Regulation of BAD by cAMP-dependent protein kinase is mediated via phosphorylation of a novel site, Ser155

The interaction of BAD (Bcl-2/Bcl-X_L-antagonist, causing cell death) with Bcl-2/Bcl-X_L is thought to neutralize the anti-apoptotic effects of the latter proteins, and may represent one of the mechanisms by which BAD promotes apoptosis. A variety of survival signals are reported to induce the phosphorylation of BAD at Ser¹¹² or Ser¹³⁶, triggering its dissociation from Bcl-2/Bcl-X_L. Ser¹³⁶ is thought to be phosphorylated by protein kinase B (PKB, also called Akt), which is activated when cells are exposed to agonists that stimulate phosphatidylinositol 3-kinase (PI3K). In contrast, Ser¹¹² is reported to be phosphorylated by mitogen-activated protein (MAP) kinase-activated protein kinase-1 (MAPKAP-K1, also called RSK) and by cAMP-dependent protein kinase (PKA). Here we identify Ser¹⁵⁵ as a third phosphorylation site on BAD. We find that Ser¹⁵⁵ is phosphorylated preferentially by PKA in vitro and is the only residue in BAD that becomes phosphorylated when cells are exposed to cAMP-elevating agents. The phosphorylation of BAD at Ser¹⁵⁵ prevents it from binding to Bcl-X_L and promotes its interaction with 14-3-3 proteins. We also provide further evidence that MAPKAP-K1 mediates the phosphorylation of Ser¹¹² in response to agonists that activate the classical MAP kinase pathway. However insulin-like growth factor 1, a potent activator of PI3K and PKB does not increase the phosphorylation of Ser¹³⁶ in BAD-transfected HEK-293 cells, and nor is the basal level of Ser¹³⁶ phosphorylation suppressed by inhibitors of PI3K.