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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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BCA2 is co-expressed with and binds to both hHR23a and 14-3-3σ. [A] The bolded black amino acids represent key residues which are imperative to the structural integrity of the BZF and RING domains. The bolded yellow residues indicate amino acids which have been modified to alanine by site-directed mutagenesis. The BZF domain (blue) binds ubiquitin and is also the site of BCA2 auto-ubiquitination (ubiquitinated lysine residues are shown in red). The AKT domain (green) is site of AKT-mediated phosphorylation of BCA2, predicted by in silico analysis. Serine residues (yellow) are likely phosphorylated, and have been mutated to alanine in the BCA2 S132, 133A mutant. The RING domain (orange) is BCA2 autoubiquitination. The yellow cysteine residues were mutated to alanine to create ligase-dead BCA2 RING mutant. [B] Immunoblots of lysates from HEK293T cells which were co-transfected with GST-tagged BCA2, BZF (C22/25A), S132/133A, RING (C228/231A) or GST-empty vector along with myc/his-tagged hHR23A. The top panel shows the result of GST-pulldown for BCA2, probed for myc-hHR23a. Whole cell GST blots are shown at 2 different exposures due to the differences in expression between GST-tagged BCA2 variants or GST alone, where the expression is substantially higher, and thus required a lower exposure. The difference in hHR23a binding is not a result of hHR23a expression, as lanes show equal loading for hHR23a and tubulin. [C] BCA2 binds to GST-tagged 14-3-3σ from bacterial cell lysates. Purified recombinant wild-type BCA2 (lane 2) as well as the RING (lane 8) mutant (mt) bind to 14-3-3σ (even lanes) but not the GST- alone protein (odd lanes). [D] Immunoblots of lysates from HEK293T cells which were co-transfected with FLAG-tagged BCA2 along with Xpress-tagged 14-3-3σ. BCA2 was introduced at 1 μg, 2 μg or 3 μg of plasmid DNA, while 14-3-3σ vector concentration was kept constant. The top panel shows increasing concentration of BCA2, the middle panel indicates a progressive decrease in expression of 14-3-3σ. β-Actin levels indicate decreasing 14-3-3σ is not an artefact of mis-loading (bottom part). [E] Immunoblots of lysates from HEK293T cells which were co-transfected with GST-tagged BCA2 along with myc/his-tagged hHR23A. BCA2 was introduced to the system in an increasing concentration, 1 μg, 2 μg or 3 μg, while the concentration of hHR23a vector was kept constant. The top panel shows increasing concentration of BCA2, the middle panel does not show any decrease in the expression of hHR23a, indicating there is no degradation. β-Tubulin levels indicate that the blot is equally loaded (third panel).
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Figure 1: BCA2 is co-expressed with and binds to both hHR23a and 14-3-3σ. [A] The bolded black amino acids represent key residues which are imperative to the structural integrity of the BZF and RING domains. The bolded yellow residues indicate amino acids which have been modified to alanine by site-directed mutagenesis. The BZF domain (blue) binds ubiquitin and is also the site of BCA2 auto-ubiquitination (ubiquitinated lysine residues are shown in red). The AKT domain (green) is site of AKT-mediated phosphorylation of BCA2, predicted by in silico analysis. Serine residues (yellow) are likely phosphorylated, and have been mutated to alanine in the BCA2 S132, 133A mutant. The RING domain (orange) is BCA2 autoubiquitination. The yellow cysteine residues were mutated to alanine to create ligase-dead BCA2 RING mutant. [B] Immunoblots of lysates from HEK293T cells which were co-transfected with GST-tagged BCA2, BZF (C22/25A), S132/133A, RING (C228/231A) or GST-empty vector along with myc/his-tagged hHR23A. The top panel shows the result of GST-pulldown for BCA2, probed for myc-hHR23a. Whole cell GST blots are shown at 2 different exposures due to the differences in expression between GST-tagged BCA2 variants or GST alone, where the expression is substantially higher, and thus required a lower exposure. The difference in hHR23a binding is not a result of hHR23a expression, as lanes show equal loading for hHR23a and tubulin. [C] BCA2 binds to GST-tagged 14-3-3σ from bacterial cell lysates. Purified recombinant wild-type BCA2 (lane 2) as well as the RING (lane 8) mutant (mt) bind to 14-3-3σ (even lanes) but not the GST- alone protein (odd lanes). [D] Immunoblots of lysates from HEK293T cells which were co-transfected with FLAG-tagged BCA2 along with Xpress-tagged 14-3-3σ. BCA2 was introduced at 1 μg, 2 μg or 3 μg of plasmid DNA, while 14-3-3σ vector concentration was kept constant. The top panel shows increasing concentration of BCA2, the middle panel indicates a progressive decrease in expression of 14-3-3σ. β-Actin levels indicate decreasing 14-3-3σ is not an artefact of mis-loading (bottom part). [E] Immunoblots of lysates from HEK293T cells which were co-transfected with GST-tagged BCA2 along with myc/his-tagged hHR23A. BCA2 was introduced to the system in an increasing concentration, 1 μg, 2 μg or 3 μg, while the concentration of hHR23a vector was kept constant. The top panel shows increasing concentration of BCA2, the middle panel does not show any decrease in the expression of hHR23a, indicating there is no degradation. β-Tubulin levels indicate that the blot is equally loaded (third panel).

