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Cell Growth & Differentiation Vol. 10, 333-342, May 1999
© 1999 American Association for Cancer Research

Fra-2-positive Autoregulatory Loop Triggered by Mitogen-activated Protein Kinase (MAPK) and Fra-2 Phosphorylation Sites by MAPK1

Masao Murakami2, Motoyasu Ui and Hideo Iba3

Department of Gene Regulation, Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan


    Abstract
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
We reported previously that activation of endogenous activator protein 1 (AP-1) in chicken embryo fibroblasts is essential for the cellular transformation induced by v-src, and we further showed that the activation of AP-1 is accompanied by elevation of Fra-2 and c-Jun expression and also high-level phosphorylation of Fra-2 by activated endogenous extracellular signal-regulated kinase [mitogen-activated protein kinase (MAPK)]. Here, we report that the transcriptional activity of Fra-2/c-Jun heterodimer was greatly enhanced by cotransfecting a constitutively active mutant of MEK1 gene (MEK-DD) into F9 cells, indicating that Fra-2 was converted into an active transactivator after phosphorylation by MAPK. High-level expression of MEK-DD alone was sufficient to induce clear cellular transformation of chicken embryo fibroblasts, which caused constitutive activation of endogenous MAPK, hyperphosphorylation of Fra-2, and elevation of fra-2 and c-jun gene expression. These results indicate that phosphorylation of Fra-2 by MAPK plays an important role in stimulating endogenous AP-1 activity in a positive autoregulation mechanism, in which phosphorylated Fra-2 induces fra-2 expression through AP-1 binding sites present in its promoter. We also localized the Fra-2 phosphorylation sites by MAPK to three threonine and three serine residues in the COOH-terminal region by means of site-directed mutagenesis and showed that the threonine residues were more susceptible to MAPK.


    Introduction
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
AP-14 is a group of transcription factors composed of Fos family proteins (c-Fos, FosB, Fra-1, and Fra-2; Refs. 1, 2, 3, 4, 5 ) and Jun family proteins (c-Jun, JunB, and JunD; Refs. 6, 7, 8, 9 ). All Fos family proteins form stable heterodimers with any of the Jun family proteins, but Jun family proteins can also form unstable dimers among themselves. These dimers bind to specific DNA sequences designated AP-1 DNA binding sites (consensus: TGAG/CTCA) to regulate gene expression (10, 11, 12, 13, 14) . By means of transient expression experiments in F9 cells, which express only a marginal level of endogenous AP-1, each Fos/Jun dimer has been shown to have a distinct transcriptional activity; the c-Fos/c-Jun heterodimer has a much higher transactivating activity whereas Fra-2/c-Jun has a lower activity than that of the c-Jun/c-Jun homodimer (14 , 15) .

By use of dominant-negative mutants of Fos or Jun, endogenous AP-1 activity was shown to be essential for the cellular transformation induced by oncogenes such as v-src, v-yes, v-fps, c-Ha-ras, and activated raf in CEFs (16 , 17) as well as in some established murine cell lines (18 , 19) . Among chicken fos/jun family genes, c-fos, fra-2, c-jun, and junD have so far been detected and cloned (4 , 7 , 20 , 21) . Biochemical analysis of AP-1 components purified by DNA-affinity chromatography as well as gel-shift analysis indicated that the major molecular component of AP-1 is the Fra-2/c-Jun heterodimer in both normal and v-src-transformed CEFs and that expression of both Fra-2 and c-Jun is elevated {approx}3-fold at the transcriptional level in v-src-transformed CEFs (17 , 22) . These studies further showed that hyperphosphorylation of Fra-2 in the v-src-transformed CEFs is the major qualitative difference in the AP-1 components between normal and v-src-transformed CEFs (22) . Several serine and threonine residues located in the COOH-terminal region of Fra-2 were shown to be phosphorylated by endogenous ERK2 (MAPK; Ref. 22 ), which is activated constitutively by high-level expression of several oncogenes, including v-src, or transiently by various growth stimuli such as addition of serum (22, 23, 24, 25) . Although unphosphorylated Fra-2 has Mr 40,000 in SDS-PAGE, the phosphorylated forms of Fra-2 show mobility shifts, resulting in multiple protein bands ranging from Mr 40,000–46,000, which probably reflect the status of phosphorylation (4 , 17 , 22 , 26) .

In this study, we showed that the transcriptional activity of Fra-2/c-Jun heterodimer is greatly elevated by introducing a constitively active mutant of MEK1 into F9 cells. We also showed that constitutive activation of MAPK is sufficient to induce cellular transformation accompanied with hyperphosphorylation of Fra-2 and the elevation of Fra-2 and c-Jun expression. We also identified a cluster of Fra-2 sites that are phosphorylated by MAPK both in vitro and in vivo and compared the COOH-terminal sequence with those of other Fos family proteins. Here, on the basis of our present and previous results, we discuss the importance of a putative Fra-2-positive autoregulatory loop mediated by activated MAPK.


