c-Jun Enhancement of Cyclic Adenosine 3',5'-Monophosphate Response Element-Dependent Transcription Induced by Transforming Growth Factor-ß Is Independent of c-Jun Binding to DNA

Patrick Pei-chih Hu1, Beth L. Harvat1, Sara S. Hook, Xing Shen, Xiao-Fan Wang and Anthony R. Means

Department of Pharmacology and Cancer Biology Duke University Medical Center Durham, North Carolina 27710


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Transforming growth factor-ß (TGFß) enhances transcription from reporter genes regulated by a single consensus cAMP-response element (CRE) upon transfection into the immortalized human keratinocyte cell line, HaCaT. Whereas both CRE-binding protein (CREB) and c-Jun present in extracts of unstimulated cells can complex with a CRE in gel-shift experiments, TGFß treatment increases the amount of c-Jun found in the complex. Overexpression of c-Jun is sufficient to increase CRE and GAL4-CREB-dependent transcription and mimics the stimulatory effects of TGFß on transcription from either reporter gene. Surprisingly, although a portion of CREB in unstimulated cells is phosphorylated on the activating serine residue, Ser-133, this level of phospho-CREB is not altered by TGFß treatment. In fact, the CREB-dependent transcriptional effects of TGFß or c-Jun do not require phosphorylation of Ser-133, although CREB-binding protein (CBP) is required as evidenced by the observation that the adenoviral oncoprotein E1A can block the effects of both agents. c-Jun enhancement of CRE or GAL4-CREB-dependent transcription neither requires the DNA-binding nor N-terminal domains of c-Jun. Collectively, these results are consistent with a model in which signaling pathways initiated by TGFß can stimulate CREB-dependent transcription by increasing the cellular concentration of c-Jun, which participates in activation of the CBP-containing transcription complex.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Transforming growth factor-ß (TGFß) is a multifunctional cytokine whose action is critical for complex biological processes such as embryonic development, wound healing, and immune system function. Loss of responsiveness to TGFß signaling is associated with diseases such as fibrosis, arthritis, and cancer. Many of the effects of TGFß involve transcriptional induction of genes whose products induce growth arrest in late G1 of the cell cycle (reviewed in Refs. 1, 2) or promote an increase in extracellular matrix deposition. Growth arrest is mediated by an inhibition of cyclin-dependent kinase (CDK) activity due to either increased synthesis of CDK inhibitors (3, 4, 5, 6, 7, 8) or a decrease in expression of the cdk activating phosphatase cdc25A (9). TGFß induces expression of two CDK inhibitors, p15Ink4b and p21WAF1/Cip1. Effects of TGFß on extracellular matrix (ECM) deposition are due to induction of both ECM structural genes such as type I collagen and inhibitors of ECM degradation such as plasminogen activator inhibitor (PAI-I). The coactivators CREB-binding protein (CBP) and p300 are required not only for TGFß-mediated transcription of p15 and p21 (10) but also for induction of PAI-1 (11, 12, 13).

In searching for additional genes that may mediate TGFß effects, Wong et al. (14) found that TGFß increases c-Jun expression. The induction of c-Jun, in turn, may serve to increase transcription of PAI-1 and type I collagen, since these genes are activated by AP-1 elements within their promoters (15, 16) and c-Jun is a common component of AP-1 protein complexes. Although it is unclear exactly how c-Jun activates transcription, it is known to bind to the coactivators p300 and CBP. The N-terminal transactivation domain of c-Jun interacts with the KIX (CREB-binding) domain of p300/CBP (17), while the C-terminal leucine zipper domain can interact with the C/H2 domain of CBP (18).

