Caspase-Dependent Proteolysis of Human Ribonucleotide Reductase Small Subunits R2 and p53R2 During Apoptosis [Protein Synthesis and Degradation]

April 15th, 2015 by Tebbi, A., Guittet, O., Tuphile, K., Cabrie, A., Lepoivre, M.

Ribonucleotide reductase (RnR) is a key enzyme synthesizing deoxyribonucleotides for DNA replication and repair. In mammals, the R1 catalytic subunit forms an active complex with either one of the two small subunits R2 and p53R2. Expression of R2 is S-phase-specific and required for DNA replication. The p53R2 protein is expressed throughout the cell cycle and in quiescent cells, where it provides dNTPs for mitochondrial DNA synthesis. Participation of R2 and p53R2 in DNA repair has been also suggested. In this study, we investigated the fate of the RnR subunits during apoptosis. The p53R2 protein was cleaved in a caspase-dependent manner in K-562 cells treated with inhibitors of the Bcr-Abl oncogenic kinase, and in HeLa 229 cells incubated with TNF-α and cycloheximide. The cleavage site was mapped between Asp342 and Asn343. Caspase attack released a C-terminal p53R2 peptide of nine residues containing the conserved heptapeptide essential for R1 binding. As a consequence, the cleaved p53R2 protein was inactive. In vitro, purified caspases 3 and 8 could release the C-terminal tail of p53R2. Knocking down these caspases, but not caspase-2, -7 and -10, also inhibited p53R2 cleavage in cells committed to die via the extrinsic death receptor pathway. The R2 subunit was subjected to caspase- and proteasome-dependent proteolysis, prevented by siRNA targeting caspase-8. Knocking down caspase-3 was ineffective. Protein R1 was not subjected to degradation. Adding deoxyribonucleosides to restore dNTP pools transiently protected cells from apoptosis. These data identify RnR activity as a pro-survival function inactivated by proteolysis during apoptosis.
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