ATM/FANC/BRCA1/NBS1 signaling pathway
Wednesday 21 April 2004
The ATM protein
The protein ATM (Ataxia Telangiectasia Mutated Protein) belongs to a family of serine-threonine kinases that have sequence homology to PI3K (Phosphoinositide 3-Kinase).
ATM is a key regulator of multiple signaling cascades which respond to DNA strand breaks induced by damaging agents IR (ionizing Radiation), radiometric agents or by normal processes.
In addition, ATM appears to function as a caretaker, suppressing tumorigenesis in specific T cell lineages.
In non-irradiated cells ATM exists as a dimer, is not phosphorylated and is present throughout the nucleus.
After irradiation, which leads to formation of DNA DSBs (DNA Double Strand Breaks), ATM becomes a monomer, it is phosphorylated on Ser1981 and some pool of it is present at sites of DNA DSBs.
ATM monitors the presence of DNA DSBs indirectly, through DNA DSB-induced changes in chromatin structure.
ATM is activated by the MRE11 (Meiotic Recombination-11)-Rad50-NBS1 (Nijmegen Breakage Syndrome-1) complex or 53BP1. The former is thought to be recruited at the DSB, whereas the latter is recruited at chromatin regions flanking the DNA DSB and extending up to a few megabases from the DSB.
ATM phosphorylates NBS1 on several residues in response to DNA damage and a functional MRE11/Rad50/NBS1 complex is required for Chk2 activation.
This links the MRE11/Rad50/NBS1 complex to DNA damage recognition. ATM activated at sites of DNA DSBs may also phosphorylate and activate ATM in the nucleoplasm.
Downstream targets of ATM
Its downstream targets include Chk1 (Cell Cycle Checkpoint Kinase-1), Chk2 (Cell Cycle Checkpoint Kinase-2), tumor suppressors like p53 and BRCA (Breast Cancer), DNA repair factors like Rad50, Rad51, GADD45 (Growth Arrest and DNA-Damage-inducible), and other signaling molecules like c-Abl and NF-KappaB (Nuclear Factor-Kappa B).
Once ATM is activated it phosphorylates multiple substrates. Two of these, Chk2 and p53, mediate many of the cell cycle effects of ATM, while two others, SMC1 (Structural Maintainance of Chromosomes-1) and histone H2AX (H2A Histone family, member X), are important for cell survival after irradiation.
Chk2 is a protein Kinase, that once activated amplifies the DNA damage signal of ATM. Two of the key substrates of Chk2 are CDC25A (Cell Division Cycle-25A) and CDC25C (Cell Division Cycle-25C).
When CDC25A and CDC25C become phosphorylated by Chk2, their function is inhibited and cells delay progression through S phase or arrest in G2. Chk2 contributes to p53 regulation in part through phosphorylation of MDMX.
The mechanism by which phosphorylation enhances MDMX-MDM2 (Mouse Double Minute-2) binding and MDMX ubiquitination by MDM2 remains to be further investigated. MDC1 (Mediator of DNA damage Checkpoint-1) localizes to sites of DNA breaks and associates with Chk2 after DNA damage. This association is mediated by the MDC1 FHA domain and the phosphorylated Thr 68 of Chk2.
Cell cycle arrest in G1 is mediated by p53, which is a substrate of both Chk2 and ATM. p53 is a transcription factor that induces expression of p21 (Cyclin Dependent Kinase Inhibitor-p21)/Waf1, GADD45, and MDM2. p21 is a CDK(Cyclin-Dependent Kinase) inhibitor.
p53 also inhibits G2-M transition by repressing the transcription of CDC2 (Cell Division Cycle-2, G1 to S and G2 to M) and Cyclin-B. p53 can also induce expression of genes that induce apoptosis; and in certain tissues, induction of p53 leads to apoptosis, rather than cell cycle arrest.
Unlike, Chk2 and p53, Phosphorylated SMC1 and Histone H2AX are found exclusively at sites of DNA DSBs. SMC1 phosphorylation is critical for cells to survive after irradiation.
Similar to SMC1 phosphorylation, histone H2AX phosphorylation also appears to be important for DNA repair. The mechanisms by which SMC1 and histone H2AX phosphorylation facilitate DNA repair are not known, but recent evidence suggests that phosphorylated histone H2AX recruits chromatin remodeling complexes to sites of DNA DSBs.
ATM also interacts with the protein product of the c-Abl proto-oncogene. This non-receptor tyrosine Kinase is involved in several stress responses including activation of the SAPK (Stress-Activated Protein Kinase), Rad51 and p73. SAPK activation by c-Abl leads to activation of c-Jun and thus plays an important role in cell survival. c-Abl also phosphorylates Rad51, which is further involved in DNA repair and recombination processes.
