GTP Cyclohydrolase I Phosphorylation and Interaction With GTP Cyclohydrolase Feedback Regulatory Protein Provide Novel Regulation of Endothelial Tetrahydrobiopterin and Nitric Oxide

Abstract

Rationale : GTP cyclohydrolase I (GTPCH-1) is the rate-limiting enzyme involved in de novo biosynthesis of tetrahydrobiopterin (BH 4 ), an essential cofactor for NO synthases and aromatic amino acid hydroxylases. GTPCH-1 undergoes negative feedback regulation by its end-product BH 4 via interaction with the GTP cyclohydrolase feedback regulatory protein (GFRP). Such a negative feedback mechanism should maintain cellular BH 4 levels within a very narrow range; however, we recently identified a phosphorylation site (S81) on human GTPCH-1 that markedly increases BH 4 production in response to laminar shear. Objective : We sought to define how S81 phosphorylation alters GTPCH-1 enzyme activity and how this is modulated by GFRP. Methods and Results : Using prokaryotically expressed proteins, we found that the GTPCH-1 phospho-mimetic mutant (S81D) has increased enzyme activity, reduced binding to GFRP and resistance to inhibition by GFRP compared to wild-type GTPCH-1. Using small interfering RNA or overexpressing plasmids, GFRP was shown to modulate phosphorylation of GTPCH-1, BH 4 levels, and NO production in human endothelial cells. Laminar, but not oscillatory shear stress, caused dissociation of GTPCH-1 and GFRP, promoting GTPCH-1 phosphorylation. We also found that both GTPCH-1 phosphorylation and GFRP downregulation prevents endothelial NO synthase uncoupling in response to oscillatory shear. Finally oscillatory shear was associated with impaired GTPCH-1 phosphorylation and reduced BH 4 levels in vivo. Conclusions : These studies provide a new mechanism for regulation of endothelial GTPCH-1 by its phosphorylation and interplay with GFRP. This mechanism allows for escape from GFRP negative feedback and permits large amounts of BH 4 to be produced in response to laminar shear stress.