leupeptins and 7-hydroxystaurosporine

Phosphorylation of Rad9A at S387 is critical for establishing a physical interaction with TopBP1, and to downstream activation of Chk1 for checkpoint activation. We have previously demonstrated a phosphorylation of Rad9A that occurs at late time points in cells exposed to genotoxic agents, which is eliminated by either Rad9A overexpression, or conversion of S387 to a non-phosphorylatable analogue. Based on this, we hypothesized that this late Rad9A phosphorylation is part of a feedback loop regulating the checkpoint. Here, we show that Rad9A is hyperphosphorylated and accumulates in cells exposed to bleomycin. Following the removal of bleomycin, Rad9A is polyubiquitinated, and Rad9A protein levels drop, indicating an active degradation process for Rad9A. Chk1 inhibition by UCN-01 or siRNA reduces Rad9A levels in cells synchronized in S-phase or exposed to DNA damage, indicating that Chk1 activation is required for Rad9A stabilization in S-phase and during checkpoint activation. Together, these results demonstrate a positive feedback loop involving Rad9A-dependend activation of Chk1, coupled with Chk1-dependent stabilization of Rad9A that is critical for checkpoint regulation.

Topics: Bleomycin; Cell Cycle Checkpoints; Cell Cycle Proteins; Checkpoint Kinase 1; DNA Damage; Enzyme Activation; Feedback, Physiological; HeLa Cells; Humans; Immunoprecipitation; Leupeptins; Models, Biological; Phosphorylation; Polyubiquitin; Proteasome Endopeptidase Complex; Protein Kinases; Protein Stability; Proteolysis; RNA, Small Interfering; Signal Transduction; Staurosporine; Ubiquitination

Cdt1 is a licensing factor for DNA replication, the function of which is tightly controlled to maintain genome integrity. Previous studies have indicated that the cell cycle-dependent degradation of Cdt1 is triggered at S phase to prevent re-replication. In this study, we found that Cdt1 is degraded upon DNA damage induced by either UV treatment or gamma-irradiation (IR). Although the IR-triggered degradation of Cdt1 was caffeine-insensitive, the UV-triggered degradation of Cdt1 was caffeine-sensitive. This indicates that the cells treated with UV utilize the checkpoint pathway, which differs from that triggered by IR. A recent study has suggested that Cdt1 is phosphorylated, ubiquitylated, and degraded at the G(1)/S boundary in the normal cell cycle. Treatment with MG132, a proteasome inhibitor, inhibited the degradation of Cdt1 and resulted in the accumulation of the phosphorylated form of Cdt1 after UV treatment. In the case of UV treatment, phosphorylation of Cdt1 induced the recruitment of Cdt1 to a SCF(Skp2) complex. Moreover, ectopic overexpression of Cdt1 after UV treatment interfered the inhibition of DNA synthesis. These results indicate that Cdt1 is a target molecule of the cell cycle checkpoint in UV-induced DNA damage.

Topics: Caffeine; Cell Cycle; Cell Cycle Proteins; Cell Line, Tumor; DNA Damage; DNA Replication; Enzyme Inhibitors; HeLa Cells; Humans; Infrared Rays; Leupeptins; Phosphorylation; Recombinant Proteins; S-Phase Kinase-Associated Proteins; Staurosporine; Ubiquitin; Ultraviolet Rays