Cleavage and degradation of Claspin during apoptosis by caspases and the proteasome

Hospital Universitario de Canarias, San Cristóbal de La Laguna, Canary Islands, Spain
Cell Death and Differentiation (Impact Factor: 8.18). 09/2007; 14(8):1433-42. DOI: 10.1038/sj.cdd.4402134
Source: PubMed


Apoptosis plays a crucial role in development and tissue homeostasis. Some key survival pathways, such as DNA damage checkpoints and DNA repair, have been described to be inactivated during apoptosis. Here, we describe the processing of the human checkpoint protein Claspin during apoptosis. We observed cleavage of Claspin into multiple fragments in vivo. In vitro cleavage with caspases 3 and 7 of various fragments of the protein, revealed cut sites near the N- and C-termini of the protein. Using mass spectrometry, we identified a novel caspase cleavage site in Claspin at Asp25. Importantly, in addition to cleavage by caspases, we observed a proteasome-dependent degradation of Claspin under apoptotic conditions, resulting in a reduction of the levels of both full-length Claspin and its cleavage products. This degradation was not dependent upon the DSGxxS phosphodegron motif required for SCF(beta-TrCP)-mediated ubiquitination of Claspin. Finally, downregulation of Claspin protein levels by short interfering RNA resulted in an increase in apoptotic induction both in the presence and absence of DNA damage. We conclude that Claspin has antiapoptotic activity and is degraded by two different pathways during apoptosis. The resulting disappearance of Claspin from the cells further promotes apoptosis.

Download full-text


Available from: Jennifer Semple, Sep 09, 2014
  • Source
    • "Primary antibodies used in this study are listed in Supplementary Table S2. The anti-Claspin antibody was a kind gift of Dr. Raimundo Freire, University of Tenerife) and was described previously (Semple et al, 2007). When indicated, a Triton X-100-insoluble (chromatin-enriched) fraction was isolated as described in Peñ a-Diaz et al (2012). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Human cells have evolved elaborate mechanisms for responding to DNA damage to maintain genome stability and prevent carcinogenesis. For instance, the cell cycle can be arrested at different stages to allow time for DNA repair. The APC/CCdh1 ubiquitin ligase mainly regulates mitotic exit but is also implicated in the DNA damage-induced G2 arrest. However, it is currently unknown whether APC/CCdh1 also contributes to DNA repair. Here, we show that Cdh1 depletion causes increased levels of genomic instability and enhanced sensitivity to DNA-damaging agents. Using an integrated proteomics and bioinformatics approach, we identify CtIP, a DNA-end resection factor, as a novel APC/CCdh1 target. CtIP interacts with Cdh1 through a conserved KEN box, mutation of which impedes ubiquitylation and downregulation of CtIP both during G1 and after DNA damage in G2. Finally, we find that abrogating the CtIP–Cdh1 interaction results in delayed CtIP clearance from DNA damage foci, increased DNA-end resection, and reduced homologous recombination efficiency. Combined, our results highlight the impact of APC/CCdh1 on the maintenance of genome integrity and show that this is, at least partially, achieved by controlling CtIP stability in a cell cycle- and DNA damage-dependent manner.
    Full-text · Article · Oct 2014 · The EMBO Journal
  • Source
    • "MCM2, MCM3, MCM4, MCM5, and Cdc45 antibodies were a gift from J. Méndez (Centro Nacional de Investigaciones Oncológicas, Madrid, Spain). Claspin, 53BP1, TopBP1, Rad9, and BLM antibodies were generated in-house and were previously described (Lee et al., 2001; Toueille et al., 2004; Semple et al., 2007; Rendtlew Danielsen et al., 2009). Selective Wee1 inhibitor II was purchased from EMD and used at 10 µM. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Correct replication of the genome and protection of its integrity are essential for cell survival. In a high-throughput screen studying H2AX phosphorylation, we identified Wee1 as a regulator of genomic stability. Wee1 down-regulation not only induced H2AX phosphorylation but also triggered a general deoxyribonucleic acid (DNA) damage response (DDR) and caused a block in DNA replication, resulting in accumulation of cells in S phase. Wee1-deficient cells showed a decrease in replication fork speed, demonstrating the involvement of Wee1 in DNA replication. Inhibiting Wee1 in cells treated with short treatment of hydroxyurea enhanced the DDR, which suggests that Wee1 specifically protects the stability of stalled replication forks. Notably, the DDR induced by depletion of Wee1 critically depends on the Mus81-Eme1 endonuclease, and we found that codepletion of Mus81 and Wee1 abrogated the S phase delay. Importantly, Wee1 and Mus81 interact in vivo, suggesting direct regulation. Altogether, these results demonstrate a novel role of Wee1 in controlling Mus81 and DNA replication in human cells.
    Full-text · Article · Aug 2011 · The Journal of Cell Biology
  • Source
    • "Our inability to observe p19 in mouse or human brain samples suggests that the usefulness of the p23 neo-epitope antibody is restricted to cells and primary neurons undergoing cellular stress. However, it should be noted that given a limited sample size, it is not particularly surprising that we did not observe the p19 fragment which may represent a transient species since it represents a caspase-cleavage product (Semple et al. 2007; Werner et al. 2007). Of note, the severe AD patients did display a consistent reduction in p23 levels. "
    [Show abstract] [Hide abstract]
    ABSTRACT: The presence of misfolded proteins elicits cellular responses including an endoplasmic reticulum (ER) stress response that may protect cells against the toxic buildup of misfolded proteins. Accumulation of these proteins in excessive amounts, however, overwhelms the "cellular quality control" system and impairs the protective mechanisms designed to promote correct folding and degrade misfolded proteins, ultimately leading to organelle dysfunction and cell death. Studies from multiple laboratories have identified the roles of several ER stress-induced cell death modulators and effectors. Earlier, we reported the role of the small co-chaperone protein p23 in preventing ER stress-induced cell death. p23 undergoes caspase-dependent cleavage to yield a 19-kD product (p19), and mutation of this caspase cleavage site not only blocks the formation of the 19-kD product but also attenuates the ER stress-induced cell death process triggered by various stressors. Thus, a critical question is whether p23 and/or p19 could serve as an in vivo marker for neurodegenerative diseases featuring misfolded proteins and cellular stress. In the present study, we used an antibody that recognizes both p23 and p19 as well as a specific neo-epitope antibody that detects only the p19 fragment. These antibodies were used to detect the presence of both these proteins in cells, primary neurons, brain samples from a mouse model of Alzheimer's disease (AD), and fixed human AD brain samples. While patients with severe AD did display a consistent reduction in p23 levels, our inability to observe p19 in mouse or human AD brain samples suggests that the usefulness of the p23 neo-epitope antibody is restricted to cells and primary neurons undergoing cellular stress.
    Preview · Article · Jun 2011 · Journal of Molecular Neuroscience
Show more