    Results
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Enhancement of Fra-2/c-Jun Transcriptional Activity by Fra-2 Phosphorylation.
Although Fra-2/c-Jun heterodimer is the major component of AP-1 in v-src-transformed CEFs, in which the transcriptional activity of endogenous AP-1 was elevated (17 , 22) , this complex was shown to have relatively low transactivating activity when F9 cells were transfected with a reporter plasmid containing a single AP-1 binding site and with expression vectors for the fra-2 and c-jun genes (14) . Because MAPK was shown to be mostly present in the nonactivated form in either growing or serum-starved F9 cells (data not shown); we thought that this discrepancy might be explained in terms of Fra-2 phosphorylation by MAPK in v-src-transformed CEFs. To test this possibility, we carried out transient expression experiments by cotransfecting a constitutively active mutant of MEK1, MEK-DD (27 , 28) , to directly activate endogenous MAPK in F9 cells. As shown in Fig. 1Citation , c-jun, c-fos, and fra-2 expression plasmids were transfected into F9 cells in various combinations together with a reporter (CAT) plasmid containing a single AP-1 DNA binding site originated from the human collagenase gene. Like mock transfection, c-fos or fra-2 transfection alone induced only marginal activity (Fig. 1)Citation . As reported previously (14) , c-jun alone significantly activated the promoter activity, and expression of both c-jun and c-fos resulted in higher activity than that of c-jun alone. In the case of c-jun and fra-2, CAT activity was less than that obtained with c-jun alone. These observations confirm previous reports that c-Fos/c-Jun heterodimer has a much higher transactivating activity whereas Fra-2/c-Jun has a lower activity than that of c-Jun/c-Jun homodimer (14 , 15) . But the CAT activity of c-jun and fra-2 was enhanced 3-fold by the coexpression of MEK-DD (Fig. 1)Citation . This strong enhancement by MEK-DD is dependent upon the expression of both fra-2 and c-jun; the CAT activity of transfectants with c-fos, fra-2, or c-fos and c-jun was not affected by MEK-DD cotransfection. These results provide strong supporting evidence that activation of MAPK by MEK-DD gene transfection causes elevation of the transcriptional activity of the Fra-2/c-Jun heterodimer. The CAT activity in the case of c-jun alone was also activated by the coexpression of MEK-DD to some extent (1.8-fold), but we can not currently explain this elevation because c-Jun is known to be a poor substrate for MAPK, as reported previously (29) .



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Fig. 1. Expression of the pcolTRE-CAT reporter plasmid (containing a single AP-1 DNA binding site) when cotransfected with expression vectors carrying jun and/or fos family genes. In each pair of transfectants, one was additionally cotransfected with the expression vector carrying MEK-DD (3 µg; ({blacksquare}), whereas the other was not ({square}). F9 cells were transfected with pcolTRE-CAT (3 µg) together with pRSV2B (empty expression vector), pRSV2B-c-jun (3 µg), pRSV2B-c-fos (3 µg), or pRSV 2B-fra-2 (3 µg) in various combinations, as shown at the bottom. In all transfections, the total amount of DNA was adjusted to 12 µg by adding pRSV 2B. Cells were disrupted 48 h after transfection, and the CAT activity in the same amount of cellular proteins was determined and normalized by taking the activity of pRSV 2B-c-jun as 100. Columns, averages of more than three independent experiments; bars, SD.

 
Constitutive Activation of MAPK in CEFs Causes Cellular Transformation Accompanied by Fra-2 Phosphorylation and Elevation of fra-2 Expression.
The biological function of Fra-2 phosphorylation in v-src-transformed CEFs could be obscured by v-src-induced activation of several independent pathways that are not related to the MAPK pathway (30, 31, 32) . For more direct analysis, we introduced two types of MEK1 mutants (27 , 28) , MEK-DD (a constitutively active mutant) and MEK-VV (kinase activity-deficient mutant), into CEFs by the use of an avian retrovirus vector (subgroup A) to modulate specifically the activation status of endogenous MAPK. To activate endogenous MAPK (ERK2) and/or JNKs, we also stimulated CEFs by serum addition (growth stimulation from G0 to G1), UV irradiation, or osmotic shock. The molecular forms of the endogenous MAPK in these cells were analyzed by Western blotting using ERK2-specific antiserum. As shown in Fig. 2aCitation , the amount of phosphorylated form (active form) of ERK2 was drastically increased in CEFs expressing MEK-DD compared with that in CEFs infected with the control virus and was much greater than that of the inactive form. In CEFs expressing MEK-VV, the active form of ERK2 was only slightly reduced, indicating that MEK-VV does not function efficiently as a dominant-negative mutant in CEFs. ERK2 activation in CEFs after growth stimulation was much higher whereas that in CEFs after osmotic shock was lower than that in CEFs exogenously expressing MEK-DD.



