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. 2010 Sep 3;285(36):28097-104.
doi: 10.1074/jbc.M110.116566. Epub 2010 Jun 17.

A-kinase-interacting protein 1 (AKIP1) acts as a molecular determinant of PKA in NF-kappaB signaling

Nan Gao  1 , Yurina Hibi, Marni Cueno, Kaori Asamitsu, Takashi Okamoto
Affiliations

A-kinase-interacting protein 1 (AKIP1) acts as a molecular determinant of PKA in NF-kappaB signaling

Nan Gao et al. J Biol Chem. .

Abstract

The cAMP-dependent protein kinase (PKA) signaling pathway plays a crucial role in the pathogenesis of many NF-kappaB-related diseases. However, there have been controversial reports with regard to the PKA actions in the regulation of NF-kappaB activity. In this study, we have demonstrated the effect of PKA on NF-kappaB activity in view of AKIP1 action; and in 293 and HeLa cells, where the endogenous AKIP1 expression is minimal, PKA-activating agents inhibited the NF-kappaB-dependent reporter gene expression, blocked the interaction of PKAc and p65 subunit of NF-kappaB, and attenuated PKA-dependent phosphorylation of p65 on Ser-276. This inhibitory function of PKAc in NF-kappaB signaling was reversed by overexpression of AKIP1 in 293 cells. In the breast cancer cell line, MDA-MB231 cells and MCF7 cells, where the endogenous AKIP1 is abundant, the PKA signal was found to be synergized with NF-kappaB activation; PKA-activating agents enhanced NF-kappaB-dependent transcriptional activity and the interaction between p65 and PKAc and augmented the phosphorylation of p65 on Ser-276. After RNAi knockdown of AKIP1 in these breast cancer cells, we observed that PKA-activating agents antagonized NF-kappaB-dependent activation. Meanwhile, PKA inhibitor suppressed NF-kappaB-induced breast cancer cell proliferation and multiple NF-kappaB-dependent anti-apoptotic gene expression. It is likely that expression of AKIP1 determines the relationship between these two signal transduction pathways. These findings explained controversial results from various independent groups regarding the action of PKA signaling on the NF-kappaB activation cascade and suggested a possible therapeutic potential of PKA inhibitor in developing anti-cancer strategies.

