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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 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).
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fig4: 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).

Mentions: Relative to the empty vector control, independent expression of STAT5A or ERBB4 resulted in insignificant levels of β-casein promoter activity (Fig. 4). Coexpression of ERBB4 and STAT5A, however, resulted in a >40-fold increase in β-casein reporter activity (Fig. 4) demonstrating for the first time that an ERBB receptor directly regulates STAT5A transactivation. As predicted activation of the β-casein promoter was abolished when STAT5A was cotransfected with an ERBB4 mutant lacking intrinsic kinase activity (ERBB4KD; Sartor et al., 2001) or when ERBB4 was cotransfected with a STAT5A protein harboring a mutation that ablates SH2 activity (STAT5AR618V) and thus disrupting the interaction between ERBB4 and STAT5A (Fig. 4; Jones et al., 1999). Strikingly, when STAT5A was cotransfected with ERBB4 harboring an inactive NLS1 (ERBB4muNLS), a significant and dramatic decrease in STAT5A transactivation was observed (Fig. 4). This ablation of STAT5A transactivation was observed despite equivalent levels of ERBB4 and ERBB4muNLS-induced STAT5A phosphorylation at the regulatory STAT5A Y694 (Fig. 4, top). Together, these results clearly indicate that nuclear translocation of 4ICD regulates STAT5A transactivation of the β-casein milk-gene promoter, implicating a physiologically important role for 4ICD nuclear translocation during milk-gene expression.


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 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).
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Related In: Results  -  Collection

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fig4: 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).
Mentions: Relative to the empty vector control, independent expression of STAT5A or ERBB4 resulted in insignificant levels of β-casein promoter activity (Fig. 4). Coexpression of ERBB4 and STAT5A, however, resulted in a >40-fold increase in β-casein reporter activity (Fig. 4) demonstrating for the first time that an ERBB receptor directly regulates STAT5A transactivation. As predicted activation of the β-casein promoter was abolished when STAT5A was cotransfected with an ERBB4 mutant lacking intrinsic kinase activity (ERBB4KD; Sartor et al., 2001) or when ERBB4 was cotransfected with a STAT5A protein harboring a mutation that ablates SH2 activity (STAT5AR618V) and thus disrupting the interaction between ERBB4 and STAT5A (Fig. 4; Jones et al., 1999). Strikingly, when STAT5A was cotransfected with ERBB4 harboring an inactive NLS1 (ERBB4muNLS), a significant and dramatic decrease in STAT5A transactivation was observed (Fig. 4). This ablation of STAT5A transactivation was observed despite equivalent levels of ERBB4 and ERBB4muNLS-induced STAT5A phosphorylation at the regulatory STAT5A Y694 (Fig. 4, top). Together, these results clearly indicate that nuclear translocation of 4ICD regulates STAT5A transactivation of the β-casein milk-gene promoter, implicating a physiologically important role for 4ICD nuclear translocation during milk-gene expression.

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