RESUMO
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.
Assuntos
Plaquetas/metabolismo , Pontos de Checagem do Ciclo Celular/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Fosfoproteínas/metabolismo , Proteína Fosfatase 2/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Doadores de Sangue , Plaquetas/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Células HEK293 , Humanos , Ácido Okadáico/farmacologia , Fosforilação/efeitos dos fármacos , Fosforilação/genética , Agregação Plaquetária/efeitos dos fármacos , Agregação Plaquetária/genética , Proteína Fosfatase 2/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , TransfecçãoRESUMO
Platelets have been recently described as an important component of the innate and adaptive immunity through their interaction with immune cells. However, information on the platelet-T cell interaction in immune-mediated diseases remains limited. Glycoprotein A repetitions predominant (GARP) expressed on platelets and on activated regulatory T cells (Treg) is involved in the regulation of peripheral immune responses by modulating the bioavailability of transforming growth factor ß (TGF-ß). Soluble GARP (sGARP) exhibits strong regulatory and anti-inflammatory capacities both in vitro and in vivo, leading to the induction of peripheral Treg. Herein, we investigated the effect of platelet-derived GARP on the differentiation, phenotype, and function of T effector cells. CD4+CD25- T cells cocultured with platelets upregulated FoxP3, the master transcription factor for Treg, were anergic, and were strongly suppressive. These effects were reversed by using a blocking anti-GARP antibody, indicating a dependency on GARP. Importantly, melanoma patients in different stages of disease showed a significant upregulation of GARP on the platelet surface, correlating to a reduced responsiveness to immunotherapy. In conclusion, our data indicate that platelets induce peripheral Treg via GARP. These findings might contribute to diseases such as cancer-associated thrombocytosis, wherein poor prognosis and metastasis are associated with high counts of circulating platelets.