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Phosphorylation of IkappaB-alpha inhibits its cleavage by caspase CPP32 in vitro
M Barkett1, D Xue, H R Horvitz
1Department of Biology, Boston University, Boston, Massachusetts 02215, USA.
Insights
The cell-death protease CPP32 (caspase-3) directly cleaves IkappaB-alpha, a key regulator of NF-kappaB. This cleavage links the NF-kappaB signaling pathway to cell death processes.
Area of Science:
- Molecular Biology
- Cell Signaling
- Biochemistry
Background:
- IkappaB proteins regulate Rel/NF-kappaB transcription factors.
- NF-kappaB signaling is crucial in immune responses and cell survival.
- Cell-death proteases, such as caspases, execute programmed cell death.
Purpose of the Study:
- To investigate the direct interaction between IkappaB proteins and caspases.
- To identify the specific caspase responsible for IkappaB cleavage.
- To elucidate the functional consequences of IkappaB cleavage in relation to NF-kappaB signaling and cell death.
Main Methods:
- In vitro cleavage assays using purified CPP32 (caspase-3) and recombinant IkappaB-alpha.
- Analysis of IkappaB-alpha cleavage in vivo in apoptotic cells.
- Comparison of CPP32 activity with other caspases (caspase-1, caspase-2).
- Investigation of the effect of IkappaB-alpha phosphorylation on CPP32-mediated cleavage.
Main Results:
- CPP32 (caspase-3) specifically cleaved chicken and human IkappaB-alpha at a conserved Asp-Ser site.
- This cleavage site is identical to the in vivo cleavage site during apoptosis.
- CPP32 also cleaved IkappaB-beta, but other caspases (caspase-1, caspase-2) did not cleave IkappaB-alpha.
- Serine phosphorylation of IkappaB-alpha inhibited CPP32-mediated cleavage.
- Cleavage could generate a constitutive inhibitor of Rel transcription complexes.
Conclusions:
- CPP32 (caspase-3) directly cleaves IkappaB-alpha and IkappaB-beta at specific sites.
- This cleavage provides a direct biochemical link between NF-kappaB signaling and cell-death protease pathways.
- Cleavage may modulate NF-kappaB activity during apoptosis, potentially by generating inhibitors of Rel transcription complexes.
Abstract:
IkappaB proteins function as direct regulators of Rel/NF-kappaB transcription complexes. We show that the cell-death protease CPP32 (caspase-3) in vitro specifically cleaved chicken and human IkappaB-alpha at a conserved Asp-Ser sequence. This cleavage site appears to be identical to the site at which chicken IkappaB-alpha is cleaved in vivo in temperature-sensitive v-Rel-transformed chicken spleen cells undergoing apoptosis. Other caspases, namely interleukin-1beta-converting enzyme (caspase-1) and Ich-1 (caspase-2), did not cleave IkappaB-alpha. CPP32 also cleaved mammalian IkappaB-beta in vitro at the analogous Asp-Ser sequence. Cleavage of IkappaB-alpha by CPP32 was blocked by serine phosphorylation of IkappaB-alpha. Cleavage of IkappaB-alpha by a CPP32- like protease could generate a constitutive inhibitor of Rel transcription complexes. This report provides evidence for a direct biochemical interaction between the NF-kappaB signaling pathway and a cell-death protease signaling pathway.