STIM1 phosphorylation at Y³¹⁶ modulates its interaction with SARAF and the activation of SOCE and I_CRAC.

Stromal interaction molecule 1 (STIM1) is one of the key elements for the activation of store-operated Ca²⁺ entry (SOCE). Hence, identification of the relevant phosphorylatable STIM1 residues with a possible role in the regulation of STIM1 function and SOCE is of interest. By performing a computational analysis, we identified that the Y³¹⁶ residue is susceptible to phosphorylation. Expression of the STIM1-Y316F mutant in HEK293, NG115-401L and MEG-01 cells resulted in a reduction in STIM1 tyrosine phosphorylation, SOCE and the Ca²⁺ release-activated Ca²⁺ current (I_CRAC). STIM1-Orai1 colocalization was reduced in HEK293 cells transfected with YFP-STIM1-Y316F compared to in cells with wild-type (WT) YFP-tagged STIM1. Additionally, the Y316F mutation altered the pattern of interaction between STIM1 and under resting conditions and upon Ca²⁺ store depletion. Expression of the STIM1 Y316F mutant enhanced slow Ca²⁺-dependent inactivation (SCDI) as compared to STIM1 WT, an effect that was abolished by duanyu1800F knockdown. Finally, in NG115-401L cells transfected with shRNA targeting expression of STIM1 Y316F induced greater SOCE than STIM1 WT. Taken together, our results provide evidence supporting the idea that phosphorylation of STIM1 at Y³¹⁶ plays a relevant functional role in the activation and modulation of SOCE.

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