Mutation of BAD within the BH3 domain impairs its phosphorylation-mediated regulation.
Pro-apoptotic functions of the BH3-only protein BAD are negatively regulated by survival signal-mediated phosphorylation at several serine residues. Recently, we found that the mutant BAD (BADD119G) with an amino acid substitution of Asp (Asp119 to Gly) within the BH3 domain displays strong pro-apoptotic activity in serum-starved COS-7 cells, although it cannot interact with Bcl-2. Here, we demonstrate that the BADD119G loses phosphorylation-mediated negative regulation. Importantly, pro-apoptotic activity of wild-type BAD (BADwt) was strongly suppressed by co-transfection with constitutively active Akt (CA-Akt) cDNA, whereas that of BADD119G was not. In these transfectants, BADD119G phosphorylation was barely detectable at serine residues (S75 and S99), although BADwt phosphorylation was clearly increased by CA-Akt. In addition, various external stimuli UV, TPA and forskolin could not phosphorylate BADD119G neither at S75, S99 nor S118 in COS-7 cells. However, in vitro kinase assay revealed that catalytic protein kinase A (PKA) strongly phosphorylated both BADs at S75 and S118, excluding the possibility that the target sequence of PKA was disrupted by mutation at S119. Furthermore, as a result of disrupted phosphorylation, BADD119G could not physically interact with 14-3-3. Taken together, disruption of phosphorylation-mediated negative regulation may explain, at least in part, the strong pro-apoptotic functions of BADD119G, and suggest a role for the BH3 domain in phosphorylation events.
Adachi
M
M
Graduate School of Medicine, Sapporo Medical University, Sapporo, Japan. adachi@sapmed.ac.jp
Zhang
Yu Bin
YB
Imai
K
K
D048948:14-3-3 Proteins; D000818:Animals; D017209:Apoptosis; D019556:COS Cells; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D006801:Humans; D009154:Mutation; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D014446:Tyrosine 3-Monooxygenase / Q000378:metabolism; D051025:bcl-Associated Death Protein
D048948:14-3-3 Proteins; C494819:BAD protein, human; D002352:Carrier Proteins; D051025:bcl-Associated Death Protein; D014446:Tyrosine 3-Monooxygenase