Abstract
Stromal interaction molecule 1 (STIM1) plays a pivotal role in store-operated Ca2+ entry (SOCE), an essential mechanism in cellular calcium signaling and in maintaining cellular calcium balance. Because O-GlcNAcylation plays pivotal roles in various cellular function, we examined the effect of fluctuation in STIM1 O-GlcNAcylation on SOCE activity. We found that both increase and decrease in STIM1 O-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 O-GlcNAcylation sites of STIM1 to determine whether the sites were O-GlcNAcylated. Co-immunoprecipitation analysis revealed that Ser621 and Thr626 were O-GlcNAcylated and that Thr626 was O-GlcNAcylated in the steady state but Ser621 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 Ser621 Treatment with the O-GlcNAcase inhibitor Thiamet G or O-GlcNAc transferase (OGT) transfection, which increases O-GlcNAcylation, reduced SOCE activity, whereas treatment with the OGT inhibitor ST045849 or siOGT transfection, which decreases O-GlcNAcylation, also reduced SOCE activity. Decrease in SOCE activity due to increase and decrease in O-GlcNAcylation was attributable to reduced phosphorylation at Ser621 These data suggest that both decrease in O-GlcNAcylation at Thr626 and increase in O-GlcNAcylation at Ser621 in STIM1 lead to impairment of SOCE activity through decrease in Ser621 phosphorylation. Targeting STIM1 O-GlcNAcylation could provide a promising treatment option for the related diseases, such as neurodegenerative diseases.
Highlights
O-Linked N-acetylglucosamine (O-GlcNAc) modification (O-GlcNAcylation) is a common posttranslational modification in numerous cytoplasmic and nuclear proteins [1,2,3]
B, D, F, and H, peak store-operated calcium entry (SOCE) activity in HEK293 cells after Ca21 addition, which was evaluated from the results shown in A, C, E, and
We focused on the potential O-GlcNAcylation sites within the Ser/Pro-rich domain of Stromal interaction molecule 1 (STIM1) because STIM1-dependent SOCE activity is accomplished by phosphorylation of STIM1 at ERK1/2 target sites within the Ser/Pro-rich domain of STIM1 [36]
Summary
O-Linked N-acetylglucosamine (O-GlcNAc) modification (O-GlcNAcylation) is a common posttranslational modification in numerous cytoplasmic and nuclear proteins [1,2,3]. Abnormal increase in O-GlcNAcylation has been implicated in diabetes mellitus [7,8,9], neurodegenerative diseases [10, 11], heart failure [12, 13], and cancer [14, 15]. O-GlcNAcylation blocks the aggregation and toxicity of the protein a-synuclein associated with Parkinson’s disease [18]. Obtaining further data on the O-GlcNAcylation of diabetes-related and neuroregulatory molecules may have important implications in developing therapeutic strategies for these diseases. O-GlcNAcylation of endogenous STIM1 for HEK293 cells treated with different glucose concentrations was examined using immunoprecipitation assays.
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