Trans-activation of the DNA-damage signalling protein kinase Chk2 by T-loop exchange

The protein kinase Chk2 (checkpoint kinase 2) is a major effector of the replication checkpoint. Chk2 activation is initiated by phosphorylation of Thr68, in the serine-glutamine/threonine-glutamine cluster domain (SCD), by ATM. The phosphorylated SCD-segment binds to the FHA domain of a second Chk2 molecule, promoting dimerisation of the protein and triggering phosphorylation of the activation segment/T-loop in the kinase domain. We have now determined the structure of the kinase domain of human Chk2 in complexes with ADP and a small-molecule inhibitor debromohymenialdisine. The structure reveals a remarkable dimeric arrangement in which T-loops are exchanged between protomers, to form an active kinase conformation in trans. Biochemical data suggest that this dimer is the biologically active state promoted by ATM-phosphorylation, and also suggests a mechanism for dimerisation-driven activation of Chk2 by trans-phosphorylation.     

Regulation of the Rad53 protein kinase in signal amplification by oligomer assembly and disassembly

Rad53, the ortholog of mammalian Chk2, is a major DNA damage checkpoint effector kinase in Saccharomyces cerevisiae. Despite extensive studies on the genetic requirements for Rad53 activation and its linkage downstream to checkpoint responses, the mechanism of Rad53 activation is not completely understood. Rad53-dependent signal amplification is thought to be a primary force that accelerates checkpoint signal transduction processes in response to DNA damage. Rad53 forms oligomers upon DNA damage in vivo. It is not clear how oligomer formation affects Rad53 activation and what is the mechanism of Rad53 oligomerization. Here, we monitor Rad53 oligomer assembly and disassembly in vitro. These processes are ATP-dependent and are regulated through phosphorylation. Mutations in FHA or SCD domains of RAD53 compromise intermolecular autophosphorylation activity and these domains are indispensable for Rad53 oligomerization. The mediator Rad9 is not necessary for Rad53 oligomerization. Rad53 kinase activity is required for disassembly of Rad53 oligomers in vivo after DNA damage. Moreover, induced oligomerization of Rad53 efficiently activates Rad53 in the absence of Mec1 in vivo. The results support the conclusions that Rad53/Chk2 homo-oligomerization is an evolutionarily conserved mechanism that drives Rad53/Chk2 activation and promotes signal amplification in DNA damage responses.     

Phosphorylation-dependent interactions between Crb2 and Chk1 are essential for DNA damage checkpoint

In response to DNA damage, the eukaryotic genome surveillance system activates a checkpoint kinase cascade. In the fission yeast Schizosaccharomyces pombe, checkpoint protein Crb2 is essential for DNA damage-induced activation of downstream effector kinase Chk1. The mechanism by which Crb2 mediates Chk1 activation is unknown. Here, we show that Crb2 recruits Chk1 to double-strand breaks (DSBs) through a direct physical interaction. A pair of conserved SQ/TQ motifs in Crb2, which are consensus phosphorylation sites of upstream kinase Rad3, is required for Chk1 recruitment and activation. Mutating both of these motifs renders Crb2 defective in activating Chk1. Tethering Crb2 and Chk1 together can rescue the SQ/TQ mutations, suggesting that the main function of these phosphorylation sites is promoting interactions between Crb2 and Chk1. A 19-amino-acid peptide containing these SQ/TQ motifs is sufficient for Chk1 binding in vitro when one of the motifs is phosphorylated. Remarkably, the same peptide, when tethered to DSBs by fusing with either recombination protein Rad22/Rad52 or multi-functional scaffolding protein Rad4/Cut5, can rescue the checkpoint defect of crb2Δ. The Rad22 fusion can even bypass the need for Rad9-Rad1-Hus1 (9-1-1) complex in checkpoint activation. These results suggest that the main role of Crb2 and 9-1-1 in DNA damage checkpoint signaling is recruiting Chk1 to sites of DNA lesions.     

