The calcineurin/nuclear factor of activated T-cell (NFAT) pathway represents a crucial transducer of cellular function. There is increasing evidence placing the sarcolemmal calcium pump, or plasma membrane calcium/calmodulin ATPase pump (PMCA), as a potential modulator of signal transduction pathways. We demonstrate a novel interaction between PMCA and the calcium/calmodulin-dependent phosphatase, calcineurin, in mammalian cells. The interaction domains were located to the catalytic domain of PMCA4b and the catalytic domain of the calcineurin A subunit. Endogenous calcineurin activity, assessed by measuring the transcriptional activity of its best characterized substrate, NFAT, was significantly inhibited by 60% in the presence of ectopic PMCA4b. This inhibition was notably reversed by the co-expression of the PMCA4b interaction domain, demonstrating the functional significance of this interaction. PMCA4b was, however, unable to confer its inhibitory effect in the presence of a calcium/calmodulin-independent constitutively active mutant calcineurin A suggesting a calcium/calmodulin-dependent mechanism. The modulatory function of PMCA4b is further supported by the observation that endogenous calcineurin moves from the cytoplasm to the plasma membrane when PMCA4b is overexpressed. We suggest recruitment by PMCA4b of calcineurin to a low calcium environment as a possible explanation for these findings. In summary, our results offer strong evidence for a novel functional interaction between PMCA and calcineurin, suggesting a role for PMCA as a negative modulator of calcineurin-mediated signaling pathways in mammalian cells. This study reinforces the emerging role of PMCA as a molecular organizer and regulator of signaling transduction pathways.
"Several studies have identified other proteins that bind to the cytosolic C‐terminal tail of ATP2B, such as membrane‐associated guanylate kinase (MAGUK) (DeMarco and Strehler, 2001, Kim et al., 1998), Ca 2+ /calmodulin-dependent serine protein kinase (CASK) (Schuh et al., 2003), PMCA-interacting single-PDZ domain protein (PISP) (Goellner et al., 2003), Ania 3/Homer (Sgambato-Faure et al., 2006), and neuronal nitrogen monoxide synthase 1 (NOS1) (Schuh et al., 2001). Calcineurin (Buch et al., 2005) and the tumor suppressor Ras-associated factor 1 (RASSF1) (Armesilla et al., 2004) bind to the intracellular loop of ATP2B1. Its N-terminal tail binds to the 14-3-3 epsilon isoform (YWHAQ), which inhibits the Ca 2+ pump activity of ATP2B (Linde et al., 2008). "
[Show abstract][Hide abstract] ABSTRACT: Plasma membrane calcium-transporting ATPase 1 (ATP2B1) is associated significantly with blood pressure in Caucasians and Asians. ATP2B1 regulates calcium homeostasis and belongs
to the P-type calcium pump family; several studies have identified diverse proteins that bind to ATP2B1. We hypothesized that
ATP2B1 regulates blood pressure through ATP2B1-interacting genes. To this end, 20 potential ATP2B1-interacting genes were
selected, 197 SNPs of which were analyzed for their association of systolic and diastolic blood pressure. These 20 genes were
categorized into 2 groups: ATP2B1-binding genes and ATP2B1-cleaving calpain family members. Three ATP2B1-binding genes (CALM1,
NOS1, and PDLIM1) were associated with blood pressure, and a SNP in CALM1 (rs2401887) generated the strongest association
signal (beta=−3.60 ±0.92, p=8.9×10−5 for systolic blood pressure and beta=−1.40 ±0.62, p=0.02 for diastolic blood pressure). Of the calpain family members, 3 genes (CAPN6, CAPN9, and CAPN10) were associated with
blood pressure, and the CAPN10 SNP rs4676348 yielded the strongest association signal (beta=−0.88 ±0.27, p=0.001 for systolic blood pressure and beta=−0.58 ±0.18, p=0.015 for diastolic blood pressure). Further, the interaction of CALM1 to ATP2B1 was examined using the blood pressure of
individuals who carried both variants of CALM1 and ATP2B1 genes. Similarly the interaction of CAPN10 to ATP2B1 was also examined.
