Chk1 activity is required for BAK multimerization in association with PUMA during mitochondrial apoptosis
© Azad and Storey; licensee Biomed Central Ltd. 2014
Received: 26 February 2014
Accepted: 18 June 2014
Published: 10 July 2014
The Bcl-2 protein BAK is a key player in mitochondrial apoptosis and responds to a myriad of different death signals. Activation of BAK is a multistep process that involves a number of conformational changes mediated by BH3-only proteins or p53 which leads to BAK multimerization and pore formation in the mitochondrial outer membrane. We previously reported that BAK activation is dependent upon dephosphorylation of both tyrosine and serine residues. Further, recent reports demonstrated that PP2A activity is required for BAK multimerization. Since Chk1, a checkpoint kinase involved in the activation of G2 checkpoint, is regulated by PP2A, we therefore hypothesized that Chk1 is involved in BAK multimerization during cell cycle arrest upon severe DNA damage.
We now show that treatment of HCT116-WT BAK cells with a Chk1 inhibitor impaired BAK dimerization and mutimerization when treated with the DNA damaging agents UV or etoposide. As a result there is a concomitant decrease of cytochrome c release from isolated mitochondria challenged with tBid protein and failure in the activation of caspase3. Interestingly, co-immunoprecipitation studies suggest that Chk1 is required for recruitment of BH3- only protein PUMA to BAK. We also showed that Chk1 is associated with BAK upon DNA damage.
These findings novelly demonstrate the involvement of a checkpoint kinase Chk1 is required for BAK activation and underscores the importance of involvement of Chk1 in mitochondrial apoptosis upon severe DNA damage.
Apoptosis block is a hallmark of cancer and may contribute to aggressive tumour progression. The Bcl-2 family proteins regulate the mitochondrial pathway of apoptosis through interactions between the pro-apoptotic proteins BAK and BAX, the anti-apoptotic Bcl-xL and MCL1, and the BH3-only proteins such as Bid, Bim, PUMA which activate BAK and BAX and or inhibit antiapoptotic proteins . BAK activation is a complex multistep process. Upon apoptotic stimuli BAK undergoes a N-terminal conformational change initiated by a ‘hit and run’ either by BH3-only proteins or p53. This is followed by transient exposure of BAK BH3 domain which then binds to the hydrophobic groove of another BAK molecule leading to dimer formation ,. The resulting symmetric homodimers then multimerize to higher order structures via α6:α6 helices  leading to the permeabilization of the mitochondrial outer membrane followed by the release of apoptogenic factors like cytochrome c, that in turn lead to downstream activation of caspase3. Recently a unified model has been proposed that suggests different affinities of BH3 proteins for both pro- and anti-apoptotic proteins .
Together with the well-characterised conformational changes required for BAK activation we recently reported that specific dephosphorylation events are also required. Firstly, tyrosine dephosphorylation at 108 (Y108) in BAK is required to convert BAK into an ‘activation-competent’ form which constitutes an obligatory first step in BAK activation . Secondly, we found that dephosphorylation at S117 residue by PP2A was required both for the BAK BH3 domain to gain access to BAK’s hydrophobic groove and permit BAK dimerization and subsequent multimerization . More recently we have shown that dephosphorylation at tyrosine 110 (Y110) is required for recruitment of Bid to BAK .
In response to DNA damage, normal cells arrest in G1 to allow time for DNA to repair or they proceed to apoptosis if the DNA damage is too severe. Robust genotoxic insults induce apoptosis rather than the cell cycle checkpoint. Checkpoint kinase Chk1, a serine/threonine kinase that was first identified in fission yeast as an essential component of the DNA damage checkpoint , and a major effector of G2/M-phase DNA damage checkpoint and S-phase replication checkpoint . Recently it has been reported that anti-apoptotic BCL-2 family protein MCL-1 regulates Chk1 . However the role of Chk1 in mitochondrial apoptosis remains to be elucidated. PP2A is known to maintain a regulatory circuit with checkpoint kinase Chk1 by continuously dephosphorylating Chk1 which is itself continuously phosphorylated by ATR (Ataxia-telangiectasia-related) . Since we found that PP2A activity was required for BAK multimerization, one particular focus was to elucidate whether Chk1 activity is involved in BAK multimerization during mitochondrial apoptosis in response to DNA damage. In the present study we found that Chk1 played a role in mitochondrial apoptosis.
Chk1 is involved in BAK multimerization upon UV irradiation
Chk1 activity is required for cytochrome c release and caspase3 activation after UV damage
The BH3-only protein PUMA is required for Chk1 mediated apoptosis
Taken together our results now identify a novel role for Chk1 in BAK activation during mitochondrial apoptosis. Using Co-IP studies, we provide a novel direct link between Chk1 and apoptotic machinery but it remains to be established whether or not BAK is the substrate of Chk1. The BH3-only protein PUMA interferes with the binding to BAK in the presence of the Chk1 inhibitor and therefore suggests that Chk1 is responsible for the recruitment of PUMA to BAK. It was previously reported that transactivation of PUMA was restored during ATR-mediated activation of Chk1 . Chk1 is regulated by PP2A and evidence for the complex feedback loop between Chk1 and PP2A has been reported . PP2A activity influences many aspects of the DNA damage response. We recently showed that PP2A activity is required for BAK multimerization . However, the role of Chk1 activity stimulating PP2A activity during mitochondrial apoptosis following DNA damage by UV or etoposide remains to be established. Thus, a dual role for Chk1 can be suggested. Chk1 maintains the DNA damage checkpoint integrity, however, if cells have sustained severe DNA damage and are no longer repairable, Chk1 may then play a role in apoptosis by interacting with BAK. Recently it has been reported that Chk1 is activated by caspase-dependent cleavage during apoptosis . In response to DNA damage Chk1 becomes phosphorylated on several serine residues (S280, S296, S317 and S345) and plays important functions in cellular processes. Thus, it would be important to investigate the role of this phosphorylation in BAK activation. Moreover, in this study we used mostly HCT116 cells which is lacking mismatch repair and Chk1 is also involved in mismatch repair (mmr). However our finding is not due to the abnormalities in mmr rather due to the inhibition of Chk1 activity as we found that caspase3 activation is also inhibited in HT1080 cells. Finally, Chk1 is may be responsible for a providing a safe guarding mechanism for the cell, so that it is poised to undergo apoptosis if DNA damage is detected and cannot be repaired.
This work was supported by funding from Cancer Research UK. We would like to thank Drs Philip Simister, Lonnie Swift, Joanna Fox, Alan McIntyre and Neil Ashley for critical reading of the manuscript, and Ghadheer Almuhaini, Peter Thomas and Samantha Oats for help.
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