeding (31,32). Moreover, partial deletion of ATG5 in mice, resulting in inhibition of autophagy, increased p16 protein levels in the liver (33).…”

Prognostic significance of p16, p21, and Ki67 expression at the invasive front of colorectal cancers

“…Besides promoter regulation, p16 is degraded by protease in the nucleus in lung cancers 32 and by autophagy in the cytosol of neurons 33 . Preliminary data revealed that FAM111, a protease responsible for p16 degradation in lung cancer 32 was expressed in CRCs (data not shown); p16 expression may be regulated by these pathways.…”