help button home button Endocrine Society Molecular Endocrinology ENDO 08 Sessions Library
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Molecular Endocrinology, doi:10.1210/me.2004-0113
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
18/10/2479    most recent
Author Manuscript (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Farrow, K. N.
Right arrow Articles by Gutierrez-Hartmann, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Farrow, K. N.
Right arrow Articles by Gutierrez-Hartmann, A.
Molecular Endocrinology 18 (10): 2479-2490
Copyright © 2004 by The Endocrine Society

Structural and Functional Analysis of the Differential Effects of c-Jun and v-Jun on Prolactin Gene Expression

Kathryn N. Farrow, Andrew P. Bradford, John J. Tentler and Arthur Gutierrez-Hartmann

Department of Medicine (K.N.F., J.J.T., A.G.-H.), Division of Endocrinology, Metabolism and Diabetes, Department of Obstetrics and Gynecology (A.P.B.), and Department of Biochemistry and Molecular Genetics (A.P.B., A.G.-H.), University of Colorado Health Sciences Center, Aurora, Colorado 80045

Address all correspondence and requests for reprints to: Dr. Gutierrez-Hartmann, MS 8106, P.O. Box 6511, Aurora, Colorado 80045. E-mail: A.Gutierrez-Hartmann{at}uchsc.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The protooncogene c-Jun and its oncogenic isoform v-Jun are members of the activator protein 1 family of transcription factors that have been shown to have differential transcriptional effects that are both promoter specific and cell type specific. Previously, we have demonstrated that whereas c-Jun inhibits pituitary-specific rat prolactin (rPRL) promoter activity, expression of v-Jun stimulates the rPRL promoter in GH4 pituitary cells. In this report, we have conducted an extensive structure-function analysis of c-Jun vs. v-Jun to determine which regions of these proteins are responsible for their differential transcriptional effects in this pituitary model system. We show that isoform-specific responses are mediated by complex interactions between the {delta}-domain, serine 243, and the amino-terminal transcriptional activation domains. Thus, in contrast to previous reports, no single domain is responsible for the differential transcriptional activities of c-Jun and v-Jun. Mutation of c-Jun serine 243 to phenylalanine and replacement of the c-Jun amino terminus with the corresponding region from v-Jun, thereby removing the {delta}-domain, are necessary and sufficient to confer a functional switch from the c-Jun-inhibitory to the v-Jun-activating phenotype. Thus, we propose that isoform-specific subdomains in c-Jun and v-Jun dictate discrete interactions with distinct protein partners, which underlie the differential Jun-dependent transcriptional responses of the rPRL promoter.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
C-JUN IS A MEMBER of the BZip family of transcription factors, all of which contain a basic DNA-binding domain (DBD) and a leucine zipper protein-dimerization domain (1, 2, 3). c-Jun is the cellular homolog of the v-Jun oncogene (4, 5, 6), and the functional consequences of the structural differences between the c-Jun and v-Jun isoforms are the subject of much investigation.

c-Jun consists of multiple functional domains (Fig. 1AGo), including two amino-terminal transactivation domains (TAD1 and TAD2), a regulatory domain ({delta}-domain), a carboxy-terminal basic DBD, and a leucine zipper protein dimerization domain (LZip) (1, 2). In the prototypical model of Jun-regulated gene activation, c-Jun dimerizes via its leucine zipper with c-Fos and then binds with high affinity to an activator protein 1 consensus DNA binding site (1, 7, 8, 9). Once the complex is bound to DNA, the transcriptional effects of Jun are mediated through the TADs (10). The c-Jun {delta}-domain serves as a binding site for Jun kinase (JNK) family members, which phosphorylate serines 63 and 73 in TAD1 of c-Jun, thereby enhancing its transcription potency (10, 11, 12, 13, 14, 15, 16, 17). The oncogenic v-Jun isoform differs structurally from its cellular homolog, c-Jun, in several of these domains, with the most obvious difference being a complete deletion of the {delta}-domain (Fig. 1AGo) (4, 18). The lack of the {delta}-domain, which binds to JNK, renders v-Jun unphosphorylated on serine 63 and serine 73 (10, 11, 12, 13, 15). There are also seven amino acid (AA) changes and six AA deletions in v-Jun in the region corresponding to the first TAD of c-Jun. Additionally, the proline-glutamine-rich or hinge region (AA 179–213), located between the two functionally defined TADs, contains 12 AA changes and 11 AA deletions in the corresponding v-Jun region (Fig. 1AGo) (4, 10). In the highly conserved carboxy terminus, v-Jun has serine 243 changed to a phenylalanine and cysteine 269 changed to a serine. Serine 243 is one of three negative regulatory phosphorylation sites in the carboxy terminus that render c-Jun unable to bind to DNA (4, 19, 20, 21). These sites have been characterized as being phosphorylated by glycogen synthase kinase 3 (GSK3), casein kinase II, and MAPK in a variety of cell systems, with serine 243 functioning as a gatekeeper site by regulating the phosphorylation of the other carboxy-terminal sites (19, 20, 21). Finally, v-Jun mRNA lacks 394 nucleotides of the 3'-untranslated region (UTR) present in c-Jun (22, 23). Within this deleted region are two mRNA-destabilizing sequence motifs, suggesting an increased stability for v-Jun mRNA (22, 23, 24).



View larger version (34K):
[in this window]
[in a new window]
  		Fig. 1. Differential Effects of c-Jun and v-Jun on the Proximal rPRL Promoter

A, GH4 rat pituitary cells were transiently transfected with 5 µg pA3PRLluc-425 or pA3{Delta}2luc and 20 µg pRSVßglobin, pc-Jun, pv-Jun, pvJun/cCT, or pcJun/vCT. Cells were lysed and assayed for luciferase activity as described in Materials and Methods. PRL promoter activity is expressed as mean fold change over control (set to 1.0) ± SEM. Effects of c-Jun and v-Jun were significantly different from control (paired t test) +, P < 0.001. Chimeric constructs were significantly different from c-Jun, *, P < 0.01;**, P < 0.001. c-Jun, n = 238; v-Jun, n = 216; vJun/cCT, n = 21; and cJun/vCT, n = 18 transfections. Regions from c-Jun are white, whereas v-Jun regions are shaded gray. See Table 1Go for details of chimeric Jun constructs. {delta}, Amino-terminal {delta}-domain; h, hinge domain; LLLLL, leucine zipper domain; P, phosphorylation site; F, Phe mutation at GSK3 site at c-Jun serine 243. B, Equal protein aliquots of extracts of transfected cells analyzed by Western blot using a polyclonal anti-c-Jun antibody (Santa Cruz Biotecnology). Blots were stripped and reprobed for actin expression as described in Materials and Methods.

 
In numerous systems, c-Jun and v-Jun have been shown to have differential effects on transcription, but the precise structural basis for these disparate effects remains unclear (10, 23, 25, 26, 27). Recent studies have shown that the distinct effects of c-Jun and v-Jun are promoter specific as well as cell type specific (28, 29, 30). To investigate the differential effects of c-Jun and v-Jun on transcription, we have used the rPRL promoter in GH4 pituitary cells as a model representing a physiologically relevant, tissue-specific gene in the context of a cell line that maintains cell type-specific functions and hormonal responses (31, 32, 33). Jun/activator protein 1 members have been shown to be critical regulators of basal and hormone-induced gene transcription in the pituitary gland, including PRL (34, 35, 36, 37, 38, 39, 40). In addition, overexpression of c-Jun has been observed in both primary pituitary adenomas (41) and in metastases derived from a prolactinoma (42). We have demonstrated that c-Jun selectively inhibits basal rPRL promoter activity, via a repressor site, footprint II (FP II), in this GH4 pituitary cell system whereas, in contrast, v-Jun activates the rPRL promoter (40). Moreover, in HeLa nonpituitary cells, both c-Jun and v-Jun stimulate rPRL promoter activity (40). Thus, the striking differential effects of c-Jun and v-Jun on rPRL transcription are both promoter and cell type specific.

