HCLK2 is essential for the mammalian S-phase checkpoint and impacts on Chk1 stability

Abstract

Here, we show that the human homologue of the Caenorhabditis elegans biological clock protein CLK-2 (HCLK2) associates with the S-phase checkpoint components ATR, ATRIP, claspin and Chk1. Consistent with a critical role in the S-phase checkpoint, HCLK2-depleted cells accumulate spontaneous DNA damage in S-phase, exhibit radio-resistant DNA synthesis, are impaired for damage-induced monoubiquitination of FANCD2 and fail to recruit FANCD2 and Rad51 (critical components of the Fanconi anaemia and homologous recombination pathways, respectively) to sites of replication stress. Although Thr 68 phosphorylation of the checkpoint effector kinase Chk2 remains intact in the absence of HCLK2, claspin phosphorylation and degradation of the checkpoint phosphatase Cdc25A are compromised following replication stress as a result of accelerated Chk1 degradation. ATR phosphorylation is known to both activate Chk1 and target it for proteolytic degradation, and depleting ATR or mutation of Chk1 at Ser 345 restored Chk1 protein levels in HCLK2-depleted cells. We conclude that HCLK2 promotes activation of the S-phase checkpoint and downstream repair responses by preventing unscheduled Chk1 degradation by the proteasome.The DNA damage response (DDR) is a complex process involving the orchestration of highly specialized cell-cycle checkpoints that need to be rapidly activated following the detection of damaged DNA. Each of these signalling cascades involves several unique and overlapping factors -classified as sensors, mediators, transducers and effectors -that ultimately lead to the spatio-temporal assembly of multi-protein complexes at the site of damage 1,2 . The functional importance of checkpoints in maintaining genome stability is highlighted by their conservation throughout eukaryotes. As such, defective checkpoint pathways in human cells leads to the accumulation of aberrant DNA and an increased risk of cancer progression, evident from the many human disease syndromes that result from defects in checkpoint factors 3,4 . Therefore, it is important to understand these complex mechanisms at the molecular level to further our knowledge of cancer progression and treatment.Checkpoints operate at the G1-S, S and G2-M boundaries of the cell cycle and are controlled by the ATM-Chk2 and ATR-Chk1 pathways. The S-phase checkpoint, which is under the control of the ATR-Chk1 pathway, has an essential role in maintaining replication-fork integrity during normal S-phase by preventing the collapse of stalled replication forks. The S-phase checkpoint also coordinates cell-cycle arrest and subsequent DNA-repair events when the replication fork encounters DNA damage 5,3,6 . Activation and recruitment of the Fanconi anaemia and homologous recombination-repair pathways to sites of replication stress requires an intact S-phase checkpoint 7 .Recent work in mouse models has highlighted a surprising, yet highly important link between biological clock proteins and the DDR 8,9 . It has been shown that two clock proteins, Per1 and Prd4, have integral roles...

Loss of autophagy causes a synthetic lethal deficiency in DNA repair

“…Previous studies have shown that Chk1 is degraded by the ubiquitinproteasome system and that this can be stimulated by phosphorylation of the protein at S345 (18)(19)(20)(21)(22). Because we had observed a decrease in phospho-Chk1 at earlier time points after Atg7 recombination, we reasoned this may be due to enhanced degradation of the protein, which over time would then have an impact on the total pool of Chk1 under conditions where DNA-damage signaling would be enhanced after a prolonged loss of autophagy.…”

Tti1 and Tel2 Are Critical Factors in Mammalian Target of Rapamycin Complex Assembly

“…In Caenorhabditis elegans, hypomorphic mutants of the Tel2 ortholog (Clk-2/Rad-5) were also reported to show extended life span and hypersensitivity to UV irradiation (29,30). Subsequent studies showed that Tel2 is essential for growth (31), and its homologs are important for DNA replication checkpoint in S. pombe, C. elegans, and mammalian cells (32)(33)(34). Recently, it was reported that mammalian Tel2 interacts with and regulates the stability of all PIKK family proteins (19).…”

Differential regulation of the PIKK kinase family by Tel2

“…The discovery that S. pombe and mammalian Tel2 interact directly with all phosphatidylinositol 3-kinase related protein kinase (PIKK) family members (which include ATM, ATR, DNA-PKcs, mTOR, FRAP and SMG1) raised the possibility that Tel2 may act as a universal regulator of the PIKK family of kinases. [3][4][5] A recent study has shown that conditional loss of Tel2 in mouse embryonic fibroblast (MEF) cells results in the dramatic reduction in protein levels of all PIKK family members. 5 This finding led de Lange and colleagues to propose that Tel2 is a highly conserved regulator of PIKK stability.…”

“…Subsequent studies in many organisms have demonstrated a conserved role for Tel2 in the DNA damage checkpoint response. [2][3][4][5] The absence of recognizable domains in Tel2 has been a major hindrance in understanding how it is able to operate at the molecular level. The discovery that S. pombe and mammalian Tel2 interact directly with all phosphatidylinositol 3-kinase related protein kinase (PIKK) family members (which include ATM, ATR, DNA-PKcs, mTOR, FRAP and SMG1) raised the possibility that Tel2 may act as a universal regulator of the PIKK family of kinases.…”

“…However, the Tel2 equivalents in fission yeast, worms and mammalian cells interact with ATR (Yeast Mec1) and are essential for its function. [3][4][5] It is notable that these studies analyzed deletion, depletion or conditional alleles that eliminate the entire Tel2 protein. Where the current study differs is in the use of the nonessential allele of Tel2 (tel2-1), which expresses the entire Tel2 protein with a single point mutant at Ser129 that compromises the Tel1 and Mec1 interaction site.…”