Phosphorylation of the pro-apoptotic protein BAD on serine 155, a novel site, contributes to cell survival

AbstractPhosphorylation of BAD, a pro-apoptotic member of the Bcl-2 protein family, on either Ser112 or Ser136 is thought to be necessary and sufficient for growth factors to promote cell survival. Here we report that Ser155, a site phosphorylated by protein kinase A (PKA), also contributes to cell survival. Ser112 is thought to be the critical PKA target, but we found that BAD fusion proteins containing Ala at Ser112 (S112A) or Ser136 (S136A) or at both positions (S112/136A) were still heavily phosphorylated by PKA in an in vitro kinase assay. BAD became insensitive to phosphorylation by PKA only when both Ser112 and Ser136, or all three serines (S112/136/155) were mutated to alanine. In HEK293 cells, BAD fusion proteins mutated at Ser155 were refractory to phosphorylation induced by elevation of cyclic AMP(cAMP) levels. Phosphorylation of the S112/136A mutant was >90% inhibited by H89, a PKA inhibitor. The S155A mutant induced more apoptosis than the wild-type protein in serum-maintained CHO-K1 cells, and apoptosis induced by the S112/136A mutant was potentiated by serum withdrawal. These data suggest that Ser155 is a major site of phosphorylation by PKA and serum-induced kinases. Like Ser112 and Ser136, phosphorylation of Ser155 contributes to the cancellation of the pro-apoptotic function of BAD

Differential phosphoprotein labeling (DIPPL), a method for comparing live cell phosphoproteomes using simultaneous analysis of P-33- and P-32- labeled proteins

We developed a differential method to reveal kinase-specific phosphorylation events in live cells. In this method, cells in which the specified kinase is inactive are labeled with 32Pi, whereas cells in which the kinase is active are labeled with 33Pi. The two cell extracts are then mixed, and proteins are separated on a single two-dimensional gel. The dried gel is exposed twice. The first exposure reveals both 32P- and 33P-labeled proteins; the kinase-specific spots are revealed because of 33P labeling. The second exposure is conducted with two acetate sheets intervening between the gel and the detection plate. This maneuver screens out the less energetic 33P-labeled proteins while allowing the more energetic 32P-labeled proteins to be detected, thus leaving only those spots that were phosphorylated independently of the specified kinase. We demonstrate the utility of this method for detecting kinase substrates in rare tissue by focusing on extracellular signal-regulated kinase-specific phosphorylation of stathmin/OP18 in primary rat sympathetic neurons