AKT Induces Transcriptional Activity of PU.1 through Phosphorylation-mediated Modifications within Its Transactivation Domain
2001; Elsevier BV; Volume: 276; Issue: 11 Linguagem: Inglês
10.1074/jbc.m007482200
ISSN1083-351X
AutoresPiotr Rieske, JaganM.R. Pongubala,
Tópico(s)NF-κB Signaling Pathways
ResumoSignal transduction by the antigen receptor complexes is critical for developmental progression of B-lymphocytes, which are defined by assembly and sequential expression of immunoglobulin genes, which in turn are regulated by the enhancer elements. Although proximal antigen-receptor signal transduction pathways are well defined, the precise nuclear factors targeted by these signals remained unknown. Previous studies have demonstrated that tissue-restricted transcription factors including PU.1 and PU. 1 interaction partner (PIP) function synergistically with c-Fos plus c-Jun to stimulate the κE3′-enhancer in 3T3 cells. In this study, we demonstrate that the functional synergy between these factors is enhanced in response to mitogen-activated protein kinase kinase kinase, in 3T3 cells, where the enhancer is inactive. However in S194 plasmacytoma cells, mitogen-activated protein kinase kinase kinase was able to stimulate the activity of PU.1 but unable to induce the κE3′-enhancer activity. We have found that Ras-phosphoinositide 3-kinase-dependent externally regulated kinase, AKT, induces κE3′-enhancer activity in both pre-B and plasmacytoma cells. AKT stimulation of the κE3′-enhancer is primarily due to PU.1 induction and is independent of PU.1 interaction with PIP. Activation of AKT had no effect on the expression levels of PU.1 or its protein-protein interaction with PIP. Using a series of deletion constructs, we have determined that the PU.1 acid-rich (amino acids 33–74) transactivation domain is necessary for AKT-mediated induction. Substitution analyses within this region indicate that phosphorylation of Ser41 is necessary to respond to AKT. Consistent with these studies, ligation of antigen receptors in A20 B cells mimics AKT activation of PU.1. Taken together, these results provide evidence that PU.1 is induced by AKT signal in a phosphoinositide 3-kinase-dependent manner, leading to inducible or constitutive activation of its target genes. Signal transduction by the antigen receptor complexes is critical for developmental progression of B-lymphocytes, which are defined by assembly and sequential expression of immunoglobulin genes, which in turn are regulated by the enhancer elements. Although proximal antigen-receptor signal transduction pathways are well defined, the precise nuclear factors targeted by these signals remained unknown. Previous studies have demonstrated that tissue-restricted transcription factors including PU.1 and PU. 1 interaction partner (PIP) function synergistically with c-Fos plus c-Jun to stimulate the κE3′-enhancer in 3T3 cells. In this study, we demonstrate that the functional synergy between these factors is enhanced in response to mitogen-activated protein kinase kinase kinase, in 3T3 cells, where the enhancer is inactive. However in S194 plasmacytoma cells, mitogen-activated protein kinase kinase kinase was able to stimulate the activity of PU.1 but unable to induce the κE3′-enhancer activity. We have found that Ras-phosphoinositide 3-kinase-dependent externally regulated kinase, AKT, induces κE3′-enhancer activity in both pre-B and plasmacytoma cells. AKT stimulation of the κE3′-enhancer is primarily due to PU.1 induction and is independent of PU.1 interaction with PIP. Activation of AKT had no effect on the expression levels of PU.1 or its protein-protein