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Fig. 2. Endogenous MAPK, Fra-2 kinase, and JNKs in CEFs exogenously expressing MEK-DD or MEK-VV as well as CEFs either before or after growth stimulation (G0 and G1) UV-irradiation [UV(-) and UV(+)] or osmotic shock [osmo-shock(-) and osmo-shock(+)]. a, CEFs infected with control virus or virus encoding a constitutively active mutant of MEK1 (MEK-DD) or a kinase activity-defective mutant of MEK1 (MEK-VV) were disrupted by boiling in SDS, and whole-cell lysates (5 µg per lane protein) were subjected to 18% SDS-PAGE and then transferred to a membrane. Amounts and molecular forms of endogenous MAPK were analyzed by Western blotting using anti-ERK2 antiserum. The same lysates (15 µg per lane protein) were used for in-gel kinase assay, with Fra-2 (b) or c-Jun (c) as the substrate. The details are described in "Materials and Methods."

 
On the basis of these results, we decided to use MEK-DD, which specifically activates endogenous ERK2 in CEFs. CEFs infected with the MEK-DD-bearing virus grew well at a low serum concentration of 0.2% and formed clear colonies in soft agar (data not shown). To analyze the endogenous Fra-2 protein, we labeled CEFs infected with the MEK-DD-bearing virus as well as the control virus with [35S]methionine, disrupted under denaturing conditions and immunoprecipitated with anti-Fos peptide 1 antiserum, which is cross-reactive to Fra-2. In MEK-DD-expressing CEFs, Fra-2 bands [especially those with higher mobility (Mr 43,000–46,000)] 43–46 kDa were denser than those in CEFs infected with the control virus (Fig. 3a)Citation . In CEFs expressing MEK-VV, the pattern and density of Fra-2 bands were similar to those of CEFs infected with the control virus (Fig. 3a)Citation . Other aliquots of the immunoprecipitates shown in Fig. 3aCitation were treated with bacterial alkaline phosphatase to dephosphorylate Fra-2 completely, so that the amounts of Fra-2 protein could be accurately compared (Fig. 3b)Citation . Fra-2 expression level was enhanced in MEK-DD-expressing CEFs. Northern blot analysis of the same series of CEFs (Fig. 4Citation , top) indicated that Fra-2 expression in MEK-DD-expressing CEFs was elevated at the mRNA level.



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Fig. 3. Analysis of endogenous AP-1 components in CEFs exogenously expressing MEK-DD or MEK-VV. a, CEFs infected with the same set of retrovirus vectors described in the legend to Fig. 2Citation were labeled with [35S]methionine for 60 min and disrupted under denaturing conditions to prepare whole-cell lysates. Endogenous Fra-2 protein in the lysate containing the same amount of 35S radioactivity was immunoprecipitated with anti-Fos pep1 antiserum (4) , which is cross-reactive to Fra-2, and analyzed by 10% SDS-PAGE. b, one-half of the Fra-2 immunoprecipitate prepared above was treated with bacterial alkaline phosphatase to completely dephosphorylate Fra-2 for accurate comparison of the amounts of Fra-2 protein. c and d, other aliquots of the labeled cell lysates prepared as described in a as well as those prepared from starved (G0) or serum-stimulated (G1) CEFs were analyzed by immunoprecipitation using anti-Fos pep1 antiserum (c) and anti-c-Jun antiserum (d).

 


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Fig. 4. Northern blot analysis of fra-2 or c-jun mRNA in CEFs exogenously expressing MEK-DD or MEK-VV. CEFs were infected with the same set of retrovirus vectors described in the legend to Fig. 2Citation , and total RNA was isolated. Each RNA sample (15 µg/lane) was subjected to 1.0% agarose gel electrophoresis and transferred to a nylon membrane. fra-2 (top) or c-jun (middle) transcripts were detected by using a 32P-labeled fra-2 or c-jun-specific cDNA probe. The ethidium bromide staining of the gel used for the Northern blotting is also shown (bottom).

 
Endogenous c-Fos was undetectable in these logarithmically growing CEFs, independently of cellular transformation, although c-Fos expression was easily detectable in this assay system after growth stimulation (Fig. 3c)Citation . Endogenous c-Jun expression was specifically elevated in MEK-DD-transformed CEFs at both the protein level and the mRNA level (Figs. 3dCitation and 4Citation , middle). The expression levels of JunD, however, were not significantly different among all these CEFs and were much lower than those of c-Jun in every case (data not shown). Thus, the major components of the AP-1 in MEK-DD-transformed CEFs were shown to be Fra-2 and c-Jun, and Fra-2 was highly phosphorylated, as reported in CEFs transformed by v-src, Ha-ras, and activated raf (17 , 22) . These results indicated that constitutive activation of MAPKinase was sufficient to cause Fra-2 hyperphosphorylation, elevation of the steady-state levels of fra-2 and c-jun expression, and cellular transformation.

