Oncogene (2001) 20, 4917 ± 4925 ã 2001 Nature Publishing Group All rights reserved 0950 ± 9232/01 $15.00 www.nature.com/onc ORIGINAL PAPERS The tumor suppressor menin interacts with NF-kB and inhibits NF-kB-mediated transactivation

Christina Heppner1,3, Karl Y Bilimoria1, Sunita K Agarwal1, MaryBeth Kester1, Leslie J Whitty1, Siradanahalli C Guru2, Settara C Chandrasekharappa2, Francis S Collins2, Allen M Spiegel1, Stephen J Marx1 and A Lee Burns*,1

1Metabolic Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland MD, USA; 2Genetics and Molecular Biology Branch, National Research Institute, National Institutes of Health, Bethesda, Maryland MD 20892, USA

Multiple endocrine neoplasia type 1 is an autosomal mutations (Marx, 2001). Somatic inactivation of both dominant tumor syndrome. Manifestations include copies of the MEN1 occurs frequently in sporadic neoplasms of the parathyroid glands, enteropancreatic parathyroid, enteropancreatic and bronchial carcinoid neuroendocrine cells, and the anterior pituitary gland. tumors (Heppner et al., 1997; Debelenko et al., 1997; The MEN1 encodes menin, a 610 Goebel et al., 2000). Germline and somatic mutations amino acid nuclear protein without to of the MEN1 gene are distributed over the entire other proteins. To elucidate menin function, we used MEN1 coding region with no mutation hot spot immunoprecipitation to identify interacting proteins. The (Marx, 2001). The majority of mutations are nonsense NF-kB proteins p50, p52 and p65 were found to interact or frameshift changes in the DNA, thus predicting speci®cally and directly with menin in vitro and in vivo. truncated or absent menin. The region of NF-kB proteins sucient for binding to Menin, the 610 amino acid protein encoded by the menin is the N-terminus. Furthermore, amino acids 305 ± MEN1 gene, contains two nuclear localization signals 381 of menin are essential for this binding. Menin (aa 479 ± 497 and aa 588 ± 608) and localizes predomi- represses p65-mediated transcriptional activation on NF- nantly to the nucleus (Guru et al., 1998). Otherwise, kB sites in a dose-dependent and speci®c manner. Also, the encoded protein sequence does not provide PMA (phorbol 12-myristate 13-acetate)-stimulated NF- recognizable information about function (Chandrase- kB activation is suppressed by menin. These observations kharappa et al., 1997). suggest that menin's ability to interact with NF-kB Subsequent to cloning the MEN1 gene, we identi®ed proteins and its modulation of NF-kB transactivation JunD as one menin interaction partner. Menin directly contribute to menin's tumor suppressor function. Onco- interacts with JunD and represses JunD mediated gene (2001) 20, 4917 ± 4925. transactivation on an AP-1 binding site (Agarwal et al., 1999). To elucidate further the function of the Keywords: MEN1; MEN1 gene; menin; NF-kB; MEN1 gene we sought additional menin interacting transcriptional repression; oncogene; Rel-homology proteins by coimmunoprecipitation and identi®ed domain transcription factors of the NF-kB family. The NF-kB transcription factors are major regula- tors of the cellular response to various types of stress. Introduction In unstimulated cells, NF-kB is present in an inactive cytoplasmic complex bound to the inhibitory protein Multiple endocrine neoplasia type 1 (MEN1) is an IkB. Following stimulation by various factors such as autosomal dominant tumor syndrome. Patients typi- ionizing radiation, mitogens, growth factors and cally present with neoplasms of the parathyroid glands cytokines, IkB is phosphorylated, and the NF-kB and less commonly with neoplasms of enteropancreatic complex translocates to the nucleus (Siebenlist et al., neuroendocrine, the anterior pituitary, or other endo- 1994; Wulczyn et al., 1996; Baldwin, 1996; May and crine and nonendocrine tissues (Marx, 1998). Ghosh, 1997). MEN1 is a tumor suppressor gene on NF-kB complexes are composed of homo- or 11q13. 94% of probands with familial MEN1 harbor heterodimers of the transcription factors p50, p52, detectable missense or truncation MEN1 germline p65 (RelA), c-Rel and RelB. Members of the NF-kB family share a highly conserved N-terminal Rel- homolgy domain of approximately 300 amino acids which mediates DNA-binding, dimerization, nuclear *Correspondence: A Lee Burns targeting and binding to IkB. While p50 and p52 are 3Current address: Klinik II und Poliklinik fuÈ r Innere Medizin der UniversitaÈ tsklinik KoÈ ln, 50924 KoÈ ln, Germany products from the precursor proteins (p105 and p100) Received 26 May 2000; revised 3 April 2001; accepted 9 April 2001 encoded by the NF-kB1 and NF-kB2 , respec- Menin interacts with NF-kB C Heppner et al 4918 tively, p65, cRel and RelB require no further processing. In contrast to the other rel family members, p50 and p52 contain no transactivation domains (Siebenlist et al., 1994; Baldwin, 1996). NF-kB family members also directly interact with other transcription factors, such as C/EBP (Stein et al., 1993), AP1 (Stein et al., 1993), SP1 (Perkins et al., 1993), NF-IL6 (LeClair 1992), the glucocorticoid receptor (Ray and Prefontaine, 1994) and nuclear coactivators (Gerritsen et al., 1997; Ravi et al., 1998; Zhong et al., 1998), allowing a broad spectrum of modi®cations of transcriptional regulation.

