Store-operated Ca2+ Entry (SOCE) Induced by Protease-activated Receptor-1 Mediates STIM1 Protein Phosphorylation to Inhibit SOCE in Endothelial Cells through AMP-activated Protein Kinase and p38β Mitogen-activated Protein Kinase

Abstract

The Ca2+ sensor STIM1 is crucial for activation of store-operated Ca2+ entry (SOCE) through transient receptor potential canonical and Orai channels. STIM1 phosphorylation serves as an “off switch” for SOCE. However, the signaling pathway for STIM1 phosphorylation is unknown. Here, we show that SOCE activates AMP-activated protein kinase (AMPK); its effector p38β mitogen-activated protein kinase (p38β MAPK) phosphorylates STIM1, thus inhibiting SOCE in human lung microvascular endothelial cells. Activation of AMPK using 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR) resulted in STIM1 phosphorylation on serine residues and prevented protease-activated receptor-1 (PAR-1)-induced Ca2+ entry. Furthermore, AICAR pretreatment blocked PAR-1-induced increase in the permeability of mouse lung microvessels. Activation of SOCE with thrombin caused phosphorylation of isoform α1 but not α2 of the AMPK catalytic subunit. Moreover, knockdown of AMPKα1 augmented SOCE induced by thrombin. Interestingly, SB203580, a selective inhibitor of p38 MAPK, blocked STIM1 phosphorylation and led to sustained STIM1-puncta formation and Ca2+ entry. Of the three p38 MAPK isoforms expressed in endothelial cells, p38β knockdown prevented PAR-1-mediated STIM1 phosphorylation and potentiated SOCE. In addition, inhibition of the SOCE downstream target CaM kinase kinase β (CaMKKβ) or knockdown of AMPKα1 suppressed PAR-1-mediated phosphorylation of p38β and hence STIM1. Thus, our findings demonstrate that SOCE activates CaMKKβ-AMPKα1-p38β MAPK signaling to phosphorylate STIM1, thereby suppressing endothelial SOCE and permeability responses.

Cooperative Signaling via Transcription Factors NF-κB and AP1/c-Fos Mediates Endothelial Cell STIM1 Expression and Hyperpermeability in Response to Endotoxin

“…Furthermore, studies have shown that reactive oxygen species can induce STIM1-mediated Ca 2ϩ entry through Orai1 channels in ECs by activating STIM1 proteins via S-glutathionylation of the N-terminal cysteine residue in the STIM1 protein (26). We have shown that thrombin-induced endothelial barrier disruption in lung ECs is mediated by STIM1 activation of SOCE (10,21). In the in vivo setting, an LPS-induced lung vascular permeability increase was abrogated in EC-restricted STIM1 knockout (Stim1 ECϪ/Ϫ ) mice (27).…”

“…Cytosolic Ca 2ϩ Measurement-The cytoplasmic Ca 2ϩ concentration ([Ca 2ϩ ] i ) in ECs was measured using the Ca 2ϩ -sensitive fluorescent dye Fura-2/AM (10,21). Cells were grown to confluence on gelatin-coated glass coverslips and then washed two times and incubated for 12 h at 37°C in medium containing 1% FBS.…”

“…In each experiment, 20 -30 cells were selected to measure the change in ([Ca 2ϩ ] i ). siRNA Transfection-ECs grown to ϳ70% confluence on gelatin-coated culture dishes were transfected with target siRNA or sc-siRNA as described previously (10). 72 h after transfection, cells were challenged with LPS (1 g/ml) and then used for Ca 2ϩ measurements or harvested for Western blot analysis.…”

“…Thirty minutes before sacrifice, mice received a 100-l bolus of Evans blue dye conjugated with albumin (EBA, 20 mg/kg) through the jugular vein. The EBA present in lung tissue was measured as described previously (10,20).…”

Centlein mediates an interaction between C-Nap1 and Cep68 to maintain centrosome cohesion

“…At 24 h after transfection, the total cell lysate was incubated with 2 mg of an antibody against GFP. The immunoprecipitated kinases were washed four times in ELB buffer and then incubated with the GST fusion N-terminal fragment of centlein (amino acid residues 89-437 of centlein) or GST fusion N-terminal fragment of Cep68 (amino acid residues 1-282 of Cep68) (500 mg/ml) in kinase buffer (50 mM Tris HCl pH 7.7, 100 mM NaCl, 25 mM bglycerophosphate, 25 mM MgCl 2 , 1 mM DTT, protease inhibitor cocktail and 200 mM ATP) for 30 min at 30˚C (Sundivakkam et al, 2013). The reaction was terminated by adding SDS-PAGE sample buffer and boiling for 5 min.…”