Phosphorylation of Bcl-2 protein by CDC2 kinase during G2/M phases and its role in cell cycle regulation.
Although it has been reported that Bcl-2 phosphorylation is associated with certain types of apoptosis, there is much controversy over the functional significance of and the kinases responsible for the phosphorylation. In this study, we examined whether Bcl-2 is phosphorylated by CDC2 kinase, a master regulator of G(2)/M transition in the eukaryotic cell cycle. When CDC2 was activated by okadaic acid in HL-60 cells, Bcl-2 phosphorylation was readily induced. The phosphorylation was correlated with the accumulation of cells in G(2)/M phases, but was not proportional to the level of apoptosis. Furthermore, we found that Bcl-2 was phosphorylated during G(2)/M phases of normal cell cycle. The ability of CDC2 to phosphorylate Bcl-2 was confirmed by in vitro kinase assay with a highly purified CDC2-cyclin B complex. Using synthetic peptides and mutant cell lines, we identified threonine 56, one of two consensus sites for CDC2 within the Bcl-2 sequence, as a residue phosphorylated by CDC2. Mutation at threonine 56 abrogated the cell cycle inhibitory effect of Bcl-2 without affecting anti-apoptotic function. These results suggest that two distinct functions of Bcl-2 (anti-apoptosis and cell cycle inhibition) are differentially regulated by post-translational mechanisms such as phosphorylation. CDC2-mediated phosphorylation of Bcl-2 may play some physiological roles in the negative regulatory events during mitosis.
Furukawa
Y
Y
Division of Molecular Hematopoiesis, Center for Molecular Medicine, the Department of Hematology, Jichi Medical School, Tochigi 329-0498, Japan. furuyu@jichi.ac.jp
Iwase
S
S
Kikuchi
J
J
Terui
Y
Y
Nakamura
M
M
Yamada
H
H
Kano
Y
Y
Matsuda
M
M
D019943:Amino Acid Substitution; D016203:CDC2 Protein Kinase / Q000378:metabolism*; D002453:Cell Cycle / Q000502:physiology*; D019926:Cyclin B / Q000378:metabolism; D016195:G2 Phase; D019254:Genes, bcl-2; D018922:HL-60 Cells; D006706:Homeostasis; D006801:Humans; D007700:Kinetics; D008938:Mitosis; D016297:Mutagenesis, Site-Directed; D019319:Okadaic Acid / Q000494:pharmacology; D010446:Peptide Fragments / Q000737:chemistry; D010748:Phosphopeptides / Q000737:chemistry; D010766:Phosphorylation; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013912:Threonine; D014162:Transfection
D019926:Cyclin B; D010446:Peptide Fragments; D010748:Phosphopeptides; D019253:Proto-Oncogene Proteins c-bcl-2; D011994:Recombinant Proteins; D019319:Okadaic Acid; D013912:Threonine; D016203:CDC2 Protein Kinase