Phosphorylation of threonine 68 promotes oligomerization and autophosphorylation of the Chk2 protein kinase via the forkhead-associated domain

Phosphorylation of Thr-68 by the ataxia telangiectasia-mutated is necessary for efficient activation of Chk2 when cells are exposed to ionizing radiation. By an unknown mechanism, this initial event promotes additional autophosphorylation events including modifications of Thr-383 and Thr-387, two amino acid residues located within the activation loop segment within the Chk2 catalytic domain. Chk2 and related kinases possess one or more Forkhead-associated (FHA) domains that are phosphopeptide-binding modules believed to be crucial for their checkpoint control activities. We show that the Chk2 FHA domain is dispensable for Thr-68 phosphorylation but necessary for efficient autophosphorylation in response to ionizing radiation. Phosphorylation of Thr-68 promotes oligomerization of Chk2 by serving as a specific ligand for the FHA domain of another Chk2 molecule. In addition, Chk2 phosphorylates its own FHA domain, and this modification reduces its affinity for Thr-68-phosphorylated Chk2. Thus, activation of Chk2 in irradiated cells may occur through oligomerization of Chk2 via binding of the Thr-68-phosphorylated region of one Chk2 to the FHA domain of another. Oligomerization of Chk2 may therefore increase the efficiency of trans-autophosphorylation resulting in the release of active Chk2 monomers that proceed to enforce checkpoint control in irradiated cells.     

Structural and functional versatility of the FHA domain in DNA-damage signaling by the tumor suppressor kinase Chk2

The Chk2 Ser/Thr kinase plays crucial, evolutionarily conserved roles in cellular responses to DNA damage. Identification of two pro-oncogenic mutations within the Chk2 FHA domain has highlighted its importance for Chk2 function in checkpoint activation. The X-ray structure of the Chk2 FHA domain in complex with an in vitro selected phosphopeptide motif reveals the determinants of binding specificity and shows that both mutations are remote from the peptide binding site. We show that the Chk2 FHA domain mediates ATM-dependent Chk2 phosphorylation and targeting of Chk2 to in vivo binding partners such as BRCA1 through either or both of two structurally distinct mechanisms. Although phospho-dependent binding is important for Chk2 activity, previously uncharacterized phospho-independent FHA domain interactions appear to be the primary target of oncogenic lesions.     

Chk2 oligomerization studied by phosphopeptide ligation: implications for regulation and phosphodependent interactions

Chk2/CHEK2/hCds1 is a modular serine-threonine kinase involved in transducing DNA damage signals. Phosphorylation by ataxia telangiectasia-mutated kinase (ATM) promotes Chk2 self-association, autophosphorylation, and activation. Here we use expressed protein ligation to generate a Chk2 N-terminal regulatory region encompassing a fork-head-associated (FHA) domain, a stoichiometrically phosphorylated Thr-68 motif and intervening linker. Hydrodynamic analysis reveals that Thr-68 phosphorylation stabilizes weak FHA-FHA interactions that occur in the unphosphorylated species to form a high affinity dimer. Although clearly a prerequisite for Chk2 activation in vivo, we show that dimerization modulates potential phosphodependent interactions with effector proteins and substrates through either the pThr-68 site, or the canonical FHA phosphobinding surface with which it is tightly associated. We further show that the dimer-occluded pThr-68 motif is released by intra-dimer autophosphorylation of the FHA domain at the highly conserved Ser-140 position, a major pThr contact in all FHA-phosphopeptide complex structures, revealing a mechanism of Chk2 dimer dissociation following kinase domain activation.     

Mdt1, a novel Rad53 FHA1 domain-interacting protein, modulates DNA damage tolerance and G(2)/M cell cycle progression in Saccharomyces cerevisiae

