Phosphorylation of NF-κB p65 by PKA Stimulates Transcriptional Activity by Promoting a Novel Bivalent Interaction with the Coactivator CBP/p300  Haihong Zhong, Reinhard E Voll, Sankar Ghosh  Molecular Cell  Volume 1, Issue 5, Pages 661-671 (April 1998) DOI: 10.1016/S1097-2765(00)80066-0

Figure 1 Stimulation of NF-κB–Dependent Transcription by CBP Is Dependent upon Phosphorylation of p65 by PKA (A) Jurkat cells were transfected with the NF-κB–dependent reporter plasmid pBIIx-Luc and the indicated constructs, using lipofectamine (GIBCO). The p65 and PKA mutants have been described previously (Zhong et al. 1997). The cells were harvested 36 hr after transfection, and the extracts were used to assay for luciferase activity. (B) Hela cells were transfected with pBIIx-Luc, p65, and CBP as indicated, and the cells were treated 6 hr before harvesting with indicated concentrations of H-89 or ML-7 (Zhong et al. 1997). Luciferase activity in the extracts was measured as in (A). Molecular Cell 1998 1, 661-671DOI: (10.1016/S1097-2765(00)80066-0)

Figure 2 Association of CBP/p300 with NF-κB Is Dependent upon Phosphorylation of p65 by PKA (A) The association of p65 with CBP/p300 in vivo was examined by stimulating 70Z/3 cells with LPS. Nuclear extracts from cells stimulated for the indicated periods of time were then immunoprecipitated with antibodies against p65. The immunoprecipitates were fractionated on SDS-PAGE and analyzed by immunoblotting with antibodies against CBP (left) or p300 (right). (B) Association of p65 and CBP is influenced by PKA. The CBP and p65 constructs were transfected into COS cells as indicated. The cells were harvested 24 hr after transfection, and immunoprecipitations were carried out on extracts using antibodies against p65. The immunoprecipitates were analyzed by immunoblotting with antibodies against CBP. (C) The association of p65 and CBP depends upon phosphorylation of p65 at serine 276. Flu-tagged p65 constructs, either WT or mutants, were transfected into COS cells with CBP and PKA. The extracts were subjected to immunoprecipitation with antibodies against CBP (left) or flu epitope (right) 24 hr after transfection. The immunoprecipitates were fractionated on SDS-PAGE and immunoblotted with anti-flu monoclonal antibodies (left) or anti-CBP antibodies (right). Molecular Cell 1998 1, 661-671DOI: (10.1016/S1097-2765(00)80066-0)

Figure 3 Phosphorylation-Independent and Phosphorylation-Dependent Bivalent Interaction between NF-κB p65 and CBP (A) Schematic outlines of CBP and p65 with different sites of truncations or mutations in the various contructs used in this and subsequent experiments are indicated. (B) Truncated forms of CBP, generated by restriction enzyme digestions (A), were translated in vitro using rabbit reticulocyte lysates. The 35S-labeled proteins produced were analyzed on SDS-PAGE (lanes 1–7) and helped determine the amount of protein used as input. 2 μl of the in vitro translated proteins were mixed with GST proteins fused to the C-terminal portion (amino acids 314–550) (lanes 8–14), the N-terminal portion (amino acids 1–313) of WT p65 (lanes 15-21 and 22-28), or S276A mutant p65 (lanes 29–35). The N-terminal fusion protein was either used directly (lanes 15-21) or phosphorylated in vitro with PKA and ATP before use (lanes 22-35). Following a 10 min incubation at room temperature, the GST proteins were precipitated with glutathione–agarose beads, and the bound proteins were analyzed by SDS-PAGE followed by fluorography. (C) In vitro translated fragments of CBP, CBP-N (amino acids 1–450), or CBP-KIX (amino acids 451–679) were mixed with GST-p65N (with and without prior phosphorylation by PKA) and GST-p65C and precipitated with glutathione–agarose beads. The precipitated proteins were analyzed as in (B). (D) Comparison of sequences surrounding serine 276 in NF-κB p65 and serine 133 of CREB. Molecular Cell 1998 1, 661-671DOI: (10.1016/S1097-2765(00)80066-0)