In this study, we have conducted a rigorous structure-function analysis of c-Jun vs. v-Jun to determine the regions critical for their differential transcriptional effects in the GH4 pituitary model system. Mutational analysis revealed that no single domain is responsible for the differential transcriptional activities of c-Jun and v-Jun, but that isoform-specific responses are mediated by complex interactions between the {delta} domain, serine 243, and the TADs. We demonstrate that mutation of c-Jun serine 243 to phenylalanine, in combination with replacement of the c-Jun amino terminus with the corresponding v-Jun region lacking the {delta}-domain, is necessary and sufficient for a full functional switch from the c-Jun-inhibitory to the v-Jun-activating phenotype. The data presented here provide important structure-function insights into the molecular mechanisms of the differential transcriptional effects of c-Jun vs. v-Jun on tissue-specific gene expression.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
We have shown previously that c-Jun and v-Jun exhibit distinctly opposite effects on basal and growth factor-inducible transcription of the PRL and GH promoters in neuroendocrine cells (40). Specifically, c-Jun inhibited the proximal –425 rPRL promoter by approximately 50%, whereas v-Jun stimulated PRL promoter activity 6.7-fold (Fig. 1AGo). Both the repressive effects of c-jun and the stimulatory response to v-jun showed dose dependency in a range of 2–30 µg of plasmid DNA (Ref. 40 , and data not shown). Similar results were obtained with a longer 2.5-kb PRL promoter construct (40). c-Jun-mediated inhibition was lost upon deletion of a repressor element FP II, the resultant {Delta}2 rPRL promoter construct showing a 1.3-fold activation. The {Delta}2 rPRL mutation also dramatically enhanced the ability of v-Jun to activate the rPRL promoter, increasing stimulation from 6.7-fold to 23-fold (Fig. 1AGo). These observations are highly reproducible and statistically significant (P < 0.001; n = 216–238) and provide the basis to examine the structural/functional domains of c-Jun and v-Jun required to mediate their strongly divergent isoform-specific effects on the tissue-specific transcription of the PRL gene.

Structural differences between c-Jun and v-Jun include loss of the {delta}-domain and the JNK phosphorylation sites, replacement of Serine 243 with phenylalanine, AA substitutions and deletions in the hinge region, and distinct 3'-untranslated regions (UTRs) (Fig. 1Go) (10, 18, 19, 20, 21, 22, 24, 43). To begin to elucidate structure-function relationships of c-Jun and v-Jun with respect to their specific transcriptional properties, chimeric proteins were constructed, precisely replacing the amino or carboxy regions of c-Jun with the corresponding sequences of v-Jun (Fig. 1Go). Details of the construction of these and subsequent constructs are given in Materials and Methods, and the resultant proteins are described in Table 1Go. Analogous domains of c-Jun and v-Jun are depicted throughout as white and gray boxes, respectively.


View this table:
[in this window]
[in a new window]
  		Table 1. Jun Mutant Expression Vectors and Their Resultant Protein Products

 
Distinct Amino and Carboxy Domains Dictate Repression vs. Activation by Jun Isoforms
Substitution of the amino terminus of c-Jun for that of v-Jun (vJun/cCT) abrogated the inhibitory effect of c-Jun on the proximal –425 rPRL promoter and conferred a 7.8-fold activation upon the {Delta}2 rPRL construct (Fig. 1Go). Similarly, exchange of the carboxy-terminal domains of c-Jun and v-Jun (cJun/vCT) also eliminated c-Jun-mediated repression of PRL promoter activity and resulted in increased stimulation of the {Delta}2 rPRL promoter (4.1-fold) in a manner comparable to that of vJun/cCT. However, unlike v-Jun, neither chimera activated the wild-type –425 rPRL promoter. Taken together, these results suggest that elements of both the amino-terminal transactivation- and carboxy-terminal DBDs are involved in the specific effects of c-Jun and v-Jun on the PRL promoter. Expression of chimeric proteins was verified by Western blot analysis using specific antibodies (Fig. 1BGo). Recombinant Jun proteins migrated according to their predicted molecular weights indicating that full-length intact proteins were expressed. The blot was reprobed with an antiactin antibody as a protein-loading control and to demonstrate that differential responses were not due to variable protein expression of the transfected constructs. These and subsequent exogenous recombinant v-Jun and c-Jun constructs were expressed at levels comparable to that of the endogenous transcription factor and regulator of the PRL promoter, Pit-1.

The {delta}-Domain Is Not Required for c-Jun-Mediated Repression of the rPRL Promoter
To further investigate the functional role of the {delta}-domain, TADs, and hinge region, a series of c-Jun/v-Jun chimeric proteins, which transposed or deleted these amino-terminal subdomains, was constructed (Fig. 2AGo). The {delta}-domain has previously been implicated as a modulator of tissue-specific transcriptional responses and transformation potency of c-Jun vs. v-Jun (10, 11, 12, 15, 23, 25, 26, 27). However, deletion of the {delta}-domain of c-Jun (cJun{Delta}{delta}) had no effect on repression of the –425 rPRL promoter and did not confer the ability to activate the {Delta}2 rPRL construct (Fig. 2AGo). Indeed, the cjun{Delta}{delta} construct functioned in a manner indistinguishable from c-Jun (Fig. 1AGo vs. Fig. 2AGo). In contrast, vJun/cCT (Fig. 1AGo), which also lacks the {delta}-domain but contains the TADs of v-Jun, did not inhibit the intact proximal rPRL promoter and significantly activated {Delta}2 rPRL. This suggests that the {delta}-domain is not essential for the c-Jun-mediated inhibition of PRL transcription and that the functional role of {delta} is context dependent with additional elements of the c-Jun and v-Jun TADs being required for repression and activation, respectively.



View larger version (28K):
[in this window]
[in a new window]
  		Fig. 2. The {delta}-Domain Is Not Required for c-Jun Inhibition of the rPRL Promoter

A, GH4 cells were transiently transfected with 5 µg pA3PRLluc-425 or pA3{Delta}2luc with 20 µg pRSVßglobin, pcJun{Delta}{delta}, pcJun/vTAD, or pcJun/vH. Data are expressed as mean fold over control ± SEM for 51 (–425) and 30 ({Delta}2) transfections for pcJun{Delta}{delta}; 12 transfections (–425 and {Delta}2) for pcJun/vTAD; and 18 (–425 and {Delta}2) transfections for pcJun/vH. Results were statistically different from c-Jun (paired t test). *, P < 0.01 as indicated. Regions of c-jun are white and v-Jun is shaded as in Fig. 1Go. B, Western blot analysis of expression of c-Jun constructs analyzed as in Fig. 1Go.

