Inhibitory interaction of the 14-3-3ε protein with isoform 4 of the plasma membrane Ca2+-ATPase pump

The isoform-specific interaction of plasma membrane Ca2+-ATPase (PMCA) pumps with partner proteins has been explored using a yeast two-hybrid technique. The 90 N-terminal residues of two pump isoforms (PMCA2 and PMCA4), which have a low degree of sequence homology, have been used as baits. Screening of 5x10(6) clones of a human brain cDNA library yielded similar to 100 LEU2- and galactoside-positive clones for both pumps. A clone obtained with the PMCA4 bait specified the epsilon-isoform of the 14-3-3 protein, whereas no 14-3-3 epsilon clone was obtained with the PMCA2 bait. The 14-3-3 epsilon protein immunoprecipitated with PMCA4 (not with PMCA2) when expressed in HeLa cells. Overexpression of 14-3-3 epsilon in HeLa cells together with targeted aequorins showed that the ability of the cells to export Ca2+ was impaired; stimulation with histamine, an inositol 1,4,5-trisphosphate-producing agonist, generated higher cytosolic [Ca2+] transients, higher post-transient plateaus of the cytosolic [Ca2+], and higher Ca2+ levels in the endoplasmic reticulum lumen and in the subplasmalemmal domain. Thus, the interaction with 14-3-3 epsilon inhibited PMCA4. Silencing of the 14-3-3 epsilon gene by RNA interference significantly reduced the expression of 14-3-3 epsilon, substantially decreasing the height of the histamine-induced cytosolic [Ca2+] transient and of the post-transient cytosolic [Ca2+] plateau.