Mutations affecting the BRCT domains of the breast cancer-associated tumor suppressor BRCA1 disrupt the recruitment of this protein to DNA double-strand breaks (DSBs). The molecular structures at DSBs recognized by BRCA1 are presently unknown. We report the interaction of the BRCA1 BRCT domain with RAP80, a ubiquitin-binding protein. RAP80 targets a complex containing the BRCA1-BARD1 (BRCA1-associated ring domain protein 1) E3 ligase and the deubiquitinating enzyme (DUB) BRCC36 to MDC1-gammaH2AX-dependent lysine(6)- and lysine(63)-linked ubiquitin polymers at DSBs. These events are required for cell cycle checkpoint and repair responses to ionizing radiation, implicating ubiquitin chain recognition and turnover in the BRCA1-mediated repair of DSBs.

DNA double strand breaks (DSBs) initiate reversible cellular checkpoint and repair activities. Whereas many of the activating events at DSBs have recently been elucidated, the mechanisms used to terminate responses at these sites are largely undefined. Here we report a pathway required to reverse RNF8-Ubc13 dependent ubiquitination events on chromatin flanking DSBs. Inhibition of the Rap80-BRCC36 de-ubiquitinating enzyme complex partially restored DSB-associated ubiquitin levels following RNF8 knockdown or proteasome inhibition. Similarly, BRCC36 knockdown or expression of a BRCC36 de-ubiquitinating enzyme-inactive mutant rescued both 53BP1 recruitment to DSBs and ionizing radiation-induced ␥H2AX ubiquitination following RNF8 depletion, and mitigated ionizing radiation sensitivity resulting from RNF8 deficiency. Thus, concomitant and opposing RNF8-Ubc13 ubiquitin ligase and Rap80-BRCC36 ubiquitin hydrolysis activities are responsible for determining steady-state ubiquitin levels at DNA DSBs. These findings reveal a Rap80-BRCC36 dependent pathway that is required for appropriate DSB recruitment and repair responses.BRCA1 ͉ DNA damage ͉ ubiquitin M ultiple, parallel signaling pathways converge upon DNA double-strand breaks (DSBs) to initiate temporal and spatial coordination of checkpoint and repair responses. These DNA damage response activities are mediated in part by accumulation of DNA repair proteins at both chromatin and non-nucleosomal DNA regions flanking DSBs (1-3). Repair factor targeting occurs within minutes of DSB induction, providing strong evidence that molecular recognition events rapidly develop at sites of DNA damage (1,4,5). Following the resolution of DNA damage, repair proteins dissociate from DSBs, thus alleviating cell cycle checkpoint responses and allowing resumption of cell proliferation. It is unclear, however, if DSB termination and activation pathways occur simultaneously in an equilibrium process during the earliest stages of repair, or if DNA damage response resolution occurs in a step-wise manner subsequent to the completion of DNA repair.Some of the recruitment events at DSBs have recently been elucidated for the breast and ovarian cancer suppressor protein BRCA1 (Breast Cancer 1, early onset) and the checkpoint and repair protein 53BP1 (p53 Binding Protein 1) (6-12). Histone H2AX phosphorylation by the PI3K-like kinase members ATM (Ataxia Telangiectasia Mutated) and DNA-PKcs (DNADependent Protein Kinase catalytic subunit) occurs at chromatin flanking DSBs to initiate a direct interaction between phosphorylated serine 139 of H2AX (␥H2AX) and the MDC1 (Mediator of DNA Damage Checkpoint Protein 1) protein (13-15). PI3K-like kinase phosphorylation of MDC1 (13, 16) creates a binding site for the E3 ubiquitin ligase RNF8 (Ring Finger 8). In conjunction with the E2 enzyme Ubc13 (ubiquitin-conjugating 13), RNF8 ubiquitinates histones H2A and H2AX in a K63-linked manner to create a docking site for DNA repair proteins to accumulate at DSBs (6-8, 17). These DSB chromatin-associated K63-link...

