IkappaB kinase alpha regulates subcellular distribution and turnover of cyclin D1 by phosphorylation

IkappaB kinases (IKKs), IKKalpha and IKKbeta, with a regulatory subunit IKKgamma/NEMO constitute a high molecular weight IKK complex that regulates NF-kappaB activity. Although IKKalpha and IKKbeta share structural and biochemical similarities, IKKalpha has been shown to have distinct biological roles. Here we show that IKKalpha plays a critical role in regulating cyclin D1 during the cell cycle. Analysis of IKKalpha-/- mouse embryo fibroblast cells showed that cyclin D1 is overexpressed and localized in the nucleus compared with parental mouse embryo fibroblasts. IKKalpha associates with and phosphorylates cyclin D1. Analysis on cyclin D1 mutants demonstrated that IKKalpha phosphorylates cyclin D1 at Thr286. Reconstitution of IKKalpha in knockout cells leads to nuclear export and increased degradation of cyclin D1. Further, RNAi-mediated knockdown of IKKalpha results in similar changes as observed in IKKalpha-/- cells. These results suggest a novel role of IKKalpha in regulating subcellular localization and proteolysis of cyclin D1 by phosphorylation of cyclin D1 at Thr286, the same residue earlier found to be a target for glycogen synthase kinase-3beta-induced phosphorylation.     

Gene expression profiling in conjunction with physiological rescues of IKKalpha-null cells with wild type or mutant IKKalpha reveals distinct classes of IKKalpha/NF-kappaB-dependent genes

Cellular responses to stress-like stimuli require the IkappaB kinase (IKK) signalsome (IKKalpha, IKKbeta, and NEMO/IKKgamma) to activate NF-kappaB-dependent genes. IKKbeta and NEMO/IKKgamma are required to release NF-kappaB p65/p50 heterodimers from IkappaBalpha, resulting in their nuclear migration and sequence-specific DNA binding; but IKKalpha was found to be dispensable for this initial phase of canonical NF-kappaB activation. Nevertheless, IKKalpha-/- mouse embryonic fibroblasts (MEFs) fail to express NF-kappaB targets in response to proinflammatory stimuli, uncovering a nuclear role for IKKalpha in NF-kappaB activation. However, it remains unknown whether the global defect in NF-kappaB-dependent gene expression of IKKalpha-/- cells is caused by the absence of IKKalpha kinase activity. We show by gene expression profiling that rescue of near physiological levels of wild type IKKalpha in IKKalpha-/- MEFs globally restores expression of their canonical NF-kappaB target genes. To prove that the kinase activity of IKKalpha was required on a genomic scale, the same physiological rescue was performed with a kinase-dead, ATP binding domain IKKalpha mutant (IKKalpha(K44M)). Remarkably, the IKKalpha(K44M) protein rescued approximately 28% of these genes, albeit in a largely stimulus-independent manner with the notable exception of several genes that also acquired tumor necrosis factor-alpha responsiveness. Thus the IKKalpha-containing signalsome unexpectedly functions in the presence and absence of extracellular signals in both kinase-dependent and -independent modes to differentially modulate the expression of five distinct classes of IKKalpha/NF-kappaB-dependent genes.