Hyperphosphorylation of replication protein A middle subunit (RPA32) in apoptosis

Replication protein A (RPA) is a trimeric single-stranded DNA (ssDNA)-bindi ng complex of eukaryotic cells that plays an important role in DNA metaboli sm by stabilising single-stranded regions of DNA. The functionally importan t binding activity towards ssDNA is mainly localised on the large subunit, RPA70, whereas the middle subunit, RPA32, appears to have a regulatory func tion. It has been shown previously that RPA32 is phosphorylated both during the S-phase of a normal cell cycle and in response to DNA damage, In this study we demonstrate that phosphorylation of RPA32 is rapidly induced durin g apoptotic cell death of Jurkat T-lymphocytes, resulting in a hyperphospho rylated form with reduced electrophoretic mobility. In contrast, the large subunit of RPA is neither modified nor cleaved during apoptosis, Phosphoryl ation of RPA32 begins in parallel to the degradation of DNA to high molecul ar weight fragments, and slowly continues until late apoptosis, Experiments with specific kinase inhibitors indicate that RPA32 hyperphosphorylation r equires the activities of DNA-dependent protein kinase and of a cyclin-depe ndent protein kinase. Interestingly, the hyperphosphorylated, but not the l ess phosphorylated forms of RPA32, sediments independently from the trimeri c complex in sucrose gradients under high ionic strength, and is not bound to the complex in immunoprecipitation assays.