Abstract
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.
Highlights
The essential role of Ca2ϩ in signal transduction pathways is well established [1]
The Region 58 –143 of Calcineurin A Is Critical for Interaction with PMCA4b—To finely map the minimal interaction domain of calcineurin A responsible for the interaction with PMCA, a series of FLAG-tagged C-terminal deletion mutants of calcineurin A ␣ were generated (Fig. 2A) and assayed by immunoprecipitation for their ability to interact with human PMCA in mammalian cells
In this work we describe a novel role for the plasma membrane Ca2ϩ/calmodulin-ATPase pump, PMCA, as an inhibitor of calcineurin signaling
Summary
The essential role of Ca2ϩ in signal transduction pathways is well established [1]. Critical to the coupling of Ca2ϩ signals to cellular responses is the serine-threonine protein phosphatase calcineurin (Cn1; called protein phosphatase 2B), which is regulated by Ca2ϩ/calmodulin [2, 3]. PMCA4b has been shown to inhibit the activity of the Ca2ϩ/calmodulin-dependent protein neuronal nitric-oxide synthase (nitric-oxide synthase I) [13] and calcium/calmodulin-dependent serine protein kinase or CASK [14], by tethering them to low Ca2ϩ/calmodulin cellular microdomains via interaction between the C-terminal end of PMCA4b and PDZ domains located in the partner proteins. This, and the calcium/calmodulin-dependent nature of calcineurin, raised the possibility that PMCA may regulate the activity of calcineurin (a protein without PDZ-domain) through interaction with a region other than the C terminus. In keeping with this hypothesis, a number of calcineurin-binding proteins have been reported to inhibit its phosphatase activity via physical interaction with the calcineurin A catalytic subunit [25]
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