Constitutive Nuclear Expression of the IκB Kinase Complex and Its Activation in Human Neutrophils

Abstract

A singular feature of human neutrophils is that they constitutively express substantial amounts of NF-κB/Rel proteins and IκB-α in the nucleus. In this study, we show that in these cells, IκB kinase α (IKKα), IKKβ, and IKKγ also partially localize to the nucleus, whereas IKK-related kinases (IKKε, TANK-binding kinase-1) are strictly cytoplasmic, and the NF-κB-inducing kinase is strictly nuclear. Following neutrophil activation, IKKβ and IKKγ become transiently phosphorylated in both the cytoplasm and nucleus, whereas IKKα transiently vanishes from both compartments in what appears to be an IKKβ-dependent process. These responses are paralleled by the degradation of IκB-α, and by the phosphorylation of RelA on serine 536, in both compartments. Although both proteins can be IKK substrates, inhibition of IKK prevented IκB-α phosphorylation, while that of RelA was mostly unaffected. Finally, we provide evidence that the nuclear IKK isoforms (α, β, γ) associate with chromatin following neutrophil activation, which suggests a potential role in gene regulation. This is the first study to document IKK activation and the phosphorylation of NF-κB/Rel proteins in primary neutrophils. More importantly, our findings unveil a hitherto unsuspected mode of activation for the IKK/IκB signaling cascade within the cell nucleus.

Activation of TAK1 by Chemotactic and Growth Factors, and Its Impact on Human Neutrophil Signaling and Functional Responses

“…3C). This was also a belated event in comparison with cells stimulated with either LPS or TNF-a, in which IKKg phosphorylation occurs within 5 min (25). Neutrophil pretreatment with 5z-7-oxozeaenol had no effect on IKKg phosphorylation elicited by fMLF (data not shown).…”

“…Neutrophils were isolated from the peripheral blood of healthy donors from whom informed consent had been obtained, under a protocol that was approved by an institutional ethics committee, as described previously (25). As determined by Wright staining and nonspecific esterase cytochemistry, the final neutrophil suspensions consistently contained fewer than 0.2% monocytes or lymphocytes, and neutrophil viability exceeded 98% after up to 4 h in culture, as determined by trypan blue exclusion.…”

“…Cells were then centrifuged and resuspended in Relaxation buffer before disruption by nitrogen cavitation, as described previously (26,27). Whole cell samples or subcellular fractions were then prepared as described previously (25)(26)(27). Samples were electrophoresed on denaturing gels prepared according to the method of Laemmli (28); equal loading was ascertained by adjusting sample volumes based on their respective protein content.…”

Acute Alcohol Exposure Exerts Anti-Inflammatory Effects by Inhibiting IκB Kinase Activity and p65 Phosphorylation in Human Monocytes

“…Recent studies have ascribed a different role for IKK␣ and IKK␤ in the nucleus and cytoplasm of various cell types (33). In this study, we investigated whether acute alcohol exposure affected IKK␣ and IKK␤ protein expression in the nucleus and cytoplasm of human monocytes.…”

Nuclear Accumulation of cRel following C-Terminal phosphorylation by TBK1/IKKε

“…IKK␣ was shown to modify histones via phosphorylation, a mechanism essential for the activation of NF-B-directed gene expression (50,51). More recently, it was reported that in primary neutrophils, IKK␣␤␥ and RelA/p65 are constitutively localized to the nucleus, whereas TBK1 and IKK remain exclusively cytoplasmic (52). It remains a possibility that a two-step modification of cRel is required for nuclear translocation and full transcriptional activation.…”