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Identification and functional characterization of phosphorylation sites on GTP cyclohydrolase I. Arterioscler Thromb Vasc Biol 2009 Dec;29(12):2161-8 PMID: 19762783 PMCID: PMC2798731

Pubmed ID

19762783

DOI

10.1161/ATVBAHA.109.194464

Abstract

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.

CONCLUSIONS: 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.

Author List

Du J, Wei N, Xu H, Ge Y, Vásquez-Vivar J, Guan T, Oldham KT, Pritchard KA Jr, Shi Y

Authors

Keith T. Oldham MD Professor in the Surgery department at Medical College of Wisconsin
Kirkwood A. Pritchard PhD Professor in the Surgery department at Medical College of Wisconsin
Jeannette M. Vasquez-Vivar PhD Professor in the Biophysics department at Medical College of Wisconsin




Scopus

2-s2.0-73849125957   8 Citations

MESH terms used to index this publication - Major topics in bold

Amino Acid Sequence
Amino Acid Substitution
Animals
Binding Sites
Biopterin
Cell Line
Cell Nucleus
GTP Cyclohydrolase
Humans
Molecular Sequence Data
Mutagenesis, Site-Directed
Phosphorylation
Protein Processing, Post-Translational
Rats
Recombinant Proteins
Transfection
jenkins-FCD Prod-332 f92a19b0ec5e8e1eff783fac390ec127e367c2b5