BRCA1 interacts with acetyl-CoA carboxylase through its ... - Nature

Oncogene (2002) 21, 6729 – 6739 ª 2002 Nature Publishing Group All rights reserved 0950 – 9232/02 $25.00 www.nature.com/onc

BRCA1 interacts with acetyl-CoA carboxylase through its tandem of BRCT domains Cle´mence Magnard1, Richard Bachelier1,3, Anne Vincent1, Michel Jaquinod2, Sylvie Kieffer2, Gilbert M Lenoir1,4 and Nicole Dalla Venezia*,1 1 Laboratoire de Ge´ne´tique, CNRS UMR 5641, Universite´ Claude Bernard Lyon I, Faculte´ de Me´decine Rockefeller, 8 Avenue Rockefeller, 69373 Lyon cedex 08, France; 2Laboratoire de Chimie des Prote´ines - DBMS, CEN - Grenoble, 17 Avenue des Martyrs, 38054 Grenoble cedex, France

Germ-line alterations in BRCA1 are associated with an increased susceptibility to breast and ovarian cancer. BRCA1 is a 220-kDa protein that contains a tandem of two BRCA1 C-Terminal (BRCT) domains. Among missense and nonsense BRCA1 mutations responsible for family breast cancer, some are located into the BRCT tandem of BRCA1 coding sequence. In an attempt to understand how BRCT is critical for BRCA1 function, we search for partners of this BRCT tandem of BRCA1. Using a glutathione-S-transferase (GST) pull-down assay with murine cells, we isolated only one major BRCA1-interacting protein, further identified as Acetyl Coenzyme A (CoA) Carboxylase a (ACCA). We showed that this interaction is conserved through murine and human species. We also delineated the minimum interacting region as being the whole tandem of BRCT domains. We demonstrated that BRCA1 interacts in vitro and in vivo with ACCA. This interaction is completely abolished by five distinct germline BRCA1 deleterious mutations affecting the BRCT tandem of BRCA1. Interestingly, ACCA originally known as a rate-limiting enzyme for fatty acids biosynthesis, has been recently shown to be overexpressed in breast cancers and considered as a potential target for anti-neoplastic therapy. Furthermore, our observation is making a bridge between the genetic factors involved in susceptibility to breast and ovarian cancers, and environmental factors such as nutrition considered as key elements in the etiology of those cancers. Oncogene (2002) 21, 6729 – 6739. doi:10.1038/sj.onc. 1205915 Keywords: BRCA; acetyl-CoA carboxylase; BRCT; GST pull-down

*Correspondence: ND Venezia; E-mail: [email protected] Current addresses: 3National Institute of Health, Building 10, 9N105, 9000 Rockville Pike, 20892 Bethesda, USA; 4Institut GustaveRoussy, 39 Rue Camille-Desmoulins, 94805 Villejuif cedex, France Received 29 April 2002; revised 17 July 2002; accepted 25 July 2002

Introduction Genetic predisposition to breast cancer accounts for about 5% of all breast cancer cases. Among the genes associated with the familial form of the disease, BRCA1 seems to account for more than half of the families with multiple breast and/or ovarian cancers (Ford et al., 1998). Frequent loss of the wild-type allele in tumors of BRCA1 mutation carriers suggests that BRCA1 acts as a tumor suppressor gene. Although somatic mutations in the BRCA1 gene have not been detected in sporadic breast cancers (Futreal et al., 1994; Merajver et al., 1995), modulation of expression of BRCA1 may play a role in these forms of cancer as suggested by some authors (Thompson et al., 1995; Magdinier et al., 1998). The BRCA1 gene (Miki et al., 1994) encodes an 1863-amino acids protein with an apparent molecular mass of 220 kDa (Chen et al., 1995; Scully et al., 1996). Small isoforms of BRCA1 referred to as BRCA1-D11 and BRCA1-D11b, which lack either all or a portion of exon 11, respectively, have also been described (Thakur et al., 1997; Wilson et al., 1999). BRCA1 protein contains a RING finger at its amino-terminal region (Miki et al., 1994), three nuclear localization signals in the central portion of the molecule (Chen et al., 1996), and a tandem of two BRCT domains at its carboxy terminal region (Koonin et al., 1996; Bork et al., 1997; Callebaut and Mornon, 1997). Several lines of evidence suggest that BRCA1 is involved in the control of cellular growth and differentiation. In transgenic mice, homozygous disruption of the Brca1 gene results in embryonic lethality (Hakem et al., 1996). In mice, Brca1 mRNA is highly expressed during embryonic development in breast epithelia (Marquis et al., 1995). BRCA1 expression is also increased during prenatal development of the human mammary gland (Magdinier et al., 1999). Association of BRCA1 with Rad51, Rad50-Mre11-p95 complex and BASC (BRCA1associated genome surveillance complex), and their colocalization in response to DNA damage indicate that BRCA1 may have a role in DNA damagedependent replication checkpoint response (Scully et al., 1997b,c; Zhong et al., 1999; Wang et al., 2000).

