Mafa Has Strong Cell Transforming Ability but Is a Weak Transactivator

MafA has strong cell transforming ability but is a weak transactivator

Makoto Nishizawa1, Kohsuke Kataoka2 and Peter K Vogtn,1

1Department of Molecular and Experimental Medicine, BCC239, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA, 92037, USA; 2Frontier Collaborative Research Center, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8503, Japan

The maf oncogene of the avian oncogenic retrovirus AS42 Kataoka et al., 1994a) and NRL (Swaroop et al., encodes a nuclear bZip protein, v-Maf, that recognizes 1992) contain a putative transactivation domain (Ka- sequences related to the AP-1 target site. The correspond- taoka et al., 1993) in their amino-terminal region. This ing cellular protein, c-Maf belongs to a family of related region is well conserved and is rich in acidic amino acids. bZip proteins together with MafA and MafB. In this Deletion of this region of v-Maf reduces cell transform- paper, we compare the transactivation and cell transforming and transactivation activities.A structure-function ing abilities of MafA and MafB along with two forms of study of v-Maf supports the idea that Maf induces the c-Maf protein. These proteins induce cellular trans- transformation by transcriptional deregulation of down- formation when expressed in chicken embryo fibroblasts. stream target genes (Kataoka et al., 1993). In reporter assays, MafA is a much less effective The cellular maf gene (c-maf) and mafB are rear- transactivator than the other Maf proteins, but unexpect- ranged and show gain of function in human tumors.In edly shows the strongest activity in cell transformation. multiple myeloma, the c-maf locus is frequently translo- Chimeras of MafA and MafB correlate the strong cell cated to either the immunoglobulin heavy or the light transforming ability of MafA with its DNA-binding chain gene, and expression is elevated, probably through domain. The DNA-binding domain of MafA is also an enhancer effect (Chesi et al., 1998; Bergsagel and correlated with weak transactivation. Additional muta- Kuehl, 2001).Similarly, the related mafB shows elevated genesis experiments show that transactivation and trans- expression in multiple myelomas that also carry a formation by MafA are also controlled by chromosomal translocation (Hanamura et al., 2001). phosphorylation of two conserved serine residues in the The small Maf proteins, MafK (Fujiwara et al., 1993), transactivation domain. Finally, we constructed MafA- MafF (Fujiwara et al., 1993) and MafG (Kataoka et al., estrogen receptor fusion molecules that show tightly 1995) lack a transactivation domain.These proteins can hormone-dependent cell transforming ability. These reg- interact with other bZip proteins to form heterodimers ulatable constructs permit a kinetic characterization of that transactivate (Andrews et al., 1993; Igarashi et al., target gene responses and facilitate discrimination be- 1994), but they fail to induce oncogenic transformation tween direct and indirect targets. on their own (Fujiwara et al., 1993). Oncogene (2003) 22, 7882–7890. doi:10.1038/sj.onc.1206526 The bZip domain is located at the C-terminus of the Maf proteins.This well-conserved region determines Keywords: oncogenic transformation; transcriptional DNA-binding and dimerization specificities.The Maf activation; regulatable construct proteins recognize 13- or 14-base palindromic sequences that are related to the recognition sequence of AP-1 transcription factors (Kataoka et al., 1994b; Kerppola and Curran, 1994).Since the Maf-recognition element (MARE) is relatively long, we have previously tried to Introduction find such elements in 50-noncoding sequences of eukaryotic genes (Kataoka et al., 1994b). Some of these The maf oncogene was identified in an acutely oncogenic MAREs were confirmed as Maf targets.For instance, avian retrovirus, AS42, isolated from a spontaneous genes for phase II enzymes, that are induced by chicken tumor (Nishizawa et al., 1989; Kawai et al., Xenobiotics, are regulated by complexes that include a 1992).It is the prototype of a family of six related genes small Maf and another bZip factor, Nrf2 (Itoh et al., that code for bZip proteins.This family encompasses 1997).c-Maf and Nrl regulate lens- and retina-specific three small and four large Maf proteins.The large Mafs, gene expression (Kumar et al., 1996; Sharon-Friling cellular and viral Maf (c-Maf, v-Maf), MafA (also et al., 1998; Kawauchi et al., 1999; Kim et al., 1999a, b; known as L-Maf) (Benkhelifa et al., 1998; Ogino and Ring et al., 2000). Gene targeting of c-maf induces Yasuda, 1998), MafB (Cordes and Barsh, 1994; severe defects in the development of several tissues, including the eye.MafA is also a positive regulator of *Correspondence: PK Vogt; E-mail: [email protected] genes expressed in eye tissue, the retina-specific QR1 Received 13 December 2002; revised 20 February 2003; accepted 26 gene and crystalline genes (Benkhelifa et al., 1998, 2001; February 2003 Ogino and Yasuda, 1998).c-Maf is also important in the Cellular transformation induced by MafA M Nishizawa et al 7883 differentiation of CD4 þ T cells into Th1 and Th2 T λM41 cells (Ho et al., 1998; Kim et al., 1999a, b; Ho and kb Glimcher, 2002).Recent results show that MafA is 0 10 involved in glucose-regulated insulin gene expression in pancreatic b-cells (Olbrot et al., 2002; Kataoka et al., BBBBBB submitted).Although several targets of the Maf family proteins have been identified, the mechanism of cell EEMM EX E E transformation by Maf remains unknown. In this study, we compare transactivation and cell 5' (A)n transforming abilities of large Mafs to examine the Figure 1 The structure of the chicken mafA gene.A genomic clone possible relationship between transactivation and cell of the chicken mafA gene, lM41 was mapped by restriction transformation.One of these Maf proteins, MafA was endonuclease and sequenced.The sequence of a 4 kb region is less effective in transactivation than the others, but was marked as a bold line on the restriction map.The transcribed much more efficient in cell transformation.We con- region is shown at the bottom.The open box indicates the coding region of the mRNA.Restriction enzyme sites are indicated as structed recombinants between MafA and MafB, and follows: E, EcoRI; B, BamHI; X, XhoI; M, MluI found that the strong transforming and weak transactivation abilities of MafA are associated with the DNA- binding/hinge region.Mutagenesis experiments showed that phosphorylation of the amino-terminal domain of MafA has a significant effect on transactivation and cell transformation as well.We also constructed fusion proteins that include the DNA-binding domain of MafA and the hormone-binding domain of the estrogen receptor (ER).These constructs induce cell transformation in a hormone-dependent manner.The inducible Maf will provide a valuable tool in the analysis of transformation.