 
Consistent with this hypothesis, c-Jun constructs harboring v-Jun TAD1 (cJun/vTAD), the hinge (h) region, and TAD2 (cJun/vH) in the presence of an intact {delta}-domain, all behaved similarly to wild-type c-Jun, exhibiting approximately 50% inhibition of the rPRL promoter (Fig. 2AGo). Because vJun/cCT did not repress the rPRL promoter and activated {Delta}2 rPRL reporter, this implies that the {delta}-domain can modulate the function of the v-Jun TADs. Hence, in the absence of the {delta}-domain, the v-Jun TADs abolished repression of rPRL promoter activity and stimulated the {Delta}2 construct (Fig. 1AGo, vJun/cCT), whereas addition of the {delta}-domain (cJun/vH, Fig. 2AGo) conferred inhibition of the proximal –425 PRL promoter and suppressed the activation of {Delta}2 rPRL. However, substitution of the carboxy-terminal region of c-Jun for that of v-Jun (Fig. 1Go, cJun/vCT), thereby replacing the hinge, DBD, leucine zipper, and 3'-UTR, resulted in loss of inhibition of the rPRL promoter and activated {Delta}2 rPRL, despite the presence of an intact {delta}-domain (Fig. 1Go). Thus, one or more of these v-Jun elements may, in turn, modulate the effects of the {delta}-domain and the consequent repression/ activation of the rPRL promoter. In accordance with these observations, whereas c-Jun has no effect on the {Delta}2 rPRL promoter (Fig. 1Go), cJun/vH demonstrated a modest but significant (P < 0.01) activation (2.4-fold) of the {Delta}2 rPRL reporter construct (Fig. 2AGo), implying that the hinge subdomain (hTAD2) of v-Jun may be important for transcriptional activation.

The observed results were not attributable to differences in expression of the constructs. All of the c-Jun amino-terminal mutations analyzed in Fig. 2AGo were expressed at comparable levels.

Mutation of Serine 243 Abrogates Inhibition of the rPRL Promoter by c-Jun
Structural changes in the carboxy-terminal domains of v-Jun include mutation of serine 243 to phenylalanine and alterations in the 3'-UTR, both of which have been implicated in v-Jun-specific responses (16, 17). To address the functional roles of these elements in the phenotypic switch from c-Jun transcriptional repression to v-Jun activation of the rPRL promoter, a series of site-specific or UTR substitution mutants were created (Fig. 3AGo).



View larger version (25K):
[in this window]
[in a new window]
  		Fig. 3. Serine 243 Is Critical for c-Jun-Mediated Inhibition

A, GH4 cells were transiently transfected with 5 µg pA3PRLluc-425 or pA3{Delta}2luc with 20 µg pRSVßglobin, pcJunS243F, pcJun/vUT, or pvJun/cUT. Data are expressed as mean fold over control ± SEM for 54 (–425) and 33 ({Delta}2) transfections for pRSVcJ/S243F; 27 (–425) and 21 ({Delta}2) transfections for pcJun/vUT; 27 (–425) and 15 ({Delta}2) transfections for pvJun/cUT; and 15 (–425) and 9 ({Delta}2) for pcJunS243F/vUT. Regions of c-jun are white and v-Jun are shaded as in Fig. 1Go. Results were statistically different from c-Jun. **, P < 0.001, as indicated. B, Western blot of carboxy-terminal Jun mutant expression.

 
As shown in Fig. 3AGo, mutation of serine 243 to phenylalanine (S243F) in c-Jun abolished the inhibition of the basal rPRL promoter and conferred a small but significant (P < 0.001) 2.8-fold activation of {Delta}2 rPRL, similar to that induced by cJun/vCT (Fig. 1AGo). Thus, S243 and/or its phosphorylation is necessary for c-Jun-mediated inhibition of transcription and may play a role in regulation of transactivation. The cJun/vUT and vJun/cUT constructs, exchanging the 3'-UTRs of v-Jun and c-Jun, behaved similarly to their respective wild-type counterparts (Fig. 3AGo). cJun/vUT inhibited the –425 rPRL promoter and did not activate {Delta}2 rPRL, whereas vJun/cUT stimulated –425 rPRL promoter activity 10.1-fold and {Delta}2 rPRL 31.9-fold (compare Fig. 1AGo and Fig. 3AGo). Hence the 3'-UTRs had minimal apparent effects on the characteristic inhibition or activation of the rPRL promoter constructs by c-Jun and v-Jun, respectively. As shown in Fig. 3BGo, the series of UTR and S243F constructs were all expressed at detectable levels and migrated at their predicted sizes.

Isoform-Specific Effects of c-Jun and v-Jun Are Mediated by Functional Interactions between the {delta}-Domain, Serine 243, and the TADs
The aforementioned results implicated three structural motifs in the isoform-specific regulation of transcription by c-Jun and v-Jun: the {delta}-domain, the TADs, and serine 243. Moreover, these elements may interact functionally in a complex, context-dependent manner, such that no single v-Jun-specific domain is sufficient to fully switch the inhibitory phenotype of c-Jun to the stimulatory action of v-Jun. Thus, we next constructed and analyzed a panel of combinatorial subdomain chimeric mutations (Fig. 4Go) to identify the cooperative structural/functional motifs underlying the divergent transcriptional responses of v-Jun and c-Jun.



View larger version (44K):
[in this window]
[in a new window]
  		Fig. 4. The Amino Terminus and Serine 243 Are Required for the Differential Effects of c-Jun and v-Jun

A, GH4 cells were transiently transfected with 5 µg pA3PRLluc-425 or pA3{Delta}2luc with 20 µg pRSVßglobin, pcJun{Delta}{delta}/vUT, pvJun/cCT/S243F, pcJunS243F/{Delta}{delta}/vUT, pcJunS243F/vTAD, pcJunS243F/vH, pcJunS243F/vTAD/vH, pcJunS243F/vTAD/vUT, and pvJun/cNT as indicated. Data are expressed as mean fold over control ± SEM for 24 (–425) and 15 ({Delta}2) transfections for pcJun{Delta}{delta}/vUT; 18 (–425 and {Delta}2) transfections for pvJun/cCT/S243F; 15 (–425) and 12 ({Delta}2) transfections for pcJun{Delta}{delta}/vUT; 12 (–425 and {Delta}2) transfections for pcJunS243F/vTAD; 15 (–425) and six ({Delta}2) transfections for pcJunS243F/vH; 15 (–425) and 12 ({Delta}2) transfections for pcJunS243F/vTAD/vH; 18 (–425) and 15 ({Delta}2) transfections for pcJunS243F/vTAD/vUT; and 15 (–425) and 12 ({Delta}2) transfections for pRSVcJ/vCT. Results were significantly different from the effects of c-Jun. **, P < 0.001, as indicated. B, Western blot of Jun mutant expression analyzed as in Fig. 1Go.

 
As described above, deletion of the c-Jun {delta}-domain (Fig. 2AGo; c-Jun{Delta}{delta}) or exchange of the c-Jun UTR for that of vJun (Fig. 3AGo; c-Jun/vUT) had no effect on the regulation of the rPRL promoter. Consistent with these results, substitution of the c-Jun UTR in the context of the {delta}-domain deletion (c-Jun{Delta}{delta}/vUT) also had no significant effects on jun-dependent –425 or {Delta}2 promoter responses (Fig. 4AGo). Mutation of serine 243 in the background of the {delta}-domain deletion and v-Jun 3'-UTR (cJunS243F/{Delta}{delta}/vUT) abolished inhibition of –425 rPRL promoter activity and conferred activation (4.8-fold) of the {Delta}2 rPRL construct, similar to that observed for cJun/vCT (Fig. 1AGo) and cJunS243F (Fig. 3Go). Thus, the effects of the S243F mutation were not significantly enhanced by the deletion of the {delta}-domain and insertion of the v-Jun 3'-UTR. This indicates that serine 243 and/or its phosphorylation is a critical factor in the isoform-specific transcriptional responses exhibited by c-Jun and v-Jun, independent of the modulatory roles of the {delta}-domain and 3'-UTRs. Conversely, mutation of serine 243 (cJunS243F) does not result in activation of the rPRL promoter and only partially (2.8-fold) activates {Delta}2rPRL compared with v-Jun (23.1-fold) (compare Fig. 3AGo and Fig. 1AGo), indicating that v-Jun-specific TADs also contribute to the phenotypic switch. Accordingly, vJun/cCT/S243F, expressing the v-Jun amino-terminal TADs in the context of the S243F mutation, exhibits significant 2-fold (P < 0.001) activation of the –425 rPRL promoter and strong (12.5-fold) stimulation of {Delta}2 rPRL, similar to that of v-Jun (Fig. 4AGo). However, an analogous construct retaining the {delta}-domain of cJun (cJunS243F/vTAD/vH) inhibited the –425 promoter, overriding the effect of the S243F mutation, and only slightly (2.7-fold) activated {Delta}2 rPRL (Fig. 4AGo). This {delta}-domain-dependent repression was not affected by substitution of the v-Jun UTR (cJunS243F/vTAD/vUT). Moreover, the ability of v-Jun to activate the rPRL promoter (Fig. 1AGo) is abrogated, and its dramatic stimulation of {Delta}2 is significantly attenuated by the addition of the {delta}-domain (Fig. 4AGo; vJun/cNT). The 6.7-fold stimulation of –425 rPRL by v-Jun is completely eliminated in cJun/vCT and the 23.1-fold activation of {Delta}2 rPRL is reduced to 4.2-fold (compare Fig. 1AGo, v-Jun, and Fig. 4AGo, vJun/cNT). Taken together, these results suggest that the phenotypic switch from c-Jun-like repression to v-Jun-like activation, mediated by S243F and the v-Jun amino-terminal TADs and hinge region, is regulated by the {delta}-domain.

