Regulation of GAP43/calmodulin complex formation via calcineurin-dependent mechanism in differentiated PC12 cells with altered PMCA isoforms composition.

Tomasz Boczek,Ludmila Zylinska,Malwina Lisek,Bozena Ferenc

doi:10.1007/s11010-015-2473-4

Tomasz Boczek, Ludmila Zylinska + Show 2 more

Open Access

https://doi.org/10.1007/s11010-015-2473-4

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Abstract

Several lines of evidence suggest the contribution of age-related decline in plasma membrane calcium pump (PMCA) to the onset of neurodegenerative diseases. From four PMCA isoforms, PMCA2, and PMCA3 respond to a rapid removal of Ca2+ and are expressed predominantly in excitable cells. We have previously shown that suppression of neuron-specific PMCAs in differentiated PC12 cells accelerated cell differentiation, but increased apoptosis in PMCA2-deficient line. We also demonstrated that altered expression of voltage-dependent calcium channels correlated with their higher contribution to Ca2+ influx, which varied between PMCA-reduced lines. Here, we propose a mechanism unique for differentiated PC12 cells by which PMCA2 and PMCA3 regulate pGAP43/GAP43 ratio and the interaction between GAP43 and calmodulin (CaM). Although down-regulation of PMCA2 or PMCA3 altered the content of GAP43/pGAP43, of paramount importance for the regulatory mechanism is a disruption of isoform-specific inhibitory PMCA/calcineurin interaction. In result, higher endogenous calcineurin (CaN) activity leads to hypophosphorylation of GAP43 in PMCA2- or PMCA3-deficient lines and intensification of GAP43/CaM complex formation, thus potentially limiting the availability of free CaM. In overall, our results indicate that both “fast” PMCA isoforms could actively regulate the local CaN function and CaN-downstream processes. In connection with our previous observations, we also suggest a negative feedback of cooperative action of CaM, GAP43, and CaN on P/Q and L-type channels activity. PMCAs- and CaN-dependent mechanism presented here, may signify a protective action against calcium overload in neuronal cells during aging, as well a potential way for decreasing neuronal cells vulnerability to neurodegenerative insults.

Highlights

Cellular function of plasma membrane calcium pump (PMCA) focuses on Ca2? extrusion and protection from calcium overload, on prevention from cell apoptosis as well on controlling of calcium signaling
The Western blotting revealed higher amount of immunoreactivity in the PMCA-reduced cell lysates probed with anti-GAP43, an antibody recognizing total GAP43 protein, and the same results were obtained in the presence of cyclosporin A (CsA) (Fig. 2b, c)
In PMCA-reduced lines lowered pGAP43 level was detected in the absence of CsA, but it significantly increased after treatment with CsA (Fig. 2b, c)

Summary

Introduction

Cellular function of plasma membrane calcium pump (PMCA) focuses on Ca2? extrusion and protection from calcium overload, on prevention from cell apoptosis as well on controlling of calcium signaling. Cellular function of plasma membrane calcium pump (PMCA) focuses on Ca2? The existence of four isoforms encoded by four different genes and tissue-specific composition of PMCAs variants in the membrane domains strongly implicate their unique function in controlling of calcium homeostasis [for ref. Some data suggest more comprehensive connection of these proteins with internal cellular event, like development, differentiation, secretion or formation of signaling complexes, but not all PMCA isoforms appear to be involved to the same extent [6, 7]. PMCA is a solely ion pump directly activated by Ca2?/CaM complex, the affinity and rate of activation varies considerably between particular isoforms [1]. Calmodulin (CaM), calcineurin (CaN), and neuromodulin (GAP43) are strongly linked with calcium. It should be noted that both, PMCA and calcineurin are the effectors for Ca2?/CaM action

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