Mentions: BCA2 contains three domains, the amino-terminal BCA2 Zinc-Finger (BZF) domain, the AKT phosphorylation domain, and the carboxy-terminal RING H2 domain (Figure 1A) [7,8]. BCA2's RING domain confers autoubiquitination activity, consistent with other E3 ubiquitin ligases such as RING proteins MDM2 and SIAH1 [2,9,10]. Touted as the "kiss of death" for proteins, ubiquitin is a highly conserved, 7 kDa protein modifier which targets proteins for proteasomal degradation. Ubiquitin conjugation to target proteins involves a number of well-coordinated steps, catalyzed by three enzyme types [11-14]. "Ubiquitination" has long had a negative connotation and in the past has been solely associated with the proteasome system. A staggering majority of enzymes that make up the UPS are particularly susceptible and seemingly promiscuously degraded not only through the actions of another ubiquitin ligase, trans-ubiquitination, but also through self-catalyzed ubiquitination. Recently, a review by de Bie and Ciechanover [15] discussed the mechanisms of regulation for E3 ligases. Both RING- and HECT-type ubiquitin ligases undergo various modifications and have multiple mechanisms that act to stabilize and/or activate these dynamic enzymes. Included in E3 modulation are substrate binding, phosphorylation and other post-translation modifications such as auto- or trans-ubiquitination for both proteolytic and non-proteolytic fates [15].


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)

BCA2 is co-expressed with and binds to both hHR23a and 14-3-3σ. [A] The bolded black amino acids represent key residues which are imperative to the structural integrity of the BZF and RING domains. The bolded yellow residues indicate amino acids which have been modified to alanine by site-directed mutagenesis. The BZF domain (blue) binds ubiquitin and is also the site of BCA2 auto-ubiquitination (ubiquitinated lysine residues are shown in red). The AKT domain (green) is site of AKT-mediated phosphorylation of BCA2, predicted by in silico analysis. Serine residues (yellow) are likely phosphorylated, and have been mutated to alanine in the BCA2 S132, 133A mutant. The RING domain (orange) is BCA2 autoubiquitination. The yellow cysteine residues were mutated to alanine to create ligase-dead BCA2 RING mutant. [B] Immunoblots of lysates from HEK293T cells which were co-transfected with GST-tagged BCA2, BZF (C22/25A), S132/133A, RING (C228/231A) or GST-empty vector along with myc/his-tagged hHR23A. The top panel shows the result of GST-pulldown for BCA2, probed for myc-hHR23a. Whole cell GST blots are shown at 2 different exposures due to the differences in expression between GST-tagged BCA2 variants or GST alone, where the expression is substantially higher, and thus required a lower exposure. The difference in hHR23a binding is not a result of hHR23a expression, as lanes show equal loading for hHR23a and tubulin. [C] BCA2 binds to GST-tagged 14-3-3σ from bacterial cell lysates. Purified recombinant wild-type BCA2 (lane 2) as well as the RING (lane 8) mutant (mt) bind to 14-3-3σ (even lanes) but not the GST- alone protein (odd lanes). [D] Immunoblots of lysates from HEK293T cells which were co-transfected with FLAG-tagged BCA2 along with Xpress-tagged 14-3-3σ. BCA2 was introduced at 1 μg, 2 μg or 3 μg of plasmid DNA, while 14-3-3σ vector concentration was kept constant. The top panel shows increasing concentration of BCA2, the middle panel indicates a progressive decrease in expression of 14-3-3σ. β-Actin levels indicate decreasing 14-3-3σ is not an artefact of mis-loading (bottom part). [E] Immunoblots of lysates from HEK293T cells which were co-transfected with GST-tagged BCA2 along with myc/his-tagged hHR23A. BCA2 was introduced to the system in an increasing concentration, 1 μg, 2 μg or 3 μg, while the concentration of hHR23a vector was kept constant. The top panel shows increasing concentration of BCA2, the middle panel does not show any decrease in the expression of hHR23a, indicating there is no degradation. β-Tubulin levels indicate that the blot is equally loaded (third panel).
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Related In: Results  -  Collection