Lysosomal-associated protein transmembrane-4 beta (LAPTM4B), a novel gene upregulated in hepatocellular carcinoma (HCC), was cloned using fluorescence differential display, RACE, and RT-PCR. It contains seven exons and encodes a 35-kDa protein with four putative transmembrane regions. Both the N-and C-termini of the protein are proline-rich, and may serve as potential ligands for the SH3 domain. Immunohistochemical analysis localized the protein predominantly to intracellular membranes. Northern blot showed that the LAPTM4B mRNAs were remarkably upregulated in HCC (87.3%) and correlated inversely with differentiation status. LAPTM4B was also overexpressed in many HCC-derived cell lines. It was also highly expressed in fetal livers and certain adult normal tissues including the heart, skeletal muscle, testis, and ovary. Promoter function assays showed a distinct difference in the gene's activities between BEL7402 and HLE cell lines, suggesting that the transcription factors responsible for regulation of the gene in the two cell lines are different, and that possible negative regulatory cis-elements may exist upstream of the promoter region. It was demonstrated that the N-terminus of LAPTM4B was essential for survival of the cells. Cells harboring the full-length LAPTM4B cDNA expression clone displayed a slightly increased efficiency in colony formation. These results suggest that LAPTM4B is a potential protooncogene, whose overexpression is involved in carcinogenesis and progression of HCC. In normal cells, it may also play important roles such as regulation of cell proliferation and survival.

Rap80 targets the breast cancer suppressor protein BRCA1 along with Abraxas and the BRCC36 deubiquitinating enzyme (DUB) to polyubiquitin structures at DNA double-strand breaks (DSBs). These DSB targeting events are essential for BRCA1-dependent DNA damage response-induced checkpoint and repair functions. Here, we identify MERIT40 (Mediator of Rap80 Interactions and Targeting 40 kD)/(C19orf62) as a Rap80-associated protein that is essential for BRCA1-Rap80 complex protein interactions, stability, and DSB targeting. Moreover, MERIT40 is required for Rap80-associated lysine 63 -ubiquitin DUB activity, a critical component of BRCA1-Rap80 G2 checkpoint and viability responses to ionizing radiation. Thus, MERIT40 represents a novel factor that links BRCA1-Rap80 complex integrity, DSB recognition, and ubiquitin chain hydrolytic activities to the DNA damage response. These findings provide new molecular insights into how BRCA1 associates with independently assembled core protein complexes to maintain genome integrity.[Keywords: C19orf62; HSPC142; MERIT40; BRCA1; Rap80; Abraxas; BRCC36] Supplemental material is available at http://www.genesdev.org.

LAPTM4B (lysosomal protein transmembrane 4 beta) is a newly identified cancer-associated gene. Both of its mRNA and the encoded LAPTM4B-35 protein are significantly upregulated with more than 70% frequency in a wide variety of cancers. The LAPTM4B-35 level in cancer is evidenced to be an independent prognostic factor and its upregulation promotes cell proliferation, migration and invasion, as well as tumorigenesis in nude mice. In contrary, knockdown of LAPTM4B-35 expression by RNA interference (RNAi) reverses all of the above malignant phenotypes. We herein reveal a new role of LAPTM4B-35 in promoting multidrug resistance of cancer cells. Upregulation of LAPTM4B-35 motivates multidrug resistance by enhancement of efflux from cancer cells of a variety of chemodrugs with variant structures and properties, including doxorubicin, paclitaxel and cisplatin through colocalization and interaction of LAPTM4B-35 with multidrug resistance (MDR) 1 (P-glycoprotein, P-gp), and also by activation of PI3K/AKT signaling pathway through interaction of PPRP motif contained in the N-terminus of LAPTM4B-35 with the p85a regulatory subunit of PI3K. The specific inhibitors of PI3K and knockdown of LAPTM4B-35 expression by RNAi eliminate the multidrug resistance effect motivated by upregulation of LAPTM4B-35. In conclusion, LAPTM4B-35 motivates multidrug resistance of cancer cells by promoting drug efflux through colocalization and interaction with P-gp, and anti-apoptosis by activating PI3K/AKT signaling. These findings provide a promising novel strategy for sensitizing chemical therapy of cancers and increasing the chemotherapeutic efficacy through knockdown LAPTM4B-35 expression by RNAi.

Loss of TGFBI, a secreted protein induced by transforming growth factor-b, has been implicated in cell proliferation, tumor progression, and angiogenesis by in vitro studies. However, in vivo antitumor functions of TGFBI as well as the underlying molecular mechanism are not well understood. To these aims, we have generated a mouse model with disruption of TGFBI genomic locus. Mice lacking TGFBI show a retarded growth and are prone to spontaneous tumors and 7,12-dimethylbenz(a)anthracene-induced skin tumors. In relation to wild-type (WT) mouse embryonic fibroblasts (MEF), TGFBI À/À MEFs display increased frequencies of chromosomal aberration and micronuclei formation and exhibit an enhanced proliferation and early S-phase entry. Cyclin D1 is up-regulated in TGFBI À/À MEFs, which correlates with aberrant activation of transcription factor cyclic AMPresponsive element binding protein (CREB) identified by chromatin immunoprecipitation and luciferase reporter assays. TGFBI reconstitution in TGFBI À/À cells by either retroviral infection with WT TGFBI gene or supplement with recombinant mouse TGFBI protein in the culture medium leads to the suppression of CREB activation and cyclin D1 expression, and further inhibition of cell proliferation. Cyclin D1 up-regulation was also identified in most of the tumors arising from TGFBI À/À mice. Our studies provide the first evidence that TGFBI functions as a tumor suppressor in vivo.

