title: Novel regulation of Skp1 by the Dictyostelium AgtA α-galactosyltransferase involves the Skp1-binding activity of its WD40 repeat domain.
abstract: The role of Skp1 as an adaptor protein that links Cullin-1 to F-box proteins in E3 Skp1/Cullin-1/F-box protein (SCF) ubiquitin ligases is well characterized. In the social amoeba Dictyostelium and probably many other unicellular eukaryotes, Skp1 is modified by a pentasaccharide attached to a hydroxyproline near its C terminus. This modification is important for oxygen-sensing during Dictyostelium development and is mediated by a HIF-α type prolyl 4-hydroxylase and five sequentially acting cytoplasmic glycosyltransferase activities. Gene disruption studies show that AgtA, the enzyme responsible for addition of the final two galactose residues, in α-linkages to the Skp1 core trisaccharide, is unexpectedly critical for oxygen-dependent terminal development. AgtA possesses a WD40 repeat domain C-terminal to its single catalytic domain and, by use of domain deletions, binding studies, and enzyme assays, we find that the WD40 repeats confer a salt-sensitive second-site binding interaction with Skp1 that mediates novel catalytic activation in addition to simple substrate recognition. In addition, AgtA binds similarly well to precursor isoforms of Skp1 by a salt-sensitive mechanism that competes with binding to an F-box protein and recognition by early modification enzymes, and the effect of binding is diminished when AgtA modifies Skp1. Genetic studies show that loss of AgtA is more severe when an earlier glycosylation step is blocked, and overexpressed AgtA is deleterious if catalytically inactivated. Together, the findings suggest that AgtA mediates non-enzymatic control of unmodified and substrate precursor forms of Skp1 by a binding mechanism that is normally relieved by switch-like activation of its glycosylation function.
authors.affiliation: From the Department of Biochemistry and Molecular Biology, Oklahoma Center for Medical Glycobiology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104.
authors.forename: Christopher M
authors.initials: CM
authors.lastname: Schafer
authors.forename: M Osman
authors.initials: MO
authors.lastname: Sheikh
authors.forename: Dongmei
authors.initials: D
authors.lastname: Zhang
authors.forename: Christopher M
authors.initials: CM
authors.lastname: West
mesh_terms: D004023:Dictyostelium / Q000201:enzymology / Q000378:metabolism*; D005700:Galactosyltransferases / Q000737:chemistry* / Q000378:metabolism*; D006031:Glycosylation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D044786:S-Phase Kinase-Associated Proteins / Q000378:metabolism*
chemical_list: D044786:S-Phase Kinase-Associated Proteins; D005700:Galactosyltransferases
keywords: Cellular Slime Mold; Development; Dictyostelium; E3 Ubiquitin Ligase; Glycosylation; Glycosyltransferases; Oxygen Sensing; Prolyl Hydroxylation; Skp1; WD40 Repeat