EFHB is a Novel Cytosolic Ca2+ Sensor That Modulates STIM1-SARAF Interaction (original) (raw)
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EFHB is a Novel Cytosolic Ca2+ Sensor That Modulates STIM1-SARAF Interaction
Cellular Physiology and Biochemistry
Background/Aims: STIM1 and Orai1 are the key components of store-operated Ca2+ entry (SOCE). Among the proteins involved in the regulation of SOCE, SARAF prevents spontaneous activation of SOCE and modulates STIM1 function. Methods: Cytosolic Ca2+ mobilization was estimated in fura-2-loaded cells using an epifluorescence inverted microscope. STIM1 interaction with Orai1, EFHB (EF-hand domain family member B, also known as CFAP21) and SARAF was detected by immunoprecipitation followed by Western blotting using specific antibodies. The involvement of EFHB in the translocation of NFAT to the nucleus was detected by confocal microscopy. Results: Here, we report the identification of EFHB as a new SOCE regulator. EFHB interacts with STIM1 upon store depletion and dissociates through a Ca2+-dependent mechanism. RNAi-mediated silencing as well as overexpression studies revealed that EFHB plays a relevant role in the interaction of STIM1 and Orai1 upon store depletion, the activation of SOC...
STIM2 Is an Inhibitor of STIM1-Mediated Store-Operated Ca 2+ Entry
Current Biology, 2006
The coupling mechanism between endoplasmic reticulum (ER) Ca 2+ stores and plasma membrane (PM) storeoperated channels (SOCs) remains elusive . STIM1 was shown to play a crucial role in this coupling process ; however, the role of the closely related STIM2 protein remains undetermined. We reveal that STIM2 is a powerful SOC inhibitor when expressed in HEK293, PC12, A7r5, and Jurkat T cells. This contrasts with gain of SOC function in STIM1-expressing cells. While STIM1 is expressed in both the ER and plasma membrane, STIM2 is expressed only intracellularly. Store depletion induces redistribution of STIM1 into distinct ''puncta.'' STIM2 translocates into puncta upon store depletion only when coexpressed with STIM1. Double labeling shows coincidence of STIM1 and STIM2 within puncta, and immunoprecipitation reveals direct interactions between STIM1 and STIM2. Independent of store depletion, STIM2 colocalizes with and blocks the function of a STIM1 EF-hand mutant that preexists in puncta and is constitutively coupled to activate SOCs. Thus, whereas STIM1 is a required mediator of SOC activation, STIM2 is a powerful inhibitor of this process, interfering with STIM1-mediated SOC activation at a point downstream of puncta formation. The opposing functions of STIM1 and STIM2 suggest they may play a coordinated role in controlling SOC-mediated Ca 2+ entry signals.
Dynamic interaction of SARAF with STIM1 and Orai1 to modulate store-operated calcium entry
Scientific reports, 2016
Ca(2+) influx by store-operated Ca(2+) channels is a major mechanism for intracellular Ca(2+) homeostasis and cellular function. Here we present evidence for the dynamic interaction between the SOCE-associated regulatory factor (SARAF), STIM1 and Orai1. SARAF overexpression attenuated SOCE and the STIM1-Orai1 interaction in cells endogenously expressing STIM1 and Orai1 while RNAi-mediated SARAF silencing induced opposite effects. SARAF impaired the association between Orai1 and the Orai1-activating small fragment of STIM1 co-expressed in the STIM1-deficient NG115-401L cells. Cell treatment with thapsigargin or physiological agonists results in direct association of SARAF with Orai1. STIM1-independent interaction of SARAF with Orai1 leads to activation of this channel. In cells endogenously expressing STIM1 and Orai1, Ca(2+) store depletion leads to dissociation of SARAF with STIM1 approximately 30s after treatment with thapsigargin, which paralleled the increase in SARAF-Orai1 inter...
Journal of Cell Science, 2019
Stromal interaction molecule 1 (STIM1) is one of the key elements for the activation of store- operated calcium entry (SOCE). Identification of the relevant phosphorylatable STIM1 residues with a possible role in the regulation of STIM1 function and SOCE might be of interest. Using computational analysis, we have identified that the Y316 residue is susceptible to be phosphorylated. Expression of the STIM1-Y316F mutant in HEK293, NG115-401L and MEG-01 cells has resulted in a reduction in STIM1 tyrosine phosphorylation, SOCE and ICRAC. STIM1-Orai1 colocalization was reduced in HEK293 cells transfected with YFP-STIM1-Y316F compared to YFP-STIM1-WT cells. Additionally, Y316F mutation altered the pattern of interaction between STIM1 and SARAF under resting conditions and upon Ca2+ store depletion. Expression of the STIM1 Y316F mutant enhanced slow Ca2+-dependent inactivation (SCDI) as compared to STIM1 WT, an effect that was abolished by SARAF knockdown. Finally, in NG115-401L cells tran...