BRCA1 interacts with acetyl CoA carboxylase C Magnard et al

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Accordingly, its ability to bind directly DNA structures may be important for its role in DNA repair (Paull et al., 2001). BRCA1 is supposed to have a role in transcription-coupled repair of oxidative damages (Gowen et al., 1998; Abbott et al., 1999). Several proteins have been reported to interact with BRCA1 and their binding sites are scattered along the whole protein (for review, see Deng and Brodie, 2000). Among these proteins, a novel BRCT domain-containing protein called BARD1 (Wu et al., 1996), the importin-a subunit of the nuclear transport signal receptor (Chen et al., 1996), a novel ubiquitin hydrolase BAP1 (Jensen et al., 1998), the tumor suppressor RB (Aprelikova et al., 1999), ATF1 (Houvras et al., 2000), and the BRG1 subunit of the SWI/SNF-related complex (Bochar et al., 2000) have been shown to bind BRCA1. Several works have suggested that the BRCT tandem of BRCA1 may be involved in transcription regulation in vitro (Chapman and Verma, 1996; Monteiro et al., 1996). This BRCA1-mediated transactivation has been shown to be enhanced by p300/ CBP (CREB binding protein) (Pao et al., 2000). Recently, it has been reported that this carboxyterminal region of BRCA1 alters chromatin structure in vivo (Hu et al., 1999). Furthermore, the tumorsuppressor p53 (Chai et al., 1999), the transcriptional repressor CtIP (Yu et al., 1998; Wong et al., 1998), and the RNA helicase A via RNA polymerase II (Scully et al., 1997a; Anderson et al., 1998) bind the carboxy-terminal region of BRCA1, supporting its role in transcription regulation. More recently, histone deacetylase (HDAC1 and HDAC2), and BACH1, a new member of the DEAH family of DNA helicases, have been shown to interact with the BRCT tandem of BRCA1 (Yarden and Brody, 1999; Cantor et al., 2001). Moreover, the majority of BRCA1 mutations responsible for family breast cancer affect the BRCT domains either directly (missense or nonsense mutations located into the BRCT coding sequence) or indirecly (truncating mutations located upstream to BRCT coding sequence). Although a common molecular function for the BRCT domain has not been elucidated yet, the tandem of two BRCT domains of BRCA1 is likely to have a central role in protein – protein interaction. To further understand the function of the tandem of BRCT domains of BRCA1, we used GST pull-down assay to identify a new BRCA1associated protein. We demonstrate here that ACCA interacts in vitro with the BRCT tandem as well as with BRCA1 in vivo. Results Isolation and identification of ACCA as a BRCA1 interacting protein, in mouse NIH3T3 cell line To isolate proteins that interact with the tandem of two BRCT domains of BRCA1, we performed a GST pull-down assay using 7.106 35S-methionine-labeled Oncogene

NIH3T3 cells, and a GST fusion protein containing mouse BRCA1 residues 1583 – 1812, namely GSTBRCT. The lysates were precleared using glutathionesepharose beads, and equilibrated with either glutathione beads alone, or with glutathione beads loaded with GST, or GST-BRCT. After extensive washing, the GST pull-down complexes were resolved by SDS-polyacrylamide gel electrophoresis (PAGE) and visualized by autoradiography. One major 210kDa band was visible exclusively in the GST-BRCT lane (Figure 1a). To identify the 210-kDa protein, a preparative GST pull-down assay representing approximately ten analytical GST pull-down assays described above, was separated by SDS – PAGE. The 210-kDa band detected with Coomassie blue, contained a sufficient amount of protein to be analysed by mass spectrometry (ms). Monoisotopic masses of peptides from the proteolytic digestion of the 210-kDa protein were compared to the masses of a peptide database calculated from NCBI’s or protein database. The top-ranked protein was Rattus norvegicus Acetyl-CoA carboxylase (EC 6.4.1.2; swiss prot access number P11497). This result was confirmed by ms/ms experiments: the 1409VEVGTEVTDYR1419, 1435EASFEYLQNEGER1447 (Figure 1b), and 98DFTVASP104 sequences showed 100% homologies with the Rattus norvegicus Acetyl-CoA carboxylase confirming the previous results. To further confirm the identity of the BRCTinteracting protein, a peptide microsequencing was also performed on a preparative GST pull-down assay. The 298KAAEEVGYP306 and 2267KDLVEWLEK2275 sequences were obtained. As expected, they showed 100% identity with the Rattus norvegicus Acetyl-CoA carboxylase (EC 6.4.1.2; swiss prot access number P11497). Altogether, the five peptide sequences obtained present 100% identities with rat ACCA. The murine Acca gene has not been cloned. Still, the rat ACCA protein presents 96% identity with the sequence of human ACCA protein, and the highly conserved biotin binding site and the putative ATP and CoA binding sites are identical in human, rat and yeast ACCs (Brownsey et al., 1997). Our results indicate that ACCA interacts in vitro with the tandem of BRCT domains from BRCA1. ACCA interacts specifically with the whole tandem of BRCT domains of BRCA1 To map the interaction domain of ACCA on BRCT repeat of BRCA1, GST pull-down assays with five fusion-proteins containing various portions of the BRCT motifs were performed on NIH3T3 cells (Figure 2a,b). Results presented in Figure 2c showed that fragment GST-BRCT-L containing BRCA1 residues 1512 – 1812, like GST-BRCT, interacted with ACCA. However, fragments GST-BRCT-A and GST-BRCT-B did not bind ACCA. GST-BRCA1-N containing the N-terminal 220 residues of BRCA1, as GST alone, failed to capture ACCA, demonstrating the specificity of the interaction between ACCA and the carboxy-


