TY - JOUR
T1 - GTP cyclohydrolase i phosphorylation and interaction with GTP cyclohydrolase feedback regulatory protein provide novel regulation of endothelial tetrahydrobiopterin and nitric oxide
AU - Li, Li
AU - Rezvan, Amir
AU - Salerno, John C.
AU - Husain, Ahsan
AU - Kwon, Kihwan
AU - Jo, Hanjoong
AU - Harrison, David G.
AU - Chen, Wei
N1 - Funding Information:
This work was supported by the National Institutes of Health (2T32-GM007347 and 5F30-AI129229, both to K. W. G.).
PY - 2010/2
Y1 - 2010/2
N2 - 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.
AB - 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.
KW - Coimmunoprecipitation
KW - Shear stress
KW - Site-directed mutagenesis
KW - Transfection
KW - eNOS uncoupling
UR - http://www.scopus.com/inward/record.url?scp=76349105015&partnerID=8YFLogxK
U2 - 10.1161/CIRCRESAHA.109.210658
DO - 10.1161/CIRCRESAHA.109.210658
M3 - Article
C2 - 19926872
AN - SCOPUS:76349105015
SN - 0009-7330
VL - 106
SP - 328
EP - 336
JO - Circulation Research
JF - Circulation Research
IS - 2
ER -