Insertion of the v-Jun TAD1 into c-Jun had no effect (data not shown). However, mutation of serine 243 in this context (cJunS243F/vTAD/vH), although retaining the inhibitory c-Jun effect on the –425 promoter, activated {Delta}2 rPRL promoter (Fig. 4AGo). Similarly, both TAD1 (S243F/vTAD) and hTAD2 (S243F/vH) can modestly activate {Delta}2 rPRL (2.9- and 5.5-fold, respectively) when serine 243 is mutated (Fig. 4AGo). However, a comparison of the transcriptional activities of the analogous constructs with an intact serine 243 site, cJun/vH and cJun/vTAD (Fig. 2AGo,) shows that serine 243 significantly attenuated the ability of these domains to stimulate {Delta}2 rPRL. Thus, S243F/vTAD activated {Delta}2 rPRL 2.9-fold (Fig. 4AGo), whereas cJun/vTAD had no effect (Fig. 2AGo). Likewise, the 2.4-fold increase in {Delta}2 rPRL activity induced by cJun/vH (Fig. 2AGo) was increased to 5.5-fold in the corresponding S243F/vH mutant (Fig. 4AGo). These data suggest that serine 243 and/or its phosphorylation, like the {delta}-domain, also suppressed the activity of the v-Jun TADs. Hence, both deletion of the {delta}-domain and mutation of serine 243 are required to permit the v-Jun TADs to activate the PRL promoter (Fig. 4AGo, vJun/cCT/S243F).

Comparison of c-Jun (Fig. 1AGo) with cJun/vUT (Fig. 3AGo) shows no apparent effect of the 3'-UTRs on the activity of c-Jun, with respect to the rPRL promoter constructs. Likewise, replacing the 3'-UTR of v-Jun with that of c-Jun (vJun/cUT) did not affect its activation of PRL transcription (Figs. 1AGo and 3AGo). Exchange of the c-Jun 3'-UTR for v-Jun 3'-UTR, in the context of the c-Jun {delta}-domain deletion, also had no significant effect (see Fig. 2AGo, cJun{Delta}{delta}; and Fig. 4AGo, cJun{Delta}{delta}/vUT) and, when S243F/vTAD and cJun/S243F/vTAD/vUT are compared (Fig. 4AGo), exchange of 3'-UTRs also had minimal effect on the ability of the v-Jun TAD1 to activate {Delta}2 rPRL. Thus, the diverse functions of c-Jun and v-Jun on the rPRL promoter appear not to be regulated by their respective 3'-UTRs. As shown in Fig. 4BGo, Western blot analysis demonstrated that all constructs were expressed and migrated consistent with their predicted molecular weights.

In summary, isoform-specific transcriptional regulation by c-Jun and v-Jun is a function of interaction of serine 243 and the {delta}-domain to modulate the activities of their respective TADs. Thus, multiple elements of both the transactivation and DBDs of c-Jun and v-Jun cooperate to mediate their differential regulation of promoter- and cell type-specific neuroendocrine gene expression.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
The effects of c-Jun and v-Jun on the activity of the rPRL promoter in GH4 pituitary cells are distinct, resulting in a strikingly divergent response. Specifically, c-Jun inhibits rPRL promoter activity, whereas v-Jun is a potent transactivator. Using this model system, we conducted an extensive structure-function analysis of c-Jun and v-Jun to map the regions that are required to mediate their differential effects on rPRL transcription. Our results indicate that, in contrast to previous reports (10, 23, 25, 26, 27), no single domain is sufficient to account for the differential transcriptional activities of c-Jun and v-Jun. Instead, isoform-specific responses appear to be dependent on complex interactions between the c-Jun {delta}-domain, serine 243, and amino-terminal TADs, motifs that are all altered or absent in v-Jun. Transcriptional repression by c-Jun is dependent on interactions of serine 243 and the {delta}-domain that modulate the activity of the TADs, resulting in inhibition of the rPRL promoter. Loss of both of these regulatory elements in v-Jun is necessary and sufficient to confer activation of PRL gene transcription.

These results are in contrast to previous studies in the literature, which implicate the {delta}-domain alone in differential effects of c-Jun and v-Jun (10, 23, 25, 26, 27). The {delta}-domain clearly modulates Jun-dependent transactivation, because the addition of the {delta}-domain to v-Jun (cJun/vCT) was sufficient to eliminate the ability of v-Jun to activate the –425 rPRL promoter (Fig. 1AGo). However, the data presented here indicate that the function of the {delta}-domain is more complex than a simple interaction of the {delta}-domain with a putative cell-specific repressor, as initially postulated (10, 27, 44). Previous data, such as the ability of c-Jun to induce differentiation (45), and to inhibit the basal activity of highly specialized promoters (46, 47, 48), suggest that the putative effects of the {delta}-domain may be governed by multiple regulatory influences, including the differentiation state of the cell, environmental cues, signaling events, repressors, coactivators, and the ubiquitination machinery. Indeed, reports have shown that the c-Jun amino-terminal TAD, including the {delta}-domain, functionally (and in some cases physically) interacts with: 1) certain transcription factors, such as MyoD, myogenin, steroid receptors, and signal transducer and activator of transcription-3ß, to either repress or activate transcription of specific target genes; and, 2) JNK (12, 13, 46, 47, 48, 49, 50). The function of the {delta}-domain has been described previously to be cell specific, being required for the transactivation of collagenase and transin promoters in chick embryo fibroblasts, but not in F9 murine embryonal carcinoma cells (27). These data imply that the {delta}-domain may contain distinct functional faces, interacting with JNK and other transacting factors (some of which may be cell specific), respectively. Our results support this hypothesis and further implicate a functional interaction between the {delta}-domain and serine 243 in the differential transcriptional responses of v-Jun and c-Jun.

Other studies have shown that another inhibitory region, termed the {epsilon}-domain, located in TAD 1, is responsible for interacting with cell type-specific repressor proteins (51). Recent studies indicate that the {epsilon}-domain is required for histone deacetylase 3-dependent suppression of the transcriptional activity of c-Jun. Interestingly, deletion of the {delta}-domain destabilized interaction with a putative histone deacetylase 3-associated repressor complex, suggesting a potential interaction between the {epsilon}- and {delta}-domains (52). However, in the pituitary system, the {epsilon}-domain appears to be insufficient to mediate the cell type-specific and promoter-specific inhibition of the rPRL promoter by c-Jun. This is best demonstrated by the fact that v-Jun, which has an intact {epsilon}-domain (51), activates the rPRL promoter in GH4 cells (Fig. 1Go).

