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Cleavage of PITSLRE kinases by ICE/CASP-1 and CPP32/CASP-3 during apoptosis induced by tumor necrosis factor
R Beyaert1, V J Kidd, S Cornelis
1Laboratory of Molecular Biology, Flanders Interuniversity Institute for Biotechnology and University of Ghent, B-9000 Ghent, Belgium.
Abstract:
Emerging evidence suggests that multiple aspartate-specific cysteine proteases (caspases (CASPs)) play a crucial role in programmed cell death. Many cellular proteins have been identified as their substrates and serve as markers to assay the activation of CASPs during the death process. However, no substrate has yet been unambiguously identified as an effector molecule in apoptosis. PITSLRE kinases are a superfamily of Cdc2-like kinases that have been implicated in apoptotic signaling and tumorigenesis. In this paper we report that tumor necrosis factor (TNF)-mediated apoptosis is associated with a CrmA- and Bcl-2-inhibitable cleavage of PITSLRE kinases, indicating a role for CASPs. Testing of seven murine CASPs for their ability to cleave p110 PITSLRE kinase alpha2-1 in vitro revealed that only CASP-1 (ICE (interleukin-1beta-converting enzyme)) and CASP-3 (CPP32) were able to produce the same 43-kDa cleavage product as observed in cells undergoing TNF-induced apoptosis. Mutational analysis revealed that cleavage of p110 PITSLRE kinase alpha2-1 occurred at Asp393 within the sequence YVPDS, which is similar to that involved in the CASP-1-mediated cleavage of prointerleukin-1beta. TNF-induced proteolysis of PITSLRE kinases was still observed in fibroblasts from CASP-1(0/0) mice. These data implicate CASP-3 as a potentially important CASP family protease responsible for the cleavage of PITSLRE kinases during TNF-induced apoptosis.
Insights
Caspases (CASPs) are key in programmed cell death. This study identifies PITSLRE kinases as substrates cleaved by CASP-3 during tumor necrosis factor (TNF)-induced apoptosis, implicating CASP-3 as an effector protease.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Caspases (CASPs) are critical aspartate-specific cysteine proteases involved in programmed cell death.
- While many CASP substrates are known, none have been definitively identified as effector molecules in apoptosis.
- PITSLRE kinases, implicated in apoptosis and tumorigenesis, are a potential target.
Purpose of the Study:
- To investigate the role of CASPs in the cleavage of PITSLRE kinases during tumor necrosis factor (TNF)-induced apoptosis.
- To identify the specific CASP(s) responsible for PITSLRE kinase proteolysis.
Main Methods:
- Analysis of PITSLRE kinase cleavage in TNF-treated cells.
- In vitro cleavage assays using seven murine CASPs against p110 PITSLRE kinase alpha2-1.
- Mutational analysis to pinpoint the cleavage site.
- Experiments using fibroblasts from CASP-1 deficient mice.
Main Results:
- TNF-mediated apoptosis involved CrmA- and Bcl-2-inhibitable cleavage of PITSLRE kinases.
- Only CASP-1 (ICE) and CASP-3 (CPP32) cleaved p110 PITSLRE kinase alpha2-1 in vitro, producing a 43-kDa product.
- Cleavage occurred at Asp393 within the YVPDS sequence.
- PITSLRE kinase proteolysis was observed in CASP-1 deficient cells, suggesting CASP-3 involvement.
Conclusions:
- CASP-3 is implicated as a key protease responsible for cleaving PITSLRE kinases during TNF-induced apoptosis.
- This identifies PITSLRE kinases as effector substrates in the apoptotic pathway.
- The findings contribute to understanding CASP-mediated apoptosis mechanisms.