Ceramide kinase/ceramide 1-phosphate signaling regulates LC3B expression and autophagosome formation.
Funou Hideki, Arai Natsuka, Nakajima Shimon, Kadowaki Ryo, Nakamaura Kenta, Uzu Miaki, Honda Takuya, Nakamura Hiroyuki
Abstract
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.
Key Findings
- Ceramide kinase (CerK) and its product ceramide 1-phosphate (C1P) regulate LC3B expression and autophagosome formation in HeLa cells.
- Loss of CerK/C1P pathway reduces LC3B-II levels and impairs autophagosome formation, which is partially restored by Nrf2 activation.
- Disruption of CerK/C1P signaling enhances nutrient starvation-induced apoptosis and decreases cell viability.
Clinical Significance
The study highlights the CerK/C1P pathway as a critical regulator of autophagy and cell survival under nutrient stress, suggesting potential therapeutic targets for diseases involving oxidative stress and impaired autophagy.
Citation
Funou Hideki, Arai Natsuka, Nakajima Shimonet al.. Ceramide kinase/ceramide 1-phosphate signaling regulates LC3B expression and autophagosome formation. Journal of lipid research. 2026-Jun-12.