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J Biol Chem, Vol. 274, Issue 2, 787-794, January 8, 1999
-inducible I
B
Proteolysis
Mediated by Cytosolic m-Calpain
B ACTIVATION
,
§,
, and
From the The cytokine tumor necrosis factor
Department of Internal Medicine, the
§ Department of Physiology and Biophysics, the
¶ Department of Microbiology & Immunology, and the
Sealy
Center for Molecular Sciences, University of Texas Medical Branch,
Galveston, Texas 77555-1060
(TNF-
)
induces expression of inflammatory gene networks by activating
cytoplasmic to nuclear translocation of the nuclear factor-
B
(NF-
B) transcription factor. NF-
B activation results from
sequential phosphorylation and hydrolysis of the cytoplasmic inhibitor,
I
B
, through the 26 S proteasome. Here, we show a parallel
proteasome-independent pathway for cytokine-inducible I
B
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-
-inducible I
B
proteolysis; pretreatment with both is
required to completely block I
B
proteolysis. Similarly, in
transient cotransfection assays, expression of the specific inhibitor,
calpastatin, partially blocks TNF-
-inducible
NF-
B-dependent promoter activity and I
B
proteolysis. In TNF-
-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 I
B
compartment. Addition of catalytically active m-calpain into broken cells was sufficient to produce ligand-independent I
B
proteolysis and NF-
B translocation. As additional evidence for
calpain-dependent I
B
proteolysis and NF-
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, I
B
proteolysis occurs in a manner
sensitive only to calpain inhibitors. Our results demonstrate that
TNF-
activates cytosolic calpains, a parallel pathway that degrades
I
B
and activates NF-
B activation independently of the
ubiquitin-proteasome pathway.
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