Funding period: 01.01.2019 - 31.12.2019
The serine/threonine protein phosphatase 2A (PP2A) is ubiquitously expressed in eukaryotic cells, accounts for the majority of serine/threonine phosphatase activity and regulates many, diverse cell functions by controlling the phosphorylation state of multiple proteins. In nucleated cells, PP2A is an important regulator of cell cycle progression together with its cellular inhibitors, the cAMP-regulated phosphoprotein isoforms α-endosulfine (ENSA) and ARPP19 when phosphorylated at S67 and S62, respectively by the Ser/Thr kinases Greatwall (Xenopus, Drosophila, yeast) or the human orthologue MASTL (HeLa cells). On the other hand, cAMP/PKA-dependent phosphorylation of ARPP19 at S104 has been shown to prevent meiosis resumption in Xenopus oocytes. PP2A has been implicated as suppressor of tumors and vascular inflammation by modulating endothelial cell and leukocyte function. Therefore, PP2A has been identified as potential drug target for cancer, rheumatoid arthritis and Alzheimer’s disease where PP2A activity is pathologically decreased. However, recent studies reveal that PP2A, dependent on its isoform, exhibits also tumor promoting properties and contributes to antiphospholipid antibody-invoked thrombosis due to inhibitory dephosphorylation of Akt and eNOS in endothelial cells. In human platelets, a comprehensive proteome analysis from our collaborating partners at ISAS Dortmund demonstrated that PP2A is well expressed as heterotrimer comprising up to 16 different isoforms due to 8 regulatory, 2 catalytic and 1 scaffolding subunit and early studies provided pharmacological evidence that PP2A regulates platelet activation. Our previous data based on phosphoproteomics of human platelets and studies with recombinant ENSA and its various phosphosite mutants in lysates of human platelets showed that the stimulation of cAMP/PKA (iloprost) or cGMP/PKG (riociguat) dependent inhibitory pathways mediate reciprocal phosphorylation of ENSA at S67 (down) and S109 (up) as well as of ARPP19 at S62 (down) and S104 (up). In addition, iloprost or riociguat induced phosphorylation of PP2A at S573 of the PP2A regulatory subunit B56δ, which is associated with increased PP2A activity induced by PKA in nucleated cells. Our preliminary data further demonstrated that ENSA S67 and ARPP19 S62 phosphorylation induced by the PP2A inhibitor okadaic acid or by recombinant MASTL kinase, mediates significant inhibition of the PP2A activity in lysates of human platelets, which is associated with diminished platelet aggregation. Our phosphoproteomics and pilot studies indicated that PP2A is a key platelet phosphatase, which is differentially regulated by ENSA and ARPP19 through multiple phosphorylations and that the ENSA/ARPP19-PP2A system represents a novel and important signaling checkpoint in human platelets. However, the regulation of different platelet PP2A isoforms by the endogenous inhibitors ENSA and ARPP19, also well expressed in human platelets, and their impact on platelet inhibitory and activating signaling pathways in relation to platelet function is unknown so far.
Therefore, the hypotheses of this project are that ENSA and ARPP19 are important regulators of different isoforms of PP2A in human platelets and that decreased PP2A activity in human and mouse platelets affect platelet MAPK signaling, distinct platelet functions and thrombus formation in vitro.