INTERLEUKIN 1-INDUCED PHOSPHORYLATION OF MAD3, THE MAJOR INHIBITOR OFNUCLEAR FACTOR KAPPA-B OF HELA-CELLS - INTERFERENCE IN SIGNALING BY THE PROTEINASE-INHIBITORS 2,4-DICHLOROISOCOUMARIN AND TOSYLPHENYLALENYLCHLOROMETHYLKETONE

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 rapidl y (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% r eduction of cellular I kappa B activity. Newly-synthesized MAD3 accumu lated to pre-stimulation levels between 60 and 90 min after stimulatio n and this coincided with the down-regulation of the phosphorylating a ctivity, The serine proteinase inhibitors 3,4-dichloroisocoumarin (DCI ) and tosylphenylalanyl chloromethylketone (TPCK) prevented phosphoryl ation and disappearance of MAD3. At the same concentrations (10-100 mu M), they also increased basal phosphorylation of the small heat shock protein (hsp27) and prevented the IL1- and phorbol 12-myristate 13-ac etate-induced increases of its phosphorylation. The inhibitors were th us 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 th e IL1-induced phosphorylation of MAD3 on serine or threonine leads to its degradation. DCI and TPCK blocked phosphorylation mechanisms and i t could not be concluded that serine proteinases were involved in the breakdown of MAD3.