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

Abstract

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.

SERCA2b Activity Is Regulated by Cyclophilins in Human Platelets

“…This finding is different from previous results, but these discrepancies might be attributed to a different effect of CsA on distinct PMCA isoforms (PMCA1 and PMCA4), with PMCA4 being the isoform predominantly expressed in platelets, more likely insensitive to CsA. 17,33,34 To identify the SERCA isoform regulated by CsA, we stimulated platelets with Thr in the presence of specific inhibitors of the different SERCA isoforms in these cells (SERCA2b and SERCA3). 18 -21 When SERCA3 was inhibited by TBHQ, CsA was still able to alter Thr-induced Ca 2ϩ mobilization, including the ability of platelets to accumulate Ca 2ϩ into intracellular stores; however, when SERCA2b was inhibited by TG, CsA was unable to modify the Thr-evoked response, thus indicating that SERCA2b activity was inhibited in the presence of CsA.…”

Plasma Membrane Ca2+ATPase Isoform 4b Is Cleaved and Activated by Caspase-3 during the Early Phase of Apoptosis

“…These workers also demonstrated the exclusive presence and co-localization of PMCA4b and SAP97 in the basolateral membrane of polarized Madin-Darby canine kidney cells. In a recent report, Zabe and Dean (60) suggested that the PMCA4b C-terminal PDZ binding sequence is involved in the association with the actin cytoskeleton of platelets and that the association in-creases dramatically upon activation with thrombin. The PDZbinding tail of PMCA4b was also shown to interact with the PDZ domain of nitric-oxide synthase and through this interaction PMCA4b dramatically inhibits nitric oxide synthesis (61).…”

Plasma Membrane Ca2+-ATPase Isoforms 2b and 4b Interact Promiscuously and Selectively with Members of the Membrane-associated Guanylate Kinase Family of PDZ (PSD95/Dlg/ZO-1) Domain-containing Proteins

“…that of SAP97 to PMCA4b in kidney epithelial cells, may serve to tether the Ca 2ϩ pump to the membrane cytoskeletal network and thereby help retain the PMCA in the proper membrane domain. Indeed, very recent work implicates a PDZ domain interaction in the association of PMCA4b with the membrane cytoskeleton in platelets (55). In these cells, the cytoskeletal attachment of the pump could be disrupted by the addition of a competitor peptide corresponding to the PMCA4b carboxyl terminus, presumably by disrupting a PDZ domain-PMCA4b interaction.…”