Effects of partner proteins on BCA2 RING ligase activity.
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.
Sunnybrook Research Institute, Toronto, ON, Canada.
Stephanie
S
Bacopulos
Yutaka
Y
Amemiya
Wenyi
W
Yang
Judit
J
Zubovits
Angelika
A
Burger
Martin
M
Yaffe
Arun K
AK
Seth
D048948:14-3-3 Proteins / Q000378:metabolism*; D014408:Biomarkers, Tumor / Q000378:metabolism*; D015153:Blotting, Western; D001943:Breast Neoplasms / Q000378:metabolism* / Q000473:pathology; D002352:Carrier Proteins / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D045643:DNA Repair Enzymes / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005092:Exonucleases / Q000378:metabolism*; D005095:Exoribonucleases; D005260:Female; D006367:HeLa Cells; D006801:Humans; D007150:Immunohistochemistry; D046228:Microarray Analysis; D009363:Neoplasm Proteins / Q000378:metabolism*; D020798:Two-Hybrid System Techniques; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism*; D054875:Ubiquitination
D048948:14-3-3 Proteins; D014408:Biomarkers, Tumor; D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D009363:Neoplasm Proteins; C086985:RAD23A protein, human; C506934:RNF115 protein, human; D044767:Ubiquitin-Protein Ligases; D005092:Exonucleases; D005095:Exoribonucleases; C413442:SFN protein, human; D045643:DNA Repair Enzymes