A functional role for the B56 α-subunit of protein phosphatase 2A in ceramide-mediated regulation of Bcl2 phosphorylation status and function

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 a 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 a 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.