Results

Menin coimmunoprecipitates with multiple proteins Immunoprecipitation was used to identify interacting partners of menin. Whole cell lysates of S-35 labeled 293 cells were immunoprecipitated with di€erent menin antibodies. Several protein bands coprecipitated with Figure 1 Coimmunoprecipitation of menin with p50 and p65. menin but not with an unrelated control antibody. (a) Nuclear extracts of HEK 293 cells co-transfected with p50 and Proteins were considered interaction candidates, if they menin were immunoprecipitated with a control antibody, two coprecipitated with at least two di€erent menin di€erent p50 antibodies and the menin antibody SQV. The membrane was probed with the menin antibody SQV. The arrow antibodies. Among the coprecipitating proteins, we points to the menin protein which is precipitated by the SQV noted four prominent bands with molecular weights of antibody and coprecipitates with two di€erent p50 antibodies but approximately 105, 100, 65 and 50 kDa (data not not with an unrelated control antibody. (b) HEK 293 wild type shown). The NF-kB family of transcription factors nuclear extracts were immunoprecipitated with a control anti- consists of proteins with molecular weights of 105 body, a p50 antibody (Ab-1), a p50 antibody (Ab-1) in the presence of its respective peptide, and the menin antibody SQV. (p105), 100 (p100), 65 (p65), 50 (p50), 52 (p52), 75 (c- In wild type nuclear extracts menin is precipitated by the menin Rel) and 68 kDa (RelB). Thus, we proceeded to assess antibody and menin coimmunoprecipitates when using the p50 if the identi®ed bands include some members of the antibody. The coimmunoprecipitation of menin with p50 is NF-kB family. abolished when the reaction is incubated in the presence of the peptide corresponding to the p50 antibody. (c) Nuclear extracts of HEK 293 cells were co-transfected with menin and p65 and NF-kB proteins immunoprecipitate with menin immunoprecipitated with a control antibody, two di€erent menin antibodies and a p65 antibody and the membrane was probed Using p50 antibodies, menin coprecipitated with p50 in with a p65 antibody. The p65 protein (arrow) precipitates when nuclear extracts from wild type cells and p50/menin using the p65 antibody and also coimmunoprecipitates with two di€erent menin antibodies SQV and AEA but not with the cotransfected cells (Figure 1a,b). Furthermore, includ- unrelated control antibody. The input lane of each panel ing an excess of the corresponding p50 peptide corresponds to 10% of total nuclear extracts used in the inhibited the coprecipitation of menin (Figure 1b), immunoprecipitations. The band below the menin band in the supporting the speci®city of the immunoprecipitation. input and a-menin SQV lane in (a) presumably corresponds to a Because the IgG heavy chain migrates at approxi- proteolytic degradation product of menin mately 50 kDa, we were unable to perform the reciprocal coimmunoprecipitation of p50 with menin (i.e. precipitate menin and immunoblot with p50). tated detectable menin, although the reproducibility In unstimulated cells, p65 is found mainly in the of this experiment was varied (data not shown). cytoplasm (Siebenlist et al., 1994; Wulczyn et al., 1996; Baldwin, 1996; May and Ghosh, 1997) while menin GST pulldown assays confirm menin/NF-kB interaction localizes predominantly to the nucleus (Guru et al., 1998). Thus, the di€erent compartmentalization of p65 Nuclear extracts of HEK 293 cells transiently trans- and menin limits the use of wild type nuclear extracts fected with menin were incubated with GST, GST-50, for coimmunoprecipitation experiments. In cells co- GST-52 and GST-65 fusion proteins coupled to transfected with menin and p65, the vast majority of glutathione beads. All three GST-NF-kB proteins were both proteins is found in the nucleus (data not shown). capable of retaining menin (data not shown). Further- Using nuclear extracts from such cells and two di€erent more, endogenous menin from HEK 293 wild type cells menin antibodies, we found that p65 coprecipitates bound to each of the GST-NF-kB proteins (Figure 2). with menin (Figure 1c). In the reverse experiment, using To validate the speci®city of the bands on the Western nuclear extracts of menin and p65 transfected cells, blots, we developed these using two di€erent menin immunoprecipitation with p65 antibodies coprecipi- antibodies (Figure 2).