Activated c-Abl may also promote apoptosis via up-regulation of p73, a proapoptotic protein and a p53 homolog. ATM also interacts with AP3B2 (Adaptor-related Protein complex-3, Beta-2 subunit), a neuronal homolog of Beta-Adaptin thought to be involved in synaptic vesicle transport in neuronal cells. ATM phosphorylates ATF2 (Activating Transcription Factor-2) on Serine 490 and 498 following IR (Ionizing Radiation). Phosphorylation of ATF2 by ATM results in its rapid colocalization with Gamma-H2AX and MRN components into IRIF (IR-Induced Foci).
ATM also phosphorylates IKappaB-Alpha and thus plays an important role in NF-KappaB activation. ATM also phosphorylates and activates Chk1, which phosphorylates TLK1 (Tousled Like Kinase-1).
TLK1 activity is rapidly suppressed by DNA damage and by inhibitors of replication. Chk1 also phosphorylate a conserved site (Ser-216) on protein phosphatase CDC25C, which results in it being inactivated and bound by the 14-3-3 protein.
The inactive CDC25C is then incapable of removing an inhibitory phosphate group on Tyr-15 of CDC2, preventing entry into mitosis. ATM also phosphorylates FANCD2 (Fanconi Anemia Complementation Group-D2) protein, which then leads to S phase arrest.
BRCA1 is also activated by the protein kinase ATM that initiates cell cycle changes after DNA damage.
BRCA1 physically interacts with Chk1 and stimulates its activity after DNA damage. Hyperphosphorylation of Rad9 induced by IR is also dependent on ATM.
Ser(272) of Rad9 is phosphorylated directly by ATM. ATM also phosphorylates CREB (cAMP Response Element-Binding Protein-1) in vitro and in vivo in response to IR and H2O2 on a stress-inducible domain.
ATM-mediated phosphorylation of CREB in response to DNA damage modulates CREB-dependent gene expression and dysregulation of the ATM-CREB pathway may contribute to neurodegeneration. ATM also phosphorylates BID (BH3 Interacting Domain Death Agonist).
This phosphorylation is required for a downstream function in cell cycle checkpoint control. ATM also seems to have a role in telomere maintenance and replication.
The disease AT (Ataxia-Telangiectasia), caused by mutations in the ATM gene, is an autosomal recessive disorder characterized by Progressive Cerebellar Ataxia, Cancer Predisposition, Oculocutaneous Telangiectasia and variable immune deficiencies.
Inhibiting ATM might sensitize repair proficient cancer cells to radiation and chemotherapy. It is possible that targeting the ATM pathway might even introduce selectivity to current cancer therapy.
However, the real test of this hypothesis will have to await the development of more potent and specific inhibitors for ATM (Ref.8, 9 & 10).
The BASC complex
BRCA1 forms a multi-subunit protein complex referred to as the BRCA1-associated genome surveillance complex (BASC), which includes DNA damage repair proteins such as MSH2-MSH6 and MLH1, as well as ATM, NBS1, MRE11, and BLM.
BRCA1 is the protein defective in some cases of hereditary breast cancer susceptibility. The recent identification of RECQL3 as part of the BRCA1-associated genome surveillance complex (BASC), links RECQL3 with a number of tumour suppressor and DNA damage repair proteins.
The BASC complex includes MSH2, MHS6, MLH1, ATM, RECQL3, the RAD50-MRE11-NBS1 complex and DNA replication factor C.
Many components of this complex have roles in recognition of DNA damage/unusual DNA structures, suggestive of this complex performing some kind of ’sensor’ role.
To examine the role of RECQL3 within BASC, the subcellular localization of RECQL3 and BRCA1 was analysed before and after exposure to DNA damaging agents. In untreated cells, RECQL3 and BRCA1 colocalization was limited to a few bright nuclear foci.
However, after treatment with hydroxyurea or ionizing radiation, colocalization was greatly enhanced in those cells that were in mid-to-late S phase or in G2. This could be indicative of specific requirement for BLM/BRCA1 in replication/repair of late replicating DNA.
Consistent with a role for BLM (possibly within BASC) in recognizing abnormal DNA structures, it has been shown that RECQL3 is able to unwind a variety of unusual DNA structures, including G-quadruplex, synthetic X-junctions (models for the Holliday junction), bubbles and forked DNA.
BRCAs (BRCA1 and BRCA2)