CREB Binding Protein Recruitment to the Transcription Complex Requires Growth Factor–Dependent Phosphorylation of Its GF Box  Kerstin Zanger, Sally Radovick, Fredric E Wondisford  Molecular Cell  Volume 7, Issue 3, Pages 551-558 (March 2001) DOI: 10.1016/S1097-2765(01)00202-7

Figure 1 CBP(312-440) Is Sufficient for Growth Factor–Dependent Activation of the AP-1 Promoter (A) Schematic of CBP indicating protein binding to specific domains (NR, nuclear receptor; NCoA, nuclear coactivator). (B) An avidin-biotin complex DNA binding assay (ABCD assay) was performed using a double-stranded biotinylated AP-1 site and whole cell extract (WCE) from nontransfected HepG2 cells, which were nontreated or treated with insulin or EGF. Full-length CBP and p300 were detected by Western blot analysis using an anti-CBP antibody (CBP A-22; Santa Cruz) and an anti-p300 antibody (p300 N-15; Santa Cruz), respectively. (C) Full-length CBP was detected in WCE from nontransfected CV-1 cells, which were nontreated or treated with insulin or EGF (12), using an anti-CBP antibody (CBP A-22; Santa Cruz) and Western blot analysis (top panel). CV-1 cells were transfected with wt CBP, CBP deletion constructs, or wt p300, AP-1 response element (middle panel), or CRE response element (bottom panel) containing reporters and were stimulated with insulin, EGF, or PDGF, respectively. (D) The transactivation domain of CBP is located within the 312–440 region, as determined by GAL4-CBP deletion constructs tested on a heterologous reporter (UAS). (E) EGF-mediated activation of the AP-1 promoter by GAL4-CBP(312–440) was inhibited by Wortmannin (W; 10 nM) and staurosporine (S; 1 nM), but not by GÖ 6976 (G; 50 nM); in contrast, the 8-bromo cAMP (cAMP)-dependent activation of a CRE by GAL4-CBP(1677-2441) was unaffected by the inhibitors Molecular Cell 2001 7, 551-558DOI: (10.1016/S1097-2765(01)00202-7)

Figure 2 Activation of the AP-1 Complex by CBP(312-440) Is Inhibited by Mutation of Putative PKC Sites within the GF Box (A) CBP (312–440) wt, CBP(312–440) S436A (m1), CBP(312–440) S317A (m2), or CBP(312–440) S436/S317A (m3) activated the AP-1 reporter 1- to 2-fold in the absence of growth factors (left panel) and the UAS reporter 20-fold (right panel). (B) m1, m2, and m3 blocked the activation of the AP-1 reporter by EGF (left panel); however, their intrinsic activity on a UAS reporter was not impaired (right panel) Molecular Cell 2001 7, 551-558DOI: (10.1016/S1097-2765(01)00202-7)

Figure 3 Mutations within the GF Box Block CBP Recruitment to the AP-1 Complex (A) Mammalian two-hybrid system with GAL4-c-jun (top panel) or GAL4-c-jun mutant S63/73A (bottom panel) indicated that intact phosphorylation sites of both CBP(312-440) and c-Jun were crucial for recruitment to DNA after growth factor–dependent stimulation. (B) Western blot analysis using an anti-FLAG M2 antibody (Sigma, St. Louis, MO) showed that VP16-wt and VP16-m3 are expressed at comparable levels in untreated or EGF-treated cells. (C) An ABCD assay was performed as in Figure 1B but with transfected FLAG-tagged VP16-wt or VP16-m3. After EGF or insulin stimulation, VP16-wt, but not the VP16-m3 mutant, bound to the AP-1 site. VP16-P300 (aa 297-425) bound to the AP-1 site in a growth factor–independent manner. Western blot analysis was performed with an anti-FLAG antibody Molecular Cell 2001 7, 551-558DOI: (10.1016/S1097-2765(01)00202-7)

Figure 4 Mutation of PKC Sites in the GF Box Blocks CBP Phosphorylation by Growth Factors (A) WCE from CBP 1-1891 and CBP 1-1891 m1 cotransfected cells was immunoprecipited with an anti-CBP antibody (CBP A-22; Santa Cruz) or an anti-p300 antibody (p300 N-15; Santa Cruz). Western blot analysis was performed with either an anti-CBP antibody (CBP A-22; Santa Cruz)/anti-p300 antibody (p300 N-15; Santa Cruz) (left panels) or an anti-phosho-serine antibody (anti-phospho-serine; Zymed) (right panels). (B) WCEs from full-length CBP wt and m1 mutant transfected and 32P-labeled cells were immunoprecipitated with an anti-CBP antibody (CBP A-22; Santa Cruz) before or after growth factor treatment. EGF and insulin phosphorylated only CBP wt but not the phosphorylation mutant m1 (top panel). On very long exposures, weak growth factor–dependent phosphorylation of endogenous CBP (left three lanes) was noted (data not shown). Western blot analysis with an anti-CBP antibody detected equal amounts in WCE of transfected CBP wt and m1 (middle panel). ABCD assay performed as in Figure 1B but with cotransfected full-length CBP wt and the m1 mutant (bottom panel). Only CBP wt bound to the AP-1 complex after growth factor treatment Molecular Cell 2001 7, 551-558DOI: (10.1016/S1097-2765(01)00202-7)

Figure 5 Replacement of S436 with Alanine in Full-Length CBP Blocks AP-1 and Pit-1-Dependent Gene Activation (A) Introduction of S436A (m1) into full-length CBP blocked growth factor–dependent activation of an AP-1 reporter as well as activation of the prolactin promoter in the presence of cotransfected Pit-1. Transfection of Pit-1 or CBP wt alone did not lead to significant transactivation of the reporter. (B) Transfection of the m1 mutant led to full activity when compared with wt CBP on a CRE reporter after 8-bromo cAMP stimulation, and on an estrogen response element (ERE) reporter after treatment with E2 alone or E2 plus insulin, respectively. (C) Model of transcriptional regulation of AP-1 and Pit-1 responsive promoters by different growth factor signaling pathways. Phosphorylation of CBP is required for activation of these promoters. Phosphorylation of Pit-1 is shown in this model but its role in signaling on a Pit-1 response element remains to be clarified Molecular Cell 2001 7, 551-558DOI: (10.1016/S1097-2765(01)00202-7)