Oncogene Menin interacts with NF-kB C Heppner et al 4919 Menin directly interacts with NF-kB Two di€erent scenarios may account for the observa- tion that menin interacts with p50, p52 and p65. First, menin may interact directly with only one NF-kB protein. This particular protein might serve as a bridging factor to other NF-kB family members. Also, the menin/NF-kB interaction could be bridged by a di€erent, unrelated protein. Second, menin may Figure 3 Menin directly interacts with NF-kB proteins. A GST interact directly with any one of the three proteins. pulldown experiment was done using three di€erent sources of Since the NF-kB proteins share the highly conserved human menin protein. GST-NF-kB proteins were incubated with Rel-homology domain, the menin interacting domain whole cell lysate of Drosophila S2 cells overexpressing human would likely reside within this region of 300 amino menin (a), anity puri®ed menin derived from menin over- expressing S2 cells (b) or menin anity puri®ed from E. coli cells acids. overexpressing menin (c). Blots were developed using the menin To test whether menin interacts directly with NF-kB antibody SQV. Menin is retained by all three GST-NF-kB fusion family members, GST pulldown experiments were proteins (though somewhat weakly by GST-50) but not by the performed using menin from sources lacking human control protein GST. Input lane shows 5% (a), 30% (b) and proteins, which might act as bridges. Human menin 100% (c) of total input overexpressed in Drosophila S2 cell lysates (S2-menin), and anity puri®ed menin from the above cells or E. coli were able to bind GST-50, GST-52 and GST-65. However, no menin was retained by the GST beads either the N- or C-terminus, or an internal segment of alone (Figure 3). These results provide evidence that menin was removed. These constructs were transiently menin binds directly and speci®cally to the NF-kB transfected into HEK 293 cells and whole cell lysates proteins (p50, p52 and p65) and virtually exclude the were used for GST pulldown experiments. Since menin involvement of a bridging protein. binds to the same region of p50, p52 and p65, pulldown experiments were done using only the GST- 65 fusion protein. Deletion of menin amino acids 276 ± The N-terminus of the Rel-homolgy domain is sufficient 479 abolished binding to GST-65 (Figure 5). The for binding to menin minimal region of menin required for binding to GST- To assess the minimal domains required for menin 65 in these experiments is estimated as amino acids binding, deletions of p65, p50 and p52 GST-fusion 305 ± 381. Probably, the preferred binding region proteins were generated. The ®rst 42 amino acids of extends slightly further towards the C-terminus, since p65 were found to be sucient to mediate binding to the binding of GST-65 to menin (1 ± 381) was already menin (Figure 4a, GST-65-4). A similarly small N- diminished as compared to menin (1 ± 476). terminal domain of p50 (Figure 4b) and p52 (Figure 4c) was sucient for the binding of menin. In addition, Menin represses NF-kB mediated transactivation menin binding was abolished when the N-terminal region of p65 was deleted (Figure 4a, GST-65-N). The p65 protein is a strong transcriptional activator on NF-kB binding sites. Both the p65 homodimer as well as the p65/p50 or p65/p52 heterodimer activate A minimal mid-region of menin is required for interaction transcription. In contrast, p50 and p52 lack transacti- with NF-kB vation domains and thus generally do not activate To assess the domains of menin required for interac- transcription alone, even though both are capable of tion with NF-kB, regions of menin were deleted at binding to NF-kB sites. Thus, p50 and p52 act mainly as transcriptional repressors, binding as homodimers to NF-kB sites and thereby decreasing access of p65 to the DNA binding site (Siebenlist et al., 1994; Wulczyn et al., 1996; Baldwin, 1996; May and Ghosh, 1997). To assess the functional implications of menin binding to NF-kB, we studied menin's e€ect on the transcriptional activity of NF-kB. Although menin or p50 alone did not activate transcription on the NF-kB site (5'-gggaatttcc-3'), p65 induced a strong transactiva- Figure 2 Interaction of menin with NF-kB by GST pulldown tion which was inhibited by cotransfection with p50. assay. Puri®ed GST, GST-50, GST-52 and GST-65 fusion When a constant amount of p65 plasmid DNA (0.1 mg) proteins were incubated with HEK 293 wild type nuclear extracts. was contransfected with increasing amounts of MEN1 Menin binding was visualized by immunoblot analysis using the plasmid DNA, p65-mediated transactivation of the menin antibodies SQV (a) or AEA (b). Menin is speci®cally NF-kB responsive reporter was decreased by menin in retained by each NF-kB protein but not by the GST control protein. The input lane corresponds to 25% of total nuclear a dose dependent manner to a level of 85 ± 90% extracts used in the experiment inhibition (Figure 6a ± c). A similar repression was