Although the cAMP response element (CRE, consensus sequence TGACGTCA) has been defined by its ability to bind to and be stimulated by the transcription factor CRE-binding protein (CREB) (19), c-Jun itself binds to the CRE-like element in its promoter that positively autoregulates transcription of the c-Jun gene (20). TGFß induction of a reporter gene regulated by the c-Jun promoter in HaCaT cells requires the CRE-like site, which only differs from the consensus CRE by a single base pair (TGACATCA). Studies in Drosophila have shown that the ability of the TGFß ortholog decapentaplegic to stimulate transcription of Ultrabithorax (Ubx) is absolutely dependent on the presence of a consensus CRE present in the Ubx promoter (21). Together these observations raised the question of whether TGFß might stimulate transcription of genes containing consensus CREs in mammalian cells and, if so, whether this response involved c-Jun.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
TGFß Stimulates Formation of a c-Jun/CRE Complex and c-Jun Potentiates CRE-Mediated Transcription in HaCaT Cells
To evaluate the ability of TGFß to affect CRE-mediated transcription through increasing c-Jun levels, we first demonstrated by electrophoretic mobility shift assay (EMSA) (Fig. 1AGo) that both CREB and c-Jun, but not ATF-2 (data not shown), can complex with a consensus CRE (TGACGTCA) in lysates from untreated HaCaT cells. While no effect of TGFß treatment on the complex was observed in the first hour, lysates from cells with longer TGFß treatment (3 h) demonstrated an increased amount of complex binding to the probe, most of which could be supershifted by the c-Jun antibody (Fig. 1BGo). The increase in complex formation occurred coordinately with increased c-Jun levels in the cells.



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Figure 1. TGFß Increases c-Jun Protein Synthesis and Incorporation into the CRE Complex

HaCaT nuclear lysates from untreated cells or cells treated with 100 pM TGF-ß for 1 h (A) or 3 h (B) were incubated with a radioactively labeled CRE probe and EMSA was performed. CREB complexes were supershifted with {alpha}-CREB (Santa Cruz Biotechnology, Inc.), and c-Jun complexes were shifted with {alpha}-c-Jun (Santa Cruz Biotechnology, Inc.). Western analysis for c-Jun from the nuclear lysates are below the EMSA (B) results.

 
TGFß treatment of HaCaT cells for 24 h caused a 5- to 9-fold increase in transcription from a luciferase reporter gene driven by one copy of a consensus CRE (1xCRE, Fig. 2AGo, left panel). We questioned whether the increase in c-Jun in response to TGFß could account for the TGFß-inducible transcription from the CRE. Increasing amounts of cotransfected c-Jun raised levels of transcription and essentially mimicked the TGFß response (Fig. 2AGo, right panel). Expression of c-Jun also increased transcription of a truncated c-Fos promoter-CAT reporter gene in which the only known response element is the CRE (Fos{Delta}80) (Fig. 2CGo). Thus, overexpression of c-Jun not only activates a consensus CRE connected to a reporter gene in an artificial context, but also activates a CRE in the context presented by the c-Fos promoter.



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Figure 2. TGFß, c-Jun, and c-Jun Mutants Stimulate CRE-Mediated Transcription

A, Luciferase activity in HaCaT cell extracts was measured after transient cotransfections of a reporter plasmid containing only a minimal TATA+Initiator without the consensus 1xCRE vs. the reporter plasmid with the consensus CRE inserted (left panel) or the 1xCRE reporter plasmid with increasing amounts of c-Jun cDNA (right panel). HaCaT cells were transfected using diethylaminoethyl-dextran as described in Materials and Methods, and then treated with TGFß for 20–22 h before harvesting. Transfection efficiency was normalized with ß-galactosidase activity. B, Luciferase activity from transient cotransfections using the 1xCRE reporter plasmid and 1 µg of either wild type c-Jun or c-Jun with mutations in the DNA-binding domain ({Delta}270) or N-terminal activation domain (TAM-67) in the absence (white bars) or presence (black bars) of TGFß. Vector refers to the parent CMV-driven expression vector for the c-Jun constructs. C, CAT assay measuring amount of c-Fos promoter, Fos-{Delta}80CAT, activated by overexpressed wild-type c-Jun, {Delta}270, or TAM-67. Identical transfections were performed as those described in panel B for luciferase assays. D, The c-Jun cDNAs that were used are depicted with their various functional domains.

