Phosphorylation of SPHK2 by AMPK couples energy stress to nuclear S1P signaling in breast cancer

AMP-activated protein kinase (AMPK) orchestrates cellular responses to energy stress, yet its nuclear substrates remain incompletely characterized. Using quantitative phosphoproteomics on nuclear extracts from AMPKα1/α2 double-knockout and control myoblasts, we identified sphingosine kinase 2 (SPHK2) as a direct AMPK substrate at Ser399. This phosphorylation relocalizes SPHK2 from membranes to cytosolic/nuclear pools, limits autophagy-lysosome degradation and stabilizes the protein, elevating nuclear sphingosine‑1‑phosphate (S1P). CRISPR‑engineered S399A knock‑in breast cancer cells exhibit reduced SPHK2 and nuclear S1P, transcriptional shifts toward stress responses, and impaired proliferation, xenograft growth and lung colonization. In human breast tumors, SPHK2 protein correlates positively with p-AMPK (Thr172), and elevated SPHK2 mRNA associates with poorer survival. Combining metformin with SPHK2 inhibitor K145 produces synergistic tumor suppression dependent on Ser399. Here, we show that an AMPK-SPHK2 axis couples energy stress to nuclear S1P signaling and tumor progression, nominating SPHK2 co-targeting to improve AMPK-activating therapies.