MDC1 maintains genomic stability by participating in the amplification of ATM-dependent DNA damage signals

MDC1 functions in checkpoint activation and DNA repair following DNA damage. To address the physiological role of MDC1, we disrupted the MDC1 gene in mice. MDC1-/- mice recapitulated many phenotypes of H2AX-/- mice, including growth retardation, male infertility, immune defects, chromosome instability, DNA repair defects, and radiation sensitivity. At the molecular level, H2AX, MDC1, and ATM form a positive feedback loop, with MDC1 directly mediating the interaction between H2AX and ATM. MDC1 binds phosphorylated H2AX through its BRCT domain and ATM through its FHA domain. Through these interactions, MDC1 accumulates activated ATM flanking the sites of DNA damage, facilitating further ATM-dependent phosphorylation of H2AX and the amplification of DNA damage signals. In the absence of MDC1, many downstream ATM signaling events are defective. These results suggest that MDC1, as a signal amplifier of the ATM pathway, is vital in controlling proper DNA damage response and maintaining genomic stability.     

Timing is everything

Cell adhesion to the extracellular matrix elicits a temporal reorganization of the actin cytoskeleton that is regulated first by Rac1 and later by RhoA. The signaling mechanisms controlling late stage RhoA activation are incompletely understood. Net1A is a RhoA/RhoB-specific guanine nucleotide exchange factor that is required for cancer cell motility. The ability of Net1A to stimulate RhoA activation is negatively regulated by nuclear sequestration. However, mechanisms controlling the plasma membrane localization of Net1A had not previously been reported. Recently we have shown that Rac1 activation stimulates plasma membrane relocalization and activation of Net1A. Net1A relocalization is independent of its catalytic activity and does not require its C-terminal pleckstrin homology or PDZ interacting domains. Rac1 activation during cell adhesion stimulates a transient relocalization of Net1A that is terminated by proteasomal degradation of Net1A. Importantly, plasma membrane localization of Net1A is required for efficient myosin light chain phosphorylation, focal adhesion maturation, and cell spreading. These data show for the first time a physiological mechanism controlling Net1A relocalization from the nucleus. They also demonstrate a previously unrecognized role for Net1A in controlling actomyosin contractility and focal adhesion dynamics during cell adhesion.     

Evasion of early cellular response mechanisms following low level radiation-induced DNA damage

DNA damage that is not repaired with high fidelity can lead to chromosomal aberrations or mitotic cell death. To date, it is unclear what factors control the ultimate fate of a cell receiving low levels of DNA damage (i.e. survival at the risk of increased mutation or cell death). We investigated whether DNA damage could be introduced into human cells at a level and frequency that could evade detection by cellular sensors of DNA damage. To achieve this, we exposed cells to equivalent doses of ionizing radiation delivered at either a high dose rate (HDR) or a continuous low dose rate (LDR). We observed reduced activation of the DNA damage sensor ataxia-telangiectasia mutated (ATM) and its downstream target histone H2A variant (H2AX) following LDR compared with HDR exposures in both cancerous and normal human cells. This lack of DNA damage signaling was associated with increased amounts of cell killing following LDR exposures. Increased killing by LDR radiation has been previously termed the "inverse dose rate effect," an effect for which no clear molecular processes have been described. These LDR effects could be abrogated by the preactivation of ATM or simulated in HDR-treated cells by inhibiting ATM function. These data are the first to demonstrate that DNA damage introduced at a reduced rate does not activate the DNA damage sensor ATM and that failure to activate ATM-associated repair pathways contributes to the increased lethality of continuous LDR radiation exposures. This inactivation may reflect one strategy by which cells avoid accumulating mutations as a result of error-prone DNA repair and may have a broad range of implications for carcinogenesis and, potentially, the clinical treatment of solid tumors.     