Although MAPK was shown to be the key molecule for the activation of endogenous AP-1, it remains possible that some autocrine factors indirectly induced by MEK-DD activate other signal transduction pathways leading to the enhancement of the endogenous AP-1 activity (32, 33, 34, 35) . To test this possibility, we analyzed protein kinase activities in the cell lysates used in Fig. 2aCitation by in-gel kinase assay using Fra-2 or c-Jun, the major components of the activated endogenous AP-1, as the substrates (Figure 2, b and c)Citation . In all the lysates, a single band of ERK2 was detected by in-gel Fra-2 kinase assay, and the density of the band was increased by the retroviral expression of MEK-DD as well as by growth stimulation or osmotic shock (Fig. 2b)Citation . MEK-DD expression did not elevate the activity of JNKs at all, whereas UV irradiation and osmotic shock as well as growth stimulation induced JNKs activities to various extents (Fig. 2c)Citation . Highly phosphorylated forms of c-Jun in UV-irradiated or serum-stimulated CEFs showed clear mobility shifts on SDS-PAGE, as reported previously (36) , but the c-Jun protein in MEK-DD-transformed CEFs was not shifted at all (Fig. 3d)Citation . These results indicate that exogenous MEK-DD expression did not induce Fra-2 kinases other than ERK-2, at least when monitored by the in-gel kinase assay used here.

Identification of Sites of Fra-2 Phosphorylation by MAPK in Vitro.
To identify the sites of Fra-2 phosphorylated by MAPK, we constructed a series of substitution mutants. As judged from the consensus phosphorylation sites for MAPK, there are six candidate phosphoacceptor sites in the Fra-2 COOH-terminal region. Two of them, Thr-271 and Ser-292 (closed circles in Fig. 5Citation ), are typical consensus phosphorylation sites of MAPK with the sequences Pro-Ala-Ile-Thr-Pro and Pro-Leu-Ser-Pro, respectively, and these sites were disrupted by replacing Thr or Ser with Ala (mutant 1T and mutant 1S, respectively). In mutant 1T1S, both residues were simultaneously substituted. Other candidates (open circles in Fig. 5Citation ) are Ser or Thr residues just before a Pro residue. All three Thr residues were changed to Ala residues in mutant 3T, whereas all three Ser residues clustering at the COOH terminus were changed to Ala residues in mutant 3S. In mutant 3T3S, all six candidate sites were changed to Ala. Wild-type Fra-2 and its derivatives were fused to GST at their NH2 termini, expressed in Escherichia coli, and purified by the use of glutathione-Sepharose beads (Fig. 6b)Citation . These substrate proteins were phosphorylated by purified activated MAPK in vitro. Wild-type Fra-2 (Lane WT) was highly phosphorylated by MAPK, as reported previously (Ref. 22 ; Fig. 6aCitation ). The extent of phosphorylation of each Fra-2 mutant was quantified by means of an image analyzer (Fig. 6c)Citation . Mutants 1T and 1S were phosphorylated, but at significantly lower levels than the wild-type, indicating that these two sites are susceptible to phosphorylation. Although the double mutant 1T1S was phosphorylated at a slightly lower level than 1T or 1S, significant phosphorylation was still detected. The results of phospho-amino acid analysis of mutant 1T1S indicated that MAPK phosphorylated not only these two typical consensus sites but also some other serine and threonine residues (data not shown). The phosphorylation level of mutant 3T was drastically reduced and that of mutant 3S was reduced to less than one-half of the wild type. Mutant 3T3S was only marginally phosphorylated by MAPK, indicating that most of the phosphorylation sites were included in these six residues substituted in this series of mutational analyses. Under these conditions of substrate excess for the kinase reaction, no significant mobility shifts were detectable (Fig. 6a)Citation , indicating that phosphorylation at multiple sites is required for mobility shift of Fra-2 protein on SDS-PAGE.



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Fig. 5. Schematic representation of structures of Fra-2 (wild type) fusion proteins and their point mutants at the putative phosphorylation sites. , representative phosphorylation consensus sequences of MAPKinase (Pro-X-X-Ser/Thr-Pro and Pro-X-Ser/Thr-Pro); {bigcirc}, the broad consensus sequence (Ser/Thr-Pro). The amino acid sequence of the COOH-terminal region of Fra-2 is shown using one-letter symbols. BD and LZ, basic domain and the leucine zipper motif, respectively.

 


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Fig. 6. Kinase assay in vitro using a series of GST-Fra-2 fusion proteins as substrates. GST-Fra-2 (WT) and its point mutants described in the legend to Fig. 5Citation were expressed in E. coli, purified, and phosphorylated by purified activated MAPK in vitro. After the kinase reaction, 32P-labeled proteins were subjected to 10% PAGE, and bands were detected by autoradiography (a). The substrate proteins used in a were subjected to 10% SDS-PAGE (6 µg/lane), and bands were detected by staining with Coomassie brilliant blue (b). c, the results in a and another two independent experiments were quantified by using an image analyzer and relative 32P incorporation is shown (WT, 100%).

