RNAi screen in mouse astrocytes identifies phosphatases that regulate NF-kappaB signaling

Abstract

Regulation of NF-kappaB activation is controlled by a series of kinases; however, the roles of phosphatases in regulating this pathway are poorly understood. We report a systematic RNAi screen of phosphatases that modulate NF-kappaB activity. Nineteen of 250 phosphatase genes were identified as regulators of NF-kappaB signaling in astrocytes. RNAi selectively regulates endogenous chemokine and cytokine expression. Coimmunoprecipitation identified associations of distinct protein phosphatase 2A core or holoenzymes with the IKK, NF-kappaB, and TRAF2 complexes. Dephosphorylation of these complexes leads to modulation of NF-kappaB transcriptional activity. In contrast to IKK and NF-kappaB, TRAF2 phosphorylation has not been well elucidated. We show that the Thr117 residue in TRAF2 is phosphorylated following TNFalpha stimulation. This phosphorylation process is modulated by PP2A and is required for TRAF2 functional activity. These results provide direct evidence for TNF-induced TRAF2 phosphorylation and demonstrate that phosphorylation is regulated at multiple levels in the NF-kappaB pathway.

Figure 1

RNAi screens of phosphatase genes that regulate NF-κB transcriptional activity. (A) The design of the pBabe-Dual vectors. The position of the puromycin resistance gene and the U6 and H1 promoters are indicated. (B) Data from representative experiments depict the modulation of NF-κB reporter activity for each of the 250 phosphatase siRNA targets in non-TNF treated astrocytes. (C) The regulation of NF-κB reporter activity after TNFα stimulation of astrocytes transfected with each pair of siRNA targeting constructs. The luciferase activity of cells transfected with control siRNA vector with TNFα was set at 1. The activity of NF-κB signaling was quantified by measurement of the log ratio of firefly luciferase activity as standardized to that of Renilla luciferase. Dashed lines indicate 2 standard deviations (2×SD) (D) Knockdown of 13 phosphatases activated NF-κB binding activity in chemiluminescent transcription factor assays (CTFA). White bars represent responses of unstimulated astrocytes; shaded bars represent responses of TNFα treated cells. Stimulation with TNFα triggered a 6 to 9 fold increase in CTFA activity; this value was normalized to 1 for comparison. Error bars represent the SD of 3 independent experiments. (E) Knockdown of 6 phosphatases suppressed NF-κB binding activity in CTFA assays. Error bars represent the SD of 3 independent experiments. (F) The fold induction of NF-κB reporter activity after treatment with with TNFα (shaded bars) or medium (open bars) in NIH3T3 cells transfected with the indicated pair of phosphatase siRNA targeting constructs

Figure 2

PP2A catalytic and structural components selectively form complexes with IKK and p65 NF-κB. (A) The indicated myc-tagged phosphatases were transfected into astrocytes and immunoprecipitated with either anti-myc or anti-IKKβ antibody. The 50 kD bands were IgG heavy chain. (B) Cell lysates were immunoprecipitated with anti-PP2A catalytic subunit (PP2A C) or anti-IKKβ antibody showing the interaction between endogenous IKK and PP2A. The 25 kD and 50 kD bands were IgG light and heavy chains. (C) PPP2CB/PP2R1A core enzyme dephosphorylates Ser¹⁸¹ of IKKβ. Astrocytes were transfected with IKKβ. 10 min after TNFα stimulation, the lysates were immunoprecipitated with anti-IKKβ antibody. In a separate transfection, 3T3 cells were transfected with myc-tagged PPP2R1A plus Flag-tagged PPP2CB or other Flag-tagged phosphatases. The phosphatase components were eluted from the Sepharose beads with Flag peptide then 2 ng were incubated with immunoprecipitated IKKβ for 1h at 30 °C, and immunoblotted with anti-phospho-IKKβ antibody. (D) PPP2CB RNAi did not synergize with the constitutively active IKKβ SSEE mutant to enhance NF-κB reporter activity. (E) PPP2CB RNAi enhanced IKKβ phosphorylation. IKKβ-myc was co-transfected with PPP2CB RNAi or control RNAi into astrocytes. After TNFα stimulation, the lysates were harvested and immunoblotted with anti-IKKβ antibody and anti-phospho-IKKβ antibody. (F) The indicated myc-tagged phosphatases were transfected into astrocytes and immunoprecipitated with either anti-myc or anti-NF-κB p65 antibody. The 50 kD band is IgG heavy chain. (G) Cell lysates were immunoprecipitated with anti-PP2A catalytic subunit (PP2A C) or anti-NF-κB p65 showing the interaction between endogenous NF-κB and PP2A. The 25 kD and 50 kD bands were IgG light and heavy chains. (H) PPP2CA /PPP2R1B complexes dephosphorylate Ser⁵³⁶ of p65 NF-κB. Astrocytes were transfected with NF-κB p65. 10 min after TNFα stimulation, cells were harvested and immunoprecipitated with anti-NF-κB p65 antibody. In a separate transfection, 3T3 cells were transfected with the Flag-tagged PPP2CA and myc-tagged PPP2R1B or other Flag-tagged phosphatases. These phosphatases were eluted from the Sepharose beads with Flag peptide, and then 2 ng were incubated with immunoprecipitated NF-κB p65 for 1h at 30°C, and immunoblotted with anti-p65 or two anti-phospho-NF-κB antibodies. The right panel depicts the protein levels of the indicated flag-tagged phosphatases.