ATM Inhibition Potentiates Death of Androgen Receptor-inactivated Prostate Cancer Cells with Telomere Dysfunction

Androgen receptor (AR) plays a role in maintaining telomere stability in prostate cancer cells, as AR inactivation induces telomere dysfunction within 3 h. Since telomere dysfunction in other systems is known to activate ATM (ataxia telangiectasia mutated)-mediated DNA damage response (DDR) signaling pathways, we investigated the role of ATM-mediated DDR signaling in AR-inactivated prostate cancer cells. Indeed, the induction of telomere dysfunction in cells treated with AR-antagonists (Casodex or MDV3100) or AR-siRNA was associated with a dramatic increase in phosphorylation (activation) of ATM and its downstream effector Chk2 and the presenceof phosphorylated ATM at telomeres, indicating activation of DDR signaling at telomeres. Moreover, Casodex washout led to the reversal of telomere dysfunction, indicating repair of damaged telomeres. ATM inhibitor blocked ATM phosphorylation, induced PARP cleavage, abrogated cell cycle checkpoint activation and attenuated the formation of γH2AX foci at telomeres in AR-inactivated cells, suggesting that ATM inhibitor induces apoptosis in AR-inactivated cells by blocking the repair of damaged DNA at telomeres. Finally, colony formation assay revealed a dramatic decrease in the survival of cells co-treated with Casodex and ATM inhibitor as compared with those treated with either Casodex or ATM inhibitor alone. These observations indicate that inhibitors of DDR signaling pathways may offer a unique opportunity to enhance the potency of AR-targeted therapies for the treatment of androgen-sensitive as well as castration-resistant prostate cancer.     

Involvement of novel autophosphorylation sites in ATM activation

ATM kinase plays a central role in signaling DNA double-strand breaks to cell cycle checkpoints and to the DNA repair machinery. Although the exact mechanism of ATM activation remains unknown, efficient activation requires the Mre11 complex, autophosphorylation on S1981 and the involvement of protein phosphatases and acetylases. We report here the identification of several additional phosphorylation sites on ATM in response to DNA damage, including autophosphorylation on pS367 and pS1893. ATM autophosphorylates all these sites in vitro in response to DNA damage. Antibodies against phosphoserine 1893 revealed rapid and persistent phosphorylation at this site after in vivo activation of ATM kinase by ionizing radiation, paralleling that observed for S1981 phosphorylation. Phosphorylation was dependent on functional ATM and on the Mre11 complex. All three autophosphorylation sites are physiologically important parts of the DNA damage response, as phosphorylation site mutants (S367A, S1893A and S1981A) were each defective in ATM signaling in vivo and each failed to correct radiosensitivity, genome instability and cell cycle checkpoint defects in ataxia-telangiectasia cells. We conclude that there are at least three functionally important radiation-induced autophosphorylation events in ATM.     

Chromatin perturbations during the DNA damage response in higher eukaryotes

The DNA damage response is a widely used term that encompasses all signaling initiated at DNA lesions and damaged replication forks as it extends to orchestrate DNA repair, cell cycle checkpoints, cell death and senescence. ATM, an apical DNA damage signaling kinase, is virtually instantaneously activated following the introduction of DNA double-strand breaks (DSBs). The MRE11-RAD50-NBS1 (MRN) complex, which has a catalytic role in DNA repair, and the KAT5 (Tip60) acetyltransferase are required for maximal ATM kinase activation in cells exposed to low doses of ionizing radiation. The sensing of DNA lesions occurs within a highly complex and heterogeneous chromatin environment. Chromatin decondensation and histone eviction at DSBs may be permissive for KAT5 binding to H3K9me3 and H3K36me3, ATM kinase acetylation and activation. Furthermore, chromatin perturbation may be a prerequisite for most DNA repair. Nucleosome disassembly during DNA repair was first reported in the 1970s by Smerdon and colleagues when nucleosome rearrangement was noted during the process of nucleotide excision repair of UV-induced DNA damage in human cells. Recently, the multi-functional protein nucleolin was identified as the relevant histone chaperone required for partial nucleosome disruption at DBSs, the recruitment of repair enzymes and for DNA repair. Notably, ATM kinase is activated by chromatin perturbations induced by a variety of treatments that do not directly cause DSBs, including treatment with histone deacetylase inhibitors. Central to the mechanisms that activate ATR, the second apical DNA damage signaling kinase, outside of a stalled and collapsed replication fork in S-phase, is chromatin decondensation and histone eviction associated with DNA end resection at DSBs. Thus, a stress that is common to both ATM and ATR kinase activation is chromatin perturbations, and we argue that chromatin perturbations are both sufficient and required for induction of the DNA damage response.     

DNA damage-induced activation of ATM and ATM-dependent signaling pathways

Ataxia-telangiectasia mutated (ATM) plays a key role in regulating the cellular response to ionizing radiation. Activation of ATM results in phosphorylation of many downstream targets that modulate numerous damage response pathways, most notably cell cycle checkpoints. In this review, we describe recent developments in our understanding of the mechanism of activation of ATM and its downstream signaling pathways, and explore whether DNA double-strand breaks are the sole activators of ATM and ATM-dependent signaling pathways.     

