Oncogene (1998) 17, 3199 ± 3202 ã 1998 Stockton Press All rights reserved 0950 ± 9232/98 $12.00 http://www.stockton-press.co.uk/onc SHORT REPORT The LMO1 and LDB1 interact in human T cell acute leukaemia with the chromosomal translocation t(11;14)(p15;q11)

V Valge-Archer1,2, A Forster1 and TH Rabbitts1

MRC Laboratory of Molecular Biology, Division of and Nucleic Acid Chemistry, Hills Road, Cambridge, CB2 2QH, UK

The ectopic expression of LMO1 or LMO2 in T cell these zinc-containing modules and data on LMO2- acute leukaemias resulting from chromosomal transloca- containing protein complexes in erythroid cells (Wad- tions t(11;14)(p15;q11) or t(11;14)(p13;q11) respectively man et al., 1994) and in T cell tumours arising in in a causal factor in tumorigenesis. LMO1 has been transgenic mice (Grutz et al., 1998) suggest that protein found as a heterodimer with a 46 Kd protein in a T cell interaction, but not direct DNA binding, is the speci®c line derived from a childhood T-acute leukaemia. This function of the LIM-only proteins LMO1 and LMO2. 46 Kd protein is the LIM-binding protein LDB1/NLI. Current molecular models propose that aberrant T The latter is a phosphoprotein and binds to LMO1 in its cell-associated protein complexes, brought together by phosphorylated state and essentially all the LMO1 and the LMO proteins after ectopic expression, contribute LDB1 protein in the T cell line is part of the complex. to tumorigenesis. The nature of the 46 Kd protein Therefore, the LMO1-LDB1 interaction is likely to be to which LMO1 binds is relevant to this issue because involved in tumorigenesis after LMO1 is ectopically it is present in a complex with LMO1 in human T cell expressed following chromosomal translocation in T cells acute leukaemia line carrying the translocation prior to development of acute leukaemias. t(11;15)(p15;q11). Thus far, the identity of the 46 Kd protein has not been determined because it is obscured Keywords: leukaemia; dimerization; tumours; chromo- by the similarity in its molecular weight of TAL1, somal translocation; transcription which also binds LMO1 (Valge-Archer et al., 1994). A candidate for the 46 Kd protein is the recently identi®ed LIM-binding protein LDB1 or NLI (Agulnick et al., 1996; Jurata et al., 1996) (herein The encoding the LMO1 and LMO2 proteins called LDB1). This protein has been shown to interact (previously known as RBTN1/TTG1 or RBTN2/ in vitro with LMO1 and LMO2 (Agulnick et al., 1996; TTG2) were discovered as transcription units adjacent Jurata et al., 1996) and in vivo LMO2 has been found to the breakpoints of chromosomal translocations in complexes with LDB1 in erythroid cells (Wadman t(11;14)(p15;q11) (Boehm et al., 1988, 1990a, 1991a; et al., 1997) and in T cells from Lmo2-transgenic mice McGuire et al., 1989) and t(11;14)(p13;q11) (Boehm et (Grutz et al., 1998). We have assessed the interaction al., 1991a; Royer-Pokora et al., 1991) respectively. of LMO1 and LDB1 in the human T cell acute Both LMO1 and LMO2 proteins (as well as the other leukaemia cell line RPMI8402 (which has been family member LMO3 (Boehm et al., 1991a; Foroni et t(11;14)(p15;q11)) using a two-step immunoprecipita- al., 1992)) are LIM-only proteins which have two zinc- tion protocol described previously (Valge-Archer et al., binding LIM domains, each comprising two LIM 1994). The T cells were labelled with 35S-methionine/ ®ngers. The chromosomal translocations activating cysteine and labelled proteins immunoprecipitated with LMO1 and LMO2 are found only in T cell tumours a rabbit antiserum raised with an LMO1-speci®c and it seems likely that ectopic expression of the LIM- peptide. The precipitated proteins were subsequently proteins contributes to leukaemogenesis by the ability analysed by fractionation on SDS ± PAGE (Figure 1a). to form aberrant protein associations in T cells This procedure precipitates the 18 Kd LMO1 protein (Rabbitts, 1994). High mRNA and protein levels can as well as the 46 Kd protein (lane 1), together with be found in T cell