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

J Biol Chem. 1999 Oct 29;274(44):31341-8. doi: 10.1074/jbc.274.44.31341.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alkaloids / pharmacology
  • Amino Acid Metabolism, Inborn Errors / enzymology
  • Amino Acid Metabolism, Inborn Errors / genetics*
  • Amino Acid Sequence
  • Animals
  • COS Cells
  • Carbazoles*
  • Consensus Sequence
  • Cyclic GMP-Dependent Protein Kinase Type II
  • Cyclic GMP-Dependent Protein Kinases / antagonists & inhibitors
  • Cyclic GMP-Dependent Protein Kinases / genetics
  • Cyclic GMP-Dependent Protein Kinases / metabolism*
  • Fibroblasts / enzymology
  • Humans
  • Indoles*
  • Mice
  • Mice, Knockout
  • Molecular Sequence Data
  • Mutation
  • Phosphorus-Oxygen Lyases / deficiency
  • Phosphorus-Oxygen Lyases / genetics
  • Phosphorus-Oxygen Lyases / metabolism*
  • Phosphorylation
  • Protein-Serine-Threonine Kinases
  • Recombinant Proteins / metabolism
  • Serine / metabolism*
  • Skin / enzymology
  • Staurosporine / pharmacology

Substances

  • Alkaloids
  • Carbazoles
  • Indoles
  • Recombinant Proteins
  • KT 5823
  • Serine
  • Protein-Serine-Threonine Kinases
  • Cyclic GMP-Dependent Protein Kinase Type II
  • Cyclic GMP-Dependent Protein Kinases
  • PRKG2 protein, human
  • Prkg2 protein, mouse
  • Phosphorus-Oxygen Lyases
  • 6-pyruvoyltetrahydropterin synthase
  • Staurosporine