Regulation of Plasma Membrane Ca2+-ATPase by Small GTPases and Phosphoinositides in Human Platelets

The plasma membrane Ca 2؉ -ATPase (PMCA) plays an essential role in maintaining low cytosolic Ca 2؉ in resting platelets. During platelet activation PMCA is phosphorylated transiently on tyrosine residues resulting in inhibition of the pump that enhances elevation of Ca 2؉ . Tyrosine phosphorylation of many proteins during platelet activation results in their association with the cytoskeleton. Consequently, in the present study we asked if PMCA interacts with the platelet cytoskeleton. We observed that very little PMCA is associated with the cytoskeleton in resting platelets but that ϳ80% of total PMCA (PMCA1b ؉ PMCA4b) is redistributed to the cytoskeleton upon activation with thrombin. Tyrosine phosphorylation of PMCA during activation was not associated with the redistribution because tyrosine-phosphorylated PMCA was not translocated specifically to the cytoskeleton. Because PMCA b-splice isoforms have C-terminal PSD-95/Dlg/ZO-1 homology domain (PDZ)-binding domains, a C-terminal peptide was used to disrupt potential PDZ domain interactions. Activation of saponin-permeabilized platelets in the presence of the peptide led to a significant decrease of PMCA in the cytoskeleton. PMCA associated with the cytoskeleton retained Ca 2؉ -ATPase activity. These results suggest that during activation active PMCA is recruited to the cytoskeleton by interaction with PDZ domains and that this association provides a microenvironment with a reduced Ca 2؉ concentration.

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confidence: 87%

Section: Plasma Membrane Ca 2ϩ -Atpase Binding To Platelet Cytoskeletonmentioning

confidence: 99%

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confidence: 99%

See 1 more Smart Citation

Plasma Membrane Ca2+-ATPase Associates with the Cytoskeleton in Activated Platelets through a PDZ-binding Domain

Zabe

Dean

2001

“…Cells loaded with fluo-4 were stimulated with 50 -420 nM ionomycin in Na ϩ /Ca 2ϩ -free PSS containing 100 M BAPTA (Molecular Probes) and 5 M cyclopiazonic acid to obtain various levels of peak [Ca 2ϩ ] i and establish a Ca 2ϩ efflux profile curve. A calcium chelator, BAPTA was included to block Ca 2ϩ influx (27), and the addition of cyclopiazonic acid prevented Ca 2ϩ sequestration into the endoplasmic reticulum by SERCA; this concentra-…”

Section: Methodsmentioning

confidence: 99%

Antisense-mediated Inhibition of the Plasma Membrane Calcium-ATPase Suppresses Proliferation of MCF-7 Cells

Lee

Robinson

Holman

et al. 2005

Alterations in Ca2؉ signaling may contribute to tumorigenesis and the mechanism of action of some anticancer drugs. The plasma membrane calcium-ATPase (PMCA) is a crucial controller of intracellular Ca 2؉ signaling. Altered PMCA expression occurs in the mammary gland during lactation and in breast cancer cell lines. Despite this, the consequences of PMCA inhibition in breast cancer cell lines have not been investigated. In this work, we used Tet-off PMCA antisense-expressing MCF-7 cells to assess the effects of PMCA inhibition in a human breast cancer cell line. At a level of PMCA inhibition that did not completely prevent PMCA-mediated Ca 2؉ efflux and did not induce cell death, a dramatic inhibition of cellular proliferation was observed. Fluorescence-activated cell sorting analysis indicated that PMCA antisense involves changes in cell cycle kinetics but not cell cycle arrest. We concluded that modulation of PMCA has important effects in regulating the proliferation of human breast cancer MCF-7 cells.

“…The results of the deletion 296 -349 suggest that, through the interaction of the A L region with the acidic lipids of the membrane, the PMCA could adopt a fourth state characterized by low V max and intermediate or high affinity for Ca 2ϩ . It has been suggested that a lower rate of Ca 2ϩ extrusion by the PMCA due to a decrease in the level of activating phosphoinositides facilitates the rapid transient elevation of intracellular Ca 2ϩ after stimulation by agonists (34,35). Because the affinity for acidic lipids at region A L would seem higher than that of the C region (9,12,13), it is tempting to speculate that under normal resting conditions when the free cytoplasmic Ca 2ϩ level decreases to a level where calmodulin activation is insignificant, the phosphoinositides keep the PMCA in an activated state similar to that exhibited by the d296 -349 enzyme, and in response to the activation induced by an agonist, the PMCA would lower its affinity for Ca 2ϩ .…”

Section: Discussionmentioning

confidence: 99%

Deletions in the Acidic Lipid-binding Region of the Plasma Membrane Ca2+ Pump

Pinto

Adamo

2002

The C-terminal segment of the loop between transmembrane helices 2 and 3 (A L region) of the plasma membrane Ca 2؉ pump (PMCA) is not conserved in other P-ATPases. Part of this region, just upstream from the third transmembrane domain, has been associated with activation of the PMCA by acidic lipids. cDNAs coding for mutants of the Ca 2؉ pump isoform h4xb with deletions in the A L region were constructed, and the proteins were successfully expressed in either COS or Chinese hamster ovary cells. Mutants with deletions in the segment 296 -349 had full Ca 2؉ transport activity, but deletions involving the segment of amino acids 350 -356 were inactive suggesting that these residues are required for a functional PMCA. In the absence of calmodulin the V max of mutant d296 -349 was similar to that of the recombinant wild type pump, but its K 0.5 for Ca