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.
Massa
Paul E
PE
Genetics Graduate Program, the Department of Biochemistry and Cell Biology, State University of New York at Stony Brook, Stony Brook, New York 11794-5215, USA.
Li
Xiang
X
Hanidu
Adedayo
A
Siamas
John
J
Pariali
Milena
M
Pareja
Jessica
J
Savitt
Anne G
AG
Catron
Katrina M
KM
Li
Jun
J
Marcu
Kenneth B
KB
D000818:Animals; D002478:Cells, Cultured; D005347:Fibroblasts / Q000166:cytology / Q000502:physiology; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D051550:I-kappa B Kinase; D051379:Mice; D018345:Mice, Knockout; D008969:Molecular Sequence Data; D005810:Multigene Family; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism*; D020411:Oligonucleotide Array Sequence Analysis; D017346:Protein Serine-Threonine Kinases / Q000235:genetics* / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism
D016328:NF-kappa B; D012333:RNA, Messenger; D017346:Protein Serine-Threonine Kinases; C496564:Chuk protein, mouse; D051550:I-kappa B Kinase; C496562:Ikbkb protein, mouse; C496566:Ikbke protein, mouse