Oncogene Menin interacts with NF-kB C Heppner et al 4920

Figure 4 De®ning the region of NF-kB proteins required for interaction with menin. (a) The C-terminus of GST-65 was progressively deleted (GST-65-1 to GST-65-4) or the N-terminus was deleted (GST-65-N). Fusion proteins were then incubated with menin (as a whole cell lysate from Drosophila S2-menin cells). Deleting the C-terminus did not a€ect GST-65 binding to menin. Furthermore, aa 1 ± 42 of p65 was sucient for the interaction. The interaction with menin was abolished when the entire N- terminus of GST-65 was deleted. Similarly, deletions of GST-50 (b) or GST-52 (c) were generated and incubated with S2-menin. Again, the N-terminus of p50 and p52 was sucient for the interaction with menin. All Western blots were developed using the menin antibody SQV. Input corresponds to 10% of lysate used for the experiment. Stippled regions of the NF-kB fusion proteins refer to all or part of the Rel-homology domain (protein sizes not drawn to linear scale)

found when menin was cotransfected with higher Menin represses PMA-induced NF-kB activity amounts of p65 (data not shown). Also, we observed a comparable repression of p65-mediated transactiva- We stimulated HeLa cells with PMA in the presence tion with a reporter vector in which ®ve copies of the or absence of menin to test whether exogenous NF-kB consensus site 5'-tggggactttccgc-3' are present activation of NF-kB is a€ected by menin. PMA upstream of a basic TATA element (data not shown). treatment without menin led to a ®vefold increase of Increasing the level of menin overexpression did not NF-kB-mediated transcription. However, this stimula- change the amount of p65 (Figure 6d) or p50 (data not tion was decreased in the presence of menin (Figure shown) expressed as indicated by Western analysis. We 7b). Similar results were obtained when Cos7 and can thus exclude the possibility that the menin- NTERA-2 cells were used or when cells were associated repression is due to an alteration of NF- stimulated with TNF-a (data not shown). The kB protein expression by menin (i.e. decreased p65 or expression of menin and its nuclear localization were increased p50 expression). una€ected by PMA or TNF-a stimulation (data not To control for the speci®city of these ®ndings, the shown). Finally, the level of nuclear p65 was the e€ect of menin on a Gal4-VP16 transactivator was same in both wild type and menin transfected cells analysed. Cotransfecting menin and a Gal4 DNA- following PMA stimulation (data not shown). We binding domain-VP16 activation domain fusion protein can therefore exclude that the menin induced did not result in a repression of Gal4-VP16 mediated repression is due to a change in p65 protein activation (Figure 7a). expression or localization.

Oncogene Menin interacts with NF-kB C Heppner et al 4921

Figure 5 Assessing the minimal domain of menin required for binding to p65. Menin deletions were generated in pCMV-sport- Menin by using appropriate restriction sites or by PCR. Menin deletion constructs were transfected into HEK 293 cells and the whole cell lysate was incubated with GST or GST-65. Membranes were developed with the menin antibodies GPN or SQV. The input lane shows 10% of lysate used in the experiments. The bottom diagram shows the region of menin binding to p65. The heavily dashed zone represents the region of menin essential for binding, the lightly dashed zone shows the extension of the preferred binding region