 
TGFß-Induced CREB-Dependent Transcription Does not Require Phosphorylation of CREB at Ser-133
Because CREB is also in complex with the CRE in HaCaT cells, we questioned whether in addition to increasing c-Jun in the complex, TGFß treatment could also alter CREB transcriptional activity. Phosphorylation of CREB on Ser-133 is known to enhance its transactivating function by mediating recruitment of the coactivator CBP (22). However, in HaCaT cells, Ser-133 phospho-CREB is readily detected in the absence of stimulus. In multiple experiments, ionomycin treatment of HaCaT cells for 1 h induced an increase (5- to 8-fold) in the phosphorylated form of CREB (Fig. 3AGo), consistent with the reported calcium-mediated phosphorylation of CREB (23, 24, 25). In contrast, TGFß treatment altered CREB phosphorylation minimally, if at all (no change to 2-fold, Fig. 3AGo). Similar results were observed during a time course of TGFß treatment from 0 to 24 h (data not shown). To confirm that a portion of CREB was phosphorylated on Ser-133, HaCaT cells were metabolically labeled by incubation with [32P]orthophosphate for 4 h in the absence or presence of TGFß. CREB was recovered by gel purification and subjected to two-dimensional peptide mapping. Autoradiograms of the peptide map in the absence (left) or presence (right) of TGFß are shown in Fig. 3BGo. In each case, the predominant 32P-labeled peptide corresponded to the one known to contain Ser-133 (25). The degree of labeling of the Ser-133-containing peptide did not change in response to TGFß treatment for 1 h (Fig. 3BGo) or between 10 min and 4 h (data not shown). The fact that it is possible to metabolically radiolabel Ser-133 within a 4-h time period shows that the phosphorylation-dephosphorylation of Ser-133 in HaCaT cells is in dynamic equilibrium. TGFß treatment does not change this equilibrium. Thus, the effect of TGFß on transcription of the 1xCRE-reporter gene does not involve a change in the steady state level of phospho-CREB.



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Figure 3. TGFß Does not Stimulate CREB Phosphorylation

A, Analysis of phosphorylated and total CREB levels by immunoblot after 1 h of treatment with 100 pM TGF-ß (ß) or 1.5 µM ionomycin (I). B, Two-dimensional tryptic peptide mapping of 32P-metabolically labeled phospho-CREB isolated from HaCaT cells in the absence or presence of TGFß treatment for 1 h. Similar results were obtained from cells treated for 10 min or 4 h.

 
To examine whether TGFß can stimulate CREB-dependent transcription, a GAL4-CREB construct lacking the bZip DNA-binding/dimerization domain of CREB was cotransfected into HaCaT cells with a 5x(GAL4)-luciferase reporter plasmid. As shown in Fig. 4AGo, this reporter gene is responsive to TGFß. To investigate whether Ser-133 phosphorylation was required for TGFß to stimulate transcription, GAL4-CREB, in which Ser-133 was mutated to Ala, was transfected into HaCaT cells. The S133A mutant was still responsive to TGFß (Fig. 4AGo), albeit somewhat less so than wild-type CREB. These results suggest that the phosphorylation of Ser-133 is not essential for the TGFß effect on GAL4-CREB transcription. On the other hand, overexpression of c-Jun potentiated transcription by GAL4-CREB and markedly decreased the fold induction in response to TGFß (Fig. 4BGo). Overexpression of c-Jun by itself had no effect on the 5xGAL4 reporter, which contains no apparent c-Jun binding sites, suggesting that the c-Jun effect on GAL4-CREB-mediated transcription may be independent of c-Jun binding directly to DNA. To test this idea, we cotransfected a c-Jun DNA binding mutant ({Delta}70) that previously had been shown to have a dominant negative effect on c-Jun/AP-1-mediated transcription (26) and determined that it could activate GAL4-CREB transcription in a manner equivalent to wild-type c-Jun (Fig. 4BGo). The latter observation prompted us to test whether DNA binding was necessary for c-Jun stimulation of transcription using the 1xCRE reporter. As shown in Fig. 2BGo, the {Delta}270 DNA-binding mutant activated the CRE-containing transgene nearly as efficiently as wild- type c-Jun. Similar results were obtained using the truncated c-Fos promoter construct (Fig. 2CGo). Collectively, these experiments suggest that the role of c-Jun in the CRE complex could be to mediate protein-protein interactions rather than to participate directly in DNA binding.



