GADD45-induced cell cycle G2-M arrest associates with altered subcellular distribution of cyclin B1 and is independent of p38 kinase activity.
In response to DNA damage, the cell cycle checkpoint is an important biological event in maintaining genomic fidelity. Gadd45, a p53-regulated and DNA damage inducible protein, has recently been demonstrated to play a role in the G2-M checkpoint in response to DNA damage. In the current study, we further investigated the biochemical mechanism(s) involved in the GADD45-activated cell cycle G2-M arrest. Using the tetracycline-controlled system (tet-off), we established GADD45-inducible lines in HCT116 (wild-type p53) and Hela (inactivated p53 status) cells. Following inducible expression of the Gadd45 protein, cell growth was strongly suppressed in both HCT116 and Hela cells. Interestingly, HCT116 cells revealed a significant G2-M arrest but Hela cells failed to arrest at the G2-M phases, indicating that the GADD45-activated G2-M arrest requires normal p53 function. The GADD45-induced G2-M arrest was observed independent of p38 kinase activity. Importantly, induction of Gadd45 protein resulted in a reduction of nuclear cyclin B1 protein, whose nuclear localization is critical for the completion of G2-M transition. The reduced nuclear cyclin B1 levels correlated with inhibition of Cdc2/cyclin B1 kinase activity. Additionally, overexpression of cyclin B1 substantially abrogated the GADD45-induced cell growth suppression. Therefore, GADD45 inhibition of Cdc2 kinase activity through alteration of cyclin B1 subcellular localization may be an essential step in the GADD45-induced cell cycle G2-M arrest and growth suppression.
Jin
Shunqian
S
Department of Radiation Oncology, Cancer Institute, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, PA 15213, USA.
Tong
Tong
T
Fan
Wenhong
W
Fan
Feiyue
F
Antinore
Michael J
MJ
Zhu
Xiaocheng
X
Mazzacurati
Lucia
L
Li
Xianxing
X
Petrik
Kimberly L
KL
Rajasekaran
Baskaran
B
Wu
Min
M
Zhan
Qimin
Q
D000071877:Checkpoint Kinase 1; D019926:Cyclin B / Q000378:metabolism*; D056744:Cyclin B1; D016195:G2 Phase / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D008938:Mitosis / Q000235:genetics*; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D011506:Proteins / Q000235:genetics*; D013347:Subcellular Fractions / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000502:physiology; D048051:p38 Mitogen-Activated Protein Kinases; D000097642:GADD45 Proteins
D000071877:Checkpoint Kinase 1; D019926:Cyclin B; D056744:Cyclin B1; D047908:Intracellular Signaling Peptides and Proteins; D020928:Mitogen-Activated Protein Kinases; D011494:Protein Kinases; D011506:Proteins; D016159:Tumor Suppressor Protein p53; D048051:p38 Mitogen-Activated Protein Kinases; D000097642:GADD45 Proteins; C534699:CCNB1 protein, human