lines derived from childhood T cell some higher molecular weight proteins (which are leukaemias with the relevant translocations (Boehm et non-speci®c since the pre-immune serum from the al., 1991b; Foroni et al., 1992; Royer-Pokora et al., animal used for peptide immunization, also precipi- 1991). Analysis of a T cell line with a t(11;14)(p15;q11), tated these bands, lane 3). Using an anti-LDB1 serum, and expressing the LMO1 , showed that LMO1 a similar pro®le of immunoprecipitated proteins interacts with at least two proteins in the nucleus of appears, notably the 18 Kd band which is most likely these cells; viz. TAL1/SCL and an uncharacterized to be LMO1 (lane 5) since it is not evident with LDB1 46 Kd protein (Valge-Archer et al., 1994). The ability pre-immune serum (lane 8). When the immunopreci- of the LIM domains of LMO1 and LMO2 to act as pitated proteins obtained with anti-LMO1 serum were surfaces for protein interaction (Valge-Archer et al., resuspended and re-immunoprecipitated with anti- 1994; Wadman et al., 1994) provides a function for LDB1 serum, we observed the speci®c immunopreci- pitation of LDB1, which co-migrates with the 46 Kd protein (lane 2). In a reciprocal experiment, anti- LMO1 immunoprecipitation of the anti-LDB1 immu- Correspondence: TH Rabbitts noprecipitate showed the LMO1 18 Kd protein (lane 2Current address: Cambridge Antibody Technology, The Science Park, Melbourn, Royston, SG8 6JJ 6). Thus the 46 Kd LMO1-interacting protein is the Received 25 June 1998; accepted 7 August 1998 LIM-binding protein LDB1. LMO1 and LDB1 interact in T-ALL VValge-Archeret al 3200 Con®rmation of the association of LMO1 with the 1991). These are neurogenic tumours which arise from LDB1 protein was obtained by immunoprecipitating nerve cells of the sympathetic nervous system, which LMO1 in the presence of the peptide used to raise the may include cells normally expressing Lmo1 (Boehm et LMO1 antiserum. In this analysis, no speci®c LMO1 al., 1991b; Greenberg et al., 1990; McGuire et al., or 46 Kd proteins were immunoprecipitated (lane 3). 1991). Thus neuroblastomas may represent a normal In addition, using the two-step immunoprecipitation cell type in which to examine Lmo1-Ldb1 interaction. procedure, when the LDB1 immune precipitate was re- Accordingly, the mouse neuroblastoma cell line 131- precipitated by anti-LMO1 serum in the presence of CCL (N2A), which expresses Lmo1 (Boehm et al., LMO1 peptide, the 18 Kd protein did not appear (lane 1991b), was incubated with [35S]methionine/cysteine for 7), demonstrating that this protein is indeed LMO1. immunoprecipitation studies (Figure 1b). This cell line Estimations of the amounts of LMO1 and LDB1 expresses low levels of Lmo1 protein (lanes 1 and 3) proteins in a complex with each other can be made by but nonetheless anti-Lmo1 antiserum co-precipitates a comparing amounts of radioactivity in each precipi- 46 Kd protein (lanes 1 and 5). The immunoprecipita- tated band. Thus it appears that almost all LMO1 tion of these proteins can be blocked by incubation protein is complexed with LDB1 in the T-ALL cell line with the Lmo1 peptide (lane 3). The identity of this as since the same amount of radiolabelled LMO1 is Ldb1 was con®rmed by the two-step immunoprecipita- observed in immunoprecipitations with either anti- tion method (lane 2) or by peptide blocking in the LMO1 and anti-LDB1 sera (Figure 1, lanes 1 and 5). second precipitation step (lane 7). Similarly, most of the LDB1 protein is co-precipitated Previous analyses of LMO1 showed that this protein with LMO1 with either antiserum. was not normally phosphorylated in vivo in RPMI8402 In an analysis of Lmo1 in mouse cells (Valge-Archer et al., 1994). The possibility that tissues and cells, it was observed that Lmo1 is LDB1 might be a phosphoprotein was investigated. 33 expressed in some neuroblastomas (Boehm et al., RPMI8402 cells were radiolabelled with Pi and 1990b, 1991b; Greenberg et al., 1990; McGuire et al., immunoprecipitation carried out (Figure 2). LDB1 is

