Phosphorylation of IκBα in the C-Terminal PEST Domain by Casein Kinase II Affects Intrinsic Protein Stability

Abstract

The NF-B/Rel transcription factors participate in the activation of immune system regulatory genes and viral early genes including the human immunodeficiency virus type 1 long terminal repeat. NF-B/Rel proteins are coupled to inhibitory molecules, collectively termed IB, which are responsible for cytoplasmic retention of NF-B. Cell activation leads to the phosphorylation and degradation of IB␣, permitting NF-B/Rel translocation to the nucleus and target gene activation. To further characterize the signaling events that contribute to IB␣ phosphorylation, a kinase activity was isolated from Jurkat T cells that specifically interacted with IB␣ in an affinity chromatography step and phosphorylated IB␣ with high specificity in vitro. By using an in-gel kinase assay with recombinant IB␣ as substrate, two forms of the kinase (43 and 38 NF-B proteins are a family of pleiotropic transcription factors that regulate expression of numerous cytokines, and viral genes (for reviews, see references 27 and 48). In cells, NF-B is present in an inactive cytoplasmic form; localization of the p50-p65 heterodimeric form of NF-B to the cytoplasm is mediated by the inhibitory IB␣ protein that binds to and masks the nuclear localization signal of the p65 subunit of NF-B (5, 7). Activating agents, such as viruses, phorbol esters, cytokines, radical oxygen intermediates, and bacterial lipopolysaccharide, promote the dissociation of the cytosolic NF-B/IB complexes, in part by activating cellular kinases that phosphorylate IB (5-7, 22, 27, 48).The DNA-binding NF-B family members share a Rel homology domain that is responsible for DNA binding, nuclear localization, and protein dimerization. DNA-binding members of NF-B/Rel include p50 (NFKB1) (9, 23, 41), p65 (RelA) (52, 58), c-Rel (13, 24), p52 (NFKB2 or Lyt-10) (8, 51, 62), RelB (I-Rel) (59, 60), and dorsal (65). p50 and p52 are synthesized as precursors of p105 and p100, respectively, that are proteolytically processed to generate active DNA-binding p50 and p52 (8,9,23,41,51,62). Different NF-B dimers bind to variant NF-B sites (44) (consensus, 5Ј-GGGANNYYCC-3Ј) present in the promoter regions of many genes (reviewed in references 2 and 48).The intracellular localization and posttranslational activity of NF-B/Rel proteins are regulated by the ankyrin repeatcontaining IB proteins (IB␣, IB␥, Bcl-3, p105, and p100) (reviewed in references 5, 25, 28, and 39). Phosphorylation by activated cellular kinases and subsequent degradation of IB␣ appear to be critical regulatory steps in the control of NF-B activation. Recent studies demonstrated that mutation of either Ser-32 or Ser-36 blocked signal-induced IB␣ phosphorylation and degradation (10,11,69), and a truncated IB␣ that was missing 45 amino acids at the N terminus corrected radiation sensitivity in ataxia telangiectasia cells (37). Furthermore, IB␣ transcription is upregulated by NF-B, since the IB␣ promoter contains functional NF-B sites. Thus, an autoregulatory loop is established, and the ultimately increased IB␣ concentrations limit the...

Oncogenic activation of c-Myb by carboxyl-terminal truncation leads to decreased proteolysis by the ubiquitin-26S proteasome pathway

“…An attractive explanation for this e ect would be that the stabilization of the mutant protein results in increased transactivation potential of mutated c-Myb protein. In fact, PEST sequences rich in proline and serine residues contain many potential sites for protein kinases and phosphorylation dependent degradation of target proteins by the 26S proteasome was already described (Kornitzer et al, 1994;Yaglom et al, 1995;Lin et al, 1996). Interestingly, there is a striking correlation between the increase in half-life of the COOHterminally truncated c-Myb (2.8-fold on average) in this paper and the increase in transactivation activity (2 ± 4 fold) by similarly truncated proteins as reported by others (Sakura et al, 1989Nakagoshi et al, 1992Dini and Lipsick, 1993).…”

“…To determine the existence of such sequences in murine cMyb we employed the algorithm PEST-FIND, which scores all sequences of ten or more amino acids residues that are enriched in P, E, S, and T and anked by the basic residues R, K, or H and combines these results with the predicted hydrophilicity of potential PEST sequence (Rogers et al, 1986). In the recent years PEST sequences were linked to the instability signal on many short-lived proteins degraded by the ubiquitin-26S proteasome pathway and their deletion signi®cantly increased stability of several (Kornitzer et al, 1994;Tsurumi et al, 1995;Yaglom et al, 1995;Lin et al, 1996). Our search located three regions in murine c-Myb that had a positive PEST-score (PEST 1, 2 3; see Figure 11a).…”

NF‐κB as a potential molecular target for cancer therapy

“…The third signaling pathway is classified as atypical because it is independent of IKK, and is activated by UV and doxorubicin, both of which cause DNA damage [105]. UV radiation induces IκBα degradation via the proteasome, but the targeted serine residues are located within a C-terminal cluster, which is recognized by the p38-activated casein kinase 2 [8,68,83]. Oxidative stress also leads to NF-κB activation via IκBα tyrosine phosphorylation [55].…”

Role for casein kinase 2 in the regulation of HIF‐1 activity

“…45 Moreover, several reports have shown that phosphorylation may affect intrinsic protein stability as it was described for the IjB inhibitor protein. 29,30,46 Indeed, stimuli such as TNF-a cause rapid phosphorylation of the C-terminal PEST domain of IjB leading to ubiquitinylation and degradation of the inhibitor subunit by the proteasome 26S. NF-jB protein then translocates to the nucleus and activates gene expression.…”