Recognition of substrate and Skp1 by the Homologue of Slimb (HOS) ubiquitin ligase receptor D role of the F-box.
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.


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.
Herter
Jason R
JR
Department of Animal Biology, University of Pennsylvania, Philadelphia, PA 19104-6046, USA. sfuks@vet.upenn.edu
Fuchs
Serge Y
SY
D020816:Amino Acid Motifs; D000818:Animals; D001665:Binding Sites; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D018797:Cell Cycle Proteins / Q000378:metabolism*; D002460:Cell Line; D006801:Humans; D051550:I-kappa B Kinase; D016328:NF-kappa B / Q000037:antagonists & inhibitors / Q000378:metabolism; D010453:Peptide Synthases / Q000378:metabolism*; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D044786:S-Phase Kinase-Associated Proteins; D044843:SKP Cullin F-Box Protein Ligases; D025801:Ubiquitin / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases; D044844:beta-Transducin Repeat-Containing Proteins
D002352:Carrier Proteins; D018797:Cell Cycle Proteins; C471645:FBXW11 protein, human; D016328:NF-kappa B; D011993:Recombinant Fusion Proteins; D044786:S-Phase Kinase-Associated Proteins; D025801:Ubiquitin; D044844:beta-Transducin Repeat-Containing Proteins; D044843:SKP Cullin F-Box Protein Ligases; D044767:Ubiquitin-Protein Ligases; D017346:Protein Serine-Threonine Kinases; C496559:CHUK protein, human; D051550:I-kappa B Kinase; C496560:IKBKB protein, human; C496565:IKBKE protein, human; D010453:Peptide Synthases