Discussion required for the formation of a menin/NF-kB complex on DNA. Since we used an isolated NF-kB binding site Menin directly interacts with the p65, p50 and p52 we would not have been able to detect a complex under proteins of the NF-kB family both in vitro and in vivo. these circumstances We excluded that a bridging protein is required for A variety of cellular stimuli lead to the nuclear the NF-kB/menin interaction. The region of NF-kB translocation of NF-kB and the subsequent transcrip- sucient for interaction encompasses the N-terminal tional activation on NF-kB promoter sites (Pahl, 1999). region of the Rel-homology domain. This region is We ®rst mimicked this mechanism by transiently also involved in DNA binding (Ghosh et al., 1995) overexpressing p65. Menin produced a signi®cant, and has been shown to participate in binding of IkB speci®c, and dose dependent repression of p65- (Malek et al., 1998; Kumar and Gelinas, 1993; mediated transactivation in three di€erent cell lines. Huxford et al., 1998). The Rel-homology domain is Moreover, menin abolished the PMA induced transac- highly conserved among all NF-kB family members, tivation. This e€ect was due neither to alterations in indicating that c-Rel, RelB and possibly the Droso- NF-kB protein levels nor protein compartmentaliza- phila homologues of NF-kB may also be capable of tion. Menin is thus a potent inhibitor of NF-kB- binding to menin. mediated transcriptional activation, and it seems likely Since the N-terminus of NF-kB proteins is involved that menin acts directly on NF-kB in the nucleus. in DNA binding we speculated that menin's binding to NF-kB plays a crucial role in the immune response, NF-kB proteins may interfere with binding of the NF- in cellular transformation and oncogenesis and serves kB complex to DNA. We thus assessed whether the as a positive mediator of cell growth. NF-kB activates presence of menin a€ects binding of NF-kB to the growth factors and their cellular receptors (Wulczyn et consensus site 5'-gggaatttcc-3'. We were neither able to al., 1996), participates in the control of cellular identify a menin-containing complex on DNA by gel adhesion molecules (Collins et al., 1995). Furthermore, shift analysis, nor observe an altered stability of the NF-kB itself can be activated through mitogenic NF-kB complex in the presence of menin (data not stimuli such as PMA, LPS, serum induction and shown). However, technical aspects may have hindered double stranded RNA (Baeuerle and Henkel, 1994; the detection of menin binding to a protein-DNA Siebenlist et al., 1994; Pahl, 1999). V-Rel is the complex in this assay. The bu€er conditions may have transforming gene of the oncogenic avian retrovirus been inappropriate or a menin containing complex Rev-T (Gilmore et al., 1996). Downregulation of the might be too unstable to allow detection by EMSA. inhibitory protein IkB-a induces malignant transforma- Also, menin binding to NF-kB on DNA may require tion (Beauparlant et al., 1994) and deregulated NF-kB the presence of proteins other than NF-kB. Possibly, activity occurs in various hematopoetic and solid factors of the basal transcription machinery are tumors (Rayet and Gelinas, 1999). NF-kB regulatory

