MMSP tumor cells expressing the EWS/ATF1 oncogene do ... - Nature

Oncogene (1998) 16, 1325 ± 1331  1998 Stockton Press All rights reserved 0950 ± 9232/98 $12.00

MMSP tumor cells expressing the EWS/ATF1 oncogene do not support cAMP-inducible transcription Kim KC Li and Kevin AW Lee Department of Biology, Hong Kong University of Science & Technology, Clear Water Bay, Kowloon, Hong Kong, P.R.C

Malignant Melanoma of Soft Parts (MMSP) is associated with the EWS/ATF1 fusion protein that arises due to chromosomal fusion of the Ewings Sarcoma oncogene (EWS) and the cellular transcription factor ATF1. EWS/ATF1 can activate several cAMP-inducible promoters, suggesting that cellular transformation in MMSP might involve constitutive activation of cAMPinducible promoters. To assess this possibility we have examined the status of the cAMP-signaling pathway in the available MMSP-derived cell lines (DTC1 and Succs-1) and ®nd that both cell lines share several features. First, in contrast to previous e€ects observed in transient assays, three chromosomal promoters containing ATF binding sites are not constitutively activated by endogenous EWS/ATF1 in MMSP cells. Second, all the components that are known to be required for cAMP-inducible transcription are present. Third, phosphorylation of the cAMP-response-element-binding protein (CREB) can be eciently induced by cAMP. Fourth, cAMP is unable to activate transcription, as assessed by a GAL4/ATF1 reporter assay and analysis of the c-fos and adenovirus early promoters. Thus, cell lines derived from MMSP have a block to cAMPsignaling that lies downstream of CREB phosphorylation. In light of the cAMP-responsiveness of almost all mammalian cell types, our ®ndings suggest that the inability to respond to cAMP might be an important feature of MMSP cells. Keywords: EWS/ATF1; cAMP; oncogene; phosphorylation

Introduction Chromosomal translocations involving fusion of the Nterminal region of the Ewings Sarcoma Oncogene (EWS) to the DNA-binding domains of a variety of transcription factors, produce dominant oncogenes that cause distinct sarcomas (Delattre et al., 1992; May et al., 1993a,b; Zucman et al., 1993a,b; Ladanyi and Gerald, 1994; Sorenson et al., 1994). The N-terminal region of EWS can function as a potent transcriptional activation domain (May et al., 1993b; Ohno et al., 1993; Bailly et al., 1994; Brown et al., 1995; Fujimura et al., 1996) suggesting that the distinct tumors referred to above arise by activation of di€erent genes, with the tumor being speci®ed by the EWS fusion partner.

Correspondence: KAW Lee Received 27 June 1997; revised 14 October 1997; accepted 14 October 1997