A second domain of interest defined by these mutational studies is the c-Jun proline-glutamine-rich domain (AA 179–213) termed the "hinge" region, which contains 12 AA substitutions and 11 deletions in the corresponding region of v-Jun (Fig. 1Go) (10). The few studies that have examined this hinge region indicate that it is not important for interaction with cell type-specific repressors or for repression of cell type-specific genes (10, 48). Accordingly, the hinge domain chimera (cJun/vH) inhibits the –425 rPRL promoter to an equivalent degree as wild-type c-Jun (Fig. 2AGo). However, the hinge domain chimera (cJun/vH) activates the {Delta}2 rPRL promoter to a greater degree than wild-type c-Jun (P < 0.01), which is reminiscent of the v-Jun phenotype. This suggests that the hinge region of c-Jun is not required for inhibition of the rPRL promoter, but that the corresponding region of v-Jun contributes to v-Jun-mediated activation of PRL transcription. Additionally, these data imply that the activating effects of the v-Jun hinge region are recessive to other inhibitory domains in the amino terminus of c-Jun, as evidenced by the ability of the hinge domain chimera (cJun/vH) to inhibit the –425 rPRL promoter (Fig. 2AGo). Thus, whereas this poorly characterized hinge region may be important for the activating effects of v-Jun on the –425 rPRL promoter, it appears able to mediate those effects only when in the context of the v-Jun amino terminus and a serine 243 to phenylalanine substitution.

The final critical c-Jun effector identified in these studies is serine 243. The AA change of serine 243 to phenylalanine, as seen in v-Jun, is sufficient to eliminate the ability of c-Jun to inhibit the –425 rPRL promoter and to switch c-Jun to an activator of the {Delta}2 rPRL promoter (Fig. 3AGo). Serine 243 is a negative regulatory phosphorylation site in c-Jun, believed to inhibit c-Jun from binding to DNA. This phosphorylation site is one of three carboxy-terminal sites that have previously been characterized as being phosphorylated by a number of different kinases, including GSK3, casein kinase II, and MAPK (19, 20, 21). However, mutation of serine 243 to phenylalanine eliminated phosphorylation on all three sites in vivo, suggesting that it may have a "gate keeper" or priming phosphate function (20, 21, 53). In the pituitary system, Western blot analysis of transiently expressed c-Jun, as compared with the serine 243 mutant (cJunS243F), demonstrates that alteration of that regulatory serine causes a downward shift of the protein on the blot (Fig. 3BGo). This downward shift may be indicative of loss of phosphorylation at the three carboxy-terminal phosphorylation sites, as previously described (20). Thus, phosphorylation of c-Jun at its carboxy terminus may play a critical role in its ability to interact with a transacting repressor protein, pituitary-specific inhibitory factor (PSIF), which we have previously hypothesized (40). Recent evidence suggests that serine 243 is phosphorylated by a proline-directed kinase distinct from GSK3, p38, MAPK, and JNK that is yet to be identified (53). The mutation of serine 243 to phenylalanine present in v-Jun would result in a carboxy-terminal dephosphorylated protein predicted to constitutively bind to DNA and thereby contribute to the oncogenicity of v-Jun. Thus, the putative novel serine 243 kinase has been speculated to function as a tumor suppressor (53). Our data support this critical role for serine 243 and further implicate this region in the formation of a protein interaction surface required for isoform-specific transcriptional response of v-Jun and c-Jun.

However, mutation of this serine 243 phosphorylation site is still not sufficient for a complete functional switch of c-Jun to become an activator of the –425 rPRL promoter, as seen with v-Jun. This conversion is only achieved with the mutation of serine 243 to phenylalanine in the context of the v-Jun amino terminus chimera (vJun/cCT/S243F, Fig. 4AGo), further supporting the idea that it is the three-dimensional structure of c-Jun that gives rise to a protein face capable of interacting with a putative PSIF. Partial disruption of this face with the v-Jun amino terminus chimera (vJun/cCT), the v-Jun carboxy-terminus chimera (cJun/vCT), or the serine 243 mutant (cJunS243F) is sufficient to alleviate the inhibitory effect of c-Jun on the rPRL promoter, but not sufficient for the functional switch to the v-Jun-activating effect. Only complete disruption of this protein face, as in the double mutant of serine 243 and the v-Jun amino terminus (vJun/cCT/S243F, Fig. 4AGo), is sufficient to block interaction of c-Jun with PSIF and allow the v-Jun-like interaction with GHF-1/Pit-1 to predominate (40), and thus activate the –425 rPRL promoter. Consistent with this hypothesis, deletion of the FP II repressor binding site ({Delta}2rPRL) enhances v-Jun stimulation of the rPRL promoter (Fig. 1Go). Interestingly, mutation of the C-terminal phosphorylation sites of murine c-Jun, including the residue corresponding to serine 243 in the human protein, rescued the defective transforming phenotype of an amino-terminal phosphorylation site mutant (54). The authors suggested that the C-terminal mutations induced a conformational change, which compensated for the inhibitory effect of the amino-terminal mutation. These data are consistent with our observations that amino-terminal TADs of Jun are also modulated by mutation of serine 243, such that substitution of both domains is required to confer transcriptional activation of the rPRL promoter (Fig. 4AGo, vJun/cCT/S243F).

Thus, we propose a model wherein c-Jun is folded in such a way that the amino terminus of the protein together with serine 243 creates a functional face that interacts with a putative PSIF (Fig. 5Go). Previous studies have postulated analogous cell type-specific c-Jun inhibitors in fibroblast cell lines (44) and cardiac myocytes (48). Disruption of the putative PSIF DNA binding site on the rPRL promoter (i.e. FP II), or mutation of serine 243 and the amino terminus, thereby reconfiguring the specific protein interaction domain topography, prevents c-Jun from mediating its inhibitory effects. Instead, mutant c-Jun or v-Jun interacts with Pit-1, thus switching to an activator of the rPRL promoter via the principal Pit-1 binding site, FP I (40). Thus, the studies presented here demonstrate that c-Jun and its oncogenic homolog, v-Jun, differentially regulate cell type-specific rPRL gene expression via isoform-specific protein interaction surfaces comprised of the {delta}-domain, amino-terminal TADs, and serine 243. Hence, divergent transcriptional responses of c-Jun and v-Jun are mediated by interactions with distinct tissue-specific trans-acting factors which then target these complexes to different cis elements of the rPRL promoter. Although this model is based upon our observations of Jun-mediated regulation of PRL gene expression in pituitary cells, it may be more widely applicable. Specifically, the structure-function relationships and interactions between the {delta}-domain, TADs, and serine 243, identified in this study, may be important in Jun-dependent tissue-specific transcriptional regulation and oncogenic transformation of other highly differentiated cell types.



View larger version (13K):
[in this window]
[in a new window]
  		Fig. 5. Model for c-Jun and v-Jun Differential Transcriptional Effects on the rPRL Promoter in GH4 Cells

c-Jun inhibits the rPRL promoter by stabilizing or enhancing the binding of a putative pituitary-specific inhibitory factor (PSIF) to FP II by a protein-protein interaction mechanism requiring the aminoterminus and serine 243 of c-Jun. Thus, mutation of the c-Jun amino terminus or serine 243, loss of PSIF, or mutation of FP II ({Delta}2rPRL) would nullify this inhibitory effect. In contrast, v-Jun does not bind to the hypothetical PSIF but instead interacts with Pit-1 (40 ) bound to FP I to activate the rPRL promoter.