Figure 2 The structure of large Maf proteins and their transactivation activities.( a) Two forms of c-Maf, MafB and MafA are Results depicted.H and G represent tracts of histidine and glycine, respectively.Transactivation activities of the proteins are listed on the right.These values are averages of three independent luciferase The structure of the chicken mafA gene assays Clones of the chicken mafA gene were isolated by screening a chicken genomic library with a v-maf probe under relaxed hybridization conditions.We studied ﬁbroblasts (CEF).The results are summarized in one of the clones, M41, by restriction mapping and Figure 2.Type I c-Maf was the most potent activator nucleotide sequence analysis.Figure 1 shows the followed by type II c-Maf and MafB.MafA was the structure of the chicken mafA gene.This gene does not least effective.The data suggest an apparent correlation contain an intron as judged by comparison to previously between activity and the size of the transactivation reported chicken (Ogino and Yasuda, 1998) and domains (cMAF: 174aa, MafB: 129aa, MafA: 125aa). Japanese quail (Benkhelifa et al., 1998) mafA cDNA. As shown below, this correlation did not hold in genetic The sequence information of the chicken mafA genomic experiments (Figure 3). clone is available from the GenBank DNA database In order to compare the cell transforming ability of (Accession number of AY152408). the three large Maf proteins in CEF, the proteins were expressed with the avian retroviral vector, pRV-9 Transactivation and cell transforming activities of the (Nishizawa et al., 2003). All large Maf proteins induced large Maf proteins cell transformation, suggesting that the structural changes seen in v-Maf are not a prerequisite of We compared the transactivation activity of MafA, transforming ability.As we reported previously, MafB MafB and two forms of c-Maf in a luciferase reporter showed reduced transforming ability compared to c- assay method.The two forms of c-Maf (types I and II) Maf.Although the numbers of foci formed by MafB are generated by alternative transcriptional termination. were comparable to those by c-Maf, they were smaller Type I has an additional 10 amino-acid residues at its and took longer to develop (Figure 3).MafA showed the carboxy-terminus.We subcloned the coding sequences highest cell transforming ability.Focus formation was of these three genes into the pHyg-EF-2 expression more rapid, and the saturation density of cells was vector (Nishizawa et al., 2003). The expression plasmids highest in MafA-transformed CEF (Figure 3).Colony for the large Maf proteins were transfected with an forming activities in soft agar suspension culture internal control plasmid (Renilla luciferase gene/pHyg- mirrored the focus assays.MafA was a potent inducer EF-2) and a luciferase reporter plasmid that includes a of anchorage-independent growth, comparable to v-Jun triplicated MARE (3x7/pRBGP) into chicken embryo (Figure 3).The other Maf proteins induced few and

