PFKP facilitates Atg4B phosphorylation during amino acid deprivation-induced autophagy

Abstract Atg4B facilitates autophagy by promoting autophagosome maturation through the reversible lipidation and delipidation of LC3. Recent reports have shown that phosphorylation of Atg4B regulates its activity and LC3 processing, leading to modulate autophagy activity. However, the mechanism about how Atg4B phosphorylation is regulated by amino acid deprivation is unclear. Here, we combined the tandem affinity purification with mass spectrometry (MS) to identify the Atg4B-interacting proteins including its well-known partner gamma-aminobutyric acid receptor-associated protein (GABARAP, a homolog of LC3) and phosphofructokinase 1 platelet isoform (PFKP). Further immunoprecipitation assays showed that amino acid deprivation strengthened the interaction between Atg4B and PFKP. By genetic depletion of PFKP using CRISPR/Cas9, we uncovered that PFKP KO reduced degradation of LC3-II and p62 due to a partial block in autophagic flux. MS analysis of Flag-tagged Atg4B identified phosphorylation of Atg4B serine 34 residue (S34) and PFKP serine 386 residue (S386) under amino acid deprivation condition. In vitro kinase assay validated that PFKP functioning as a protein kinase phosphorylated Atg4B at S34. This phosphorylation could enhance Atg4B activity and p62 degradation. In addition, PFKP S386 phosphorylation abundance correlates with Atg4B S34 phosphorylation abundance and autophagy in HEK293T cells. In brief, our findings described that PFKP, a rate-limiting enzyme in the glycolytic pathway, functions as a protein kinase for Atg4B to regulate Atg4B activity and autophagy under amino acid deprivation condition.