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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2009;29:2161.)
© 2009 American Heart Association, Inc.

Cell Biology/Signaling

Identification and Functional Characterization of Phosphorylation Sites on GTP Cyclohydrolase I

Jianhai Du; Na Wei; Hao Xu; Ying Ge; Jeannette Vásquez-Vivar; Tongju Guan; Keith T. Oldham; Kirkwood A. Pritchard, Jr; Yang Shi

From the Division of Pediatric Surgery (J.D., N.W., H.X., T.G., K.T.O., K.A.P., Y.S.), Children’s Research Institution (J.D., N.W., H.X., T.G., K.T.O., K.A.P., Y.S.), and the Department of Biophysics (J.V.-V.), Medical College of Wisconsin, Milwaukee; and the Human Proteomics Program (Y.G.), School of Medicine and Public Health, University of Wisconsin-Madison.

Correspondence to Yang Shi, PhD, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226. E-mail yangshi{at}mcw.edu

Objective— The posttranslational regulation of GTP cyclohydrolase I (GCH-1), the rate-limiting enzyme for tetrahydrobiopterin (BH4) synthesis, remains elusive. Here, we identified specific phosphorylation sites on GCH-1 and characterized the function of these sites.

Methods and Results— Mass spectrometry studies showed overexpressed rat GCH-1 was phosphorylated at serine (S) 51, S167, and threonine (T) 231 in HEK293 cells, whereas a computational analysis of GCH-1 revealed 8 potential phosphorylation sites (S51, S72, T85, T91, T103, S130, S167 and T231). GCH-1 activity and BH4 were significantly decreased in cells transfected with the phospho-defective mutants (S72A, T85A, T91A, T103A, or S130A) and increased in cells transfected with the T231A mutant. BH4 and BH2 were increased in cells transfected with S51E, S72E, T85E, T91E, T103D, or T130D mutants, but decreased in cells transfected with the T231D mutant, whereas cells transfected with the S167A or the S167E mutant had increased BH2. Additionally, cells transfected with the T231A mutant had reduced GCH-1 nuclear localization and nuclear GCH-1 activity.

Conclusion— Our data suggest GCH-1 activity is regulated either positively by phosphorylation S51, S72, T85, T91, T103, and S130, or negatively at T231. Such information might be useful in designing new therapies aiming at improving BH4 bioavailability.

The regulation of GCH-1 is not fully understood. We identified 8 putative phosphorylation sites on GCH-1. Our study suggests that GCH-1 is phosphorylated at S51, S167, and T231 and GCH-1 activity is regulated by phosphorylation positively at sites S51, S72, T85, T91, T103, and S130 and negatively at site T231.

Key Words: GTP cyclohydrolase I • tetrahydrobiopterin • phosphorylation