Figure 1 Association of LMO1 with LDB1 in a T cell acute leukaemia and in a neuroblastoma. Exponentially growing cells were labelled with a mixture of 35S-methionine and 35S-cysteine. Total cellular proteins from RPMI8402 T cell acute leukaemia cells (a) or N2A (131-CCL) neuroblastoma cells (b) were immunoprecipitated with the indicated antisera. In the case of two-step immunoprecipitation, the ®rst antiserum is indicated followed by an arrow and the second antiserum. Each lane 3 is a two-step immunoprecipitation carried out with pre-immune serum used for the second step. LMO1+peptide indicates an immunoprecipitation carried with anti-LMO1 antiserum in the presence of 50-fold excess of the immunizing peptide. In one case for each panel, the second LMO1 immunoprecipitation was carried out with blocking LMO1 peptide. pi indicates pre-immune serum from the LMO1 antiserum. Commercially available size markers were used to estimate size in Kd, as shown between panels. Methods: Cells were grown to mid-log phase, labelled for 4 h at about 26107 cells/ml, in the presence of 200 mCi 35S-cysteine and 35S-methionine. Protein extracts were prepared as previously described (Valge-Archer et al., 1994) and one- and two-step immunoprecipitations carried out with N-terminal anti-peptide anti-LMO1 antiserum (Valge-Archer et al., 1994). Peptide blocking was carried out by pre-incubating the serum with peptide for 1 h prior to immunoprecipitation. The rabbit polyclonal antiserum was raised against an NH2-terminal peptide of LMO. Rabbit antiserum for Ldb1 has been described (Wadman et al., 1997) (note the sequence of mouse Ldb1 and human LDB1 are identical in the region of the peptide) LMO1 and LDB1 interact in T-ALL VValge-Archeret al 3201 by the pre-immune serum (lane 2). Further, anti-LMO1 serum also precipitates the 46 Kd LDB1 protein (lane 3) but no LMO1 protein is evident (as it is not phosphorylated). Finally, the LDB1 phospho-protein can be found in complex with LMO1 by the two-step protocol (lane 4) and this co-precipitation is blocked by LMO1 peptide (lane 5). Our results provide further formal proof that endogenous LDB1 protein interacts with LIM-only proteins, in this case LMO1. The functional signifi- cance of this heterodimerization is unclear but at least some of this complex must also involve TAL1 as this also co-precipitates with LMO1 in RPMI8402 cells (Valge-Archer et al., 1994), although a relatively small amount appears to be part of the complex. One potential function for the Lmo1-related protein Lmo2 is in the formation or maintenance of a complex of proteins capable of binding to distinct bipartite DNA recognition sites in erythroid cells and in transgenic T cells tumours after enforced Lmo2 expression in thymus cells (Grutz et al., 1998; Wadman et al., 1997). Thus ectopic LMO1 expression may similarly cause aberrant T cell complexes to form, including heterodimerization between LMO1 and LDB1. At present evidence for the existence of an LMO1- containing oligomeric complex, which would be the analogue of those found with Lmo2, is restricted to data with LDB1 and TAL1. Other proteins may also be present in the LMO1 complex. Whether LMO1 functions in the development of T-ALL by facilitating the formation of a DNA binding complex or by sequestration of components within this complex preventing their function (Grutz et al., 1998), the involvement of protein-protein interactions not usually Figure 2 LDB1 is a phosphoprotein in association with LMO1 in T cell acute leukaemia. RPMI8402 T cells were labelled with found in T cells presumably accounts for the 33 Pi for 2 h and whole cells proteins were extracted and pathogenic e€ects of the t(11;14)(p15;q11), underscor- immunoprecipitated with the indicated antisera. p.i.=pre- ing the importance of protein interactions in the modes immune LMO1 antiserum. Commercially available size markers of action of translocation-activated oncogenes. were used to estimate size in Kd. Methods: RPMI8402 cells were labelled with 1 mCi 33P-orthophosphate prior to extraction of protein and immunoprecipitation analysis as described before (Valge-Archer et al., 1994)

