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

Williams CC, Allison JG, Vidal GA, Burow ME, Beckman BS, Marrero L, Jones FE - J. Cell Biol. (2004)

Bottom Line: We have identified an intrinsic ERBB4 NLS (residues 676-684) within the ERBB4 intracellular domain (4ICD) that is essential for nuclear accumulation of 4ICD.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Tulane University Health Sciences Center, Tulane Cancer Center, New Orleans, LA 70112, USA.

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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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.
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fig2: 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.

Mentions: We next examined the contribution of NLS1 to ERBB4 nuclear translocation by deconvolution microscopy of MCF-7B human breast cancer cells (Burow et al., 2001) transiently transfected with ERBB4-EGFP or ERBB4muNLS-EGFP. In untreated cells, the majority of ERBB4-EGFP was localized to the cellular membrane and perinuclear region (Fig. 2, A–C). Treatment of transfected cells with the ERBB4 ligand heregulin β1 (HRG) dramatically enhanced both perinuclear and nuclear accumulation of ERBB4/4ICD (Fig. 2, G–I and Y). ERBB4 also encodes three putative nuclear export signals harbored within 4ICD (Ni et al., 2001). Therefore, we treated transfected cells with the inhibitor of chromatin region maintenance 1/exportin 1 nuclear export protein, leptomycin B (LMB; Kudo et al., 1999). LMB treatment resulted in nuclear retention of >90% of basal-activated ERBB4/4ICD (Fig. 2, M–O and Y) and >80% of HRG-activated ERBB4/4ICD (Fig. 2, S–V and Y). Strikingly, mutation of NLS1 completely abolished ERBB4/4ICD nuclear translocation under each condition tested (Fig. 2 Y), indicating that NLS1 of ERBB4 is a functionally active intrinsic NLS mediating nuclear translocation of 4ICD. In another experimental system, NIH 3T3 cells were stably transfected to express physiological levels of ERBB4-Flag or ERBB4muNLS-Flag. Despite equivalent levels of HRG-induced tyrosine phosphorylation, nuclear accumulation of ERBB4/4ICD was only observed in cells expressing ERBB4-Flag (Fig. S3, available at http://www.jcb.org/cgi/content/full/jcb.200403155/DC1).


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

Williams CC, Allison JG, Vidal GA, Burow ME, Beckman BS, Marrero L, Jones FE - J. Cell Biol. (2004)

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.
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Related In: Results  -  Collection

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fig2: 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.
Mentions: We next examined the contribution of NLS1 to ERBB4 nuclear translocation by deconvolution microscopy of MCF-7B human breast cancer cells (Burow et al., 2001) transiently transfected with ERBB4-EGFP or ERBB4muNLS-EGFP. In untreated cells, the majority of ERBB4-EGFP was localized to the cellular membrane and perinuclear region (Fig. 2, A–C). Treatment of transfected cells with the ERBB4 ligand heregulin β1 (HRG) dramatically enhanced both perinuclear and nuclear accumulation of ERBB4/4ICD (Fig. 2, G–I and Y). ERBB4 also encodes three putative nuclear export signals harbored within 4ICD (Ni et al., 2001). Therefore, we treated transfected cells with the inhibitor of chromatin region maintenance 1/exportin 1 nuclear export protein, leptomycin B (LMB; Kudo et al., 1999). LMB treatment resulted in nuclear retention of >90% of basal-activated ERBB4/4ICD (Fig. 2, M–O and Y) and >80% of HRG-activated ERBB4/4ICD (Fig. 2, S–V and Y). Strikingly, mutation of NLS1 completely abolished ERBB4/4ICD nuclear translocation under each condition tested (Fig. 2 Y), indicating that NLS1 of ERBB4 is a functionally active intrinsic NLS mediating nuclear translocation of 4ICD. In another experimental system, NIH 3T3 cells were stably transfected to express physiological levels of ERBB4-Flag or ERBB4muNLS-Flag. Despite equivalent levels of HRG-induced tyrosine phosphorylation, nuclear accumulation of ERBB4/4ICD was only observed in cells expressing ERBB4-Flag (Fig. S3, available at http://www.jcb.org/cgi/content/full/jcb.200403155/DC1).

Bottom Line: We have identified an intrinsic ERBB4 NLS (residues 676-684) within the ERBB4 intracellular domain (4ICD) that is essential for nuclear accumulation of 4ICD.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Tulane University Health Sciences Center, Tulane Cancer Center, New Orleans, LA 70112, USA.

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.

Show MeSH
Related in: MedlinePlus