interaction with PIP. Using a series of deletion constructs, we have determined that the PU.1 acid-rich (amino acids 33–74) transactivation domain is necessary for AKT-mediated induction. Substitution analyses within this region indicate that phosphorylation of Ser41 is necessary to respond to AKT. Consistent with these studies, ligation of antigen receptors in A20 B cells mimics AKT activation of PU.1. Taken together, these results provide evidence that PU.1 is induced by AKT signal in a phosphoinositide 3-kinase-dependent manner, leading to inducible or constitutive activation of its target genes. B cell receptor PU. 1 interaction partner phosphoinositide 3-kinase polymerase chain reaction chloramphenicol acetyltransferase green fluorescent protein mitogen-activated protein kinase kinase kinase IκB kinase signalsome Signaling components of the antigen receptor complexes on the surface of B cell progenitors (pre-BCR)1 and B cells (BCR) are necessary for the developmental progression of B-lymphocytes (1Kitamura D. Rajewsky K. Nature. 1992; 356: 154-156Crossref PubMed Scopus (320) Google Scholar, 2Kitamura D. Kudo A. Schaal S. Muller W. Melchers F. Rajewsky K. 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Sakumi K. Nakamura H. Kingsbury L. David C. Hardy R. Yamamura K. Sakano H. Cell. 1995; 83: 1113-1123Abstract Full Text PDF PubMed Scopus (57) Google Scholar). Previous studies have demonstrated that transcriptional activity of the κE3′-enhancer is critically dependent on binding components of the κE3′-CRE (binds c-Fos, c-Jun, CREM, and ATF1), PU.1 plus PIP (binds PU.1 and PIP) and E2A (binds E12/47) sites. Mutation of any one of these binding sites greatly reduces enhancer activity (22Pongubala J.M.R. Nagulapalli S. Klemsz M. McKercher S. Maki R. Atchison M. Mol. Cell. Biol. 1992; 12: 368-378Crossref PubMed Scopus (309) Google Scholar, 23Pongubala J.M.R. Van Beveren C. Nagulapalli S.K.M.J. McKercher S. Maki R. Atchison M. Science. 1993; 259: 1622-1625Crossref PubMed Scopus (240) Google Scholar, 24Pongubala J.M.R. Atchison M. J. Biol. Chem. 1995; 270: 10304-10313Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 25Pongubala J.M.R. Atchison M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 127-132Crossref PubMed Scopus (77) Google Scholar). Among the core binding proteins, the expression of PU.1 and PIP is restricted to cells of hematopoietic lineages. Within the κE3′-enhancer, PU.1 recruits the binding of PIP, through protein-protein interaction to its adjacent DNA-binding site (22Pongubala J.M.R. Nagulapalli S. Klemsz M. McKercher S. Maki R. Atchison M. Mol. Cell. Biol. 1992; 12: 368-378Crossref PubMed Scopus (309) Google Scholar, 23Pongubala J.M.R. Van Beveren C. Nagulapalli S.K.M.J. McKercher S. Maki R. Atchison M. Science. 1993; 259: 1622-1625Crossref PubMed Scopus (240) Google Scholar). The recruitment of PIP requires phosphorylation of PU.1 at the amino acid, Ser148. Mutation of this serine residue to alanine (S148A) prevents interaction of PU.1 with PIP as well as the binding of PIP to its adjacent DNA binding sequences. This phosphorylation-mediated interaction of PU.1 appears to induce a conformational change in PIP, thereby allowing it to recognize DNA-binding sequences (26Perkel J.M. Atchison M.L. J. Immunol. 1998; 160: 241-252PubMed Google Scholar). Similar protein-protein interaction between PU.1 and PIP has been detected in the Ig λ light chain enhancer and the CD20 promoter (27Brass A. Kehrli E. Eisenbeis C. Storb U. Singh H. Genes Dev. 1996; 10: 2335-2347Crossref PubMed Scopus (207) Google Scholar, 28Brass A.L. Zhu A.Q. Singh H. EMBO J. 1999; 18: 977-991Crossref PubMed Scopus (147) Google Scholar, 29Himmelmann A. Riva A. Wilson G.L. Lucas B.P. Thevenin C. Kehrl J.H. Blood. 