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Effects of partner proteins on BCA2 RING ligase activity.

Bacopulos S, Amemiya Y, Yang W, Zubovits J, Burger A, Yaffe M, Seth AK - BMC Cancer (2012)

Bottom Line: BCA2 expression showed a statistically significant correlation with tumor grade.Binding to BCA2 by hHR23a and 14-3-3sigma was confirmed in vitro using tagged partner proteins and BCA2. hHR23a and 14-3-3sigma effect the autoubiquitination and auto-degradation activity of BCA2.Ubiquitination of hHR23a-bound BCA2 was found to be dramatically lower than that of free BCA2, suggesting that hHR23a promotes the stabilization of BCA2 by inactivating its autoubiquitination activity, without degradation of hHR23a.

View Article: PubMed Central - HTML - PubMed

Affiliation: Sunnybrook Research Institute, Toronto, ON, Canada.

ABSTRACT

Background: BCA2 is an E3 ligase linked with hormone responsive breast cancers. We have demonstrated previously that the RING E3 ligase BCA2 has autoubiquitination activity and is a very unstable protein. Previously, only Rab7, tetherin, ubiquitin and UBC9 were known to directly interact with BCA2.

Methods: Here, additional BCA2 binding proteins were found using yeast two-hybrid and bacterial-II-hybrid screening techniques with Human breast and HeLa cDNA libraries. Co-expression of these proteins was analyzed through IHC of TMAs. Investigation of the molecular interactions and effects were examined through a series of in vivo and in vitro assays.

Results: Ten unique BCA2 interacting proteins were identified, two of which were hHR23a and 14-3-3sigma. Both hHR23a and 14-3-3sigma are co-expressed with BCA2 in breast cancer cell lines and patient breast tumors (n = 105). hHR23a and BCA2 expression was significantly correlated (P = < 0.0001 and P = 0.0113) in both nucleus and cytoplasm. BCA2 expression showed a statistically significant correlation with tumor grade. High cytoplasmic hHR23a trended towards negative nodal status. Binding to BCA2 by hHR23a and 14-3-3sigma was confirmed in vitro using tagged partner proteins and BCA2. hHR23a and 14-3-3sigma effect the autoubiquitination and auto-degradation activity of BCA2. Ubiquitination of hHR23a-bound BCA2 was found to be dramatically lower than that of free BCA2, suggesting that hHR23a promotes the stabilization of BCA2 by inactivating its autoubiquitination activity, without degradation of hHR23a. On the other hand, phosphorylated BCA2 protein is stabilized by interaction with 14-3-3sigma both with and without proteasome inhibitor MG-132 suggesting that BCA2 is regulated by multiple degradation pathways.

Conclusions: The interaction between BCA2 and hHR23a in breast cancer cells stabilizes BCA2. High expression of BCA2 is correlated with grade in breast cancer, suggesting regulation of this E3 ligase is important to cancer progression.

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Endogenous expression and localization of BCA2 and partner proteins. [A] Western blot comparing expressed protein levels of BCA2, hHR23a, 14-3-3σ and β-Tubulin in ER-positive and ER-Negative cell lines. Normalized relative intensities of each protein are displayed below their respective blots. Microscopy images, taken at 100 × magnification, of endogenous IF staining for [B] BCA2 and hHR23a or [C] BCA2 and 14-3-3σ, tagged with either secondary antibody conjugated to FITC-dye or Cy3, in MCF7 breast cancer cells. The last panel shows the merged image and co-localization of the proteins.
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Figure 2: Endogenous expression and localization of BCA2 and partner proteins. [A] Western blot comparing expressed protein levels of BCA2, hHR23a, 14-3-3σ and β-Tubulin in ER-positive and ER-Negative cell lines. Normalized relative intensities of each protein are displayed below their respective blots. Microscopy images, taken at 100 × magnification, of endogenous IF staining for [B] BCA2 and hHR23a or [C] BCA2 and 14-3-3σ, tagged with either secondary antibody conjugated to FITC-dye or Cy3, in MCF7 breast cancer cells. The last panel shows the merged image and co-localization of the proteins.

Mentions: Whole cell lysates from a panel of breast cancer cell lines were analyzed via Western blotting. Blots were probed to investigate the endogenous expression of hHR23a, 14-3-3σ and BCA2. BCA2 has been shown to be transcriptionally up-regulated in ER-positive mammary epithelial cell lines [2,16], protein expression levels shown in Figure 2A are consistent with those observations [2,16]. ER-positive cell lines ZR751, BT474 and MCF7 (Figure 2A, lanes 1-3) have elevated expression of the BCA2 protein compared with ER-negative cell line MDA MB 231 (Figure 2A, lane 4). Expression of hHR23a was marginally increased in ER positive cells as determined by densitometry. Analysis further showed that BCA2 was upregulated in cells expressing high amounts of 14-3-3σ. The expression of 14-3-3σ was variable between cell lines, likely due the epigenetic regulation of this protein in cancer cells.


