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

• Type of Material: Article
• Authors: Li Li, Emory University; Amir Rezvan, Emory University; John C. Salerno, 3 Department of Biology and Physics, Kennesaw State University, Kennesaw, GA 30144; Ahsan Husain, Emory University; Kihwan Kwon, Emory University; Hanjoong Jo, Emory University; David G Harrison, Emory University; Wei Chen, Emory University
• Language: English
• Date: 2010-02-05
• Publisher: American Heart Association
• Publication Version: Author Accepted Manuscript (After Peer Review)
• Copyright Statement: © 2010 American Heart Association, Inc.
• Final Published Version (URL): http://dx.doi.org/10.1161%2FCIRCRESAHA.109.210658
• Title of Journal or Parent Work: Circulation Research
• ISSN: 0009-7330
• Volume: 106
• Issue: 2
• Start Page: 328
• End Page: 328
• Grant/Funding Information: This work was supported by NIH Grants P01HL58000, R01HL39006, P01HL58000 and a Department of Veterans Affairs merit grant (to D.G.H.) and an American Heart Association Predoctoral Grant (to L.L.).
• Keywords: high performance liquid chromatography; transfection; co-immunoprecipitation; eNOS uncoupling; electron spin resonance; shear stress; site directed mutagenesis
• Research Categories: Engineering, Biomedical; Health Sciences, Pharmacology

Abstract

Rationale GTP cyclohydrolase I (GTPCH-1) is the rate-limiting enzyme involved in de novo biosynthesis of tetrahydrobiopterin (BH4), an essential cofactor for nitric oxide synthases and aromatic amino acid hydroxylases. GTPCH-1 undergoes negative feedback regulation by its end-product BH4 via interaction with the GTP cyclohydrolase feedback regulatory protein (GFRP). Such a negative feedback mechanism should maintain cellular BH4 levels within a very narrow range; however, we recently identified a phosphorylation site (S81) on human GTPCH-1 that markedly increases BH4 production in response to laminar shear. Objective 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 siRNA or overexpressing plasmids, GFRP was shown to modulate phosphorylation of GTPCH-1, BH4 levels and nitric oxide (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 down-regulation prevents eNOS uncoupling in response to oscillatory shear. Finally oscillatory shear was associated with impaired GTPCH-1 phosphorylation and reduced BH4 levels in vivo. Conclusion 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 BH4 to be produced in response to laminar shear stress.