JunB is a member of the AP-1 (activator protein-1) family of dimeric transcription factors. It exerts a dual action on the cell cycle. It is best known as a cell proliferation inhibitor, a senescence inducer and a tumour suppressor. As for the molecular mechanisms involved, they largely involve both positive actions on genes such as the p16INK4α cyclin-dependent kinase inhibitor and negative effects on genes such as cyclin D1 during the G1-phase of the cell cycle. However, JunB is also endowed with a cell-division-promoting activity, in particular via stimulation of cyclin A2 gene expression during S-phase. Strikingly, its role in G2 and M has received little attention so far despite its possible role in the preparation of mitosis. This review addresses the known and possible mechanisms whereby JunB is implicated in the control of the different phases of the cell cycle.
The AP-1 (activator protein-1) transcription complex consists of a whole collection of dimeric bZip (basic region leucine zipper motif)-containing transcription factors. Its best-studied components are the members of the Fos (c-Fos, FosB, Fra-1 and Fra-2) and Jun (c-Jun, JunB and JunD) families, which bind to DNA owing to a basic motif [DBD (DNA-binding domain)] and dimerize via an adjacent LZ (leucine zipper) domain. AP-1 dimers bind to the so-called AP-1/TRE [PMA (‘TPA’)-responsive element] and CRE (cAMP-response element) DNA motifs. They act either positively or negatively on transcription depending on their composition, their post-translational modifications, the target gene, the cell context and the environmental signals. AP-1/TRE and CRE motifs are found in many genes. Hence, AP-1 regulates many fundamental cell processes, including proliferation, differentiation, apoptosis and responses to stresses and is essential for many physiological functions at the whole organism level. AP-1 is also implicated in various pathologies, notably tumorigenesis due to its multiple effects on cell fate [1–7]. Although certain AP-1 proteins can be oncogenic on their own in certain situations, the major contribution of AP-1 to tumorigenesis is as a downstream effector of various oncogenes. In particular, abnormal Jun and Fos expressions are associated with a number of human neoplasia , consistently with their physiological actions on the expression of cell-cycle regulators such as cyclins and CKIs (cyclin-dependent kinase inhibitors). However, some of the AP-1 components can also display oncosuppressor activity in certain circumstances. This is illustrated not only by c-Fos , but also by JunB. The case of the latter protein is particularly interesting. Although it is best known as an inhibitor of cell division [10,11], an inducer of senescence  and a tumour suppressor, at least in the myeloid lineage [12,13], there is accumulating evidence that JunB has not only cell-division-inhibiting but also cell-division-promoting activities. The manifestation of these two opposite properties depends on both the cell-cycle stage and the environmental conditions, as reviewed below.
Cell-cycle expression of JunB
Like c-Fos and c-Jun, JunB expression is very low in quiescent cells but reaches a peak during the G0/G1 transition before returning to an intermediate level in response to mitogenic stimulations. This induction is instrumental for cell progression towards S-phase, as demonstrated by microinjection of a specific antibody into cells exiting from quiescence after stimulation by growth factors [14,15]. In cycling cells, JunB levels strongly increase as cells progress through G1-phase and enter S-phase, whereas in G2/M- and G1-phases, JunB levels are very low. In contrast, c-Jun levels remain constant during the same period of time with, however, a progressive increase in transcription potential during early G1 resulting from N-terminal phosphorylation  (Figure 1).
Fluctuations of c-Jun and JunB levels during the cell cycle
Cell-division-inhibitory activity of JunB
Early studies based on antibody microinjection experiments showed that JunB antagonizes the transforming activity of c-Jun . Further experiments support these results and suggest that JunB is a negative regulator of cell proliferation. In particular, primary MEFs (mouse embryonic fibroblasts) derived from JunB-overexpressing transgenic mice show reduced proliferation and, after immortalization, JunB-expressing fibroblasts display proliferation defects due to extended G1. Interestingly, JunB accumulation leads to cell-cycle arrest in G1 via induction of the cell-cycle kinase inhibitor p16INK4α gene . In addition, overexpression of JunB antagonizes c-Jun-mediated induction of cyclin D1 in G1 . Cells are consequently blocked before they can enter S-phase, which can be followed by senescence . The idea of antiproliferative function of JunB is further supported by the observation that transgenic mice specifically lacking JunB expression in the myeloid lineage are affected by a myeloproliferative disease progressing to blast crisis. Consistent with this observation, the absence of JunB expression results in down-regulation of p16INK4α expression and increased levels of c-Jun . In line with these results obtained in an experimental mouse model, JunB expression is down-regulated in both human CML (chronic myeloid leukaemia)  and AML (acute myeloid leukaemia) . Moreover, recent results showing that JunB negatively regulates the proliferation of haemopoietic stem cells suggests that the oncogenic process leading to myeloid tumours starts in these cells . Also supporting the notion that JunB can manifest inhibitory effects on the cell cycle, its overexpression inhibits transformation of B-cells by the v-abl oncogene in the mouse .
