(A) CREB enhances p53 binding to the KIX domain of CBP. Purified p53 (5 pmol) was incubated with either GST alone (1 pmol) (lanes 2 and 3) or GST-KIX (aa 588 to 683) (1 pmol) (lanes 4 to 7) in the absence (−) and presence (+) of the indicated amount of purified, PKA-phosphorylated CREB. Bound p53 protein was detected by Western blot analysis. Onput protein (6%) is shown in lane 1 and protein standards (in kilodaltons) are indicated at left. (B) pCREB, but not unphosphorylated CREB, enhances p53 binding to the KIX domain of CBP. Purified p53 (5 pmol) was incubated with GST-KIX (1 pmol) in the absence or presence of the indicated amounts of either purified pCREB (lanes 3 to 5) or purified, mock-phosphorylated CREB (lanes 6 to 8). p53 (upper panel) was detected by Western blot analysis, and bound protein is indicated. The blot was reprobed using an anti-CREB antibody, and bound protein is indicated in the lower panel. Onput protein (2%) is shown in lane 1 and protein standards (in kilodaltons) are indicated at left. (C) p53 does not enhance the binding of CREB to the KIX domain of CBP. Purified, phosphorylated or mock-phosphorylated CREB (10 pmol) was incubated with GST-KIX (10 pmol) in the absence or presence of the indicated amount of purified p53 (lanes 3 to 5 and 7 to 9). CREB (upper panel) was detected by Western blot analysis. The blot was reprobed using an anti-p53 antibody, and bound protein is indicated in the lower panel. Onput protein (2%) is shown in lane 1, and protein standards (in kilodaltons) are indicated at left.

(A) Wild-type (wt) and mutant (mut) (L22Q-W23S) p53 directly interact with CREB in the absence of KIX. Purified wt or mutant p53 protein (10 pmol each) was incubated with either GST alone (10 pmol) (lanes 3 and 4), or GST-CREB-His₆ (10 pmol) (lanes 5 and 6). Bound p53 proteins were detected by Western blot analysis. Onput protein (10%) is shown in lanes 1 and 2, and protein standards (in kilodaltons) are shown at left. (B) PKA-phosphorylated or mock-phosphorylated CREB (25 pmol) was incubated with either GST alone (100 pmol) (lanes 1 and 3) or GST-p53 (100 pmol) (lanes 2 and 4). Bound CREB was detected by Western blot analysis. Output proteins (10%) (lanes 5 and 6) and protein standards (in kilodaltons) are indicated. (C) CREB (5 pmol) was incubated with either GST alone (10 pmol) (lane 2) or GST-p53 produced and purified from baculovirus-infected Sf9 cells (10 pmol) (lane 3). Bound CREB was detected by Western blot analysis. Onput protein (10%) (lane 1) and protein standards (in kilodaltons) are indicated.

Induction of apoptosis by forskolin. (A) Molt 4 cells were treated with the indicated concentrations of forskolin for 24 h. (B) H24 p53-3 cells (10) were removed from tetracycline-containing medium and immediately treated with forskolin for 24 h. Apoptotic cells (upper panels) were detected under fluorescent microscopy using YO-PRO-1. Lower panels correspond to bright-field microscopy of the cells presented in the upper panel. (C) Western blot analysis of H24 p53-3 extracts derived from cells grown in the presence (uninduced) (+) and absence (induced) (−) of tetracycline. Extracts from Molt4 cells were run in an adjacent lane for comparison. p53 protein was detected by Western blot analysis (DO-1).

(A) Forskolin induces expression of the p21 gene. Shown is the northern blot analysis of total RNA (15 μg) isolated from untreated or 20 μM forskolin-treated MCF7 cells. RNA was hybridized with a p21-specific probe or a GAPDH-specific probe as a loading control. Specific transcripts are indicated. An RNA kilobase ladder and 18S and 28S rRNAs are shown at left. (B) CREB phosphorylation correlates with p21 expression. Western blot analysis of pCREB (anti-phosphoserine 133-CREB) (upper panel), p21 (gift from J. Wade Harper) (middle panel), and p53 (DO-1) (lower panel) was performed following a time course of 20 μM forskolin treatment of MCF7 cells. Protein standards (in kilodaltons) are shown at left.

