Analysis of the cell cycle regulatory system MASTL-ENSA/ARPP19-PP2A in human platelets
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Abstract
Small non-nucleated platelets, circulating in the blood, are activated upon vessel injury, to immediately stop the blood loss via thrombus formation. Under resting conditions, circulating platelets are inhibited through nitric oxide (NO, activating protein kinase G (PKG)) or prostacyclin (PGI2, activating protein kinase A (PKA)). These inhibitors induce phosphorylation of multiple proteins inside the platelet to inhibit platelet activation and signaling pathways. Platelet signal transduction upon activation or inhibition is regulated through protein kinases and phosphatases.
A full phosphoproteomic study headed by PD Dr. K. Jurk and Dr. R. Zahedi with inhibited human platelets (data unpublished), revealed a high number of proteins regulated upon PKA and PKG activation. Two proteins, α-endosulfine (ENSA or ARPP19e) and cAMP-regulated phosphoprotein 19 (ARPP19), were strongly phosphorylated at S109/S104 (ENSA/ARPP19) in response to PKA and PKG activation (PKG effect was unknown before). The functional role of the PKA phosphorylation of ENSA and ARPP19 (identified years ago in different cell systems) is still unknown. The phosphorylation of ENSA/ARPP19 at S67/S62 via the Greatwall kinase in Drosophila, Xenopus and yeast and in mammalian cells by its homolog, the microtubule-associated serine/threonine kinase-like (MASTL), converts ENSA and ARPP19 to strong inhibitors of the protein phosphatase PP2A resembling the pharmacological inhibitor okadaic acid (OA). In dividing cells, this PP2A regulation is important for the cell cycle control; it is not reported for platelets so far.
The overall aim of this thesis was to elucidate the regulation and function of ENSA S67/S109 and ARPP19 S62/S104 phosphorylation in human platelets and the relation to the MASTL-ENSA/ARPP19-PP2A regulatory mechanism. As experimental approaches, ENSA and ARPP19 phosphorylation was analyzed by recombinant MASTL, recombinant PKG and purified PKA, as well as ENSA and ARPP19 phosphorylation at S67/S62 in intact human platelets and platelet lysates. The influence of recombinant pS67 ENSA/pS62 ARPP19 on platelet PP2A activity and the functional relevance of PP2A inhibition for platelet activation was investigated.
ENSA, ARPP19 and PP2A, but not MASTL, were detected in human platelets at significant protein levels. ENSA, cloned from human platelets, was expressed in HEK293 cells and E.coli BL21, on top, recombinant HisENSA was purified from BL21. ENSA and ARPP19 phosphorylation was studied with intact human platelets, platelet lysates and recombinant proteins as well as with ENSA phosphosite-mutants. ENSA S67/ARPP19 S62 phosphorylation was elevated upon phosphatase PP1/PP2A inhibition with OA for endogenous ENSA, recombinant ARPP19 and wild type ENSA in human platelet lysates, and in intact platelets. These data clearly demonstrated the presence of a MASTL-like kinase activity in human platelets. A broad spectrum of PP2A holoenzymes was shown to be present in human platelets. pS67 HisENSA and pS62 GST-ARPP19 reduced platelet PP2A activity of about 20% (compared to unphosphorylated control) in platelet lysates, using a PP2A specific non-radioactive serine/threonine phosphatase activity assay. With 1 nM of OA, platelet PP2A activity was completely inhibited. The phosphorylation of PP2A substrates (vasodilator-stimulated phosphoprotein, VASP, and mitogen-activated protein kinase (MAPK), p38) was increased in low dose OA treated isolated human platelets. The same concentration of OA decreased platelet activation/aggregation upon thrombin stimulation, revealing a regulatory role of PP2A upon platelet activation mechanisms in human platelets.
Based on these results, it can be concluded that human platelets contain all components of the [MASTL-like]–ENSA/ARPP19–PP2A (B55/B56) pathway, which is essential for the cell cycle control but must have other function(s) in non-dividing platelets. In the future it will be important to elucidate the molecular and functional targets of the [MASTL-like]-ENSA/ARPP19-PP2A pathway in human platelets.