Cell-division-promoting activity of JunB
There is accumulating evidence that JunB has both cell-division-promoting and -inhibiting activities, the manifestation of which depends on both the cell-cycle stage and the environmental conditions. In this sense, replacement of c-Jun by JunB in mice abrogates the proliferation defects and deregulated expression of cyclin D1, p53 and p21 associated with the loss of c-Jun in fibroblasts . These results suggest that in the absence of Jun, JunB may function as a positive regulator and that the antiproliferative activity of JunB requires the formation of growth-inhibitory cJun–JunB heterodimers. It has also been shown that JunB can activate the expression of IL-4 (interleukin-4) and drive helper T-lymphocytes differentiation towards the Th2 lineage . Furthermore, JunB has been shown to have cell-cycle-promoting activity through cyclin A2 activation. Fibroblasts with a targeted null mutation in the junB gene show an increase in the number of cells entering S-phase, whereas during the period extending from S to the G2/M transition, the loss of JunB leads to delay in cell-cycle progression. It was concluded from these analyses that rapid progression through S-phase depends on JunB, whose expression increases at the G1/S transition, to positively regulate transcription of the cyclin A2 gene (Ccna2) . In line with this cell-division-promoting activity, JunB can contribute to the tumour phenotype. For example, it co-operates with c-Jun in the development of mouse fibrosarcoma , and its increased expression seems essential for the pathogenesis of human anaplastic large-cell lymphoma and Hodgkin's lymphoma through induction of the CD30 promoter . Moreover, our own recent results indicate that perturbation of JunB abundance in G2 provokes mitotic abnormalities, which also supports the idea that JunB may exert oncogenic actions .
Regulation of JunB activity
Among the different cellular mechanisms controlling AP-1 activity, post-translational modifications and regulation of protein turnover are crucial. Post-translational modifications of the Jun proteins are most extensively documented in the case of mitogen- and cellular stress-induced hyperphosphorylation, with a particular emphasis on the activation of c-Jun by the JNK (c-Jun N-terminal kinase; reviewed by Shaulian and Karin  and Karin and Gallagher ). In contrast, JunB phosphorylations have hardly been studied. The fact that the specific JNK phosphorylation sites found in c-Jun are not conserved in JunB  suggests that the latter protein is not a substrate for JNKs. However, the work of Li et al.  shows that JNK MAPKs (mitogen-activated protein kinases) can phosphorylate JunB on Thr-102 and Thr-104 to stimulate IL-4 gene expression.
More recently, Bakiri et al.  have reported that a variety of mouse and human mitotic cells express low levels of JunB. Notably, JunB appears in an electrophoretically retarded form in these cells. This retardation is suppressed either by phosphatase treatment or by point mutation of three residues (Ser-23, Thr-150 and Ser-186) into non-phosphorylatable alanine residues in the mouse JunB . Interestingly, these residues are located within S/T-P motifs that are potential target sites for cyclin-containing CDK (cyclin-dependent kinase) complexes. Moreover, JunB co-immunoprecipitates with CDK1 from cell extracts and is phosphorylated by CDK1–cyclin B1 complexes on these residues in vitro , which we could confirm in in vitro phosphorylation assays (V. Baldin and R. Farràs, unpublished work). Consequently, Bakiri et al.'s  work raised the interesting hypothesis that CDK1–cyclin B1 complexes would phosphorylate JunB on these residues during mitosis to destabilize it during this specific period of the cell cycle in order to ensure low levels at the onset of the following G1 phase and, thereby, to permit cell-cycle progression . Some of our recent results, however, support an alternative scenario. Although not questioning the necessity to have low JunB levels in early G1, it suggests that phosphorylation-triggered degradation of JunB occurs earlier than mitosis . This also questions the fact that the destabilization-inducing kinase would be cyclin B/CDK1 as this kinase is activated abruptly only at the end of G2 and shows maximal activity during mitosis . Moreover, cyclin B is predominantly cytoplasmic during interphase , whereas JunB is essentially nuclear. We, however, do not exclude that the remnant of JunB found throughout mitosis might be phosphorylated by cyclin B1/CDK1, especially after nuclear envelope breakdown, which might possibly be useful to fully repress JunB transcriptional activity.
