NFBD 1 / MDC 1 Regulates Cav 1 and Cav 2 Independently of DNA Damage and p 53

NFBD1/MDC1 is involved in DNA damage checkpoint signaling and DNA repair. NFBD1 binds to the chromatin component gH2AX at sites of DNA damage, causing amplification of ataxia telangiectasia-mutated gene (ATM) pathway signaling and recruitment of DNA repair factors. Residues 508–995 of NFBD1 possess transactivation activity, suggesting a possible role of NFBD1 in transcription. Furthermore, NFBD1 influences p53-mediated transcription in response to adriamycin. We sought to determine the role of NFBD1 in ionizing radiation (IR)–responsive transcription and if NFBD1 influences transcription independently of p53. Using microarray analysis, we identified genes altered upon NFBD1 knockdown. Surprisingly, most NFBD1 regulated genes are regulated in both the absence and presence of IR, thus pointing toward a novel function for NFBD1 outside of the DNA damage response. Furthermore, NFBD1 knockdown regulated genes mostly independent of p53 knockdown. These genes are involved in pathways including focal adhesion signaling, carbohydrate metabolism, and insulin signaling. We found that CAV1 and CAV2 mRNA and protein levels are reduced by both NFBD1 knockdown and knockout independently of IR and p53. NFBD1-depleted cells exhibit some similar phenotypes to Cav1-depleted cells. Furthermore, like Cav1-depletion, NFBD1 shRNA increases Erk phosphorylation. Thus, Cav1 could act as a mediator of the DNA-damage independent effects of NFBD1 in mitogenic signaling. Mol Cancer Res; 9(6); 766–81. 2011 AACR. Introduction NFBD1 was discovered by a computational screen for genes encoding proteins with BRCA1 carboxy-terminal (BRCT) domains (1). NFBD1 is also known as MDC1 (mediator of DNA damage checkpoint 1). NFBD1 is a DNA damage response factor that modulates the ataxia telangiectasia-mutated gene (ATM)–Chk2 signaling pathway (2–6). Previous work indicates that NFBD1 amplifies ATM signals by binding directly to gH2AX, thus recruiting active ATM and other DNA damage response factors to sites of DNA damage. H2AX is a chromatin component that is phosphorylated by ATM and other factors to form gH2AX (7). The interaction between NFBD1 and gH2AX is important for homologous recombination in addition to checkpoint signaling (8). Another mechanism by which NFBD1 influences checkpoint signaling is by binding to the MRE11/Rad50/NBS1 (MRN) complex, thus allowing for sustained MRN complex association with damaged chromatin (3, 5, 9–14). The MRN complex is essential for activation of ATM following DNA damage (15–22). Furthermore, Mre11 possesses nuclease activity that is important for DNA repair and genomic stability (23). Although the best characterized function for NFBD1 is in DNA damage signaling, there is also evidence that NFBD1 possesses transactivation activity. Residues 508–995 activate transcription of a chloramphenicol acetyl transferase reporter in yeast and mammalian cells (24). That region of NFBD1 has not been reported to contain additional motifs or functions. Another study establishes a relationship between NFBD1 and p53. Treatment of A549 lung carcinoma cells with adriamycin (ADR) and other DNA damaging agents reduces NFBD1 protein and mRNA at late time points (6–12 hours). The decrease in NFBD1 levels occurs simultaneously with increased p53 phosphorylation on S15 and increased abundance of p53 targets. Forcing greater NFBD1 expression decreases p53 S15 phosphorylation and reduces expression of p53 targets at late time points after ADR treatment. Luciferase reporter assays using NFBD1 overexpression and knockdown show that NFBD1 Authors' Affiliations: Department of Pathology, Graduate Program in Genetics, and Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut Note: Supplementary data for this article are available at Molecular Cancer Research Online (http://mcr.aacrjournals.org/). K. A. Wilson and S. A. Colavito have contributed equally to this work. Corresponding Author:David F. Stern, Yale University, 333 Cedar Street, P.O. Box 208023, New Haven, CT 06520. Phone: 203-785-4832; Fax: 1-203-785-7467; E-mail: df.stern@yale.edu doi: 10.1158/1541-7786.MCR-10-0317 2011 American Association for Cancer Research. Molecular Cancer Research Mol Cancer Res; 9(6) June 2011 766 on July 10, 2017. © 2011 American Association for Cancer Research. mcr.aacrjournals. org Downloaded from Published OnlineFirst May 6, 2011; DOI: 10.1158/1541-7786.MCR-10-0317