Inhibitory interaction of the 14-3-3{epsilon} protein with isoform 4 of the plasma membrane Ca(2+)-ATPase pump.
The isoform-specific interaction of plasma membrane Ca(2+)-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 5 x 10(6) clones of a human brain cDNA library yielded approximately 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 14-3-3epsilon clone was obtained with the PMCA2 bait. The 14-3-3epsilon protein immunoprecipitated with PMCA4 (not with PMCA2) when expressed in HeLa cells. Overexpression of 14-3-3epsilon in HeLa cells together with targeted aequorins showed that the ability of the cells to export Ca(2+) was impaired; stimulation with histamine, an inositol 1,4,5-trisphosphate-producing agonist, generated higher cytosolic [Ca(2+)] transients, higher post-transient plateaus of the cytosolic [Ca(2+)], and higher Ca(2+) levels in the endoplasmic reticulum lumen and in the subplasmalemmal domain. Thus, the interaction with 14-3-3epsilon inhibited PMCA4. Silencing of the 14-3-3epsilon gene by RNA interference significantly reduced the expression of 14-3-3epsilon, substantially decreasing the height of the histamine-induced cytosolic [Ca(2+)] transient and of the post-transient cytosolic [Ca(2+)] plateau.
Rimessi
Alessandro
A
Department of Biochemistry, University of Padova, Viale G. Colombo 3, 35121 Padova, Italy.
Coletto
Luisa
L
Pinton
Paolo
P
Rizzuto
Rosario
R
Brini
Marisa
M
Carafoli
Ernesto
E
D048948:14-3-3 Proteins / Q000235:genetics / Q000378:metabolism*; D000331:Aequorin / Q000378:metabolism; D001921:Brain / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000235:genetics / Q000378:metabolism*; D027682:Cation Transport Proteins / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000201:enzymology*; D004721:Endoplasmic Reticulum / Q000378:metabolism; D015723:Gene Library; D020868:Gene Silencing; D006367:HeLa Cells; D006632:Histamine / Q000494:pharmacology; D006801:Humans; D047468:Immunoprecipitation; D053499:Plasma Membrane Calcium-Transporting ATPases; D010957:Plasmids; D020033:Protein Isoforms; D034741:RNA, Small Interfering / Q000494:pharmacology; D012441:Saccharomyces cerevisiae; D020798:Two-Hybrid System Techniques
D048948:14-3-3 Proteins; D027682:Cation Transport Proteins; D020033:Protein Isoforms; D034741:RNA, Small Interfering; D000331:Aequorin; D006632:Histamine; D053499:Plasma Membrane Calcium-Transporting ATPases; C506958:ATP2B2 protein, human; D000252:Calcium-Transporting ATPases; D002118:Calcium