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. 2004 Nov 8;167(3):469-78.
doi: 10.1083/jcb.200403155.

The ERBB4/HER4 receptor tyrosine kinase regulates gene expression by functioning as a STAT5A nuclear chaperone

Christopher C Williams  1 , June G Allison, Gregory A Vidal, Matthew E Burow, Barbara S Beckman, Luis Marrero, Frank E Jones
Affiliations

The ERBB4/HER4 receptor tyrosine kinase regulates gene expression by functioning as a STAT5A nuclear chaperone

Christopher C Williams et al. J Cell Biol. .

Abstract

In the lactating breast, ERBB4 localizes to the nuclei of secretory epithelium while regulating activities of the signal transducer and activator of transcription (STAT) 5A transcription factor essential for milk-gene expression. We have identified an intrinsic ERBB4 NLS (residues 676-684) within the ERBB4 intracellular domain (4ICD) that is essential for nuclear accumulation of 4ICD. To determine the functional significance of 4ICD nuclear translocation in a physiologically relevant system, we have demonstrated that cotransfection of ERBB4 and STAT5A in a human breast cancer cell line stimulates beta-casein promoter activity. Significantly, nuclear localization of STAT5A and subsequent stimulation of the beta-casein promoter requires nuclear translocation of 4ICD. Moreover, 4ICD and STAT5A colocalize within nuclei of heregulin beta 1 (HRG)-stimulated cells and both proteins bind to the endogenous beta-casein promoter in T47D breast cancer cells. Together, our results establish a novel molecular mechanism of transmembrane receptor signal transduction involving nuclear cotranslocation of the receptor intracellular domain and associated transcription factor. Subsequent binding of the two proteins at transcription factor target promoters results in activation of gene expression.

