Limits...
Specificity and heregulin regulation of Ebp1 (ErbB3 binding protein 1) mediated repression of androgen receptor signalling.

Zhang Y, Hamburger AW - Br. J. Cancer (2005)

Bottom Line: Downregulation of Ebp1 expression in LNCaP cells using siRNA resulted in activation of AR in the absence of androgen.Ebp1 associated with ErbB3 in LNCaP cells in the absence of HRG, but HRG induced the dissociation of Ebp1 from ErbB3.These studies suggest that Ebp1 is an AR corepressor whose biological activity can be regulated by the ErbB3 ligand, HRG.

View Article: PubMed Central - PubMed

Affiliation: Greenebaum Cancer Center, University of Maryland School of Medicine, Baltimore, MD, USA.

ABSTRACT
Although ErbB receptors have been implicated in the progression of prostate cancer, little is known about proteins that may mediate their interactions with the androgen receptor (AR). Ebp1, a protein cloned via its association with the ErbB3 receptor, binds the AR and inhibits androgen-regulated transactivation of wild-type AR in COS cells. As the complement of coregulators in different cells are important for AR activity, we determined the effect of Ebp1 on AR function in prostate cancer cell lines. In addition, we examined the regulation of Ebp1 function by the ErbB3/4 ligand heregulin (HRG). In this study, we demonstrate, using several natural AR-regulated promoters, that Ebp1 repressed transcriptional activation of wild-type AR in prostate cancer cell lines. Downregulation of Ebp1 expression in LNCaP cells using siRNA resulted in activation of AR in the absence of androgen. Ebp1 associated with ErbB3 in LNCaP cells in the absence of HRG, but HRG induced the dissociation of Ebp1 from ErbB3. In contrast, HRG treatment enhanced both the association of Ebp1 with AR and also the ability of Ebp1 to repress AR transactivation. These studies suggest that Ebp1 is an AR corepressor whose biological activity can be regulated by the ErbB3 ligand, HRG.

Show MeSH

Related in: MedlinePlus

In vivo interaction of Ebp1 with ErbB3 (A) Ebp1 associates with ErbB3 in LNCaP cells. LNCaP cell lysates were immunoprecipitated with mouse isotype control IgG (lane 1) or an anti-ErbB-3 monoclonal antibody (lane 2) and analysed by sequential immunoblotting (IB) with either Ebp1 (top panel) or ErbB3 (bottom panel) antibodies. (B) HRG-induced dissociation of Ebp1. LNCaP cells were left untreated (0) or stimulated with HRG (20 ng ml−1) for the times indicated. ErbB-3 associated Ebp1 was analysed by ErbB-3 immunoprecipitation (IP) and sequential immunoblotting (IB) with antibodies against Ebp1 (top) and ErbB-3 (bottom).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2361729&req=5

fig4: In vivo interaction of Ebp1 with ErbB3 (A) Ebp1 associates with ErbB3 in LNCaP cells. LNCaP cell lysates were immunoprecipitated with mouse isotype control IgG (lane 1) or an anti-ErbB-3 monoclonal antibody (lane 2) and analysed by sequential immunoblotting (IB) with either Ebp1 (top panel) or ErbB3 (bottom panel) antibodies. (B) HRG-induced dissociation of Ebp1. LNCaP cells were left untreated (0) or stimulated with HRG (20 ng ml−1) for the times indicated. ErbB-3 associated Ebp1 was analysed by ErbB-3 immunoprecipitation (IP) and sequential immunoblotting (IB) with antibodies against Ebp1 (top) and ErbB-3 (bottom).

Mentions: We next determined if ErbB3 could bind Ebp1 in human prostate cell lines as it does in breast carcinoma cells (Yoo et al, 2000) and if HRG could affect this binding. Lysates of serum starved LNCaP cells were incubated with either a mouse monoclonal antibody to ErbB3 or control IgG. Proteins were resolved by SDS–PAGE and immunoblotted with antibody to Ebp1. Ebp1 was found in ErbB3, but not control, immunoprecipitates (Figure 4A). Next, we determined if the binding of Ebp1 to ErbB3 could be regulated by HRG. LNCaP cells were serum starved and treated with HRG (20 ng ml−1) for 0, 15, 60 and 120 min and 24 h. Cell lysates were immunoprecipitated with antibody to ErbB3 as described. Ebp1 was found in ErbB3 immunoprecipitates of untreated cells (Figure 4B). There was a decrease in the level of Ebp1 associated with ErbB3 starting at 15 min. No Ebp1 was found in the Erb3 immunoprecipitates 60 min after treatment. Binding was increased 2 h after treatment and by 24 h after HRG treatment, Ebp1 binding to ErbB3 was restored.