Effects of partner proteins on BCA2 RING ligase activity.

Bacopulos S, Amemiya Y, Yang W, Zubovits J, Burger A, Yaffe M, Seth AK - BMC Cancer (2012)

Endogenous expression and localization of BCA2 and partner proteins. [A] Western blot comparing expressed protein levels of BCA2, hHR23a, 14-3-3σ and β-Tubulin in ER-positive and ER-Negative cell lines. Normalized relative intensities of each protein are displayed below their respective blots. Microscopy images, taken at 100 × magnification, of endogenous IF staining for [B] BCA2 and hHR23a or [C] BCA2 and 14-3-3σ, tagged with either secondary antibody conjugated to FITC-dye or Cy3, in MCF7 breast cancer cells. The last panel shows the merged image and co-localization of the proteins.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3298473&req=5

Figure 2: Endogenous expression and localization of BCA2 and partner proteins. [A] Western blot comparing expressed protein levels of BCA2, hHR23a, 14-3-3σ and β-Tubulin in ER-positive and ER-Negative cell lines. Normalized relative intensities of each protein are displayed below their respective blots. Microscopy images, taken at 100 × magnification, of endogenous IF staining for [B] BCA2 and hHR23a or [C] BCA2 and 14-3-3σ, tagged with either secondary antibody conjugated to FITC-dye or Cy3, in MCF7 breast cancer cells. The last panel shows the merged image and co-localization of the proteins.
Mentions: Whole cell lysates from a panel of breast cancer cell lines were analyzed via Western blotting. Blots were probed to investigate the endogenous expression of hHR23a, 14-3-3σ and BCA2. BCA2 has been shown to be transcriptionally up-regulated in ER-positive mammary epithelial cell lines [2,16], protein expression levels shown in Figure 2A are consistent with those observations [2,16]. ER-positive cell lines ZR751, BT474 and MCF7 (Figure 2A, lanes 1-3) have elevated expression of the BCA2 protein compared with ER-negative cell line MDA MB 231 (Figure 2A, lane 4). Expression of hHR23a was marginally increased in ER positive cells as determined by densitometry. Analysis further showed that BCA2 was upregulated in cells expressing high amounts of 14-3-3σ. The expression of 14-3-3σ was variable between cell lines, likely due the epigenetic regulation of this protein in cancer cells.

Bottom Line: BCA2 expression showed a statistically significant correlation with tumor grade.Binding to BCA2 by hHR23a and 14-3-3sigma was confirmed in vitro using tagged partner proteins and BCA2. hHR23a and 14-3-3sigma effect the autoubiquitination and auto-degradation activity of BCA2.Ubiquitination of hHR23a-bound BCA2 was found to be dramatically lower than that of free BCA2, suggesting that hHR23a promotes the stabilization of BCA2 by inactivating its autoubiquitination activity, without degradation of hHR23a.

View Article: PubMed Central - HTML - PubMed

Affiliation: Sunnybrook Research Institute, Toronto, ON, Canada.

ABSTRACT

Background: BCA2 is an E3 ligase linked with hormone responsive breast cancers. We have demonstrated previously that the RING E3 ligase BCA2 has autoubiquitination activity and is a very unstable protein. Previously, only Rab7, tetherin, ubiquitin and UBC9 were known to directly interact with BCA2.

Methods: Here, additional BCA2 binding proteins were found using yeast two-hybrid and bacterial-II-hybrid screening techniques with Human breast and HeLa cDNA libraries. Co-expression of these proteins was analyzed through IHC of TMAs. Investigation of the molecular interactions and effects were examined through a series of in vivo and in vitro assays.

Results: Ten unique BCA2 interacting proteins were identified, two of which were hHR23a and 14-3-3sigma. Both hHR23a and 14-3-3sigma are co-expressed with BCA2 in breast cancer cell lines and patient breast tumors (n = 105). hHR23a and BCA2 expression was significantly correlated (P = < 0.0001 and P = 0.0113) in both nucleus and cytoplasm. BCA2 expression showed a statistically significant correlation with tumor grade. High cytoplasmic hHR23a trended towards negative nodal status. Binding to BCA2 by hHR23a and 14-3-3sigma was confirmed in vitro using tagged partner proteins and BCA2. hHR23a and 14-3-3sigma effect the autoubiquitination and auto-degradation activity of BCA2. Ubiquitination of hHR23a-bound BCA2 was found to be dramatically lower than that of free BCA2, suggesting that hHR23a promotes the stabilization of BCA2 by inactivating its autoubiquitination activity, without degradation of hHR23a. On the other hand, phosphorylated BCA2 protein is stabilized by interaction with 14-3-3sigma both with and without proteasome inhibitor MG-132 suggesting that BCA2 is regulated by multiple degradation pathways.

Conclusions: The interaction between BCA2 and hHR23a in breast cancer cells stabilizes BCA2. High expression of BCA2 is correlated with grade in breast cancer, suggesting regulation of this E3 ligase is important to cancer progression.

Show MeSH
Related in: MedlinePlus