THE INHIBITOR-OF-APOPTOSIS PROTEIN SURVIVIN INCREASES P34CDC2 PHOSPHORYLATION AND ENHANCES CELL SURVIVAL AND PROLIFERATION BY PROTECTING THE WEE1 KINASE FROM DEGRADATION BY CASPASE-3

Abstract

The anti-apoptotic protein Survivin and the cyclin-dependent kinase p34Cdc2 are involved in cell cycle progression and apoptosis. Activation of Cdc2 is required for its pro-apoptotic activity, which can be inhibited by phosphorylation at Tyrosine-15 (Tyr15). In transduced IL-3-dependent murine BaF3 hematopoietic cells, over-expression of wild-type-(wt)-Survivin increased Cdc2-Tyr15 phosphorylation, while over-expression of a dominant-negative-(dn)-T34A-Survivin construct decreased its phosphorylation. The increased phospho-Tyr15 levels associated with ectopic Survivin directly correlated with enhanced BaF3 cell survival in the absence of growth factors, and low phospho-Tyr15 levels observed in cells expressing ectopic dn-Survivin correlated with decreased survival. BaF3 cells transduced with Internal Tandem Duplication (ITD) mutations of the Flt3 receptor that results in increased Survivin levels, also contained increased levels of Tyr15 phosphorylated Cdc2. In BaF3 cells over-expressing wt-Survivin, 2-fold higher levels of Wee1 protein were observed compared to cells expressing control vector alone. Treatment of control BaF3 cells with the caspase-3 inhibitor Ac-DEVD-CHO increased both Cdc2-Tyr15 phosphorylation and Wee1 protein levels. In a similar fashion over-expression of wt-Survivin in these cells maintained high levels of Tyr15 phosphorylated Cdc2 and Wee1 protein. In MCF7 human breast cancer cells that lack caspase-3, increase of Tyr15 phosphorylated Cdc2 and Wee1 kinase protein by caspase-3, -7 or a pan-caspase inhibitor was absent, linking Survivin and caspase-3 to the increase of Wee1 and Tyr15 phosphorylation of Cdc2. To further link Survivin and Cdc2, we treated cells with AICAR and 17-AAG that inhibit Hsp90, which is known to be required for Survivin stability. Treatment of BaF3 cells expressing wt-Survivin with AICAR and 17-AAG decreased Cdc2-Tyr15 phosphorylation compared to vehicle-treated control cells. Taken together, these results indicate that Survivin protects the Cdc2-Tyr15-targeting kinase Wee1 from degradation by caspase-3 which leads to increased inhibitory Cdc2-Tyr15 phosphorylation resulting in reduced apoptosis and enhanced survival.