RNA helicase A is a DNA-binding partner for EGFR-mediated transcriptional activation in the nucleus

Abstract

EGF induces the translocation of EGF receptor (EGFR) from the cell surface to the nucleus where EGFR activates gene transcription through its binding to an AT-rich sequence (ATRS) of the target gene promoter. However, how EGFR, without a DNA-binding domain, can bind to the gene promoter is unclear. In the present study, we show that RNA helicase A (RHA) is an important mediator for EGFR-induced gene transactivation. EGF stimulates the interaction of EGFR with RHA in the nucleus of cancer cells. The EGFR/RHA complex then associates with the target gene promoter through binding of RHA to the ATRS of the target gene promoter to activate its transcription. Knockdown of RHA expression in cancer cells abrogates the binding of EGFR to the target gene promoter, thereby reducing EGF/EGFR-induced gene expression. In addition, interruption of EGFR-RHA interaction decreases the EGFR-induced promoter activity. Consistently, we observed a positive correlation of the nuclear expression of EGFR, RHA, and cyclin D1 in human breast cancer samples. These results indicate that RHA is a DNA-binding partner for EGFR-mediated transcriptional activation in the nucleus.

Fig. 1.

Association of EGFR with RHA in the nucleus. (A) Endogenous association of EGFR with RHA in A431 cells. Cells with 80–85% confluence were serum starved overnight before EGF treatment. Equal amounts of cellular fractionated proteins were immunoprecipitated with an anti-EGFR antibody and loaded for Western blotting. Input samples from equal amounts of proteins blotted for EGFR, RHA, lamin B, and α-tubulin are shown as loading and fractionation controls. C, cytoplasmic fraction; N, nuclear fraction; −, without EGF treatment; +, with EGF treatment. (B) Endogenous association of EGFR with RHA in MDA-MB-468 cells. Quantification of the band's density was performed using ImageJ 1.41 (National Institutes of Health). The density of the band in lane 5 was set as 1. The numbers under the band in lane 6 indicate the relative density of that band as compared with the density of the band in lane 5. (C) Time-dependent association of EGFR with RHA in the nucleus. Nuclear proteins from A431 were immunoprecipitated with an anti-EGFR antibody and then were immunoblotted to detect RHA. Input nuclear fraction samples blotted for EGFR, RHA, lamin B, and tubulin are shown as the loading and fractionation controls. (D) (Left) Colocalization of EGFR and RHA in MDA-MB-468 cells. Cells treated with EGF (50 ng/mL for 30 min) or left untreated were stained with indicated antibodies. Colocalization of EGFR and RHA is shown as yellow in the merged image and is indicated by arrows in the Inset. Scale bar, 10 μm. (Right) The bar graph shows the percentage of the 50 counted cells with colocalized EGFR and RHA.

Fig. 2.

Coactivation of gene expression by EGFR and RHA. (A) Costimulation of cyclin D1 (pCCD1-Luc, Left) and iNOS (piNOS-Luc, Right) promoter activity by EGFR and RHA. P values calculated from Student's t test are shown above paired bars. (B) Knockdown of RHA expression diminishes EGFR-induced promoter activity. HeLa cells with stable expression of indicated shRNAs were transfected with plasmids. Luciferase assay was performed after 5 h EGF stimulation. The expression levels of EGFR and RHA are shown in the lower panel. The density of the RHA band was quantified using ImageJ with the density of the basal level band (i.e., lane 1 without EGF) from control shRNA set as 100. The numbers under other bands are the relative band densities as compared with the density of lane 1. Ctrl, control; IB, immunoblotting. (C) ATRS-dependent activation of the cyclin D1 promoter by EGFR and RHA. Relative luciferase activities (i.e., percentage of wild-type ATRS promoter activity) are presented as the mean results ± SD (n = 3). (D) Association of EGFR/RHA with the cyclin D1 gene promoter. (Top) Binding of EGFR and RHA to the cyclin D1 gene promoter in A431 cells. The band's density was quantified using ImageJ with the band density in lane 3 set as 1. The numbers under other bands are the relative densities as compared with the density of the band in lane 3. (Middle) Sequential ChIP-PCR analysis of the association of EGFR and RHA with the cyclin D1 promoter. (Bottom) Reduced binding of EGFR to the cyclin D1 promoter after RHA knockdown. (E) Reduced association of EGFR with the iNOS promoter in A431 cells after RNA knockdown. (Upper) Normal ChIP-PCR. (Lower) Quantitative ChIP-PCR.

