Oncogene (2001) 20, 2691 ± 2703 ã 2001 Nature Publishing Group All rights reserved 0950 ± 9232/01 $15.00 www.nature.com/onc Identi®cation of functional domains involved in BTG1 cell localization A Rodier1, P Rochard1, C Berthet2, JP Rouault2, F Casas1, L Daury1, M Busson1, JP Magaud2, C Wrutniak-Cabello1 and G Cabello*,1 1UMR DieÂrenciation Cellulaire et Croissance (INRA, Universite Montpellier II, ENSAM), Unite d'Endocrinologie Cellulaire, INRA, 2 place Viala, 34060 Montpellier Cedex 1, France; 2Unite INSERM U453, Centre LeÂon BeÂrard, 69373 Lyon Cedex 08, France We have previously shown that BTG1 stimulates the basis of their sequence homology (Gue henneux et myoblast dierentiation. In addition, this protein displays al., 1997). Their predicted peptidic sequence displays a a major nuclear localization in con¯uent myoblasts, high degree of conservation in two regions of 22 and decreasing during the early steps of dierentiation, and is 20 amino acids named boxes A and B (Gue henneux et essentially detected in the cytoplasm of mature myo- al., 1997) which constitute the two signature motifs of tubes. To identify the domains involved in the cellular this family. tracking of BTG1, we observed the localization of A previous study demonstrated that BTG1 expres- several BTG1 sequences fused to bGalactosidase. The sion is easily detected in quiescent adult tissue (Rouault highly conserved B box among all members of the BTG et al., 1992). In agreement with this observation, BTG1 family induces a signi®cant nuclear localization of the mRNA is preferentially induced at the G0/G1 phase bGal moiety, enhanced by presence of the BTG1 transition of the cell cycle (Rouault et al., 1992) and carboxy-terminal sequence. In addition, a functional decreases as the cells progress throughout G1. More- Nuclear Export Signal (NES) overlaps the B box. over BTG1 and its related proteins inhibit proliferation Moreover, presence of the ®rst 43 NH2-terminal amino of several cell lines including NIH3T3, PC12 and QM7 acids reduced the nuclear localization of each chimeric quail myoblast lines (Rouault et al., 1992; Montagnoli protein tested. Last, the BTG1 amino-terminal domain et al., 1996; Matsuda et al., 1996; Rodier et al., 1999). bears an LxxLL motif favouring nuclear accumulation, Therefore, it is believed that these proteins are and another region encompassing the A box inhibiting members of an antiproliferative protein family. nuclear localization. In contrast to a BTG1 mutant However, their molecular role is poorly understood. exclusively localized in the cytoplasm, transient expres- The previous observation that Tob (Transducer Of sion of a mutant displaying a nuclear localization erbB-2) interacts with the membrane-bound growth enhanced myoblasts withdrawal from the cell cycle and factor receptor erbB2 suggests that it could be terminal dierentiation, thus mimicking the myogenic involved in growth factor-signalling pathway (Matsu- in¯uence of BTG1. In conclusion, several regions of da et al., 1996). More recent studies using a system of BTG1 are implicated in its cellular localization, and two hybrid interactions demonstrated that BTG1 and BTG1 myogenic activity is induced at the nuclear level. BTG2 (Tis21/PC3) interact with other proteins. First, Oncogene (2001) 20, 2691 ± 2703. Herschman's group reported an interaction with PRMT1 (Protein-Arginin N-MethylTransferase) (Lin Keywords: BTG1; NLS; NES; myoblast; cell localiza- et al., 1996), which asymmetrically methylates arginine tion residues occurring in the glycine- and arginine-rich domain (RGG) of hnRNP substrates (Rajpurohit et al., 1994) or histones (Ghosh et al., 1988). Further- Introduction more, by using CARM1, homologous to protein arginine methyltransferase PRMT1, Chen et al. The btg1 (B-cell Translocation Gene 1) gene coding (1999) demonstrated that arginine methylation con- sequence was isolated from a translocation break point tributes to transcriptional regulation. Other interac- t(8; 12) (q24; q22) in a case of B-cell Chronic tions have been subsequently reported with CAF1 Lymphocytic Leukaemia (CLL-B) (Rimokh et al., (Rouault et al., 1998; Bogdan et al., 1998) and the 1991; Rouault et al., 1992) and shared high homology homeoprotein Hoxb9 (Prevot et al., 2000). CAF1 is with the previously identi®ed tis21 (Fletcher et al., the murine homologue of a yeast component of the 1991) and pc3 (Bradbury et al., 1991) genes. To date, CCR4 transcriptional regulatory system (Draper et seven genes have been classi®ed in the same family on al., 1995) interacting with BTG1 and BTG2 through the B box (Rouault et al., 1998). More recently, Ikematsu et al. (1999) described such an interaction between Tob2 and the human homologue of CAF1, *Correspondence: G Cabello Received 19 December 2000; revised 23 January 2001; accepted 12 stressing a possible functional importance of this February 2001 association for BTG family proteins. In agreement