(A) A p53 mutant, defective for KIX binding, binds KIX in the presence of pCREB. Purified wild-type (wt) (10 pmol) (lanes 2 and 3) or mutant (mut) (L22Q-W23S) (10 pmol) (lanes 4 and 5) p53 was incubated with GST-KIX (2 pmol) in the absence or presence of 30 pmol of purified, phosphorylated CREB. p53 and CREB were detected simultaneously by Western blot analysis using an anti-His–anti-CREB antibody mixture. Onput wt p53 (2%) is shown in lane 1, and protein standards (in kilodaltons) are indicated at left. At the exposure presented, p53 binding to KIX was not detected. (B) The PKA-phosphorylated kinase-inducible domain of CREB does not support enhanced binding of p53 to KIX. Purified p53 (10 pmol) was incubated with GST-KIX (10 pmol) in the absence or presence of 30 pmol of phosphorylated full-length CREB (lane 2) or pKID domain of CREB (lane 3). p53 was detected by Western blot analysis. Protein standards (in kilodaltons) are indicated at left.

(A) Schematic of the CREB deletion mutants. (B) CREB deletion mutants are competent for p53 binding. The indicated CREB deletion mutants (10 pmol) were incubated with GST-p53 (25 pmol) (lanes 1 to 6). Bound CREB proteins were detected by Western blot analysis. Protein standards (in kilodaltons) are indicated. (C) The bZIP domain is sufficient for interaction with p53. The indicated amounts of either full-length or bZIP (aa 254 to 327) CREB were incubated with GST alone (100 pmol) (lanes 1 and 4) or GST-p53 (100 pmol) (lanes 2, 3, 5, and 6). Bound CREB was detected by Western blot analysis. Onput proteins (5 pmol of CREB, 10 pmol of bZIP) (lanes 7 and 8), and molecular mass standards (in kilodaltons) are indicated. (D) Anti-p53 immunoprecipitates ATF/CREB proteins from a T-cell lysate. The upper panel shows a CREB Western blot of the proteins bound to p53 immobilized using either the Ab-1 or DO-1 anti-p53 antibodies (lanes 2 and 3). Purified, recombinant CREB was electrophoresed as a control (lane 1). The lower panel shows a Western blot of p53 bound to the immobilized anti-p53 antibodies (lanes 2 and 3).

(A) Forskolin activates p53-dependent transcription in vivo. The p53-responsive pG13-Luc reporter (400 ng) was cotransfected into p53-null Jurkat T cells with the p53 expression plasmid (pC53-SN3) (400 ng), as indicated. Forskolin (20 μM) and/or H-89 (5 μM) was added (+) or not added (−) to the indicated transfection reactions. (B) Forskolin had no effect on transfected p53 protein levels. Western blot analysis of p53 derived from cells transfected with pC53-SN3 (400 ng), in the absence (−) and presence (+) of forskolin (20 μM). (C) A CREB-responsive promoter responds appropriately to forskolin and H-89. The reporter plasmid CRE-Luc (1 μg) (24) was transfected in Jurkat T cells in the presence (+) or absence (−) of forskolin (20 μM) and/or H-89 (5 μM) as indicated. (D) Forskolin activates the p53-responsive Bax promoter. The Bax-Luc reporter plasmid (400 ng) was cotransfected in Jurkat T-cells with the p53 expression plasmid (pC53-SN3) (400 ng) in the presence (+) or absence (−) of forskolin (20 μM), as indicated. (E) The amino terminus of p53 is sufficient for forskolin responsiveness. The Gal4 reporter plasmid (pGalTK-Luc) (400 ng) was cotransfected in Jurkat T cells with the Gal4-p53 expression plasmids carrying either full-length p53 (pGal4-wtp53) or the amino terminus of p53 (aa 1 to 52) (pGal4-p53N) (800 ng each) (12). Reactions were either cotransfected with the catalytic subunit of PKA (100 ng) or treated with forskolin (20 μM) as indicated. Reported values for each transient-transfection experiment are the average luminescence + the standard error (error bar) from one experiment performed in duplicate. Each experiment was performed at least twice.

Model showing the ternary complex, containing CBP, pCREB, and p53, assembled on a p53-responsive promoter.