GTP cyclohydrolase I phosphorylation and interaction with GTP cyclohydrolase feedback regulatory protein provide novel regulation of endothelial tetrahydrobiopterin and nitric oxide.
BACKGROUND
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.


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.
Li
Li
L
Division of Cardiology, Emory University School of Medicine, 1639 Pierce Dr WMBR 319, Atlanta, GA 30322, USA.
Rezvan
Amir
A
Salerno
John C
JC
Husain
Ahsan
A
Kwon
Kihwan
K
Jo
Hanjoong
H
Harrison
David G
DG
Chen
Wei
W
D000818:Animals; D001665:Binding Sites / Q000235:genetics; D001708:Biopterins / Q000031:analogs & derivatives* / Q000378:metabolism; D015153:Blotting, Western; D002339:Carotid Arteries / Q000503:physiopathology / Q000601:surgery; D047390:Casein Kinase II / Q000378:metabolism; D002460:Cell Line; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000166:cytology / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006136:GTP Cyclohydrolase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D009154:Mutation; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D009569:Nitric Oxide / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III / Q000037:antagonists & inhibitors / Q000378:metabolism; D010766:Phosphorylation; D034622:RNA Interference; D013314:Stress, Mechanical
D004791:Enzyme Inhibitors; C101092:GCHFR protein, human; D047908:Intracellular Signaling Peptides and Proteins; D001708:Biopterins; D009569:Nitric Oxide; D052250:Nitric Oxide Synthase Type III; D047390:Casein Kinase II; D006136:GTP Cyclohydrolase; C003402:sapropterin; D019331:NG-Nitroarginine Methyl Ester