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Figure 4. Phosphorylation of Ser-133 on CREB Is not Essential for TGFß or c-Jun Stimulation of Transcription Driven by a GAL4-CREB Fusion Protein Whereas p300/CBP May Be Required

A, Luciferase activity from HaCaT cells cotransfected with a 5xGAL4 reporter gene and 2 µg of either GAL4-wild-type CREB or GAL4-CREB-S133A. Both CREB cDNAs lack the bZip DNA binding/dimerization domain. Transfections were performed as in Fig. 2Go in the presence or absence of TGFß. B, HaCaT cells were cotransfected with 1 µg of wild type c-Jun cDNA, mutant c-Jun that cannot bind DNA ({Delta}270), or the N-terminal deletion mutant TAM-67, GAL4-CREB, and the 5xGAL4 reporter. C, Transient cotransfections were performed using wild- type E1A or mutant E1A ({Delta}2–36), which cannot bind CBP, with the 5xGAL4 reporter gene and GAL4-CREB. D, HaCaT cells were transfected with wild-type E1A or mutant E1A ({Delta}2–36), the 5xGAL4 reporter gene, GAL4-CREB-S133A, and c-Jun. E, HaCaT cells were transfected with wild-type c-Jun cDNA, TAM-67, or TAM-67{Delta}zip and either the 1xCRE reporter or the 5xGAL4 reporter gene and GAL4-CREB.

 
TGFß and c-Jun Transcriptional Responses Blocked by E1A
A protein complex that could be influenced by c-Jun might include CBP, as both CREB and c-Jun are known to bind to this transcriptional coactivator. To determine whether CBP was necessary for TGFß regulation of GAL4-CREB, we performed similar experiments as shown in Fig. 4Go, A and B, but in the presence of E1A, an adenoviral oncoprotein that binds to and inactivates p300/CBP (27, 28, 29, 30, 31, 32). Both basal and TGFß-induced transcription from either GAL4-CREB or GAL4-CREB-S133A could be abrogated by coexpression of wild-type E1A but not by a mutant of E1A that is unable to bind CBP, {Delta}2–36 E1A (Fig. 4CGo). Similarly, the ability of c-Jun to potentiate CREB-mediated transcription could be blocked by cotransfection of E1A but not {Delta}2–36 E1A (Fig. 4DGo). Thus, the effects of TGFß and c-Jun on transcription of GAL4-CREB require CBP.