Oncogene Cellular transformation induced by MafA M Nishizawa et al 7884

Figure 3 Focus and colony formation by the large Maf proteins.The proteins were expressed in CEF with the avian retroviral vector pRV-9.( a) Indicated amounts of virus stocks were inoculated into the CEF.After 2 weeks of culture under soft agar, the cells were stained with 3% crystal violet-30% methanol.( b) CEF were infected with the recombinant viruses which expressed Maf family proteins, and were incubated in soft agar suspension culture for 3 weeks

small agar colonies.This high transforming ability of These fragments contain the DNA-binding domain and MafA contrasts with its low performance in luciferase hinge regions that connects to the zipper domain.We assays. tested transactivation and cell transforming abilities after subcloning into the appropriate expression vectors. Exchange of the N-terminal half containing the The DNA-binding domain is a major determinant of the major transactivation domain (MafA-S-B and MafB- strong cell transforming ability of MafA S-A in Figure 4a) had only a minor effect on In order to investigate the discrepancy between tran- transactivation.The size of the transactivation domain scriptional activation and transformation in MafA, we does therefore not determine transactivation activity. constructed MafA/MafB chimeras (Figure 4a).Plasmids Exchange of the zipper domain had an equally negligible coding MafA-S-B and MafB-S-A, were constructed effect on transactivation potential (MafA-X-B and using the SacI site that is conserved in both genes. MafB-X-A in Figure 4a): The leucine zipper of MafB We then introduced an XhoI site at the ﬁrst leucine transplanted onto MafA did not enhance the transacti- residue of the zipper structure of MafA and MafB, vation potential of the hybrid, and the leucine zipper of without changing amino-acid sequences, and exchanged MafA did not affect the high transactivation of the restriction fragments to generate MafA-X-B MafB.However, if the SacI/XhoI fragments, containing and Maf B-X-A.Finally, we built MafA-B-A and the DNA-binding domain and hinge regions were MafB-A-B by exchanging the SacI–XhoI fragments. swapped, the transactivation potentials were exchanged

