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J. Biol. Chem., Vol. 280, Issue 15, 14645-14655, April 15, 2005

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The Skp1 Prolyl Hydroxylase from Dictyostelium Is Related to the Hypoxia-inducible Factor- Class of Animal Prolyl 4-Hydroxylases^*

Hanke van der Wel, Altan Ercan, and Christopher M. West

From the Department of Biochemistry and Molecular Biology, Oklahoma Center for Medical Glycobiology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104

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 Pro¹⁴³ and subsequent O-glycosylation by -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 [³H]GlcNAc to recombinant Skp1 by recombinant (Skp1-protein)-hydroxyproline -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 O₂, -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- (HIF) 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-type P4Hs suggest that hydroxylation of Skp1 may, like that of animal HIF, be regulated by availability of O₂, -ketoglutarate, and ascorbate, which might exert novel control over Skp1 glycosylation.

Received for publication, January 18, 2005 , and in revised form, February 7, 2005.

^* This work was supported in part by National Institutes of Health Grant R01-GM37539. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) AY768817.

To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, 940 Stanton L. Young Blvd., BMSB 937, Oklahoma City, OK 73104. Tel.: 405-271-2227 (ext. 1247); Fax: 405-271-3139; E-mail: Cwest2{at}ouhsc.edu.

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