The Rad53 kinase plays a central role in yeast DNA damage checkpoints. Rad53 contains two FHA phosphothreonine-binding domains that are required for Rad53 activation and possibly downstream signaling. Here we show that the N-terminal Rad53 FHA1 domain interacts with the RNA recognition motif, coiled-coil, and SQ/TQ cluster domain-containing protein Mdt1 (YBl051C). The interaction of Rad53 and Mdt1 depends on the structural integrity of the FHA1 phosphothreonine-binding site as well as threonine-305 of Mdt1. Mdt1 is constitutively threonine phosphorylated and hyperphosphorylated in response to DNA damage in vivo. DNA damage-dependent Mdt1 hyperphosphorylation depends on the Mec1 and Tel1 checkpoint kinases, and Mec1 can directly phosphorylate a recombinant Mdt1 SQ/TQ domain fragment. MDT1 overexpression is synthetically lethal with a rad53 deletion, whereas mdt1 deletion partially suppresses the DNA damage hypersensitivity of checkpoint-compromised strains and generally improves DNA damage tolerance. In the absence of DNA damage, mdt1 deletion leads to delayed anaphase completion, with an elongated cell morphology reminiscent of that of G(2)/M cell cycle mutants. mdt1-dependent and DNA damage-dependent cell cycle delays are not additive, suggesting that they act in the same pathway. The data indicate that Mdt1 is involved in normal G(2)/M cell cycle progression and is a novel target of checkpoint-dependent cell cycle arrest pathways.     

The hCds1 (Chk2)-FHA domain is essential for a chain of phosphorylation events on hCds1 that is induced by ionizing radiation

hCds1 (Chk2) is an evolutionarily conserved kinase that functions in DNA damage response and cell cycle checkpoint. The Cds1 family of kinases are activated by a family of large phosphatidylinositol 3-kinase-like kinases. In humans, ataxia telangiectasia-mutated (ATM) and ataxia-telangiectasia and Rad3-related kinases activate hCds1 by phosphorylating Thr(68) . hCds1 and Cds1-related kinases contain the FHA (forkhead-associated) domain, which appears to be important for integrating the DNA damage signal. It is not known how ATM phosphorylation activates hCds1 function and whether the phosphorylation is linked to the FHA. Here, we demonstrate that the hCds1-FHA domain is essential for Thr(68) phosphorylation. Thr(68) phosphorylation, in turn, is required for ionizing radiation-induced autophosphorylation of two amino acid residues in hCds1, Thr(383) and Thr(387). These two amino acid residues, located in the activation loop of hCds1, are conserved in hCds1-related kinases and are essential for hCds1 activity. Thus, the hCds1-FHA domain mediates a chain of phosphorylation events on hCds1, which includes phosphorylation by ATM and hCds1 autophosphorylation, in response to DNA damage.     

The FHA domain determines Drosophila Chk2/Mnk localization to key mitotic structures and is essential for early embryonic DNA damage responses

DNA damage responses, including mitotic centrosome inactivation, cell-cycle delay in mitosis, and nuclear dropping from embryo cortex, maintain genome integrity in syncytial Drosophila embryos. A conserved signaling kinase, Chk2, known as Mnk/Loki, is essential for the responses. Here we demonstrate that functional EGFP-Mnk expressed from a transgene localizes to the nucleus, centrosomes, interkinetochore/centromere region, midbody, and pseudocleavage furrows without DNA damage and in addition forms numerous foci/aggregates on mitotic chromosomes upon DNA damage. We expressed EGFP-tagged Mnk deletion or point mutation variants and investigated domain functions of Mnk in vivo. A triple mutation in the phosphopeptide-binding site of the forkhead-associated (FHA) domain disrupted normal Mnk localization except to the nucleus. The mutation also disrupted Mnk foci formation on chromosomes upon DNA damage. FHA mutations and deletion of the SQ/TQ-cluster domain (SCD) abolished Mnk transphosphorylations and autophosphorylations, indicative of kinase activation after DNA damage. A potent NLS was found at the C-terminus, which is required for normal Mnk function. We propose that the FHA domain in Mnk plays essential dual functions in mediating embryonic DNA damage responses by means of its phosphopeptide-binding ability: activating Mnk in the nucleus upon DNA damage and recruiting Mnk to multiple subcellular structures independently of DNA damage.     

SQ/TQ cluster domains: concentrated ATM/ATR kinase phosphorylation site regions in DNA-damage-response proteins

ATM/ATR-like protein kinases play central roles in the maintenance of genome stability and phosphorylate numerous substrates in response to DNA damage, preferentially on SQ or TQ motifs. ATM/ATR substrates often contain several closely spaced SQ/TQ motifs in regions that have been termed SQ/TQ cluster domains (SCDs). SCDs are now considered a structural hallmark of DNA-damage-response proteins. Mutational analyses of a number of SCD-containing proteins indicate that multisite phosphorylation of SQ/TQ motifs is required for normal DNA-damage responses, most commonly by mediating protein-protein interactions in the formation of DNA-damage-induced complexes. SCD sequences are highly diverse and these domains may be largely unfolded in their native state rather than adopting a common three-dimensional fold. Structural disorder of SCDs could be advantageous for efficient phosphorylation by ATM/ATR kinases and also enable them to be molded into distinct conformations to facilitate flexible interactions with multiple binding partners.     