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Figure 1 Isolation of a BRCA1 interacting protein and identification by mass spectrometry. (a) GST pull-down assay. Whole cell lysates prepared from 7.106 35S-methionine labeled NIH3T3 cells were incubated with glutathione beads alone (7), or loaded with GST (GST) or loaded with GST fusion protein containing BRCA1 residues 1583 – 1812 (GST-BRCT). Bound proteins were resolved on a 6% SDS – PAGE and visualized by autoradiography. A 210-kDa band is present when GST-BRCT was used in the assay. (b) Reconstruct ms/ms spectrum from the tryptic digest of 210-kDa protein. Throughout ms/ms analysis, the identity of the protein was confirmed as the Rattus norvegicus Acetyl-CoA carboxylase (ACCA)

Figure 2 Mapping of interacting region of BRCA1 to ACCA. (a) Schematic diagram of the full-length mouse BRCA1 protein and the six fusion proteins generated in E.coli. The amino-terminal RING domain and the two BRCT domains are indicated by dense stippling. BRCA1 residues are marked relative to the translation initiation site. (b) Synthesis of GST fusion proteins in E.coli. Equivalent amount of fusion proteins were resolved on a 10% SDS – PAGE and visualized by Coomassie blue staining. Note that GST-BRCT-B and GST-BRCT-M were unstable. (c) GST pull-down assay using NIH3T3 cells. Cells were incubated with glutathione beads loaded with comparable amounts of GST fusion proteins. Bound proteins were resolved on a 6% SDS – PAGE and immunoblotted using streptavidin-coupled peroxidase (Witters et al., 1994). Three biotinyl proteins are visualized in NIH3T3 lysate (Input): Acetyl-CoA-carboxylase (ACC), Pyruvate carboxylase (PC) and Propionyl-CoA carboxylase (PCC). Input represents 5% of the total cellular lysates used in binding reactions. The figure shows that ACCA specifically associated with GST-BRCT and GST-BRCT-L. Positions of proteins are indicated on the right and sizes on the left

terminal region of BRCA1. Thus the minimal BRCA1 sequence required for association with ACCA corresponds to the tandem of BRCT domains, indicating that both BRCT domains are involved in the interaction with ACCA.

Conservation of the BRCA1-ACCA interaction in human mammary epithelial cells The protein sequences of the two BRCT domains of mouse BRCA1 share 75 and 58% identity with those Oncogene


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of human BRCA1, respectively (Callebaut and Mornon, 1997), and ACCA is highly conserved among species. Therefore, we examined whether the interaction between BRCA1 and ACCA was conserved among mouse and human species. As seen in Figure 3a,b, the human BRCT tandem binds human ACCA in HBL100 cells; the potential of mouse BRCT tandem to bind ACCA is conserved in human mammary epithelial cells and, reciprocally, the human BRCT tandem binds ACCA in mouse fibroblasts. Given that BRCA1 gene is associated with familial breast cancer, we searched for the interaction between the BRCT tandem and ACCA in mammary cells. As we have shown that the BRCT tandem of BRCA1 bound the endogenous ACCA in mouse fibroblasts, we examined whether this interaction was conserved in four mouse epithelial mammary cell lines, namely, Be4a, I3G2, Tac-2 and J3B1 (Montesano et al., 1997). As seen in Figure 3c, the ability of BRCT tandem to bind ACCA is retained in all of these cell lines.

Figure 3 Conservation of the BRCT-ACCA interaction in human and mouse mammary epithelial cells. (a) GST pull-down assay using two human mammary epithelial cell lines: HBL100 and MCF7. Cells were incubated with GST or mouse GST-BRCT fusion proteins loaded on glutathione beads. (b) GST pull-down assay using mouse fibroblasts NIH3T3 and human mammary epithelial cells HBL100. Cells were incubated with glutathione beads alone (7), or loaded with GST or the GST fusion protein containing human BRCA1 residues 1640 – 1863 (GST-BRCTh). (c) GST pull-down assay using four mouse mammary epithelial cell lines: Be4a, I3G2, Tac-2 and J3B1. Cells were incubated with GST or mouse GST-BRCT fusion proteins loaded on glutathione beads. Bound protein ACCA was detected using streptavidin Oncogene

Subcellular localization of the BRCA1-ACCA interaction Because BRCA1 is believed to be predominantly nuclear, our results obtained with the cytoplasmic localized ACCA raised the question about the physiological significance of this interaction. Therefore, to confirm the subcellular localization of the BRCA1ACCA interaction detected in GST pull-down assay, NIH3T3 cells were fractionated into nuclear and cytoplasmic fractions. The fractionation of these cells was checked with antibodies raised against nuclear p300 and cytoplasmic b-tubulin. A GST pull-down assay performed on these two fractions was analysed by SDS – PAGE followed by Western blotting. The results showed that the BRCT-interacting protein is mainly cytoplasmic (Figure 4). Full-length human BRCA1, mouse BRCA1 and mouse short isoform BRCA1-D11, associate ACCA in vivo To determine whether human form of BRCA1 can interact with ACCA in vivo, lysates prepared from the transfected Bosc cells with expression plasmid encoding human full-length BRCA1 were immunoprecipitated with BRCA1 antibody OP92 and analysed by immunoblotting. As shown in Figure 5a, human BRCA1 coprecipitates with endogenous ACCA. We further investigated whether ectopical mouse full-length BRCA1 or short BRCA1-D11 isoform could associate with endogenous ACCA. Immmunoprecipitation of BRCA1 or BRCA1-D11 with antibody D16 resulted in the coprecipitation of endogenous ACCA (Figure 5c). To perform the reciprocal experiment, it was necessary to generate immunological reagent that

