Serine 19 of human 6-pyruvoyltetrahydropterin synthase is phosphorylated by cGMP protein kinase II

Abstract

6-Pyruvoyltetrahydropterin synthase (PTPS) participates in tetrahydrobiopterin cofactor biosynthesis. We previously identified in a PTPS-deficient patient an inactive PTPS allele with an Arg16 to Cys codon mutation. Arg16 is located in the protein surface exposed phosphorylation motif Arg16-Arg-Ile-Ser, with Ser19 as the putatire phosphorylation site for serine-threonine protein kinases. Purification of recombinant PTPS-S19A from bacterial cells resulted in an active enzyme (k(cat)/K(m) = 6.4 x 103 M-1 s-1), which was similar to wild-type PTPS (k(cat)/K(m) = 4.1 x 103 M-1 s-1). In assays with purified enzymes, wild-type but not PTPS-S19A was a specific substrate for the cGMP-dependent protein kinase (cGK) type I and II. Upon expression in COS-1 cells, PTPS-S19A was stable but not phosphorylated and had a reduced activity of ~33% in comparison to wild-type PTPS. Extracts from several human cell lines, including brain, contained a kinase that bound to and phosphorylated immobilized wild-type, but not mutant PTPS. Addition of cGMP stimulated phosphotransferase activity 2-fold. Extracts from transfected COS-1 cells overexpressing cGKII stimulated Ser19 phosphorylation more than 100-fold, but only 4-fold from cGKI overexpressing cells. Moreover, fibroblast extracts from mice lacking cGKII exhibited significantly reduced phosphorylation of PTPS. These results suggest that Ser19 of human PTPS may be a substrate for cGKII phosphorylation also in vivo, a modification that is essential for normal activity.