BRCC36 is a JAMM (JAB1/MPN/Mov34 metalloenzyme) domain, lysine 63-ubiquitin (K63-Ub)-specific deubiquitinating enzyme (DUB) and a member of two protein complexes: the DNA damage-responsive BRCA1-RAP80 complex, and the cytoplasmic BRCC36 isopeptidase complex (BRISC). The presence of several identical constituents in both complexes suggests common regulatory mechanisms and potential competition between K63-Ub-related signaling in cytoplasmic and nuclear compartments. Surprisingly, we discover that BRCC36 DUB activity requires different interactions within the context of each complex. Abraxas and BRCC45 were essential for BRCC36 DUB activity within the RAP80 complex, whereas KIAA0157/Abro was the only interaction required for DUB activity within the BRISC. Poh1 also required protein interactions for activity, suggesting a common regulatory mechanism for JAMM domain DUBs. Finally, BRISC deficiency enhanced formation of the BRCA1-RAP80 complex in vivo, increasing BRCA1 levels at DNA double strand breaks. These findings reveal that JAMM domain DUB activity and K63-Ub levels are regulated by multiple mechanisms within the cell.The mammalian genome is remarkably stable despite an estimated 10 5 mutagenic events/cell cycle (1). This exquisite fidelity can be attributed to the multiple and varied activities of the DNA damage response (DDR). 4 To maintain genome integrity, eukaryotic cells activate the DDR, a complex signaling network that integrates and coordinates DNA damage recognition, cell cycle checkpoints, and DNA repair (2, 3). Recent evidence implicates ubiquitin chain formation, recognition, and breakdown at the site of the genomic lesion as an essential component of the DDR. Ubiquitin is a 76-amino acid protein that can be attached to a target protein via an isopeptide linkage between the ⑀-amino lysine residue of a target protein and the C-terminal glycine residue within ubiquitin. Protein ubiquitination can have quite complex outcomes resulting from the considerable structural information embedded within ubiquitin polymers. Specifically, a single ubiquitin monomer can be extended through the ubiquitination of any one of seven lysines or through the N terminus, creating polyubiquitin chains (4). These different ubiquitin topologies result in the formation of diverse structures resulting in vastly different biological outcomes. The canonical Lys 48 -linked polyubiquitination of proteins signals for proteasomal degradation (5); conversely, Lys 63 -linked polyubiquitin has been implicated in non-degradative signals in response to both cytoplasmic and nuclear cues. Specifically, Lys 63 -linked polyubiquitin is involved in both the recruitment and retention of DNA repair factors at sites of DNA damage (6 -8).BRCA1 is central to the DDR and forms a number of mutually exclusive macromolecular complexes, each with discrete activities (9, 10). Recently, it has been shown that the core RAP80 complex plays an important role in facilitating BRCA1 localization to DNA double strand breaks (DSBs). The RAP80 complex is a five-...

Ribosomal proteins have emerged as novel regulators of the Mdm2-p53 feedback loop, especially in the context of ribosomal stress. RPS26 is a recently identified Diamond-Blackfan Anemia-related ribosomal protein and its role in p53 activation has not been previously explored. In this study we found knockdown of RPS26 induced p53 stabilization and activation via a RPL11-dependent mechanism, resulting in p53-dependent cell growth inhibition. Moreover, RPS26 has the ability to interact with Mdm2 and inhibits Mdm2-mediated p53 ubiquitination that leads to p53 stabilization upon overexpression. Importantly, we discovered that RPS26 knockdown impaired p53's ability to transcriptionally activate its target genes in response to DNA damage, without affecting its stability. Accordingly, the cells lost the ability to induce G2/M cell cycle arrest. We further found that upon RPS26 knockdown, the DNA damage induced recruitment of p53 to the promoters of its target genes and p53 acetylation were both greatly reduced. In addition, RPS26 can interact with p53 independent of Mdm2 and coexist in a complex with p53 and p300. These data establish a role of RPS26 in DNA damage response by directly influencing p53 transcriptional activity, and suggest that RPS26 acts distinctively in different scenarios of p53 activation. Our finding also implicates p53 transcriptional activity control as an important mechanism of p53 regulation by ribosomal proteins.