1997; 90: 3984-3995Crossref PubMed Google Scholar). Interestingly, PIP can bind independently to the interferon-stimulated response element, ISRE (30Matsuyama T. Grossman A. Mittrucker H. Siderovski D. Kiefer F. Kawakami T. Richardson C. Taniguchi T. Yoshinaga S. Mak T. 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Gene targeting studies indicate that PU.1 is essential for development of both B cells and macrophages (13Scott E. Simon M. Anastasi J. Singh H. Science. 1994; 265: 1573-1577Crossref PubMed Scopus (1266) Google Scholar, 14McKercher S. Torbett B. Anderson K. Henkel G. Vestal O. Baribault H. Klemsz M. Feeney A. Wu G. Paige C. Maki R. EMBO J. 1996; 15: 5647-5658Crossref PubMed Scopus (914) Google Scholar), whereas PIP function is necessary for maturation of B and T lymphocytes (34Mittrucker H.W. Matsuyama T. Grossman A. Kundig T. Potter J. Shahinian A. Wakeham A. Patterson B. Ohashi P. Mak T. Science. 1997; 275: 540-543Crossref PubMed Scopus (4) Google Scholar). PU.1−/− mutants completely lack both lymphoid and myeloid progenitors, whereas PIP−/− animals exhibit a block in peripheral maturation of B cells and fail to produce antibodies in response to antigenic stimulation. Similarly, T cells of the mutant animals (PIP−/−) lack proliferative and cytotoxic responses (34Mittrucker H.W. Matsuyama T. Grossman A. Kundig T. Potter J. Shahinian A. Wakeham A. Patterson B. Ohashi P. Mak T. Science. 1997; 275: 540-543Crossref PubMed Scopus (4) Google Scholar). Our recent studies indicate that both PU.1 and PIP interact with additional factors and function synergistically within the κE3′-enhancer. Such factors include c-Fos plus c-Jun, which binds to the κE3′-CRE site, and E2A proteins (E12/E47), which binds to the E2A site (see Fig. 1). Through these interactions, PU.1 participates in the assembly of an enhanceosome, a higher order nucleoprotein complex (25Pongubala J.M.R. Atchison M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 127-132Crossref PubMed Scopus (77) Google Scholar). Despite the ability to interact with various transcription factors, PU.1 was found to be a weak transactivator. Studies of gene regulation have suggested that transcriptional activity of some factors requires phosphorylation-mediated modifications (35Hill C. Treisman R. Cell. 1995; 80: 199-211Abstract Full Text PDF PubMed Scopus (1194) Google Scholar, 36Marshall C. Cell. 1995; 80: 179-185Abstract Full Text PDF PubMed Scopus (4213) Google Scholar). For instance, phosphorylation of serine residues at positions 63 and 73 of c-Jun is important for its activity. These residues are rapidly phosphorylated in response to oncoproteins (v-Sis and Raf) or exposure to UV radiation (37Devary Y. Gottlieb R. Lau L. Karin M. Mol. Cell. Biol. 1991; 11: 2804-2811Crossref PubMed Scopus (597) Google Scholar), growth factors (38Minden A. Lin A. Smeal T. Derijard B. Cobb M. Davis R. Karin M. Mol. Cell. Biol. 1994; 14: 6683-6688Crossref PubMed Scopus (435) Google Scholar), or cytokines (tumor necrosis factor α) (39Westwick J.K. Bielawska A.E. Dbaibo G. Hannun Y.A. Brenner D.A. J. Biol. Chem. 1995; 270: 22689-22692Abstract Full Text Full Text PDF PubMed Scopus (362) Google Scholar). Both Ser63 and Ser73 of c-Jun are preferentially phosphorylated by JNK1, which is sequentially regulated as result of activation of the Ras-PI3K-responsive protein kinase, MEKK1. In fact, target disruption studies suggest that MEKK1 is essential for JNK activation (40Xia Y. Makris C. Su B. Li E. Yang J. Nemerow G. Karin