Oncogene Cellular transformation induced by MafA M Nishizawa et al 7885

Figure 4 Chimeric constructs of MafA and MafB.( a) Structures of the MafA and MafB chimeras.The SacI and XhoI restriction sites, which were used to make the chimeric molecules are marked.Results of two transactivation experiments with different primary cultures are shown.The fold activations induced by the constructs are averages of three independent luciferase assays.The cell transforming abilities of the chimeric constructs are summarized in the right of the ﬁgure.Transformation was graded from 1 þ to 4 þ , standardized according to the results summarized in Figure 3: 2 þ corresponding to MafB and 3 þ to MafA.( b) Comparison of amino-acid sequences of the 45 amino-acid residues encoded by the SacI–XhoI fragments of mafA and mafB.Amino-acid residues that are different are boxed.The ﬁrst leucine residue of the zipper motif is indicated by arrows

with them (MafA-B-A and MafB-A-B in Figure 4a).We transactivation by MafA with a minor role played by the conclude that the domain coded for by the SacI–XhoI N-terminal regions. fragment exerts a major control on transactivation potential.Within the DNA-binding/hinge region, MafA No detectable difference in the DNA-binding ability and MafB differ by only five amino acids as shown in between MafA and MafB Figure 4b. The cell transforming activities of the chimeras are The controlling effect of the DNA-binding domain on summarized in Figures 4a and 5.As is in the parental transactivation suggested a possible difference between Maf proteins, high transactivation potentials are MafA and MafB in binding to the DNA target generally correlated with low transforming ability and sequence.In order to test this possibility, we synthesized vice versa.The most actively transforming construct MafA, MafB and two of the chimeric constructs by in was MafB-S-A, which also induced a rapid loss of vitro transcription/translation and tested them in gel cell adhesion.Constructs that include the DNA-binding mobility shift assays.A 32P-labeled double-stranded domain and hinge regions of MafA were associated MARE oligonucleotide was used as a probe. with weak transactivation activity and a high level Equal translation efficiency of the recombinant of cell transformation.The N-terminal domains proteins was verified by using 35S-methionine label in showed minor effects on transformation.The presence separate reactions (Figure 6a).The multiple bands seen of the N-terminal region derived from MafB was in the figure are probably derived from alternative starts associated with enhanced transformation of hybrid and premature terminations.The DNA-binding effi- constructs (compare MafA-S-B to MafB-S-A or ciency of MafA and MafB were about equal, and MafA-B-A to MafB-A-B).We conclude that the exchange of the DNA-binding domain did not alter DNA-binding domain and hinge regions are the main binding significantly.The DNA-binding and hinge determinants for strong cell transformation and weak regions of MafA and MafB must therefore have an

Oncogene Cellular transformation induced by MafA M Nishizawa et al 7886

Figure 5 Morphology of CEF transformed by the chimeric Maf proteins.CEF were infected with pKV-9 retroviral vectors expressing the chimeric constructs depicted in Figure 4.Cells were transferred twice at 2-day intervals and then photographed.Transformation by MafA-S-B was marginal; MafB-S-A and MafB-A-B were strong transformers

additional function that affects transactivation and cell reporter that contains the Maf-binding A box of the transformation. quail retina-specific QR1 gene.The A box element is similar to a half site of the palindromic MARE sequence and is recognized by Maf proteins, probably at reduced Effect of phosphorylation of MafA on its transactivation efficiency.We used a triplicated palindromic MARE and cell transforming activities sequence as binding site of the reporter plasmid to Two serine residues in the transactivation domain of achieve higher efficiency of binding.Mutation of Ser 14 MafA are phosphorylated by ERK2 kinase in vivo,and slightly reduced transactivation activity of MafA the phosphorylation status of the two serine residues (Figure 7a).If both Ser 14 and Ser65 were converted to greatly upregulates transactivation activity of MafA alanine (MafA S14AS65A), transactivation was nearly (Benkhelifa et al., 2001). In order to test the effect of abolished.The single mutation of Ser 65 (MafA S65A) phosphorylation on the cell transforming ability of unexpectedly increased transactivation activity. MafA, we introduced point mutations in the phosphor- The mutated genes were introduced into the pRV-9 ylation sites.These mutants are similar to the ones of retroviral expression vector, and recombinant viruses Benkhelifa et al.(2001) working with MafA of Japanese were recovered after DNA transfection into CEF.The quail.They tested transactivation activity with a recovered viruses were then used for infection of CEF to

