Lysine crotonylation regulates leucine-deprivation-induced autophagy by a 14-3-3ε-PPM1B axis

Lysine crotonylation as a protein post-translational modification regulates diverse cellular processes and functions. However, the role of crotonylation in nutrient signaling pathways remains unclear. Here, we find a positive correlation between global crotonylation levels and leucine-deprivation-induced autophagy. Crotonylome profiling identifies many crotonylated proteins regulated by leucine deprivation. Bioinformatics analysis dominates 14-3-3 proteins in leucine-mediated crotonylome. Expression of 14-3-3ε crotonylation-deficient mutant significantly inhibits leucine-deprivation-induced autophagy. Molecular dynamics analysis shows that crotonylation increases molecular instability and disrupts the 14-3-3ε amphipathic pocket through which 14-3-3ε interacts with binding partners. Leucine-deprivation-induced 14-3-3ε crotonylation leads to the release of protein phosphatase 1B (PPM1B) from 14-3-3ε interaction. Active PPM1B dephosphorylates ULK1 and subsequently initiates autophagy. We further find that 14-3-3ε crotonylation is regulated by HDAC7. Taken together, our findings demonstrate that the 14-3-3ε-PPM1B axis regulated by crotonylation may play a vital role in leucine-deprivation-induced autophagy. [Display omitted] •Global lysine crotonylation and autophagy levels are enhanced by leucine deprivation•Crotonylome profiling identifies many proteins regulated by leucine deprivation•14-3-3ε crotonylation affects its binding to PPM1B, which is a phosphatase for ULK1 Zheng et al. find the link between lysine crotonylation and leucine-deprivation-induced autophagy. Leucine deprivation induces crotonylation of 14-3-3ε protein, which releases PPM1B to dephosphorylate ULK1 and activate autophagy. The crotonylation-regulated 14-3-3ε-PPM1B axis is critical for the initiation of autophagy.