The Cell Cycle Checkpoint System MAST(L)-ENSA/ARPP19-PP2A is Targeted by cAMP/PKA and cGMP/PKG in Anucleate Human Platelets

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.

Platelet-Derived GARP Induces Peripheral Regulatory T Cells—Potential Impact on T Cell Suppression in Patients with Melanoma-Associated Thrombocytosis

“…Citrated blood was supplemented with 2 mM EGTA and centrifuged for 10 min at 200× g. The resulting PRP was diluted with CGS buffer (120 mM NaCl, 12.9 mM trisodium citrate dihydrate, 30 mM d-glucose, pH 6.5) at a ratio of 1:1. For leukocyte depletion, the PRP was centrifuged for 10 min at 69× g. The resulting supernatant was subsequent centrifuged for 10 min at 400× g. The resulting pellet was resuspended in CGS buffer and again centrifuged for 10 min at 400× g. The platelets were resuspended in 1× PBS for flow cytometry [56].…”

Cryptochrome 1 Alleviates the Antiproliferative Effect of Isoproterenol on Human Gastric Cancer Cells

“…The activation of the cAMP/PKA pathway generally leads to the suppression of the MAPK pathway. 13 - 17 And the phosphorylation of ERK1/2 is in line with the activation of the MAPK pathway. In our experiments, it was observed that the phosphorylation level of ERK1/2 was obviously decreased in the HGC-27 control cells after treatment of ISO, meanwhile the phosphorylation level of ERK1/2 was retained in the CRY1o cells ( Figure 4B ).…”

“… 9 - 12 The activation of the cAMP/PKA pathway generally inhibits the MAPK pathway, leading to inhibition of cell growth and proliferation. 13 - 17 Nevertheless, the levels of intracellular cAMP was altered by changes in β-adrenergic receptors, and then cell growth and differentiation were affected. 18 The β2-adrenergic antagonists was reported to suppress invasion and proliferation in pancreatic cancer cell by inhibiting cAMP/PKA pathway, which could regulate activation of the MAPK pathway.…”

Knockdown of Microtubule Associated Serine/threonine Kinase Like Expression Inhibits Gastric Cancer Cell Growth and Induces Apoptosis by Activation of ERK1/2 and Inactivation of NF-κB Signaling

“…A bioinformatical tool analyzer showed that MASTL could be stimulated by many TFs, such as SPI1 and KLF5 in stomach tissues [39] . In the other hand, MASTL could activate downstream signal pathway, such as AKT/mTOR and Wnt/b-catenin oncogenic kinase signaling [13] , the PKA, MAPK and PI3K/Akt signaling in human platelets [40] . PP1 regulatory subunit 3B (PPP1R3B) was found as a cell cycle regulator by governing MASTL dephosphorylation [41] .…”