Functional domains involved in BTG1 localization A Rodier et al 2692 Figure 1 The B box and the carboxy terminus domain of BTG1 are involved in the nuclear localization of the protein. Cells were transiently transfected with the vector pCH110 encoding the wild type bGalactosidase (a); or encoding the following sequences fused to bGal: box B, B/bGal (b); full length BTG1, BTG1/bGal (c); BTG1 without the B box, BTG1DB/bGal (d); B box and the carboxy-terminal region of BTG1, B-C-ter/bGal (e); carboxy-terminal domain of BTG1 (aa: 119 ± 171), C-ter/bGal (f); B box and Oncogene Functional domains involved in BTG1 localization A Rodier et al 2693 with these data suggesting that a part of their exclusive nuclear localization enhances myoblast with- antiproliferative activity involves the control of gene drawal from the cell cycle and their terminal expression, Guardavaccaro et al. (2000) reported that dierentiation, whereas a cytoplasmic mutant was cyclin D1 transcription is decreased by BTG2 devoid of any myogenic activity. These data suggest expression, leading to the accumulation of the that regulation of BTG1 cellular tracking partly hypophosphorylated, growth inhibitory forms of drives its in¯uence on myoblast dierentiation. pRb. In addition, it has been shown that CAF1 is capable of binding cyclin-dependent kinases (Ikematsu et al., 1999). Results In previous works (Marchal et al., 1995; Rodier et al., 1999) we demonstrated that the BTG1 protein is The B box and the carboxy terminus domain are involved located in the nucleus of con¯uent QM7 avian in the nuclear localization of BTG1 myoblasts. Its nuclear accumulation was increased by the presence of triiodothyronine (T3) or 8-Br-cAMP The well-conserved B box has been shown to mediate which also reduced proliferation and stimulated the interaction of BTG1 with the nuclear protein dierentiation in this cell line (Marchal et al., 1993, CAF1 in yeast (Rouault et al., 1998). This result raised 1995). Interestingly, we also observed that the nuclear the question of a possible involvement of this domain localization of BTG1 displays a transient feature since in the nuclear localization of BTG1. To test this in the early dierentiation steps, amounts of the hypothesis, we expressed a B box/bGal fusion protein protein decrease in the nucleus and BTG1 becomes in QM7 cells. Whereas bGal alone was detected in the essentially located in the cytoplasm of mature nuclei of a reduced number of transfected cells myotubes (Rodier et al., 1999). This cellular tracking (percentage of cells harbouring exclusive nuclear is probably associated with substantial changes in the staining N+percentage of cells harbouring simulta- activity of the protein. neous nuclear and cytoplasmic staining N/C=N+N/ The major aim of this study was to identify BTG1 C: 7%; Figure 1a), the B box drove signi®cant amounts sequences involved in the cellular tracking of the of this protein into the nuclei, re¯ected by an 11.5-fold protein, using chimeric constructs composed of BTG1 increase in the number of cells displaying nuclear domains fused to the bGalactosidase enzyme. We ®rst staining (N+N/C: 81%, P50.001; Figure 1b). Con- observed that BTG1 does not bear any Nuclear versely, deletion of the B box in the BTG1 integral Localization Sequence (NLS) similar to that pre- protein fused to bGal fully abrogated its nuclear viously identi®ed on SV40TAg (Newmeyer and localization (N+N/C: 0% vs 11% for BTG1/bGal, Forbes, 1988) or nucleoplasmin (Dingwall et al., P50.001; Figure 1d and c respectively). 1982) and we found that several sequences are In addition, although it was unable to aect by itself speci®cally involved in the determination of each the cell localization of the bGal moiety (Figure 1f), the BTG1 cellular localization. We concluded that the B presence of the BTG1 carboxy-terminal strongly box is sucient to drive signi®cant amounts of bGal potentiated the frequency of nuclear staining induced in the nucleus, and that its in¯uence is strongly by the B box (Figure 1e). Not only was bGal detected increased in the presence of the BTG1 carboxy- in the nuclei of all cells (N+N/C: 100% vs 81% for B terminal domain. Moreover, we identi®ed a functional box/bGal, P50.05; Figure 1e and b respectively), but Nuclear Export Signal (NES) overlapping the B box, in majority they displayed an exclusive nuclear staining closely related to that described in the viral Rev (N: 76% vs 4% for B box/ûGal, P50.001; Figure 1e protein (Fischer et al., 1995), cellular protein PKI and b respectively), thus underlining a major in¯uence (Wen et al., 1995) or MAPKK (Fukuda et al., 1996). of this sequence. In addition, 43 residues of BTG1 amino-terminus These data clearly suggested the involvement of a inhibit the nuclear accumulation of several chimeric cooperation between the B box and the carboxy proteins. We also characterized two other domains of terminal domain in the nuclear localization of BTG1.