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. 2001 May;21(10):3482-90.
doi: 10.1128/MCB.21.10.3482-3490.2001.

Interaction between hnRNPA1 and IkappaBalpha is required for maximal activation of NF-kappaB-dependent transcription

D C Hay  1 , G D Kemp, C Dargemont, R T Hay
Affiliations

Interaction between hnRNPA1 and IkappaBalpha is required for maximal activation of NF-kappaB-dependent transcription

D C Hay et al. Mol Cell Biol. 2001 May.

Abstract

Transcriptional activation of NF-kappaB is mediated by signal-induced phosphorylation and degradation of its inhibitor, IkappaBalpha. NF-kappaB activation induces a rapid resynthesis of IkappaBalpha which is responsible for postinduction repression of transcription. Following resynthesis, IkappaBalpha translocates to the nucleus, removes template bound NF-kappaB, and exports NF-kappaB to the cytoplasm in a transcriptionally inactive form. Here we demonstrate that IkappaBalpha interacts directly with another nucleocytoplasmic shuttling protein, hnRNPA1, both in vivo and in vitro. This interaction requires one of the N-terminal RNA binding domains of hnRNPA1 and the C-terminal region of IkappaBalpha. Cells lacking hnRNPA1 are defective in NF-kappaB-dependent transcriptional activation, but the defect in these cells is complemented by ectopic expression of hnRNPA1. hnRNPA1 expression in these cells increased the amount of IkappaBalpha degradation, compared to that of the control cells, in response to activation by Epstein-Barr virus latent membrane protein 1. Thus in addition to regulating mRNA processing and transport, hnRNPA1 also contributes to the control of NF-kappaB-dependent transcription.

