Autoubiquitination of BCA2 RING E3 ligase regulates its own stability and affects cell migration.
Accumulating evidence suggests that ubiquitination plays a role in cancer by changing the function of key cellular proteins. Previously, we isolated BCA2 gene from a library enriched for breast tumor mRNAs. The BCA2 protein is a RING-type E3 ubiquitin ligase and is overexpressed in human breast tumors. In order to deduce the biochemical and biological function of BCA2, we searched for BCA2-binding partners using human breast and fetal brain cDNA libraries and BacterioMatch two-hybrid system. We identified 62 interacting partners, the majority of which were found to encode ubiquitin precursor proteins including ubiquitin C and ubiquitin A-52. Using several deletion and point mutants, we found that the BCA2 zinc finger (BZF) domain at the NH(2) terminus specifically binds ubiquitin and ubiquitinated proteins. The autoubiquitination activity of BCA2, RING-H2 mutant, BZF mutant, and various lysine mutants of BCA2 were investigated. Our results indicate that the BCA2 protein is strongly ubiquitinated and no ubiquitination is detected with the BCA2 RING-H2 mutant, indicating that the RING domain is essential for autoubiquitination. Mutation of the K26 and K32 lysines in the BZF domain also abrogated autoubiquitination activity. Interestingly, mutation of the K232 and K260 lysines in and near the RING domain resulted in an increase in autoubiquitination activity. Additionally, in cellular migration assays, BCA2 mutants showed altered cell motility compared with wild-type BCA2. On the basis of these findings, we propose that BCA2 might be an important factor regulating breast cancer cell migration/metastasis. We put forward a novel model for BCA2 E3 ligase-mediated cell regulation.
Amemiya
Yutaka
Y
Division of Molecular and Cellular Biology, Sunnybrook Research Institute, Toronto, Ontario, Canada M4N 3M5.
Azmi
Peter
P
Seth
Arun
A
D015153:Blotting, Western; D001921:Brain / Q000378:metabolism; D001943:Breast Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D002465:Cell Movement / Q000502:physiology*; D002478:Cells, Cultured; D004795:Enzyme Stability; D005333:Fetus / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D006801:Humans; D007668:Kidney / Q000378:metabolism / Q000473:pathology; D009154:Mutation / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D020349:Surface Plasmon Resonance; D014162:Transfection; D020798:Two-Hybrid System Techniques; D025801:Ubiquitin / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism*; D054875:Ubiquitination; D014945:Wound Healing; D016335:Zinc Fingers
D011994:Recombinant Proteins; D025801:Ubiquitin; C506934:RNF115 protein, human; D044767:Ubiquitin-Protein Ligases