Induced stabilization of IkappaBalpha can facilitate its re-synthesis and prevent sequential degradation.
The transcription factor NF-kappaB is responsible for regulating genes that can profoundly impact cell proliferation, apoptosis, inflammation, and immune responses. The NF-kappaB inhibitor IkappaBalpha is rapidly degraded and then re-synthesized after an NF-kappaB stimulus. We have found that the re-synthesis of IkappaBalpha in a human colon-derived cell line (HT-29) includes the post-translational stabilization of newly synthesized IkappaBalpha. The TNF-alpha-induced stabilization of newly synthesized IkappaBalpha involves the C-terminal PEST region of the protein: N-terminal deletion mutants (lacking the IkappaB kinase phosphorylation sites) were readily stabilized by TNF-alpha, whereas deletion of the C-terminus resulted in a constitutively stable protein. The role of the C-terminus in stabilization was further supported by the finding that fusion of the IkappaBalpha C-terminus to GFP generated a protein that could also be stabilized by TNF-alpha. The p38 mitogen-activated protein (MAP) kinase inhibitor SB203580 prevented stabilization of IkappaBalpha and delayed the re-emergence of IkappaBalpha following TNF-alpha-induced degradation. The IkappaBalpha stabilization pathway could prevent sequential rounds of IkappaBalpha degradation without preventing IkappaBalpha phosphorylation. Analysis of two other cell lines (SW480 and THP-1) revealed similarities and cell-specific differences in the regulation of IkappaBalpha stabilization. We propose that cytokine stabilization of newly synthesized IkappaBalpha in some cell types is a critical homeostatic mechanism that limits inflammatory gene expression.
Place
Robert F
RF
Department of Molecular and Cell Biology, University of Connecticut, Storrs 06269, USA.
Haspeslagh
Derek
D
Giardina
Charles
C
D002460:Cell Line; D019073:HT29 Cells; D006801:Humans; D020672:I-kappa B Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D007375:Interleukin-1 / Q000494:pharmacology; D007700:Kinetics; D020928:Mitogen-Activated Protein Kinases / Q000502:physiology; D000072000:NF-KappaB Inhibitor alpha; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000032:analysis; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000037:antagonists & inhibitors / Q000494:pharmacology; D048051:p38 Mitogen-Activated Protein Kinases
D020672:I-kappa B Proteins; D007375:Interleukin-1; C000606204:NFKBIA protein, human; D011993:Recombinant Fusion Proteins; D014409:Tumor Necrosis Factor-alpha; D000072000:NF-KappaB Inhibitor alpha; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases