Phosphoproteomics identifies pathways underlying the role of receptor-interaction protein kinase 3 in alcohol-associated liver disease and uncovers apoptosis signal-regulating kinase 1 as a target.

Receptor-interaction protein kinase 3 (RIP3), a critical determinant of the necroptotic pathway of programmed cell death, contributes to injury in murine models of alcohol-associated liver disease (ALD); however, the underlying mechanisms are unknown. We investigated the effect of chronic ethanol feeding on the hepatic phosphoproteome in C57BL/6 and RIP3-deficient (Rip3-/- ) mice, focusing on death receptor (DR) signaling pathways. C57BL/6 and Rip3-/- mice were fed an ethanol-containing liquid diet or pair-fed control diet. A label-free mass spectrometry-based approach identified differentially phosphorylated proteins that were mapped to pathways affected by ethanol and Rip3 genotype. Identified targets were validated in both the murine model of ALD and in liver tissue from patients with alcohol-associated hepatitis (AH) and healthy controls. Chronic ethanol dysregulated hepatic tumor necrosis factor-induced DR signaling pathways. Of particular importance, chronic ethanol feeding to C57BL/6 mice decreased the phosphorylation of apoptosis signal-regulating kinase 1 (ASK1) at serine (S)1036/S1040 (S1029/S1033 human), sites linked with the inhibition of ASK1 death-promoting activity. This decrease in phosphorylation of inhibitory sites was muted in Rip3-/- mice. Decreased phosphorylation at S1033 was also lower in liver of patients with severe AH compared to healthy controls, and phosphorylation at the ASK1 activation site (threonine [Thr]-838) was increased in patients with AH. The net impact of these changes in phosphorylation of ASK1 was associated with increased phosphorylation of p38, a downstream target of ASK1, in patients with AH and C57BL/6 but not Rip3-/- mice. Similarly, chronic ethanol feeding affected the c-Jun N-terminal kinase pathway in C57BL/6 but not Rip3-/- mice. Taken together, our data indicate that changes in inhibitory phosphorylation of ASK1 are an important target in ALD and suggest the involvement of noncanonical functions of Rip3 in ALD.