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

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Title: The RhoA regulators Myo9b and GEF-H1 are targets of cyclic nucleotide-dependent kinases in platelets
Other Titles: RhoA regulation by Myo9b and GEF-H1 in platelets
Authors: Comer, ShaneNagy, ZoltanBolado, AlfonsoSmolenski, Albert al.
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Date: Nov-2020
Online since: 2021-11-10T10:38:54Z
Abstract: 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 nucleotide-dependent 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.
Funding Details: University College Dublin
Funding Details: Russian Foundation For Basic Research
Deutsche Forschungsgemeinschaft
Type of material: Journal Article
Publisher: Wiley
Journal: Journal of Thrombosis and Haemostasis
Volume: 18
Issue: 11
Start page: 3002
End page: 3012
Copyright (published version): 2020 International Society on Thrombosis and Haemostasis
Keywords: Peripheral vascular disease14-3-3 proteinsCyclic AMP-dependent protein kinasesCyclic GMP-dependent protein kinasesGTPase-activating proteinsGuanine nucleotide exchange factorsPhosphorylationGTPase-activating proteinShape changeMyosin IXBSignaling pathwayPhosphorylationPhosphataseRevealsBindingContractionThrombin
DOI: 10.1111/jth.15028
Language: en
Status of Item: Peer reviewed
ISSN: 1538-7933
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Appears in Collections:Conway Institute Research Collection
Medicine Research Collection

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