Here, we show that ATM regulates the PPP by inducing G6PD activity. This pathway is conserved in vertebrate organisms as shown by experiments performed in Xenopus eggs and several human cell lines. These observations might help to understand the molecular basis underlying the pathogenesis of A-T. In normal cells, ATM might contribute to maintain the reducing power of the cellular environment by promoting NADPH production. In the absence of ATM, cells would not be able to counteract oxidative stress. Consistent with this, previous studies showed a correlation between redox state, measured as reduced levels of glutathione and severity of the A-T phenotype (Buoni et al, 2006
; Broccoletti et al, 2008
; Russo et al, 2009
). These and our findings are in agreement with earlier observations reporting defects in A-T cells of re-synthesis of glutathione, which requires NADPH (Meredith and Dodson, 1987
Low levels of NADPH were also found in the cerebellum of the ATM−/− mice. The absence of ATM mediated increase in G6PD activity could explain the impaired oxidative stress response in A-T cells (Stern et al, 2002
). Considering that the cerebellum is the area of the brain affected by neurodegeneration in A-T patients, it is possible that the absence of ATM also leads to low levels of NADPH in the human brain. Neuron metabolism produces high level of ROS, which in normal conditions might activate ATM directly or indirectly and in turn promote G6PD activity restoring the redox state of the cells. In A-T patients this feedback might be compromised, leading to the accumulation oxidative stress. Consistent with this hypothesis it has recently been shown that ATM is directly activated by ROS and that a mutation in ATM, which impairs ATM ability to respond to ROS but not to DNA damage is responsible for A-T (Guo et al, 2010
). On the other hand deficiency in DSB repair is known to cause defects similar to A-T. Therefore, it is possible that the A-T phenotype is the result of a defect in the response to both DNA damage and ROS.
Interestingly, an X chromosome linked human disease with partial deficiency in G6PD activity can cause haemolytic anaemia but not any of the symptoms found in A-T (Cappellini and Fiorelli, 2008
). In this case it is possible that compensatory mechanisms leading to an increased production of NADPH are activated in the absence of constitutive levels of G6PD. NADPH can indeed be produced by additional enzymes not active in erythrocytes, which for this reason are particularly sensitive to low levels of G6PD (Cappellini and Fiorelli, 2008
). We cannot exclude that these enzymes are also under the control of ATM. These observations might explain the absence of A-T symptoms associated with G6PD deficiency.
We also show that the activation of G6PD is required for efficient DSB repair. The activation of G6PD correlates with an increased activity of the PPP and this might be required to increase the dNTPs pool needed to repair DNA. In yeast, it is well established that upon DNA damage, cells increase their dNTPs pool (Lee and Elledge, 2006
). In yeast as well as in mammalian cells, the regulation of the dNTPs pool relies mostly on the ribonucleotide reductase (RNR), whose expression is controlled by p53 in mammalian cells (Pontarin et al, 2007
) and is dependent on NADPH for its activity (Avval and Holmgren, 2009
). The defect we observe in the repair of DSBs might be due to an impairment of RNR activity as a consequence of low levels of NADPH and to an imbalance in the dNTPs pool, whose de novo
synthesis depends on the PPP and RNR.
As far as the activation of G6PD and, in turn, the PPP is concerned we provide evidence that this is mediated by Hsp27. A role for the small heat shock protein Hsp27 in oxidative stress response has already been proposed (Preville et al, 1999
). However, the mechanism underlying Hsp27 action was unclear. In humans, cells treated with hydrogen peroxide Hsp27 promotes an increase of the G6PD protein levels (Preville et al, 1999
). In mice, instead, downregulation of Hsp25, the homologue of Hsp27, affects G6PD activity but not G6PD protein levels (Yan et al, 2002
). In our system G6PD protein levels are unaffected. We instead observe that ATM activation promotes the interaction between Hsp27 and G6PD (), and that Hsp27 directly increases G6PD activity in vitro
. Importantly, we show that Hsp27 inactivation abolishes ATM-dependent stimulation of G6PD activity. It is possible that Hsp27 stabilizes the active conformation of G6PD.
It is known that human Hsp27 is phosphorylated mainly on three serines by p38–MK2 complex (Kostenko and Moens, 2009
). Hsp27 forms large oligomers. In general, phosphorylation of Hsp27 regulates its oligomerization level and localization in response to different stimuli (Bruey et al, 2000
). A study on human skin fibroblasts showed that following IR Hsp27 is phosphorylated on serine 78/82 or ser15, depending on the dose (Yang et al, 2006
). In our system, following exposure of cells to IR, Hsp27 is phosphorylated on serine 78, a target of p38–MK2 pathway. p38–MK2 is responsible for the ‘cytoplasmic' branch of the ATM-dependent checkpoint (Reinhardt et al, 2010
). Once activated in the nucleus, p38–MK2 re-localizes to the cytoplasm where it can phosphorylate cytoplasmic targets such as Hsp27. It is possible that this phosphorylation increases Hsp27 affinity for G6PD and in turn increases the activity of the latter. The phosphomimic mutant of Hsp27 can form large oligomers and protect the cells from several forms of stress (Rogalla et al, 1999
). ATM might favour the formation of the large oligomers of Hsp27 by inducing serine 78 phosphorylation and these large oligomers could mediate G6PD activation ().
Figure 7 Schematic representation of G6PD regulation and downstream effect. DSBs activate ATM, which in turn promotes the interaction between Hsp27 and G6PD. This association leads to increased activity of G6PD and stimulation of the PPP. ATM also regulates glycolysis (more ...)
ATM-dependent control of cellular metabolism and ROS production is probably not limited to the events described here. Previous studies have reported a late activation of the PPP and a contemporary inhibition of the glycolysis sustained by p53 through TIGAR and NF-κB (Bensaad et al, 2006
; Kawauchi et al, 2008
). ATM-mediated inhibition of glycolysis might be important to reduce ROS produced by glycolytic metabolism (). Interestingly, cancer cells, in which ATM is frequently mutated, have a high energetic demand but nonetheless rely on glycolysis rather than oxidative phosphorylation for the ATP supply, even when oxygen is present. This phenomenon is known as Warburg effect (Hsu and Sabatini, 2008
). It is possible that the molecular mechanisms underlying this phenomenon include a deficient activation of the PPP, which normally counteracts glycolysis. In addition to this, two glycolytic enzymes, glyceraldeyde-3-phosphate dehydrogenase and pyruvate kinase M2 were recently found to be ATM/ATR substrate (Matsuoka et al, 2007
; Stokes et al, 2007
). These observations, together with our own, indicate that metabolic control might be an important event in the ATM-dependent DNA damage response.
Overall our data indicate that ATM is a major player in the control of redox metabolism and nucleotide production. The lack of these ATM-dependent functions might contribute to the numerous clinical manifestations of A-T disease. Therapeutic strategies aimed at direct stimulation of anti-oxidant pathways might be helpful to promote cell survival in A-T patients bypassing the requirement for ATM protein.