M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5243-5248Crossref PubMed Scopus (230) Google Scholar). Therefore, the possibility arose that externally regulated signals could play an important role in stimulation of Ig κE3′-enhancer activity. Therefore, we have focused on the role of the Ras-PI3K-dependent signal molecules, MEKK1 and AKT, on κE3′-enhancer activity and target sites within the enhancer in the current studies. Here we demonstrate that MEKK1 stimulates the synergy between PU.1 plus PIP and c-Fos plus c-Jun in 3T3 cells. In S194 plasmacytoma cells, where the κE3′-enhancer is active, MEKK1 weakly stimulated activity of the PU.1 plus PIP site but failed to induce enhancer activity. Interestingly, the PI3K-dependent signal molecule, AKT, stimulated activity of the PU.1 plus PIP site and induced κE3′-enhancer activity in both pre-B and plasmacytoma cells. Activation of the PU.1 plus PIP site in response to the AKT signal is higher when compared with the stimulation observed in the presence of MEKK1. Mutational analyses of the PU.1 plus PIP site indicated that PU.1 responds to the AKT signals, and this response is independent of its interaction with PIP, suggesting that AKT induction of the κE3′-enhancer is primarily due to PU.1 stimulation. By mutational analyses, we determined that AKT stimulation of PU.1 is mediated through the acid-rich (amino acids 33–74) region. Activation of PU.1 in response to AKT appears to be due to phosphorylation-mediated modifications within this region. Mutation of PU.1 serine 41 (which is phosphorylated in vivo) to alanine impaired PU.1 induction by AKT signal. Consistent with these studies, we found that cross-linking the BCR mimicked AKT activation of PU.1. These studies indicate that PU.1 serves as a target molecule for Ras-PI3K-mediated signals in B cells providing a role for PU.1 in B-cell proliferation and humoral immunity. Plasmid constructs containing the TK promoter driving the expression of the enhancer core, κE3′-CRE, PU.1/PIP, and E2A were previously described (41Pongubala J.M.R. Atchison M. Mol. Cell. Biol. 1991; 11: 1040-1047Crossref PubMed Scopus (98) Google Scholar). Mutations of the PU.1 plus PIP binding sites were reported earlier (24Pongubala J.M.R. Atchison M. J. Biol. Chem. 1995; 270: 10304-10313Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Wild type or deletion mutants of c-Fos and c-Jun expression plasmids were kindly supplied by Dr. Frank Rauscher (Wistar Institute, Philadelphia, PA). Plasmids expressing MEKKΔ, MEKKΔ (K432M) were kindly provided by Dr. Michael Karin (University of California, San Diego, CA). The expression plasmids of AKT including HA-Wt AKT, HA-Myr AKT, and HA-Myr AKT (K−) and retroviral vectors, pBabe GFP and pBabe GFP Myr AKT were generously provided by Dr. Philip Tsichlis (Thomas Jefferson University, Philadelphia, PA). The reporter plasmid, pT81-Luc containing the minimal TK promoter driving expression of the luciferase gene was kindly provided by Dr. Daniel Tanen (Harvard University, Boston, MA). The pT81 Luc-M5.6 reporter plasmid containing the multimerized PU.1 binding site was constructed by transferring M5.6 from the TK-CAT plasmid by BamHI and HindIII digestion followed by cloning as a blunt end fragment into a blunt endHindIII site of pT81-Luc. The PU.1 deletion mutants, Δ2–30, Δ33–74, and Δ75–100 were constructed by isolating them from PURI plasmids (42Fisher R. Olson M. Pongubala J. Perkel J. Atchison M. Scott E. Simon M. Mol. Cell. Biol. 1998; 18: 4347-4357Crossref PubMed Scopus (46) Google Scholar) by EcoRI and ligating them into theEcoRI site of an expression plasmid pCB6+containing the CMV promoter (kindly supplied by Dr. Frank Rauscher, Wistar Institute, PA). The serine to alanine mutant S41A was isolated from Bluescript KS+ by EcoRI digest and cloned into the EcoRI site of the pCB6+ expression vector, whereas serine to alanine substitution mutants including S37A, S45A, S41A/S45A, and S37A/S41A/S45A were prepared by the overlap extension PCR method (43Ho S. Hunt H. Horton R. Pullen J. Pease R. Gene ( Amst. ). 