Interleukin 1-induced phosphorylation of MAD3, the major inhibitor of nuclear factor kappa B of HeLa cells. Interference in signalling by the proteinase inhibitors 3,4-dichloroisocoumarin and tosylphenylalanyl chloromethylketone.
The regulation of the inhibitor of nuclear factor kappa B (I kappa B) by interleukin 1 (IL1) was investigated in HeLa cells. Two forms of I kappa B were resolved by ion-exchange chromatography. The major form (75%) was identified as MAD3 by specific antisera. IL1 generated rapidly (6 min) an electrophoretically retarded form of MAD3 that was stable in acid and was converted into the unmodified form by phosphatase 2A. It thus corresponded to a phosphorylation of the protein on serine or threonine. IL1 also caused the disappearance of MAD3 from the cells, which was complete 15 min after stimulation and coincided with a 46% reduction of cellular I kappa B activity. Newly-synthesized MAD3 accumulated to pre-stimulation levels between 60 and 90 min after stimulation and this coincided with the down-regulation of the phosphorylating activity. The serine proteinase inhibitors 3,4-dichloroisocoumarin (DCI) and tosylphenylalanyl chloromethylketone (TPCK) prevented phosphorylation and disappearance of MAD3. At the same concentrations (10-100 microM), they also increased basal phosphorylation of the small heat shock protein (hsp27) and prevented the IL1- and phorbol 12-myristate 13-acetate-induced increases of its phosphorylation. The inhibitors were thus interfering with protein kinases when blocking degradation of MAD3. Recombinant MAD3 phosphorylated in vitro by protein kinase C was not electrophoretically retarded, suggesting that MAD3 was phosphorylated by another kinase in IL1-stimulated cells. Our results suggest that the IL1-induced phosphorylation of MAD3 on serine or threonine leads to its degradation. DCI and TPCK blocked phosphorylation mechanisms and it could not be concluded that serine proteinases were involved in the breakdown of MAD3.
Guesdon
F
F
Department of Development and Signalling, Babraham Institute, Cambridge, U.K.
Ikebe
T
T
Stylianou
E
E
Warwick-Davies
J
J
Haskill
S
S
Saklatvala
J
J
D001483:Base Sequence; D002852:Chromatography, Ion Exchange; D003374:Coumarins / Q000494:pharmacology; D004268:DNA-Binding Proteins / Q000276:immunology / Q000378:metabolism* / Q000494:pharmacology; D006367:HeLa Cells / Q000187:drug effects / Q000378:metabolism*; D006360:Heat-Shock Proteins / Q000378:metabolism; D006801:Humans; D020672:I-kappa B Proteins; D007375:Interleukin-1 / Q000494:pharmacology*; D049934:Isocoumarins; D008969:Molecular Sequence Data; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000037:antagonists & inhibitors* / Q000235:genetics; D009363:Neoplasm Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000276:immunology; D010766:Phosphorylation / Q000187:drug effects; D011493:Protein Kinase C / Q000378:metabolism; D011499:Protein Processing, Post-Translational / Q000187:drug effects*; D011518:Proto-Oncogene Proteins / Q000037:antagonists & inhibitors / Q000145:classification / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000276:immunology; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology*; D015398:Signal Transduction / Q000187:drug effects*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014108:Tosylphenylalanyl Chloromethyl Ketone / Q000494:pharmacology; D051841:Transcription Factor RelB; D014157:Transcription Factors
D003374:Coumarins; D004268:DNA-Binding Proteins; D006360:Heat-Shock Proteins; D020672:I-kappa B Proteins; D007375:Interleukin-1; D049934:Isocoumarins; D016328:NF-kappa B; C000606204:NFKBIA protein, human; D009363:Neoplasm Proteins; D011518:Proto-Oncogene Proteins; C497593:RELB protein, human; D011993:Recombinant Fusion Proteins; D015842:Serine Proteinase Inhibitors; D014157:Transcription Factors; D000072000:NF-KappaB Inhibitor alpha; D051841:Transcription Factor RelB; D014108:Tosylphenylalanyl Chloromethyl Ketone; C046235:3,4-dichloroisocoumarin; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate