ATP-independent phosphate recycling on AGC kinase activation loops induced by alkali metal ions

Changes in extracellular Na ⁺ and K ⁺ concentrations have traditionally been considered to influence intracellular signal transduction through alterations in cell volume or membrane potential. However, whether intracellular ion concentration changes directly regulate signaling molecules, independent of these conventional pathways, remains largely unexplored. In this study, we demonstrate that even in the absence of cellular membranes, an increase in Na ⁺ or K ⁺ concentration rapidly reduces activation-loop phosphorylation of multiple AGC kinases, including PKN, PKCζ/λ, and p70 S6 kinase. When ion concentrations were reduced, the activation-loop phosphorylation, which had initially decreased, recovered within a short period. Notably, this recovery occurred in the absence of PDK1, a known kinase responsible for the phosphorylation of these activation loops, and did not require ATP or Mg ²⁺ in lysate assays. ³² P tracing experiments revealed a novel 'reacquisition of phosphate group' mechanism, in which phosphate groups transiently dissociate from the activation loops under high Na ⁺ or K ⁺ conditions and are subsequently re-incorporated into the activation loops when ion concentrations are reduced. These findings indicate that elevated Na ⁺ or K ⁺ concentrations directly and rapidly reduce the activity of multiple AGC kinases, and that the activity rapidly recovers upon reduction in ion concentrations, through an unconventional phosphate transfer mechanism distinct from canonical protein phosphorylation reactions. Our study suggests the existence of a robust phosphorylation homeostasis mechanism independent of conventional kinase-phosphatase systems, providing new insights into signaling pathways regulated by intracellular Na ⁺ /K ⁺ ion dynamics.