An NF-kappaB-specific inhibitor, IkappaBalpha, binds to and inhibits cyclin-dependent kinase 4.
IkappaBalpha, a protein composed of six ankyrin repeats, is a specific inhibitor of nuclear factor kappaB (NF-kappaB) 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 IkappaBalpha binds to cyclin-dependent kinase 4 (CDK4) specifically and inhibits its kinase activity. The potencies of binding and inhibition of IkappaBalpha are comparable to those of INK4 proteins, the specific CDK4 inhibitors that also contain ankyrin repeats. Furthermore, we showed that INK4 proteins and IkappaBalpha compete with each other for binding to CDK4. These results led us to propose a hypothesis that there is cross talk between the NF-kappaB/IkappaBalpha pathway and the p16/CDK4/Rb pathway in cells, and that IkappaBalpha could substitute for the CDK4-inhibiting function of p16, a tumor suppressor frequently inactivated in human tumors. To further understand the structural basis of IkappaBalpha-CDK binding, we used different mutants of CDK4 to show that there are notable differences between IkappaBalpha and INK4 proteins in CDK4 binding since the binding is affected differently by different CDK4 mutations. We also demonstrated that the interaction of IkappaBalpha with CDK4 is different from that with its NF-kappaB. While most of the contacts contributing to NF-kappaB 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.
Li
Junan
J
Department of Biochemistry, The Ohio State University, Columbus, Ohio 43210, USA.
Joo
Sang Hoon
SH
Tsai
Ming-Daw
MD
D000595:Amino Acid Sequence; D017089:Ankyrin Repeat; D018797:Cell Cycle Proteins; D051358:Cyclin-Dependent Kinase 4; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000235:genetics / Q000378:metabolism; D050763:Cyclin-Dependent Kinase Inhibitor p18; D018844:Cyclin-Dependent Kinases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D020672:I-kappa B Proteins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000037:antagonists & inhibitors; D011233:Precipitin Tests; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011518:Proto-Oncogene Proteins; D011869:Radioligand Assay; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000494:pharmacology; D017386:Sequence Homology, Amino Acid; D025521:Tumor Suppressor Proteins / Q000235:genetics / Q000378:metabolism; D020798:Two-Hybrid System Techniques; D015003:Yeasts / Q000235:genetics
C493971:CDKN2C protein, human; D018797:Cell Cycle Proteins; D019941:Cyclin-Dependent Kinase Inhibitor p16; D050763:Cyclin-Dependent Kinase Inhibitor p18; D020672:I-kappa B Proteins; D016328:NF-kappa B; C000606204:NFKBIA protein, human; D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins; D025521:Tumor Suppressor Proteins; D000072000:NF-KappaB Inhibitor alpha; C495900:CDK4 protein, human; D051358:Cyclin-Dependent Kinase 4; D018844:Cyclin-Dependent Kinases