title: Caspase-dependent Proteolysis of Human Ribonucleotide Reductase Small Subunits R2 and p53R2 during Apoptosis.
abstract: 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 also been 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 Asp(342) and Asn(343). 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 caspase-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, which was 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 prosurvival function inactivated by proteolysis during apoptosis.
authors: From the Université Paris Sud, Institute of Molecular and Cellular Biochemistry and Biophysics, UMR 8619, 91405 Orsay, France, CNRS, 91405 Orsay, France, and Department of Virology, Institut Pasteur, Pathogenesis of Hepatitis B Virus, 75015 Paris, France.
authors: Ali
authors: A
authors: Tebbi
authors: From the Université Paris Sud, Institute of Molecular and Cellular Biochemistry and Biophysics, UMR 8619, 91405 Orsay, France, CNRS, 91405 Orsay, France, and.
authors: Olivier
authors: O
authors: Guittet
authors: From the Université Paris Sud, Institute of Molecular and Cellular Biochemistry and Biophysics, UMR 8619, 91405 Orsay, France, CNRS, 91405 Orsay, France, and.
authors: Karine
authors: K
authors: Tuphile
authors: From the Université Paris Sud, Institute of Molecular and Cellular Biochemistry and Biophysics, UMR 8619, 91405 Orsay, France, CNRS, 91405 Orsay, France, and.
authors: Aimeric
authors: A
authors: Cabrié
authors: From the Université Paris Sud, Institute of Molecular and Cellular Biochemistry and Biophysics, UMR 8619, 91405 Orsay, France, CNRS, 91405 Orsay, France, and michel.lepoivre@u-psud.fr.
authors: Michel
authors: M
authors: Lepoivre
mesh_terms: D017209:Apoptosis; D020169:Caspases / Q000378:metabolism*; D018797:Cell Cycle Proteins / Q000378:metabolism*; D002460:Cell Line; D049109:Cell Proliferation; D004247:DNA / Q000737:chemistry; D004260:DNA Repair; D004261:DNA Replication; D003854:Deoxyribonucleotides / Q000737:chemistry*; D006801:Humans; D017434:Protein Structure, Tertiary; D059748:Proteolysis; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D012264:Ribonucleotide Reductases / Q000378:metabolism*; D015398:Signal Transduction
chemical_list: D018797:Cell Cycle Proteins; D003854:Deoxyribonucleotides; D034741:RNA, Small Interfering; D004247:DNA; C405947:RRM2B protein, human; D012264:Ribonucleotide Reductases; C108427:ribonucleotide reductase R2 subunit; D020169:Caspases
keywords: apoptosis; caspase; nucleoside/nucleotide biosynthesis; protein degradation; ribonucleotide reductase