Cyclic nucleotide‐dependent inhibitory signaling interweaves with activating pathways to determine platelet responses

Nagy, Z.; Smolenski, Albert

doi:10.1002/rth2.12122

Cited by 31 publications

(44 citation statements)

References 119 publications

(234 reference statements)

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“…Subsequently, in our analysis of the iloprost/cAMP-stimulated phosphoproteome of human platelets, more than 130 cAMP/PKA regulated phosphoproteins were detected including ENSA (phosphorylated at S109) and ARPP19 (at S104) [35]. Platelets are strongly inhibited by both cAMP-and cGMP-elevating agents mediated by the corresponding cAMP-and cGMP-dependent protein kinases (PKA/PKG), which have overlapping specificity [18,19]. Therefore, we investigated the effect of the cGMP/PKG-pathway on ENSA/ARPP19 phosphorylation using various NO-donors [S-nitrosocysteine (SNC), sodium-nitroprusside (SNP), diethylamine NONOate (DEA-NO)] and the soluble guanylyl cyclase (sGC)-stimulator riociguat in comparison to the iloprost (cAMP) pathway, using vasodilator-stimulated phosphoprotein (VASP) as established PKA/PKG substrate and marker.…”

Section: Resultsmentioning

confidence: 99%

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

Kumm

Pagel

Gambaryan

et al. 2020

Cells

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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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Section: Resultsmentioning

confidence: 99%

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

Kumm

Pagel

Gambaryan

et al. 2020

Cells

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“…We observed that cAMP-and cyclic guanosine monophosphate (cGMP)-elevating platelet inhibitors strongly inhibited GPIbα-/GPVI-mediated platelet activation but enhanced the initial Syk activation [28]. These phosphoproteomic and functional approaches suggest that there is a network of interacting protein kinases at the level of Syk in platelets [29,30].…”

Section: Introductionmentioning

confidence: 95%

“…This agonist-induced Syk S297 phosphorylation is also inhibited by the cAMP/PKA pathway, which is known to strongly inhibit receptor-stimulated Ca2 + responses and PKC activation [30,36,37].…”

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confidence: 99%

Feedback Regulation of Syk by Protein Kinase C in Human Platelets

Makhoul

Dorschel

Gambaryan

et al. 2019

IJMS

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The spleen tyrosine kinase (Syk) is essential for immunoreceptor tyrosine-based activation motif (ITAM)-dependent platelet activation, and it is stimulated by Src-family kinase (SFK)-/Syk-mediated phosphorylation of Y352 (interdomain-B) and Y525/526 (kinase domain). Additional sites for Syk phosphorylation and protein interactions are known but remain elusive. Since Syk S297 phosphorylation (interdomain-B) was detected in platelets, we hypothesized that this phosphorylation site regulates Syk activity via protein kinase C (PKC)-and cyclic adenosine monophosphate (cAMP)-dependent pathways. ADP, the GPVI-agonist convulxin, and the GPIbα-agonist echicetin beads (EB) were used to stimulate human platelets with/without effectors. Platelet aggregation and intracellular messengers were analyzed, along with phosphoproteins, by immunoblotting using phosphosite-specific antibodies or phos-tags. ADP, convulxin, and EB upregulated Syk S297 phosphorylation, which was inhibited by iloprost (cAMP pathway). Convulxin-stimulated Syk S297 phosphorylation was stoichiometric, transient, abolished by the PKC inhibitor GF109203X, and mimicked by the PKC activator PDBu. Convulxin/EB stimulated Syk S297, Y352, and Y525/526 phosphorylation, which was inhibited by SFK and Syk inhibitors. GFX and iloprost inhibited convulxin/EB-induced Syk S297 phosphorylation but enhanced Syk tyrosine (Y352/Y525/526) and substrate (linker adaptor for T cells (LAT), phospholipase γ2 (PLC γ2)) phosphorylation. GFX enhanced convulxin/EB-increases of inositol monophosphate/Ca2+. ITAM-activated Syk stimulates PKC-dependent Syk S297 phosphorylation, which is reduced by SFK/Syk/PKC inhibition and cAMP. Inhibition of Syk S297 phosphorylation coincides with enhanced Syk activation, suggesting that S297 phosphorylation represents a mechanism for feedback inhibition in human platelets.

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“…These factors contribute to the regulation of platelet function and haemostasis, by maintaining platelets in their inactive state. [1][2][3] PGI 2 binds to a Gs-coupled receptor, thus stimulating cAMP synthesis by adenylate cyclase. NO diffuses through the cellular membrane and activates soluble guanylate cyclase to produce cGMP.…”

Section: Introductionmentioning

confidence: 99%

The RhoA regulators Myo9b and GEF‐H1 are targets of cyclic nucleotide‐dependent kinases in platelets

Comer

Nagy

Bolado

et al. 2020

Journal of Thrombosis and Haemostasis

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Background: Circulating platelets are maintained in an inactive state by the endothelial lining of the vasculature. Endothelium-derived prostacyclin and nitric oxide stimulate cAMP-and cGMP-dependent kinases, PKA and PKG, to inhibit platelets. PKA and PKG effects include the inhibition of the GTPase RhoA, which has been suggested to involve the direct phosphorylation of RhoA on serine 188. Objectives: We wanted to confirm RhoA S188 phosphorylation by cyclic nucleotidedependent kinases and to identify possible alternative mechanisms of RhoA regulation in platelets. Methods: Phosphoproteomics data of human platelets were used to identify candidate PKA and PKG substrates. Phosphorylation of individual proteins was studied by Western blotting and Phos-tag gel electrophoresis in human platelets and transfected HEK293T cells. Pull-down assays were performed to analyze protein interaction and function. Results: Our data indicate that RhoA is not phosphorylated by PKA in platelets. Instead, we provide evidence that cyclic nucleotide effects are mediated through the phosphorylation of the RhoA-specific GTPase-activating protein Myo9b and the guanine nucleotide exchange factor GEF-H1. We identify Myo9b S1354 and guanine nucleotide exchange factor-H1 (GEF-H1) S886 as PKA and PKG phosphorylation sites. Myo9b S1354 phosphorylation enhances its GTPase activating protein function leading to reduced RhoA-GTP levels. GEF-H1 S886 phosphorylation stimulates binding of 14-3-3β and has been shown to inhibit GEF function by facilitating binding of GEF-H1 to microtubules. Microtubule disruption increases RhoA-GTP levels confirming the importance of GEF-H1 in platelets. Conclusion: Phosphorylation of RhoA regulatory proteins Myo9b and GEF-H1, but not RhoA itself, is involved in cyclic nucleotide-mediated control of RhoA in human platelets. | 3003 COMER Et al.

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Cyclic nucleotide‐dependent inhibitory signaling interweaves with activating pathways to determine platelet responses

Cited by 31 publications

References 119 publications

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

Feedback Regulation of Syk by Protein Kinase C in Human Platelets

The RhoA regulators Myo9b and GEF‐H1 are targets of cyclic nucleotide‐dependent kinases in platelets

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