Phosphatases and regulation of cell death

Reversible phosphorylation, catalyzed by kinases and phosphatases, regulates many apoptosis molecules. Phosphorylation inactivates, whereas dephosphorylation activates, the pro-apoptotic functions of select apoptotic regulators, including the BCL2 family, such as BAD, and transcription factors such as FOXOs. The apoptotic function of the BH3 molecule BAD is exquisitely regulated by phosphorylation. Although phosphorylated BAD is sequestered in the cytosol, dephosphorylated BAD translocates to the mitochondria and inactivates BCL-xL and BCL2. Analogously, Akt-phosphorylated FOXO1 is cytosolic and inactive as a transcription factor, but dephosphorylated FOXO1 translocates to the nucleus, where it regulates the expression of pro-apoptotic Bim and cell cycle inhibitors. By use of inhibitor experiments and a combination of immunoprecipitations and tagged pull-downs in interaction studies, we identified PP2A enzymes as BAD and FOXO1 phosphatases. PP2A dephosphorylation of BAD is regulated by competitive interaction of 14-3-3, PP2A, and BAD. On survival factor deprivation, PP2A dephosphorylation of pSer112 plays the gatekeeper role for subsequent dephosphorylation at pSer136 and pSer155 by multiple phosphatases. In contrast, PP2A and 14-3-3 can interact with FOXO1 concomitantly, but PP2A dephosphorylates the pThr24 and pSer256 only once 14-3-3 dissociates. Functional assays of cell death, Bim upregulation by FOXO1, and FOXO1 nuclear translocation in the presence of phosphatase inhibitors and phosphatase siRNAs revealed the physiologic significance of PP2A activity on BAD and FOXO1. Demonstrating the role of PP2A in regulating the function of two very different cell death molecules, a BH3 protein and a transcription factor, suggests that activation of pro-apoptotic factors by protein phosphatases may be a general regulatory mechanism in apoptosis.