 
Phosphorylation of Thr Residues Induced Fra-2 Mobility Shifts on SDS-PAGE in Vivo and in Vitro.
Because most of Fra-2 is present in the formes of heterodimers with c-Jun in CEFs, Fra-2 phosphorylation in vivo could be different from that detected in vitro using purified Fra-2 alone as the substrate. In fact, such a difference between in vivo and in vitro results has been reported for c-Fos protein (37) . Kinases other than MAPK may also contribute to the phosphorylation in vivo. To analyze Fra-2 phosphorylation sites in vivo, we tagged Fra-2 point mutants with HA at their NH2 termini and introduced them into CEFs by the use of the avian retrovirus vector (Fig. 5)Citation . CEFs infected with these vectors were serum-starved and labeled with [35S]methionine before or just after the serum addition. Fra-2 proteins were immunoprecipitated by anti-Fra-2 pep1 antiserum and the immunoprecipitates were solubilized by denaturing in SDS, renatured by reducing the SDS concentration, and reimmunoprecipitated with anti-HA monoclonal antibody to eliminate the endogenous Fra-2 protein. As shown in Fig. 7aCitation , HA-Fra-2 (Lane WT) formed smear bands in the serum-starved condition but gave a sharp band (50 kDa) after serum stimulation. Mutants 1T, 1S, and 1T1S were also phosphorylated and underwent a mobility shift upon serum stimulation. But, unlike wild-type Fra-2 and mutant 1S, which showed almost complete mobility shifts, mutants 1T and 1T1S left several bands with lower apparent molecular weights even after serum stimulation (Fig. 7a)Citation . This result indicated that phosphorylation of Thr-271 contributes greatly to the Fra-2 mobility shift on SDS-PAGE. Although mutant 3S was shifted like the wild type, the shifts of mutant 3T and mutant 3T3S were greatly retarded (Fig. 7a)Citation , indicating that phosphorylation of the cluster of threonine residues is mainly responsible for the Fra-2 mobility shifts in vivo. It is noteworthy that HA-Fra-2 wild type and derivatives formed multiple bands even in serum-starved CEFs and that the patterns were different among the mutants (Fig. 7a)Citation . This would be partly explained by the presence of the activated form of ERK2 even in growth-arrested CEFs (Fig. 2a)Citation .



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Fig. 7. a, mobility shifts of HA-tagged Fra-2 proteins in CEFs after growth stimulation. CEFs expressing HA-tagged Fra-2 (Lane WT) or its mutants were serum-starved and pulse-labeled with [35S]methionine for 20 min just before or after the serum addition. Whole-cell lysates were prepared under denaturing conditions and immunoprecipitated with anti-Fra-2 pep1 antiserum. The immunoprecipitates were boiled in SDS, renatured by diluting the SDS, and subsequently reimmunoprecipitated with anti-HA monoclonal antibody (12CA5). After resolution by 10% SDS-PAGE, protein bands were detected by fluorography. b, mobility shifts of Fra-2 point mutants after phosphorylation by MAPK in vitro. GST-Fra-2 (Lanes WT) and its point mutants were incubated in the presence (Lanes +) or absence (Lanes -) of purified activated MAPK and phosphorylated in vitro, as described in "Materials and Methods." After the kinase reaction, each sample was subjected to 10% SDS-PAGE and analyzed by Western blotting using anti-Fra-2 pep1 antiserum. c, the same assays as in b were performed in the presence of [{gamma}-32P]ATP. The 32P-labeled proteins were subjected to 10% SDS-PAGE, transferred to a nylon membrane, and detected by autoradiography.

 
To test whether phosphorylation of the cluster of threonine residues by MAPK could cause the Fra-2 mobility shift, we next phosphorylated Fra-2 in vitro using a modified condition for the MAPK reaction, in which smaller amounts of Fra-2 proteins were phosphorylated by larger amounts of activated MAPK. After the kinase reaction, the series of GST-Fra-2 substrates was analyzed by Western blotting using anti-Fra-2 antiserum. While a clear mobility shift of GST-Fra-2 (WT) was detected as described previously (22) , mutant 3T3S showed no mobility shift, and phosphorylation was negligible (Fig. 7, b and c)Citation . Mobility shifts and phosphorylation of mutant 3T were much more affected than those of mutant 3S, indicating that the phosphorylation of Thr residues contributes prominently to the mobility shifts even in vitro. Therefore, the migration patterns of GST-Fra-2 mutants phosphorylated in vitro were similar to those of the corresponding HA-Fra-2 mutants detected in serum-stimulated CEFs, although mobility shifts of the HA-Fra-2 derivatives in SDS-PAGE were detected more sensitively because of the loss of the long fusion protein of GST (Fig. 7, a and b)Citation .


    Discussion
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Here, we have shown that the activation of MAPK by the transfection of a constitutively active mutant of the MEK1 gene into F9 cells causes activation of the transcriptional activity of the Fra-2/c-Jun heterodimer. We have also shown that constitutive activation of endogenous MAPK in CEFs is sufficient for Fra-2 hyperphosphorylation, elevation of fra-2 and c-jun gene expression, and induction of cellular transformation.