One of the above sarcomas (Malignant Melanoma of Soft Parts (MMSP)) is a rare aggressive tumor, that is typically associated with tendons and aponeuroses and is thought to be of neuroectodermal origin (Chung and Enzinger, 1983; Epstein et al., 1984). In MMSP, a balanced t(12 : 22) translocation results in production of a fusion protein (EWS/ATF1) in which the N-terminal 325 amino acids of EWS is fused to the C-terminal 206 amino acids of Activating Transcription Factor 1 (ATF1) (Zucman et al., 1993a). The reciprocal ATF1/ EWS fusion protein is not produced in tumor cells due to an in-frame stop codon immediately C-terminal to the ATF1 sequence (Zucman et al., 1993a). ATF1 is a member of a sub-group of bZIP transcription factors that includes the cAMP-response-element-binding protein (CREB) and activator forms of the cAMPresponse-element-modulator (CREM) (Lee and Masson, 1993). ATF1 homodimerizes and forms heterodimers with CREB (Hurst et al., 1990), binds directly to cAMP-inducible promoters and activates transcription upon phosphorylation by the cAMP-dependent protein kinase, PKA (Ribeiro et al., 1994). In contrast to ATF1, EWS/ATF1 functions as a potent constitutive activator of several cAMP-inducible promoters when assayed by transfection in cells that lack EWS/ATF1 (Brown et al., 1995; Fujimura et al., 1996). Moreover, promoters that can be activated by exogenous EWS/ATF1 are constitutively active when introduced transiently into tumor-derived cell lines (Su-ccs-1 and DTC1, hereafter collectively referred to as MMSP cells) that express endogenous EWS/ATF1 (Brown et al., 1995). Thus, EWS/ATF1 has the potential to up-regulate cAMPinducible promoters in MMSP cells and this, in turn, might contribute to cellular transformation. To date however, the activity of endogenous cAMP-inducible promoters and the status of the cAMP-signaling pathway in MMSP cells has not been studied. To gain insight into the cellular events that might be de-regulated in MMSP, we have employed several approaches to monitor the response of MMSP cells to cAMP. Using three independent assays (induction of a GAL4/ATF1 fusion protein, c-fos promoter activity and adenovirus early promoter activity) we ®nd that MMSP cells are unable to support cAMP-inducible transcription. We also ®nd that the known positive acting components of the cAMP-signaling pathway are present in MMSP cells and that in vivo phosphorylation of CREB by PKA occurs normally. The defect in the cAMP-pathway in MMSP cells therefore lies downstream of CREB phosphorylation. Since the cAMP-pathway is operative in most mammalian cell types but inoperative in MMSP cells, our results suggest that the inability to respond to cAMP at the transcriptional level, might be an important feature of cells that are transformed by EWS/ATF1.

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GAL4/ATF1 dependent activation of a CAT reporter (pGAL4CAT) containing GAL4 binding sites. In contrast cAMP is unable to activate GAL4/ATF1 in MMSP cells (Figure 1a). Intracellular cAMP is elevated under the experimental conditions employed as shown later (Figure 4). The above result indicates that the cAMP-signaling pathway, vis a vis activation of ATF1/CREB, is inoperative in MMSP cells. To examine whether the inability to respond to cAMP involves a lack of PKA, we tested the e€ect of overexpressing the catalytic sub-unit of PKA (cPKA) on GAL4/ATF1 activity (Figure 1b). Western blot analysis using an antibody against cPKA (Hemmings, 1986) con®rmed expression of cPKA in transfected cells (data not shown). Again, in contrast to activation of GAL4/ATF1 by cPKA in JEG3 cells, cPKA is unable to activate GAL4/ATF1 in MMSP cells. Taken together, the above results indicate that there is a defect in the cAMP-signaling pathway in MMSP cells and that this defect lies downstream of PKA.

Analysis of cAMP-inducible transcription in MMSP cells Previous studies employing cAMP-inducible promoters (including somatostatin and c-fos) demonstrated that such promoters are constitutively activated by endogenous factors (probably EWS/ATF1) when transiently introduced into MMSP cells (Brown et al., 1995). A priori therefore this seemed to rule out the use of transient assays based on natural promoters for monitoring the cAMP-pathway in MMSP cells. Consistent with this, expression of the catalytic subunit of PKA (cPKA) by transient transfection of MMSP cells (Figure 1b) does not further increase the activity of a reporter (D(-71)Som) containing the somatostatin promoter linked to CAT. As a control, cPKA strongly activates D(-71)Som (*100-fold) in the cAMP-responsive JEG3 cell line. The di€erence in signal observed for D(-71)Som in Su-ccs-1 cells compared with DTC1 cells is due to a low transfection eciency for Su-ccs-1 and does not re¯ect lower activity of the somatostatin promoter (Brown et al., 1995). Considering the above, we initially used a GAL4/ ATF1 fusion reporter assay (Hurst et al., 1991) to test the endogenous cAMP-signaling pathway in MMSP cells (Figure 1a). In JEG3 cells, addition of cAMP to the cell culture medium strongly stimulates (*40-fold)

Activity of the c-fos promoter in MMSP cells As an alternative means to monitor cAMP-signaling in MMSP cells we analysed the activity of the endogenous c-fos promoter (Figure 2). We chose the c-fos promoter because it is highly responsive to cAMP (Sassone-Corsi et al., 1988; Berkowitz et al., 1989; Fisch et al., 1989;

