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 stress‐response proteins and has also been shown to attenuate age‐related pathologies, but it is unclear whether p16 is involved in this pathway. 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 serum starvation, etoposide, and hydrogen peroxide stimulate autophagy and drive p16 recruitment to acidic cytoplasmic vesicles within 4 hr. Blocking lysosomal proteases with leupeptin and ammonium chloride resulted in the accumulation of p16 within lysosomes and increased total p16 levels suggesting that p16 is degraded by this pathway. Furthermore, autophagy blockers chloroquine and bafilomycin A1 caused p16 aggregation within stalled vesicles containing autophagosome marker LC3. Increase of p16 within these vesicles coincided with the accumulation of LC3‐II. Knockdown of autophagosome chaperone p62 attenuated the formation of p16 aggregates in lysosomes, 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 age‐related diseases.

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.

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