Figure 4 Subcellular localization of the BRCA1-ACCA interaction. Nuclear (Nu) and cytosolic (Cy) fractions were prepared from NIH3T3 cells. Equivalent aliquots of each fraction were subjected to a GST pull-down assay with GST or mouse GSTBRCT, and analysed by SDS – PAGE followed by Western blotting with streptavidin specific for biotinyl proteins or with antibodies specific for p300 and b-tubulin. p300 served as a marker for nuclear distribution and b-tubulin for cytoplasmic distribution. ACCA is purified in the cytoplasmic fraction


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Figure 5 BRCA1 and ACCA associate in vivo. (a) and (b) Coimmunoprecipitation of ACCA with human BRCA1. Bosc cells were transfected with expression plasmid encoding BRCA1. Additional cultures were transfected with the parental plasmid (7). After 2 days, the cells were lysed and each lysate was subjected to immunoprecipitation with anti-BRCA1 antibody OP92 (a) or anti-ACCA antibody L3J74 (b). Pellets (P) and supernatants (S) were resolved on a 6% SDS – PAGE. The presence of endogenous ACCA was determined by immunoblotting with streptavidin (a) or anti-ACCA antibody (b), and the presence of ectopically expressed BRCA1 was determined by immunoblotting with anti-BRCA1 antibody. (c) and (d) Coimmunoprecipitation of ACCA with mouse BRCA1 and short BRCA1-D11 isoform. Separate cultures of Bosc cells were transfected with expression plasmids encoding mouse BRCA1 or BRCA1-D11. Coimmunoprecipitation was performed as above, using anti-BRCA1 antibody D16 (c) or anti-ACCA antibody (d). ACCA was detected by immunoblotting with streptavidin (c) or L3J74 antibody (d), and mouse BRCA1 and BRCA1-D11 with D16 antibody

specifically recognizes ACCA (see Materials and methods). The rabbit polyclonal L3J74 antibody raised against ACCA immunoprecipitated specifically endogenous ACCA on Bosc and HBL100 cell lysates (data not shown). As shown in Figure 5b,d, immunoprecipitation of endogenous ACCA with L3J74 antibody in lysates of transfected Bosc cells, resulted in the coprecipitation of full-length human BRCA1, mouse BRCA1, or mouse short BRCA1-D11 isoform. In vivo association of endogenous BRCA1 and ACCA To test whether ACCA associate with BRCA1 in vivo, under physiological conditions, we performed coimmunoprecipitation analysis of the endogenous proteins in HBL100 cells. Immunoprecipitation of BRCA1 with OP92 or OP107 antibodies resulted in the coprecipitation of ACCA (Figure 6a and data not shown). The negative controls, an unrelated polyclonal antibody anti-HA and a mouse pre-immune serum, were unable to coprecipitate ACCA. In reciprocal experiments,

ACCA antibody coprecipitated BRCA1 (Figure 6b), but not the negative controls, an unrelated polyclonal antibody anti-JUND and an assay performed in the absence of antibody. Therefore, the in vivo interaction of ACCA and BRCA1 was observed by coimmunoprecipitation using either ACCA-specific or BRCA1specific antibodies as the immunoprecipitating agent. We also performed the same experiment with HCC1937 cells (Tomlison et al., 1998). In these BRCA17/7 cells, no coimmunoprecipitation was observed, demonstrating that coimmunoprecipitation depends upon the presence of BRCA1 and is not due to non-specific binding of the antibodies (Figure 6c). These data strongly suggest that BRCA1 and ACCA form an endogenous complex in vivo. Germline BRCA1 mutations located in BRCT domains disrupt the association with ACCA Several germline BRCA1 mutations are found in the region coding for the carboxy-terminal segment of the Oncogene


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Figure 7 Germline BRCA1 mutations located in BRCT domains disrupt the association with ACCA. (a) Schematic diagram of the BRCT tandem from BRCA1 (GST-BRCTh) and the five mutations analysed in GST pull-down assay. (b) GST pull-down assays using HBL100 cells were performed as in Figure 3b, with wildtype (GST-BRCTh) or mutation-containing BRCT polypeptides as indicated. Negative control was performed with beads alone (7). Upper panel: Equivalent amount of fusion proteins were resolved on 10% SDS – PAGE and visualized by Western blotting using anti-GST antibody. Note that GST-BRCT-M1775R was unstable. Lower panel: Bound protein (ACCA) was resolved on a 6% SDS – PAGE and visualized by immunoblotting using streptavidin

