Abstract
BackgroundCalcium 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.ObjectivesHere, we investigated whether PKC-β controls intracellular calcium dynamics through Stim1.MethodsSeveral 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.ResultsOur 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.ConclusionIn sum, our data suggest a repressive role of PKC in regulating calcium entry through SOCE.
Highlights
The Ca2+ ions are essential regulator of many normal cellular processes, including muscle contraction, gene transcription, and cell migration (Bong and Monteith 2018; Carafoli 2002; Clapham 2007; Kim et al 2014; Wei et al 2012)
During this calcium depletion process, the N-terminal domain of stromal interaction molecule 1 (Stim1) dissociates from intra endoplasmic reticulum (ER) calcium ions and undergoes conformational changes to promote self-oligomerization, after which it localizes to the plasma membrane (PM) for interaction with the calcium channel Orai1
Using immunoprecipitation and C a2+ mobilization and confocal assays, we discovered that (1) Protein kinase C (PKC)-β interacts with and phosphorylates the C-terminal region of Stim1, whereby residue Ser660 represents an important target site, (2) mutation of Ser660 to Ala restores store-operated calcium entry (SOCE) activity in response to PKC activation, (3) PKC activity is inversely related to SOCE activity, and (4) Stim1 oligomerization induced by ionomycin is affected by either chemical activators or inhibitors of PKC isozymes
Summary
The Ca2+ ions are essential regulator of many normal cellular processes, including muscle contraction, gene transcription, and cell migration (Bong and Monteith 2018; Carafoli 2002; Clapham 2007; Kim et al 2014; Wei et al 2012). Intracellular Ca2+ levels are tightly controlled in nonexcitable cells by ligand-gated C a2+ channels in the plasma membrane (PM) and endoplasmic reticulum (ER). Genes & Genomics (2022) 44:571–582 triphosphate (IP3) and IP3 binding to ER-resident calcium channels, resulting in the release of Ca2+ from the ER store (Liou et al 2005; Prakriya et al 2015; Roos et al 2005). During this calcium depletion process, the N-terminal domain of Stim dissociates from intra ER calcium ions and undergoes conformational changes to promote self-oligomerization, after which it localizes to the PM for interaction with the calcium channel Orai. Conclusion In sum, our data suggest a repressive role of PKC in regulating calcium entry through SOCE
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