Oncogene Cellular transformation induced by MafA M Nishizawa et al 7887 test transforming abilities.The double mutant, MafA S14AS65A, showed clearly decreased cell transforming ability (Figure 7b).Introduction of mutation on either one of the two phosphorylation sites showed different effects on transformation.Mutation of Ser 14 signiﬁ- cantly reduced the cell transforming ability.Mutation of Ser65 activated cell transformation.

Construction of an inducible cell transformation system To take advantage of the strong transforming ability of MafA, we designed an inducible cell transformation system.We constructed several fusion proteins that include the DNA-binding domain of MafA and the hormone-binding domain of the human ER (Kumar et al., 1986). We have found that v-Maf and MafA with the zipper domain replaced by the yeast GCN4 retain transforming ability (Kataoka et al., 2001; Nishizawa, 2002, unpublished observation).We used the MafA- GCN4 chimera for the ER fusions because the GCN4 Figure 6 DNA-binding activity of the MafA/MafB chimeric zipper domain only homodimerizes, eliminating poten- constructs.( a) Maf family proteins and chimeric proteins composed of MafA and MafB were translated in vitro in the tially complicating interactions with other bZip pro- presence of 35S-methionine and were separated by SDS–polyacry- teins.We also deleted the amino-terminal domain of lamide gel electrophoresis.The gel was immersed in EN3HANCE MafA and replaced it with the nonconditional transac- (NEN life science, Boston, MA, USA) was dried under vacuum tivation domain of the herpes simplex virus activator and exposed to X-ray ﬁlm.The Maf proteins appear as heavily labeled doublets.The smaller forms are probably due to internal VP16 or with the estrogen-regulatable transactivation initiation or proteolytic degradation.( b) Gel mobility shift assay of domain of ER.This exchange eliminates the effect of the MafA/MafB chimeric proteins.Indicated Maf proteins were protein kinases and protein phosphatases that regulate translated in vitro, and were used in gel mobility shift assays the MafA-transactivation domain.

Figure 7 Point mutants of MafA and their biological activities.( a) Schematic representation of phosphorylation site mutants of MafA and their transactivation activities.The transactivation activities of the mutants are summarized on the right.( b) Effect of overexpression of the MafA mutants on the morphology of CEF.The double mutant S14AS65A showed greatly reduced transforming ability.The single mutant S14A transformed, but the activity was lower than that of MafA; the single mutant S65A showed enhanced transformation

Oncogene Cellular transformation induced by MafA M Nishizawa et al 7888

Figure 8 Inducible cell transformation by MafA-ER fusion proteins.The structures of the fusion proteins are represented.The fusion proteins were expressed in CEF with the pRV-9 expression vector, and the cells were incubated for 3 weeks in the presence of b- estradiol or 4-HT.Control cells treated with solvent (ethanol) are also shown

