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

Abstract

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 [(3)H]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 CoCl(2), 3,4-dihydroxybenzoate, and 3,4-dihydroxyphenyl acetate, as observed for known animal cytoplasmic P4Hs of the hypoxia-inducible factor-alpha (HIFalpha) 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 HIFalpha-type P4Hs suggest that hydroxylation of Skp1 may, like that of animal HIFalpha, be regulated by availability of O(2), alpha-ketoglutarate, and ascorbate, which might exert novel control over Skp1 glycosylation.

• Authors: Hanke van der Wel, Altan Ercan, Christopher M West
• Journal: The Journal of biological chemistry (J Biol Chem) Vol. 280 Issue 15 Pg. 14645-55 (Apr 15 2005) ISSN: 0021-9258 [Print] United States
• PMID: 15705570 (Publication Type: Journal Article, Research Support, N.I.H., Extramural, Research Support, U.S. Gov't, P.H.S.)

Chemical References

• DNA, Complementary
• Hypoxia-Inducible Factor 1, alpha Subunit
• Ketoglutaric Acids
• Phytochrome A
• Protein Isoforms
• Recombinant Proteins
• Transcription Factors
• Phytochrome
• Procollagen-Proline Dioxygenase
• SKP Cullin F-Box Protein Ligases
• Protein Serine-Threonine Kinases
• Ascorbic Acid
• Oxygen

Topics

• Amino Acid Sequence
• Animals
• Ascorbic Acid (chemistry)
• Base Sequence
• Blotting, Western
• Cloning, Molecular
• Computational Biology
• Cytosol (metabolism)
• DNA, Complementary (metabolism)
• Dictyostelium (enzymology)
• Dose-Response Relationship, Drug
• Electrophoresis, Polyacrylamide Gel
• Escherichia coli (metabolism)
• Exons
• Genetic Vectors
• Glycosylation
• Hydroxylation
• Hypoxia-Inducible Factor 1, alpha Subunit
• Introns
• Ketoglutaric Acids (chemistry)
• Kinetics
• Models, Genetic
• Molecular Sequence Data
• Mutation
• Oxygen (metabolism)
• Phytochrome (chemistry)
• Phytochrome A
• Procollagen-Proline Dioxygenase (chemistry)
• Protein Isoforms
• Protein Serine-Threonine Kinases (chemistry)
• Recombinant Proteins (chemistry)
• Recombination, Genetic
• SKP Cullin F-Box Protein Ligases (chemistry)
• Transcription Factors (metabolism)