 

   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Plasmids
The pA3PRLluc-425 promoter-luciferase construct and the site-specific mutant FP II (pA3{Delta}2luc) have been described previously (32, 55, 56). The plasmids, pRSVc-Jun and pRSVv-Jun, were from Dr. Michael Karin (43, 57). pRC/CMVcjunAK-3 (deletion of AA 34–60) was provided by Dr. Andrew Kraft (University of Colorado Health Sciences Center) (58).

To construct the 3'-UTR exchange mutants, pcJun/vUT and pvJun/cUT, HindIII/BspHI fragments from pRSVc-Jun and pRSVv-Jun, respectively, were ligated, with BspHI/BamHI fragments containing the converse 3'-UTRs, into HindIII/BamHI cut pRSV. pcJun{Delta}{delta} was derived by insertion of a HindIII/PstI fragment of pRcCMVcjunAK-3 into pRSVc-Jun digested with HindIII/PstI. The plasmid pcJun{Delta}{delta}/vUT was constructed by ligation of the above HindIII/PstI c-JunAK-3 insert into pcJun/vUT digested with HindIII/PstI.

The plasmid pcJun/vH contains the c-Jun cDNA with AA 169–223, encompassing the hinge region of c-Jun, replaced by the corresponding v-Jun hinge region, AA 136–179. The specified region of plasmid pRSVv-Jun was amplified by PCR with the sense oligonucleotide 5'-CCCCGGTCTACGCCAATC-3' and the antisense oligonucleotide 5'-TCAGAGCCTGCAGCCTGG-3', which code for v-Jun with point mutations to restore AccI and PstI sites, respectively. The PCR product was cloned into pCR2.1 (Original T/A Cloning Kit; Invitrogen, Carlsbad, CA), generating pCR2.1vhinge, digested with AccI/PstI, and subcloned into pRSVc-Jun. pcJunS243F was obtained by PCR amplification of pRSVc-Jun with the oligonucleotides 5'-GTCAACGGGGCAGGCATGGTG-3' (sense) and 5'-GGACTCCATGTCGATGGGGAACAGGG-3' (antisense) encoding the desired serine to phenylalanine mutation at AA 243. The resulting PCR fragment was digested with AccI and BstXI and cloned into pRSVc-Jun. Primers were obtained from Life Technologies, Inc. (Gaithersburg, MD). The plasmid pcJunS243F/{Delta}{delta}/vUT was generated by digestion of pcJun{Delta}{delta} with HindIII/PstI, and ligation of the resulting insert into HindIII/PstI cut pcJunS243F/vUT. The plasmid pcJunS243F/vH, containing the S243F mutation and the v-Jun hinge region (AA 136–179) substituted for the c-Jun hinge region (AA 169–223), was constructed by cloning of the AccI/PstI insert from pCR2.1vhinge into pcJunS243F. Constructs were verified by dideoxy sequencing performed by the sequencing core facility of the Colorado Cancer Center.

The plasmid pcJun/vCT was obtained by ligation of a pv-Jun BstXI/BamHI fragment into pcJun/vH. The corresponding pvJun/cCT chimera was generated by insertion of a pv-Jun HindIII/BstXI fragment into pcJun/vH. The latter was partially digested with BstX1in the presence of ethidium bromide at 37 C to slow the rate of enzymatic digestion. The desired fragment was used for subsequent HindIII digests before ligation of the v-Jun HindIII/BstXI insert. The plasmid pvJun/cCT/S243F was obtained by digestion of pcJunS243F with HindIII/BstXI as described above and ligation with the HindIII/BstXI fragment of pv-Jun.

The plasmid pCRvTAD was created by PCR amplification of the specified region of pRSVv-Jun using the sense oligonucleotide 5'-GCCTCCCCCGAGCTGGAAC-3' and the antisense oligonucleotide 5'-ATTGGCGTAGACCGGGGGCT-3', which code for v-Jun with point mutations to restore AvaI and AccI sites, respectively. The PCR-generated insert was then cloned into the vector pCR2.1 to create pCRvTAD. The sequence of the AvaI/AccI v-Jun insert generated by PCR was verified by dideoxy cycle sequencing as described above. To construct pcJun/vTAD, pRSVc-Jun was digested with AccI followed by HindIII/AvaI. The resulting HindIII/AvaI c-Jun insert was then ligated with HindIII/AccI cut pRSVc-Jun and the AvaI/AccI insert derived from pCRvTAD. The plasmid pcJunS243F/vTAD was constructed by digestion of pcJunS243F as above to generate HindIII/AvaI and HindIII/AccI vector fragments, which were then ligated with the AvaI/AccI insert of pCRvTAD.

To generate pcJunS243F/vTAD/vH, pcJunS243F/vH was digested with HindIII/AccI and ligated with the HindIII/AccI insert of pcJun/vTAD. pcJun/vCT was digested with HindIII/AccI and ligated with the HindIII/AccI insert of pcJun/vTAD forming pvJun/cNT.

Plasmid DNAs were purified and quantitated as described previously (40, 55). Descriptions of the protein products encoded by these Jun mutant constructs are summarized in Table 1Go.

Cell Culture, Transient Transfection, and Luciferase Assay
The GH4 T2 rat pituitary tumor cells were grown in 5% CO2 at 37 C in DMEM (Life Technologies, Inc.) containing 10% fetal calf serum (HyClone Laboratories, Inc., Logan, UT) and 50 µg/ml of penicillin and streptomycin. Cells were prepared and transfected by electroporation as previously described (40, 59). The total amount of DNA was kept constant with pRSVß-globin, which also controls for nonspecific effects of RSV expression vectors. Electroporations were performed in triplicate for each condition within an experiment, and experiments were repeated multiple times using different plasmid preparations of each construct. Cells were harvested at 24 h after transfection, and cell extracts were prepared as previously described. Luciferase assays were performed as previously described using 30 µl cell extract, and all samples were assayed in duplicate. Luciferase light units of the control value were set to 1, and the data were expressed as fold effect relative to control. All data was expressed as the mean ± SEM for replicated experiments (40, 59).

Western Blot Analysis
GH4 cells transiently transfected with the rPRL promoter-reporter and various effector plasmids were harvested and lysed as previously described (40). Equal volumes of whole-cell lysate, typically 10 µl, from equivalent numbers of cells were resolved on a sodium dodecyl sulfate 10% polyacrylamide gel, transferred to nitrocellulose and processed as described (40). Membranes were probed with a rabbit polyclonal Jun antibody directed against AA 95–105 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) or a mouse monoclonal actin antibody, clone C4 (Roche Clinical Laboratories, Indianapolis, IN), at 1:1000 dilution. Antibody complexes were detected using goat antirabbit or goat antimouse secondary antibodies linked to horseradish peroxidase (Life Technologies, Inc.) and developed using an enhanced chemiluminescence kit from Amersham Life Sciences (Arlington Heights, IL). Between probes with different antibodies, the nitrocellulose membranes were stripped and reblocked as described previously (40).


   ACKNOWLEDGMENTS
 
We thank Kelley Fantle and Duane Mata for technical assistance; Michael Karin and Andrew Kraft for Jun constructs; Scott Diamond for assistance in mutant construction; and Drs. William Wood, Paul Jedlicka, and Twila Jackson for critical reading and discussion of the manuscript.


   FOOTNOTES
 
This work was supported by an Alexander Foundation Scholarship and Medical Scientist Training Program training support [National Institutes of Health (NIH) Grant T32 GM 08497] (to K.N.F.) and NIH Grant R01 DK 37667 (to A.G.-H.). Core Facilities of the Colorado Cancer Center provided tissue culture media and sequencing analysis (NIH Grant P30 CA 46934).