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Figures

Figure 1.
Figure 1.
Identification of a minimal ERBB4 sequence that enhances EGFP nuclear accumulation. (A) ERBB4 residues representing putative NLSs fused to the amino terminus of EGFP. (B–E) Localization by deconvolution microscopy of indicated NLS-EGFP constructs 48 h after transfection of HEK 293T cells. (F) Basic residues (red) altered in NLS1-EGFP fusions. (G–J) Localization by deconvolution microscopy of indicated NLS1-EGFP mutants 48 h after transfection of HEK 293T cells.
Figure 2.
Figure 2.
ERBB4 nuclear translocation requires an intact NLS1. MCF-7B cells were transfected with ERBB4-EGFP (A–C, G–I, M–O, and S–U) or ERBB4muNLS-EGFP (D–F, J–L, P–R, and V–X) harboring base substitutions (K681E, K682I, K683M, R684G) within NLS1. Immediately before fixation in 4% PFA at 48 h after transfection cells were treated by (A–F) mock stimulation, (G–L) stimulation with 50 ng/ml of HRG for 30 min at RT (HRG), (M–R) incubation with 10 ng/ml of LMB for 12 h (LMB), or (S–X) a combination of HRG and LMB treatments (HRG/LMB). After fixation, nuclei were stained with Hoechst dye and observed by deconvolution microscopy. (Y) The mean percentage of EGFP fluorescence in the cytoplasmic or nuclear compartments for each treatment was determined by analyzing EGFP intensities from 10 transfected cells. Bar, 8 μm.
Figure 3.
Figure 3.
Nuclear accumulation of the 4ICD requires an intact NLS1. Transfected MCF-7B cells were mock stimulated or treated with 50 ng/ml of HRG for 30 min. Cell lysates were prepared and separated into membrane/cytosolic (Mem/Cyto) and nuclear fractions. The lysates were analyzed by PAGE and Western blot using an antibody directed against the carboxyl-terminal Flag epitope tags of ERBB4 and ERBB4muNLS. The 80-kD ERBB4 cleavage product, 4ICD, was detected in Mem/Cyto fractions of ERBB4 (lanes 2 and 8) and ERBB4muNLS (lanes 3 and 9) transfected cells but only in the nuclear fractions of HRG-stimulated ERBB4 transfected cells (lane 11). A high molecular mass nonspecific band was detected in Mem/Cyto extracts prepared from vector control transfected cells (lanes 1 and 7) and nuclear extracts prepared from HRG-stimulated ERBB4 and ERBB4muNLS transfected cells (lanes 11 and 12). Each experiment detected a variable but low level of full-length ERBB4 contamination in nuclear extracts prepared from HRG-stimulated cells (lanes 11 and 12).
Figure 4.
Figure 4.
ERBB4 nuclear translocation modulates STAT5A stimulation of the β-casein promoter. MCF-7B cells were cotransfected with the bovine β-casein promoter fused to luciferase and plasmids expressing the indicated cDNAs. Cell lysates were prepared at 2 d after transfection and luciferase activity was determined using standard methods. Results are reported as fold increase in luciferase activity relative to β-casein promoter luciferase cotransfected with empty vector controls. ERBB4/STAT5A stimulation of the β-casein promoter was significantly greater than each of the other treatments (* indicates P < 0.05). ERBB4muNLS/STAT5A stimulation of the β-casein promoter was significantly greater than the ERBB4 kinase-dead (ERBB4KD/STAT5A) and STAT5A SH2 domain mutant (ERBB4-Flag/STAT5AR618V) negative controls (** indicates P < 0.05). Each treatment was preformed in duplicate and the entire experiment was repeated three times. STAT5A was immunoprecipitated from lysates prepared for luciferase assay and analyzed by Western blot for STAT5A expression and activation by phosphorylation at the regulatory Y694 (top).
Figure 5.
Figure 5.
Activated ERBB4 mediates nuclear translocation of coexpressed STAT5A. MCF-7B cells were cotransfected with (A and B) Red-STAT5A and ERBB4-EGFP or (C and D) Red-STAT5A and ERBB4muNLS-EGFP. Immediately before fixation in 4% PFA at 48 h after transfection, cells were mock stimulated (Mock) or stimulated with 50 ng/ml of HRG for 30 min at RT (HRG). After fixation, cells were stained with Hoechst dye and observed by deconvolution microscopy. Cytoplasmic and nuclear colocalization of ERBB4 (green) and STAT5A (red) of 10 cotransfected cells in the merged image (Merge w/Hoechst) was measured using a pixel correlation algorithm (B and D). Bar, 5 μm.
Figure 6.
Figure 6.
The 4ICD and STAT5A associate in vivo and bind to the endogenous β-casein promoter. (A) 4ICD is associated with STAT5A. COS-7 cells were transfected with the indicated expression plasmids and at 2 d after transfection cells were mock stimulated or stimulated with 50 ng/ml of HRG for 30 min at RT. ERBB4 and STAT5A immunoprecipitates were prepared from cell lysates, resolved by PAGE, and probed for ERBB4 and/or STAT5A by Western blot. White lines indicate that intervening lanes have been spliced out. (B) Schematic of β-casein gene indicating positions of primers for semi-quantitative PCR of distal (−4719/−4276) and proximal (−294/−1) upstream regulatory regions and a promoter region lacking STAT5A GAS binding sites (−923/−590). Gray boxes indicate positions of STAT5A GAS binding sites (Winklehner-Jennewein et al., 1998). (C) Semi-quantitative PCR amplification of DNA bound to ERBB4 and STAT5A isolated by ChIP assay. T47D breast cancer cells were mock stimulated, stimulated with 5 μg/ml of ovine Prl, or stimulated with 50 ng/ml of HRG for 30 min at RT. PFA cross-linked chromatin was immunoprecipitated using control rabbit IgG or antibodies directed against ERBB4 and STAT5A and subjected to 35 cycles of PCR. Input chromatin was prepared from cross-linked and cleared cell lysates using standard DNA precipitation procedures and amplified by PCR as above. PCR amplified samples were resolved on a 2% agarose gel and stained with ethidium bromide.
Figure 7.
Figure 7.
A model for ERBB4 regulation of STAT5A-stimulated gene expression. Growth factor–stimulated ERBB4 undergoes sequential proteolytic processing at the cell membrane by TACE and presenilin-dependent γ-secretase to release 4ICD. 4ICD accumulates in the perinuclear region where a perinuclear/nuclear equilibrium is established favoring perinuclear over nuclear 4ICD. Cytosolic STAT5A associates with activated ERBB4/4ICD in an SH2 domain–dependent manner and STAT5A is phosphorylated at multiple residues including the regulatory Y694 (Jones et al., 1999, 2003). Nuclear cotranslocation of 4ICD and STAT5A requires an intact ERBB4 NLS. The two nuclear proteins bind to STAT5A target promoters containing GAS thereby stimulating expression of STAT5A regulated milk-genes including β-casein and whey acidic protein (Long et al., 2003). STAT5A mediates nuclear retention of 4ICD resulting in a dramatic shift from perinuclear and nuclear accumulation of 4ICD. Interestingly, 4ICD harbors transactivation activity (TA) which may directly augment STAT5A target gene expression.
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