c-Jun binds to two regions of CBP. The N-terminal end of c-Jun associates with the KIX domain of CBP (17). On the other hand, the C-terminal leucine zipper domain of c-Jun associates with the C/H2 domain of CBP (18). To determine the relative importance of the two CBP binding regions of c-Jun on CRE and GAL4-CREB-mediated transcription, we evaluated the effects of a mutant form of c-Jun from which the N terminus (residues 3–122) had been deleted. As shown in Fig. 2BGo, TAM-67 markedly stimulates transcription of the 1xCRE reporter gene and mimics the effect of TGFß, even though its expression level detected by immunoprecipitation-Western analysis appears to be slightly lower than the other Jun constructs (data not shown). In addition, TAM-67 is a potent agonist of the reporter gene regulated by the truncated c-Fos promoter, Fos {Delta}80CAT (Fig. 2CGo). Finally, the GAL4-CREB reporter gene is also markedly stimulated by TAM-67 in either the absence or presence of TGFß as demonstrated in Fig. 4BGo. It should be pointed out that the data in Fig. 4BGo were obtained using 1 µg of TAM-67 cDNA. Increasing the amount of this mutant causes an additional increase in transcription and completely mimics the effects of TGFß. Conversely, a mutant of TAM-67 from which the leucine zipper has been deleted was unable to stimulate transcription from either the CRE or GAL4-CREB reporter gene (Fig. 4EGo). Full-length c-Jun lacking only the leucine zipper was also inactive with these reporter genes (data not shown). These data suggest that the interaction of the leucine zipper region of c-Jun with the C/H2 region of CBP could be involved in the stimulation of CREB/CBP-dependent transcription.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Our data show that c-Jun overexpression in HaCaT cells can stimulate CRE and CREB-mediated transcription in a manner that requires CBP, presumably through recruitment of c-Jun to CBP. Bannister et al. (17) showed that transactivation by a Jun/CBP complex requires phosphorylation of Ser63 and Ser73 in c-Jun, and Hocevar et al. (33) recently demonstrated in a fibrosarcoma HT1080-derived cell line that TGFß signals can activate Jnk, the kinase responsible for the phosphorylation of Jun. It was surprising, therefore, that when the N terminus of c-Jun was deleted (residues 3–122; TAM-67), the mutant c-Jun activated transcription from the CRE or through GAL4-CREB even better than wild-type c-Jun. Since stimulation of the CRE and GAL4-CREB is inhibited by E1A and therefore appears to utilize endogenous CBP, we hypothesized that the activation by TAM-67 was due to the recently reported interaction of the leucine zipper domain of c-Jun with the C/H2 domain of CBP (18). In support of this, we found that a C-terminal truncation of full-length c-Jun or TAM-67 to remove the leucine zipper resulted in proteins that could not stimulate transcription. Thus, the leucine zipper is required for the CRE/CREB response, even in the context of GAL4-CREB{Delta}bzip where Jun cannot form a heterodimer on the DNA. We cannot rule out, however, that the leucine zipper may mediate essential interactions with other, as yet unidentified, proteins involved in CRE transactivation. The dispensability of Ser63/73 phosphorylation in certain situations is supported by recent findings that a LexA-c-Jun fusion protein in which both residues are mutated to Leu is transcriptionally active in a CBP-dependent manner during calcium influx (34).

Additionally, while the amount of c-Jun increases in complex with the consensus CRE upon TGFß treatment as measured by EMSA, Jun does not appear to bind to DNA directly since the DNA binding mutant activates CRE-dependent transcription as well as wild-type Jun does. This finding is in direct contrast to numerous reports in which DNA binding mutants or the TAM-67 mutant of c-Jun act as dominant negatives for Jun-mediated transcription on AP-1 sites (35, 36, 37, 38). Both DNA binding and the Jnk phosphorylation sites are necessary for transcriptional activity by c-Jun as a heterodimer either with c-Fos on AP-1 sites or with ATF-2 on CRE-like sites (17, 39). Indeed, we originally used the DNA-binding and transactivation domain mutant c-Jun constructs in an attempt to block the Jun effects on the consensus CRE, to demonstrate that c-Jun directly participates in TGFß stimulation. Instead, we uncovered a novel mechanism by which Jun activation of CRE-mediated transcription requires the leucine zipper, but neither DNA binding nor the N-terminal phosphorylation sites for JNK. Unfortunately, these results precluded using the mutant constructs as proof of principle that TGFß signals to the CRE through up-regulation of Jun. However, it is striking that cotransfection of either full-length CREB or the TGFß-activated transcription factor Smad 3, which can bind to and thus titrate out endogenous Jun from a GAL4-CREB DNA-bound complex, acts to squelch TGFß enhancement of GAL4-CREB-mediated transcription (B. L. Harvat, unpublished results). Cotransfection of the various Jun constructs and the 1xCRE reporter in HeLa cells gave identical results as they did in HaCaT cells (B. L. Harvat, unpublished observations), suggesting that the CBP-dependent mechanism that depends on the leucine zipper of Jun, but not DNA-binding or Jnk phosphorylation, may be used by other epithelial-type cells. Additional signals may also impact the CBP/Jun complex proposed here, as we have observed a similar ability of c-Jun to substitute for ionomycin stimulation of CRE and GAL4-CREB-mediated transcriptional activation in HaCaT cells (B .L. Harvat and A. R. Means, unpublished observations).

