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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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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).
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fig3: 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).

Mentions: The contribution of NLS1 to 4ICD nuclear localization was confirmed by Western blot analysis of nuclear extracts isolated from transiently transfected and HRG-stimulated MCF-7B cells. Despite high levels of ERBB4 and 4ICD protein within the membrane/cytosolic fractions from ERBB4-Flag and ERBB4muNLS-Flag–transfected cells (Fig. 3, lanes 2 and 3), 4ICD was not detected within the nuclei of mock-stimulated cells (Fig. 3, lanes 5 and 6). As predicted, 4ICD was detected in nuclear extracts from HRG-stimulated ERBB4-Flag transfected cells (Fig. 3, lane 11), however, HRG failed to drive nuclear translocation of 4ICD in ERBB4muNLS–transfected cells (Fig. 3, lane 12). Some membrane ERBB4 contamination was observed in nuclear extracts of HRG-treated cells (Fig. 3, lanes 11 and 12). Together, our cellular microscopy and biochemical data strongly implicate NLS1 as a functional ERBB4 NLS mediating nuclear translocation of 4ICD.


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)

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).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172499&req=5

fig3: 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).
Mentions: The contribution of NLS1 to 4ICD nuclear localization was confirmed by Western blot analysis of nuclear extracts isolated from transiently transfected and HRG-stimulated MCF-7B cells. Despite high levels of ERBB4 and 4ICD protein within the membrane/cytosolic fractions from ERBB4-Flag and ERBB4muNLS-Flag–transfected cells (Fig. 3, lanes 2 and 3), 4ICD was not detected within the nuclei of mock-stimulated cells (Fig. 3, lanes 5 and 6). As predicted, 4ICD was detected in nuclear extracts from HRG-stimulated ERBB4-Flag transfected cells (Fig. 3, lane 11), however, HRG failed to drive nuclear translocation of 4ICD in ERBB4muNLS–transfected cells (Fig. 3, lane 12). Some membrane ERBB4 contamination was observed in nuclear extracts of HRG-treated cells (Fig. 3, lanes 11 and 12). Together, our cellular microscopy and biochemical data strongly implicate NLS1 as a functional ERBB4 NLS mediating nuclear translocation of 4ICD.

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