Identification and functional characterization of phosphorylation sites on GTP cyclohydrolase I.
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.


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.
Du
Jianhai
J
Division of Pediatric Surgery, Medical College of Wisconsin, Milwaukee, WI 53226, USA.
Wei
Na
N
Xu
Hao
H
Ge
Ying
Y
Vásquez-Vivar
Jeannette
J
Guan
Tongju
T
Oldham
Keith T
KT
Pritchard
Kirkwood A
KA
Shi
Yang
Y
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D001708:Biopterins / Q000031:analogs & derivatives / Q000096:biosynthesis; D002460:Cell Line; D002467:Cell Nucleus / Q000201:enzymology; D006136:GTP Cyclohydrolase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014162:Transfection
D011994:Recombinant Proteins; D001708:Biopterins; D006136:GTP Cyclohydrolase; C003402:sapropterin