Plasma membrane Ca(2+)-ATPase associates with the cytoskeleton in activated platelets through a PDZ-binding domain.
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 approximately 80% 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.
Department of Biochemistry and Molecular Biology, School of Medicine, University of Louisville, Louisville, Kentucky 40292, USA.
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Dean
D000595:Amino Acid Sequence; D001792:Blood Platelets / Q000201:enzymology*; D000252:Calcium-Transporting ATPases / Q000737:chemistry / Q000378:metabolism*; D002462:Cell Membrane / Q000201:enzymology; D003599:Cytoskeleton / Q000201:enzymology*; D006801:Humans; D010766:Phosphorylation; D015539:Platelet Activation; D014443:Tyrosine / Q000378:metabolism
D014443:Tyrosine; D000252:Calcium-Transporting ATPases