Citation

  • Authors: Knapp ML. et al.
  • Year: 2022
  • Journal: EMBO Rep 23 e53135
  • Applications: in vitro / DNA, siRNA / INTERFERin, jetPRIME
  • Cell types:
    1. Name: MEF
      Description: Murine embryonic fibroblast cells 
    2. Name: Primary murine hippocampal astrocytes
    3. Name: SH-SY5Y
      Description: Human neuroblastoma cells
      Known as:

Method

All cell lines were cultivated at 37°C, 5% CO2 in a humidified incubator in respective medium (HEK293 minimum essential medium, HEK293STIM1/2−/− Dulbecco’s modified Eagle’s medium, MEFStim1/2−/− Dulbecco’s modified Eagle’s medium, HEKO1 minimum essential medium, and SH-SY5Y STIM1−/− and SH-SY5Y STIM1−/−;STIM2−/− Dulbecco’s modified Eagle’s medium with 1% non-essential amino acids; NEAA, Gibco). All culture media were supplemented with 10% fetal calf serum. For passaging, cells were detached using trypsin. For transfections, HEK cells were transfected via electroporation using the Amaxa® Nucleofector II® (Lonza) according to the user manual. Amount of DNA used was 1µg/1.000.000 cells. MEF/SH-SY5Y cells were transfected using jetPRIME® (Polyplus transfections) transfection reagent following the manufacturer’s protocol. siRNA (#1+#2, 20 nM total) or siCTL (20 nM) were transfected 24h before recording using INTERFERin® (Polyplus transfections). Cells were analyzed 20-24 h after transfection of siRNA or with specified DNA constructs as listed in Table EV1; primers and siRNA are listed in Table EV2.

Abstract

Alternative splicing is a potent modifier of protein function. Stromal interaction molecule 1 (Stim1) is the essential activator of store-operated Ca2+ entry (SOCE) triggering activation of transcription factors. Here, we characterize Stim1A, a splice variant with an additional 31 amino acid domain inserted in frame within its cytosolic domain. Prominent expression of exon A is found in astrocytes, heart, kidney, and testes. Full-length Stim1A functions as a dominant-negative regulator of SOCE and ICRAC, facilitating sequence-specific fast calcium-dependent inactivation and destabilizing gating of Orai channels. Downregulation or absence of native Stim1A results in increased SOCE. Despite reducing SOCE, Stim1A leads to increased NFAT translocation. Differential proteomics revealed an interference of Stim1A with the cAMP-SOCE crosstalk by altered modulation of phosphodiesterase 8 (PDE8), resulting in reduced cAMP degradation and increased PIP5K activity, facilitating NFAT activation. Our study uncovers a hitherto unknown mechanism regulating NFAT activation and indicates that cell-type-specific splicing of Stim1 is a potent means to regulate the NFAT signalosome and cAMP-SOCE crosstalk.

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