Tumor necrosis factor-alpha-inducible I kappa B alpha proteolysis mediatedby cytosolic m-calpain - A mechanism parallel to the ubiquitin-proteasome pathway for nuclear factor-kappa B activation
Yq. Han et al., Tumor necrosis factor-alpha-inducible I kappa B alpha proteolysis mediatedby cytosolic m-calpain - A mechanism parallel to the ubiquitin-proteasome pathway for nuclear factor-kappa B activation, J BIOL CHEM, 274(2), 1999, pp. 787-794
The cytokine tumor necrosis factor alpha (TNF-alpha) induces expression of
inflammatory gene networks by activating cytoplasmic to nuclear translocati
on of the nuclear factor-kappa B (NF-kappa B) transcription factor. NF-kapp
a B activation results from sequential phosphorylation and hydrolysis of th
e cytoplasmic inhibitor, I kappa B alpha, through the 26 S proteasome, Here
, we show a parallel proteasome-independent pathway for cytokine-inducible
I kappa B alpha proteolysis in HepG2 liver cells mediated by cytosolic calc
ium-activated neutral protease (calpains). Pretreatment with either calpain
- or proteasome-selective inhibitors partially blocks up to 50% of TNF-alph
a-inducible I kappa B alpha proteolysis; pretreatment with both is required
to completely block I kappa B alpha proteolysis. Similarly, in transient c
otransfection assays, expression of the specific inhibitor, calpastatin, pa
rtially blocks TNF-alpha-inducible NF-kappa B-dependent promoter activity a
nd I kappa B alpha proteolysis. In TNF-alpha-stimulated cells, a rapid (wit
hin 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 redistributi
on of the catalytic m-calpain subunit into the I kappa Ba alpha compartment
, Addition of catalytically active m-calpain into broken cells was sufficie
nt to produce ligand-independent I kappa B alpha proteolysis and NF-kappa B
translocation. As additional evidence for calpain-dependent I kappa B alph
a proteolysis and NF-kappa B activation, we demonstrate that this process o
ccurs in a cell line (ts20b) deficient in the ubiquitin-proteasome pathway,
Following inactivation of the temperature-sensitive ubiquitin-activating e
nzyme, I kappa B alpha proteolysis occurs in a manner sensitive only to cal
pain inhibitors. Our results demonstrate that TNF-alpha activates cytosolic
calpains, a parallel pathway that degrades I kappa B alpha and activates N
F-kappa B activation independently of the ubiquitin-proteasome pathway.