Figure 8 represents structures of fusion molecules that large Maf proteins.Expression profiles and functional contain the DNA-binding domain of MafA, the zipper properties of these proteins indicate that they are dimerization domain of GCN4, and the hormone- regulators of tissue specific gene expression and differ- binding domain of ER.Two of the fusion constructs entiation.Yet all large cellular Maf proteins rapidly also contain the strong transactivation domain of VP16. induce transformation of CEF without structural These fusion constructs were expressed in CEF using the changes probably because the expression by the retrovirus vector, pRV-9.The infected cells were treated pRV-9 retroviral vector in CEF is both ectopic and with b-estradiol or with a homologue of estrogen, 4- excessive. hydroxytamoxifen (4-HT).The hormone-binding do- In chicken neuroretina cells, expression of MafA by a main of ER regulates cellular localization of the retroviral expression vector induces differentiation into chimeric molecule and provides an estrogen-dependent lens cells (Ogino and Yasuda, 1998), in contrast to the transactivation activity.4-HT also induces nuclear clear oncogenic effect of similarly expressed MafA in localization but is defective in the induction of the CEF.It will be interesting to determine the mechanism transactivation function of the hormone-binding do- that governs the choice between terminal differentiation main.Two of the ER fusions, NDMafA-GCN4-ER and and unregulated growth. VP16-NDMafA-GCN4-ER induced cell transformation We have previously constructed a series of deletion in a b-estradiol-dependent manner.The latter construct mutants of v-Maf, and found that there is a correlation was particularly tight, it induced transformation only in between transactivation and transformation (Kataoka the presence of b-estradiol.Although this construct et al., 1993). However, this correlation does not hold if contains the nonconditional VP16 transactivation do- we compare the large Maf proteins with each other. main, treatment with 4-HT did not induce cell trans- MafA is a weak transactivator, but a potent transfor- formation, suggesting dependence on the transactivation mer.In chimeric constructs between MafA and MafB, activity of the ER hormone-binding domain. we identified the DNA-binding and hinge regions as the domain responsible for low transactivation and high transformation.A possible explanation for the transforming activity of MafA could be greatly elevated Discussion expression relative to MafB.We could not rule out this possibility by Western blot analysis because of the lack Proto-oncogenes often require structural changes to of antibodies that recognize these proteins and their acquire cell transforming ability.An example is the v- chimeras with equal efficiency.However, the Maf B-A-B Jun protein whose high oncogenic activity results from chimera differs from MafB in only five amino acids; the changes in its primary structure (Bos et al., 1990). In this two proteins show different levels of transactivation and study, we investigated the transforming ability of the transformation, and they are translated in vitro with

