ABSTRACT
Chk1 protein kinase plays a critical role in checkpoints that restrict progression through the cell cycle if DNA replication has not been completed or DNA damage has been sustained. ATR-dependent activation of Chk1 is mediated by Claspin. Phosphorylation of Claspin at two sites (Thr916 and Ser945 in humans) in response to DNA replication arrest or DNA damage recruits Chk1 to Claspin. Chk1 is subsequently phosphorylated by ATR and fully activated to control cell cycle progression. We show that ablation of Chk1 by siRNA in human cells or its genetic deletion in chicken DT40 cells does not prevent phosphorylation of Claspin at Thr916 (Ser911 in chicken). Chk1, however, does play other roles, possibly indirect, in the phosphorylation of Claspin and its induction. These results demonstrate that phosphorylation of Claspin within the Chk1-binding domain is catalysed by an ATR-dependent kinase distinct from Chk1.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , DNA Damage , DNA Replication , Protein Kinases/metabolism , Animals , Ataxia Telangiectasia Mutated Proteins , Catalysis , Cell Cycle Proteins/metabolism , Cell Line , Checkpoint Kinase 1 , Chickens/genetics , Chickens/metabolism , DNA/drug effects , DNA/radiation effects , DNA Replication/drug effects , DNA Replication/radiation effects , Humans , Phosphorylation , Protein Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Protein Structure, Tertiary , RNA, Small Interfering/pharmacology , Threonine/metabolismABSTRACT
Claspin is involved in ATR-dependent activation of Chk1 during DNA replication and in response to DNA damage. We show that degradation of Claspin by the ubiquitin-proteosome pathway is regulated during the cell cycle. Claspin is stabilized in S-phase but is abruptly degraded in mitosis and is absent from early G(1) cells in which the phosphorylation of Chk1 by ATR is abrogated. In response to hydroxyurea, UV or aphidicolin, Claspin is phosphorylated in the Chk1-binding domain and its protein levels are increased in an ATR-dependent manner. Thus, the Chk1 pathway is regulated through both phosphorylation of Claspin and its controlled degradation.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Cell Cycle Proteins/metabolism , G1 Phase , Proteasome Endopeptidase Complex/metabolism , Protein Serine-Threonine Kinases/metabolism , S Phase , Ubiquitin/metabolism , Antineoplastic Agents/pharmacology , Aphidicolin/pharmacology , Ataxia Telangiectasia Mutated Proteins , Cell Line, Tumor , Checkpoint Kinase 1 , Enzyme Inhibitors/pharmacology , G1 Phase/drug effects , G1 Phase/radiation effects , Humans , Hydroxyurea/pharmacology , Mitosis/drug effects , Mitosis/radiation effects , Phosphorylation/drug effects , Phosphorylation/radiation effects , Protein Kinases/metabolism , Protein Processing, Post-Translational/drug effects , Protein Processing, Post-Translational/radiation effects , S Phase/drug effects , S Phase/radiation effects , Signal Transduction/drug effects , Signal Transduction/radiation effects , Ultraviolet RaysABSTRACT
Claspin is required for the phosphorylation and activation of the Chk1 protein kinase by ATR during DNA replication and in response to DNA damage. This checkpoint pathway plays a critical role in the resistance of cells to genotoxic stress. Here, we show that human Claspin is cleaved by caspase-7 during the initiation of apoptosis. In cells, induction of DNA damage by etoposide at first produced rapid phosphorylation of Chk1 at a site targeted by ATR. Subsequently, etoposide caused activation of caspase-7, cleavage of Claspin, and dephosphorylation of Chk1. In apoptotic cell extracts, Claspin was cleaved by caspase-7 at a single aspartate residue into a large N-terminal fragment and a smaller C-terminal fragment that contain different functional domains. The large N-terminal fragment was heavily phosphorylated in a human cell-free system in response to double-stranded DNA oligonucleotides, and this fragment retained Chk1 binding activity. In contrast, the smaller C-terminal fragment did not bind Chk1, but did associate with DNA and inhibited the DNA-dependent phosphorylation of Chk1 associated with its activation. These results indicate that cleavage of Claspin by caspase-7 inactivates the Chk1 signaling pathway. This mechanism may regulate the balance between cell cycle arrest and induction of apoptosis during the response to genotoxic stress.