1989; 77: 51-59Crossref PubMed Scopus (6771) Google Scholar). Two substitute mutant primers were generated for each desired amino acid, one on the top strand and one on the bottom strand. The sequences of the mutant primers were as follows: F37, 5′-GACTACTACGCCTTCGTGGGC-3′; R37, 5′-GCCCACGAAGGCGTAGTAGTC; F45, 5′-GATGGAGAAGCCCATAGCGAT; and R45, ATCGCTATGGGCTTCTCCATC. Two external primers containing EcoRI sites were also used, corresponding to the 5′ and 3′ ends of PU.1. The sequence of 5′ and 3′ end primers of PU.1 were, 5′-GCGGAATTCAGCTGGATGTTACAGGCG-3′ and 5′-GCGGAATTCTCAGTGGGGCGGGAGGCG-3′, respectively. Typically, the first PCR amplification was carried out with each mutant primer and corresponding external primer to generate two DNA fragments, each with a newly substituted amino acid. The amplified fragments were gel purified and subjected to the second PCR reaction in the presence of 5′ and 3′ external primers of PU.1, and full-length cDNAs were generated. The amplified products were digested with EcoRI and ligated into the EcoRI site of the pCB6+expression plasmid. Each PCR reaction was performed by using 2.5 units of Taq Polymerase (Roche Molecular Biochemicals), 2.5 units of Taq Extender (Stratagene), a deoxynucleoside triphosphate mix in a final concentration of 0.25 mm, 10 ng of template DNA, and 250 ng of each primer. The wild type PU.1 cDNA was used as a template to generate the S37A and S45A mutants. The S41A/S45A mutant was generated using S45A mutant primers and a plasmid containing S41A mutant cDNA as a template. Subsequently, the PU.1 mutant containing serine to alanine mutations S37A/S41A/S45A was generated using S37A mutant primers and S41A/S45A PU.1 cDNA as a template during PCR amplification. DNA sequences of all inserts were determined to confirm the mutations and to verify that no new mutations were introduced by PCR. S1 94 plasmacytoma cells were grown in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with 10% horse serum and antibiotics (100 units/ml penicillin and 0.1 mg/ml streptomycin, henceforth known as Pen-Strep), whereas A20 B cells were maintained in RPMI 1640 supplemented with 10% fetal calf serum, 5 μmβ-mercaptoethanol and Penn Strep. 3T3 cells were maintained in Dulbecco's modified Eagle's medium containing 10% fetal calf serum and Pen-Strep. Transfections in S194 cells were performed by the DEAE-dextran (Amersham Pharmacia Biotech Inc.) procedure (44Grosschedl R. Baltimore D. Cell. 1985; 41: 885-897Abstract Full Text PDF PubMed Scopus (287) Google Scholar). Transfections contained 4–5 μg of reporter plasmid, 1 μg of β-galactosidase expression plasmid pCH110 (45Hall C. Jacob P. Ringold G. Lee F. J. Mol. Appl. Genet. 1983; 2: 101-110PubMed Google Scholar), and varying concentrations of MEKK1, AKT, or PU.1 expression plasmids. The maximum amount of DNA was kept between 6 and 7 μg during all transfections. 3T3 cells were transfected by the calcium phosphate coprecipitation method of Graham and Van der Eb (46Graham F. Van der Eb A. Virol. 