Ceramide kinase/ceramide 1-phosphate signaling regulates LC3B expression and autophagosome formation.

Ceramide kinase (CerK) generates ceramide 1-phosphate (C1P), a bioactive sphingolipid involved in diverse cellular responses, but its role in autophagy is not fully understood. Here, we examined whether the CerK/C1P pathway regulates LC3B expression and autophagosome formation in HeLa cells. Proteomics analysis of cerebellum from Cerk-KO mice identified reduced levels of multiple autophagy-related proteins. In HeLa cells, genetic ablation, siRNA-mediated knockdown, and pharmacological inhibition of CerK consistently reduced LC3B-II levels. This effect was reversed by extracellular C1P and by re-expression of wild-type, but not kinase-dead, CerK, indicating that CerK-generated C1P is required for maintenance of LC3B-II. LC3B-II levels remained lower in CERK-KO cells in the presence of bafilomycin A1, and two-step flux analysis showed that disruption of the CerK/C1P pathway preferentially impaired the LC3B-associated autophagosome formation parameter. MAP1LC3B mRNA and Nrf2 protein levels were reduced in CERK-KO cells, and pharmacological activation of Nrf2 tended to restore MAP1LC3B mRNA levels and significantly increased LC3B-II protein levels. Finally, loss of the CerK/C1P pathway enhanced nutrient starvation-induced apoptotic responses and loss of viability. Together, these results identify the CerK/C1P pathway as a positive lipid signaling mechanism that maintains LC3B expression, supports LC3B-associated autophagosome formation, and promotes cell survival under nutrient-deprived conditions.