Molecular dissection of the interactions among IκBα, FWD1, and Skp1 required for ubiquitin-mediated proteolysis of IκBα

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 I kappa B alpha FWD1 associates with Skp1 through the F-box domain and also recognizes the conserved DS-GXXS motif of I kappa B alpha. The structural requirements for the interactions of FWD1 with I kappa B alpha and with Skp1 have now been investigated further. The D31A mutation (but not the G33A mutation) in the DSGXXS motif of I kappa B alpha abolished the binding of I kappa B alpha to FWD1 and its subsequent ubiquitination without affecting the phosphorylation of I kappa B alpha. The I kappa B alpha 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 I kappa B alpha. 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 I kappa B alpha, FWD1, and Skp1 that underlie substrate recognition by the SCF ubiquitin ligase complex.