Related Experiment Videos
Phosphorylation of PITSLRE p110 isoforms accompanies their processing by caspases during Fas-mediated cell death
D Tang1, R Gururajan, V J Kidd
1Department of Tumor Cell Biology, St. Jude Children's Research Hospital, Memphis, Tennessee 38101, USA.
Abstract:
A number of cellular proteins have been identified as caspase targets during cell death, including the PITSLRE protein kinases. These targets generally fall into one of three possible categories: 1) other caspases, 2) proteins that are inactivated during apoptosis, and 3) proteins that are required for execution of the cell death program. However, not all proteins are cleaved by caspases during apoptosis. Why only specific proteins are destined to be processed by caspases during cell death is currently not clear. Here we show that multiple caspase-like activities are involved in the processing of the PITSLRE p110 isoforms during Fas-induced apoptosis in Jurkat T-cells. Three p110 caspase cleavage sites have been mapped to the amino-terminal domain of p110 and verified by site-directed mutagenesis. Curiously, the mutagenesis studies revealed that cleavage of two juxtaposed caspase sites is necessary for the complete processing of this protein during cell death in vivo. Finally, we demonstrate that the PITSLRE p110 protein is rapidly phosphorylated during Fas-induced apoptosis in Jurkat cells and that phosphorylation of an amino-terminal portion of the protein may enhance caspase cleavage in this region.
Insights
Multiple caspase-like activities process PITSLRE p110 during Fas-induced apoptosis. Phosphorylation may enhance caspase cleavage, revealing specific protein processing during cell death.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Caspase-mediated cleavage targets specific proteins during apoptosis.
- The precise mechanisms determining caspase substrate specificity remain unclear.
- PITSLRE protein kinases are known caspase targets during programmed cell death.
Purpose of the Study:
- To investigate the caspase-like processing of PITSLRE p110 isoforms during Fas-induced apoptosis.
- To identify and characterize caspase cleavage sites within the PITSLRE p110 protein.
- To explore the role of phosphorylation in regulating PITSLRE p110 caspase processing.
Main Methods:
- Site-directed mutagenesis to verify caspase cleavage sites.
- Analysis of PITSLRE p110 processing in Jurkat T-cells undergoing Fas-induced apoptosis.
- Phosphorylation assays to assess post-translational modifications.
Main Results:
- Three caspase cleavage sites were mapped to the amino-terminal domain of PITSLRE p110.
- Complete processing of PITSLRE p110 in vivo requires cleavage at two adjacent caspase sites.
- PITSLRE p110 undergoes rapid phosphorylation during Fas-induced apoptosis, potentially enhancing caspase cleavage.
Conclusions:
- Multiple caspase-like activities contribute to PITSLRE p110 processing during apoptosis.
- Specific cleavage site recognition and phosphorylation are critical for PITSLRE p110 processing.
- This study elucidates mechanisms of caspase substrate selection during programmed cell death.