Transient nuclear factor kappaB (NF-kappaB) activation stimulated by interleukin-1beta may be partly dependent on proteasome activity, but not phosphorylation and ubiquitination of the IkappaBalpha molecule, in C6 glioma cells. Regulation of NF-kappaB linked to chemokine production.
We previously reported that several stresses can induce cytokine-induced neutrophil chemoattractant expression in a nuclear factor kappaB (NF-kappaB)-dependent manner. In this study, we focused further on the regulation of NF-kappaB. The activation of NF-kappaB and the subsequent cytokine-induced neutrophil chemoattractant induction in response to interleukin-1beta (IL-1beta) were inhibited by proteasome inhibitors, MG132 and proteasome inhibitor I. Translocation of NF-kappaB into nuclei occurs by the phosphorylation, multi-ubiquitination, and degradation of IkappaBalpha, a regulatory protein of NF-kappaB. Nascent IkappaBalpha began to degrade 5 min after treatment with IL-1beta and disappeared completely after 15 min. However, IkappaBalpha returned to basal levels after 45-60 min. Interestingly, resynthesized IkappaBalpha was already phosphorylated at Ser-32. These results suggest that 1) the upstream signals are still activated, although the translocation of NF-kappaB peaks at 15 min; and 2) the regulated protein(s) acts downstream of IkappaBalpha phosphorylation. Western blotting showed that the resynthesized and phosphorylated IkappaB molecules were also upward-shifted by multi-ubiquitination in response to IL-1beta treatment. On the other hand, ATP-dependent Leu-Leu-Val-Tyr cleaving activity transiently increased, peaked at 15 min, and then decreased to basal levels at 60 min. Furthermore, the cytosolic fraction that was stimulated by IL-1beta for 15 min, but not for 0 and 60 min, could degrade phosphorylated and multi-ubiquitinated IkappaBalpha. These results indicate that the transient translocation of NF-kappaB in response to IL-1beta may be partly dependent on transient proteasome activation.
Uehara
T
T
Department of Pharmacology, Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo 060-0812, Japan.
Matsuno
J
J
Kaneko
M
M
Nishiya
T
T
Fujimuro
M
M
Yokosawa
H
H
Nomura
Y
Y
D000818:Animals; D016227:Benzoquinones; D019743:Chemokines, CXC; D002630:Chemotactic Factors / Q000235:genetics; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004789:Enzyme Activation; D005786:Gene Expression Regulation / Q000187:drug effects; D005910:Glioma; D006133:Growth Substances / Q000235:genetics; D020672:I-kappa B Proteins; D036341:Intercellular Signaling Peptides and Proteins; D007375:Interleukin-1 / Q000378:metabolism*; D047029:Lactams, Macrocyclic; D007976:Leupeptins / Q000494:pharmacology; D009097:Multienzyme Complexes / Q000378:metabolism*; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000378:metabolism*; D009842:Oligopeptides / Q000378:metabolism; D010766:Phosphorylation; D011480:Protease Inhibitors / Q000494:pharmacology; D046988:Proteasome Endopeptidase Complex; D011809:Quinones / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017828:Rifabutin / Q000031:analogs & derivatives; D015533:Transcriptional Activation / Q000187:drug effects; D014407:Tumor Cells, Cultured; D014452:Ubiquitins / Q000378:metabolism*
D016227:Benzoquinones; D019743:Chemokines, CXC; D002630:Chemotactic Factors; D004268:DNA-Binding Proteins; D006133:Growth Substances; D020672:I-kappa B Proteins; D036341:Intercellular Signaling Peptides and Proteins; D007375:Interleukin-1; D047029:Lactams, Macrocyclic; D007976:Leupeptins; D009097:Multienzyme Complexes; D016328:NF-kappa B; C000606206:Nfkbia protein, rat; D009842:Oligopeptides; D011480:Protease Inhibitors; D011809:Quinones; D012333:RNA, Messenger; D014452:Ubiquitins; D000072000:NF-KappaB Inhibitor alpha; D017828:Rifabutin; C020754:herbimycin; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde