Lea M. Starita

BRCA1-Dependent Ubiquitination of γ-Tubulin Regulates Centrosome Number

ABSTRACT Proper centrosome duplication and spindle formation are crucial for prevention of chromosomal instability, and BRCA1 plays a role in this process. In this study, transient inhibition of BRCA1 function in cell lines derived from mammary tissue caused rapid amplification and fragmentation of centrosomes. Cell lines tested that were derived from nonmammary tissues did not amplify the centrosome number in this transient assay. We tested whether BRCA1 and its binding partner, BARD1, ubiquitinate centrosome proteins. Results showed that centrosome components, including γ-tubulin, are ubiquitinated by BRCA1/BARD1 in vitro. The in vitro ubiquitination of γ-tubulin was specific, and function of the carboxy terminus was necessary for this reaction; truncated BRCA1 did not ubiquitinate γ-tubulin. BRCA1/BARD1 ubiquitinated lysines 48 and 344 of γ-tubulin in vitro, and expression in cells of γ-tubulin K48R caused a marked amplification of centrosomes. This result supports the notion that the modification of these lysines in living cells is critical in the maintenance of centrosome number. One of the key problems in understanding the biology of BRCA1 has been the identification of a specific target of BRCA1/BARD1 ubiquitination and its effect on mammary cell biology. The results of this study identify a ubiquitination target and suggest a biological impact important in the etiology of breast cancer.

Global analysis of phosphorylation and ubiquitylation cross-talk in protein degradation

Cross-talk between different types of post-translational modifications on the same protein molecule adds specificity and combinatorial logic to signal processing, but it has not been characterized on a large-scale basis. We developed two methods to identify protein isoforms that are both phosphorylated and ubiquitylated in the yeast Saccharomyces cerevisiae, identifying 466 proteins with 2,100 phosphorylation sites co-occurring with 2,189 ubiquitylation sites. We applied these methods quantitatively to identify phosphorylation sites that regulate protein degradation via the ubiquitin-proteasome system. Our results demonstrate that distinct phosphorylation sites are often used in conjunction with ubiquitylation and that these sites are more highly conserved than the entire set of phosphorylation sites. Finally, we investigated how the phosphorylation machinery can be regulated by ubiquitylation. We found evidence for novel regulatory mechanisms of kinases and 14-3-3 scaffold proteins via proteasome-independent ubiquitylation.

The multiple nuclear functions of BRCA1: transcription, ubiquitination and DNA repair.

Interest in BRCA1 stems from its role as a tumour suppressor in breast and ovarian cancer. Intensive research in BRCA1 has revealed little about its specific role in cancer; rather, this protein has been implicated in a multitude of important cellular processes. The diverse biochemical activities of BRCA1 combine to protect the genome from damage. New data reveal that BRCA1 transcriptionally regulates some DNA-repair genes, and, in addition, new roles for BRCA1 have been identified in heterochromatin formation on the X chromosome, double-strand-break repair, and ubiquitination. These diverse activities of BRCA1 may be linked in a single pathway, or BRCA1 might function in multiple nuclear processes.

Centrosomal Microtubule Nucleation Activity Is Inhibited by BRCA1-Dependent Ubiquitination

ABSTRACT In this study we find that the function of BRCA1 inhibits the microtubule nucleation function of centrosomes. In particular, cells in early S phase have quiescent centrosomes due to BRCA1 activity, which inhibits the association of γ-tubulin with centrosomes. We find that modification of either of two specific lysine residues (Lys-48 and Lys-344) of γ-tubulin, a known substrate for BRCA1-dependent ubiquitination activity, led to centrosome hyperactivity. Interestingly, mutation of γ-tubulin lysine 344 had a minimal effect on centrosome number but a profound effect on microtubule nucleation function, indicating that the processes regulating centrosome duplication and microtubule nucleation are distinct. Using an in vitro aster formation assay, we found that BRCA1-dependent ubiquitination activity directly inhibits microtubule nucleation by centrosomes. Mutant BRCA1 protein that was inactive as a ubiquitin ligase did not inhibit aster formation by the centrosome. Further, a BRCA1 carboxy-terminal truncation mutant that was an active ubiquitin ligase lacked domains critical for the inhibition of centrosome function. These experiments reveal an important new functional assay regulated by the BRCA1-dependent ubiquitin ligase, and the results suggest that the loss of this BRCA1 activity could cause the centrosome hypertrophy and subsequent aneuploidy typically found in breast cancers.

Overexpression of a protein fragment of RNA helicase A causes inhibition of endogenous BRCA1 function and defects in ploidy and cytokinesis in mammary epithelial cells.

The breast- and ovarian-specific tumor suppressor, BRCA1, has been implicated to function in many nuclear processes, including DNA damage repair, recombination, transcription, ubiquitination, cell cycle checkpoint enforcement, and centrosome regulation. Utilizing a previously described interaction between BRCA1 and RNA helicase A (RHA), we have developed a dominant-negative approach to block BRCA1 function in human breast epithelial cells. Overexpression of a truncated RHA peptide that can bind to the BRCA1 carboxy-terminus prevents normal BRCA1 function, such as BRCA1 association with nuclear foci following DNA damage. Overexpression of this dominant-negative protein induces pleomorphic nuclei, aberrant mitoses with extra centrosomes, and tetraploidy. This model system allows us to observe changes to mammary epithelial cells that occur acutely following loss of BRCA1 function. Furthermore, inhibition of BRCA1 via overexpressing the RHA fragment coincides with a reduction in PARP-1 protein expression, suggesting a possible mechanism for BRCA1 in the maintenance of genomic integrity.

