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Oncogene (2002) 21, 7923 – 7932 ª 2002 Nature Publishing Group All rights reserved 0950 – 9232/02 $25.00 www.nature.com/onc B-Myb overcomes a p107-mediated cell proliferation block by interacting with an N-terminal domain of p107 Manel Joaquin1,4, Maria Bessa1,2,4, Mark K Saville1,3 and Roger J Watson*,1 1Ludwig Institute for Cancer Research, Section of Virology and Cell Biology, Faculty of Medicine, Imperial College of Science, Technology and Medicine, Norfolk Place, London W2 1PG, UK B-Myb is a cell-cycle regulated transcription factor myb expression is transcriptionally repressed during G0 which is implicated in cell proliferation and has an and G1 phases of the cell cycle by p107 and p130 E2F essential role in early embryonic development. In this complexes, and is transcribed maximally during early S study we examined the functions of B-Myb required to phase (Hurford et al., 1997; Lam et al., 1994; Lam and overcome G1 arrest in Saos-2 cells induced by the Watson, 1993). In addition to this level of control, the retinoblastoma-related p107 protein. Our results demon- B-Myb protein undergoes a specific post-translational strated that this activity was independent of B-Myb modification during S phase (Robinson et al., 1996) transactivation function, but correlated with its capacity which results from phosphorylation by the cyclin A2/ to form an in vivo complex with p107. A large proportion cdk2 protein kinase (Ansieau et al., 1997; Bartsch et of B-Myb formed complexes with p107 in cotransfected al., 1999; Johnson et al., 1999b; Lane et al., 1997; Sala cells, however, B-Myb bound weakly to the related p130 et al., 1997; Saville and Watson, 1998b; Ziebold et al., protein and not at all to pRb. In contrast to the E2F 1997). The major cyclin A2/cdk2 phosphorylation sites transcription factors, which bind the p107 C-terminal map within the C-terminal half of B-Myb, and lie pocket domain, B-Myb recognizes an N-terminal p107 within or adjacent to a negative-regulatory domain region which overlaps the larger cyclin-binding domain. (NRD) which restricts B-Myb DNA-binding and B-Myb and cyclin A2 formed mutually exclusive transactivation activities (Ansieau et al., 1997; Lane complexes with p107, and B-Myb enhanced the activity et al., 1997; Watson et al., 1993; Ziebold et al., 1997). of co-transfected cyclin E kinase activity, implying that Significantly, ectopic expression of cyclin A2 markedly B-Myb affects the cell cycle by preventing sequestration enhances B-Myb transactivation activity (Ansieau et of active cyclin/cdk2 complexes. This study defines a al., 1997; Lane et al., 1997; Sala et al., 1997; Ziebold et novel function of B-Myb and further suggests that the al., 1997), suggesting that phosphorylation counteracts p107 N-terminus provides an interaction domain for the inhibitory effect of the NRD. transcription factors involved in cell cycle control. The findings that transcriptional and post-transla- Oncogene (2002) 21, 7923 – 7932. doi:10.1038/sj.onc. tional mechanisms combine to direct high levels of 1206001 hyperactivated B-Myb protein to S phase strongly suggest that B-Myb plays a critical role at this stage of Keywords: B-Myb; p107 pocket protein; cell cycle; the cell cycle. B-myb expression is ubiquitous within cyclin E/Cdk2; cyclin A2/Cdk2 proliferating cell lines, suggesting that this requirement for B-Myb function might be common to a wide spectrum of cell types. In support of this notion, B-myb Introduction antisense oligonucleotides were found to inhibit prolif- eration of certain myeloid, lymphoid, glioblastoma, In common with the related c-Myb onco-protein and fibroblast and neuroblastoma cell lines (Arsura et al., A-Myb, the B-Myb transcription factor is implicated in 1992; Lin et al., 1994; Raschella et al., 1995; Sala and regulating cell growth and development (reviewed in Calabretta, 1992), albeit not all studies were able to Sala and Watson, 1999; Saville and Watson, 1998a). B- demonstrate these effects (Robinson et al., 1996). Conversely, constitutive expression of B-myb prevented cell cycle arrest associated with culture of BALB/c 3T3 *Correspondence: RJ Watson, Section of Virology and Cell Biology, fibroblasts in low serum-containing medium (Sala and Faculty of Medicine, Imperial College of Science, Technology and Calabretta, 1992) or IL-6-induced differentiation of M1 Medicine, St Mary’s Campus, Norfolk Place, London W2 1PG, UK; myeloid leukaemia cells (Bies et al., 1996). An essential E-mail: [email protected] Current addresses: 2School of Biological Sciences, Stopford Building, and ubiquitious role for B-Myb is also suggested by the University of Manchester, Oxford Road, Manchester M13 9PT, UK; findings that B-myb is expressed throughout mammalian 3Department of Surgery and Molecular Oncology, University of embryogenesis (Sitzmann et al., 1996), and that homo- Dundee, Ninewells Hospital and Medical School, Dundee DD1 9SY, zygous