Department of Pharmacology and Center for Lung and Vascular Biology, College of Medicine, University of Illinois, Chicago, Illinois 60612-7343.
Department of Pharmacology and Center for Lung and Vascular Biology, College of Medicine, University of Illinois, Chicago, Illinois 60612-7343.
J Biol Chem. 2013 Jun 7;288(23):17030-17041. doi: 10.1074/jbc.M112.411272. Epub 2013 Apr 26.
The Ca(2+) sensor STIM1 is crucial for activation of store-operated Ca(2+) 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 Ca(2+) 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 Ca(2+) 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.
钙（Ca2+）传感器 STIM1 对于通过瞬时受体电位经典型和 Orai 通道激活储存操纵的 Ca2+内流（SOCE）至关重要。STIM1 磷酸化可作为 SOCE 的“关闭开关”。然而，STIM1 磷酸化的信号通路尚不清楚。在这里，我们表明 SOCE 激活 AMP 激活的蛋白激酶（AMPK）；其效应物 p38β 丝裂原激活的蛋白激酶（p38β MAPK）磷酸化 STIM1，从而抑制人肺微血管内皮细胞中的 SOCE。使用 5-氨基咪唑-4-甲酰胺-1-β-D-核糖呋喃糖苷（AICAR）激活 AMPK 会导致 STIM1 丝氨酸残基磷酸化，并防止蛋白酶激活受体-1（PAR-1）诱导的 Ca2+内流。此外，AICAR 预处理可阻断 PAR-1 诱导的小鼠肺微血管通透性增加。用凝血酶激活 SOCE 会导致 AMPK 催化亚基的同工型 α1 而不是 α2 磷酸化。此外，敲低 AMPKα1 增强了凝血酶诱导的 SOCE。有趣的是，p38 MAPK 的选择性抑制剂 SB203580 阻断了 STIM1 磷酸化，并导致持续的 STIM1 斑点形成和 Ca2+内流。在内皮细胞中表达的三种 p38 MAPK 同工型中，p38β 敲低可防止 PAR-1 介导的 STIM1 磷酸化并增强 SOCE。此外，抑制 SOCE 下游靶标钙调蛋白激酶激酶 β（CaMKKβ）或敲低 AMPKα1 可抑制 PAR-1 介导的 p38β 和 STIM1 磷酸化。因此，我们的研究结果表明，SOCE 激活 CaMKKβ-AMPKα1-p38β MAPK 信号通路磷酸化 STIM1，从而抑制内皮 SOCE 和通透性反应。