Association of CNK1 with Rho guanine nucleotide exchange factors controls signaling specificity downstream of Rho

BACKGROUND: Rho is a small GTPase that controls signal transduction pathways in response to a large number of extracellular stimuli. With over 15 potential Rho target proteins identified to date, however, it is not clear how distinct signaling outputs can be generated downstream of a particular stimulus. RESULTS: Several of the known Rho targets are structurally reminiscent of scaffold proteins, which are generally thought to play an important role in controlling signaling specificity. Here, we show that the Rho target CNK1 is a scaffold protein that interacts with Net1 or p115RhoGEF, two Rho-specific guanine nucleotide exchange factors (GEFs), as well with MLK2 and MKK7, two of the kinase components in the JNK MAP kinase cascade. CNK1 acts cooperatively with the two GEFs to activate JNK MAP kinase, but not other Rho-mediated pathways. In HeLa cells, serum or sphingosine-1-phosphate stimulate Rho-dependent activation of the JNK MAP kinase cascade, and this requires endogenous CNK1. CONCLUSIONS: We conclude that CNK1 couples a subset of Rho exchange factors to activation of the JNK MAP kinase pathway and that signaling specificity is achieved through complexes containing both upstream activators and downstream targets of Rho.     

SNM1A acts downstream of ATM to promote the G1 cell cycle checkpoint

We have shown previously that SNM1A colocalizes with 53BP1 at sites of double-strand breaks (DSBs) induced by IR, and that these proteins interact with or without DNA damage. However, the role of SNM1A in the DNA damage response has not been elucidated. Here, we show that SNM1A is required for an efficient G1 checkpoint arrest after IR exposure. Interestingly, the localization of SNM1A to sites of DSBs does not require either 53BP1 or H2AX, nor does the localization of 53BP1 require SNM1A. However, the localization of SNM1A does require ATM. Furthermore, SNM1A is shown to be a phosphorylation substrate of ATM in vitro, and to interact with ATM in vivo particularly after exposure of cells to IR. In addition, in the absence of SNM1A the activation of the downstream ATM target p53 is reduced. These findings suggest that SNM1A acts with ATM to promote the G1 cell cycle checkpoint.     

UV-induced ataxia-telangiectasia-mutated and Rad3-related (ATR) activation requires replication stress

Ataxia-telangiectasia-mutated and Rad3-related (ATR) plays an essential role in the maintenance of genome integrity and cell viability. The kinase is activated in response to DNA damage and initiates a checkpoint signaling cascade by phosphorylating a number of downstream substrates including Chk1. Unlike ataxia-telangiectasia-mutated (ATM), which appears to be mainly activated by DNA double-strand breaks, ATR can be activated by a variety of DNA damaging agents. However, it is still unclear what triggers ATR activation in response to such diverse DNA lesions. One model proposes that ATR can directly recognize DNA lesions, while other recent data suggest that ATR is activated by a common single-stranded DNA (ssDNA) intermediate generated during DNA repair. In this study, we show that UV lesions do not directly activate ATR in vivo. In addition, ssDNA lesions created during the repair of UV damage are also not sufficient to activate the ATR-dependent pathway. ATR activation is only observed in replicating cells indicating that replication stress is required to trigger the ATR-mediated checkpoint cascade in response to UV irradiation. Interestingly, H2AX appears to be required for the accumulation of ATR at stalled replication forks. Together our data suggest that ssDNA at arrested replication forks recruits ATR and initiates ATR-mediated phosphorylation of H2AX and Chk1. Phosphorylated H2AX might further facilitate ATR activation by stabilizing ATR at the sites of arrested replication forks.     

The small GTPase RhoA localizes to the nucleus and is activated by Net1 and DNA damage signals

Background

Rho GTPases control many cellular processes, including cell survival, gene expression and migration. Rho proteins reside mainly in the cytosol and are targeted to the plasma membrane (PM) upon specific activation by guanine nucleotide exchange factors (GEFs). Accordingly, most GEFs are also cytosolic or associated with the PM. However, Net1, a RhoA-specific GEF predominantly localizes to the cell nucleus at steady-state. Nuclear localization for Net1 has been seen as a mechanism for sequestering the GEF away from RhoA, effectively rendering the protein inactive. However, considering the prominence of nuclear Net1 and the fact that a biological stimulus that promotes Net1 translocation out the nucleus to the cytosol has yet to be discovered, we hypothesized that Net1 might have a previously unidentified function in the nucleus of cells.

