Abstract 1152: Laminar Shear Stress Increases Endothelial Cell Tetrahydrobiopterin Levels by Stimulating Phosphorylation of GTP Cyclohydrolase I on Serine 81
Laminar shear stress inhibits while oscillatory shear stimulates vascular lesion formation. Laminar shear also stimulates endothelial NO production and nitric oxide synthase (eNOS) expression An essential co-factor for eNOS is tetrahydrobiopterin (H4B). In the present study, we show that laminar shear stress increases H4B levels and enzymatic activity of GTP cyclohydrolase 1 (GPTCH-1) the first step of H4B biosynthesis in human endothelial cells by 30 fold. In contrast to this striking change in enzyme activity shear had no effect on protein levels of GTPCH-1 suggesting post-translational regulation. Shear did not change protein expression or activity of the downstream enzymes, 6-pyruvoyltetrahydropterin synthase and sepiapterin reductase and decreased protein levels of the salvage enzyme dihydrofolate reductase. Oscillatory shear only modestly affected H4B levels and GPTCH-1 activity. GTPCH1 has 5 putative casein kinase 2 phosphorylation sites, and we found that the CK2 inhibitor 4,5,6,7-tetrabromobenzotriazole and siRNA against CK2 alpha’ prevented the increase in H4B and GTPCH1 activity caused by shear. We raised antiphosphoantibodies against each of the potential CK2 phosphorylation sites and found no evidence of phosphorylation of serines 60 and 139 or of threonine 112 and 240 either at baseline or in response to shear. In contrast, serine 81 showed faint phosphorylation in unsheared cells and a striking increase in phosphorylation in response to laminar shear. This was blocked by siRNA against CK2α’. Of note, oscillatory shear did not stimulate phosphorylation of serine 81. Analysis of the GTPCH1 structure indicates that serine 81 resides in an extension of the protein overlying the GTP binding site, and it is likely that phosphorylation of serine 81 leads to opening of this site, permitting access of GTP. These studies provide new evidence of how GTPCH1 activity can be regulated by phosphorylation. Furthermore, the fact that oscillatory shear does not stimulate this event likely explains why H4B and GTPCH1 activity are only modestly affected by this proatherogenic mechanical force.