In the presence of DNA damage or incomplete DNA replication, eukaryotic cells activate cell cycle checkpoints that temporarily halt the cell cycle to permit DNA repair or completion of DNA replication to take place. In the presence of extensive damage or absence of timely repair, these checkpoint-signaling pathways may also trigger a pathway that effects programmed cell death or apoptosis (Ref.1). DNA damage-activated cell cycle checkpoints are regulated in part by the phosphoinositide kinase family of checkpoint components, including the yeast Rad3 in Schizosaccharomyces pombe, Mec1/Tel1 in Saccharomyces cerevisiae, mammalian ATM (Ataxia Telangiectasia-Mutated), ATR (ATM/Rad3-related), MEI-41 in Drosophila, and X-ATM and X-ATR in Xenopus. These checkpoint kinases regulate the activities of two downstream effector serine/threonine kinases, CDS1 and Chk1 (Csk Homologous Kinase) that are evolutionarily conserved. The CDS1 family includes conserved representatives from the yeasts (CDS1 in fission yeast and Rad53 in budding yeast) to man (hCDS1, also known as CDS1/CHEK2/Chk2) (Ref.2).
The Chk originally referred to as MATK (Megakaryocyte-Associated Tyrosine Kinase), is highly restricted in its expression to brain and hematopoietic cells (Ref.3). Both Chks, Chk1 and Chk2 are structurally unrelated yet functionally overlapping serine/threonine kinases which relay the checkpoint signals from the proximal checkpoint kinases of the PI3K (Phosphatidylinositol-3-Kinase) family, particularly ATM and ATR. Ionizing radiation, telomere erosion, and radiomimetic drugs generate DNA DSB (Double-Strand Breaks) and activate the ATM kinase, which phosphorylates Chk2 at Thr68. This in turn promotes homodimerization and intermolecular transphosphorylation of Chk2 on its C-terminal kinase domain, a modification required for a full activation of Chk2 toward heterologous substrates. The ATM-dependent phosphorylation of Chk2 cannot occur freely in the nucleoplasm, but requires a specific DSB-associated adaptor protein(s). In addition, other checkpoint proteins may coregulate the Chk2 activation. These factors include a DSB-interacting protein 53BP1 (Tumor Protein p53 Binding Protein-1), DNA ends-processing MRN nuclease complex (MRE11/Rad50/NBS1), and its newly identified binding partner MDC1 (Mediator of DNA Damage Checkpoint-1) (Ref.1). Chk2 has a key role in delaying cell cycle progression in response to DNA damage. Upon activation Chk2 phosphorylate the mitosis-inducing phosphatase CDC25C (Cell Division Cycle 25C) on an inhibitory site, blocking the progression from G2 to M-phase, and p53 on a regulatory site, which induces transcription of WAF1 (Wildtype P53-Activated Fragment-1)/p21/CIP1 (Cyclin-Dependent Kinase Inhibitor-1), resulting in arrest of G1-phase of the cell cycle (Ref.4). After DNA damage, Chk2 phosphorylates p53 on Ser20, attenuating the binding of p53 to MDM2 (Mouse Double Minute-2) that targets p53 for degradation in the proteasome, and allowing accumulation and subsequent activation of WAF1/p21 and G1 arrest. The oncosuppressor protein BRCA1 (Breast Cancer Susceptibility Protein-1), the core of the BRCA1-associated super complex physically interacts with Chk2 protein and is also a putative target of Chk2 activity (Ref.5). Phosphorylation of BRCA1 by Chk2 in response to DNA damage is required for survival after DNA damage.
Ultraviolet light, stalled replication, and some drugs activate ATR, the major upstream kinase phosphorylating and activating Chk1. Its recruitment to DNA requires ATRIP, a DNA-interacting adaptor protein. ATM also phosphorylates Chk1 in cells exposed to ionizing radiation and both ATR and ATM target the SQ-rich C-terminus of Chk1, including Ser317 and Ser345, respectively. These phosphorylations directly lead to Chk1 activation. Optimal activation of Chk1 also requires a cooperative action of other factors including the multifunctional BRCA1 tumor suppressor, the claspin adaptor molecule, and the PCNA (Proliferating Cell Nuclear Antigen)-like DNA sliding clamp (Rad9/Rad1/HUS1) (Ref.1). Rad3-dependent activation of Chk1 leads to negative regulation of CDC25A. This negative regulation may occur by direct inhibition of CDC25 activity, prevention of the activation of CDC25 that occurs at the G2-M transition, or interference in the interaction between CDC25 and CDC2. Inhibitory phosphorylation of CDC2 is crucial for G2 DNA damage arrest in mammalian cells (Ref.6). Chk1 phosphorylation of CDC25C promotes the binding of a 14-3-3 protein, which sequesters CDC25C in the cytoplasm. CDS1 arrest cells in G2 by phosphorylating CDC25C on amino acid residues also targeted by Chk1 (Ref.2).
Following their activation, Chk1 and Chk2 phosphorylate unique and overlapping downstream effectors that further propagate the checkpoint signaling. Depending on the type of stress, velocity of DNA damage, and cellular context, this leads to (1) switching of the stress-induced transcription program (E2F1, BRCA1, p53), (2) direct or indirect initiation of DNA repair (BRCA1, p53), (3) acute delay (degradation of CDC25A) and/or sustained block (CDC25C, p53, PLK3) of cell cycle progression, (4) apoptosis (PML1, p53, E2F1), and (5) modulation of the chromatin remodeling pathways (TLK1/2) (Ref.1). Despite their overlapping roles in checkpoint signaling, the biological requirements for Chk1 and Chk2 function are strikingly different, as Chk1 is essential for mammalian development and viability. Chk2 has several other important functions apart from targeting p53; so in the absence of either Chk2 or p53, the other protein remains engaged in multiple checkpoint pathways (Ref.1).
Cell cycle checkpoints are regulatory pathways that control the order and timing of cell cycle transitions and ensure that critical events such as DNA replication and chromosome segregation are completed with high fidelity. Mutations of Chks results in diverse types of sporadic human malignancies, including carcinomas of the breast, lung, vulva, urinary bladder, colon, and ovary, osteosarcomas, and lymphomas. The majority of these mutations are missense or truncation mutations, clustered in three domains of Chk2: the N-terminal SQ/TQ-rich regulatory domain, the protein-protein interaction FHA (Forkhead Associated) domain, or the C-terminal catalytic domain (Ref.1).