Oncogene Cellular transformation induced by MafA M Nishizawa et al 7889 equal efficiency (Figure 6b).MafA and MafA-B-A are with rearranged c-maf or mafB Sebolt-Leopold (2000). also likely to be expressed at similar levels, but are However, preliminary results show that mutation of the different in their biological activities. conserved phosphorylation sites of human c-Maf do not Among the five amino-acid differences within the affect transforming ability in CEF (Nishizawa, 2002, short region of MafA and MafB, two are located in unpublished observation). hinge region that connects the DNA-interacting surface The hormone-regulatable MafA fusions may become and the leucine zipper (Figure 4b).The differences in the a valuable tool in target identification.This kind of hinge region are reminiscent of a v-Maf mutant, Q5H, conditional transcription factor can become active in the that show higher transforming ability than v-Maf presence of an inhibitor of protein synthesis, cyclohex- (Kataoka et al., 1993). In this mutant, Gln297 of the imide, allowing the distinction between direct and hinge region is replaced by His.This mutation does not indirect targets (Kruse et al., 1997). It is best used in affect on transactivation potential of v-Maf. conjunction with other transforming constructs that Since DNA binding of MafA and MafB are not address the same DNA target sequence.Candidate significantly different, some other function must account target genes may then be sought among the differentially for the differences in transformation and transactiva- expressed genes that are affected by all of these tion.One possibility is differential interaction with an transforming constructs.We have followed this reason- inhibitor or activator.We recently reported that a set of ing in a recent study that describes transforming hybrids Hox proteins can interact with the DNA-binding containing sequences from GCN4 and VP16 (Nishizawa domain of c-Maf and inhibit its activity.In the case of et al., 2003). This combinatorial use of different MafA and MafB, the unknown partner would have to regulatable constructs with identical or near-identical affect transactivation and transformation without alter- DNA targets could be useful in focusing on transforma- ing DNA binding.Another possible explanation for the tion-relevant genes. discrepancy between transactivation and transformation in MafA and MafB may be provided by the reporter assays.MafA may recognize and strongly activate transformation-relevant promoters, while showing weak Materials and methods activity with standard reporter constructs.Reporter Molecular cloning and plasmid construction assays probably do not accurately reflect transformation-relevant transcriptional regulation.If MafA speci- A chicken genomic DNA library was purchased from fically activates targets relevant for cell transformation Clontech.Genomic clones of the mafA gene were isolated by and affects other target genes only minimally, screens screening the library with a 0.74-kb v-maf specific probe for differential gene expression in MafA transformed excised from the pSae-2 plasmid (Fujiwara et al., 1993) under low stringency conditions.The insert of one of the genomic cells would show reduced noise from irrelevant genes. clones, lM41 was subcloned into a plasmid vector and was Such screens could also identify variant promoter mapped by restriction endonucleases and sequenced.The sequences that respond preferentially to transforming whole coding sequences of mafA, mafB and two forms of c-maf Maf.Incorporation of such sequences into reporter genes were subcloned into two expression vectors, pRV-9 and constructs would allow a better comparison between pHyg-EF-2 (Nishizawa et al., 2003). The pRV-9 is an avian transactivation and transformation. retroviral vector derived from RCAS (Hughes et al., 1987; The importance of the transactivation function of Nishizawa et al., 2003), and subclones of this vector were Maf proteins is strengthened by studies of the transacti- used to recover recombinant viruses which express the Maf vation domain.A deletion of the transactivation domain proteins, The pHyg-EF-2 vector contains the strong pro- from MafA resulted in a nontransforming construct moter of human elongation factor-1a gene (Mizushima and Nagata, 1990), and the subclones in this vector were used for (Nishizawa, 2001, unpublished observation), suggesting transient transfection assays.In order to improve expression that transactivation is important for oncogenicity. efficiency, we replaced sequences just before the initiator Further, transactivation by MafA is stimulated by methionine codon with the 50-noncoding sequence of v-src phosphorylation, and mutation of both phosphoryla- gene. tion sites negatively affects both transactivation and transformation.Unexpectedly, a single site mutation, S65A, enhanced both transactivation and transforma- Transient transfection assays tion.This seemingly contradictory observation under- Transactivation was measured with a reporter plasmid that lines the inadequacy of our current understanding of contained three copies of the MARE sequence upstream of the transactivation and its relationship to transformation. minimal promoter of rabbit b-globin gene.The MARE Replacement of the MafA transactivation domain with sequence used in the reporter was TGCTTACTAAGCA.This that of the herpes simplex virus protein VP16 sequence is a variant of the consensus sequence (TGCTGACT- greatly enhances both transactivation and transforma- CAGCA), it is efficiently recognized by Maf but, unlike the consensus sequence, it is not recognized by AP-1 thus tion activities of the proteins (Nishizawa, 2001, eliminating AP-1 background activity (Kataoka et al., unpublished observation).If phosphorylation of the 1994b).Transfection and measurement of luciferase activities conserved serine residues also regulates activities of were performed as described previously using Lipofectamine other large Maf proteins, the kinase responsible for Maf Plus transfection reagent (Invitrogen, Carlsbad, CA, USA) phosphorylation could be a promising target of novel and Dual-luciferase reporter assay system (Promega, Madison, anticancer drugs effective in human multiple myeloma WI, USA).

Oncogene Cellular transformation induced by MafA M Nishizawa et al 7890 Cells and viruses Acknowledgements This work was supported by National Institutes of Health Primary CEF cultures were prepared from embryos supplied Research Grants CA42564, CA78230, and CA79616.This is by SPAFAS (Preston, CT, USA) and were maintained as manuscript number 15330-MEM of the Department of described previously (Vogt, 1969).For recovery of infectious Molecular and Experimental Medicine, the Scripps Research viruses, pRV-9 and its derived constructs were transfected into Institute. CEF by the DMSO transfection method (Kawai and Nishizawa, 1984).

References

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Oncogene