Specificity and heregulin regulation of Ebp1 (ErbB3 binding protein 1) mediated repression of androgen receptor signalling.

Zhang Y, Hamburger AW - Br. J. Cancer (2005)

In vivo interaction of Ebp1 with ErbB3 (A) Ebp1 associates with ErbB3 in LNCaP cells. LNCaP cell lysates were immunoprecipitated with mouse isotype control IgG (lane 1) or an anti-ErbB-3 monoclonal antibody (lane 2) and analysed by sequential immunoblotting (IB) with either Ebp1 (top panel) or ErbB3 (bottom panel) antibodies. (B) HRG-induced dissociation of Ebp1. LNCaP cells were left untreated (0) or stimulated with HRG (20 ng ml−1) for the times indicated. ErbB-3 associated Ebp1 was analysed by ErbB-3 immunoprecipitation (IP) and sequential immunoblotting (IB) with antibodies against Ebp1 (top) and ErbB-3 (bottom).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2361729&req=5

fig4: In vivo interaction of Ebp1 with ErbB3 (A) Ebp1 associates with ErbB3 in LNCaP cells. LNCaP cell lysates were immunoprecipitated with mouse isotype control IgG (lane 1) or an anti-ErbB-3 monoclonal antibody (lane 2) and analysed by sequential immunoblotting (IB) with either Ebp1 (top panel) or ErbB3 (bottom panel) antibodies. (B) HRG-induced dissociation of Ebp1. LNCaP cells were left untreated (0) or stimulated with HRG (20 ng ml−1) for the times indicated. ErbB-3 associated Ebp1 was analysed by ErbB-3 immunoprecipitation (IP) and sequential immunoblotting (IB) with antibodies against Ebp1 (top) and ErbB-3 (bottom).
Mentions: We next determined if ErbB3 could bind Ebp1 in human prostate cell lines as it does in breast carcinoma cells (Yoo et al, 2000) and if HRG could affect this binding. Lysates of serum starved LNCaP cells were incubated with either a mouse monoclonal antibody to ErbB3 or control IgG. Proteins were resolved by SDS–PAGE and immunoblotted with antibody to Ebp1. Ebp1 was found in ErbB3, but not control, immunoprecipitates (Figure 4A). Next, we determined if the binding of Ebp1 to ErbB3 could be regulated by HRG. LNCaP cells were serum starved and treated with HRG (20 ng ml−1) for 0, 15, 60 and 120 min and 24 h. Cell lysates were immunoprecipitated with antibody to ErbB3 as described. Ebp1 was found in ErbB3 immunoprecipitates of untreated cells (Figure 4B). There was a decrease in the level of Ebp1 associated with ErbB3 starting at 15 min. No Ebp1 was found in the Erb3 immunoprecipitates 60 min after treatment. Binding was increased 2 h after treatment and by 24 h after HRG treatment, Ebp1 binding to ErbB3 was restored.

Bottom Line: Downregulation of Ebp1 expression in LNCaP cells using siRNA resulted in activation of AR in the absence of androgen.Ebp1 associated with ErbB3 in LNCaP cells in the absence of HRG, but HRG induced the dissociation of Ebp1 from ErbB3.These studies suggest that Ebp1 is an AR corepressor whose biological activity can be regulated by the ErbB3 ligand, HRG.

View Article: PubMed Central - PubMed

Affiliation: Greenebaum Cancer Center, University of Maryland School of Medicine, Baltimore, MD, USA.

ABSTRACT
Although ErbB receptors have been implicated in the progression of prostate cancer, little is known about proteins that may mediate their interactions with the androgen receptor (AR). Ebp1, a protein cloned via its association with the ErbB3 receptor, binds the AR and inhibits androgen-regulated transactivation of wild-type AR in COS cells. As the complement of coregulators in different cells are important for AR activity, we determined the effect of Ebp1 on AR function in prostate cancer cell lines. In addition, we examined the regulation of Ebp1 function by the ErbB3/4 ligand heregulin (HRG). In this study, we demonstrate, using several natural AR-regulated promoters, that Ebp1 repressed transcriptional activation of wild-type AR in prostate cancer cell lines. Downregulation of Ebp1 expression in LNCaP cells using siRNA resulted in activation of AR in the absence of androgen. Ebp1 associated with ErbB3 in LNCaP cells in the absence of HRG, but HRG induced the dissociation of Ebp1 from ErbB3. In contrast, HRG treatment enhanced both the association of Ebp1 with AR and also the ability of Ebp1 to repress AR transactivation. These studies suggest that Ebp1 is an AR corepressor whose biological activity can be regulated by the ErbB3 ligand, HRG.

Show MeSH
Related in: MedlinePlus