Principal Findings

Using an affinity precipitation method to pulldown the active form of Rho GEFs from different cellular fractions, we show here that nuclear Net1 does in fact exist in an active form, contrary to previous expectations. We further demonstrate that a fraction of RhoA resides in the nucleus, and can also be found in a GTP-bound active form and that Net1 plays a role in the activation of nuclear RhoA. In addition, we show that ionizing radiation (IR) specifically promotes the activation of the nuclear pool of RhoA in a Net1-dependent manner, while the cytoplasmic activity remains unchanged. Surprisingly, irradiating isolated nuclei alone also increases nuclear RhoA activity via Net1, suggesting that all the signals required for IR-induced nuclear RhoA signaling are contained within the nucleus.

Conclusions/Significance

These results demonstrate the existence of a functional Net1/RhoA signaling pathway within the nucleus of the cell and implicate them in the DNA damage response.     

ATM-dependent phosphorylation of MRE11 controls extent of resection during homology directed repair by signalling through Exonuclease 1

The MRE11/RAD50/NBS1 (MRN) complex plays a central role as a sensor of DNA double strand breaks (DSB) and is responsible for the efficient activation of ataxia-telangiectasia mutated (ATM) kinase. Once activated ATM in turn phosphorylates RAD50 and NBS1, important for cell cycle control, DNA repair and cell survival. We report here that MRE11 is also phosphorylated by ATM at S676 and S678 in response to agents that induce DNA DSB, is dependent on the presence of NBS1, and does not affect the association of members of the complex or ATM activation. A phosphosite mutant (MRE11S676AS678A) cell line showed decreased cell survival and increased chromosomal aberrations after radiation exposure indicating a defect in DNA repair. Use of GFP-based DNA repair reporter substrates in MRE11S676AS678A cells revealed a defect in homology directed repair (HDR) but single strand annealing was not affected. More detailed investigation revealed that MRE11S676AS678A cells resected DNA ends to a greater extent at sites undergoing HDR. Furthermore, while ATM-dependent phosphorylation of Kap1 and SMC1 was normal in MRE11S676AS678A cells, there was no phosphorylation of Exonuclease 1 consistent with the defect in HDR. These results describe a novel role for ATM-dependent phosphorylation of MRE11 in limiting the extent of resection mediated through Exonuclease 1.     

Rad17 recruits the MRE11‐RAD50‐NBS1 complex to regulate the cellular response to DNA double‐strand breaks

The MRE11‐RAD50‐NBS1 (MRN) complex is essential for the detection of DNA double‐strand breaks (DSBs) and initiation of DNA damage signaling. Here, we show that Rad17, a replication checkpoint protein, is required for the early recruitment of the MRN complex to the DSB site that is independent of MDC1 and contributes to ATM activation. Mechanistically, Rad17 is phosphorylated by ATM at a novel Thr622 site resulting in a direct interaction of Rad17 with NBS1, facilitating recruitment of the MRN complex and ATM to the DSB, thereby enhancing ATM signaling. Repetition of these events creates a positive feedback for Rad17‐dependent activation of MRN/ATM signaling which appears to be a requisite for the activation of MDC1‐dependent MRN complex recruitment. A point mutation of the Thr622 residue of Rad17 leads to a significant reduction in MRN/ATM signaling and homologous recombination repair, suggesting that Thr622 phosphorylation is important for regulation of the MRN/ATM signaling by Rad17. These findings suggest that Rad17 plays a critical role in the cellular response to DNA damage via regulation of the MRN/ATM pathway.     