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Figures

FIGURE 1.
FIGURE 1.
Expression of endogenous AKIP1 in various cells. 293, HeLa, MCF7, and MDA-MB231 cells were cultured and lysed for Western blotting using polyclonal antibody against AKIP1. Note the different expression levels of endogenous AKIP1 in these cell lines. The protein expression levels of α-tubulin indicate that the same amounts of cell lysates were loaded.
FIGURE 2.
FIGURE 2.
Interaction of p65 and PKAc in 293 cells and MDA-MB231 cells. A, p65-PKAc interaction and p65 Ser-276 phosphorylation in wild type 293. Cells incubated with Bt2cAMP (3 mm) for 12 h and TNFα (1 ng/ml) for an additional 6 h were subjected to IP (p65)-WB (PKA) or (phospho-p65-Ser-276). B, effect of AKIP1 overexpression in 293 cells. The p65-PKAc interaction and p65 Ser-276 phosphorylation were examined in AKIP1-overexpressed 293 cells. 293 cells were transiently transfected with FLAG-AKIP1 and treated with Bt2cAMP and TNFα 24 h after the transfection. Then cells were subjected to IP-WB assay. C, p65-PKAc interaction and p65 Ser-276 phosphorylation in wild type MDA-MB231 cells. The cells were treated with Bt2cAMP and TNFα as above, and the cell lysates were subjected to IP-WB assay. D, effects of AKIP1 knockdown in MDA-MB231 cells. After transfecting RNAi-AKIP1 to MDA-MB231 cells, the p65-PKAc interaction and p65 Ser-276 phosphorylation were similarly examined. Equal amount of p65 was immunoprecipitated with anti-p65 antibody and subjected to the following Western blotting assays; 1:50 input of PKA expression was also indicated. The amounts of whole cell lysates were adjusted by examining the level of α-tubulin expression. The results were reproducibly observed at least three times, and the representative data are shown.
FIGURE 3.
FIGURE 3.
Selective recruitment of coactivator protein CBP by AKIP1. A and B, 293 cells were treated with Bt2cAMP (3 mm) and TNFα (1 ng/ml). These cells were collected and immunoprecipitated with anti-p65 antibody (A) or anti-CREB antibody (B), and then an equal amount of immunocomplexes was blotted with anti-CBP antibody to detect the interaction of CBP with either p65 or CREB. The phosphorylated CREB at Ser-133 (S133) was also monitored (B). C and D, AKIP1-overexpressing 293 cells were treated Bt2cAMP and TNFα, and then IP-WB was similarly performed as in A and B. The 1:50 input of CBP expression was also indicated, and the α-tubulin expression levels were monitored as an internal control.
FIGURE 4.
FIGURE 4.
Effects of Bt2cAMP on the NF-κB-dependent transcription and IL-8. A, AKIP1 switches the role of PKA signaling in NF-κB-dependent luciferase expression. Left panel, 293 (closed columns) and MDA-MB231 cells (open columns) were treated with Bt2cAMP (1 and 3 mm) for 12 h and TNFα (1 ng/ml) for an additional 6 h. Cells were lysed and subjected to luciferase assay and normalized with the Renilla luciferase activity as an internal control. The vertical axis indicates the relative luciferase activity in fold activation. Values are representative of triplicate experiments (mean ± S.D.). Right panel, 293 cells transfected with (open columns) or without FLAG-AKIP1 (closed columns) were treated with Bt2cAMP and TNFα and subjected to luciferase assay. Values are representative of triplicate experiments (mean ± S.D.). The inset in the right panel indicates the expanded representation of results in lanes 1–3 (the vertical axis indicates the fold activation). B, effects of AKIP1 on the NF-κB-dependent expression of IL-8. 293 and MDA-MB231 cells were cultured in 24-well plates and incubated with Bt2cAMP and TNFα. The concentration of IL-8 was measured by ELISA. The vertical axis indicates the secretion of IL-8 in the culture medium, and the values are representative of triplicate experiments (mean ± S.D.).
FIGURE 5.
FIGURE 5.
Effects of PKA inhibitor on the proliferation of MDA-MB231 cells. A, MDA-MB231 cells were cultured for 24 h, treated with PKA inhibitor H89 (1 and 3 μm) for 4 h, and then they were subjected to WST-1 assay to evaluate the cell viability. B, MDA-MB231 cells were treated with H89 (3 μm) for 4 h and further incubated with IKK inhibitor BAY11-7082 (5, 10, and 20 μm) for an additional 20 h. Then the proliferating cells were evaluated by WST-1 assay. Note the synergism between inhibition of PKA signaling and that of NF-κB activation signaling. C, 293 cells treated with H89 (3 μm) for 4 h and further incubated with IKK inhibitor BAY11-7082 (0.5, 2, 5, and 10 μm) for an additional 20 h, and WST-1 assay was performed.
FIGURE 6.
FIGURE 6.
Expression of the NF-κB-dependent anti-apoptotic genes is regulated by AKIP1. 293 cells, transfected with AKIP1 or control vehicle vector, were treated with Bt2cAMP for 12 h and TNFα for an additional 6 h. The total RNA was prepared from cells, and cDNA was reverse-transcribed using 0.3 μg of total RNA of each sample. A–E, real time-PCR was performed in triplicate to analyze the relative expression levels of anti-apoptotic genes (cyclin D1, bcl-2, bcl-xL, survivin, and xiap); gene expression is expressed relative to β-actin (mean ± S.E. presented). F, semi-quantitative reverse transcription-PCR was performed with 0.3 μg for each sample.
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