In mammalian cells, two PI-3 kinase-related kinases called ATM and ATR initiate the cascade of phosphorylation events that result in cell cycle arrest and DNA repair. Chk1 and Chk2 kinases phosphorylated by ATM and ATR and mediate cell cycle arrest and DNA repair by phosphorylating p53, BRCA1, Cdc25A, B and C. In the presence of excessive DNA damage, Chk2 phosphorylate p53 and E2F1 resulting in programmed cell death called apoptosis. Failure to execute programmed cell death in the presence of excessive DNA damage can promote cancer. This notion is supported by the observation that carriers of p53 or Chk2 mutation are prone to develop cancer. The mechanism by which Chk2 induces ionizing irradiation-induced apoptosis is poorly understood, but is thought to involve stabilization of p53. However, ionizing irradiation-induced apoptosis also occurs via p53-independent mechanisms. In order to understand how Chk2 kinase activates programmed cell death in a p53-independent manner, the possibility that Chk2 may be functionally linked to the product of the PML gene was investigated. The promyelocytic leukemia gene (PML), which is translocated in most acute promyelocytic leukemias, encodes a tumor suppressor. Although the molecular mechanism remains largely unknown, PML is involved in multiple apoptotic pathways. PML -/- mice and PML -/- cells are resistant to the lethal effects of ionizing irradiation suggesting that PML plays an important role in DNA damage-induced apoptosis. However, the signaling cascade of the PML-mediated apoptosis after DNA damage is not understood. hCds1/Chk2 mediates ionizing irradiation-induced apoptosis in a p53-independent manner by an ATM-Chk2-PML pathway. The apoptotic function of PML is regulated by phosphorylation of serine 117 by hCds1/Chk2 after ionizing radiation. Chk2 and PML also colocalize in nuclear bodies along with BRCA1 suggesting that the nuclear bodies may be a storage body for DNA damage response proteins. We recently discovered that the presence of the nuclear bodies may be important for triggering autophosphorylation of Chk2 which is required for Chk2 activation. Acute promyelocytic leukemias are often treated with Arsenic trioxide, which triggers apoptosis of the leukemic cells. Our findings suggest that Arsenic trioxide may utilize the Chk2-dependent pathway to trigger apoptosis. Interestingly, unlike ionizing radiation, ATR appears to be the upstream kinase that regulates Chk2. Prior to phosphorylating its substrates such as PML, Chk2 phosphorylates itself (i.e. autophosphorylation). This is an essential step for Chk2 activation and requires Chk2-Chk2 interaction. We discovered that the Chk2-Chk2 interaction and thus Chk2 activation can be blocked with a threonine-phosphorylated peptide that binds to one of the conserved domains in Chk2 called the FHA domain. The use of such peptide may allow one to chemically regulate Chk2 activity thereby modulate the extent of apoptosis after severe DNA damage.
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