Proteasome inhibition suppresses DNA-dependent protein kinase activation caused by camptothecin

The ubiquitin–proteasome pathway plays an important role in DNA damage signaling and repair by facilitating the recruitment and activation of DNA repair factors and signaling proteins at sites of damaged chromatin. Proteasome activity is generally not thought to be required for activation of apical signaling kinases including the PI3K-related kinases (PIKKs) ATM, ATR, and DNA-PK that orchestrate downstream signaling cascades in response to diverse genotoxic stimuli. In a previous work, we showed that inhibition of the proteasome by MG-132 suppressed 53BP1 (p53 binding protein1) phosphorylation as well as RPA2 (replication protein A2) phosphorylation in response to the topoisomerase I (TopI) poison camptothecin (CPT). To address the mechanism of proteasome-dependent RPA2 phosphorylation, we investigated the effects of proteasome inhibitors on the upstream PIKKs. MG-132 sharply suppressed CPT-induced DNA-PKcs autophosphorylation, a marker of the activation, whereas the phosphorylation of ATM and ATR substrates was only slightly suppressed by MG-132, suggesting that DNA-PK among the PIKKs is specifically regulated by the proteasome in response to CPT. On the other hand, MG-132 did not suppress DNA-PK activation in response to UV or IR. MG-132 blocked the interaction between DNA-PKcs and Ku heterodimer enhanced by CPT, and hydroxyurea pre-treatment completely abolished CPT-induced DNA-PKcs autophosphorylation, indicating a requirement for ongoing DNA replication. CPT-induced TopI degradation occurred independent of DNA-PK activation, suggesting that DNA-PK activation does not require degradation of trapped TopI complexes. The combined results suggest that CPT-dependent replication fork collapse activates DNA-PK signaling through a proteasome dependent, TopI degradation-independent pathway. The implications of DNA-PK activation in the context of TopI poison-based therapies are discussed.     

Differential localization of ATM is correlated with activation of distinct downstream signaling pathways

ATM, the gene mutated in the genetic disease ataxia telangiectasia (AT), is a well-known protein involved in the DNA double-strand break response, where it plays an important role in sensing damage and signaling to DNA repair machinery and cell cycle checkpoints. However, a number of recent papers, including ours have found that ATM also plays important roles outside of the nucleus, which may explain some of the phenotypic features seen in AT patients. Our research into mechanisms of TSC2 regulation helped uncover a pathway upstream of TSC2 that is regulated by cytoplasmic ATM in response to ROS initiated by ATM activation of LKB1 and AMPK. We found that TSC2 activation results in mTORC1 repression and subsequent induction of autophagy. Elucidation of this stress response pathway provides a molecular mechanism for ATM signaling in the cytoplasm, and lays the groundwork for further studies on how ATM activity is regulated beyond DNA damage in different cellular compartments.     

NEMO and RIP1 control cell fate in response to extensive DNA damage via TNF-α feedforward signaling

Upon DNA damage, ataxia telangiectasia mutated (ATM) kinase triggers multiple events to promote cell survival and facilitate repair. If damage is excessive, ATM stimulates cytokine secretion to alert neighboring cells and apoptosis to eliminate the afflicted cell. ATM augments cell survival by activating nuclear factor (NF)-κB; however, how ATM induces cytokine production and apoptosis remains elusive. Here we uncover a p53-independent mechanism that transmits ATM-driven cytokine and caspase signals upon strong genotoxic damage. Extensive DNA lesions stimulated two sequential NF-κB activation phases, requiring ATM and NEMO/IKK-γ: The first phase induced TNF-α-TNFR1 feedforward signaling, promoting the second phase and driving RIP1 phosphorylation. In turn, RIP1 kinase triggered JNK3/MAPK10-dependent interleukin-8 secretion and FADD-mediated proapoptotic caspase-8 activation. Thus, in the context of excessive DNA damage, ATM employs NEMO and RIP1 kinase through autocrine TNF-α signaling to switch on cytokine production and caspase activation. These results shed light on cell-fate regulation by ATM.     

The Rad9 protein enhances survival and promotes DNA repair following exposure to ionizing radiation

Following DNA damage cells initiate cell cycle checkpoints to allow time to repair sustained lesions. Rad9, Rad1, and Hus1 proteins form a toroidal complex, termed the 9-1-1 complex, that is involved in checkpoint signaling. 9-1-1 shares high structural similarity to the DNA replication protein proliferating cell nuclear antigen (PCNA) and 9-1-1 has been shown in vitro to stimulate steps of the repair process known as long patch base excision repair. Using a system that allows conditional repression of the Rad9 protein in human cell culture, we show that Rad9, and by extension, the 9-1-1 complex, enhances cell survival, is required for efficient exit from G2-phase arrest, and stimulates the repair of damaged DNA following ionizing radiation. These data provide in vivo evidence that the human 9-1-1 complex participates in DNA repair in addition to its previously described role in DNA damage sensing.