Figure 6 Identification of endogenous BRCA1/ACCA complex in vivo. (a) and (b) Lysates from HBL100 cells were subjected to immunoprecipitation with monoclonal anti-BRCA1 antibody OP92 (a) or anti-ACCA polyclonal antibody L3J74 (b). The protein complexes associated with the antibody were resolved on 6% SDS – PAGE and then probed with streptavidin or L3J74 antibody to detect ACCA, and with OP92 antibody to detect BRCA1. Negative controls were precipitates with unrelated monoclonal antibody (anti-HA) or preimmune mouse IgG (Pre) (a), and with unrelated polyclonal antibody (anti-JUND) or beads alone (7) (b). Input represents 5% of total cellular lysate used in immunoprecipitates. (c) Lysates from BRCA17/7 cells (HCC1937) were subjected to immunoprecipitation with antiBRCA1 (OP92) or anti-ACCA (L3J74) antibody. Immunoprecipitates were resolved on 6% SDS – PAGE followed by immunoblot analysis with L3J74 to detect ACCA or with anti-BRCA1 antibody OP92. No coimmunoprecipitation was observed

protein (Figure 7a). We investigated the effect of five BRCT mutations on in vitro binding with ACCA, using GST pull-down assay on human epithelial mammary cells (HBL100). Three of the mutations produce single amino acid substitutions in BRCT-A (A1708E), BRCT-B (M1775R), or between the two Oncogene

domains (P1749R). The two others, R1835X and Y1853X, eliminate the last 29 and 11 amino acid residues of BRCA1 respectively, and therefore disrupt the second BRCT domain. As shown in Figure 7b, the interaction with ACCA was abolished by each of the five tumor-associated BRCA1 mutations. Similarly, the mouse W1777X mutation that mimics the R1835X mutation (Bachelier et al., 2001) disrupted the association with ACCA (Figure 2c). These results demonstrate that tumorigenic lesions of the BRCT tandem prevent the association of BRCA1 with ACCA. Discussion The BRCT domain was first identified as a highly conserved structural domain among more than 50 proteins implicated in the response to cellular damage (Koonin et al., 1996; Bork et al., 1997; Callebaut and Mornon, 1997). Although a common molecular function for this domain has not yet been found, BRCT domains are known to be motifs of protein – protein interactions: i.e. XRCC4 and DNA ligase IV (Critchlow et al., 1997), XRCC1 and DNA ligase III (Nash et al., 1997) and PARP (Masson et al., 1998) interact via their BRCT domains; BRCT is also responsible for the XRCC1 homodimerization (Zhang


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et al., 1998). Several germline BRCA1 mutations are found in the sequence coding for BRCT tandem, suggesting that the function of the tandem of BRCT domains of BRCA1 is important. A recent report indicated that the whole BRCT tandem is requiered for BRCA1 function in the mouse (Hohenstein et al., 2001). Here, using a GST pull-down assay with murine cells, we isolated one major BRCA1-interacting protein, further identified as ACCA. We delineated the minimum interacting region as being the whole tandem of BRCT domains. We also showed that this interaction is conserved through mouse and human species. By coimmunoprecipitation assays, we demonstrated that BRCA1 interacts with ACCA in vivo. Interestingly, three missense and two nonsense germline BRCA1 mutations located in the BRCT tandem completely abolish the interaction. Previous works indicated that some of the mutations tested here interfere with the role of the BRCT domain in activating transcription (Chapman and Verma, 1996; Monteiro et al., 1996) and block the ability of the BRCT domain to interact with CtIP (Yu et al., 1998; Li et al., 1999), histone deacetylases (Yarden and Brody, 1999), and BACH1 (Cantor et al., 2001). More recently, the mutations A1708E, M1775R and Y1853X have been shown to impair the folding of the BRCT domain (Williams et al., 2001). Accordingly, the endogenous complex reported here and its disruption by germ line BRCA1 mutations suggest that this interaction BRCA1/ACCA is important for BRCA1 function. Because BRCA1 is believed to be predominantly nuclear (Scully et al., 1996; Ruffner and Verma, 1997; Wilson et al., 1999), our results obtained with the cytoplasmic localized ACCA (Abu-Elheiga et al., 2000) raised the question about the physiological significance of this interaction. Transient transfection experiments suggest that human BRCA1-D11 or mouse BRCA1D11 isoforms were localized in cytoplasm (Thakur et al., 1997; Bachelier et al., 2000). We and others have previously shown that ectopically expressed full-length mouse or human BRCA1 are located predominantly in the cytoplasm (Bachelier et al., 2000; Wilson et al., 1999). Therefore, in an attempt to delineate the location of the BRCA1/ACCA complex, the analysis of the subcellular location of transiently expressed human BRCA1 and mouse BRCA1-D11 isoform which bound ACCA confirmed their cytoplasmic location (data not shown). Furthermore, the results obtained under physiological conditions, clearly demonstrate that endogenous BRCA1 interacts with ACCA. In this context, it is interesting to note that a recent report identified a functional nuclear export sequence in BRCA1 (Rodriguez and Henderson, 2000) suggesting that, although the major fraction of the cellular pool of BRCA1 is nuclear, endogenous BRCA1 is not restricted to the nucleus, but is able to shuttle between the nucleus and the cytoplasm. Furthermore, the interaction between the full-length BRCA1 and a cytoplasmic protein namely BRAP2 suggests that BRAP2 may retain newly synthetized BRCA1 protein

