title: 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 Arg(16) to Cys codon mutation. Arg(16) is located in the protein surface exposed phosphorylation motif Arg(16)-Arg-Ile-Ser, with Ser(19) as the putative 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 10(3) M(-1) s(-1)), which was similar to wild-type PTPS (k(cat)/K(m) = 4.1 x 10(3) 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 approximately 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 Ser(19) 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 Ser(19) of human PTPS may be a substrate for cGKII phosphorylation also in vivo, a modification that is essential for normal activity.
authors.affiliation: Department of Pediatrics, Division of Clinical Chemistry and Biochemistry, University of Zürich, Steinwiesstrasse 75, CH-8032 Zürich, Switzerland.
authors.forename: T
authors.initials: T
authors.lastname: Scherer-Oppliger
authors.forename: W
authors.initials: W
authors.lastname: Leimbacher
authors.forename: N
authors.initials: N
authors.lastname: Blau
authors.forename: B
authors.initials: B
authors.lastname: Thöny
mesh_terms: D000470:Alkaloids / Q000494:pharmacology; D000592:Amino Acid Metabolism, Inborn Errors / Q000201:enzymology / Q000235:genetics*; D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002227:Carbazoles; D016384:Consensus Sequence; D062967:Cyclic GMP-Dependent Protein Kinase Type II; D017869:Cyclic GMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D005347:Fibroblasts / Q000201:enzymology; D006801:Humans; D007211:Indoles; D051379:Mice; D018345:Mice, Knockout; D008969:Molecular Sequence Data; D009154:Mutation; D019761:Phosphorus-Oxygen Lyases / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases; D011994:Recombinant Proteins / Q000378:metabolism; D012694:Serine / Q000378:metabolism*; D012867:Skin / Q000201:enzymology; D019311:Staurosporine / Q000494:pharmacology
chemical_list: D000470:Alkaloids; D002227:Carbazoles; D007211:Indoles; D011994:Recombinant Proteins; C073601:KT 5823; D012694:Serine; D017346:Protein Serine-Threonine Kinases; D062967:Cyclic GMP-Dependent Protein Kinase Type II; D017869:Cyclic GMP-Dependent Protein Kinases; C569729:PRKG2 protein, human; C569730:Prkg2 protein, mouse; D019761:Phosphorus-Oxygen Lyases; C043497:6-pyruvoyltetrahydropterin synthase; D019311:Staurosporine