Massively Parallel Functional Analysis of BRCA1 RING Domain Variants

Interpreting variants of uncertain significance (VUS) is a central challenge in medical genetics. One approach is to experimentally measure the functional consequences of VUS, but to date this approach has been post hoc and low throughput. Here we use massively parallel assays to measure the effects of nearly 2000 missense substitutions in the RING domain of BRCA1 on its E3 ubiquitin ligase activity and its binding to the BARD1 RING domain. From the resulting scores, we generate a model to predict the capacities of full-length BRCA1 variants to support homology-directed DNA repair, the essential role of BRCA1 in tumor suppression, and show that it outperforms widely used biological-effect prediction algorithms. We envision that massively parallel functional assays may facilitate the prospective interpretation of variants observed in clinical sequencing.

Massively parallel single-amino-acid mutagenesis

Random mutagenesis methods only partially cover the mutational space and are constrained by DNA synthesis length limitations. Here we demonstrate programmed allelic series (PALS), a single-volume, site-directed mutagenesis approach using microarray-programmed oligonucleotides. We created libraries including nearly every missense mutation as singleton events for the yeast transcription factor Gal4 (99.9% coverage) and human tumor suppressor p53 (93.5%). PALS-based comprehensive missense mutational scans may aid structure-function studies, protein engineering, and the interpretation of variants identified by clinical sequencing.

Activity-enhancing mutations in an E3 ubiquitin ligase identified by high-throughput mutagenesis

Significance Ubiquitin is a 76 residue protein that is attached to target proteins as a posttranslational modification. This modification is dependent on the successive activity of three enzymes, designated E1, E2, and E3. We developed a high-throughput mutagenesis strategy to probe the mechanism of E3-catalyzed transfer of ubiquitin from the E2 to the target protein. By scoring the effect of nearly 100,000 mutations in an E3, we identified mutations that affect direct and allosteric interactions between the E3 and the E2. These results highlight the general utility of high-throughput mutagenesis in delineating the molecular basis of enzyme activity. Although ubiquitination plays a critical role in virtually all cellular processes, mechanistic details of ubiquitin (Ub) transfer are still being defined. To identify the molecular determinants within E3 ligases that modulate activity, we scored each member of a library of nearly 100,000 protein variants of the murine ubiquitination factor E4B (Ube4b) U-box domain for auto-ubiquitination activity in the presence of the E2 UbcH5c. This assay identified mutations that enhance activity both in vitro and in cellular p53 degradation assays. The activity-enhancing mutations fall into two distinct mechanistic classes: One increases the U-box:E2-binding affinity, and the other allosterically stimulates the formation of catalytically active conformations of the E2∼Ub conjugate. The same mutations enhance E3 activity in the presence of another E2, Ube2w, implying a common allosteric mechanism, and therefore the general applicability of our observations to other E3s. A comparison of the E3 activity with the two different E2s identified an additional variant that exhibits E3:E2 specificity. Our results highlight the general utility of high-throughput mutagenesis in delineating the molecular basis of enzyme activity.

Identification of Domains of BRCA1 Critical for the Ubiquitin-Dependent Inhibition of Centrosome Function

The breast and ovarian cancer specific tumor suppressor BRCA1, bound to BARD1, has multiple functions aimed at maintaining genomic stability in the cell. We have shown earlier that the BRCA1/BARD1 E3 ubiquitin ligase activity regulates centrosome-dependent microtubule nucleation. In this study, we tested which domains of BRCA1 and BARD1 were required to control the centrosome function. In the present study, (a) we confirmed that the ubiquitination activity of BRCA1 regulates centrosome number and function in Hs578T breast cancer cells; (b) we observed that both the amino and carboxyl termini of BRCA1 are required for regulation of centrosome function in vitro; (c) an internal domain (770-1,290) is dispensable for centrosome regulation; (d) BARD1 is required for regulation of centrosome function and protein sequences within the terminal 485 amino acids are necessary for activity; and (e) BARD1 is localized at the centrosome throughout the cell cycle. We conclude that the BRCA1-dependent E3 ubiquitin ligase functions to restrain centrosomes in mammary cells, and loss of BRCA1 in the precancerous breast cell leads to centrosomal hypertrophy, a phenotype commonly observed in incipient breast cancer.

BRCA1 DNA-binding activity is stimulated by BARD1.

The breast- and ovarian-specific tumor suppressor BRCA1 has been implicated in numerous cellular processes, including transcription, ubiquitination, and DNA repair. Its tumor suppression activity is tightly linked to that of BARD1, a protein that heterodimerizes with BRCA1. It has been previously shown that BRCA1 binds to DNA, an interesting functional observation in light of the genetic data linking BRCA1 to DNA repair pathways. In this work, we reexamine the DNA-binding properties of BRCA1, comparing them with the DNA-binding properties of the BRCA1/BARD1 heterodimer. Because nuclear BRCA1 exists as a heterodimer with BARD1, it is likely that in vitro studies of the heterodimer will provide a more accurate model of physiologic conditions. Our results indicate that whereas BARD1 cannot directly bind DNA, it does enhance DNA binding by BRCA1. This is a surprising observation as both DNA-binding domains are distal to the BARD1-interacting RING domain of BRCA1. Further analysis of the dimerization reveals that the BRCA1/BARD1 interaction is not limited to the amino-terminal RING domains of each protein. The carboxyl terminus of BRCA1 contributes significantly to the stability of the heterodimer. We also show that the presence of BARD1 has a secondary effect, as autoubiquitination of BRCA1/BARD1 heterodimers additionally enhances the affinity of BRCA1 for DNA. Together, these data suggest that BRCA1 and BARD1 heterodimerization is stabilized via domains not previously thought to interact and that BARD1 acts in both ubiquitination-dependent and ubiquitination-independent ways to influence the role of BRCA1 in DNA repair.