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Figure 1 Transcriptional activation by cAMP and PKA in control cells and MMSP cells. (a) E€ect of cAMP. Di€erent cell lines (indicated at the top) were cotransfected with a reporter plasmid pG5E4CAT (GAL4CAT) and an activator plasmid pGAL/ATF1 (GAL4ATF1). After 24 h intracellular cAMP was increased as described in Materials and methods and CAT assays performed 24 h later. (b) E€ect of over-expressing PKA. Di€erent cell lines (indicated at the top) were co-transfected with reporter plasmids (pG5E4CAT or pD(-71)SomCAT) and activator plasmids (pGAL/ATF1 or pCaEV (PKA)) as indicated at the bottom of the ®gure. CAT assays were performed at 40 h post-transfection

Transformation by EWS/ATF1 KKC Li and KAW Lee

other cell types (Treisman, 1985). Thus the c-fos promoter exhibits normal serum inducibility and there is no indication that endogenous EWS/ATF1 (or any other factors) leads to increased constitutive activity of the c-fos promoter in MMSP cells. The above result enables testing of the c-fos promoter for response to cAMP (Figure 2b). As is the case in other cell types (Berkowitz et al., 1989; Fisch et al., 1989) cAMP rapidly and transiently activates the c-fos promoter in JEG3 cells (Figure 2b). In MMSP cells however, cAMP does not increase c-fos RNA levels above the low basal level. Thus, the endogenous c-fos promoter does not respond to intracellular cAMP despite the presence of multiple cAMP-responsive promoter elements in its regulatory region.

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Figure 2 Analysis of c-fos expression (a) Serum response of the endogenous c-fos promoter. Di€erent cell lines (as indicated at the top) were serum starved for 24 h and then stimulated by addition of 10% serum. Total RNA was extracted at the indicated times in minutes following serum stimulation (bottom) and analysed by RNAse protection assay using 32P-labeled anti-sense probes for cfos and g-actin as described in the materials and methods. g-actin serves as invariant control. Bands indicated to the left of the autoradiogram are the protected fragments corresponding to c-fos and g-actin mRNAs. (b) cAMP response of the endogenous c-fos promoter. Intracellular cAMP was increased for the di€erent cell lines (top) as described in Materials and methods. Total RNA was extracted at the indicated times in minutes following cAMPinduction (bottom) and c-fos RNA analysed as described above

Metz and Zi€, 1991) and because c-fos is ubiquitously expressed and unlikely to be dominantly repressed in MMSP cells. The c-fos promoter is also of signi®cance to MMSP because it is a possible target for EWS/ ATF1 (Brown et al., 1995) and is activated by a variety of mitogenic agents (Janknecht and Nordheim, 1996; Bannister et al., 1995; Nakajima et al., 1996; Ginty et al., 1994; Sheng et al., 1990; Dash et al., 1991; Bhattacharya et al., 1996). We examined the e€ects of EWS/ATF1 and cAMP on expression of the endogenous c-fos gene in MMSP cells. Initially, we tested the response of the c-fos promoter to serum stimulation (Figure 2a). c-fos transcripts were detected by RNAse protection analysis using g-actin as an invariant control for RNA preparation and gel loading. Surprisingly, the c-fos promoter has a very low basal activity in serumstarved MMSP cells (Figure 2a) or normally growing cells (data not shown) and exhibits a strong and transient serum response, typical of that seen for most

