Dual phosphorylation of glycogen synthase kinase 3β differentially integrates metabolic programs to determine T cell immunity across vertebrates

The integration of metabolic programs with T cell signaling establishes a molecular foundation for immune metabolism. As a key metabolic regulator, GSK3β’s activity is dynamically modulated by phosphorylation at Ser9 and Tyr216. However, the contribution of these phosphorylation sites on metabolism-driven T cell response remains unclear. Using tilapia and mouse models, we investigated the regulation of GSK3β on T cell metabolism and its evolutionary variation. In tilapia, T cell activation induces GSK3β signaling, linking to both glycolysis and oxidative phosphorylation (OXPHOS). Tyr216 phosphorylation preferentially promotes glycolysis, facilitating T cell activation, proliferation, and antibacterial immunity; while inhibition of Ser9 phosphorylation specifically enhances OXPHOS to sustain T cell responses. Differently, Tyr216 phosphorylation supports both glycolysis and OXPHOS in mouse, ensuring CD4⁺ T and CD8⁺ T cell activation, proliferation, and cytokine production. Although Ser9 phosphorylation controls OXPHOS, its inhibition impairs rather than enhances OXPHOS and CD4⁺ T cell responses in mouse. We thus revealed a previously unknown mechanism underlying T cell metabolism and proposed that, through evolution, GSK3β has restructured the regulatory strategy, enabling bidirectional control of T cell metabolism and immunity in mammals and enhancing the flexibility of the adaptive immune system.