Identification of a Functional Domain in a GADD45-mediated G2/M Checkpoint

Cell cycle checkpoints are essential for the maintenance of genomic stability in response to DNA damage. We demonstrated recently that GADD45, a DNA damage-inducible protein, activates a G2/M checkpoint induced by either UV radiation or alkylating agents. GADD45 can interact in vivowith the G2 cell...

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Bibliographic Details
Published in:The Journal of biological chemistry 2000-11, Vol.275 (47), p.36892-36898
Main Authors: Yang, Qin, Manicone, Anne, Coursen, Jill D., Linke, Steven P., Nagashima, Makoto, Forgues, Marshonna, Wang, Xin Wei
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Amino Acid Substitution
CDC2 Protein Kinase - metabolism
CDC2-CDC28 Kinases
Cyclin B - metabolism
Cyclin B1
Cyclin-Dependent Kinase Inhibitor p21
Cyclin-Dependent Kinases - antagonists & inhibitors
Cyclins - metabolism
G2 Phase
GADD45 Proteins
Humans
Intracellular Signaling Peptides and Proteins
Mitosis
Molecular Sequence Data
Mutagenesis, Site-Directed
Peptide Mapping
Point Mutation
Proliferating Cell Nuclear Antigen - metabolism
Proteins - chemistry
Proteins - genetics
Proteins - metabolism
Tumor Cells, Cultured
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Summary:Cell cycle checkpoints are essential for the maintenance of genomic stability in response to DNA damage. We demonstrated recently that GADD45, a DNA damage-inducible protein, activates a G2/M checkpoint induced by either UV radiation or alkylating agents. GADD45 can interact in vivowith the G2 cell cycle-specific kinase, Cdc2, proliferating cell nuclear antigen (PCNA), and the cell cycle kinase inhibitor p21 waf1 . The ability of GADD45 to induce a G2/M arrest may be caused in part by the inhibition of Cdc2 kinase activity. Here, we report the identification of a region of GADD45 that is involved in this G2/M checkpoint. Mutants of GADD45 that lacked either the first 35 or the last 80 residues still retained an ability to induce G2/M arrest. A mutant with a deletion of the central region (residues 50–76), which is conserved in the family members GADD45β and GADD45γ, lacked such activity. This mutant also lacked an ability to bind to Cdc2, PCNA, and p21 waf1in vivo. Consistently, either GADD45β or GADD45γ bind to Cdc2 in vivo. However, unlike GADD45, neither GADD45β nor GADD45γ inhibited the Cdc2 kinase or induced G2/M arrest. The unique effect of GADD45 may be caused by the presence of a region containing DEDDDR residues. Alanine substitutions in the region abolished GADD45 induction of a G2/M arrest and its inactivation of the Cdc2 kinase but not its binding to Cdc2, PCNA, or p21 waf1 . Therefore, the binding of GADD45 to Cdc2 was insufficient to induce a G2/M arrest, and additional activity contributed by the DEDDDR residues may be necessary to regulate the G2/M checkpoint.
ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.M005319200