On the basis of these results and our previous observations (15 , 17 , 22 , 38) , the positive autoregulatory loop of Fra-2 triggered by constitutively activated endogenous MAPK could be schematically presented as shown in Fig. 8Citation . This model would explain the molecular mechanisms involved in cellular transformation caused by several oncogenes. In nontransformed cells, the levels of either phosphorylation or expression of Fra-2 were relatively low because the heterodimer formed between hypophosphorylated Fra-2 and c-Jun has lower transcriptional activity than that of c-Jun homodimer. However, constitutive expression of an oncogene such as src, ras, raf, or a constitutively active mutant of MEK, causes endogenous MAPK to be constitutively activated. The activated MAPK translocates from the cytoplasm to the nucleus and phosphorylates the COOH-terminal region of the Fra-2 protein. Because the heterodimer formed between phosphorylated Fra-2 and c-Jun has very high transcriptional activity, it would induce expression of a wide range of cellular genes through AP-1 DNA binding sites, including those present in the fra-2 promoter. Induced Fra-2 would subsequently be phosphorylated by MAPK, and this positive circuit would further enhance endogenous AP-1 activity in these transformed cells. Like the transformation by v-src, Ha-ras, or activated raf, MEK-DD-induced transformation was suppressed by a dominant negative mutant of AP-1, supJunD (Ref. 39 ; data not shown), indicating this Fra-2-positive autoregulatory loop plays an important role in the cellular transformation induced by these oncogenes.



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Fig. 8. Possible mechanisms involved in the activation of endogenous AP-1 in the transformed cells induced by several oncogenes. High-level expression of v-Src, c-Ha-Ras, or constitutively active mutants of Raf or MEK cause elevation of the endogenous MAPK activity in CEFs. Although endogenous Fra-2 in normal CEFs is poorly phosphorylated and expressed at a basel level, this protein is now efficiently phosphorylated by MAPK. Like hypophosphorylated Fra-2, hyperphosphorylated Fra-2 forms a stable heterodimer with endogenous c-Jun. The resultant heterodimer transactivates several genes through AP-1 DNA binding sites, including those present in the fra-2 promoter region. In these transformed cells, this autoregulation causes accumulation of hyperphosphorylated forms of Fra-2, and this is the cause of enhancement of endogenous AP-1 activity.

 
Although AP-1 proteins purified by DNA affinity chromatography contained either non-phosphorylated or phosphorylated forms of Fra-2 (22) , the DNA binding activity of Fra-2/c-Jun was suggested to be enhanced by the phosphorylation by MAPK in vitro (40) . This enhancement of DNA binding activity might partly explain the activation mechanism of transcriptional activity of this heterodimer.

We observed that c-jun expression was also elevated in CEFs expressing a constitutively active mutant of MEK1, as well as in v-src-transformed CEFs (17 , 22) . We believe that the autoregulation model for fra-2 would also be applicable to this elevation of c-jun expression because the c-jun promoter was reported to have two enhancer sequences that respond to AP-1 (29 , 41) . Because JNK activity was not elevated in MEK-DD-transformed CEFs (Fig. 2c)Citation or v-src-transformed CEF (22 , 42) as compared to nontransformed CEFs, Fra-2 phosphorylation by MAPK rather than c-Jun or ATF-2 phosphorylation by JNK (29 , 43, 44, 45, 46, 47, 48, 49) seems to be responsible for the enhancement of c-jun expression in CEFs.

By means of in vitro and in vivo analyses, we identified sites of Fra-2 phosphorylation by MAPK. Although a cluster composed of both serine and threonine residues was phosphorylated, threonine residues were shown to be more efficient phosphoacceptor sites, and phosphorylation of threonine residues contributed predominantly to the Fra-2 mobility shifts on SDS-PAGE. It should be pointed out that the Fra-2 mutant 3T3S formed three bands in vivo in growth-arrested CEFs. Because this mutant lacks all the major phosphorylation sites of MAPK, we believe that some other kinases must be involved in Fra-2 phosphorylation independently of growth stimulation. However, the lowest band of 3T3S was shifted after serum stimulation, so it is possible that some serum-inducible kinases, such as RSK, are also involved in Fra-2 phosphorylation because the site of c-Fos phosphorylation by RSK is conserved in Fra-2 (Ref. 50 ; Ser-305 in Fig. 9Citation ).



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Fig. 9. Sequence alignment of Fos family proteins in the COOH-terminal region. The COOH-terminal region of Fos family proteins contains conserved regions III and IV. *, indicate serine or threonine residues just before a proline residue. Underlined letters, RSK phosphorylation sites reported previously (50) .

