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 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.
Linde
Cristina I
CI
Venetian Institute of Molecular Medicine, Via G. Orus 2, 35129 Padova, Italy.
Di Leva
Francesca
F
Domi
Teuta
T
Tosatto
Silvio C E
SC
Brini
Marisa
M
Carafoli
Ernesto
E
D048948:14-3-3 Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D000331:Aequorin; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism*; D002120:Calcium Channel Agonists / Q000494:pharmacology; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology*; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism*; D006224:Cricetinae; D003412:Cricetulus; D006367:HeLa Cells; D006706:Homeostasis / Q000187:drug effects / Q000235:genetics; D006801:Humans; D007202:Indicators and Reagents; D008969:Molecular Sequence Data; D010766:Phosphorylation; D053499:Plasma Membrane Calcium-Transporting ATPases / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D020033:Protein Isoforms / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D020798:Two-Hybrid System Techniques
D048948:14-3-3 Proteins; D002120:Calcium Channel Agonists; D007202:Indicators and Reagents; D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins; D000331:Aequorin; D053499:Plasma Membrane Calcium-Transporting ATPases; D002118:Calcium