JBC Invitrogen Ultrasensitive Cytokine Assays

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J Biol Chem, Vol. 274, Issue 2, 787-794, January 8, 1999

Tumor Necrosis Factor-alpha -inducible Ikappa Balpha Proteolysis Mediated by Cytosolic m-Calpain
A MECHANISM PARALLEL TO THE UBIQUITIN-PROTEASOME PATHWAY FOR NUCLEAR FACTOR-kappa B ACTIVATION

Youqi HanDagger , Steven WeinmanDagger §, Istvan Boldogh, Randall K. Walkerparallel , and Allan R. BrasierDagger parallel

From the Dagger  Department of Internal Medicine, the § Department of Physiology and Biophysics, the  Department of Microbiology & Immunology, and the parallel  Sealy Center for Molecular Sciences, University of Texas Medical Branch, Galveston, Texas 77555-1060

The cytokine tumor necrosis factor alpha  (TNF-alpha ) induces expression of inflammatory gene networks by activating cytoplasmic to nuclear translocation of the nuclear factor-kappa B (NF-kappa B) transcription factor. NF-kappa B activation results from sequential phosphorylation and hydrolysis of the cytoplasmic inhibitor, Ikappa Balpha , through the 26 S proteasome. Here, we show a parallel proteasome-independent pathway for cytokine-inducible Ikappa Balpha proteolysis in HepG2 liver cells mediated by cytosolic calcium-activated neutral protease (calpains). Pretreatment with either calpain- or proteasome-selective inhibitors partially blocks up to 50% of TNF-alpha -inducible Ikappa Balpha proteolysis; pretreatment with both is required to completely block Ikappa Balpha proteolysis. Similarly, in transient cotransfection assays, expression of the specific inhibitor, calpastatin, partially blocks TNF-alpha -inducible NF-kappa B-dependent promoter activity and Ikappa Balpha proteolysis. In TNF-alpha -stimulated cells, a rapid (within 1 min), 2.2-fold increase in cytosolic calpain proteolytic activity is measured using a specific fluorescent assay. Inducible calpain proteolytic activity occurs coincidentally with the particulate-to-cytosol redistribution of the catalytic m-calpain subunit into the Ikappa Balpha compartment. Addition of catalytically active m-calpain into broken cells was sufficient to produce ligand-independent Ikappa Balpha proteolysis and NF-kappa B translocation. As additional evidence for calpain-dependent Ikappa Balpha proteolysis and NF-kappa B activation, we demonstrate that this process occurs in a cell line (ts20b) deficient in the ubiquitin-proteasome pathway. Following inactivation of the temperature-sensitive ubiquitin-activating enzyme, Ikappa Balpha proteolysis occurs in a manner sensitive only to calpain inhibitors. Our results demonstrate that TNF-alpha activates cytosolic calpains, a parallel pathway that degrades Ikappa Balpha and activates NF-kappa B activation independently of the ubiquitin-proteasome pathway.


Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.



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