As a transcriptional cointegrator, p300/CBP binds to multiple transcription factors that, in turn, bind to their respective DNA promoter elements (reviewed in Ref. 40). Genes containing DNA binding sites for three of these factors, CREB, nuclear factor-{kappa}B, and AP-1, are TGFß responsive in HaCaT cells (14, 41, 42). In addition, TGFß induces transcription of p15 and p21 CDK inhibitors through Sp1-binding elements and, although direct association between p300/CBP and Sp1 has not been demonstrated, the up-regulation of these genes is blocked by E1A, implicating involvement of p300/CBP (10). The mechanisms by which TGFß stimulates transcription through these p300/CBP-associated factors are still unclear. However, it seems possible that the proposed increase in c-Jun-bound p300/CBP could have similar effects on other TGFß-responsive genes whose transcription is p300/CBP dependent.

TGFß is also known to signal through the phosphorylation and nuclear translocation of Smad-2 and Smad-3 proteins (reviewed in Refs. 2, 43), which can then bind to response elements in DNA, recruit p300 and CBP (11, 12, 13, 44), and stimulate transcription. Overexpression of Smad-3 and Smad-4 greatly increases TGFß-independent transcription of plasminogen activator inhibitor (PAI-1), a known TGFß target (45), presumably because overexpression can force localization in the nucleus and binding to DNA in the absence of Smad 3 phosphorylation by TGFß receptor. We investigated the ability of overexpressed Smad-3 and Smad-4 to similarly stimulate GAL4-CREB and CRE-dependent transcription or enhance the c-Jun effect, even though our CRE reporters lack Smad-binding elements in the promoters. In contrast to c-Jun, overexpression of Smad-3 and Smad-4 did not substitute for the ability of TGFß to stimulate CRE/CREB-mediated transcription, but rather squelched transcription (P. Hu., B. L. Harvat., X.-F. Wang, and A.R. Means, unpublished observations). The most direct interpretation of these results would be that Smads are unable to participate in the formation or stabilization of the transcription complex on consensus CRE or GAL4-CREB elements in the absence of DNA binding sites for Smads and thus cannot stimulate Jun activity or affinity for the complex. However, because of their ability to bind to p300/CBP (11, 12, 13, 44) or Jun (46, 47), when overexpressed, Smads can squelch transcription by binding and sequestering these proteins that appear essential for CRE/CREB activity. In contrast, in the context of promoters that contain both Smad-binding elements and a CRE, such as the Jun promoter (14) or the Drosophila ultrabithorax promoter (48), Smads may stabilize the CRE-dependent complex and thus contribute to Jun activation of these promoters.