Mdt1/ASCIZ: A new DNA damage response protein family

DNA damage response pathways are crucial for genome stability and prevention of cancer, and are overall remarkably conserved from yeast to mammals. Two novel DNA damage response proteins, yeast Mdt1 (Modifier of DNA damage tolerance 1) and human ASCIZ (ATM/ATR-substrate Chk2-interacting Zn2+-finger protein), were recently identified based on their interactions with the N-terminal FHA domains of the conserved checkpoint kinases Rad53 and Chk2, respectively, and ASCIZ was subsequently re-isolated as an ATM-interacting protein (ATMIN). Mdt1 and ASCIZ share remarkable sequence similarity (36% highly conserved residues, 17% identity) and extended SQ/TQ cluster domains (SCDs) typical of DNA damage response proteins. However, despite their structural similarities and conserved interactions with the checkpoint machinery, the two proteins seem to respond to different DNA lesions: the strongest phenotypes of ASCIZ deficiency are increased sensitivity to DNA base damaging agents and altered immunoglobulin gene diversification following enzyme-induced base damage in B lymphocytes, whereas absence of Mdt1 leads to hypersensitivity to 3'-blocked DNA double-strand breaks and inefficient recombinational maintenance of telomeres. The Mdt1/ASCIZ family may function as structurally related scaffolds that facilitate efficient DNA repair, albeit with diverged lesion specificity.     

Regulation of Chk1 by its C-terminal domain

Chk1 is a protein kinase that is the effector molecule in the G2 DNA damage checkpoint. Chk1 homologues have an N-terminal kinase domain, and a C-terminal domain of ~200 amino acids that contains activating phosphorylation sites for the ATM/R kinases, though the mechanism of activation remains unknown. Structural studies of the human Chk1 kinase domain show an open conformation; the activity of the kinase domain alone is substantially higher in vitro than full-length Chk1, and coimmunoprecipitation studies suggest the C-terminal domain may contain an autoinhibitory activity. However, we show that truncation of the C-terminal domain inactivates Chk1 in vivo. We identify additional mutations within the C-terminal domain that activate ectopically expressed Chk1 without the need for activating phosphorylation. When expressed from the endogenous locus, activated alleles show a temperature-sensitive loss of function, suggesting these mutations confer a semiactive state to the protein. Intragenic suppressors of these activated alleles cluster to regions in the catalytic domain on the face of the protein that interacts with substrate, suggesting these are the regions that interact with the C-terminal domain. Thus, rather than being an autoinhibitory domain, the C-terminus of Chk1 also contains domains critical for adopting an active configuration.     

Inhibition of Chk1 by activated PKB/Akt

We have shown recently that DNA damage effector kinase Chk1 is phosphorylated in vitro by protein kinase B/Akt (PKB/Akt) on serine 280. Activation of Chk1 by DNA damage in vivo is suppressed in presence of activated PKB. In this study we show that Chk1 is phosphorylated by PKB in vivo, and that increased phosphorylation by PKB on serine 280 correlates with impairment of Chk1 activation by DNA damage. Our results indicate a likely mechanism for the negative effects that phosphorylation of serine 280 has on activation of Chk1. The Chk1 protein phosphorylated by PKB on serine 280 does not enter into protein complexes after replication arrest. Moreover, Chk1 phosphorylated by PKB fails to undergo activating phosphorylation on serine 345 by ATM/ATR. Phosphorylation by ATM/ATR and association with other checkpoint proteins are essential steps in activation of Chk1. Inhibition of these steps provides a plausible explanation for the observed attenuation of Chk1 activation by activated PKB after DNA damage.     

