Abstract 4035: The breast cancer associated gene 2 (BCA2) binds to Ubc9 and is involved in sumoylation
Bibliographic record
Abstract
Abstract SUMOylation is a common post-translational modification in which small ubiquitin-like modifier (SUMO) protein moiety is attached to a lysine residue of the target protein and alters its stability, protein-protein interactions, protein-DNA interactions and subcellular localization. Recently, there is an increased recognition for this process as SUMO conjugation plays a critical role in basic cellular processes like proliferation, intracellular signaling and stabilization of transcriptional factors and chromatin remodeling factors. We are studying the Breast Cancer Associated Gene 2 (BCA2), a well characterized ubiquitin E3 ligase which we discovered by subtractive hybridization cloning. In order to identify substrates for the E3 ligase, we performed bacterial two-hybrid screening and found that the SUMO conjugating enzyme Ubc9 as a binding partner. In this study, we examined, whether the BCA2 ligase might be involved in sumoylation.Bioinformatic analyses of BCA2 sequences by SUMOplotTM prediction revealed Lysine 32 as the possible SUMO modification site. To establish direct protein-protein interactions, we performed pull down assays using GST- tagged Ubc9 expressed by the pGEX4T3 expression vector and purified recombinant wild-type BCA2. Results were analyzed by SDS-PAGE Western blotting. To assess if BCA2 and Ubc9 co-localize, we transfected Hek293T cells with BCA2 and Ubc9 constructs and analyzed them using Immunoflorescence staining. We found that BCA2 interacts with Ubc9 and they are co-localized in both the cytoplasm and nucleus. To investigate whether BCA2 is sumoylated by SUMO 1 or SUMO 2/3 we performed in vitro sumoylation assays. SUMO E1 and E2 were incubated along with recombinant BCA2, SUMO1 or SUMO 2/3 respectively for 2 hours at 37°C. The Western blots were probed with SUMO1, SUMO 2/3 and BCA2 antibodies. We also performed in vivo sumoylation with exogenous BCA2 by transfecting pCMV BCA2-Flag vector into Hek293T cells by using Fugene transfection reagent and after 48 hours of transfection, cells were lysed with denaturing lysis buffer and pull downs were performed using anti Flag beads followed by Western blots. These in vitro and in vivo sumoylation studies reveal that BCA2 is sumoylated by SUMO 2/3, but not SUMO1. In the recent past it has become more clear that both the ubiquitination and sumoylation systems communicate and jointly affect the properties of common substrate proteins and there is an increasing evidence in breast cancer development that deregulation of these processes play a vital role. Our findings of a possible involvement of BCA2 in both of these processes opens up avenues that should be further exploited in order to understand the regulatory role of BCA2 in breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4035. doi:10.1158/1538-7445.AM2011-4035
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.001 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.001 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.006 | 0.004 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".