To further examine cAMP-signaling in MMSP cells we tested the adenovirus E2 and E3 promoters for response to cAMP (Figure 3). Both of these promoters contain ATF/CREB binding sites (Lee et al., 1987) and have previously been shown to respond to cAMP in PC12 cells (Sassone-Corsi, 1988; Lee et al., 1989) and we therefore used PC12 cells as a positive control for our experiments. MMSP cells (Figure 3b) were infected with wild-type (WT) adenovirus (plus E1A) and adenovirus dl1500 and dl312 (which do not express the E1a protein required for activation of the E2 and E3 promoters). At 24 h post-infection cells were stimulated with cAMP and E2/E3 RNA was analysed using a primer extension assay as previously described (Lee and Green, 1987). In MMSP cells infected with WT virus, correctly initiated E2 and E3 transcripts are readily detected, while in dl1500 nd dl312 infected cells, transcript levels are very low. As expected, E2 and E3 transcripts are readily detected in dl1500- and dl312infected 293 cells (that express endogenous E1A) con®rming the infectivity of the mutant viruses used (Figure 3a). Thus (as is the case for most other mammalian cell types) in the absence of E1A the E2 and E3 promoters have low activity in MMSP cells. Treatment of dl1500- and dl312-infected MMSP cells with cAMP does not elevate E2 and E3 RNAs above the low basal levels. As a control for cAMP-induction, E2 RNA levels are strongly induced by cAMP in PC12 cells (Figure 3a) as previously shown (Lee et al., 1989). Thus in the context of virally infected MMSP cells, cAMP is unable to activate transcription of the adenovirus E2 and E3 promoters. CREB phosphorylation in MMSP cells The inability of MMSP cells to respond to cAMP is not due to the absence of ATF1/CREB (Brown et al., 1995). cAMP-induced phosphorylation of ser133 of CREB is required for transcriptional activation (Gonzalez and Montminy, 1989; Gonzalez et al., 1991. We therefore used a previously described antibody (Ab5322) that can recognise PKA-phosphorylated CREB (Hagiwara et al., 1993) to examine whether cAMP is able to induce CREB phosphorylation in MMSP cells (Figure 4). To control for total CREB levels the same samples were analysed using an antibody that recognizes both phosphorylated and

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Figure 3 cAMP-induction of adenovirus early promoters. (a) Control experiments in 293 and PC12 cells. (b) cAMP-induction in MMSP cells. Cells were infected with wild-type (WT), dl1500 or dl312 adenoviruses as described in Materials and methods. At 24 h post-infection, dl500- and dl312- infected cells were treated by addition of cAMP to the cell culture medium and total RNA was extracted at the indicated times (hours) following cAMP addition (as indicated at the bottom). Correctly initiated E2/E3 transcripts (indicated to the side) were detected by primer extension using 32P-labeled primers

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Figure 4 E€ect of cAMP on CREB/ATF1 phosphorylation in MMSP cells. Di€erent cell lines (top) were treated with cAMP for the indicated times in minutes (bottom), nuclear extracts were prepared and analysed by Western blotting using anti-CREB (above) or Ab5322 (below). Phospho-CREB (pCREB) and phospho-ATF1 (pATF1) are indicated to the right

unphosphorylated CREB (see Materials and methods). For JEG3 cells, basal levels of CREB phosphorylation are barely detectable and maximum phosphorylation is achieved 40 min after addition of cAMP to the cell culture medium. cAMP-induced CREB phosphorylation occurs at a similar rate and to a similar extent in both DTC1 and Su-ccs-1 cells, although the basal level of phosphorylation is higher for both Su-ccs-1 and DTC1 cells (Figure 4). cAMP-induced phosphorylation of a protein in the size range of ATF1 also occurs normally in DTC1 cells but is not detected in Su-ccs-1 cells. This may be due to low levels of ATF1 in these cells (Brown et al., 1995) although this remains to be determined. The above results show that PKA is present and active in MMSP cells and that the inability of these cells to respond to cAMP is not due to a defect in cAMP-mediated CREB phosphorylation.