Oncogene Menin interacts with NF-kB C Heppner et al 4922

Figure 6 Menin represses p65-mediated transactivation. Cos7, HeLa or NTERA-2 cells were transfected with 0.5 mg of the pNF- kB-luciferase reporter vector, p65 and increasing concentrations of p50 or Flag-menin (a±c). Cotransfection of menin induced a signi®cant, dose-dependent repression of p65-mediated transactivation in all three cell lines. For comparison, the repression achieved by the NF-kB protein p50 is shown. Each experiment was repeated at least three times. Data shown are mean and standard deviation from four points (two transfections in duplicate) representing one of three experiments. In addition, bars showing menin- mediated repression derive from three independent transfections using independent preparations of the Flag-menin plasmid. (d) Western blot analysis showing that p65 levels are una€ected by cotransfected menin. Cos7 cells were cotransfected with 0.5 mg pNF- kB-luciferase reporter vector, 0.1 mg p65 and increasing concentrations of menin. Whole cell lysates were prepared, resolved on a SDS gel, transferred to nitrocellulose and incubated with a p65 antibody or the a-menin antibody SQV

Oncogene Menin interacts with NF-kB C Heppner et al 4923 transcriptional mediator complex. Recently, Smad3 has been identi®ed to interact with menin (Kaji et al., 2001). Menin represses NF-kB mediated transactivation. Also, menin interaction with JunD induced a sig- ni®cant repression of JunD transactivity (Agarwal et al., 1999). Thus, menin may use a common mechan- ism to exert its repressive e€ect with menin itself acting as a repressor or possibly recruiting repressors such as histone deacetylases (Gobl et al., 1999). Further studies are needed to establish the functional role of menin/NF-kB interaction and its signi®cance in endocrine tumorigenesis. In conclusion, we have identi®ed NF-kB as a direct interaction partner of menin and shown that menin speci®cally inhibits p65- mediated transactivation. This information will be useful in further illuminating menin's role in cellular growth and oncogenesis.