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Figures

FIG. 1
FIG. 1
Affinity purification of IκBα complexes from a B-cell extract. A protein extract from Namalwa cells was first passed through a column of protein A Sepharose and then through a column of preimmune IgG linked to protein A Sepharose (PI) and finally through a column of anti-IκBα IgG linked to protein A Sepharose (α-IκBα). The cell extract prior to passage over the affinity columns (Load) and after passage over the affinity columns (FT) was analyzed by Western blotting (WB) with an IκBα antibody (A). Proteins bound to the immunoaffinity matrices were eluted with acetic acid (HAc), and each of the fractions (lanes 1 to 4) was analyzed by Western blotting with antibodies to IκBα (B), NF-κB p65 (C), NF-κB p50 (D), or Coomassie blue staining of the polyacrylamide gel (E). Stained polypeptides were subjected to in-gel trypsin digestion, and peptides were sequenced by Edman degradation. The sequence output and identification of the peptides are indicated (F).
FIG. 2
FIG. 2
Interaction between IκBα and hnRNPA1 in HeLa cells. (A) Extracts from HeLa cells were immunoprecipitated with preimmune IgG (PI), antibodies to NF-κB p65 (α-p65), or antibodies to IκBα (α-IκBα), and immunoprecipitates were analyzed by Western blotting with the 4B10 monoclonal antibody to hnRNPA1 (WB α-hnRNPA1) or the 10B monoclonal antibody to IκBα (WB α-IκBα). (B) Extracts from HeLa cells were immunoprecipitated with the 4B10 monoclonal antibody (α-hnRNPA1) or an irrelevant monoclonal antibody (α-SV5) and analyzed by Western blotting with the 4B10 monoclonal antibody to hnRNPA1 (WB α-hnRNPA1) or the 10B monoclonal antibody to IκBα (WB α-IκBα).
FIG. 3
FIG. 3
hnRNPA1 interacts directly with IκBα in vitro. (A) Recombinant IκBα (present in all lanes) and NF-κB p50 and NF-κB p65 (as indicated) were incubated with either GST, GST-NFIII, GST-IκBγ, GST-p65, or GST-hnRNPA1 immobilized on glutathione agarose. Bound proteins were eluted and analyzed by Western blotting with the 10B monoclonal antibody to IκBα. (B) In vitro-translated 35S-labeled p65 was incubated with GST-IκBα, GST-hnRNPA1 fusion proteins, and GST immobilized on glutathione agarose beads. Bound proteins were analyzed by SDS-PAGE and phosphorimaging.
FIG. 4
FIG. 4
hnRNPA1 binds to the C terminus of IκBα. (A) Diagrammatic representation of IκBα and truncated versions of IκBα, with their abilities to bind to hnRNPA1 indicated. (B) In vitro-translated, [35S]methionine-labeled hnRNPA1 was incubated with GST, GST-NFIII, GST-IκBγ, GST-IκBα wild type (WT), and GST-IκBα truncation mutants immobilized on glutathione agarose beads. Bound proteins were analyzed by SDS-PAGE and phosphorimaging. The location of 35S-labeled hnRNPA1 is indicated (A1). (C) In vitro-translated 35S-labeled β-Gal or fusions with either the N terminus of IκBα (N-T), the C terminus of IκBα (C-T), or both the N and C termini of IκBα (CT + NT) were incubated with either GST or GST-hnRNPA1 immobilized on glutathione agarose beads. Bound proteins were analyzed by SDS-PAGE and phosphorimaging. (D) In vitro-translated, 35S-labeled IκBα wild type (WT) or truncation mutants were incubated with either GST or GST-hnRNPA1 immobilized on glutathione agarose beads. Bound proteins were analyzed by SDS-PAGE and phosphorimaging. The input in vitro-translated products are shown in the right-hand panel.
FIG. 5
FIG. 5
An hnRNPA1 RNA binding domain is required for interaction with IκBα. (A) Diagrammatic representation of the hnRNPA1 molecule with the two RNA binding domains (RBD), the RGG box, and the M9 nuclear transport signal indicated. Truncation mutants used in this study and their abilities to bind IκBα are indicated. (B and C) In vitro-translated, 35S-labeled hnRNPA1 wild type (WT) or truncation mutants were incubated with either GST, GST-IκBα, or GST-IκBα265–317 immobilized on glutathione agarose beads. Bound proteins were analyzed by SDS-PAGE and phosphorimaging. (D) In vitro-translated, 35S-labeled hnRNPA1 wild type (WT) or truncation mutants were incubated with GST or GST-IκBα immobilized on glutathione agarose beads. Bound proteins were analyzed by SDS-PAGE and phosphorimaging. (E) In vitro-translated 35S-labeled IκBα was incubated with GST, GST-hnRNPA1 wild type (WT), or GST-hnRNPA1 truncation mutants immobilized on glutathione agarose beads. Bound proteins were analyzed by SDS-PAGE and phosphorimaging.
FIG. 6
FIG. 6
hnRNPA1 enhances NF-κB-dependent transcriptional activation. (A and B) The NF-κB-dependent luciferase reporter 3 enh conA luc and the RSV-lacZ reporter were electroporated with pCDNA3 empty vector or pCDNA3 expression constructs containing either the wild-type (WT) hnRNPA1 cDNA (A1) or the indicated truncation mutants into CB3 cells which do not express hnRNPA1. (A) Expression levels of hnRNPA1 were determined by Western blotting. (B) To provide an NF-κB activation signal, cells were electroporated with an expression construct containing the cDNA for EBV LMP-1 or empty vector. Sixteen hours after electroporation, cells were lysed for determination of luciferase and LacZ activity. The activity of the Rous sarcoma virus LacZ reporter was used as an internal control, and the values indicated represented the ratio of luciferase activity to LacZ activity. Assays were performed in triplicate, and error bars represent 1 standard deviation. (C) The control experiments using ConA Luc and AP1 ConA Luc were performed as described above, in duplicate, and results are quoted in relative light units per milligram of protein.
FIG. 7
FIG. 7
hnRNPA1 enhances IκBα processing in response to LMP-1 activation. (A, B, and C) IκBα wild type (WT) and the S32A S36A, 1–292, and 1–303 constructs were electroporated with pcDNA 3 empty vector or pcDNA 3 expression constructs containing either hnRNPA1 cDNA or the indicated truncation mutants into CB3 cells which do not express hnRNPA1. To provide an NF-κB activation signal, cells were electroporated with an expression construct containing the cDNA for EBV LMP-1 or empty vector. To control for the level of transfection between the different conditions, the cells were transfected with an expression construct containing the cDNA for pyruvate kinase with a myc tag. Sixteen hours posttransfection the cells were lysed and separated by SDS–10% PAGE. Following separation, cells were transferred to a polyvinylidene difluoride membrane and analyzed using the SV5 monoclonal antibody to SV5-tagged proteins and the myc monoclonal antibody to myc-tagged proteins.
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