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 G(2)/M checkpoint induced by either UV radiation or alkylating agents. GADD45 can interact in vivo with the G(2) 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 G(2)/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 G(2)/M checkpoint. Mutants of GADD45 that lacked either the first 35 or the last 80 residues still retained an ability to induce G(2)/M arrest. A mutant with a deletion of the central region (residues 50-76), which is conserved in the family members GADD45beta and GADD45gamma, lacked such activity. This mutant also lacked an ability to bind to Cdc2, PCNA, and p21(waf1) in vivo. Consistently, either GADD45beta or GADD45gamma bind to Cdc2 in vivo. However, unlike GADD45, neither GADD45beta nor GADD45gamma inhibited the Cdc2 kinase or induced G(2)/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 G(2)/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 G(2)/M arrest, and additional activity contributed by the DEDDDR residues may be necessary to regulate the G(2)/M checkpoint.
Yang
Q
Q
Laboratory of Human Carcinogenesis, DBS, NCI, National Institutes of Health, Bethesda, Maryland 20892, USA.
Manicone
A
A
Coursen
J D
JD
Linke
S P
SP
Nagashima
M
M
Forgues
M
M
Wang
X W
XW
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D016203:CDC2 Protein Kinase / Q000378:metabolism; D042846:CDC2-CDC28 Kinases; D019926:Cyclin B / Q000378:metabolism; D056744:Cyclin B1; D050759:Cyclin-Dependent Kinase Inhibitor p21; D018844:Cyclin-Dependent Kinases / Q000037:antagonists & inhibitors; D016213:Cyclins / Q000378:metabolism; D016195:G2 Phase; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008938:Mitosis; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010449:Peptide Mapping; D017354:Point Mutation; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism; D011506:Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D000097642:GADD45 Proteins
D016203:CDC2 Protein Kinase; D042846:CDC2-CDC28 Kinases; D019926:Cyclin B; D056744:Cyclin B1; D050759:Cyclin-Dependent Kinase Inhibitor p21; D018844:Cyclin-Dependent Kinases; D016213:Cyclins; D047908:Intracellular Signaling Peptides and Proteins; D018809:Proliferating Cell Nuclear Antigen; D011506:Proteins; D000097642:GADD45 Proteins; C534699:CCNB1 protein, human; C493956:CDKN1A protein, human