Integrative Phosphoproteomic Characterization of ULK1 Phosphosites Reveals Co-Regulatory Networks in Autophagy and Metabolic Adaptation.

Unc-51-like autophagy-activating kinase 1 (ULK1), a serine/threonine kinase, is the central regulator of autophagy initiation and plays a crucial role in cellular homeostasis, metabolic adaptation, and stress responses. We performed a large-scale phosphoproteomic data analysis of ULK1 by systematically integrating and analyzing 891 profiling and 245 differential human phosphoproteomic datasets. S450, S623, and S638 emerged as the predominant phosphosites in ULK1, with the highest frequency of detection across multiple cellular phosphoproteomics datasets. Conservation analysis showed that these phosphosites are conserved across mammalian species and human ULK1 and ULK2 (Unc-51-like autophagy-activating kinase 2). The co-regulatory networks associated with these phosphosites, including interacting proteins, upstream kinases, and downstream substrates, were systematically analyzed. The phosphosite co-regulated ULK1 interactome revealed a network of associations linking ubiquitin-dependent cargo recognition to autophagosome biogenesis, trafficking, and maturation, indicating that ULK1 phosphoregulation may extend beyond its established role in autophagy initiation and be associated with multiple stages of selective autophagy. Notably, ULK1 phosphosites were linked to key metabolic enzymes in glycogen, sphingolipid, phospholipid, and glycolytic pathways, as well as classical lipolysis. Protein-normalized analysis across 12 cancer cohorts showed significant upregulation of the predominant ULK1 phosphosites, most frequently at S450 and S623. Comparative analysis with an independent ULK1/2 knockout phosphoproteomic dataset further supported the relevance of these identified co-regulated protein phosphosites. Collectively, this study provides a phosphosite-resolved view of ULK1 regulation and identifies candidate phosphosites and associated proteins for future functional and mechanistic studies.