STIM1 Phosphorylation at Y361 Recruits Orai1 to STIM1 Puncta and Induces Ca2+ Entry

Abstract

Store-operated Ca²⁺ entry (SOCE) mediates the increase in intracellular calcium (Ca²⁺) in endothelial cells (ECs) that regulates several EC functions including tissue-fluid homeostasis. Stromal-interaction molecule 1 (STIM1), upon sensing the depletion of (Ca²⁺) from the endoplasmic reticulum (ER) store, organizes as puncta that trigger store-operated Ca²⁺ entry (SOCE) via plasmalemmal Ca²⁺-selective Orai1 channels. While the STIM1 and Orai1 binding interfaces have been mapped, signaling mechanisms activating STIM1 recruitment of Orai1 and STIM1-Orai1 interaction remains enigmatic. Here, we show that ER Ca²⁺-store depletion rapidly induces STIM1 phosphorylation at Y361 via proline-rich kinase 2 (Pyk2) in ECs. Surprisingly, the phospho-defective STIM1-Y361F mutant formed puncta but failed to recruit Orai1, thereby preventing. SOCE Furthermore, studies in mouse lungs, expression of phosphodefective STIM1-Y361F mutant in ECs prevented the increase in vascular permeability induced by the thrombin receptor, protease activated receptor 1 (PAR1). Hence, Pyk2-dependent phosphorylation of STIM1 at Y361 is a critical phospho-switch enabling recruitment of Orai1 into STIM1 puncta leading to SOCE. Therefore, Y361 in STIM1 represents a novel target for limiting SOCE-associated vascular leak.

Figure 1. Thapsigargin induces phosphorylation of STIM1 at Y361 residue to facilitate SOCE.

(a) STIM1 was immunoprecipitated (IP) with anti-STIM1 antibodies from lysates of HPAE cell treated with 2 μM thapsigargin for indicated time points. Resulting precipitates were probed with the phosphotyrosine antibodies. A representative immunoblot (top) and densitometry of bar graph of mean ± SD (bottom) demonstrate transient changes in STIM1 phosphorylation on tyrosine residues at various times after thapsigargin stimulation. Fold change was expressed as a ratio between phosphotyrosine and total STIM1 density normalized to 0 times. Data are from 3 independent experiments. *p < 0.05, and ***p < 0.001 indicates significant increase as compared to 0 time points. (b) Schematic representation of STIM1 domains and putative tyrosine phosphorylation sites. Note, Y316 and 361 are located in Cα2 and SOAR domains, respectively. (c,d) Phosphorylation of STIM1-Y361 residue is required for inducing Ca²⁺ entry. HPAE cells were transfected with vector (YFP), WT-STIM1, Y316F-STIM1 or Y361F-STIM1 mutants. Cells were stimulated with thapsigargin in Ca²⁺ -free medium followed by addition of 2 mM Ca²⁺ at ~300 s. (c) Representative traces are shown.. (d) Individual data points (from 5–10 cells) and mean ± SD are plotted from 3 independent experiments. **p < 0.01 compared to unstimulated cells or cells stimulated with thapsigargin in Ca²⁺ -free medium.

Figure 2. Pyk2 phosphorylates STIM1.

(a) Western blot analysis of a time-course of Pyk2 phosphorylation using anti-Y-402-phospho-Pyk2 in HPAE cells treated with thapsigargin at indicated time points. Note, Pyk2 undergoes phosphorylation after treatment with thapsigargin. A representative blot is shown from experiments that were repeated at least three times. (b) Depletion of Pyk2 blocks STIM1 tyrosine phosphorylation. Western blot analysis of STIM1 phosphorylation (as described in Fig. 1a) in HPAE cells depleted of Pyk2 and treated with thapsigargin at indicated time points. Western blot with anti-Pyk2 was used to determine efficiency of Pyk2 depletion. siSC, scramble siRNA; siPyk2, Pyk2-targeting siRNA. A representative blot is shown from experiments that were repeated at least three times. (c,d) Overexpression of phospho-mimetic Y361D-STIM1 mutant but not WT-STIM1 or phosho-defective Y361F-STIM1 mutant rescues SOCE in Pyk2-depleted cells. Effect of Pyk2 depletion on SOCE was assessed as in Fig. 1. Representative traces are shown (c). (d) Summary results. Individual data points (from 5–10 cells) and mean ± SD are plotted from 3 independent experiments. **p < 0.01, ***p < 0.001 compared to unstimulated cells or cells stimulated with thapsigargin in Ca²⁺-free medium.

