Identification and functional characterization of phosphorylation sites on GTP cyclohydrolase I. Arterioscler Thromb Vasc Biol 2009 Dec;29(12):2161-8
Date
09/19/2009Pubmed ID
19762783Pubmed Central ID
PMC2798731DOI
10.1161/ATVBAHA.109.194464Scopus ID
2-s2.0-73849125957 (requires institutional sign-in at Scopus site) 11 CitationsAbstract
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 YAuthors
Kirkwood A. Pritchard PhD Professor in the Surgery department at Medical College of WisconsinJeannette M. Vasquez-Vivar PhD Professor in the Biophysics department at Medical College of Wisconsin
MESH terms used to index this publication - Major topics in bold
Amino Acid SequenceAmino Acid Substitution
Animals
Binding Sites
Cell Line
Cell Nucleus
GTP Cyclohydrolase
Humans
Molecular Sequence Data
Mutagenesis, Site-Directed
Phosphorylation
Protein Processing, Post-Translational
Rats
Recombinant Proteins
Transfection