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

Shane Comer1,2, Zoltan Nagy1,3, Alfonso Bolado4

  • 1UCD School of Medicine and Conway Institute, University College Dublin, Belfield, Dublin 4, Ireland.

Journal of Thrombosis and Haemostasis : JTH
|July 22, 2020
PubMed

Insights

Platelet inhibition by cyclic nucleotides does not involve RhoA phosphorylation. Instead, it is mediated by phosphorylating Myo9b and GEF-H1, key regulators of RhoA activity in human platelets.

Area of Science:

  • Platelet biology and signaling
  • Molecular mechanisms of cell regulation
  • GTPase signaling pathways

Background:

  • Platelets remain inactive due to endothelial factors like prostacyclin and nitric oxide.
  • These factors activate kinases PKA and PKG, which inhibit platelets by affecting RhoA signaling.
  • RhoA phosphorylation at serine 188 was previously hypothesized as a mechanism for this inhibition.

Purpose of the Study:

  • To confirm if cyclic nucleotide-dependent kinases phosphorylate RhoA at S188 in platelets.
  • To explore alternative pathways for RhoA regulation by cyclic nucleotides in platelets.

Main Methods:

  • Utilized phosphoproteomics data from human platelets to identify potential PKA and PKG targets.
  • Confirmed protein phosphorylation using Western blotting and Phos-tag gel electrophoresis in human platelets and HEK293T cells.
  • Employed pull-down assays to investigate protein interactions and functions.

Main Results:

  • RhoA is not phosphorylated by PKA in human platelets.
  • Cyclic nucleotide effects are mediated by phosphorylating Myo9b (at S1354) and GEF-H1 (at S886).
  • Myo9b phosphorylation enhances its GTPase-activating protein activity, reducing RhoA-GTP levels; GEF-H1 phosphorylation inhibits its GEF function, impacting RhoA activity.

Conclusions:

  • Cyclic nucleotide-mediated control of RhoA in human platelets involves the phosphorylation of its regulators, Myo9b and GEF-H1.
  • RhoA itself is not directly phosphorylated by these kinases in platelets.
  • These findings reveal a novel regulatory mechanism for RhoA in platelet function.
Abstract

Keywords:
14-3-3 proteinsGTPase-activating proteinscyclic AMP-dependent protein kinasescyclic GMP-dependent protein kinasesguanine nucleotide exchange factorsphosphorylation

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