Phosphorylation of Bcl-2 protein by CDC2 kinase during G(2)/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 thi s study, we examined whether Bcl-2 is phosphorylated by CDC2 kinase, a mast er regulator of G(2)/M transition in the eukaryotic cell cycle. When CDC2 w as activated by okadaic acid in HL-60 cells, Bcl-2 phosphorylation was read ily induced. The phosphorylation was correlated with the accumulation of ce lls in G(2)/M phases, but was not proportional to the level of apoptosis. F urthermore, 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. Usi ng synthetic peptides and mutant cell lines, we identified threonine 56, on e of two consensus sites for CDC2 within the Bcl-2 sequence, as a residue p hosphorylated by CDC2. Mutation at threonine 56 abrogated the cell cycle in hibitory effect of Bcl-2 without affecting anti-apoptotic function. These r esults suggest that two distinct functions of Bcl-2 (anti-apoptosis and cel l cycle inhibition) are differentially regulated by post-translational mech anisms such as phosphorylation. CDC2-mediated phosphorylation of Bcl-2 may play some physiological roles in the negative regulatory events during mito sis.