Materials and methods

Antibodies The menin antibodies a-SQV and a-AEA have been described (Guru et al., 1998). In addition, the antibody a-GPN, raised against amino acids 187 ± 211 of menin, was used. The NF- kB antibodies [p50: sc-114 (Ab-1), sc-1190 (Ab-2); p65: (sc- Figure 7 (a) Menin does not repress Gal4-VP16 mediated activation. HeLa cells were transfected with 0.5 mg of the Gal4- 372)] and the p50 peptide (sc-114P) were purchased from Luciferase vector (pNF-Gal4). Cotransfection of a Gal4-VP16 Santa Cruz Biotechnology Inc. The Anti-Flag M2 antibody transactivator plasmid (pM3-VP16) induced a signi®cant transac- was purchased from Sigma. tivation which was not a€ected by cotransfection of menin. (b) Menin represses phorpbol 12-myristate 13-acetate (PMA)-induced transactivation on NF-kB sites. HeLa cells were transfected with Mammalian expression plasmids the pNF-kB-luciferase reporter vector either in the absence (open For transfection experiments the menin expression vector bars) or in the presence of menin (shaded bars). 36 h after pCMV-sport-Menin (Chandrasekharappa et al., 1997) or transfection cells were stimulated with PMA for 2, 4, 6 and 8 h. Flag-tagged menin, engineered as described below were In the presence of menin the PMA-induced transactivation was signi®cantly diminished. Data (mean and standard deviation) are used. The myc-tagged full length menin construct and the representative for three independent experiments. Baseline lucifer- menin deletion construct menin (1 ± 276, 479 ± 610) have ase expression in the absence of PMA or menin is arbitrarily set been described (Guru et al., 1998). Additional deletion at 1.0 on the y-axis constructs were generated by taking advantage of appro- priate restriction sites: MscI for menin (1 ± 567), SmaI for menin (1 ± 476) and BstXI for menin (1 ± 381). The N- terminal deletions menin (100 ± 610), menin (200 ± 610), menin (305 ± 610) were generated by PCR using Expand elements are found in several oncogenes such as c-myc polymerase (Boehringer). All PCR generated constructs (La Rosa et al., 1994) and cyclin D1 (Hinz et al., 1999). were sequenced on at least one strand by the Taq Dideoxy Loss of menin-mediated repression of NF-kB activity Terminator Cycle sequencing method (Applied Biosystems). Constructs generated by restriction and ligation were may thus contribute to oncogenesis. veri®ed by checking the insert size on agarose gels and We previously reported that menin interacts with the sequencing the cloning sites. AP-1 factor JunD (Agarwal et al., 1999). AP-1 and Full length p65 and p50 (amino acids 1 ± 436) were NF-kB family members physically interact and enhance ampli®ed out of human leucocyte cDNA (Clontech) and DNA binding and transcriptional activation via both cloned into the pCI vector (Promega). The p52 construct the AP-1 and the NF-kB response element (Stein et al., (amino acids 1 ± 443) was cut out of a p100 containing vector 1993). Many transcriptional regulators function in and cloned into the pCI vector. large mediator complexes (Thanos and Maniatis., For reporter assays, the vector pNF-kB-Luciferase (Clon- 1995; Wolberger, 1998; Wathelet et al., 1998; Kingston, tech), in which four copies of the NF-kB consensus site (5'- 1999), some of which contain both AP-1 and NF-kB gggaatttcc-3') are cloned in front of a minimal thymidine kinase promoter, was used. To generate the Gal4-Luciferase factors (Thanos and Maniatis, 1995). Considering the vector (pNF-Gal4), the NF-kB binding sites of the pNF-kB- size of the menin protein and the ®nding that missense Luciferase vector were excised using NheI and BglII and mutations of the MEN1 gene occur throughout the replaced by ®ve copies of the Gal4 binding site (5'- open reading frame, it is possible that menin has agcggagtactgtcctccg-3'). The Gal4-VP16 transactivator plas- additional interacting and modifying partners within a mid was obtained from Clontech.