c-Jun stimulation of GAL4-CREB-mediated transcription suggests that CREB has a role in directing the TGFß response. We know that TGFß treatment does not significantly alter the amount of CREB phosphorylated on Ser-133 in HaCaT cells. Indeed, in untreated HaCaT cells, a substantial fraction of CREB is phosphorylated on Ser-133, suggesting that a constitutive phospho-CREB/CBP association might exist. Furthermore, since Ser-133 of endogenous CREB can be metabolically phosphorylated within a 4-h labeling period, there must be a continual turnover of Ser-133 phosphorylation. This suggests there may be a dynamic equilibrium of CBP/CREB association at CRE sites in HaCaT cells. If so, such transient complexes could be stabilized by the elevated levels of c-Jun that occur as a rapid response to TGFß. In our experiments, CREB cannot recruit c-Jun through heterodimerization, since the leucine zipper domain is missing in the GAL4-CREB constructs. However, CREB association with CBP through the KIX domain could recruit CBP-bound c-Jun to the transcription complex via the association of c-Jun with another region of CBP. The C/H2 region is the only other region of CBP that has been shown to bind Jun to date, and we also have seen that GST-CBP-C/H2 region fusion proteins can pull down Jun from HaCat lysates (B. L. Harvat, unpublished results). Since TGFß can also stimulate transcription mediated by CREB-S133A, and this transcription can be prevented by E1A, it is possible that either lower affinity contacts of CREB with CBP or interaction of CREB with components of transcription factor IID (TFIID) (49, 50, 51) acting as an indirect bridge between CREB and CBP may still allow the mutant CREB to recruit c-Jun through CBP. An equally plausible possibility is that the increase in c-Jun bound to CBP upon TGFß treatment could lead to stabilization of a CREB/CBP complex, even without an increase in Ser-133 phosphorylation, and subsequently increase CRE-dependent transcription. In addition, we cannot rule out the possibility that other cell type-specific accessory factors or TGF-ß-mediated phosphorylation of Jun may be involved in these transcriptional effects. At any rate, the question of a universal role for the CREB-binding/KIX domain in CBP in integrating signals for proliferation, as opposed to growth arrest or differentiation, is an interesting one. c-Jun has an important role in regulating keratinocyte proliferation and differentiation (52), and keratinocytes may be particularly sensitive to c-Jun levels. Finally, the discovery that c-Jun can stimulate CBP-mediated transcription in a manner that is independent of its ability to bind DNA represents a novel mechanism by which signal transduction pathways can be integrated through CBP to influence transcription.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Plasmids/Constructs
For 1xCRE-luc, a consensus CRE (5'-GCT CTC TGA CGT CAG GCA AT-3') was subcloned into pgl2-basic (Promega Corp., Madison, WI). Insertion of only one copy of the CRE was verified by sequencing. Tam67{Delta}zip was created by inserting two stop codons in the Tam67 construct in place of the codons for amino acids K283 and K285, which are at the 5'-end of the leucine zipper. The mutagenesis was done using the QuikChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA) and the following primers: sense, –5'-GGAGGAATAAGTGTAAACCTTG-3', and antisense, -5'-CAAGGTTTACACTTATTCCTCC-3'. The underlined nucleotides are the ones that were changed from the wild-type sequence.

Cell Culture
HaCaT cells were grown in {alpha}-MEM (Life Technologies, Inc., Gaithersburg, MD), containing 10% FBS, 2 mM L-glutamine (Life Technologies, Inc.) and 50 U/ml Penicillin-G and 50 µg/ml Streptomycin Sulfate (Life Technologies, Inc.).

EMSA
EMSAs were performed using 1–3 µl of nuclear extracts prepared from cells untreated or treated with 100 pM TGFß1 for 1 h or 3 h. The 1xCRE (5'-GCT CTC TGA CGT CAG GCA AT-3') construct was radiolabeled with 32P-{gamma}ATP with T4 Polynucleotide kinase and purified using a spin column (QIAGEN, Chatsworth, CA). Gel shift conditions are exactly as described in Yingling et al. (41). For supershift analysis of CREB or c-Jun, we used 2 µl of anti-CREB (24H24-X) and c-Jun (KM-1-X), respectively (Santa Cruz Biotechnology, Inc., Santa Cruz, CA).

Cotransfections and Luciferase Assays
HaCaT cells were plated in six-well plates either at 175,000 cells per well and grown overnight or at 50,000 cells per well and grown for 2 days. The media were then replaced with {alpha}-MEM containing 100 µM chloroquine. The indicated amounts of DNA were diluted to a total volume of 225 µl in Hanks solution, mixed with 75 µl of diethylaminoethyl-dextran, and added dropwise to each well. Cells were incubated with the DNA mix for 3 h, then rinsed once in PBS, glycerol shocked for 2 min, rinsed again, and incubated in growth media overnight. The following day, cells were treated with either 100 pM TGFß or left untreated and harvested 20–24 h later. Soluble extracts were prepared, centrifuged at 14,000 rpm for 5 min at 4 C, and analyzed for luciferase activity using either a Berthold or Labsystems Luminoskan luminometer. ß-Galactosidase activity was assayed using the substrate, chlorophenolred-ß-D-galactospyranoside (Roche Molecular Biochemicals, Nutley, NJ), and determining the optical density at 575 nm. The results were used to normalize the luciferase activity for transfection efficiency. Each transfection experiment was repeated two to five times. Individual transfections within a single experiment were done independently in duplicate or triplicate as were the luciferase and ß-galactosidase assays for each sample lysate.

pCREB Western
HaCaT cells were plated and grown overnight in culture media. The next day, cells were treated with TGFß or ionomycin for 1 h or left untreated. Cells were harvested as previously described (53) in boiling 2x sample buffer. The samples were then subjected to immunoblot analysis using phospho-specific (Dr. D. Ginty) and total CREB rabbit polyclonal antibodies (Upstate Biotechnology, Inc., Lake Placid, NY).

