Molecular dissection of the interactions among IkappaBalpha, FWD1, and Skp1 required for ubiquitin-mediated proteolysis of IkappaBalpha.
The SCF complex containing Skp1, Cul1, and the F-box protein FWD1 (the mouse homologue of Drosophila Slimb and Xenopus beta-TrCP) functions as the ubiquitin ligase for IkappaBalpha. FWD1 associates with Skp1 through the F-box domain and also recognizes the conserved DSGXXS motif of IkappaBalpha. The structural requirements for the interactions of FWD1 with IkappaBalpha and with Skp1 have now been investigated further. The D31A mutation (but not the G33A mutation) in the DSGXXS motif of IkappaBalpha abolished the binding of IkappaBalpha to FWD1 and its subsequent ubiquitination without affecting the phosphorylation of IkappaBalpha. The IkappaBalpha mutant D31E still exhibited binding to FWD1 and underwent ubiquitination. These results suggest that, in addition to site-specific phosphorylation at Ser(32) and Ser(36), an acidic amino acid at position 31 is required for FWD1-mediated ubiquitination of IkappaBalpha. Deletion analysis of Skp1 revealed that residues 61-143 of this protein are required for binding to FWD1. On the other hand, the highly conserved residues Pro(149), Ile(160), and Leu(164) in the F-box domain of FWD1 were dispensable for binding to Skp1. Together, these data delineate the structural requirements for the interactions among IkappaBalpha, FWD1, and Skp1 that underlie substrate recognition by the SCF ubiquitin ligase complex.
Hattori
K
K
Department of Molecular Biology, Medical Institute of Bioregulation, Kyushu University, Fukuoka 812-8582, Japan.
Hatakeyama
S
S
Shirane
M
M
Matsumoto
M
M
Nakayama
K
K
D000595:Amino Acid Sequence; D000818:Animals; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D020672:I-kappa B Proteins; D051379:Mice; D016297:Mutagenesis, Site-Directed; D000072000:NF-KappaB Inhibitor alpha; D044786:S-Phase Kinase-Associated Proteins; D017386:Sequence Homology, Amino Acid; D014452:Ubiquitins / Q000378:metabolism*; D044844:beta-Transducin Repeat-Containing Proteins
C471644:BTRC protein, human; C471643:Btrc protein, mouse; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D020672:I-kappa B Proteins; C000606204:NFKBIA protein, human; C000606205:Nfkbia protein, mouse; D044786:S-Phase Kinase-Associated Proteins; D014452:Ubiquitins; D044844:beta-Transducin Repeat-Containing Proteins; D000072000:NF-KappaB Inhibitor alpha; D019204:GTP-Binding Proteins