1973; 52: 456-467Crossref PubMed Scopus (6464) Google Scholar). The total amount of DNA per transfection in 3T3 cells varied between 16 and 21 μg. Both S194 and 3T3 cells were harvested 48 h post-transfection, cells were lysed by freezing and thawing and the β-galatosidase activity was determined from each cellular extract. CAT assays were carried out according to Gorman et al. (47Gorman C. Moffat L. Howard B. Mol. Cell. Biol. 1982; 2: 1044-1051Crossref PubMed Scopus (5286) Google Scholar) using normalized cell extracts. Transfections in A20 B cells were performed using FuGene-6 reagent (Roche Molecular Biochemicals) with 3 μg of pT81 Luc reporter alone or containing multimerized PU.1 binding site (pT81 Luc-M5.6) along with 1 μg of β-gal expression plasmid. Following transfection, cells were either unstimulated or stimulated with 5 μg/ml of F(ab′)2 anti-mouse IgG (Jackson ImmunoResearch Laboratories, PA). To block AKT activation, cells were incubated for 30 min in the presence of 100 nm wortmannin (PI3K inhibitor) prior to addition of antibodies for stimulation. After 36 h, cells were harvested, and luciferase activity was determined by reading in a luminometer. The luciferase activities were corrected for transfection efficiency by using the β-galactosidase activities. Metabolic labeling and immunoprecipitation of PU.1 proteins was performed essentially as described previously (25Pongubala J.M.R. Atchison M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 127-132Crossref PubMed Scopus (77) Google Scholar). Briefly, 3T3 cells were transfected by the calcium phosphate method with 5 μg of plasmid expressing either wild type or various PU.1 mutants. 24 h post-transfection, cells were pulsed with 0.2 mCi/ml35S-protein labeling mix (EXPRE35S35S Protein Labeling Mixl-35S-Met; PerkinElmer Life Sciences) for 2 h and then chased with cold methionine (0.5 mm). Cells were washed twice with phosphate-buffered saline, harvested in a lysis buffer (containing 20 mm Tris, pH 7.4, 50 mm NaCl, 0.5% SDS, 0.5% deoxycholate, 1 mmdithiothreitol, 10 μg/ml leupeptin, 1 μg/ml pepstatin), and sonicated, and clear cellular lysates were collected following centrifugation. Cell lysates were normalized by trichloroacetic acid precipitation of the labeled proteins. Approximately 10 × 106 cpm counts of each cell lysate were incubated with anti-PU.1 antibodies (Santa Cruz Biotechnology Inc.) for 2 h at room temperature, and the immune complexes were separated by protein A-Sepharose CL-4B (Amersham Pharmacia Biotech Inc.). The beads were washed three times with a RIPA buffer (10 mm Tris, pH 7.4, 0.15 m NaCl, 1.0% IGEPAL CA-630, 1% deoxycholate, 0.1% SDS, and 0.5% aprotinin) and once with a high salt buffer (2m NaCl, 10 mm Tris, pH 7.4, 1.0% IGEPAL CA-630, and 0.5% deoxycholate). The immune-complexes were eluted by boiling with SDS-polyacrylamide gel electrophoresis sample buffer, resolved on 10% SDS-polyacrylamide gels, and subjected to autoradiography. 293T retroviral packaging cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 1 mm sodium pyruvate, and Pen-Strep. The 293T cells were transiently transfected at >50% confluence on 100-mm dishes with 5 μg of retroviral vector, pBabe GFP, or pBabe GFP-Myr AKT along with 5 μg of Ecotropic packaging vector by the calcium phosphate coprecipitation method as described above. 36 h post-transfection, viral supernatants were collected and filtered, and the viral titers were determined by infecting 3T3 cells. 70Z/3 pre-B cells were grown in RPMI 1640 (Life Technologies, Inc.) supplemented with 10% heat-inactivated fetal calf serum, 5 μm β-mercaptoethanol, and Pen-Strep. Prior to retroviral infection, cells were washed and treated with DEAE-dextran (1 mg/ml) for 30 min and exposed to retroviral supernatants expressing GFP alone, or GFP-Myr AKT, for 4 h at 37 °C. Viral supernatants were removed, and cells were plated in regular growth medium. 