Serine phosphorylation of the RhoGEF Trio stabilizes endothelial cell-cell junctions

• Published in: Small GTPases 2023-12, Vol.14 (1), p.45-54
• Main Authors: Daniel, Anna E, van der Meer, Werner J, Wester, Lynn, de Waard, Vivian, van den Biggelaar, Maartje, van Buul, Jaap D
• Format: Article
• Language: English
• Subjects: Brief Report; Endothelial Cells - metabolism; Intercellular Junctions - metabolism; Phosphorylation; Protein Serine-Threonine Kinases - metabolism; rac1 GTP-Binding Protein - metabolism; Rho Guanine Nucleotide Exchange Factors - genetics; Rho Guanine Nucleotide Exchange Factors - metabolism; Thrombin
• ISSN: 2154-1248; 2154-1256
• DOI: 10.1080/21541248.2023.2242166

The RhoGEF Trio is a large multi-domain protein and an activator of the small GTPases Rac1, RhoG, and RhoA. Although Trio has been implicated in many cellular mechanisms like leukocyte transendothelial migration, cell-cell junction stability, lamellipodia formation, axon outgrowth, and muscle fusion, it remains unclear how Trio is activated. Using stable isotope labelling by amino acids in cell culture (SILAC)-based mass spectrometry analysis of endothelial cells, we identified two serine residues (S1785/S1786) located in between the two exchange domains of Trio that were highly phosphorylated upon short thrombin treatment. Using phosphomimetic Trio S1785D/S1786D double mutants, we did not find an increase in Rac1/RhoG activity, indicating that the phosphorylation events do not increase Trio exchange activity. However, we found that the Trio mutants localized more strongly at cell-cell junctions and prevented junction destabilization upon thrombin treatment, judged by junction linearity. Our data suggest that serine phosphorylation of Trio potentiates the localization of Trio to junctional regions, resulting in locally promoting the exchange for Rac1 at junction regions and increasing endothelial cell-cell junction stability upon permeability-inducing reagents such as thrombin.