Autophagic flux: Is p62 a good indicator?

By Christina Towers, PhD

Is p62 a good indicator of autophagic flux? The short answer: Yes … but … SQSTM1 encodes the cargo adaptor protein, p62, which interacts with autophagic substrates and delivers them to autophagosomes for degradation. In the process, p62 is itself degraded and when autophagy is induced, a corresponding decrease in p62 levels is observed. Congruently, when autophagy is potently inhibited, i.e. genetic knock out of ATG core autophagy proteins like ATG5 or ATG7, p62 accumulates in the cell. These changes can be observed by immunofluorescent staining, western blotting, and immunohistochemistry in the case of intact tissue samples.

p62 is a versatile adaptor protein with multiple functions

Interestingly, p62 itself is thought to be dispensable for canonical autophagy. In contrast to almost all of the core ATG proteins whose loss in mice results in embryonic or neonatal lethality, the only adverse phenotype displayed in p62-/- mice is mature-onset obesity¹. A number of recent publications have shown that p62 is a multifunctional scaffolding protein that interacts with a variety of proteins to regulate diverse processes including apoptosis and other forms of cell death like necroptosis, as well as redox state via regulation of the KEAP1-NRF2 pathway. p62 has also been shown to interact with ERK1 to regulate adipogenesis and protein kinase C (PKC zeta) to regulate NF-κB signaling².

Always consider alternative assays to monitor autophagic flux

These diverse roles suggest that homeostatic regulation may affect p62 levels independent of autophagy. For this reason, it is important to validate changes in autophagic flux as measured by p62 levels, with additional assays. Most commonly, conjugated levels of LC3, known as LC3-II, can be monitored by western blot in the presence of a lysosomal inhibitor such as bafilomycin-A1. Lysosomal delivery of specific autophagic cargo can also be monitored with the use of tandem tagged proteins that contain a pH stable mCherry fluorophore and a pH sensitive GFP fluorophore.

An advantage to monitoring p62 to measure autophagic flux is that lysosomal inhibitors are not necessary, because unlike LC3-II, p62 does not usually increase when autophagy is induced. However, changes in p62 can often be subtle compared to LC3-II flux, probably because of additional mechanisms of regulation. For these reasons, it is important to always use multiple assays to measure autophagy flux³.