Division of Biochemistry, College of Medicine, Chungbuk National University, Ch'ongju, 28644, Korea.
Biomedical Research Center for Signal Transduction Networks, Department of Chemistry, Inha University, Incheon, 402-751, Korea.
Genes Genomics. 2022 May;44(5):571-582. doi: 10.1007/s13258-022-01230-3. Epub 2022 Mar 7.
Calcium ions play a pivotal role in cell proliferation, differentiation, and migration. Under basal conditions, the calcium level is tightly regulated; however, cellular activation by growth factors increase the ion level through calcium pumps in the plasma membrane and endoplasmic reticulum for calcium signaling. Orai1 is a major calcium channel in the cell membrane of non-excitable cells, and its activity depends on the stromal interaction molecule 1 (Stim1). Several groups reported that the store-operated calcium entry (SOCE) can be modulated through phosphorylation of Stim1 by protein kinases such as extracellular signal-regulated kinase (ERK), protein kinase A (PKA), and p21-activated kinase (PAK). PKC is a protein kinase that is activated by calcium and diacylglycerol (DAG), but it remains unclear what role activated PKC plays in controlling the intracellular calcium pool.
Here, we investigated whether PKC-β controls intracellular calcium dynamics through Stim1.
Several biochemical methods such as immune-precipitation, site directed mutagenesis, in vitro kinase assay were employed to investigate PKC interaction with and phosphorylation of Stim1. Intracellular calcium mobilization, via Stim1 mediated SOCE channel, were studied using in the presence of PKC activator or inhibitor under a confocal microscope.
Our data demonstrate that PKC interacts with and phosphorylates Stim1 in vitro. phosphorylation of Stim1 at its C-terminal end appears to be important in the regulation of SOCE activity in HEK293 and HeLa cells. Additionally, transient intracellular calcium mobilization assays demonstrate that the SOCE activity was inhibited by PKC activators or activated by PKC inhibitors.
In sum, our data suggest a repressive role of PKC in regulating calcium entry through SOCE.
钙离子在细胞增殖、分化和迁移中起着关键作用。在基础条件下，钙离子水平受到严格调节；然而，生长因子激活细胞会通过质膜和内质网中的钙泵增加离子水平，以进行钙信号传递。Orai1 是非兴奋性细胞细胞膜中的主要钙离子通道，其活性依赖于基质相互作用分子 1（Stim1）。有几个研究小组报告称，通过细胞外信号调节激酶（ERK）、蛋白激酶 A（PKA）和 p21 激活激酶（PAK）等蛋白激酶对 Stim1 的磷酸化，可以调节钙库操纵性钙内流（SOCE）。PKC 是一种被钙离子和二酰基甘油（DAG）激活的蛋白激酶，但目前尚不清楚激活的 PKC 在控制细胞内钙库方面发挥什么作用。
本研究旨在探讨 PKC-β 是否通过 Stim1 来控制细胞内钙离子动力学。
采用免疫沉淀、定点突变、体外激酶测定等多种生化方法，研究 PKC 与 Stim1 的相互作用及其磷酸化。在共聚焦显微镜下，使用 PKC 激活剂或抑制剂研究 Stim1 介导的 SOCE 通道引起的细胞内钙离子动员。
我们的数据表明 PKC 在体外与 Stim1 相互作用并磷酸化它。Stim1 的 C 端末端的磷酸化似乎在调节 HEK293 和 HeLa 细胞中 SOCE 活性方面起着重要作用。此外，瞬时细胞内钙动员实验表明，SOCE 活性被 PKC 激活剂抑制或被 PKC 抑制剂激活。
综上所述，我们的数据表明 PKC 在调节通过 SOCE 的钙内流中起抑制作用。
