DOMAIN ORGANIZATION OF I-KAPPA-B-ALPHA AND SITES OF INTERACTION WITH NF-KAPPA-B P65

The DNA-binding activity and cellular distribution of the transcriptio n factor NP-kappa B are regulated by the inhibitor protein I kappa B a lpha. I kappa B alpha belongs to a family of proteins that contain mul tiple repeats of a 30- to 35-amino acid sequence that was initially re cognized in the erSthrocyte protein ankyrin. Partial proteolysis has b een used to study the domain structure of I kappa B alpha and to deter mine the sites at which it interacts with NF-kappa B. The data reveal a tripartite structure for I kappa B alpha in which a central, proteas e-resistant domain composed of five ankyrin repeats is flanked by an u nstructured N-terminal extension and a compact, highly acidic C-termin al domain that is connected to the core of the protein by a flexible l inker. Functional analysis of V8 cleavage products indicates that I ka ppa B alpha molecules lacking the N terminal region can interact with and inhibit the DNA-binding activity of the p65 subunit of NF-kappa B, whereas I kappa B alpha molecules which lack both the N- and C-termin al regions are incapable of doing so. Protease cleavage of the N termi nus of I kappa B alpha was unaffected by the presence of the p65 subun it of NF-kappa B, whereas bound p65 blocked cleavage of the flexible l inker connecting the C-terminal domain to the ankyrin repeat containin g core of the protein. This linker region is highly conserved within t he human, rat, pig, and chicken homologs of I kappa B alpha, and while it has been suggested that it represents a sixth ankyrin repeat, it i s also likely that this is a flexible region of the protein that inter acts with NF-kappa B.