Abstract
The biosynthesis of hypusine [Nepsilon-(4-amino-2-hydroxybutyl)-lysine] occurs in the eIF-5A precursor protein through two step posttranslational modification involving deoxyhypusine synthase which catalyzes transfer of the butylamine moiety of spermidine to the epsilon-amino group of a designated lysine residue and subsequent hydroxylation of this intermediate. This enzyme is exclusively required for cell viability and growth of yeast (Park, M.H. et al., J. Biol. Chem. 273: 1677-1683, 1998). In an effort to understand structure-function relationship of deoxyhypusine synthase, posttranslational modification(s) of the enzyme by protein kinases were carried out for a possible cellular modulation of this enzyme. And also twelve deletion mutants were constructed, expressed in E. coli system, and enzyme activities were examined. The results showed that deoxyhypusine synthase was phosphorylated by PKC in vitro but not by p56lck and p60c-src. Treatment with PMA specifically increased the relative phosphorylation of the enzyme supporting PKC was involved. Phosphoamino acid analysis of this enzyme revealed that deoxyhypusine synthase is mostly phosphorylated on serine residue and weakly on threonine. Removal of Met1-Glu10 (deltaMet1-Glu10) residues from amino terminal showed no effect on the catalytic activity but further deletion (deltaMet1-Ser20) caused loss of enzyme activity. The enzyme with internal deletion, deltaGln197-Asn212 (residues not present in the human enzyme) was found to be inactive. Removal of 5 residues from carboxyl terminal, deltaLys383-Asn387, retained only slight activity. These results suggested that deoxyhypusine synthase is substrate for PKC dependent phosphorylation and requires most of the polypeptide chains for enzyme activity except the first 15 residues of N-terminal despite of N- and C-terminal residues of the enzyme consist of variable regions.
Highlights
IntroductionThe biosynthesis of hypusine [Nε-(4-amino-2-hydroxybutyl)-lysine] occurs exclusively in one cellular protein, the precursor of eukaryotic translation initiation factor 5A (eIF-5A) through a unique two-step posttranslational modification (Park et al, 1993a; Park et al, 1993b)
The biosynthesis of hypusine [Nε-(4-amino-2-hydroxybutyl)-lysine] occurs exclusively in one cellular protein, the precursor of eukaryotic translation initiation factor 5A through a unique two-step posttranslational modification (Park et al, 1993a; Park et al, 1993b)
Yeast deoxyhypusine synthase contains multiple potential phosphorylation sequence motifs for casein kinase II (CKII), protein kinase C (PKC), and tyrosine kinases. This suggests that yeast deoxyhypusine synthase could be phosphorylated on tyrosine as well as serine and threonine
Summary
The biosynthesis of hypusine [Nε-(4-amino-2-hydroxybutyl)-lysine] occurs exclusively in one cellular protein, the precursor of eukaryotic translation initiation factor 5A (eIF-5A) through a unique two-step posttranslational modification (Park et al, 1993a; Park et al, 1993b). Deoxyhypusine synthase catalyzes NADdependent transfer of the butylamine moiety of the polyamine spermidine to the ε-amino group of a specific lysine residue of eIF-5A precursor (Lys in the yeast protein) to form a deoxyhypusine [Nε-(4-aminobutyl) lysine] residue (Park et al, 1982; Wolff et al, 1995). Subsequent hydroxylation of this intermediate by deoxyhypusine hydroxylase completes hypusine conformation and eIF5A maturation (Park et al, 1993a; Park et al, 1993b). The arrest in cell proliferation by inhibitors of polyamine biosynthetic enzymes has been attributed to depletion of eIF-5A following depletion of spermidine (Byers et al, 1992)
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