Figure 4 Bivalent Interaction between p65 and CBP Is Required for Efficient Transcription by NF-κB (A) Different fragments of CBP were subcloned into pcDNA3 and translated in vitro. The amounts in lanes 1–5 represent approximately 25% of the input used for the pull-down experiment using GST-p65C (lanes 6–10). The proteins precipitated with the GST proteins were analyzed as before. (B) Progressive truncations of p65 from the C terminus were generated using restriction enzyme digestions and produced by translation in vitro. The 35S-labeled p65 proteins were mixed with GST-CBP (amino acids 313–450) fusion protein and precipitated using glutathione-agarose. The amounts in lanes 1–5 represent approximately 25% of the input used for the pull-down experiments (lanes 6–10). (C) C-terminally truncated forms of flu-tagged p65 (which remove the site for phosphorylation-independent interaction with CBP) and serine 276 to alanine mutants of p65 (which removes the site for phosphorylation-dependent interaction with CBP) were tested by cotransfection into Jurkat cells along with a pBIIx-luciferase reporter construct. Molecular Cell 1998 1, 661-671DOI: (10.1016/S1097-2765(00)80066-0)

Figure 5 Squelching of NF-κB–Dependent Transcription by p65-Interacting Domains of CBP (A) The indicated fragments of CBP and the pBIIX-luc reporter plasmid were transfected into Jurkat cells. Approximately 24 hr after transfection, the cells were stimulated with P/P for 4 hr, harvested, and assayed for luciferase activity. (B) The different flu-tagged fragments of CBP (A) were transfected along with p65 and PKAC into COS cells. As a control, one transfection was carried out with flu-IκB-β and p65. Extracts were prepared and immunoprecipitated with the anti-flu antibody 24 hr after transfection, followed by immunoblotting with the p65 antibody. Molecular Cell 1998 1, 661-671DOI: (10.1016/S1097-2765(00)80066-0)

Figure 6 Phosphorylation of NF-κB p65 by PKA Relieves Intramolecular Masking of the p65 N Terminus by the C Terminus and Allows Accessibility to CBP (A) DNA binding by full-length and C-terminally truncated forms of p65 produced by in vitro translation was tested in an electrophoretic mobility shift assay (EMSA). (B) Summary of results using yeast two-hybrid assay system. The p65 C-terminal region fused to the GAL4 DNA binding domain could not be tested as it gave a strong positive signal by itself. (C) The abilty of full-length WT and S276A mutant p65 produced in bacteria to bind DNA was tested with and without prior phosphorylation by PKA. (D) In vitro translated p65C (amino acids 314–550) was mixed together with GST-p65N (amino acids 1–313) fusion protein and precipitated with glutathione–agarose beads. The GST-p65N protein was used by itself or after phosphylation in vitro by PKA. (E) Full-length p65 was produced in bacteria and tested for association with in vitro translated, full-length CBP. After allowing the two proteins to mix with each other, the p65 protein was immunoprecipitated and the immunoprecipitates were analyzed by SDS-PAGE. Molecular Cell 1998 1, 661-671DOI: (10.1016/S1097-2765(00)80066-0)

Figure 7 Model Depicting How Phosphorylation of p65 by PKA Stimulates the Transcriptional Activity of NF-κB The inactive, cytoplasmic complex of NF-κB:IκB:PKAc responds to external signals such as interleukin-1, tumor necrosis factor α, or LPS by phosphorylation and degradation of the IκB protein. The removal of IκB activates PKAc, which phosphorylates the NF-κB p65 subunit on serine 276. The phosphorylation of p65 weakens the interaction of the p65 N-terminal region with the C-terminal region, unmasking the phosphorylation-independent, CBP-interaction domain in p65. The phosphorylated p65 then associates with CBP through a bivalent interaction, CBP-KIX (amino acids 450–679) with phospho-serine 276, and CBP amino acids 313–450 with p65C (amino acids 477–504). Molecular Cell 1998 1, 661-671DOI: (10.1016/S1097-2765(00)80066-0)