Hyperphosphorylation of replication protein A middle subunit (RPA32) in apoptosis.
Replication protein A (RPA) is a trimeric single-stranded DNA (ssDNA)-binding complex of eukaryotic cells that plays an important role in DNA metabolism by stabilising single-stranded regions of DNA. The functionally important binding activity towards ssDNA is mainly localised on the large subunit, RPA70, whereas the middle subunit, RPA32, appears to have a regulatory function. 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 during apoptotic cell death of Jurkat T-lymphocytes, resulting in a hyperphosphorylated form with reduced electrophoretic mobility. In contrast, the large subunit of RPA is neither modified nor cleaved during apoptosis. Phosphorylation of RPA32 begins in parallel to the degradation of DNA to high molecular weight fragments, and slowly continues until late apoptosis. Experiments with specific kinase inhibitors indicate that RPA32 hyperphosphorylation requires the activities of DNA-dependent protein kinase and of a cyclin-dependent protein kinase. Interestingly, the hyperphosphorylated, but not the less phosphorylated forms of RPA32, sediments independently from the trimeric complex in sucrose gradients under high ionic strength, and is not bound to the complex in immunoprecipitation assays.
Treuner
K
K
Laboratory of Molecular Genetics, Department of Biology, University of Konstanz, 78434 Konstanz, Germany and Banyu Tsukuba Research Institute, Okubo 3, Tsukuba 300-26, Japan.
Okuyama
A
A
Knippers
R
R
Fackelmayer
F O
FO
D017209:Apoptosis; D018844:Cyclin-Dependent Kinases / Q000378:metabolism; D051747:DNA-Activated Protein Kinase; D004268:DNA-Binding Proteins / Q000378:metabolism*; D006801:Humans; D019169:Jurkat Cells; D009687:Nuclear Proteins; D010766:Phosphorylation; D011487:Protein Conformation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D051716:Replication Protein A; D013601:T-Lymphocytes / Q000378:metabolism* / Q000473:pathology
D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; C497167:RPA1 protein, human; D051716:Replication Protein A; D051747:DNA-Activated Protein Kinase; C497263:PRKDC protein, human; D017346:Protein Serine-Threonine Kinases; D018844:Cyclin-Dependent Kinases