Cdc25 PHOSPHATASES AND CHECKPOINT CONTROLS

Giulio Draetta
Department of Experimental Oncology
European Institute of Oncology
Via Ripamonti, 435
20141 Milan, Italy

The Cdc25A phosphatase has a critical role in cell cycle progression due to its role in dephosphorylating cyclin-dependent protein kinases. In response to DNA damage or to stalled replication, activation of the ATM and ATR protein kinases leads to Chk1 and Chk2 activation and Cdc25A hyperphosphorylation. These events stimulate the ubiquitin-mediated proteolysis of Cdc25A and contribute to delaying cell cycle progression, thereby preventing genomic instability. This mechanism is independent of, and occurs prior to the induction of sustained cell cycle arrest mediated by p53 activation.

We have found that Cdc25A is able to bind the beta-TrCP F-Box protein both in vitro and in vivo. Cdc25A recruitment by beta-TrCP and its subsequent ubiquitylation require the presence of a DSG motif in the sequence of Cdc25A. Down regulation of beta-TrCP1 and beta-TrCP2 expression by small interfering RNA (siRNA) causes Cdc25A accumulation in cells progressing through S-phase and prevents ionizing radiation-induced Cdc25A degradation. Consistent with this hypothesis, suppression of beta-TrCP expression results in defective S-phase checkpoint activation in response to DNA damage. Our results highlight a crucial role for beta-TrCP in mediating the response to DNA damage through Cdc25A degradation, leading to efficient activation of the intra-S-phase checkpoint.

We had previously shown that the ubiquitin-mediated degradation of the Cdc25A phosphatase at the exit of mitosis is mediated by the APC/CCdh1 (Anaphase Promoting Complex/Cyclosome) ubiquitin ligase through recognition of a specific KEN box sequence. We have demonstrated that Cdc25A mutants resistant to APC-mediated ubiquitylation remain short lived in interphase as the wild-type protein does, and are degraded in response to ionizing radiation treatment. Furthermore, Cdc25A degradation both in cycling cells and in response to DNA damage depends on phosphorylation events, a requirement for efficient target recruitment to SCF (Skp1/Cullin/F-box) ubiquitin ligases by F-box proteins. We have found that beta-TrCP is required for regulating the accumulation of Cdc25A in cycling and DNA damaged cells. Cdc25A is phosphorylated on Ser82 and Ser88 and then recruited by beta-TrCP to the SCF and then degraded. This mechanism should maintain Cdc25A under a threshold level that is required for correct cell cycle progression by preventing premature activation of cyclin-dependent kinases. This timely degradation should allow for efficient Cdc25A removal in response to DNA damage, thus delaying cell cycle progression in view of repairing the defect and preventing genomic instability.

Our findings are in line with previous evidence of separate ubiquitin-dependent mechanisms regulating the abundance of Cdc25A at the exit of mitosis, in S phase and in response to checkpoint activation. Recently, it has been shown that Cdc25A is phosphorylated on specific serine residues by the Chk1 protein kinase in cycling cells and by both Chk1 and Chk2 in checkpoint activated cells and that disruption of the Chk1/Cdc25A pathway abrogates ionizing radiation-induced S and G2 checkpoints. Combined mutation of these residues confers stability to the protein, thus suggesting that multiple phosphorylation sites contribute to Cdc25A degradation in vivo. These findings would indicate that multiple phosphorylation events contribute to enhancing the interaction and to stimulate polyubiquitylation. Given the fact that mutations in the ATM and ATR pathways, upstream negative effectors of Cdc25A, are common in cancer, an assessment of the tissue and the subcellular distribution as well as of the occurrence of mutations in the beta-TrCP sequence in normal and tumor tissue should be warranted.

References

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