Priming phosphorylation of Chk2 by polo-like kinase 3 (Plk3) mediates its full activation by ATM and a downstream checkpoint in response to DNA damage

The tumor suppressor gene Chk2 encodes a serine/threonine kinase that signals DNA damage to cell cycle checkpoints. In response to ionizing radiation, Chk2 is phosphorylated on threonine 68 (T68) by ataxia-telangiectasia mutated (ATM) protein leading to its activation. We have previously shown that polo-like kinase 3 (Plk3), a protein involved in DNA damage checkpoint and M-phase functions, interacts with and phosphorylates Chk2. When Chk2 was immunoprecipitated from Daudi cells (Plk3-deficient), it had weak kinase activity towards Cdc25C compared with Chk2 derived from T47D cells (Plk3-expressing cells). This activity was restored by addition of recombinant Plk3 in a dose-dependent manner. Plk3 phosphorylates Chk2 at two residues, serine 62 (S62) and serine 73 (S73) in vitro, and this phosphorylation facilitates subsequent phosphorylation of Chk2 on T68 by ATM in response to DNA damage. When the Chk2 mutant construct GFP-Chk2 S73A (serine 73 mutated to alanine) is transfected into cells, it no longer associates with a large complex in vivo, and manifests a significant reduction in kinase activity. It is also inefficiently activated by ATM by phosphorylation at T68 and, in turn, is unable to phosphorylate the Cdc25C peptide 200-256, which contains the inhibitory S216 target phosphorylation residue. As a consequence, tyrosine 15 (Y15) on Cdc2 remains hypophosphorylated, and there is a loss of the G2/M checkpoint. These data describe a functional role for Plk3 in a pathway linking ATM, Plk3, Chk2, Cdc25C and Cdc2 in cellular response to DNA damage.     

Inhibition of Chk1 by Activated PKB/Akt

We have shown recently that DNA damage effector kinase Chk1 is phosphorylated invitro by protein kinase B/Akt (PKB/Akt) on serine 280. Activation of Chk1 by DNAdamage in vivo is suppressed in presence of activated PKB. In this study we show thatChk1 is phosphorylated by PKB in vivo, and that increased phosphorylation by PKB onserine 280 correlates with impairment of Chk1 activation by DNA damage. Our resultsindicate a likely mechanism for the negative effects that phosphorylation of serine 280has on activation of Chk1. The Chk1 protein phosphorylated by PKB on serine 280 doesnot enter into protein complexes after replication arrest. Moreover, Chk1 phosphorylatedby PKB fails to undergo activating phosphorylation on serine 345 by ATM/ATR.Phosphorylation by ATM/ATR and association with other checkpoint proteins areessential steps in activation of Chk1. Inhibition of these steps provides a plausibleexplanation for the observed attenuation of Chk1 activation by activated PKB after DNAdamage.     

Characterization of tumor-associated Chk2 mutations

The integrity of the DNA damage response pathway is essential for prevention of neoplastic transformation. Several proteins involved in this pathway including p53, BRCA1, and ATM are frequently mutated in human cancer. Checkpoint kinase 2 (Chk2) is a DNA damage-activated protein kinase that lies downstream of ATM in this pathway. Recently, heterozygous germline mutations in Chk2 have been identified in a subset of patients with Li-Fraumeni syndrome, a highly penetrant familial cancer phenotype, suggesting that Chk2 is a tumor suppressor gene. In this study, we have reported the biochemical characterization of the four tumor-associated Chk2 mutants. Two of the reported Chk2 mutations identified in Li-Fraumeni syndrome result in loss of Chk2 kinase activity. Whereas one mutation within the Chk2 forkhead homology-associated (FHA) domain, R145W, retains some basal kinase activity, this mutant cannot be phosphorylated at an ATM-dependent phosphorylation site (Thr-68) and cannot be activated following gamma radiation. Wild-type Chk2 exists mainly in a protein complex of M(r) approximately 200,000 whereas the R145W mutant forms a larger, presumably inactive complex in the cell. The other FHA domain mutant, I157T, behaves as wild-type Chk2 in all the assays used here. Because the FHA domain is involved in protein-protein interactions, this mutation may affect associations of Chk2 with other proteins. Additionally, we have shown that Chk2 can also be inactivated by down-regulation of its expression in cancer cells. Thus, Chk2 may be inactivated by multiple mechanisms in the cell.