In Vivo Labeling and 2-D Electrophoresis
Exponentially growing HaCaT cells (2 x 106) were washed in phosphate-free DMEM (Life Technologies, Inc.) and preincubated 1–2 h. The media were then aspirated, and approximately 2 mCi of [32P]orthophosphate (ICN Biochemicals, Inc., Cleveland, OH) in 2 ml of fresh media were added. Cells were labeled for 4 h, during which TGFß was added for either 10 min, 1 h, or 4 h before harvesting or cells were left untreated. Cells were then washed and lysed in Universal Lysis Buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.5% Nonidet P-40, 50 mM NaF, 1 mM sodium orthovanadate, 0.2 mM sodium molybdate, 20 mM ß-glycerophosphate, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, and protease inhibitors.

32P-labeled proteins were separated by SDS-PAGE and transferred to Immobilon (Millipore Corp., Bedford, MA). The membrane was rinsed in H2O and exposed at -80 C for 30 min to visualize labeled proteins. After excision, the bands were immediately incubated in 0.5% PVP-360 in 100 mM acetic acid for 30 min at 37 C. The bands were then washed five times with H2O and twice with 50 mM NH4HCO3. Two hundred microliters of 50 mM NH4HCO3 were added to cover the membrane, and digestion was started with 10 µl of 1 mg/ml TPCK-treated trypsin (Worthington Biochemical Corp., Freehold, NJ). Two hours later an additional 10 µl of trypsin were added and incubated overnight. After removing the membrane and lyophilizing the sample, 50 µl of performic acid (900 µl formic acid, 100 µl H2O2) were added for 1 h on ice; 1 ml of H2O was added and the sample was lyophilized. Cherenkov counts were determined and the sample was resuspended in a small volume of H2O. Similar to the procedure used by Sun et al. (25), 1–2 µl (~2000–5000 cpm) were loaded on a TLC plate and electrophoresed for 20 min at 1,000 V by use of the Hunter Thin Layer Electrophoresis System (HTLE-7000, CBS Scientific Co., Delmar, CA), followed by TLC with n-butanol-pyridine-acetic acid-water in volume ratios of 0.375, 0.25, 0.075, and 0.30, respectively. Autoradiography was used to visualize the phosphopeptides.


    ACKNOWLEDGMENTS
 
We thank R & D Systems (Minneapolis, MN) for supplying TGFß1. We are grateful to Dr. R. Maurer for the GAL4-CREB and GAL4-CREB 133A DNA constructs; Dr. D. Ginty for his phospho-serine 133-CREB antibody; Dr. S. Shenolikar and H. Quan for purified CREB; Dr. T. Curran and Dr. M. Birrer for the c-Jun expression plasmids; Dr. M. Gilman for the c-Fos{Delta}80 construct; and Dr. J. Nevins for E1A plasmids. We are grateful to Carolyn Wong and Elissa M. Rougier-Chapman for sharing unpublished information as well as technical advice. We would like to thank members of the Wang and Means laboratories for helpful discussions, Charles Mena and Yong Yu for excellent technical assistance, and Tso-Pang Yao for critical reading of this manuscript.


    FOOTNOTES
 
Address requests for reprints to: Dr. Anthony R. Means, Department of Pharmacology, Duke University Medical Center, C238 Levine Science Research Center, Durham, North Carolina 27710.

S.S.H. is supported by a predoctoral fellowship from the Department of the Army DAMD17–97-1–7101. X.-F. W. is a Leukemia Society Scholar. This work was supported by NIH grants to A. R. M. (HD-07503; GM-33976) and X.-F. W. (DK-45746).

1 These authors contributed equally to this work. Back

Received for publication June 2, 1999. Revision received September 16, 1999. Accepted for publication September 21, 1999.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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