Fluctuation in -GlcNAcylation inactivates STIM1 to reduce store-operated calcium ion entry via down-regulation of Ser phosphorylation.

Stromal interaction molecule 1 (STIM1) plays a pivotal role in store-operated Ca entry (SOCE), an essential mechanism in cellular calcium signaling and in maintaining cellular calcium balance. Because -GlcNAcylation plays pivotal roles in various cellular function, we examined the effect of fluctuation in STIM1 -GlcNAcylation on SOCE activity. We found that both increase and decrease in STIM1 -GlcNAcylation impaired SOCE activity. To determine the molecular basis, we established STIM1-knockout HEK293 (STIM1-KO-HEK) cells using the CRISPR/Cas9 system and transfected STIM1 WT (STIM1-KO-WT-HEK), S621A (STIM1-KO-S621A-HEK), or T626A (STIM1-KO-T626A-HEK) cells. Using these cells, we examined the possible -GlcNAcylation sites of STIM1 to determine whether the sites were -GlcNAcylated. Co-immunoprecipitation analysis revealed that Ser and Thr were -GlcNAcylated and that Thr was -GlcNAcylated in the steady state but Ser was not. The SOCE activity in STIM1-KO-S621A-HEK and STIM1-KO-T626A-HEK cells was lower than that in STIM1-KO-WT-HEK cells because of reduced phosphorylation at Ser Treatment with the -GlcNAcase inhibitor Thiamet G or -GlcNAc transferase (OGT) transfection, which increases -GlcNAcylation, reduced SOCE activity, whereas treatment with the OGT inhibitor ST045849 or siOGT transfection, which decreases -GlcNAcylation, also reduced SOCE activity. Decrease in SOCE activity due to increase and decrease in -GlcNAcylation was attributable to reduced phosphorylation at Ser These data suggest that both decrease in -GlcNAcylation at Thr and increase in -GlcNAcylation at Ser in STIM1 lead to impairment of SOCE activity through decrease in Ser phosphorylation. Targeting STIM1 -GlcNAcylation could provide a promising treatment option for the related diseases, such as neurodegenerative diseases.