Presence of CBP in MMSP cells Lack of a cAMP-response in MMSP cells could be due to the absence or limitation of positive factors, or the presence of dominant repressors. In addition to PKA and ATF1/CREB, the only other known component of the cAMP-pathway is the CREB co-activator CBP (Kwok et al., 1994) that directly interacts with PKAphosphorylated CREB (Chrivia et al., 1993; Parker et al., 1996). The role of CBP in MMSP is also of interest in light of the role of CBP as a target for several mitogenic signals (Sheng et al., 1990; Dash et al., 1991; Ginty et al., 1994; Bannister et al., 1995; Bhattacharya et al., 1996; Janknecht and Nordheim, 1996; Nakajima et al., 1996 and reviewed in Janknecht and Hunter, 1996). We probed for CBP in nuclear extracts from MMSP cells by Western blot analysis (Figure 5) using

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Transformation by EWS/ATF1 KKC Li and KAW Lee

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Figure 5 Expression of the CREB co-activator CBP in MMSP cells. Nuclear extracts from di€erent cell lines (top) were analysed by Western blotting using anti-CBP-NT (UBI) as described in Materials and methods. Multiple bands representing di€erent isoforms of CBP and molecular weight standards (kDa, Biorad pre-stained low molecular weight range) are indicated to the left

an antibody (CBP-NT) that recognises the N-terminus of CBP and does not cross-react with the closely related p300 (Arany et al., 1994). Two major high molecular weight polypeptides, including the *265 kDa polypeptide that corresponds to intact CBP, are detected using CBP-NT as previously described (Arias et al., 1994). Levels of CBP in MMSP cells are comparable to JEG3 and HeLa cells. Thus all of the known positive factors required for cAMP-inducible transcription (PKA, CREB and CBP) are present in MMSP cells. Discussion One working model for the role of EWS/ATF1 in cellular transformation involves constitutive activation of cAMP-inducible genes. The main objective of the experiments described here was to evaluate this model by determining the status of the cAMP-signaling pathway in MMSP cells. In light of the wide variety of genes that are activated by cAMP in mammalian cells, it is of interest that, according to the assays that we have employed, both of the available MMSP cell lines do not respond to cAMP. Thus, although our data does not demonstrate any connection between cAMP-signaling and transformation, our ®ndings suggest that lack of cAMP-responsiveness may be an important feature of MMSP cells. Whether this feature is inherent to the natural target cells that give rise to MMSP or whether it re¯ects a secondary event required for tumorigenesis remains to be addressed. One further possibility is that EWS/ATF1 actually causes down-regulation of the cAMP-response. However, we view this latter alternative as unlikely since EWS/ATF1 can activate most cAMP-inducible promoters tested in transient assays (Brown et al., 1995). The inability of cAMP to activate transcription in MMSP cells is not due to the absence of any known positive factors or due to lack of PKA-mediated phosphorylation of CREB. There remain several possibilities to account for the lack of activity. First,

although phosphorylation of CREB on ser 133 is required for interaction with CBP and transcriptional activation (Chrivia et al., 1993; Kwok et al., 1994), it is, at least in some circumstances, not sucient for either of these functions (Ginty et al., 1991; Brindle et al., 1995; Sun and Mauere, 1995). CBP itself is phosphorylated and activated by PKA (Janknecht and Nordheim, 1996) raising the possibility that a defect in PKA-mediated CBP phosphorylation could account for the lack of response to cAMP in MMSP cells. This idea remains to be directly tested. Other mechanisms for lack of cAMP-responsiveness could involve competition for a limiting component or the presence of dominant inhibitors. In the former case, CBP is of interest, since it has already been shown to allow mutually exclusive signaling by di€erent activators (Kamei et al., 1996). Either steric or functional e€ects might prevent a productive CREB/CBP interaction and there is accumulating evidence from other systems for both possibilities. First, factors that might directly compete for CREB binding to CBP, include c-jun (Bannister et al., 1995) and c-myb (Dai et al., 1996). Second, factors that might functionally inhibit CREB/CBP-dependent activation include nuclear hormone receptors (Kamei et al., 1996) and the mitogen stimulated kinase pp90RSK (Nakajima et al., 1996). It will be of interest to identify proteins that associate with CBP in MMSP cells and thereby identify potential modulators of the cAMP-reponse. To date we have not studied the cAMP-response of cellular promoters other than c-fos in MMSP cells. It therefore remains to be determined whether the inability to respond to cAMP is global or whether there is some degree of promoter speci®city. Overall however our data suggest a global e€ect. First, there are multiple independent cAMP-responsive elements within the cfos promoter that are not all CREB-dependent (Sassone-Corsi et al., 1988; Berkowitz et al., 1989; Fisch et al., 1989; Metz and Zi€, 1991). Second, the inability of GAL4/ATF1 to respond to PKA demonstrates that the defect in cAMP-signaling is not dependent on a speci®c mammalian promoter element. Lack of a transformation assay for EWS/ATF1 has limited progress in understanding the precise role that EWS/ATF1 plays in cellular transformation. Since c-fos (Janknecht et al., 1995), CREB and CBP (Sheng et al., 1990; Dash et al., 1991; Ginty et al., 1994; Bannister et al., 1995; Bhattacharya et al., 1996; Janknecht and Nordheim, 1996; Nakajima et al., 1996) are targets for a wide range of mitogenic signals, CBP is likely to play a key role in integration of mitogenic signals at the c-fos promoter. CBP can function as a coactivator for CREB and SAP-1a (Janknecht and Nordheim, 1996), a member of the TCF family that interacts with the c-fos SRE and activates the c-fos promoter as part of the MAP/ERK kinase pathway (Treisman, 1994). In relation to our results, this strongly suggests that CBP can function as a co-activator for SAP-1a but not for CREB in MMSP cells and therefore allow distinct responses to serumversus cAMP-induced mitogenic signals in these cells. This would be similar to the opposing e€ects of the MAP-kinase and cAMP-pathways on proliferation in many other cell types (Cook and McCormick, 1993; Graves et al., 1993; Sevetson et al., 1993; Wu et al., 1993). Interestingly, growth factor stimulation of two