in the cytoplasm, preceeding signaling such as phosphorylation (Li et al., 1998). Interestingly, fulllength BRCA1 has been located in the cytoplasm of breast cell lines and tumors (Chen et al., 1995, 1996; Fan et al., 2001; Zhang et al., 2000), and short BRCA1-D11 form in nuclear foci in fibroblasts (Huber et al., 2001). In total, our findings that BRCA1 and ACCA form an endogenous complex in vivo raise the possibility that ACCA binds a cytoplasmic pool of fulllength BRCA1. Furthermore, the BRCA1-D11 form which functions are largely unknown, may find here a role through its association with ACCA. ACC is a biotinyl-enzyme and catalyses the rate limiting step in the biogenesis of long-chain fatty-acids (for reviews, see Brownsey et al., 1997; Kim, 1997). Two forms of ACC encoded by separate genes, have been identified: the 265-kDa ACCA and the 280-kDa ACCB (Thampy, 1989; Bianchi et al., 1990). The human ACCA gene coding sequence (Abu-Elheiga et al., 1995) is well conserved among species and shows approximately 90% identity with rat ACCA gene (Lopez-Casillas et al., 1988). ACCA is expressed in all cell types but demonstrates elevated expression and is the major form in the lipogenic tissues of adipose tissue and liver, and in mammary gland during lactation. In addition, mutant Schizosaccharomyces pombe that lacks ACC exhibits a defect in nuclear division that causes cell death during mitosis. If the level of intracellular fatty acid is affected, the progression of nuclear and cell division is impaired, leading to the production of daughter nuclei whose sizes are dramatically different (Saitoh et al., 1996). These results may suggest that an unidentified aspect of fatty acid synthesis would be crucial in mitosis. It is interesting to speculate that ACC might participate, in association with BRCA1, to the control of cell proliferation. ACCA is highly expressed in human breast carcinoma cell lines and breast carcinoma (Witters et al., 1994; Milgraum et al., 1997). Hence, fatty acid synthesis modification in cancer progression might result in change in the lipid composition of cell membrane. It is also possible that changes in the expression of transacting factors such as sterol regulatory elementbinding protein-1 (SREBP-1) (Pai et al., 1998) or Spot 14 (Moncur et al., 1998) contribute to the overexpression of genes involved in fatty acid synthesis and are the basis of the activation of the lipogenic program. Inhibition of fatty acid synthesis by cerulenin delays disease progression in a xenograft model of ovarian cancer (Pizer et al., 1996b) and induces apoptosis in breast carcinoma cells (Pizer et al., 1996a). The ACCA inhibitor, TOFA, contributes to mimick the cytotoxic effect of cerulenin (Thupari et al., 2001). These pharmacological actions on cancers suggest that the fatty acid synthesis pathway, and more specifically ACCA, may be exploited as novel targets for anti-neoplastic therapy (Kuhajda, 2000). From an epidemiological point of view, breast cancer incidence is regularly increasing in North America and Northern Europe. The major causative environmental factors associated with this increase are reproductive Oncogene


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and nutritional factor. It is striking that history of weight gain in early adult life is associated with increased breast cancer risk (Carroll, 1998; Stoll, 1995). A large prospective study coordinated by the International Agency for Research on Cancer is trying to elucidate the rational basis of this increase in relation to nutrition (Riboli, 2001). Most studies on etiology of multifactorial common diseases such as obesity, diabetes, cancer or cardiovascular diseases, emphasize a diet-gene interaction (Patterson et al., 1999). We suggest that ACCA/BRCA1 interaction may play a key role in breast cancer risk through control of energy inbalance (Ruderman and Flier, 2001). Our observation finally might lead to the elucidation of the tissue specificity of BRCA1 related cancer predisposition (Venkitaraman, 2002). The direct interaction reported here between ACCA and BRCA1 via the BRCT tandem, adds to the biological diversity of the BRCA1 functions.

Materials and methods Plasmid constructs Bacterial expression vectors The pGEX-BRCT plasmid was generated by polymerase chain reaction amplification of nucleotides 4747 – 5436 of Brca1 using the cDNA of mouse Brca1 cloned as the template, and the following primers: 5’-GCGAATTCACATCTTCAGAAGAAAGAGC-3’ and 5’-GCGTCGACTTAATCATTGGAGTCTTGTGG-3’. The 0.7-kb polymerase chain reaction product was cloned into the EcoRI/SalI sites of pGEX-4T-1 (Amersham Pharmacia Biotech). Shorter constructs with the first or second module of the BRCT tandem were obtained with the following primers: 5’-GCGAATTCACATCTTCAGAAGAAAGAGC-3’ and 5’-GCGTCGACTCAGCCCTTGAAGAGCTTTTCC-3’ for the pGEX-BRCT-A construct, 5’-GCGAATTCCGGGAAAAGCTCTTCAAGG-3’ and 5’-GCGTCGACTTAATCATTGGAGTCTTGTGG-3’ for the pGEX-BRCT-B construct. A longer form pGEX-BRCT-L containing nucleotides 4534 – 5436 of Brca1 was also prepared using the following primers: 5’-GCGAATTCGAAGGAACCCCATACCTG-3’ and 5’-GCGTCGACTTAATCATTGGAGTCTTGTGG-3’. A pGEX-BRCT mutant (pGEX-BRCT-M) was produced with the same primers than for wild-type GST-BRCT using a Brca1 cDNA with the nonsense mutation W1777X that was prepared in our laboratory (Bachelier et al., 2001) as the template. The pGEX-BRCA1-N construct was produced by polymerase chain reaction amplification of nucleotides 63 – 661 of Brca1 using the cDNA of mouse Brca1 and the following primers: 5’-GCGAATTCATGGATTTATCTGCCGTCCA-3’ and 5’-GCGTCGACTGCAGAGTGCAGCTTGC-3’. The pGEX-BRCTh plasmid was generated by polymerase chain reaction amplification of nucleotides 4917 – 5592 of BRCA1 using the cDNA of human BRCA1 cloned in pCDNA3b, and the following primers: 5’-GCGGATCCACAGCTTCAACAGAAAGGG-3’ and 5’-GCGTCGACTCAGTAGTGGCTGTGGGG-3’. The constructs were verified by DNA sequencing. The pGEX-BRCTh mutants, namely GSTBRCT-A1708E, GST-BRCT-P1749R, GST-BRCT-M1775R, GST-BRCT-R1835X and GST-BRCT-Y1853X, were generated by site-directed mutagenesis (GeneEditor in vitro siteOncogene