 
With regard to Fra-2 functions, we have previously reported that it has transforming activity when exogenously expressed at high levels ({approx}12 times the endogenous Fra-2 level) in CEFs. We believe this transformation would be caused by different mechanisms from those observed in cellular transformation induced by v-src or MEK-DD because no elevation of Fra-2 phosphorylation was observed in CEFs exogenously expressing Fra-2. This is consistent with our observation that Fra-2 transforming activity was not significantly affected by the point mutants of the phosphorylation sites constructed here.5

Mammalian Fos family proteins consist of c-Fos, FosB, Fra-1, and Fra-2 and have five conserved regions (regions 0, I, II, III, and IV; Ref. 4 ). The six amino acid residues in chicken Fra-2 mutated here were fully conserved in mouse Fra-2 (Fig. 9)Citation . The three threonine residues of Fra-2 are located in the conserved region III and are well conserved in all Fos family proteins except for FosB (Fig. 9)Citation . Although the three serine residues in Fra-2 are not well conserved, it is noteworthy that Ser-317 of chicken Fra-2 is completely conserved in all Fos family proteins. Chen et al. (50) reported previously that the counterpart of this serine residue in c-Fos (Ser-374) was phosphorylated by MAPK. Fra-1 protein was reported to be phosphorylated after serum stimulation, with consequent mobility shifts on SDS-PAGE (39 , 51) . Because five of six Fra-2 phosphorylation sites are conserved in Fra-1, similar phosphorylation patterns by MAPK would be expected in Fra-1 and Fra-2.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Plasmid Construction.
To construct Fra-2 point mutants, we carried out site-directed mutagenesis using the U.S.E. mutagenesis kit (Pharmacia) and pGEX-F2 (215–323) as the template. Five mutated primers (X, Y, A, B, and C) and one selection primer were synthesized as follows; X, 5'-GTTGGAGGAACCAGGCGCGATGGCTGGTGTG-3'; Y, 5'-GACTCGGA-CGGCGCCAGAGGAGACTCCTG-3'; A, 5'-GGAGGAACCAGGCGCGATGGCTGGTGCCGAGGTCACCACGATGGGAGCGTTGAGTGCCTC-3'; B, 5'-GGACTCGGACGGCGCCAGAGGAGCCTCCTGATCCAA-3'; C, 5'-CA-GCAAGGTGGGCGCGTTCAAGGAATCCGA-3' (underlined letters indicate the mutated nucleotides); and the selection primer, 5'-CGTTGTTGCCATTGCCGCGGGCATGGTGGTGTCACGG-3'. pGEX-F2 (215–323) -1T, -1S, -1T1S, -3T, -3S, and -3T3S were constructed by using: X; Y; X and Y; A; B and C; and A, B, and C, respectively. The mutation sites were confirmed by direct DNA sequencing using dideoxynucleotide methods. The AvaII-XhoI fragments, which contain all the mutation sites and the terminal codons, were inserted into the AvaII-XhoI sites of pGEX-F2 (1–323) to generate full-length Fra-2 point mutants fused to GST at their NH2 termini. To construct retrovirus vectors encoding Fra-2 point mutants tagged with HA, we listed the NcoI-SalI fragments of pGEX-F2 (1–323) derivatives (containing full-length Fra-2) with the NcoI-XhoI fragment of pDS3-HA (22) . To construct retrovirus vectors that encode two mutants of MEK1, XbaI-BamHI fragments of pVL1392-MEK1-DD and -VV (containing full-length MEK1) were each blunt-ended with Klenow fragment and ligated into the single EcoRV site of pDS3-RV. A linker composed of two oligonucleotides (5'-GATCTCCCTCTAGACCCA-3' and 5'-GATCTGGGTCTAGAGGGA-3') containing a XbaI site (underlined letters) was inserted into the single BamHI site of pVL1392-MEK1-DD. To generate pRSV2-MEK-DD, the XbaI fragment containing the complete MEK-DD sequence was inserted into the XbaI site of pRSV2.

Preparation of Recombinant Protein.
GST-fused Fra-2 was expressed in E. coli as described previously (22) . The product was purified from E. coli by using glutathione-Sepharose beads (Pharmacia) and eluted with PBS containing 20 mM glutathione, 120 mM NaCl, and protease inhibitors. Samples were dialyzed in 10 mM Tris-HCl (pH 8.0), and the amounts of protein were determined using a Bio-Rad protein assay kit.

Cell Culture and Preparation of Cell Lysates.
CEFs were prepared from 10-day-old embryos and kept in MEM supplemented with 5% CS, 10% Tryptose phosphate broth, and 1% DMSO, as described previously (4 , 26) . For starvation, cultures were kept in 0.2% CS for 16 h and then growth-stimulated by addition of CS to a final concentration of 10% and further incubated for 20 min. For UV irradiation, we irradiated logarithmically growing CEFs with UV using a germicidal 254-nm UV lamp at the dose of 20 J/m2 (a fluence rate of 1 J/m2 • s-1; Ref. 22 ). For osmotic stress stimulation, we stimulated logarithmically growing CEFs by addition of NaCl to a final concentration of 0.7 M. At 20 min after irradiation or at 30 min after osmotic stimulation, whole-cell lysates were prepared as described previously (22) . CEFs were fully infected with high-titer virus (subgroup A) encoding HA-tagged Fra-2 (WT), its point mutants, or DS3-HA (the control vector encoding HA tag alone), as described previously (22 , 52) . Cultures (60-mm dishes) were metabolically labeled with 0.5 mCi of [35S]methionine for 1 h, as described previously (17 , 22 , 26) . For immunoprecipitation analysis, cell lysates were prepared under denaturing conditions in the presence of phosphatase inhibitors (5 mM Na3VO4 and 10 mM NaF).

