Cellular checkpoints are signaling pathways evolved in eukaryotes to maintain genomic integrity. Checkpoint activation by genotoxins results in cell cycle arrest and repair of the damage, senescence or apoptosis. Malfunctioning checkpoints lead to an accumulation of genetic alterations which is directly associated with cancerogenesis. p21 and Chk2 are important components of DNA damage checkpoints connected by the tumor suppressor p53, which is an inducer of p21 and a substrate of Chk2. p21 induces arrest in G1
by inactivating G1
-specific cyclin/Cdk complexes. Chk2 phosphorylates and inactivates Cdc25C, which is required for the activation of Cdk1 at the G2
/M boundary, resulting in a G2
arrest. Phosphorylation of Chk2 at Thr68 by ATM is the first step in its activation.28,29
It is followed by homodimerization and trans-phosphorylation at Thr383 and Thr387 resulting in full activation.30
EAPP is an E2F-binding protein that stimulates E2F-dependent transcription and is frequently overexpressed in human tumor cells.21
We have shown recently that EAPP is not only essential for the survival of a cell but also an important regulator of p21 activity.23
We show here that EAPP also interacts with Chk2 and seems to interfere with its activity.
shows a model that could explain our observations. Following DNA damage, ATM phosphorylates Chk2 at Thr68, a step required for full activation of Chk2. Activated Chk2 phosphorylates substrates including Cdc25C, p53, E2F1 and PML, resulting in cell cycle arrest and DNA repair or apoptosis. EAPP also accumulates during DNA damage. It binds to phosphorylated Chk2 and the resulting dimeric or oligomeric complex recruits a phosphatase like Wip1, PP2A or PP1 that dephosphorylates Chk2 and this in turn could result in the disassembly of the oligomeric complex. The emergence of P-Chk2 in EAPP knockdown cells would thus be the consequence of inefficient dephosphorylation. The reduction of Wip1 (and possibly other phosphatases) levels would also contribute. In addition to the downregulation of p21, this activation of Chk2 may contribute to apoptosis observed in EAPP knockdown cells.23
Chk2 has been shown to phosphorylate E2F1 on Ser364, a modification that enhances E2F1 stability31
and seems to alter its promoter specificity32
resulting in the expression of pro-apoptotic genes like p73.33
According to this model EAPP would be required for the inactivation of Chk2 during checkpoint recovery and a certain level of EAPP would be crucial for the proper regulation of Chk2 activity. Since the accumulation of EAPP as a result of DNA damage occurs slower than Chk2 phosphorylation, premature Chk2 inactivation is avoided. Two different scenarios are conceivable: in the first one EAPP interacts only with the newly Thr68 phosphorylated, monomeric, Chk2, stimulating its dephosphorylation and thus preventing dimerization and full activation. In this case already active Chk2 would not be affected; only its replenishment would be inhibited. Inactivation of Chk2 during checkpoint recovery could be achieved by Plk1 through phosphorylation of the FHA domain of Chk2.25
In the second scenario EAPP interacts with both monomeric and dimeric Chk2, stimulating their dephosphorylation and thus contributing to their inactivation.
Figure 6 Proposed model for the interaction of EAPP and phosphorylated Chk2. Without DNA damage a fraction of Chk2 becomes phosphorylated by the existing small amounts of active ATM. This active Chk2 is important for maintaining genomic integrity. EAPP binds to (more ...)
A DNA damage independent function of Chk2 during mitosis has been described recently. Chk2 mediated phosphorylation of Ser988 on BRCA1 and this is required for correct formation of the mitotic spindle.34
The mitotic activity Chk2 was similar to DNA damage induced activity. This is somewhat surprising since its activator ATM seems to be only modestly more active in mitosis than in interphase. We have shown previously that EAPP levels go down during mitosis.21
Our model predicts that EAPP is required for Chk2 dephosphorylation. If it is correct, it could explain high Chk2 activity in mitosis. Reduced dephosphorylation resulting from lower EAPP and steady phosphorylation of Chk2 would lead to more active P-Chk2. Its abrogation has been shown to induce chromosomal instability (CIN) leading to gain or loss of whole chromosomes.34
The resulting aneuploidy is a hallmark of cancer and contributes to tumorigenesis and tumor progression.35
This might be one of the reasons why so many tumors have elevated EAPP levels. The concomitant reduction of active Chk2 favors aneuploidy possibly followed by cancer.