Survivin Inhibition of p34Cdc2 Via Protection of Wee1 Correlates with Survival.

The inhibitor of apoptosis protein Survivin and p34^Cdc2 (the cyclin-dependent kinase, Cdk1) are involved in cell cycle progression and apoptosis. Cdc2 and Survivin are tightly regulated in normal cells but deregulated in cancer. We have previously shown that Survivin regulates entry of hematopoietic stem cells into cell cycle. It has been established that Cdc2 phosphorylates Survivin at threonine 34 (T34), which is believed to be a stabilizing event necessary for normal Survivin function and anti-apoptotic activity. Activation of Cdc2 is required for its apoptotic activity and inhibition of the proapoptotic activity of Cdc2 can be achieved by phosphorylation at tyrosine 15 (Tyr15). Stable overexpression of a wild-type (wt) mouse Survivin-IRES-GFP construct in IL-3-dependent mouse BaF3 cells resulted in increased Tyr15 phosphorylation of Cdc2 and enhanced cell proliferation, while cells transduced with a threonine 34 to alanine (T34A) dominant negative (DN) Survivin construct showed reduced Cdc2 Tyr15 phosphorylation and accelerated apoptosis. Furthermore, when apoptosis was induced in stably transduced BaF3 cells by IL-3 withdrawal, Survivin expression and phospho-Tyr15 levels correlated with enhanced survival and decreased survival and low phospho-Tyr15 levels correlated with Survivin disruption by T34A-DN-Survivin. Since Survivin does not contain intrinsic kinase activity we sought to determine what kinase(s) could be involved in stabilization or increase in phosphoTyr-15-Cdc2 by Survivin. Since Survivin inhibits caspase 3 activity and the Cdc2-targeting kinase Wee1 is a degradation target of active caspase 3, we performed immunoblotting assays on Survivin wt and T34A-transduced BaF3 cells and showed that cells over-expressing wt Survivin contain 2-fold higher levels of Wee1 protein as compared to those expressing vector or DN-Survivin. In addition, cells overexpressing wt Survivin contain >2-fold higher intact PARP protein levels which is another target of active caspase 3, as compared to cells transduced with MIEG vector. In order to test whether mimicking wt Survivin inhibition of caspase 3 could lead to increased Cdc2-Tyr15 phosphorylation, we evaluated the specific caspase 3 inhibitor Ac-DEVD-CHO. Inhibition of caspase 3 in BaF3 cells produced an increase in Cdc2Tyr15 phosphorylation in a dose-dependent manner, with 25 mM giving >4-fold higher intact PARP and >6-fold higher phospho-Tyr15 protein levels. Taken together, these results suggest that the antiapoptotic effect of Survivin is mediated through inactivation of Cdc2 by phosphorylation on Tyr-15 as a consequence of protection of the Wee1 kinase from degradation by caspase 3.