Dissecting ATM-mediated signaling pathways in the cellular response to DNA damage can provide important insights into how the loss of ATM function causes such a devastating disease, ataxia-telangiectasia (A-T), in humans. Upon DNA damage, ATM binds strongly to damaged sites and its kinase activity is enhanced. Activated ATM in turn phosphorylates a list of substrates in pathways that together ensure cellular survival and recovery. A number of ATM-mediated signaling pathways have been revealed, and the functional significance of these pathways has been studied extensively. However, due to the complexity of the A-T phenotypes, detailed mechanisms on how the loss of ATM leads to a variety of A-T phenotypes remains to be further explored. In this report, we highlight a novel signaling pathway that involves ATM, PP1, and I-2. We demonstrate that I-2 is a substrate of ATM and that ATM phosphorylation of I-2 at serine 43 is required for the activation of PP1 in response to DNA damage.
Previously, we reported a rapid and ATM-dependent G2
/M checkpoint that correlates with the down-regulation of histone H3 serine 10 phosphorylation in response to IR (30
). However, how ATM links to regulators of histone H3 serine 10 phosphorylation was not known. It was reported previously that the enzymatic activities of PP1 are activated in response to IR in an ATM-dependent manner (9
) and that Cdk2-mediated PP1 threonine 320 phosphorylation is attenuated after DNA damage. However, a detailed mechanism of ATM-mediated PP1 activation in response to DNA damage remained unknown. Starting with investigations of IR-induced PP1 activity, we found that IR induced an ATM-dependent dissociation of the PP1-I-2 complex. Further studies showed that ATM phosphorylated I-2 on serine 43 and that this phosphorylation led to the dissociation of the complex and the activation of PP1. This effect, in turn, resulted in the inhibition of Aurora-B, the down-regulation of histone H3 serine 10 phosphorylation, and the activation of the G2
/M checkpoint (Fig. ).
FIG. 7. Proposed role of ATM-mediated I-2 phosphorylation in the activation of PP1 and the signaling cascade in G2/M checkpoint regulation in response to IR-induced DNA damage. I-2S43p, I-2 phosphorylated at serine 43; H3 Ser10p, H3 serine 10 phosphorylation. (more ...)
Our data also demonstrate that ATM-mediated I-2 phosphorylation is an essential step for the attenuation of IR-induced threonine 320 phosphorylation. One possible explanation is that, after the dissociation of the PP1-I-2 complex, PP1 initiates autodephosphorylation which eventually activates the phosphatase. More-detailed investigations are required to determine whether threonine 320 dephosphorylation may also play a role in ATM-mediated I-2 phosphorylation and PP1-I-2 dissociation.
PP1 activity is also controlled by other regulatory subunits, such as I-1, NIPP1, and DARPP32 (21
). Whether these regulators are involved in the DNA damage response and whether they dissociate from PP1 are not known. It is reasonable to suspect that some inhibitors are also involved in regulating PP1 activity in response to DNA damage. For example, I-1 has been shown previously to regulate cell growth and has been linked to PP1 in the G1
cell cycle control (21
Histone H3 serine 10 phosphorylation is critical for chromosome condensation and segregation, and it has been used previously as a mitotic marker for studying the activation of the G2
/M checkpoint. Our data demonstrate that the activation of PP1 governed by ATM phosphorylation of I-2 leads to the down-regulation of H3 serine 10 phosphorylation. We also found that activated PP1 leads to the inhibition of the Aurora-B kinase. Therefore, PP1 may prevent H3 phosphorylation to delay the transition from G2
to M, thereby activating the G2
/M checkpoint. However, it is also possible that activated PP1 may directly dephosphorylate the phosphorylated H3 when cells are already in the M phase. Therefore, PP1 and I-2 serine 43 phosphorylation may also have a role to facilitate the mitotic exit. The latter scenario is supported by the evidence that yeast PP1 homolog Dis2 can down-regulate Chk1 activity for a checkpoint release (6
). Therefore, the detailed mechanisms of ATM-mediated PP1 activation in the regulation of histone H3 serine 10 phosphorylation remain to be further investigated.
The functional significance of ATM-mediated phosphorylation of I-2 and activation of PP1 activity may extend beyond the roles of these processes in histone H3 modification and cell cycle checkpoint regulation. Since PP1 is a major eukaryotic protein serine/threonine phosphatase that regulates a variety of cellular functions, the regulation of PP1 through ATM phosphorylation of I-2 may have a significant impact on many cellular responses to DNA damage. Dephosphorylation by phosphatases can turn signals off or regulate the degradation of phosphorylated substrates, thus balancing the physiological effects of kinases (19
One of the known physiological roles of I-2 is to control sperm motility (28
). A testis-specific isoform of PP1 forms an inactive complex with I-2, and GSK-3-mediated I-2 phosphorylation which activates the PP1-I-2 complex results in an increase in the PP1 activity seen in nonmotile immature sperm. The exposure of the immature sperm to phosphatase inhibitors, such as okadaic acid and calyculin A, induces motility, suggesting that I-2 inhibits PP1 activity in mature mammalian sperm cells to facilitate their motility. The PP1-I-2 complex is also involved in insulin signaling (5
). These observations are particularly interesting since both A-T patients and A-T mice are sterile and have glucose intolerance and insulin resistance (26
), suggesting a physiologically important link between ATM and PP1-I-2. Indeed, we have observed that ATM phosphorylates I-2 at serine 43 in response to insulin stimulation (unpublished data). The establishment of a serine 43 phosphorylation mutant knock-in mouse model to study the physiological significance of ATM-mediated I-2 phosphorylation is under way.
In summary, our data provide mechanistic insights into the activation process of PP1 in DNA damage response pathways in mammalian cells. The results of these studies also provide a foundation for future studies of the ATM-PP1-I-2 pathway in regulating cellular responses to stress.