Differential phosphorylation of the signal-responsive domain of I kappa B alpha and I kappa B beta by I kappa B kinases

NF-kappa B activity is regulated by its association with the inhibitory I kappa B proteins, among which I kappa B alpha and I kappa B beta are the most abundant. I kappa B proteins are widely expressed in different cells and tissues and bind to similar combinations of NF-kappa B proteins. The degradation of I kappa B proteins allows nuclear translocation of NF-kappa B and hence plays a critical role in NF-kappa B activation. Previous studies have demonstrated that, although both I kappa B proteins are phosphorylated by the same I kappa B kinase (IKK) complex, and their ubiquitination and degradation following phosphorylation are carried out by the same ubiquitination/degradation machinery, their kinetics of degradation are quite different. To better understand the underlying mechanism of the differences in degradation kinetics, we have carried out a systematic, comparative analysis of the ability of the IKK catalytic subunits to phosphorylate I kappa B alpha and I kappa B beta. We found that, whereas IKK alpha is a weak kinase for the N-terminal serines of both I kappa B isoforms, IKK beta is an efficient kinase for those residues in I kappa B alpha. However, IKK beta phosphorylates the N-terminal serines of I kappa B beta far less efficiently, thereby providing an explanation for the slower rate of degradation observed for I kappa B beta. Mutational analysis indicated that the regions around the two N-terminal serines collectively influence the relative phosphorylation efficiency, and no individual residue is critical. These findings provide the first systematic analysis of the ability of I kappa B alpha and I kappa B beta to serve as substrates for IKKs and help provide a possible explanation for the differential degradation kinetics of I kappa B alpha and I kappa B beta.     

Role of unphosphorylated, newly synthesized I kappa B beta in persistent activation of NF-kappa B.

Stimulation with inducers that cause persistent activation of NF-kappa B results in the degradation of the NF-kappa B inhibitors, I kappa B alpha and I kappa B beta. Despite the rapid resynthesis and accumulation of I kappa B alpha, NF-kappa B remains induced under these conditions. We now report that I kappa B beta is also resynthesized in stimulated cells and appears as an unphosphorylated protein. The unphosphorylated I kappa B beta forms a stable complex with NF-kappa B in the cytosol; however, this binding fails to mask the nuclear localization signal and DNA binding domain on NF-kappa B, and the I kappa B beta-NF-kappa B complex enters the nucleus. It appears therefore that during prolonged stimulation, I kappa B beta functions as a chaperone for NF-kappa B by protecting it from I kappa B alpha and allowing it to be transported to the nucleus.     

Cyclooxygenase (COX)-2 inhibitor celecoxib abrogates TNF-induced NF-kappa B activation through inhibition of activation of I kappa B alpha kinase and Akt in human non-small cell lung carcinoma: correlation with suppression of COX-2 synthesis

The cyclooxygenase 2 (COX-2) inhibitor celecoxib (also called celebrex), approved for the treatment of colon carcinogenesis, rheumatoid arthritis, and other inflammatory diseases, has been shown to induce apoptosis and inhibit angiogenesis. Because NF-kappa B plays a major role in regulation of apoptosis, angiogenesis, carcinogenesis, and inflammation, we postulated that celecoxib modulates NF-kappa B. In the present study, we investigated the effect of this drug on the activation of NF-kappa B by a wide variety of agents. We found that celecoxib suppressed NF-kappa B activation induced by various carcinogens, including TNF, phorbol ester, okadaic acid, LPS, and IL-1 beta. Celecoxib inhibited TNF-induced I kappa B alpha kinase activation, leading to suppression of I kappa B alpha phosphorylation and degradation. Celecoxib suppressed both inducible and constitutive NF-kappa B without cell type specificity. Celecoxib also suppressed p65 phosphorylation and nuclear translocation. Akt activation, which is required for TNF-induced NF-kappa B activation, was also suppressed by this drug. Celecoxib also inhibited the TNF-induced interaction of Akt with I kappa B alpha kinase (IKK). Celecoxib abrogated the NF-kappa B-dependent reporter gene expression activated by TNF, TNF receptor, TNF receptor-associated death domain, TNF receptor-associated factor 2, NF-kappa B-inducing kinase, and IKK, but not that activated by p65. The COX-2 promoter, which is regulated by NF-kappa B, was also inhibited by celecoxib, and this inhibition correlated with suppression of TNF-induced COX-2 expression. Besides NF-kappa B, celecoxib also suppressed TNF-induced JNK, p38 MAPK, and ERK activation. Thus, overall, our results indicate that celecoxib inhibits NF-kappa B activation through inhibition of IKK and Akt activation, leading to down-regulation of synthesis of COX-2 and other genes needed for inflammation, proliferation, and carcinogenesis.     

