GTPCH Serine81 phosphorylation modulates GTPCH–GFRP complex formation and GTPCH activity in aged endothelial cells under static and shear stress conditions

In dysfunctional endothelium, the decreased BH4 production is attributed to a reduced GTPCH activity although molecular mechanisms involved are not yet clearly elucidated. Phosphorylation at serine81 site (Ser81-P) and interaction with the feedback regulatory protein GFRP are two mechanisms that have been described to modulate GTPCH activity. In this study, we demonstrated in aged endothelial cells, a model of endothelial dysfunction as shown by the decreased NOS3 activity (−75%) and cGMP production (−50%), that BH4 production and GTPCH activity were decreased (42% and 54%, respectively). In parallel, the GTPCH–GFRP complex was increased (25%) while the ser81-P-GTPCH form was decreased (−80%) compared to native cells. This indicated that most of the GTPCH protein (74%) was associated with GFRP in aged endothelial cells while the phosphorylated form was never found to be linked with this regulatory protein. The role of GTPCH phosphorylation in the interaction of GTPCH and GFRP was further demonstrated when using sanguinarine, a PP2C inhibitor, which raised GTPCH phosphorylation by 58% and reduced complex formation by 18%. Under physiological shear stress (SS), which has already been shown to increase GTPCH activity, the phosphorylation of this enzyme first increased transiently (64%) and was then followed by a sustained dissociation of GTPCH–GFRP complex (−30%). These results suggest that GTPCH activity (in static conditions or under SS) first depends on its phosphorylation which then dissociates GTPCH–GFRP complex in dysfunctional endothelial cells.