ZNF185 prevents stress fiber formation through the inhibition of RhoA in endothelial cells

Abstract

Signaling through cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) promotes endothelial barrier function to prevent plasma leakage induced by inflammatory mediators. The discovery of novel PKA substrates in endothelial cells has increased our understanding of the molecular mechanisms involved in vessel maturation. In this study, we evaluated a cAMP inducer, forskolin, and a phospho-PKA substrate antibody to identify ZNF185 as a PKA substrate. ZNF185 interacted with PKA and a deletion mutant of ZNF185, which lacks the PKA-interacting domain, was not phosphorylated by PKA. In addition to PKA, ZNF185 colocalized with F-actin in endothelial cells. Both phospho-ZNF185 and F-actin accumulated in the plasma membrane region in response to forskolin to stabilize the cortical actin structure. By contrast, ZNF185 knockdown disrupted actin filaments and promoted stress fiber formation without inflammatory mediators. Constitutive activation of RhoA was induced by ZNF185 knockdown, which resulted in forskolin-resistant endothelial barrier dysfunction. ZNF185 was essential for cAMP/PKA/RhoA signaling for the suppression of endothelial hyperpermeability. Thrombin protease was used as a positive control to assemble stress fibers via RhoA activation. Unexpectedly, ZNF185 was cleaved by thrombin, resulting in an N-terminal actin-targeting domain and a C-terminal PKA-interacting domain. Irreversible dysfunction of ZNF185 protein potentially causes RhoA-dependent stress fiber formation by thrombin.