The CALM1 variant (rs2401887) and CAPN10 variants (rs4676348) appear to decrease blood pressure further in addition to the
decrease by the variant (rs17249754) of ATP2B1, which suggests that ATP2B1 might regulate blood pressure through the ATP2B1-interacting
genes CALM1 and CAPN10.
"A number of studies have identified proteins that bind to the cytosolic C-terminal tail of the PMCA pump, i.e., the membrane-associated guanylate kinase (MAGUK)  , the Ca 2+ /calmodulin-dependent serine protein kinase (CASK) , the PMCA-interacting single-PDZ domain protein (PISP) , the Ania 3/Homer protein , the neuronal nitrogen monoxide synthase 1 (NOS1) . Other studies have focused on the main intracellular loop of the pump, identifying calcineurin  and the tumor suppressor Ras-associated factor 1 (RASSF1) as partners . "
[Show abstract][Hide abstract] ABSTRACT: A previous study has demonstrated that the ubiquitous plasma membrane Ca(2+) pump PMCA4 interacted with isoform epsilon of the 14-3-3 protein, whereas the nervous tissue-specific PMCA2 did not. The 14-3-3 proteins are widely expressed small acidic proteins, which modulate cell signaling, intracellular trafficking, transcription and apoptosis. The investigation has been extended to the other tissue-restricted pump (PMCA3) and to the other ubiquitous pump (PMCA1). At variance with PMCA2, PMCA3 interacted with the 14-3-3epsilon protein in a two-hybrid system assay, which could not be used for PMCA1. The 14-3-3epsilon protein immunoprecipitated with both PMCA3 and PMCA1 when expressed in HeLa cells. Pull-down experiments using GST-PMCA1 and GST-PMCA3 fusion products confirmed the interaction of both pumps with the 14-3-3epsilon protein. The binding was phosphorylation-independent with both PMCA3 and PMCA1. The 14-3-3zeta isoform also interacted with PMCA3; however, it did not interact with PMCA1. The effect of the interaction on the activity of the two pumps, and thus on the homeostasis of Ca(2+), was investigated by co-expressing the 14-3-3epsilon protein and PMCA3 or PMCA1 in CHO cells together with the recombinant Ca(2+) indicator aequorin: the ability of cells to re-establish the basal Ca(2+) concentration following a Ca(2+) transient induced by an InsP(3)-producing agonist was substantially decreased with both pumps, indicating that the interaction with the 14-3-3 protein inhibited the activity of both PMCA3 and PMCA1.
"Samples were boiled and resolved as previously described . Western blot membranes were incubated at room temperature with a 0.1% (v/v) solution of anti-Flag M2 peroxidase-conjugated monoclonal antibody (Sigma–Aldrich) in Tris-buffered saline plus Tween 20 (TBS-T) (for Flag-epitope detection) or with a 0.25% (v/v) solution of isoform specific rabbit polyclonal anti-PMCA antibody (Swant) for detection of the corresponding isoform of PMCA for 3 h. "
[Show abstract][Hide abstract] ABSTRACT: Plasma membrane calcium/calmodulin-dependent ATPases (PMCAs) are high affinity calcium pumps that extrude calcium from the cell. Emerging evidence suggests a novel role for PMCAs as regulators of calcium/calmodulin-dependent signal transduction pathways via interaction with specific partner proteins. In this work, we demonstrate that endogenous human PMCA2 and -4 both interact with the signal transduction phosphatase, calcineurin, whereas, no interaction was detected with PMCA1. The strongest interaction was observed between PMCA2 and calcineurin. The domain of PMCA2 involved in the interaction is equivalent to that reported for PMCA4b. PMCA2-calcineurin interaction results in inhibition of the calcineurin/nuclear factor of activated T-cells signalling pathway.
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