Oncogene Menin interacts with NF-kB C Heppner et al 4924 Prokaryotic expression plasmids Metabolic labeling of cells The GST-50 plasmid was provided by U Siebenlist. HEK 293 cells were metabolically labeled at a con¯uency of Constructs of GST-65 and GST-52 were generated in 90 ± 95%. Labeling with S-35 was carried out as follows: Cells pGEX-5X (Amersham Pharmacia Biotech). The GST-65 were exposed to methionine and cysteine depleted DMEM deletion constructs GST-65-1, GST-65-2, GST-65-3, GST- for 2 h. The medium was removed, fresh methionine/cysteine 65-4 and GST-65-N were generated by restriction digest using depleted DMEM supplemented with Trans35S-label (100 mCi/ NdeI, BspeI, BlpI, SacII or EcoR, respectively. GST-50 ml) (NEN Life Science Products, Boston, MA, USA) was deletions were engineered using SpeI (GST-50-1) or AccI added and cells were exposed for 16 h. Cells were washed (GST-50-2). GST-50-3 and GST-52-3 were generated by twice with DPBS, harvested by scraping and a whole cell PCR. The GST-52 deletions GST-52-1 and GST-52-2 were lysate was made as described above. generated using BspEI or DraIII, respectively. Immunoprecipitation Expression and purification of proteins in E. coli and Immunoprecipitation was carried out as described (Agarwal GST-pulldown assays et al., 1999). Immunoprecipitation of S-35 labeled cells was E. coli fusion proteins were expressed in BL21 (DE3)pLysS carried out similarly with minor modi®cations. The precipita- (Stratagene) following standard protocols (Kaelin et al., tion reactions were washed with a high salt lysis bu€er 1991). GST-proteins were recovered on gluthathione- (500 mM NaCl, 20 mM Tris-HCl (pH 8.0), 10% glycerol, sepharose beads (Amersham Pharmacia). 0.1% Igepal, 1 mM NaF and 1 mM Na3VO4). Following the For GST-pulldown experiments, approximately 2 mgof separation of proteins on SDS gels, gels were ®xed in 7% fusion proteins were incubated with nuclear extracts, whole acetic acid, 7% methanol, 1% glycerol, dried at 648C for cell lysate, puri®ed E. coli menin, or Drosophila S2-menin for 90 min and exposed to ®lm for 24 ± 48 h. 14 h at 48C on a rotating shaker. Gluthathione-sepharose beads were then washed four to six times with lysis bu€er Reporter assays (137 mM NaCl, 20 mM Tris-HCl (pH 8.0), 10% glycerol, 0.1% Igepal (Sigma), 1 mM NaF and 1 mM Na3VO4). Cells were plated in six well plates 1 day prior to transfection. Subsequently, beads were boiled in SDS loading bu€er and 0.5 mg reporter vector was used and the total amount of separated on SDS polyacrylamide gels. Proteins were DNA for transfection was kept constant at 3.5 mg in all transferred to nitrocellulose membranes and probed with transfection reactions by adding appropriate amounts of the appropriate antibody. All GST-pulldown experiments empty pCMV-sport vector. 36 ± 48 h after transfection, cells were repeated at least three times. were harvested and luciferase was measured using the luciferase assay system (Promega). Luciferase counts were corrected for equal amount of protein. All experiments were Human menin expression in Drosophila done at least three times in duplicate and with independent Stable, inducible expression of human menin was accom- plasmid preparation as indicated. plished with the Drosophila Expression System (Invitrogen) Stimulation with phorbol 12-myristate 13-acetate (PMA) (Valle et al., 2001). was carried out as follows: Cells were transfected with mammalian expression plasmids as indicated. Thirty-six hours after transfection cells were exposed to medium Cell culture, transient transfection and preparation of cell lysates containing 0.1 mg PMA previously dissolved in DMSO and Human embryonic kidney (HEK) 293, Cos7, NTERA-2 or harvested after 2, 4, 6 or 8 h. HeLa cells were split one day prior to transfection. Cells were transfected at a cell density of 50 ± 60% using Superfect (Qiagen) and harvested 36 ± 48 h later. For the preparation of Acknowledgments whole cell lysates for immunoprecipitation or pulldown We thank Dr Paul K Goldsmith (NIDDK) for puri®ed assays, cells were washed once with ice-cold DPBS and menin antibodies, Dr George Poy (NIDDK) for DNA harvested by scraping. The cell pellet was lysed in a bu€er sequencing and Dr Regina M Collins (NIDDK) for cell containing 137 mM NaCl, 20 mM Tris-HCl (pH 8.0), 10% culture assistance. Furthermore, we thank Dr Ulrich glycerol, 0.1% Igepal, 1 mM NaF and 1 mM Na3VO4 Siebenlist (NIAID) for helpful discussion and providing Preparation of nuclear extracts was described previously the GST-50 construct and Dr Nancy R Rice (NCl) for the (Agarwal et al., 1999). p100 construct.

References

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Oncogene