Figure 3

Dephosphorylation of TRAF2 by PP2A holoenzyme inhibits NF-κB activity. (A) The indicated myc-tagged phosphatases were transfected into astrocytes and immunoprecipitated with either anti-myc or anti-TRAF2 antibody. (B) Cell lysates were immunoprecipitated with anti-PP2A catalytic subunit (PP2A C) or anti-TRAF2 showing the interaction between endogenous IKK and TRAF2. The 25 kD and 50 kD bands were IgG light and heavy chains. (C) PPP2R5C-myc was co-transfected with Flag-tagged TRAF2 into astrocytes and immunoprecipitated with either anti-Flag or anti-myc antibody. (D) Map of various TRAF2 constructs and their ability to associate with PPP2R5C. (E) Indicated phosphatase genes were co-transfected with TRAF2 into astrocytes. Over-expression of PPP2R5C inhibited TRAF2 induced NF-κB reporter activity. Error bars represent the SD of 3 independent experiments. (F) After IL-1β stimulation PPP2R5C RNAi failed to stimulate NF-κB reporter activity in astrocytes.

Figure 4

TNF induced TRAF2 Thr¹¹⁷ phosphorylation. (A) Amino acid sequence alignment of a portion of the first zinc finger domain of TRAF2. (B) The fold induction of NF-κB reporter activity in astrocytes transfected with TRAF2 and different Ser/Thr to Ala mutants. (C) The fold induction of NF-κB reporter activity in TRAF2^-/- MEFs transfected with TRAF2 and different Ser/Thr to Ala mutants. Error bars represent the SD of 3 independent experiments. (D) The zinc finger domain (residues 99-271) showed two bands by electrophoresis in a 4-20% SDS-PAGE gel. The upper band was sensitive to CIP phosphatase treatment. (E) Thr¹¹⁷ to Ala mutation abolished the upper band of the first finger domain. (F) Specificity of anti-phospho-TRAF2 (Thr¹¹⁷) antibody. (G) Time course of TNF induced TRAF2 Thr¹¹⁷ phosphorylation. (H) PPP2R5C inhibited Thr¹¹⁷ phosphorylation. Astrocytes were transfected Flag-TRAF2 with or without PPP2R5C-myc. Cells were treated with or without TNFα for 15 min before harvest and immunoprecipitation with anti-IKKβ or anti-NF-κB p65 antibodies and immunoblotted with the indicated antibodies. (I) PPP2R5C RNAi enhanced TRAF2 phosphorylation. TRAF2-Flag was co-transfected with PPP2R5C RNAi or control RNAi into astrocytes. After TNFα stimulation, the lysates were harvested and immunoblotted with anti-Flag antibody and anti-phospho-TRAF2 (Thr¹¹⁷) antibody.

Figure 5

Phosphatases regulate chemokine and cytokine expression. (A) The relative mRNA levels of MCP-1 (open bars), KC (shaded bars), and IL-6 (solid bars) in RNAi transfected astrocytes without stimulation. Astrocytes were transfected with phosphatase RNAi vectors and cultured for 72 h. The cDNAs were analyzed by real-time PCR. All phosphatase mRNA levels were normalized with the housekeeping gene, β-glucuronidase. The dashed lines represent a 4 fold increase and 70% reduction, respectively. Error bars represent the SD of 3 independent experiments. (B) One hr after TNFα stimulation cells were collected for RNA isolation and subsequent reverse transcription. The relative mRNA levels of MCP-1, KC and IL-6 in phosphatase RNAi transfected astrocytes after TNFα stimulation. The dashed lines represent a 3 fold increase and 70% reduction, respectively. Error bars represent the SD of 3 independent experiments. (C) Summary of phosphatase interactions characterized in this report. NF-κB signaling was regulated by dephosphorylation of the TRAF2, IKK and NF-κB complexes by the indicated PP2A cofactors.