Effects of plasma membrane Ca2+‐ATPase tyrosine phosphorylation on human platelet function

Abstract

Summary. Background: The plasma membrane Ca 2+ -ATPase (PMCA) plays an essential role in maintaining low intracellular Ca 2+ ([Ca 2+ ] i ) in resting platelets. Earlier studies demonstrated that platelet activation by thrombin results in tyrosine phosphorylation of PMCA, which inhibits pump activity. Objectives: The objective was to determine the functional consequences of PMCA tyrosine phosphorylation. Methods: A decapeptide including the tyrosine phosphorylation site of PMCA and a scrambled version were synthesized and introduced into human platelets using saponin. Fura-2 calcium monitoring and aggregometry were used to characterize the effects of inhibition of tyrosine phosphorylation. Results: Western blot analysis of immunoprecipitates showed that introduction of the inhibitory peptide decreased tyrosine phosphorylation of PMCA by nearly 60% in saponin-permeabilized, thrombin-treated platelets as compared with the scrambled control peptide. Concomitant with inhibition of PMCA tyrosine phosphorylation was a significant decrease in [Ca 2+ ] i during thrombin-mediated platelet activation. The functional consequence of reduced PMCA tyrosine phosphorylation and decreased [Ca 2+ ] i was a significant delay in the onset of thrombin-mediated platelet aggregation. Conclusions: The results demonstrate that PMCA tyrosine phosphorylation regulates [Ca 2+ ] i during platelet activation, which affects downstream events in the activation process. Moreover, PMCA tyrosine phosphorylation and resultant inhibition of PMCA activity produces a positive feedback loop mechanism by enhancing the increase in [Ca 2+ ] i accompanying platelet activation.

Inhibition of the Plasma Membrane Ca2+Pump by CD44 Receptor Activation of Tyrosine Kinases Increases the Action Potential Afterhyperpolarization in Sensory Neurons

“…Coordinated modulation of PMCA-mediated Ca 2+ clearance might help to shape [Ca 2+ ] i microdomains needed for localized adhesion. FAK phosphorylation of PMCA4 in platelets contributes to thrombin-induced platelet adhesion (Bozulic et al, 2007), consistent with the idea that FAK regulation of Ca 2+ clearance modulates cell motility.…”

“…The modulation of PMCA4 by PTKs in platelets and B cells (Chen et al, 2004; Bozulic et al, 2007) provides a precedent for the Ca 2+ pump modulation described here. The loss of tonic inhibition of Ca 2+ clearance in PMCA4 knockdown cells supports this contention (Fig.…”

“…This is the first suggestion that HA might control the excitability of sensory neurons, although enzymatic breakdown of the ECM increases the excitability of interneurons (Dityatev et al, 2007). Other Ca 2+ -dependent processes that might be particularly sensitive to the rate of PMCA-mediated Ca 2+ clearance include cell adhesion and migration (Bozulic et al, 2007), neurotransmitter release (Jensen et al, 2007), synaptic plasticity (Simons et al, 2009), and survival (Fernandes et al, 2007). …”

“…Antigen cross-linking of the B cell receptor produces an increase in intracellular Ca 2+ concentration ([Ca 2+ ] i ) that is inhibited following PMCA activation by the tyrosine phosphatase SHP-1 (Chen et al, 2004). PMCA isoform 4 is phosphorylated during platelet activation, probably by focal adhesion kinase (FAK) (Wan et al, 2003), resulting in slowed Ca 2+ clearance (Bozulic et al, 2007). …”

Calcium Transport Mechanisms of PC12 Cells

“…It is already known that components of transporters can be regulated through changes of phosphorylation state (Toyofuku et al, 1993; Bozulic et al, 2007). The summed model equations we give describe the clearance after 3-s well.…”