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protein kinases pp90RSK (Nakajima et al., 1996) and ERK-1 (Janknecht and Nordheim, 1996) can also repress the cAMP-pathway by binding to or modifying CBP. It will be of interest to determine whether either of the above factors contribute to down-regulation of the cAMP-response in MMSP cells. Previous studies (Brown et al., 1995; Fujimura et al., 1996) have suggested that EWS/ATF1 is likely to be a potent constitutive activator of endogenous cAMP-inducible promoters in MMSP cells. The study described here is the ®rst to examine the e€ect of EWS/ATF1 on promoters present in a normal chromosomal context, either cellular (c-fos) or viral (E2 and E3). The inability of EWS/ATF1 to activate the above promoters is surprising and the underlying reason(s) remains to be elucidated. Irrespective of the mechanism however, this issue is of signi®cance for one potential therapeutic approach to MMSP. The identi®cation of promoters that can be activated by EWS/ATF1 suggested that they might function as tumor speci®c promoters and could be exploited to express toxic genes and thereby target MMSP cells for the action of cytotoxic drugs (Huber et al., 1991; Gutierrez et al., 1992). Considering our ®ndings it will be important to verify the activity of EWS/ ATF1-responsive promoters when they are introduced into cells in a context that is appropriate for therapeutic application. In this regard, our data suggests that it may not be possible to use adenovirus (a commonly used vector for gene therapy experiments) to introduce EWS/ATF1 responsive promoters into MMSP cells. Materials and methods Plasmids and constructions pD(-71)SomCAT contains the somatostatin promoter to position minus 71, fused to the chloramphenicol acetyl transferase (CAT) coding sequences (Montminy et al., 1986). pG5E4CAT and pGALATF1 (Hurst et al., 1991) and pCaEV (Masson et al., 1992) are previously described. pSP64Fos5' contains a BamHI/XmnI fragment from pF4 (Treisman, 1985) inserted into BamHI/HincII digested pSP64. Cell culture and adenovirus infections JEG3 (Kohler and Bridson, 1971), Su-ccs-1 (Epstein et al., 1984) and DTC1 (Brown et al., 1995) cells lines were maintained as monolayers in Dulbecco's modi®cation of Eagles medium (DMEM) containing 10% FCS. PC12 cells were maintained as monolayers in DMEM containing 10% FCS and 5% horse serum. Conditions for adenovirus infections were as follows. Cells in a 60 mm culture dish at *50% con¯uence were infected at a multiplicity of infection of 50. Adsorption of the virus was carried out in 0.5 mls of DMEM bu€ered with 20 m M HEPES pH 7.2 for 45 min, followed by dilution with fresh growth media. For cAMPinduction, cells were stimulated by simultaneous addition of 30 mM forskolin and 400 mM cptcAMP to the culture medium. Transfections and CAT assays All transfections were carried out by calcium phosphate coprecipitation and CAT assays were performed as previously described (Gorman et al., 1982). Precipitates contained 5 mg of reporter plasmid, the indicated amount of activator plasmid(s) and 20 mg total DNA made up with