In Vitro Kinase Assay.
GST-fused Fra-2 proteins (12 µg) were suspended in 45 µl of kinase buffer [10 mM HEPES (pH 7.9), 10 mM MgCl2, 20 mM ß-glycerophosphate, 0.5 mM Na3VO4, 1 mM NaF, and 2 mM DTT] containing 10 µM ATP and 5 µCi of [{gamma}-32P]ATP, and then 1 ng of purified activated MAPK (p44mpk; Upstate Biotechnology, Inc., Lake Placid, NY) was added and the mixture was incubated at 4°C for 60 min. The reaction mixture was boiled in the sample gel buffer for 5 min to stop the kinase reaction. Proteins were resolved by 10% SDS-PAGE, and protein bands were detected by autoradiography. For the assay of Fra-2 mobility shifts caused by phosphorylation, 300 ng of each substrate were incubated with 20 ng of MAPK at 30°C for 120 min to complete the kinase reaction.

In-Gel Kinase Assay.
The procedure of in-gel kinase assay was described in detail previously (22) . Whole-cell lysate (15 µg per lane) was resolved on a 10% polyacrylamide gel polymerized with GST-fused Fra-2 COOH-terminal region (40 µg/ml) or His-tagged c-Jun NH2-terminal region (40 µg/ml) as the substrate (22) . After the kinase reaction and washing, kinase activity was detected by autoradiography.

Immunoprecipitation and Western Blotting.
35S-labeled cell lysates were immunoprecipitated with anti-Fos pep 1 antiserum (4) or anti-Fra-2 pep 1 antiserum (4 , 26) in the presence of phosphatase inhibitors. These immunoprecipitates were solubilized by boiling in the denaturation buffer for 5 min. The samples were renatured by adding the radioimmunoprecipitation assay buffer without SDS to reduce the final SDS concentration to 0.15% and reimmunoprecipitated with anti-HA monoclonal antibody (12CA5), purchased from Boehringer Mannheim. After SDS-PAGE, protein bands were detected by fluorography. For Western blotting, proteins separated by SDS-PAGE were transferred to a polyvinylidene difluoride membrane (Immobilon P; Millipore); Immunoblots were treated with 5% skim milk and incubated with anti-Fra-2 peptide 1 antisera (4 , 26) . The membrane was incubated with antirabbit IgG antibody conjugated to horseradish peroxidase and the protein bands were visualized by use of the ECL Western Blotting Detection System (Amersham).

Northern Blot Analysis.
Total RNA of CEFs infected with viruses was extracted by the guanidinium thiocyanate-acid phenol method using an RNA preparation kit (Isogen; Nippon Gene). Total RNAs (15 µg per lane) were electrophoresed on 1.0% agarose-formaldehyde gel and transferred onto a nylon membrane (Schleicher & Schuell). fra-2 or c-jun transcripts were detected by using 32P-labeled fra-2 (38) or c-jun-specific probe (39) .

Transfection and CAT Assay.
DNA transfection into F9 cells was performed as described previously (14 , 15 , 53) , and 48 h after the end of transfection, cells were disrupted for the determination of CAT activity. CAT activity was normalized with respect to the total amount of protein in the lysate. The 14C radioactivity in the spots on TLC plates was quantitated by means of an image analyzer (Fuji BAS 2000).


    Acknowledgments
 
We thank Dr. R. L. Erikson for a gift of the cDNA of murine MEK1 and its mutants MEK-VV and -DD. We also thank Y. Yoshikawa and E. Endo for assistance in the preparation of this manuscript.


    Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported in part by grants and endowments from Eisai Co., Ltd., and by a Grant-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Science and Culture. Back

2 Present address: Division of Molecular Genetics, National Institute of Infectious Disease, Shinjuku-ku, Tokyo 162-8640, Japan. Back

3 To whom requests for reprints should be addressed, at Department of Gene Regulation, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan. Phone: 81-3-5449-5730; Fax: 81-3-5449-5449; E-mail: iba{at}ims.u-tokyo.ac.jp Back

4 The abbreviations used are: AP-1, activator protein 1; CEF, chicken embryo fibroblast; ERK, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; CAT, chloramphenicol acetyltransferase; JNK, c-Jun NH2-terminal kinase; GST, glutathione S-transferase; HA, hemagglutinin; RSK, ribosomal S6 kinase; CS, calf serum. Back

5 M. Murakami and H. Iba, unpublished results. Back

Received for publication 11/ 5/98. Revision received 2/26/99. Accepted for publication 3/22/99.


    References
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 

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