directed mutagenesis system, Promega) with the following primers: 5’-CTGAAATATTTTCTAGGAATTGAGGGAGGAAAATGGGTAG-3’ for A1708E, 5’-GAAACCACCAAGGTCGAAAGCGAGCAAGAG-3’ for P1749R, 5’-CCCTTCACCAACAGGCCCACAGATCAAC-3’ for M1775R, 5’-GCAC CTGTGGT GAC CTG AGAG TGG GTGTTGG-3’ for R1835X and 5’-GGAGCTGGACACCTAACTGATACCCCAG-3’ for Y1853X. The constructs were sequenced to verify the mutations. Mammalian expression vectors The pUHD 10.3 plasmids expressing mouse BRCA1 wild-type or the D11 form were prepared in our laboratory as previously described (Bachelier et al., 2000). The pCDNA3b plasmid expressing human BRCA1 has been previously described (Scully et al., 1997c). Cell culture In general, cells were grown in DMEM supplemented with 10% fetal calf serum. MCF7 cells were cultivated in the same medium with 5 mg/ml insulin. For radiolabeling, cells were starved for 1 h in DMEM without methionine and then cultivated for 3 h in DMEM without methionine containing 250 mCi of 35S-labeled methionine. For transfection, the standard calcium phosphate precipitation method was used (Gibco BRL), with 5 mg of total plasmid DNA. Cells were collected 48 h after transfection. GST pull-down assay GST-fusion proteins were synthesized in Escherischia coli strain JM109 (Promega) transformed with pGEX-4T-1, pGEX-BRCA1-N, pGEX-BRCT, pGEX-BRCT-A, pGEXBRCT-B, pGEX-BRCT-L, pGEX-BRCT-M or pGEXBRCTh, pGEX-BRCT-A1708E, pGEX-BRCT-P1749R, pGEX-BRCT-M1775R, pGEX-BRCT-R1835X, pGEXBRCT-Y1853X. They were purified by affinity chromatography on glutathione-Sepharose beads. The in vitro GST pull-down binding assay was performed by incubating cell lysates with no protein, non-recombinant GST or GST-fusion proteins, and glutathione-Sepharose beads. 10 mg of GST or GST-fusion proteins were used in each binding assay. When GST-fusion proteins of mutated BRCT domains were used (pGEX-BRCT-A1708E, pGEX-BRCT-P1749R, pGEXBRCT-M1775R, pGEX-BRCT-R1835X, pGEX-BRCTY1853X), 2 mg of each were incubated with cell lysates. Proteins complexes were extensively washed (50 mM Tris HCl pH 7.6, 100 mM KCl, 0.05% Tween 20, 1 mM DTT, 0.2 mM PMSF) to eliminate non-specific protein interactions and released by heating at 1008C in 56SDS – PAGE loading buffer and 100 mM DTT. Proteins were analysed by SDS – PAGE and visualized by autoradiography or Coomassie-blue staining, in accordance with the nature of the lysate used. Preparation of whole cell extracts and subcellular fractionation Cells were softly broken by heat shock (nitrogen/378C) in the following buffer: 25 mM Tris HCl pH 7.6, 1 mM EDTA, 1 mM EGTA, 0.2 mM PMSF, 10 mg/ml leupeptin, pepstatin A and aprotinin. Cytoplasmic and nuclear extracts were prepared as before (Bachelier et al., 2000). Immunoprecipitation, Western blot analysis and antibodies Immunoprecipitations of in vivo overexpressed or endogenous proteins were performed in lysis buffer, with 0.2 – 0.5 mg specific antibody and protein G-sepharose for 2 h at 48C.


