beta-Arrestin inhibits NF-kappaB activity by means of its interaction with the NF-kappaB inhibitor IkappaBalpha

Abstract

In addition to their roles in desensitization and signaling of seven-membrane-spanning receptors, beta-arrestins have been more recently implicated in regulating non-seven-membrane-spanning receptor pathways. By using a yeast two-hybrid screen, we identified the inhibitor of NF-kappaB, IkappaBalpha, as a binding partner of beta-arrestin 1. Both beta-arrestin 1 and 2 interact with IkappaBalpha in transfected cells as assessed by immunoprecipitation experiments. Additionally, upstream kinases known to regulate the function of IkappaBalpha, such as IkappaB kinase alpha and beta and NF-kappaB-inducing kinase, were also shown to interact with beta-arrestin. Overexpression of either beta-arrestin 1 or beta-arrestin 2 led to marked inhibition of NF-kappaB activity, as measured by reporter gene activity. Inhibition of NF-kappaB activity was independent of the type of stimulus used for NF-kappaB activation. Conversely, suppression of beta-arrestin 1, but not beta-arrestin 2, expression by using RNA interference led to a 3-fold increase in tumor necrosis factor-stimulated NF-kappaB activity as measured by NF-kappaB mobility-shift analysis. These data uncover a role of beta-arrestins in the regulation of NF-kappaB-mediated gene regulation.

Fig. 1.

Identification of IκBα as a β-arrestin-interacting protein. (A) Yeast two-hybrid screening of a human heart cDNA library with β-arrestin 1 identified four independent clones of IκBα. The basic structure of IκBα and the positions of the 5′ end of the identified clones are depicted. (B) HeLa cells overexpressing either FLAG β-arrestin 1 (β-arr1) or 2 (β-arr2) along with IκBα were immunoprecipitated (IP) with anti-FLAG antibodies. Western blots (WB) depict total cell lysates probed with anti-FLAG antibody (Left) and FLAG-immunoprecipitates analyzed with anti-IκBα antibody (Right). (C) HeLa cells overexpressing FLAG IκBα along with either β-arrestin 1 or 2 were immunoprecipitated with anti-FLAG antibodies. Western blots are immunoprecipitates stained with anti-β-arrestin antibody. Data shown are representative of four independent experiments.

Fig. 2.

β-arrestin (β-arr1/2) interacts with members of the IKK complex. HeLa cells were transfected with the indicated plasmids. After transfection (48 h), cells were lysed and immunoprecipitated (IP) by using anti-FLAG M2 beads. Western blot analysis was performed by using anti-β-arrestin antibody. Data shown are representative of four independent experiments.

Fig. 3.

Attenuation of NF-κB activation by β-arrestins. (A) HeLa or Jurkat T cells were transfected with an NF-κB reporter gene along with β-arrestin 1 (β-arr1, gray), β-arrestin 2 (β-arr2, white), or pcDNA3 (black) as a control. For carbachol-stimulated experiments, cells were also transfected with plasmid for the M1 muscarinic receptor. After overnight serum starving, cells were stimulated for 4 h with 10 ng/ml TNF-α or 100 μM carbachol. (B) HeLa cells were treated as in A with the exception of being transfected with FLAG β-arrestin 1 and FLAG β-arrestin 2 constructs. Levels of β-arrestin expression are shown by Western blot (WB) detection with FLAG antibody (Inset). Cells were treated with either 10 ng/ml TNF-α or 100 μM angiotensin for 4 h. Data represent the mean ± SE from three independent experiments performed in triplicate. Statistical significance was determined by using a one-way ANOVA to compare control with β-arrestin 1 and β-arrestin 2 transfected cells by using prism software. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Fig. 4.

Transfection of β-arrestin 1, but not β-arrestin 2, siRNA leads to an increase in NF-κB activity. (A) HeLa cells were transfected with siRNA for β-arrestin 1(βarr1), β-arrestin 2 (βarr2), or control (CTL), treated with 10 ng/ml TNF-α, and subjected to gel-shift mobility assays as described in Materials and Methods. A representative mobility shift experiment (Left) and a supershift experiment with antibodies to the p50 and p65 subunits of NF-κB(Right) are shown. (B) The NF-κB band intensity was quantified by using a PhosphorImager. The data represent the mean ± SE (n = 7). Statistical significance was determined by using a one-way ANOVA to compare control-transfected cells to β-arrestin 1 and β-arrestin 2 siRNA-transfected cells after 30 min of stimulation. *, P < 0.05. (C) Western blot of lysates from cells transfected with control, β-arrestin 1, and β-arrestin 2 siRNAs probed with anti-β-arrestin antibody.