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Show All Figures
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Figure 1: BCA2 is co-expressed with and binds to both hHR23a and 14-3-3σ. [A] The bolded black amino acids represent key residues which are imperative to the structural integrity of the BZF and RING domains. The bolded yellow residues indicate amino acids which have been modified to alanine by site-directed mutagenesis. The BZF domain (blue) binds ubiquitin and is also the site of BCA2 auto-ubiquitination (ubiquitinated lysine residues are shown in red). The AKT domain (green) is site of AKT-mediated phosphorylation of BCA2, predicted by in silico analysis. Serine residues (yellow) are likely phosphorylated, and have been mutated to alanine in the BCA2 S132, 133A mutant. The RING domain (orange) is BCA2 autoubiquitination. The yellow cysteine residues were mutated to alanine to create ligase-dead BCA2 RING mutant. [B] Immunoblots of lysates from HEK293T cells which were co-transfected with GST-tagged BCA2, BZF (C22/25A), S132/133A, RING (C228/231A) or GST-empty vector along with myc/his-tagged hHR23A. The top panel shows the result of GST-pulldown for BCA2, probed for myc-hHR23a. Whole cell GST blots are shown at 2 different exposures due to the differences in expression between GST-tagged BCA2 variants or GST alone, where the expression is substantially higher, and thus required a lower exposure. The difference in hHR23a binding is not a result of hHR23a expression, as lanes show equal loading for hHR23a and tubulin. [C] BCA2 binds to GST-tagged 14-3-3σ from bacterial cell lysates. Purified recombinant wild-type BCA2 (lane 2) as well as the RING (lane 8) mutant (mt) bind to 14-3-3σ (even lanes) but not the GST- alone protein (odd lanes). [D] Immunoblots of lysates from HEK293T cells which were co-transfected with FLAG-tagged BCA2 along with Xpress-tagged 14-3-3σ. BCA2 was introduced at 1 μg, 2 μg or 3 μg of plasmid DNA, while 14-3-3σ vector concentration was kept constant. The top panel shows increasing concentration of BCA2, the middle panel indicates a progressive decrease in expression of 14-3-3σ. β-Actin levels indicate decreasing 14-3-3σ is not an artefact of mis-loading (bottom part). [E] Immunoblots of lysates from HEK293T cells which were co-transfected with GST-tagged BCA2 along with myc/his-tagged hHR23A. BCA2 was introduced to the system in an increasing concentration, 1 μg, 2 μg or 3 μg, while the concentration of hHR23a vector was kept constant. The top panel shows increasing concentration of BCA2, the middle panel does not show any decrease in the expression of hHR23a, indicating there is no degradation. β-Tubulin levels indicate that the blot is equally loaded (third panel).
Mentions: BCA2 contains three domains, the amino-terminal BCA2 Zinc-Finger (BZF) domain, the AKT phosphorylation domain, and the carboxy-terminal RING H2 domain (Figure 1A) [7,8]. BCA2's RING domain confers autoubiquitination activity, consistent with other E3 ubiquitin ligases such as RING proteins MDM2 and SIAH1 [2,9,10]. Touted as the "kiss of death" for proteins, ubiquitin is a highly conserved, 7 kDa protein modifier which targets proteins for proteasomal degradation. Ubiquitin conjugation to target proteins involves a number of well-coordinated steps, catalyzed by three enzyme types [11-14]. "Ubiquitination" has long had a negative connotation and in the past has been solely associated with the proteasome system. A staggering majority of enzymes that make up the UPS are particularly susceptible and seemingly promiscuously degraded not only through the actions of another ubiquitin ligase, trans-ubiquitination, but also through self-catalyzed ubiquitination. Recently, a review by de Bie and Ciechanover [15] discussed the mechanisms of regulation for E3 ligases. Both RING- and HECT-type ubiquitin ligases undergo various modifications and have multiple mechanisms that act to stabilize and/or activate these dynamic enzymes. Included in E3 modulation are substrate binding, phosphorylation and other post-translation modifications such as auto- or trans-ubiquitination for both proteolytic and non-proteolytic fates [15].

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