Abbreviations: AA, Amino acids; DBD, DNA binding domain; FP II, footprint II; GSK3, glycogen synthase kinase 3; JNK, Jun kinase; PRL, prolactin; PSIF, pituitary-specific inhibitory factor; TAD, transactivation domain; UTR, untranslated region.

Received for publication March 16, 2004. Accepted for publication June 24, 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Karin M, Smeal T 1992 Control of transcription factors by signal transduction pathways: the beginning of the end. Trends Biochem Sci 17:418–422[CrossRef][Medline]
  2. Vinson CR, Sigler PB, McKnight SL 1989 Scissors-grip model of DNA recognition by a family of leucine zipper proteins. Science 246:911–916[Abstract/Free Full Text]
  3. Vogt PK 2001 Jun, the oncoprotein. Oncogene 20:2365–2377[CrossRef][Medline]
  4. Angel P, Allegretto EA, Okino ST, Hattori K, Boyle WJ, Hunter T, Karin M 1988 Oncogene jun encodes a sequence-specific trans-activator similar to AP-1. Nature 332:166–171[CrossRef][Medline]
  5. Bohmann D, Bos TJ, Admon A, Nishimura T, Vogt PK, Tjian R 1987 Human proto-oncogene c-jun encodes a DNA binding protein with structural and functional properties of transcription factor AP-1. Science 238:1386–1392[Abstract/Free Full Text]
  6. Maki Y, Bos TJ, Davis C, Starbuck M, Vogt PK 1987 Avian sarcoma virus 17 carries the jun oncogene. Proc Natl Acad Sci USA 94:2848–2852
  7. Halazonetis TD, Georgopoulos K, Greenberg ME, Leder P 1988 c-Jun dimerizes with itself and with c-Fos, forming complexes of different DNA binding affinities. Cell 55:917–924[CrossRef][Medline]
  8. Hunter T, Karin M 1992 The regulation of transcription by phosphorylation. Cell 70:375–387[CrossRef][Medline]
  9. Smeal T, Angel P, Meek J, Karin M 1989 Different requirements for formation of Jun:Jun and Jun:Fos complexes. Genes Dev 3:2091–2100[Abstract/Free Full Text]
  10. Baichwal VR, Tjian R 1990 Control of c-Jun activity by interaction of a cell-specific inhibitor with regulatory domain {delta}: differences between v- and c-Jun. Cell 63:815–825[CrossRef][Medline]
  11. Dai T, Rubie E, Franklin CC, Kraft A, Gillespie DAF, Avruch J, Kyriakis JM, Woodgett JR 1995 Stress-activated protein kinases bind directly to the {delta} domain of c-Jun in resting cells: implications for repression of c-Jun function. Oncogene 10:849–855[Medline]
  12. Derijard B, Hibi M, Wu I-H, Barrett T, Su B, Deng T, Karin M, Davis R 1994 JNK1: a protein kinase stimulated by UV light and Ha-Ras that binds and phosphorylates the c-Jun activation domain. Cell 76:1025–1037[CrossRef][Medline]
  13. Hibi M, Lin A, Smeal T, Minden A, Karin M 1993 Identification of an oncoprotein- and UV-responsive protein kinase that binds and potentiates the c-Jun activation domain. Genes Dev 7:2135–2148[Abstract/Free Full Text]
  14. Karin M 1992 Signal transduction from cell surface to nucleus in development and disease. FASEB J 6:2581–2590[Abstract]
  15. Kyriakis JM, Banerjee P, Nikolakaki E, Dai T, Rubie EA, Ahmad MF, Avruch J, Woodgett JR 1994 The stress-activated protein kinase subfamily of c-Jun kinases. Nature 369:156–160[CrossRef][Medline]
  16. Smeal T, Binetruy B, Mercola DA, Birrer M, Karin M 1991 Oncogenic and transcriptional cooperation with Ha-Ras requires phosphorylation of c-Jun on serines 63 and 73. Nature 354:494–496[CrossRef][Medline]
  17. Smeal T, Binetruy B, Mercola D, Grover-Bardwick A, Heidecker G, Rapp UR, Karin M 1992 Oncoprotein-mediated signalling cascade stimulates c-Jun activity by phosphorylation of serines 63 and 73. Mol Cell Biol 12:3507–3513[Abstract/Free Full Text]
  18. Bos TJ, Bohmann D, Tsuchie H, Tjian R, Vogt PK 1988 v-jun Encodes a nuclear protein with enhancer binding properties of AP-1. Cell 52:705–712[CrossRef][Medline]
  19. Chou S, Baichwal V, Ferrell Jr JE 1992 Inhibition of c-Jun DNA binding by mitogen-activated protein kinase. Mol Biol Cell 3:1117–1130[Abstract]
  20. Boyle WJ, Smeal T, Defize LHK, Angel P, Woodgett JR, Karin M, Hunter T 1991 Activation of protein kinase C decreases phosphorylation of c-Jun at sites that negatively regulate its DNA-binding activity. Cell 64:573–584[CrossRef][Medline]
  21. Lin A, Frost J, Deng T, Smeal T, Al-Alawi N, Kikkawa U, Hunter T, Brenner D, Karin M 1992 Casein kinase II is a negative regulator of c-Jun DNA binding and AP-1 activity. Cell 70:777–789[CrossRef][Medline]
  22. Nishimura T, Vogt PK 1988 The avian cellular homolog of the oncogene jun. Oncogene 3:659–663[Medline]
  23. Bos T, Monteclaro F, Mitsunobu F, Ball Jr AR, Chang C, Nishimura T, Vogt P 1990 Efficient transformation of chicken embryo fibroblasts by c-Jun requires structural modification in coding and noncoding sequences. Genes Dev 4:1677–1687[Abstract/Free Full Text]
  24. Peng SS-Y, Chen C-YA, Shyu A-B 1996 Functional characterization of a non-AUUUA AU-rich element from the c-jun proto-oncogene mRNA: evidence for a novel class of AU-rich elements. Mol Cell Biol 16:1490–1499[Abstract]
  25. Alani V, Brown P, Binetruy B, Dosaka H, Rosenberg R, Angel P, Karin M, Birrer M 1991 The transactivation domain of the c-Jun proto-oncoprotein is required for cotransformation of rat embryo cells. Mol Cell Biol 12:6276–6285
  26. Bohmann D, Tjian R 1989 Biochemical analysis of transcriptional activation by Jun: differential activity of c- and v-Jun. Cell 59:709–717[CrossRef][Medline]
  27. Havarstein L, Morgan I, Wong W-Y, Vogt P 1992 Mutations in the Jun {delta} domain suggest an inverse correlation between transformation and transcription. Proc Natl Acad Sci USA 89:618–622[Abstract/Free Full Text]
  28. Gao M, Morgan I, Vogt PK 1996 Differential and antagonistic effects of v-Jun and c-Jun. Cancer Res 56:4229–4235[Abstract/Free Full Text]
  29. Hussain S, Kilbey A, Gillespie DA 1998 v-Jun represses c-jun proto-oncogene expression in vivo through a 12-O-tetradecanoylphorbol-13-acetate-responsive element in the proximal gene promoter. Cell Growth Differ 9:677–686[Abstract]
  30. Sevcikova S, Soucek K, Kubala L, Bryja V, Smarda J 2002 Differential effects of v-Jun and c-Jun proteins on v-myb-transformed monoblasts. Cell Mol Life Sci 59:1690–1705[Medline]
  31. Elsholtz HP 1992 Molecular biology of prolactin: cell-specific and endocrine regulators of the prolactin gene. Semin Reprod Endocrinol 10:183–195
  32. Keech CA, Jackson SM, Siddiqui SK, Ocran KW, Gutierrez-Hartmann A 1992 Cyclic AMP activation of the rat prolactin promoter is restricted to the pituitary-specific cell type. Mol Endocrinol 6:2059–2070[Abstract]