Acknowledgements phosphorylated in these cells, as the 46 Kd protein is We would like to thank Dr Alan Agulnick for anti-Ldb1 precipitated with the anti-LDB1 serum (lane 1) but not antiserum. VVA was supported by a Merck Fellowship.

References

Agulnick AD, Taira M, Breen JJ, Tanaka T, Dawid IB and Greenberg JM, Boehm T, Sofroniew MV, Keynes RJ, Barton Westphal H. (1996). Nature, 384, 270 ± 272. SC, Norris ML, Surani MA, Spillantini M-G and Rabbitts Boehm T, Baer R, Lavenir I, Forster A, Waters JJ, Nacheva TH. (1990). Nature, 344, 158 ± 160. E and Rabbitts TH. (1988). EMBO J., 7, 385 ± 394. Grutz G, Bucher K, Lavenir I, Larson R, Larson T and Boem T, Foroni L, Kaneko Y, Perutz MP and Rabbitts TH. Rabbitts TH. (1998). EMBO J., 17, 459 ± 4605. (1991a). Proc. Natl. Acad. Sci. USA, 88, 4367 ± 4371. Jurata LW, Kenny DA and Gill GN. (1996). Proc. Natl. Boehm T, Foroni L, Kennedy M and Rabbitts TH. (1990a). Acid. Sci. USA, 93, 11693 ± 11698. Oncogene, 5, 1103 ± 1105. McGuire EA, Davis AR and Korsmeyer SJ. (1991). Blood, Boehm T, Greenberg JM, Buluwela L, Lavenir I, Forster A 77, 599 ± 606. and Rabbitts TH. (1990b). EMBO J., 9, 857 ± 868. McGuire EA, Hockett RD, Pollock KM, Bartholdi MF, Boehm T, Spillantini M-G, Sofroniew MV, Surani MA and O'Brien SJ and Korsmeyer SJ. (1989). Mol. Cell. Biol., 9, Rabbitts TH. (1991b). Oncogene, 6, 695 ± 703. 2124 ± 2132. Foroni L, Boehm T, White L, Forster A, Sherrington P, Liao Rabbitts TH. (1994). Nature, 372, 143 ± 149. XB, Brannan CI, Jenkins NA, Copeland NG and Rabbitts Royer-Pokora, B., Loos U and Ludwig W-D. (1991). TH. (1992). J. Mol. Biol., 226, 747 ± 761. Oncogene, 6, 1887 ± 1893. LMO1 and LDB1 interact in T-ALL VValge-Archeret al 3202 Valge-Archer VE, Osada H, Warren AJ, Forster A, Li J, Wadman IA, Osada H, Grutz GG, Agulnick AD, Westphal Baer R and Rabbitts TH. (1994). Proc. Natl. Acad. Sci. H, Forster A and Rabbitts TH. (1997). EMBO J., 16, USA, 91, 8617 ± 8621. 3145 ± 3157. Wadman I, Li J, Bash RO, Forster A, Osada H, Rabbitts TH and Baer R. (1994). EMBO J., 13, 4831 ± 4839.