The Cell Cycle Checkpoint System MAST(L)-ENSA/ARPP19-PP2A is Targeted by cAMP/PKA and cGMP/PKG in Anucleate Human Platelets
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 proteo...
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MDPI AG
2020-02-01
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| Online Access: | https://www.mdpi.com/2073-4409/9/2/472 |
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| author | Elena J. Kumm Oliver Pagel Stepan Gambaryan Ulrich Walter René P. Zahedi Albert Smolenski Kerstin Jurk |
| author_facet | Elena J. Kumm Oliver Pagel Stepan Gambaryan Ulrich Walter René P. Zahedi Albert Smolenski Kerstin Jurk |
| author_sort | Elena J. Kumm |
| collection | DOAJ |
| description | 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. |
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| last_indexed | 2024-03-12T06:41:03Z |
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| series | Cells |
| spelling | doaj.art-5586b17b8ff842898b521527567836392023-09-03T01:02:34ZengMDPI AGCells2073-44092020-02-019247210.3390/cells9020472cells9020472The Cell Cycle Checkpoint System MAST(L)-ENSA/ARPP19-PP2A is Targeted by cAMP/PKA and cGMP/PKG in Anucleate Human PlateletsElena J. Kumm0Oliver Pagel1Stepan Gambaryan2Ulrich Walter3René P. Zahedi4Albert Smolenski5Kerstin Jurk6Center for Thrombosis and Hemostasis (CTH), University Medical Center of the Johannes Gutenberg-University Mainz, 55131 Mainz, GermanyLeibniz-Institut für Analytische Wissenschaften—ISAS—e.V., 44227 Dortmund, GermanyCenter for Thrombosis and Hemostasis (CTH), University Medical Center of the Johannes Gutenberg-University Mainz, 55131 Mainz, GermanyCenter for Thrombosis and Hemostasis (CTH), University Medical Center of the Johannes Gutenberg-University Mainz, 55131 Mainz, GermanyLeibniz-Institut für Analytische Wissenschaften—ISAS—e.V., 44227 Dortmund, GermanyUCD Conway Institute, UCD School of Medicine and Medical Science, University College Dublin, D04 V1W8 Dublin, IrelandCenter for Thrombosis and Hemostasis (CTH), University Medical Center of the Johannes Gutenberg-University Mainz, 55131 Mainz, GermanyThe 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.https://www.mdpi.com/2073-4409/9/2/472plateletsserine/threonine protein phosphatasescyclic ampcyclic gmpensaarpp19map kinase |
| spellingShingle | Elena J. Kumm Oliver Pagel Stepan Gambaryan Ulrich Walter René P. Zahedi Albert Smolenski Kerstin Jurk The Cell Cycle Checkpoint System MAST(L)-ENSA/ARPP19-PP2A is Targeted by cAMP/PKA and cGMP/PKG in Anucleate Human Platelets Cells platelets serine/threonine protein phosphatases cyclic amp cyclic gmp ensa arpp19 map kinase |
| title | The Cell Cycle Checkpoint System MAST(L)-ENSA/ARPP19-PP2A is Targeted by cAMP/PKA and cGMP/PKG in Anucleate Human Platelets |
| title_full | The Cell Cycle Checkpoint System MAST(L)-ENSA/ARPP19-PP2A is Targeted by cAMP/PKA and cGMP/PKG in Anucleate Human Platelets |
| title_fullStr | The Cell Cycle Checkpoint System MAST(L)-ENSA/ARPP19-PP2A is Targeted by cAMP/PKA and cGMP/PKG in Anucleate Human Platelets |
| title_full_unstemmed | The Cell Cycle Checkpoint System MAST(L)-ENSA/ARPP19-PP2A is Targeted by cAMP/PKA and cGMP/PKG in Anucleate Human Platelets |
| title_short | The Cell Cycle Checkpoint System MAST(L)-ENSA/ARPP19-PP2A is Targeted by cAMP/PKA and cGMP/PKG in Anucleate Human Platelets |
| title_sort | cell cycle checkpoint system mast l ensa arpp19 pp2a is targeted by camp pka and cgmp pkg in anucleate human platelets |
| topic | platelets serine/threonine protein phosphatases cyclic amp cyclic gmp ensa arpp19 map kinase |
| url | https://www.mdpi.com/2073-4409/9/2/472 |
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