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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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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.
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fig6: 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.

Mentions: Perinuclear colocalization of ERBB4/4ICD and STAT5A (Fig. 5) suggests that the two proteins exist in a cytosolic complex before nuclear translocation. We have reported previously an interaction between the ERBB4 holoreceptor and STAT5A (Jones et al., 1999). To determine if 4ICD also interacts with STAT5A we cotransfected COS-7 cells with ERBB4-Flag or ERBB4muNLS-Flag and STAT5A and probed STAT5A immunoprecipitates for ERBB4/4ICD using a Flag antibody. Consistent with our previous results, the ERBB4 holoreceptor was detected in STAT5A immunoprecipitates from lysates of cells transfected with ERBB4-Flag/STAT5A and ERBB4muNLS-Flag/STAT5A (Fig. 6 A, lanes 5, 6, 11, and 12). Significantly, 4ICD was detected in the same samples indicating that STAT5A and 4ICD exist in a cytosolic complex. An interaction between cytosolic 4ICD and STAT5A supports the contention that perinuclear 4ICD chaperones associated STAT5A across the nuclear membrane. Due to the high levels of ERBB4 constitutive activation in transient transfected cells (Fig. S2) HRG treatment had little impact on the association between ERBB4/4ICD and STAT5A.


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)

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.
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fig6: 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.
Mentions: Perinuclear colocalization of ERBB4/4ICD and STAT5A (Fig. 5) suggests that the two proteins exist in a cytosolic complex before nuclear translocation. We have reported previously an interaction between the ERBB4 holoreceptor and STAT5A (Jones et al., 1999). To determine if 4ICD also interacts with STAT5A we cotransfected COS-7 cells with ERBB4-Flag or ERBB4muNLS-Flag and STAT5A and probed STAT5A immunoprecipitates for ERBB4/4ICD using a Flag antibody. Consistent with our previous results, the ERBB4 holoreceptor was detected in STAT5A immunoprecipitates from lysates of cells transfected with ERBB4-Flag/STAT5A and ERBB4muNLS-Flag/STAT5A (Fig. 6 A, lanes 5, 6, 11, and 12). Significantly, 4ICD was detected in the same samples indicating that STAT5A and 4ICD exist in a cytosolic complex. An interaction between cytosolic 4ICD and STAT5A supports the contention that perinuclear 4ICD chaperones associated STAT5A across the nuclear membrane. Due to the high levels of ERBB4 constitutive activation in transient transfected cells (Fig. S2) HRG treatment had little impact on the association between ERBB4/4ICD and STAT5A.

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