Dual Phosphorylation of Thr175 and Ser176 Is Essential for SnRK1α1 Activation.

SnRK1 protein kinases play a pivotal role in regulating plant development, growth signaling, and stress responses by managing cellular responses to energy fluctuations. SnRK1 activation was thought to depend mainly on the phosphorylation of threonine at position 175 (Thr175) within the activation loop. However, recent phosphoproteomic studies have identified additional phosphorylation sites. We explored the functional significance of these modifications, focusing on serine at position 176 (Ser176), adjacent to Thr175 in SnRK1α1. Our results reveal that dual phosphorylation of Ser176 and Thr175 is vital for optimal SnRK1 activity. Structural modeling and thermodynamic analyses highlight the critical role of these modifications in optimising substrate positioning and enzymatic efficiency. Furthermore, only the wild-type SnRK1α1, which can be phosphorylated at both sites, retains full functionality in in vivo experiments with yeast and Arabidopsis. Interestingly, pSer176 exhibits greater stability than pThr175 at various times throughout the day. Mutant proteins with substitutions at these sites (T175A/S176A mutants) accumulate in cytoplasmic aggregates after heat shock, suggesting a strong link between phosphorylation status, protein stability, and SnRK1 degradation pathways.