Identification of Kinase-Mediated Signaling Pathways Regulating Autophagy in Response to Cellular Stresses

Abstract

Autophagy is a conserved and highly regulated catabolic process essential for cellular homeostasis, particularly under stress conditions such as nutrient starvation and infection. While bulk autophagy facilitates the indiscriminate degradation of cytosolic components during starvation, selective autophagy targets specific organelles and intracellular pathogens for turnover. A critical component of autophagy is the ATG16L1-containing E3-like enzyme, which drives autophagosome formation. Polymorphisms in ATG16L1, such as the Crohn's disease-associated T300A variant (caATG16L1), have been linked to autophagy dysfunction. Our findings demonstrate that ULK1 kinase directly phosphorylates ATG16L1 in response to infection and starvation, facilitating its localization to bacterial entry sites and promoting anti-bacterial autophagy. However, ULK1-mediated phosphorylation of caATG16L1 leads to its destabilization under stress, highlighting a dual role of ULK1 signaling - enhancing wild-type ATG16L1 function while compromising the stability of its disease-associated variant.

We next sought to further dissect the kinase-mediated regulation of selective autophagy targeting specific organelles. We proposed that selective autophagy may be regulated by distinct upstream signaling from starvation-induced autophagy to promote organelle turn-over. To investigate this hypothesis, we developed a customized kinome-wide CRISPR screening platform designed for the parallel interrogation of multiple stress-induced autophagy pathways. Utilizing a reporter cell line expressing DsRed-IRES-GFP-p62, we were able to monitor basal autophagy, starvation-induced autophagy, ER-phagy, and pexophagy. The Brunello kinome library was employed to maximize on-target gene disruption while minimizing off-target effects, enhancing the overall robustness of the screen. Through this approach, we identified both known and novel kinases involved in autophagy regulation, including condition-specific regulators such as CDK11A and NME3 for ER-phagy, and PAN3 and CDC42BPG for pexophagy.

Overall, our study emphasizes the central role of kinase signaling in coordinating selective autophagy. We demonstrate that ULK1 differentially affects autophagic outcomes depending on the ATG16L1 allele, revealing an additional layer of regulatory complexity. Additionally, the datasets generated from our optimized screening strategy provide valuable insight into the distinct regulatory networks governing bulk versus selective autophagy, offering a foundation for future mechanistic and therapeutic studies.