JunB appears to undergo ubiquitination-mediated proteasomal degradation , at least in various transfection assays, including our own unpublished results, and upon activation of mouse helper T-cells [32–34]. Nevertheless, under other conditions, it might be possible that only a fraction of JunB would be a target for proteasomal degradation, as treatment of exponentially growing mouse fibroblasts with a proteasome inhibitor only led to partial stabilization of JunB . Concerning ubiquitination- and proteasome-dependent degradation, it has been reported that the E3 HECT (homologous to E6-associated protein C-terminus) protein Itch is responsible for ubiquitination of JunB in mouse T-cells [32,34]. Recent RNAi (RNA interference) experiments against AIP4 (which is the human homologue of Itch) conducted in asynchronously growing HeLa cells have failed to detect any stabilization of JunB, suggesting that there might exist several E3 factors targeting JunB (R. Farràs, unpublished work). In fact, this would not be surprising, as this has already been shown for other proteins, including c-Jun [24,34–37]. As far as ubiquitination is concerned, it is important to keep in mind that it may serve purposes other than targeting proteins to the proteasome [39–42] and that other AP-1 proteins, albeit ubiquitinatable, can be degraded by the proteasome without being ubiquitinated either in vitro (c-Jun; ) or in vivo (c-Fos and Fra-1; [44–47]).
Finally, like c-Fos and c-Jun , JunB can undergo modification by the small ubiquitin-like modifiers of the SUMO (small ubiquitin-related modifier) family . SUMOylation is generally responsible for transcriptional inactivation of transcription factors. However, JunB SUMOylation in T-cells may result in transcriptional activation of the AP-1-responsive gene, although this effect may depend on the targeted promoter .
JunB implication in cancer is twofold and context-dependent. In addition to its well-described cell proliferation inhibition- and senescence-promoting activities whereby it can act as a tumour suppressor, JunB can also contribute to the tumour phenotype. JunB has unique functions in cell-cycle regulation, which cannot be compensated by other AP-1 members. Moreover, it can display antagonistic functions (i.e. be a transcriptional repressor or a transcriptional activator, depending on the conditions and the gene) that are both required for progression through the cell cycle (Figure 2). Elucidating the still largely elusive molecular mechanisms governing JunB protein level and activity throughout the cell cycle may help understand the biological outcomes it is responsible for. In particular, modification by phosphorylation and/or SUMOylation, as well as its ubiquitin/proteasome-dependent destabilization, deserves further investigation through complementary biological and biochemical approaches.
Model for cell-cycle-inhibiting and -promoting activities of JunB
Third Intracellular Proteolysis Meeting: A joint Biochemical Society and INPROTEOLYS Network Focused Meeting held at Auditorio de Tenerife, Santa Cruz de Tenerife, Canary Islands, Spain, 5–7 March 2008. Organized and Edited by Rosa Farràs (Centro de Investigación Príncipe Felipe, Valencia, Spain), Gemma Marfany (Barcelona, Spain), Manuel Rodríguez (CICbioGUNE, Derio, Spain), Eduardo Salido (La Laguna, Tenerife, Spain) and Dimitris Xirodimas (Dundee, U.K.).
M.P.'s laboratory is an ‘Equipe Labellisée’ supported by the Ligue Nationale contre le Cancer. This work has also been supported by grants from the Fondo de Investigaciones Sanitarias (PI05/2139) and the Conselleria Valenciana D'Empresa, Universitat i Ciència (GV05/154). R.F. is supported by the Fondo de Investigaciones Sanitarias (CP03/00106).