The BRCT repeats of BRCA1 are essential for tumor suppression. Phospho-peptide affinity proteomic analysis identified a novel protein, Abraxas, that directly binds the BRCA1 BRCT repeats through a phospho-SXXF motif. Abraxas binds BRCA1 mutually exclusively with BACH1 and CTIP, forming a third Brca1 complex. Abraxas recruits the ubiquitin-interacting motif (UIM) containing protein Rap80 to BRCA1. Both Abraxas and Rap80 are required for DNA damage resistance, G2/M checkpoint control and DNA repair. Rap80 is required for a subset of Brca1-foci formation in response to IR and the UIM domains alone are capable of foci formation. The Rap80/Abraxas complex may help recruit Brca1 to DNA damage sites in part through recognition of ubiquitinated proteins.The Brca1 tumor suppressor is associated with hereditary breast and ovarian cancer and has significant roles in DNA repair, cell cycle checkpoint control and maintenance of genomic stability (1, 2). Brca1 contains a N-terminal RING domain, a SQ cluster domain phosphorylated by DNA damage signaling kinases ATM/ATR(3), and two C-terminal BRCT repeats. Brca1 forms a heterodimeric complex with Bard1 (4) that exhibits E3 ubiqutin ligase activity(5-7). Many tumorigenic mutations in BRCA1 disrupt the BRCT repeats which constitute a phospho-peptide recognition domain that binds peptides containing a pSxxF motif (8-11).To identify proteins that bind the BRCT domains of Brca1, we combined peptide affinity purification and stable isotope labeling with amino acids in cell culture (SILAC) (12-14) to identify phospho-peptides that directly bind to the BRCT domain of Brca1 and to quantify their abundance in the presence of DNA damage by mass spectrometry. Proteins from cells grown in heavy medium and cells grown in light medium treated with 10 Gy IR were prepared, mixed (1:1) and digested with Trypsin. Tryptic peptides bound to Gst-Brca1-BRCT were identified by mass spectrometry analysis. We searched for phospho-peptides that contained pSXXF motif and compared the peptides to the list of proteins we recently identified in an ATM/ATR substrate screen (Matsuoka et al., in preparation (Fig. 1B). We named this protein Abraxas for the Greek god and the gene Abra1. SILAC quantification analysis also indicated a doubly phosphorylated peptide, GEGFYS#RS#PTF containing p-S404 and p-S406, was enriched approximately 8-fold after DNA damage (Fig. S1). Both p-S406, and doubly, p-S404 p-S406, phosphorylated Abraxas peptides bound purified GST-Brca1-BRCT, while p-S404 and unphosphorylated peptides did not (Fig. 1C), a result confirmed using BIACORE analysis (Fig. S2A).Abraxas is well conserved in vertebrates (Fig. S3). Bioinformatics analysis also revealed KIAA0157, which is 39% identical to Abra1 in the N-terminal two-thirds of the protein (AA 1-260) we call the "ABR" domain. This protein, now named Abro1 (Abraxas Brother 1) ( Fig. 1D), is also conserved in vertebrates ( Fig. S3) but lacks the pSXXF motif and does not bind to Brca1 (data not shown).Full length Abraxas binds the BRCT-repeats...

The MYC proto-oncogene encodes a transcription factor that has been implicated in the genesis of many human tumours. Here, we used a bar-code short hairpin RNA (shRNA) screen to identify multiple genes that are required for MYC function. One of these genes encodes USP28, an ubiquitin-specific protease. USP28 is required for MYC stability in human tumour cells. USP28 binds to MYC through an interaction with FBW7alpha, an F-box protein that is part of an SCF-type ubiquitin ligase. Therefore, it stabilizes MYC in the nucleus, but not in the nucleolus, where MYC is degraded by FBW7gamma. High expression levels of USP28 are found in colon and breast carcinomas, and stabilization of MYC by USP28 is essential for tumour-cell proliferation.