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. 2003 Sep;23(18):6350–6362. doi: 10.1128/MCB.23.18.6350-6362.2003

FIG. 1.

FIG. 1.

14-3-3 prevents in vitro dephosphorylation of BAD pSer112, pSer136, and pSer155, but only pSer136 is the major 14-3-3 recruitment site. (A) Lysates were prepared from NIH 3T3 cells expressing wild-type (Wt) BAD in phosphatase buffer as described previously (6) and incubated at 30°C for 20 min in the absence or presence of 25 μM R18 peptide. Dephosphorylation reactions terminated by adding SDS sample buffer and boiling for 5 min were analyzed by SDS-PAGE and Western blotting with phospho-specific antibodies to pSer112, pSer136, and pSer155 and a mouse antibody against total BAD. (B) Specificity of antiphosphoserine residues was confirmed by Western blotting of lysates from parental NIH 3T3 cells (3T3), NIH 3T3 cells expressing wild-type BAD (3T3/WT), and the 112A, 136A, and 155A mutants (3T3/112A, 3T3/136A, and 3T3/155A, respectively) with antibodies to pSer112 (upper), pSer136 (middle), and pSer155 (lower) and an antibody against total BAD (Santa Cruz; 31420). Asterisks indicate nonspecific bands. (C) Alanine substitution of Ser136 results in significant loss of 14-3-3 association with BAD. BAD was immunoprecipitated with anti-BAD C-20 antibody from NIH 3T3 or FL5.12 cells expressing wild-type BAD or BAD with alanine substitutions at one, two, or three (3A) phosphorylation sites, as indicated. The BAD immunocomplexes were separated by SDS-PAGE and immunoblotted with anti-14-3-3 antibody (upper) or anti-BAD 36420 antibody (lower). The asterisk indicates a species immunoreactive with BAD antibodies. (D) Top, phosphoserine 136 peptide displaces 14-3-3 from BAD immunocomplexes. BAD was immunoprecipitated from FL5.12 cells expressing wild-type BAD in the absence (−) or presence of 25 μM pSer112, pSer136, or pSer155 peptides (pS112p, pS136P, and pS155p, respectively) and immunoblotted for 14-3-3 and BAD as above. Bottom, phospho-Ser136 peptide blocks 14-3-3 association with phosphorylated GST-BAD. In vitro protein kinase A-phosphorylated GST-BAD was incubated with NIH 3T3 lysates in the presence of pSer112, pSer136, or pSer155 peptide, pulled down by glutathione agarose, and immunoblotted for 14-3-3 and BAD as above.

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