Files
Abstract
Most eukaryotes depend on the SCF (Skp1/cullin-1/F-box protein) family of E3 ubiquitin ligases to regulate its proteome. The SCF binds to specific substrates and poly-ubiquitinates them as a signal for subsequent degradation by the 26S proteasome. The importance of proper regulation of the assembly and activity of this complex is evident in the cancers and other disease states that result from its dysregulation. Here we used Dictyostelium discoideum as a model to study how an oxygen dependent post-translational modification can influence Skp1 binding to F-box proteins (FBP) of both substrate receptor (SR) types and those lacking substrate receptor domains. This modification consists of prolyl hydroxylation by PhyA and subsequent glycosylation by a series of 5 glycosylation reactions. Immunoprecipitation of Skp1 from vegetative and slug cells revealed dramatic remodeling of Skp1’s interactome between the two different stages of its life cycle. At both stages, Skp1 had higher levels of interaction with a number of FBPs when it was glycosylated that was not simply the result of differential abundance. Two of these FBPs, JcdI and FbxwD, were further characterized. JcdI, a JmjC non-heme dioxygenase and an FBP conserved throughout Protista, has been shown to serve as a putative oxygen-sensor in an SCF independent manner. JcdI’s F-box domain may serve to regulate its degradation as its levels are significantly increased in developing slugs that lack the Skp1 glycan. FbxwD, a WD40 SR-type FBP, negatively impacted Dictyostelium’s ability to successfully differentiate during development when overexpressed. Interestingly, this inhibition was dependent on FbxwD having an intact RING domain and an intact copy of the Vwa1 locus. Vwa1, a homologue to the tumor suppressor VWA5A/BCSC-1, has no effect on development when knocked-out or over-expressed but expression of individual domains of the protein cause dramatic arrests of development only when the endogenous copy was expressed. Vwa1 may engage in a form of auto-inhibition that prevents it from forming active complexes with FbxwD, a model that could be used to understand the role of its poorly understood human homolog.