Inhibitory interaction of the 14-3-3 proteins with ubiquitous (PMCA1) and tissue-specific (PMCA3) isoforms of the plasma membrane Ca2+ pump

A previous study has demonstrated that the ubiquitous plasma membrane Ca2+ pump PMCA4 interacted with isoform ɛ 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 14-3-3ɛ protein in a two-hybrid system assay, which could not be used for PMCA1. The 14-3-3ɛ 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 14-3-3ɛ protein. The binding was phosphorylation-independent with both PMCA3 and PMCA1. The 14-3-3ζ 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 Ca2+, was investigated by co-expressing the 14-3-3ɛ protein and PMCA3 or PMCA1 in CHO cells together with the recombinant Ca2+ indicator aequorin: the ability of cells to re-establish the basal Ca2+ concentration following a Ca2+ transient induced by an InsP3-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.