The Skp1 prolyl hydroxylase from Dictyostelium is related to the hypoxia-inducible factor-alpha class of animal prolyl 4-hydroxylases

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VAN DER WEL H., Ercan A., WEST C. M.

JOURNAL OF BIOLOGICAL CHEMISTRY, vol.280, no.15, pp.14645-14655, 2005 (SCI-Expanded) identifier identifier

  • Publication Type: Article / Article
  • Volume: 280 Issue: 15
  • Publication Date: 2005
  • Doi Number: 10.1074/jbc.m500600200
  • Journal Indexes: Science Citation Index Expanded (SCI-EXPANDED), Scopus
  • Page Numbers: pp.14645-14655
  • Abdullah Gül University Affiliated: No


Skp1 is a cytoplasmic and nuclear protein of eukaryotes best known as an adaptor in SCF ubiquitin-protein isopeptide ligases. In Dictyostelium, Skp1 is subject to 4-hydroxylation at Pro(143) and subsequent O-glycosylation by alpha-linked GlcNAc and other sugars. Soluble cytosolic extracts have Skp1 prolyl 4-hydroxylase (P4H) activity, which can be measured based on hydroxylation-dependent transfer of [H-3]GlcNAc to recombinant Skp1 by recombinant (Skp1-protein)-hydroxyproline alpha-N-acetyl-D-glucosaminyltransferase. The Dictyostelium Skp1 P4H gene (phyA) was predicted using a bioinformatics approach, and the expected enzyme activity was confirmed by expression of phyA cDNA in Escherichia coli. The purified recombinant enzyme (P4H1) was dependent on physiological concentrations of O-2, alpha-ketoglutarate, and ascorbate and was inhibited by CoCl2, 3,4-dihydroxybenzoate, and 3,4-dihydroxyphenyl acetate, as observed for known animal cytoplasmic P4Hs of the hypoxia-inducible factor-alpha (HIF alpha) class. Overexpression of phyA cDNA in Dictyostelium yielded increased enzyme activity in a soluble cytosolic extract. Disruption of the phyA locus by homologous recombination resulted in loss of detectable activity in extracts and blocked hydroxylation-dependent glycosylation of Skp1 based on molecular weight analysis by SDS-PAGE, demonstrating a requirement for P4H1 in vivo. The sequence and functional similarities of P4H1 to animal HIF alpha-type P4Hs suggest that hydroxylation of Skp1 may, like that of animal HIF alpha, be regulated by availability of O-2, alpha-ketoglutarate, and ascorbate, which might exert novel control over Skp1 glycosylation.