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Ionizing radiation-dependent and independent phosphorylation of the 32-kDa subunit of replication protein A during mitosis.
Stephan, Holger Concannon, Claire Kremmer, Elisabeth Carty, Michael P Nasheuer, Heinz-Peter
Stephan, Holger
Concannon, Claire
Kremmer, Elisabeth
Carty, Michael P
Nasheuer, Heinz-Peter
Advisors
Editors
Other Contributors
Date
2009-10
Date Submitted
Keywords
Other Subjects
Subject Mesh
Cell Cycle Proteins
DNA Damage
DNA-Activated Protein Kinase
DNA-Binding Proteins
Humans
Mitosis
Phosphorylation
Protein Subunits
Protein-Serine-Threonine Kinases
Radiation, Ionizing
Replication Protein A
Serine
Tumor Suppressor Proteins
DNA Damage
DNA-Activated Protein Kinase
DNA-Binding Proteins
Humans
Mitosis
Phosphorylation
Protein Subunits
Protein-Serine-Threonine Kinases
Radiation, Ionizing
Replication Protein A
Serine
Tumor Suppressor Proteins
Planned Date
Start Date
Collaborators
Principal Investigators
Files
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19671522.pdf
Adobe PDF, 2.76 MB
Alternative Titles
Publisher
Abstract
The human single-stranded DNA-binding protein, replication protein A (RPA), is regulated by the N-terminal phosphorylation of its 32-kDa subunit, RPA2. RPA2 is hyperphosphorylated in response to various DNA-damaging agents and also phosphorylated in a cell-cycle-dependent manner during S- and M-phase, primarily at two CDK consensus sites, S23 and S29. Here we generated two monoclonal phospho-specific antibodies directed against these CDK sites. These phospho-specific RPA2-(P)-S23 and RPA2-(P)-S29 antibodies recognized mitotically phosphorylated RPA2 with high specificity. In addition, the RPA2-(P)-S23 antibody recognized the S-phase-specific phosphorylation of RPA2, suggesting that during S-phase only S23 is phosphorylated, whereas during M-phase both CDK sites, S23 and S29, are phosphorylated. Immunofluorescence microscopy revealed that the mitotic phosphorylation of RPA2 starts at the onset of mitosis, and dephosphorylation occurs during late cytokinesis. In mitotic cells treated with ionizing radiation (IR), we observed a rapid hyperphosphorylation of RPA2 in addition to its mitotic phosphorylation at S23 and S29, associated with a significant change in the subcellular localization of RPA. Our data also indicate that the RPA2 hyperphosphorylation in response to IR is facilitated by the activity of both ATM and DNA-PK, and is associated with activation of the Chk2 pathway.
Language
en
ISSN
1362-4962
eISSN
ISBN
DOI
10.1093/nar/gkp605
PMID
19671522