pGEM3 as carrier. One third of the precipitate was added to *50% con¯uent JEG3 cells in a 60 mm culture dish. For quantitation of results, % conversion of unacetylated to acetylated 14C-chloramphenicol under linear assay conditions was determined by excision of spots from the TLC plate and quantitation of radioactivity using a liquid scintillation counter. For experiments with pGAL/ATF1 and pCaEV, cells were co-transfected and CAT assays performed at 40 h post-transfection. For cAMP induction experiments, cells were transfected with pGAL/ATF1 and 8-chlorophenylthio-cAMP pG5E4CAT and 400 mM (cptcAMP) was added to the culture medium 24 h later. CAT assays were performed 48 h post-transfection. Serum/cAMP-induction and RNA analysis For serum stimulation of c-fos, all cells were placed in 0.5 % serum for 24 h and then stimulated by addition of 10% fetal bovine serum to the culture medium. For cAMPinduction, sub-con¯uent monolayers of cells were stimulated by simultaneous addition of 30 mM forskolin and 400 mM cptcAMP to the culture medium. Preparation of total cellular RNA (Favalaro et al., 1980) and detection of speci®c transcripts by RNAse protection analysis (Zinn et al., 1983) were as previously described. A high speci®c activity probe for human c-fos was produced by linearisation of pSP64fos5' with BssHII and transcription by SP6 polymerase (Boehringer) to yield an *700 nt c-fos antisense transcript containing sequences between +617 (XmnI) and 7101 (BssHII) of the c-fos gene. This yields a protected fragment of 290 nt corresponding to exon 1 of c-fos. RNA analysis by primer extension was as previously described (Lee and Green, 1987). ATF1/CREB phosphorylation cAMP induction was carried out as described for induction of c-fos transcription. Small-scale nuclear extracts were prepared as previously described (Hurst et al., 1990) but with the addition of a cocktail of phosphatase inhibitors (2 mM sodium ¯uoride, 0.5 mM sodium ortho-vanadate and 1mM okadaic acid) to the cell lysis bu€er and the high salt nuclear extraction bu€er. Protein concentration was determined using a protein stain (Biorad 500 ± 0111) according to the manufacturers instructions. Antibodies and Western blotting Anti-mouse CBP-NT antibody (Upstate Biotechnology Incorporated, Cat #06-297) was raised in rabbits against a 22aa peptide present at the amino terminus of human CBP. Anti-CBP does not cross react with p300. For Western blotting, CBP-NT antibody was used at 2 mg/ml and detected using alkaline-phosphatase-conjugated anti-rabbit antibody. Anti-phospho-CREB antibody (Ab5322) was raised in rabbits against an in vitro phosphorylated synthetic peptide containing the phospho-acceptor site of CREB (Hagiwara et al., 1993). Anti-CREB antibody was obtained from New England Biolabs (Cat # 9192). For Western blotting, puri®ed Ab5322 was used at a 1 : 2000 dilution and anti-CREB at a 1 : 1000. Detection was carried out using peroxidaseconjugated donkey anti-rabbit antibodies (Amersham NA934) and ECL detection reagents (Amersham RPN2106) according to the manufacturers instructions.

Acknowledgements We are very grateful to Marc Montminy for supplying anti-phospho-CREB antibody and Richard Treisman for pSP64fos5'. This work was supported by Hong Kong Government Research Grants Council grant (award #HKUST 645/96M) to KAWL.

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