The I kappa B kinase (IKK) complex is tripartite and contains IKK gamma but not IKAP as a regular component

A critical step in the activation of NF-kappa B is the phosphorylation of I kappa Bs by the I kappa B kinase (IKK) complex. IKK alpha and IKK beta are the two catalytic subunits of the IKK complex and two additional molecules, IKK gamma/NEMO and IKAP, have been described as further integral members. We have analyzed the function of both proteins for IKK complex composition and NF-kappa B signaling. IKAP and IKK gamma belong to distinct cellular complexes. Quantitative association of IKK gamma was observed with IKK alpha and IKK beta. In contrast IKAP was complexed with several distinct polypeptides. Overexpression of either IKK gamma or IKAP blocked tumor necrosis factor alpha induction of an NF-kappa B-dependent reporter construct, but IKAP in addition affected several NF-kappa B-independent promoters. Whereas specific down-regulation of IKK gamma protein levels by antisense oligonucleotides significantly reduced cytokine-mediated activation of the IKK complex and subsequent NF-kappa B activation, a similar reduction of IKAP protein levels had no effect on NF-kappa B signaling. Using solely IKK alpha, IKK beta, and IKK gamma, we could reconstitute a complex whose apparent molecular weight is comparable to that of the endogenous IKK complex. We conclude that while IKK gamma is a stoichiometric component of the IKK complex, obligatory for NF-kappa B signaling, IKAP is not associated with IKKs and plays no specific role in cytokine-induced NF-kappa B activation.     

Protein kinase C-associated kinase (PKK) mediates Bcl10-independent NF-kappa B activation induced by phorbol ester

Protein kinase C-associated kinase (PKK) is a recently described kinase of unknown function that was identified on the basis of its specific interaction with PKC beta. PKK contains N-terminal kinase and C-terminal ankyrin repeats domains linked to an intermediate region. Here we report that the kinase domain of PKK is highly homologous to that of two mediators of nuclear factor-kappa B (NF-kappa B) activation, RICK and RIP, but these related kinases have different C-terminal domains for binding to upstream factors. We find that expression of PKK, like RICK and RIP, induces NF-kappa B activation. Mutational analysis revealed that the kinase domain of PKK is essential for NF-kappa B activation, whereas replacement of serine residues in the putative activation loop did not affect the ability of PKK to activate NF-kappa B. A catalytic inactive PKK mutant inhibited NF-kappa B activation induced by phorbol ester and Ca(2+)-ionophore, but it did not block that mediated by tumor necrosis factor alpha, interleukin-1 beta, or Nod1. Inhibition of NF-kappa B activation by dominant negative PKK was reverted by co-expression of PKC beta I, suggesting a functional association between PKK and PKC beta I. PKK-mediated NF-kappa B activation required IKK alpha and IKK beta but not IKK gamma, the regulatory subunit of the IKK complex. Moreover, NF-kappa B activation induced by PKK was not inhibited by dominant negative Bimp1 and proceeded in the absence of Bcl10, two components of a recently described PKC signaling pathway. These results suggest that PKK is a member of the RICK/RIP family of kinases, which is involved in a PKC-activated NF-kappa B signaling pathway that is independent of Bcl10 and IKK gamma.