gene knockout mutants died early in UK 4These two authors contributed equally to this publication development at around E4.5 – E6.5 (Tanaka et al., 1999). Received 27 February 2002; revised 22 August 2002; accepted 29 Although these studies indicate an essential role for August 2002 B-Myb in cell growth, neither the functions required Interaction of B-Myb with p107 M Joaquin et al 7924 nor the stage of the cell cycle at which B-Myb acts have been definitively established. Some studies suggest, however, that ectopic expression of B-myb can promote transition of cells from the G1 to the S phase of the cell cycle, most notably in cell lines in which the G1 checkpoint was activated by p53 or p107 (Lin et al., 1994; Sala et al., 1996a). This conclusion is supported by evidence that overexpression of B-myb was sufficient by itself to promote entry of T98G glioblastoma cells into S phase (Sala et al., 1996a), whilst in the U-2 OS osteosarcoma cell line B-Myb acted synergistically with cyclin A2 to induce S phase (Lane et al., 1997). Although it has been tacitly assumed that promotion of cell proliferation by B- Myb reflects its role as a transcriptional activator, a transactivation domain mutant (MUT-7) was still partially active in overcoming p53-induced growth arrest in Saos-2 cells (Lin et al., 1994). Moreover, we recently found that the ability of ectopically expressed B-Myb to overcome a p107-induced G1 arrest was dependent upon a direct physical interaction between these proteins (Bessa et al., 2001a), and notably a transcriptionally activated B-Myb protein was deficient in abrogating p107 activity. We have extended these observations by mapping the interaction domains between B-Myb and p107, and by examining whether B-Myb transactivation activity is required to abrogate the p107 block. Our findings reveal that B-Myb binds to an N-terminal p107 domain that overlaps with the cyclin-binding domain. Results Figure 1 Bypass of a p107 G1 block by B-Myb does not corre- late with transcriptional activity. (a) The B-Myb DNA binding B-Myb transactivation activity is not required to domain, Acidic Region and Conserved Region (CR) are indicated overcome a p107-mediated G1 block together with the locations of the cyclin A2/cdk2 phosphorylation sites mapped to date (represented by lollipops). Indicated below To explore whether B-Myb transactivation function is are the interstitial deletions introduced into B-Myb within the required to overcome a p107-induced G1 arrest, we Acidic Region and in evolutionarily conserved sequences immedi- ately downstream. (b) Transactivation activities of wt B-Myb and first introduced a number of small deletions into the the B-Myb AR mutants were compared by transfection of B-Myb Acidic Region (AR: aa 205 – 308), which pcDNA3 expression vectors encoding these proteins together with constitutes the major transactivation domain (Naka- the Myb-responsive luciferase reporter pT81luc 3xA into U-2 OS goshi et al., 1993). Notably, the B-Myb AR is quite cells. Luciferase activities were assayed 2 days post-transfection and were corrected with respect to b-galactosidase activity direc- distinct from the c-Myb transactivation domain, which ted by a cotransfected reporter. (c) Saos-2 cells were transfected shares a weakly acidic character but little if any with the empty pcDNA3 vector or pcDNA3 encoding either wt homology. Additional deletions were also introduced B-Myb or the AR mutants. Duplicate plates also received either downstream of the AR coding sequence in a region the empty pCMV expression or pCMV encoding p107. The per- that is highly conserved between mouse and human B- centage of cells in G1 phase in the duplicate plates was deter- mined by flow cytometry (transfected cells were discriminated Myb (aa 333 – 388). The transactivation activities of by virtue of a cotransfected CD20 plasmid) and are presented this set of mutants (Figure 1a) were then compared as a stack bar chart. The increase in G1 cells upon pCMVp107 with wild-type B-Myb by transient transfection in U-2 transfection (indicated by the shaded bar) resulted largely from OS cells using the pT81luc 3xA luciferase reporter a concomitant decrease in S phase cells (Ness et al., 1989). In this cell line, transfected B-Myb is partially phosphorylated by endogenous cyclin A2/ cdk2 (Saville and Watson, 1998b), resulting in MybD289 – 308) demonstrated enhanced activity enhanced transactivation activity. Deletion of the (Figure 1b). most N-terminal AR sequence (as in B-MybD205 – We next compared the abilities of wt B-Myb and the 243) abolished transactivation activity almost comple- B-Myb mutants to overcome a p107-mediated G1 tely, whereas deletion of other segments of the AR arrest in Saos-2 cells, with the goal of testing whether and the downstream sequences resulted in retention of this cell cycle effect correlated with transactivation residual transactivation activity (Figure 1b).

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