An NF-κB-Specific Inhibitor, IκBα, Binds to and Inhibits Cyclin-Dependent Kinase 4†

I BR, a protein composed of six ankyrin repeats, is a specific inhibitor of nuclear factor B (NF-B) and functions in signal transductions in many different cell types. Using both in vivo yeast two-hybrid assays and in vitro activity and binding assays, we showed that IBR binds to cyclin-dependent kinase 4 (CDK4) specifically and inhibits its kinase activity. The potencies of binding and inhibition of IBR are comparable to those of INK4 proteins, the specific CDK4 inhibitors that also contain ankyrin repeats. Furthermore, we showed that INK4 proteins and IBR compete with each other for binding to CDK4. These results led us to propose a hypothesis that there is cross talk between the NF-B/IBR pathway and the p16/CDK4/Rb pathway in cells, and that IBR could substitute for the CDK4-inhibiting function of p16, a tumor suppressor frequently inactivated in human tumors. To further understand the structural basis of IBR-CDK binding, we used different mutants of CDK4 to show that there are notable differences between IBR and INK4 proteins in CDK4 binding since the binding is affected differently by different CDK4 mutations. We also demonstrated that the interaction of IBR with CDK4 is different from that with its NF-B. While most of the contacts contributing to NF-B binding are located within the last two C-terminal ankyrin repeats and the loop region bridging them, the first four ankyrin repeats at the N-terminus are responsible for CDK4 binding and inhibition.