A Functional Role for the B56 α-Subunit of Protein Phosphatase 2A in Ceramide-mediated Regulation of Bcl2 Phosphorylation Status and Function

Peter P Ruvolo,Warren Clark,Marc Mumby,Fengqin Gao,W Stratford May

doi:10.1074/jbc.m201830200

Abstract

Recently it has been shown that the potent apoptotic agent ceramide activates a mitochondrial protein phosphatase 2A (PP2A) and promotes dephosphorylation of the anti-apoptotic molecule Bcl2 (Ruvolo, P. P., Deng, X., Ito, T., Carr, B. K., and May, W. S. (1999) J. Biol. Chem. 274, 20296-20300). In cells expressing Bcl2, dephosphorylation of Bcl2 appears to be required for ceramide-induced cell death because treatment of cells with low doses of the PP2A inhibitor okadaic acid blocks Bcl2 dephosphorylation and promotes cell survival. Furthermore, the non-phosphorylatable (i.e. PP2A-resistant) gain-of-function S70E mutant Bcl2 can protect cells from ceramide-induced apoptosis. These findings support a model whereby Bcl2 function is regulated by PP2A. PP2A is a heterotrimer that contains a catalytic C-subunit, a structural A-subunit, and a regulatory B-subunit. The A- and C-subunits are fairly conserved and ubiquitously expressed, and they form the catalytic complex of the phosphatase. In contrast, there are at least three families of diverse B-subunit molecules that vary in expression temporally and by tissue type. It is hypothesized that ceramide regulates PP2A via the B-subunit. Thus, understanding the mechanism of how PP2A regulates Bcl2 phosphorylation status and how ceramide might regulate this process requires identification of the regulatory B-subunit of PP2A that comprises the Bcl2 phosphatase. Results indicate that the B56 alpha-subunit is a candidate regulatory subunit of the physiologic Bcl2 phosphatase since (a) B56 alpha associates with Bcl2 as evidenced by pull-down experiments, (b) B56 alpha co-localizes with Bcl2 in mitochondrial membranes, (c) ceramide promotes translocation of B56 alpha to mitochondrial membranes, and (d) overexpression of B56 alpha promotes mitochondrial PP2A activity and Bcl2 dephosphorylation and potentiates cell killing with ceramide. These findings suggest a role for B56 alpha in regulating the Bcl2 phosphatase.

Highlights

Introduction ofB56 ␣ into HL60 cells resulted in reduced levels of basal Bcl2 phosphorylation
Consistent with previous findings correlating Bcl2 phosphorylation status and sensitivity to ceramide in HL60 cells [12], the B56 ␣ transfectants were more sensitive to stress induced by ceramide treatment compared with parental cells
These results strongly suggest that overexpression of B56 ␣ in HL60 cells promotes mitochondrial phosphatase 2A (PP2A) activity resulting in reduced levels of phosphorylated Bcl2, which leads to greater chemosensitivity

Summary

Introduction

It has been shown that the potent apoptotic agent ceramide activates a mitochondrial protein phosphatase 2A (PP2A) and promotes dephosphorylation of the anti-apoptotic molecule Bcl2 Results indicate that the B56 ␣-subunit is a candidate regulatory subunit of the physiologic Bcl2 phosphatase since (a) B56 ␣ associates with Bcl2 as evidenced by pull-down experiments, (b) B56 ␣ co-localizes with Bcl2 in mitochondrial membranes, (c) ceramide promotes translocation of B56 ␣ to mitochondrial membranes, and (d) overexpression of B56 ␣ promotes mitochondrial PP2A activity and Bcl2 dephosphorylation and potentiates cell killing with ceramide.

Results

Conclusion