Figure 3. Tyrosine phosphorylation of STIM1 at Y361 residue mediates recruitment of Orai1 to STIM1 puncta.

(a,d) HPAE cells co-expressing either WT-YFP-STIM1 and mCherry-Orai1 or Y361F-YFP-STIM1 and mCherry-Orai1 were stimulated with 2 μM thapsigargin for the indicated time points, fixed and visualized with confocal LSM 880 microscope. Images showing STIM1 puncta and recruitment of Orai1 to STIM1 puncta (a), number of STIM1 (b) and Orai1 clusters (c), colocalization index for Orai1 (d). Individual data points (from 5–10 cells) and mean ± SD are plotted. ***p < 0.001 compared to unstimulated cells (b–d) or thapsigargin stimulated STIM1-Y361F mutant expressing cells (c,d).

Figure 4. Tyrosine phosphorylation of STIM1 at Y361 residue mediates STIM1-Orai1 interaction and activation of Orai1.

(a) HEK cells co-expressing YFP, WT-YFP-STIM1, or YFP-Y361F-STIM1, YFP-Y361D-STIM1 mutant together with mCherry-Orai1 were stimulated with thapsigargin for 5 min or left unstimulated. Exogenous STIM1 was immunoprecipitated with anti-GFP antibodies followed by immunoblotting using both anti-Orai1 and anti-GFP antibodies. A representative blot is shown from experiments that were repeated multiple times. (b) Western blot analysis of Orai1 expression in HPAE cells depleted of Orai1. A representative blot is shown from experiments that were repeated multiple times. siSC, scramble siRNA; siOrai1, Orai1-targeting siRNA. (c,d) Overexpression of Y361D-STIM1 mutant failed to rescue SOCE in Orai1 depleted ECs. Effect of Orai1 depletion on SOCE was assessed as in Fig. 1c,d; (c) Representative traces are shown; (d) Summary results. Individual data points (from 10–15 cells) and mean ± SD are plotted from 3 independent experiments. ***p < 0.001 compared to unstimulated cells or thapsigargin stimulated cells as indicated (c,d).

Figure 5. STIM1 phosphorylation at Y361 residue contributes to increased lung vascular permeability.

(a) Western blot analysis of a time-course of Pyk2 phosphorylation using anti-Y-402-phospho-Pyk2 in HPAE cells treated with thrombin as determined in Fig. 1. Image shown are cropped. Full immunoblot is uploaded in Supplementary Figure S3. A representative blot is shown from experiments that were repeated multiple times (b) Thrombin induces tyrosine phosphorylation of STIM1. Lysates were IP with anti-STIM1 antibody. Resulting precipitates were probed with the phosphotyrosine antibodies. A representative blot is shown from experiments that were repeated multiple times. (c,d) Phosphorylation of STIM1 on Y361 is required for α-thrombin-induced SOCE. A representative trace (c). (d) Individual data points (from 10–15 cells) and mean ± SD are plotted from 3 independent experiments. **p < 0.01 compared to unstimulated cells or thrombin stimulated STIM1-Y361F mutant expressing cells. (e) Liposomes containing either YFP, WT-STIM1 or Y361F-STIM1 were injected into WT mouse ice through retroorbital sinus 40 h prior to experiments. In parallel experiment, STIM^fl/fl and EC-Stim1^−/− were used. To induce vascular permeability, PAR1 agonist peptide was given i.v. 30 min prior to harvesting lungs for measurement of lung wet/dry ratio. Individual data points and mean ± SD are plotted from 5 mice. Experiments were repeated two times. **p < 0.01 compared to mice receiving control peptide or PAR1 activated mice expressing STIM1-Y361F mutant.