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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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Immunohistochemistry of breast cancer TMA (n = 105). [A] An example of how tumors were scored for protein localization. The top panel is staining positively in the cytoplasm with no nuclear staining, the bottom panel shows a tissue which is staining weakly in the cytoplasm and stongly in the nuclear compartment. [B] Shows examples of the criteria for staining intensity, where 3+ (top left) has the strongest staining/most intense and 1+ (top right) which has the weakest staining/least intense. A tissue staining negatively (0), is also shown (bottom right). [C] Shows a typical staing pattern for a serial section of the same tumor at 5 × magnification and 20 × magnification for BCA2, hHR23a and 14-3-3σ immunostaining, aswell as H&E staining.
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Figure 3: Immunohistochemistry of breast cancer TMA (n = 105). [A] An example of how tumors were scored for protein localization. The top panel is staining positively in the cytoplasm with no nuclear staining, the bottom panel shows a tissue which is staining weakly in the cytoplasm and stongly in the nuclear compartment. [B] Shows examples of the criteria for staining intensity, where 3+ (top left) has the strongest staining/most intense and 1+ (top right) which has the weakest staining/least intense. A tissue staining negatively (0), is also shown (bottom right). [C] Shows a typical staing pattern for a serial section of the same tumor at 5 × magnification and 20 × magnification for BCA2, hHR23a and 14-3-3σ immunostaining, aswell as H&E staining.

Mentions: The prevalence of co-expression of BCA2 and either hHR23a or 14-3-3σ was evaluated in multiple breast cancer cases. Serial sections of two breast cancer tissue microarrays (TMA) totalling 105 case were were assessed. TMA immunostaining was visualized with DAB following probing with antibodies against BCA2, hHR23a and 14-3-3σ. Sections were scored for percentage of cells staining in the cytoplasmic or nuclear compartments of cells. An average of the triplicate punches was used to score each case. Cells were designated as having nuclear staining, if the nuclear compartment was significantly darker than the surrounding cytoplasmic staining, to rule out mis-scoring due to cytoplasm overlaying the nucleus. Percent abundance of staining as it pertained to each cellular compartment was defined by the number of tumor cells which expressed the proteins in question in each location, independent of expression of proteins in the other compartments (examples of staining are shown in Figure 3A). Sections were further scored for the intensity of DAB staining, and were classified as either 3+, 2+, 1+ or 0 (Figure 3B), corresponding to strong, medium, weak or negative staining (staining verified by J.Z). Cells were considered to have low staining if intensity was scored at 0 or 1+ or if 10% or less of tumor cells were staining. Cores were considered to be staining highly if more than 10% of tumor cells were staining at an intensity of 2+ or 3+.


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)

Immunohistochemistry of breast cancer TMA (n = 105). [A] An example of how tumors were scored for protein localization. The top panel is staining positively in the cytoplasm with no nuclear staining, the bottom panel shows a tissue which is staining weakly in the cytoplasm and stongly in the nuclear compartment. [B] Shows examples of the criteria for staining intensity, where 3+ (top left) has the strongest staining/most intense and 1+ (top right) which has the weakest staining/least intense. A tissue staining negatively (0), is also shown (bottom right). [C] Shows a typical staing pattern for a serial section of the same tumor at 5 × magnification and 20 × magnification for BCA2, hHR23a and 14-3-3σ immunostaining, aswell as H&E staining.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Immunohistochemistry of breast cancer TMA (n = 105). [A] An example of how tumors were scored for protein localization. The top panel is staining positively in the cytoplasm with no nuclear staining, the bottom panel shows a tissue which is staining weakly in the cytoplasm and stongly in the nuclear compartment. [B] Shows examples of the criteria for staining intensity, where 3+ (top left) has the strongest staining/most intense and 1+ (top right) which has the weakest staining/least intense. A tissue staining negatively (0), is also shown (bottom right). [C] Shows a typical staing pattern for a serial section of the same tumor at 5 × magnification and 20 × magnification for BCA2, hHR23a and 14-3-3σ immunostaining, aswell as H&E staining.
Mentions: The prevalence of co-expression of BCA2 and either hHR23a or 14-3-3σ was evaluated in multiple breast cancer cases. Serial sections of two breast cancer tissue microarrays (TMA) totalling 105 case were were assessed. TMA immunostaining was visualized with DAB following probing with antibodies against BCA2, hHR23a and 14-3-3σ. Sections were scored for percentage of cells staining in the cytoplasmic or nuclear compartments of cells. An average of the triplicate punches was used to score each case. Cells were designated as having nuclear staining, if the nuclear compartment was significantly darker than the surrounding cytoplasmic staining, to rule out mis-scoring due to cytoplasm overlaying the nucleus. Percent abundance of staining as it pertained to each cellular compartment was defined by the number of tumor cells which expressed the proteins in question in each location, independent of expression of proteins in the other compartments (examples of staining are shown in Figure 3A). Sections were further scored for the intensity of DAB staining, and were classified as either 3+, 2+, 1+ or 0 (Figure 3B), corresponding to strong, medium, weak or negative staining (staining verified by J.Z). Cells were considered to have low staining if intensity was scored at 0 or 1+ or if 10% or less of tumor cells were staining. Cores were considered to be staining highly if more than 10% of tumor cells were staining at an intensity of 2+ or 3+.

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