Fig. 3.

Requirement of EGFR tyrosine kinase for EGFR/RHA-induced promoter activity. (A) Abrogation of EGFR/RHA-induced cyclin D1 promoter activity by EGFRkd mutant. (B) Abrogation of EGFR/RHA-induced promoter activity by Gefitinib (10 μM) treatment. (C) Abrogation of EGFR/RHA-induced promoter activity by AG1478 (10 μM) treatment. (D) Abrogation of EGFR/RHA-induced promoter activity by Erlotinib (2.5 μM) treatment. (E) EGFR tyrosine kinase-independent interaction between EGFR and RHA. A total cell lysate from HEK293T cells cotransfected with indicated plasmids was immunoprecipitated with an anti-Myc antibody followed by blotting with an anti-Flag antibody. IB, immunoblotting; IP, immunoprecipitation; WCL, whole-cell lysate. (F) Effects of Gefitinib treatment on the binding of EGFR to cyclin D1 promoter. A431 cells treated with EGF without or with Gefitinib were crosslinked, fractionated, and submitted to ChIP-PCR analysis. (G) There was no detectable tyrosine phosphorylation of endogenous RHA upon EGF stimulation. A431 cell lysate was immunoprecipitated with an anti-EGFR or anti-RHA antibody followed by detection with a tyrosine phosphorylation antibody 4G10.

Fig. 4.

Reducing EGFR-induced cyclin D1 promoter activity by interruption of EGFR–RHA Interaction. (A) Schematic of RHA deletion constructs. +, binding to EGFR; −, no binding to EGFR. RGG, RHA RGG domain. (B) Disturbance of EGFR–RHA interaction by deleting the helicase domain of RHA. HEK293T cells were cotransfected with indicated plasmids followed by cell lysis, immunoprecipitation, and Western blot. Expression levels of whole-cell lysates blotted for RHA (Flag), EGFR, and tubulin are shown. IB, immunoblotting; IP, immunoprecipitation; WCL, whole-cell lysate. (C) Reduction of EGFR-induced cyclin D1 promoter activity by deleting the helicase domain of RHA. Luciferase assay was performed in HeLa cells with stable knockdown of endogenous RHA. (D) Reduction of EGFR–RHA interaction by EGFR_mNLS. HEK293T cells were transfected with indicated plasmids and lysed for immunoprecipitation-Western blot. (E) Abrogation of EGFR/RHA-induced cyclin D1 promoter activity by EGFR_mNLS.

Fig. 5.

Correlation of the expression of nuclear EGFR, RHA, and cyclin D1 in human breast tumor samples. (A) Relationships among the expression of nuclear EGFR, RHA, and cyclin D1 in human breast tumor samples. High, high level of nuclear staining (>31% for RHA and >21% for cyclin D1); Low&−, low level of nuclear staining (0–30% for RHA and 0–20% for cyclin D1); M^+/−, nuclear staining negative but membrane staining either positive or negative for EGFR; N⁺, nuclear staining positive for EGFR. (B) Relationship between the nuclear expression levels of RHA and cyclin D1 in these tumor samples. (C) Representative human breast tumor samples showing positive correlation among the expression levels of nuclear EGFR (nEGFR), RHA (nRHA), and cyclin D1 (nCCD1). (Magnification: 400×.)