Molecular Determinants for STIM1 Activation During Store- Operated Ca2+ Entry
Current Molecular Medicine, 2017
Background-STIM/ORAI-mediated store-operated Ca 2+ entry (SOCE) mediates a myriad of Ca 2+-dependent cellular activities in mammals. Genetic defects in STIM1/ORAI1 lead to devastating severe combined immunodeficiency; whereas gain-of-function mutations in STIM1/ ORAI1 are intimately associated with tubular aggregate myopathy. At molecular level, a decrease in the Ca 2+ concentrations within the lumen of endoplasmic reticulum (ER) initiates multimerization of the STIM1 luminal domain to switch on the STIM1 cytoplasmic domain to engage and gate ORAI channels, thereby leading to the ultimate Ca 2+ influx from the extracellular space into the cytosol. Despite tremendous progress made in dissecting functional STIM1-ORAI1 coupling, the activation mechanism of SOCE remains to be fully characterized. Objective and Methods-Building upon a robust fluorescence resonance energy transfer assay designed to monitor STIM1 intramolecular autoinhibition, we aimed to systematically dissect the molecular determinants required for the activation and oligomerization of STIM1. Results-Here we showed that truncation of the STIM1 luminal domain predisposes STIM1 to adopt a more active conformation. Replacement of the single transmembrane (TM) domain of
Novel Role for STIM1 as a Trigger for Calcium Influx Factor Production
Journal of Biological Chemistry, 2008
STIM1 has been recently identified as a Ca 2؉ sensor in endoplasmic reticulum (ER) and an initiator of the store-operated Ca 2؉ entry (SOCE) pathway, but the mechanism of SOCE activation remains controversial. Here we focus on the early ERdelimited steps of the SOCE pathway and demonstrate that STIM1 is critically involved in initiating of production of calcium influx factor (CIF), a diffusible messenger that can deliver the signal from the stores to plasma membrane and activate SOCE. We discovered that CIF production is tightly coupled with STIM1 expression and requires functional integrity of its intraluminal sterile ␣-motif (SAM) domain. We demonstrate that 1) molecular knockdown or overexpression
Role of STIM1 in the surface expression of SARAF
Channels (Austin, Tex.), 2016
The store-operated Ca(2+) entry-associated regulatory factor (SARAF), a protein expressed both in the endoplasmic reticulum and the plasma membrane, has been presented as a STIM1-interacting protein with the ability to modulate intracellular Ca(2+) homeostasis. SARAF negatively modulates store-operated Ca(2+) entry (SOCE) by preventing STIM1 spontaneous activation and regulating STIM1-Orai1 complex formation. In addition, SARAF is a negative regulator of Ca(2+) entry through the arachidonate-regulated Ca(2+) (ARC) channels. Here we explored the possible role of the surface expression of SARAF on the location of STIM1 in the plasma membrane. In NG115-401L cells, lacking a detectable expression of native STIM1, transfection with pHluorin-STIM1, which is able to translocate to the cell surface, enhances the plasma membrane location of SARAF as compared to cells transfected with YFP-STIM1, lacking the ability to translocate to the cell surface. These findings suggest that the surface lo...
SERAF, a Novel Regulator of Store Operated Calcium Entry
Biophysical Journal, 2012
Store operated calcium entry (SOCE) is a principal cellular process by which cells regulate basal calcium, refill intracellular Ca 2+ stores, and execute a wide range of specialized activities. STIM and Orai proteins have been identified as the essential components enabling the reconstitution of Ca 2+ release-activated Ca 2+ (CRAC) channels that mediate SOCE. Here, we report the molecular identification of SARAF as a negative regulator of SOCE. Using heterologous expression, RNAi-mediated silencing and site directed mutagenesis combined with electrophysiological, biochemical and imaging techniques we show that SARAF is an endoplasmic reticulum membrane resident protein that associates with STIM to facilitate slow Ca 2+ -dependent inactivation of SOCE. SARAF plays a key role in shaping cytosolic Ca 2+ signals and determining the content of the major intracellular Ca 2+ stores, a role that is likely to be important in protecting cells from Ca 2+ overfilling.
Signalling to transcription: Store-operated Ca2+ entry and NFAT activation in lymphocytes
Cell Calcium, 2007
In cells of the immune system that are stimulated by antigen or antigen-antibody complexes, Ca 2+ entry from the extracellular medium is driven by depletion of endoplasmic reticulum Ca 2+ stores and occurs through specialized store-operated Ca 2+ channels known as Ca 2+release-activated Ca 2+ (CRAC) channels. The process of store-operated Ca 2+ influx is essential for short-term as well as long-term responses by immune-system cells. Short-term responses include mast cell degranulation and killing of target cells by effector cytolytic T cells, whereas long-term responses typically involve changes in gene transcription and include T and B cell proliferation and differentiation. Transcription downstream of Ca 2+ influx is in large part funneled through the transcription factor nuclear factor of activated T cells (NFAT), a heavily phosphorylated protein that is cytoplasmic in resting cells, but that enters the nucleus when dephosphorylated by the calmodulin-dependent serine/threonine phosphatase calcineurin. The importance of the Ca 2+ /calcineurin/NFAT signalling pathway for lymphocyte activation is underscored by the finding that the underlying defect in a family with a hereditary severe combined immune deficiency (SCID) syndrome is a defect in CRAC channel function, store-operated Ca 2+ entry, NFAT activation and transcription of cytokines, chemokines and many other NFAT target genes whose transcription is essential for productive immune defence.
Cancers
Breast cancer is among the most common malignancies in women. From the molecular point of view, breast cancer can be grouped into different categories, including the luminal (estrogen receptor positive (ER+)) and triple negative subtypes, which show distinctive features and, thus, are sensitive to different therapies. Breast cancer cells are strongly dependent on Ca2+ influx. Store-operated Ca2+ entry (SOCE) has been found to support a variety of cancer hallmarks including cell viability, proliferation, migration, and metastasis. The Ca2+ channels of the Orai family and the endoplasmic reticulum Ca2+ sensor STIM1 are the essential components of SOCE, but the extent of Ca2+ influx is fine-tuned by several regulatory proteins, such as the STIM1 modulators SARAF and EFHB. Here, we show that the expression and/or function of SARAF and EFHB is altered in breast cancer cells and both proteins are required for cell proliferation, migration, and viability. EFHB expression is upregulated in ...