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Identification and Functional Characterization of Phosphorylation Sites on GTP cyclohydrolase I
Author(s) -
Du Jianhai,
Wei Na,
Xu Hao,
VasquezVivar Jeannette,
Pritchard Kirkwood A,
Shi Yang
Publication year - 2009
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.23.1_supplement.628.3
Subject(s) - tetrahydrobiopterin , gtp cyclohydrolase i , mutant , phosphorylation , biochemistry , microbiology and biotechnology , serine , alanine , chemistry , threonine , enzyme , aspartic acid , biology , amino acid , cofactor , gene
GTP cyclohydrolase I (GCH‐1) is the rate‐limiting enzyme for the biosynthesis of tetrahydrobiopterin (BH4), an essential cofactor for nitric oxide synthases (NOS) and aromatic amino acid hydrolases. In this study, we aim to identify specific phosphorylation sites on GCH‐1 and characterize their function. Rat GCH‐1 with FLAG tag was subcloned into a Tet‐On plasmid which was stably expressed in Flp‐in 293 cells. Mass spectrometry studies revealed GCH‐1 was phosphorylated in vivo at serine(S) 167 and threonine (T) 231. Sequence analysis of GCH‐1 protein showed 8 potential phosphorylation sites [S51, S72, T85, T91, T103, S130, S167 and T231]. Functional studies demonstrated that GCH‐1 activity and BH4 levels were significantly decreased in cells expressing phospho‐defective alanine (A) mutants S72 A, T85A, T91A, T103A or S130A while they were increased in cells expressing the T231A mutant. Furthermore, GCH1 activity, and BH4 levels were increased in cells expressing the phospho‐mimic glutamic acid (E)/aspartic acid (D) mutants S72E, T85E, T91E, T103D or T130D, while they were decreased in cells expressing the T231D mutant. Interestingly, cells expressing the S51E, but not S51A had increased cellular BH4 production, while cells expressing S167A or S167E mutant did not affect BH4 synthesis. In summary, our study suggests that GCH‐1 is positively and negatively regulated by multiple phosphorylation sites.