Li Junan, Joo Sang Hoon, Tsai Ming-Daw
Department of Biochemistry, The Ohio State University, Columbus, Ohio 43210, USA.
Biochemistry. 2003 Nov 25;42(46):13476-83. doi: 10.1021/bi035390r.
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.
IκBα是一种由六个锚蛋白重复序列组成的蛋白质,是核因子κB(NF-κB)的特异性抑制剂,在许多不同细胞类型的信号转导中发挥作用。通过体内酵母双杂交试验以及体外活性和结合试验,我们发现IκBα能特异性结合细胞周期蛋白依赖性激酶4(CDK4)并抑制其激酶活性。IκBα的结合和抑制能力与INK4蛋白相当,INK4蛋白也是特异性的CDK4抑制剂,同样含有锚蛋白重复序列。此外,我们还发现INK4蛋白和IκBα在与CDK4的结合上相互竞争。这些结果使我们提出一个假说:细胞中NF-κB/IκBα信号通路与p16/CDK4/Rb信号通路之间存在相互作用,并且IκBα可以替代p16的CDK4抑制功能,p16是一种在人类肿瘤中经常失活的肿瘤抑制因子。为了进一步了解IκBα与CDK结合的结构基础,我们使用了不同的CDK4突变体,结果表明IκBα和INK4蛋白在与CDK4结合方面存在显著差异,因为不同的CDK4突变对结合的影响不同。我们还证明了IκBα与CDK4的相互作用不同于其与NF-κB的相互作用。虽然大多数与NF-κB结合有关的接触位点位于C末端最后两个锚蛋白重复序列及其之间的环区,但N末端的前四个锚蛋白重复序列负责与CDK4的结合和抑制。