36 h post-infection, cells were washed with phosphate-buffered saline, and mini-nuclear extracts were prepared according to the method described by Schreiber et al.(48Schreiber E. Matthias P. Muller M. Schaffner W. Nucleic Acids Res. 1989; 17: 6419Crossref PubMed Scopus (3903) Google Scholar). Binding reactions were performed as previously described (41Pongubala J.M.R. Atchison M. Mol. Cell. Biol. 1991; 11: 1040-1047Crossref PubMed Scopus (98) Google Scholar). Briefly, various concentrations of nuclear extracts were pre-incubated with 2 μg of poly(dI-dC) in a binding buffer (10 mm Tris, pH 7.6, 50 mm NaCl, 20% glycerol, 1 mm dithiothreitol, and 0.5 mm EDTA) for 5 min at room temperature. Approximately 10,000 cpm of 32P-labeled DNA containing the PU.1 plus PIP DNA-binding site (from the κE3′-enhancer) was then added to the binding reaction, and incubation was continued for an additional 25 min. The bound complexes were separated on 4% nondenaturing polyacrylamide gels and exposed for autoradiography. Previous studies have demonstrated that the κE3′-enhancer requires multiple core binding proteins for its activity (25Pongubala J.M.R. Atchison M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 127-132Crossref PubMed Scopus (77) Google Scholar). Such factors include c-Fos plus c-Jun, which binds to the κE3′-CRE site, PU.1 plus PIP, which bind to PU.1, and PIP sites and E2A proteins (E12/E47), which binds to the E2A site (Fig. 1). Cotransfection of the enhancer with either c-Fos plus c-Jun, PU.1 plus PIP, or E2A alone resulted in no enhancer activity in 3T3 cells. However, mixture of c-Fos, c-Jun, PU.1, and PIP caused a dramatic induction in enhance activity in 3T3 cells, where it is normally inactive. Removal of any one of these factors resulted in a loss of enhancer activity (25Pongubala J.M.R. Atchison M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 127-132Crossref PubMed Scopus (77) Google Scholar). Transcriptional activation by c-Jun requires phosphorylation at serines 63 and 73. These sites are rapidly phosphorylated by a protein kinase, JNK1 (49Derijard B. Hibi B. Wu H. Barrett T. Su B. Deng T. Karin M. Davis R. Cell. 1994; 76: 1025-1037Abstract Full Text PDF PubMed Scopus (2941) Google Scholar, 50Hibi M. Lin A. Smeal T. Minden A. Karin M. Genes Dev. 1993; 7: 2135-2148Crossref PubMed Scopus (1697) Google Scholar), whose activity is regulated through the Ras-responsive protein kinase, MEKK1 (51Lange-Carter C. Pleiman C. Gardner A. Blumer K. Johnson G. Science. 1993; 260: 315-319Crossref PubMed Scopus (869) Google Scholar). Because c-Jun is important for the activity of the κE3′-enhancer, we sought to determine the role of MEKK1 in functional synergy and enhancer activity. If MEKK1 is important to activity of the κE3′-enhancer, it should stimulate the enhancer when expressed along with PU.1 plus PIP and c-Fos plus c-Jun. To test this, transfections were carried out in 3T3 cells with a reporter plasmid containing the enhancer core fragment along with PU.1, PIP, c-Fos, and c-Jun either in the absence or presence of various concentrations of catalytically active MEKK1, MEKKΔ. Parallelly, transfections were carried out with the kinase inactive mutant, MEKKΔ (KM). Expression of MEKK1 caused a significant increase in the functional synergy between PU.1, PIP, c-Fos, and c-Jun and led to stimulation of activity of the enhancer in a concentration-dependent manner. On the other hand, expression of MEKKΔ (KM) resulted in a loss of enhancer activity (Fig. 2). Because MEKK1 stim
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