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After precipitation, beads were washed three times with lysis buffer, and proteins were eluted by heating for 5 min in SDSloading buffer. Proteins were separated on 6 – 10% SDS – PAGE, and blotted to poly(vinylidene difluoride) (PVDF) membranes (Immobilon-P, Millipore). Membranes were blocked in TBS containing 0.05% Tween 20 and 5% dry milk. Primary and secondary antibody reactions were performed in TBS containing 0.05% Tween 20 and 2% dry milk, and proteins were detected by chemiluminescence (ECL, Amersham Pharmacia Biotech). The primary antibodies used in this study were as follows. Polyclonal antibody L3J74 was raised in rabbits against both N- and C-terminal peptides of human ACCA: MDEPSPLAQPLELNQ and AEVIRILSTMDSPST conjugated to KLH. The serum was affinity-purified using the two peptides. The specificity of the antibody was analysed on three human mammary epithelial cell lines (HBL100, MCF7, BT20), Bosc cells and also on tissues (mammary gland, ovary, pancreas) using immunoblotting (data not shown). Commercial antibodies were mouse monoclonal anti-BRCA1 (OP92 and OP107, Oncogene Research Products), goat polyclonal antiBRCA1 (D16, Santa Cruz Biotechnology), rabbit polyclonal anti-p300 (N-15, Santa Cruz Biotechnology), mouse monoclonal anti-b-tubulin (Roche Molecular Biochemicals), rabbit polyclonal anti-JUND (Santa Cruz Biotechnology), mouse monoclonal anti-HA (Roche Molecular Biochemicals) and goat polyclonal anti-GST (Amersham Pharmacia Biotech). Secondary antibodies were peroxidase-conjugated anti-goat (Dako), anti-mouse (Amersham Pharmacia Biotech) or antirabbit (Amersham Pharmacia Biotech) immunoglobulins. Streptavidin-horseradish peroxidase conjugate (Amersham Pharmacia Biotech) had a high affinity for biotin. Peptide sequencing by mass spectrometry In gel digestion The Coomassie stained 210-kDa protein feature of interest was excised from SDS – PAGE gel, washed with 50 mM ammonium acetate pH 7 buffer for 1 h, then washed in a 50/50% (v/v) acetonitrile/25 mM ammonium acetate pH 7.5 solution for 1 h and finally with pure water before complete dehydration in a vacuum centrifuge. Digestion occured at 378C for 4 h by a sequenced grade modified porcine trypsin (Promega) solution containing 0.25 mg of protease. MALDI-MS Mass spectra of the tryptic digests were acquired on a Voyager Elite Xl (Perseptive Biosystems) MALDI-TOF mass spectrometer equipped with a delayed extraction. The instrument was operated in the reflectron mode. 1 ml of digest was deposited directly onto the sample probe and mixed with 1 ml of 2.5-dihydroxybenzoic acid satured solution. A mass list of peptides was obtained for each protein digest. This peptide mass fingerprint was then submitted to an appropriated software in order to identify the proteins (MS-FIT: http://prospector.ucsf.edu/ucsfhtml3.2/ msfit.htm, or PROFOUND: http://129.85.19.192/prowl-cgi/ ProFound.exe). The tryptic peptides of the 210-kDa protein

were identified from its tryptic peptide mass map. The tryptic digests were extracted twice with a 50/50% (v/v) acetonitrile/ 25 mM ammonium acetate pH 7.5 solution. The digest solution and the extracts were then pooled, dried in a vacuum centrifuge and desalted with ZipTip C18 (Millipore, Bedford, MA, USA) before nanospray MS/MS analysis. Nanospray-MS/MS A Q-Tof instrument (Micromass, Manchester, UK) was used with a Z-Spray ion-source working in the nanospray mode. About 3 – 5 ml of the desalted sample was introduced into a needle (medium sample needle, PROTANA Inc. Odense, DK) to run MS and MS/MS experiments. The capillary voltage was set to an average voltage of 1000 V and the sample cone to 50 V. Glufibrinopeptide was used to calibrate the instrument in the MS/MS mode. MS/MS spectra were transformed using MaxEnt3 (MassLynx, Micromass Ltd), and amino acid sequences were analysed using PepSeq (BioLynx, Micromass Ltd). Amino acid sequences, sequence Tags or peptide ion fragments that could be detemined were used to screen the protein and the EST databases with dedicated softwares: Pepfrag (http://prowl1.rockefeller.edu/prowl/pepfragch.html), Scan (http//dna.standford.edu/scan) or BLAST for homologies searching (http://www.ncbi.nlm.nih.gov/blast/blast). Peptide sequencing by Edman degradation A preparative GST pull-down assay was prepared from 5.107 NIH3T3 cells and separated by SDS – PAGE, followed by amidoblack staining. In these conditions, the band of interest contained a sufficient amount of protein (25 pmoles corresponding to 5 mg of the 210-kDa protein) to be analysed by peptide sequencing. Polyacrylamide gel slice corresponding to the 210-kDa band was excised and incubated in a 0.05 M Tris HCl pH 8.6/0.03% SDS solution containing 0.4 mg of endolysin-c at 358C for 18 h. The endolytic peptides were separated by HPLC on a DEAE-C18 column with a acetonitrile-0.1% TFA gradient. The sequencing of isolated peptides was performed according to Edman degradation on an Applied Biosystems Procise Sequencer. Amino acid sequences were used to screen the protein database for homologies searching: BLAST (http://www.ncbi.nlm.nih.gov/ blast/blast), ExPASy (http://www.expasy.ch).

Acknowledgements We thank Jean Feunteun for the pCDNA3b-BRCA1wt. We would like to thank Marc Billaud and Jean-Pierre Rouault for advice and discussion. We are also very grateful to Marc Billaud and Robert Dante for critical reading of the manuscript. Cle´mence Magnard is the recipient of a fellowship from the Ligue Nationale contre le Cancer, Comite´ De´partemental de la Haute-Savoie. The present work was supported by the Ligue Nationale contre le Cancer, Comite´ De´partemental du Rhoˆne and the CNRS (grant PCV97-102).

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