Abstract
The cell cycle is controlled by microtubule-associated serine/threonine kinase-like (MASTL), which phosphorylates the cAMP-regulated phosphoproteins 19 (ARPP19) at S62 and 19e/α-endosulfine (ENSA) at S67and converts them into protein phosphatase 2A (PP2A) inhibitors. Based on initial proteomic data, we hypothesized that the MASTL-ENSA/ARPP19-PP2A pathway, unknown until now in platelets, is regulated and functional in these anucleate cells. We detected ENSA, ARPP19 and various PP2A subunits (including seven different PP2A B-subunits) in proteomic studies of human platelets. ENSA-S109/ARPP19–S104 were efficiently phosphorylated in platelets treated with cAMP- (iloprost) and cGMP-elevating (NO donors/riociguat) agents. ENSA-S67/ARPP19-S62 phosphorylations increased following PP2A inhibition by okadaic acid (OA) in intact and lysed platelets indicating the presence of MASTL or a related protein kinase in human platelets. These data were validated with recombinant ENSA/ARPP19 and phospho-mutants using recombinant MASTL, protein kinase A and G. Both ARPP19 phosphorylation sites S62/S104 were dephosphorylated by platelet PP2A, but only S62-phosphorylated ARPP19 acted as PP2A inhibitor. Low-dose OA treatment of platelets caused PP2A inhibition, diminished thrombin-stimulated platelet aggregation and increased phosphorylation of distinct sites of VASP, Akt, p38 and ERK1/2 MAP kinases. In summary, our data establish the entire MASTL(like)–ENSA/ARPP19–PP2A pathway in human platelets and important interactions with the PKA, MAPK and PI3K/Akt systems.
Highlights
Platelets are small, anucleate blood cells, which are essential in physiological and pathological haemostasis and have important roles in inflammation, atherosclerosis and cancer [1,2,3,4,5]
Satoru Mochida (Kumamoto University, Kurokami, Chuo Ward, Japan); PAN vasodilator-stimulated phosphoprotein (VASP) antibody and antibody against ENSA pS109/ARPP19 pS104 were custom prepared by ImmunoGlobe®, Himmelstadt, Germany; HisTrap columns (1 mL) and PD-10 desalting columns were from GE Healthcare, Chicago, IL USA; E. coli BL21 (DE3) were from New England Biolabs (NEB), Frankfurt am Main, Germany; pET28a vector was from Novagen/Merck KGaA, Darmstadt, Germany; human embryonic kidney cells
This work shows that the well-established microtubule-associated serine/threonine kinase-like (MASTL)-ENSA/ARPP19-phosphatase 2A (PP2A) pathway, which is essential for the cell cycle control of most dividing eukaryotic cells, is present and regulated in non-dividing, anucleate human platelets (Figure 8)
Summary
Anucleate blood cells, which are essential in physiological and pathological haemostasis and have important roles in inflammation, atherosclerosis and cancer [1,2,3,4,5]. Either inherited or acquired dysfunctions of human platelets may cause serious, even lethal bleeding or thrombotic complications [3,6] In addition to their essential regulation of haemostasis and coagulation, activated platelets release and secrete, primarily from their α- and δ-granules, more than 300 biomolecules and proteins, which affect other platelets, blood, vascular and tissue cells [7,8]. These secreted platelet factors regulate multiple physiological and pathophysiological processes such as microvascular integrity, wound healing, inflammation, tumour stability and metastasis [7,8]. Targeting and blocking of enhanced platelet activation by TXA2 synthesis inhibitors (aspirin) and/or ADP-receptor antagonists (thienopyridines) have been well established as the most effective intervention to prevent and attenuate complications of various acute and chronic cardiovascular diseases [9]
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