ABSTRACT
BACKGROUND: SCFHOS-Roc1 E3 ubiquitin ligase is an enzymatic complex, which mediates ubiquitination and subsequent proteasome-dependent degradation of phosphorylated inhibitor of NF-kB (IkB) and b-catenin. HOS is a WD40 repeats/F-box-containing protein that actually associates with the substrates and binds to Skp1 via the F-box. MATERIAL/METHODS: Here, we have studied the structural determinants of the substrate recognition and ligase recruitment by HOS. The binding (pull-down and immunoprecipitation assays) and ubiquitination assays were performed in vitro with purified or partially purified recombinant proteins obtained via expression in bacteria or mammalian cells or by in vitro translation. RESULTS: We identified specific amino acid residues (I143 and L152) within the F-box of HOS that play a critical role in maintaining the hydrophobic interface of HOS-Skp1 interaction and found substantial similarity between interaction of Skp1 with HOS and with another F-box protein Skp2. Binding of Skp1 augments the ability of HOS to recognize the phosphorylated IkBa. CONCLUSIONS: These observations indicate the role of the F-box of HOS in both recruitment of ubiquitin ligase activity and substrate recognition as well as identify the structural elements that are important for both functions of HOS F-box domain.
Subject(s)
Carrier Proteins/metabolism , Cell Cycle Proteins/metabolism , Peptide Synthases/metabolism , beta-Transducin Repeat-Containing Proteins , Amino Acid Motifs , Animals , Binding Sites , Carrier Proteins/genetics , Cell Line , Humans , I-kappa B Kinase , NF-kappa B/antagonists & inhibitors , NF-kappa B/metabolism , Protein Binding , Protein Serine-Threonine Kinases/metabolism , Recombinant Fusion Proteins/metabolism , S-Phase Kinase-Associated Proteins , SKP Cullin F-Box Protein Ligases , Ubiquitin/metabolism , Ubiquitin-Protein LigasesABSTRACT
NF-kappaB transcription factor is activated upon ubiquitination and subsequent proteolysis of its inhibitor IkappaB. The phosphorylation-dependent ubiquitination is mediated by SCF E3 ubiquitin ligase. In this study, we identified a novel murine F-box/WD40 repeat-containing protein, mHOS (a homologue of HOS/betaTrCP2). mHOS efficiently binds Skp1 protein (a 'core' component of SCF ubiquitin ligase), and phosphorylated IkappaB(alpha). We found that mHOS associates with SCF-ROC1 E3 ubiquitin ligase activity. We have also observed that mHOS is overexpressed in chemically-induced mouse skin tumors, and its overexpression (but not accelerated IkappaB phosphorylation) coincides with the accelerated degradation of IkappaB in vivo. The role of mHOS in the constitutive activation of NF-kappaB in skin carcinogenesis is discussed.