  33. Tashjian AHJ, Yasumura Y, Levine L, Sato GH, Parker ML 1968 Establishment of clonal strains of rat pituitary tumor cells that secrete growth hormone. Endocrinology 82:342–352[Medline]
  34. Norwitz ER, Xu S, Xu J, Spiryda LB, Park JS, Jeong KH, McGee EA, Kaiser UB 2002 Direct binding of AP-1 (Fos/Jun) proteins to a SMAD binding element facilitates both gonadotropin-releasing hormone (GnRH)- and activin-mediated transcriptional activation of the mouse GnRH receptor gene. J Biol Chem 277:37469–37478[Abstract/Free Full Text]
  35. Anouar Y, Lee HW, Eiden LE 1999 Both inducible and constitutive activator protein-1-like transcription factors are used for transcriptional activation of the galanin gene by different first and second messenger pathways. Mol Pharmacol 56:162–169[Abstract/Free Full Text]
  36. Luo LG, Jackson IM 1998 Antisense oligomers of cfos and cjun block glucocorticoid stimulation of thyrotropin-releasing hormone (TRH) gene expression in cultured anterior pituitary cells. Peptides 19:1295–1302[CrossRef][Medline]
  37. Zhu YS, Pfaff DW 1998 Differential regulation of AP-1 DNA binding activity in rat hypothalamus and pituitary by estrogen. Brain Res Mol Brain Res 55:115–125[Medline]
  38. Autelitano DJ, Cohen DR 1996 CRF stimulates expression of multiple fos and jun related genes in the AtT-20 corticotroph cell. Mol Cell Endocrinol 119:25–35[CrossRef][Medline]
  39. Passegue E, Richard J-L, Boulla G, Gourdji D 1995 Multiple intracellular signallings are involved in thyrotropin-releasing hormone (TRH)-induced c-fos and jun B mRNA levels in clonal prolactin cells. Mol Cell Endocrinol 107:29–40[CrossRef][Medline]
  40. Farrow KN, Manning N, Schaufele F, Gutierrez-Hartmann A 1996 The c-Jun {delta}-domain inhibits neuroendocrine promoter activity in a DNA sequence- and pituitary-specific manner. J Biol Chem 271:17139–17146[Abstract/Free Full Text]
  41. Korbonits M, Chahal HS, Kaltsas G, Jordan S, Urmanova Y, Khalimova Z, Harris PE, Farrell WE, Claret FX, Grossman AB 2002 Expression of phosphorylated p27(Kip1) protein and Jun activation domain-binding protein 1 in human pituitary tumors. J Clin Endocrinol Metab 87:2635–2643[Abstract/Free Full Text]
  42. Hurel SJ, Harris PE, McNicol AM, Foster S, Kelly WF, Baylis PH 1997 Metastatic prolactinoma: effect of octreotide, cabergoline, carboplatin and etoposide; immunocytochemical analysis of proto-oncogene expression. J Clin Endocrinol Metab 82:2962–2965[Abstract/Free Full Text]
  43. Angel P, Hattori K, Smeal T, Karin M 1988 The jun proto-oncogene is positively autoregulated by its product, Jun/AP-1. Cell 55:875–885[CrossRef][Medline]
  44. Baichwal VR, Park A, Tjian R 1991 v-Src and EJ Ras alleviate repression of c-Jun by a cell-specific inhibitor. Nature 352:165–168[CrossRef][Medline]
  45. Yamaguchi-Iwai Y, Satake M, Murakami Y, Sakai M, Muramatsu M, Ito Y 1990 Differentiation of F9 embryonal carcinoma cells induced by the c-jun and activated c-Ha-ras oncogenes. Proc Natl Acad Sci USA 87:8670–8674[Abstract/Free Full Text]
  46. Bois-Joyeux B, Denissenko M, Thomassin H, Guesdon S, Ikonomova R, Bernuau D, Feldmann G, Danan JL 1995 The c-jun proto-onocogene down-regulates the rat {alpha}-fetoprotein promoter in HepG2 hepatoma cells without binding to DNA. J Biol Chem 270:10204–10211[Abstract/Free Full Text]
  47. Li L, Chambard J, Karin M, Olson EN 1992 Fos and Jun repress transcriptional activation by myogenin and MyoD: the amino terminus of Jun can mediate repression. Genes Dev 6:676–689[Abstract/Free Full Text]
  48. McBride K, Robitaille L, Tremblay S, Argentin S, Nemer M 1993 fos/jun Repression by cardiac-specific transcription in quiescent and growth-stimulated myocytes is targeted at a tissue-specific cis element. Mol Cell Biol 13:600–612[Abstract/Free Full Text]
  49. Schaeffer T, Sanders L, Nathans D 1995 Cooperative transcriptional activity of Jun and Stat3ß, a short form of Stat3. Proc Natl Acad Sci USA 92:9097–9101[Abstract/Free Full Text]
  50. Yang-Yen H, Chambard J, Sun Y, Smeal T, Schmidt TJ, Drouin J, Karin M 1990 Transcriptional interference between c-Jun and the glucocorticoid receptor: mutual inhibition of DNA binding due to direct protein-protein interaction. Cell 62:1205–1215[CrossRef][Medline]
  51. Baichwal VR, Park A, Tijan R 1992 The cell-type-specific activator region of c-Jun juxtaposes constitutive and negatively regulated domains. Genes Dev 6:1493–1502[Abstract/Free Full Text]
  52. Weiss C, Schneider S, Wagner EF, Zhang X, Seto E, Bohmann D 2003 JNK phosphorylation relieves HDAC3-dependent suppression of the transcriptional activity of c-Jun. EMBO J 22:3686–3695[CrossRef][Medline]
  53. Morton S, Davis RJ, McLaren A, Cohen P 2003 A reinvestigation of the multisite phosphorylation of the transcription factor c-Jun. EMBO J 22:3876–3886[CrossRef][Medline]
  54. Bost F, Caron L, Vial E, Montreau N, Marchetti I, Dejong V, Defize L, Castellazzi M, Binetruy B 2001 The defective transforming phenotype of c-Jun Ala(63/73) is rescued by mutation of the C-terminal phosphorylation site. Oncogene 20:7425–7429[CrossRef][Medline]
  55. Rajnarayan S, Chiono M, Alexander LM, Gutierrez-Hartmann A 1995 Reconstitution of protein kinase A regulation of the rat prolactin promoter in HeLa nonpituitary cells: identification of both GHF-1/Pit-1 dependent and independent mechanisms. Mol Endocrinol 9:502–512[Abstract]
  56. Jackson SM, Keech CA, Williamson DJ, Gutierrez-Hartmann A 1992 Interaction of basal positive and negative transcription elements controls repression of the proximal rat prolactin promoter in nonpituitary cells. Mol Cell Biol 12:2708–2719[Abstract/Free Full Text]
  57. Theill LE, Castrillo JL, Wu D, Karin M 1989 Dissection of functional domains of the pituitary-specific transcription factor GHF-1. Nature 342:945–948[CrossRef][Medline]
  58. Adler V, Polotskaya A, Wagner F, Kraft AS 1992 Affinity-purified c-Jun amino-terminal protein kinase requires serine/threonine phosphorylation for activity. J Biol Chem 267:17001–17005[Abstract/Free Full Text]
  59. Keech CA, Gutierrez-Hartmann A 1989 Analysis of rat prolactin promoter sequences that mediate pituitary-specific and 3',5'-cyclic adenosine monophosphate-regulated gene expression in vivo. Mol Endocrinol 3:832–839[Abstract]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
18/10/2479    most recent
Author Manuscript (PDF)