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Molecular Cancer Research 1:48-55 (2002)
© 2002 American Association for Cancer Research

Signaling and Regulation

Physical and Functional Interaction Between the Transcriptional Cofactor CBP and the KH Domain Protein Sam681

Wei Hong1, Ross J. Resnick3, Carrie Rakowski1, David Shalloway3, Steven J. Taylor4 and Gerd A. Blobel1,2

1 Division of Hematology, Children's Hospital of Philadelphia, Philadelphia, PA;
2 University of Pennsylvania School of Medicine, Philadelphia, PA;
3 Section of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, NY; and
4 Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, CA

Requests for reprints: Gerd A. Blobel, Division of Hematology, Children's Hospital of Philadelphia, PA. Phone: (215) 590-3988; Fax: (215) 590-4834. E-mail: blobel{at}email.chop.edu


   Abstract
Top
 Notes
 Abstract
Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
CBP is a multifunctional transcriptional cofactor with tumor suppressor activity. The CH3 domain of CBP binds numerous transcription factors and several viral oncoproteins. We identified the Src substrate and RNA-binding protein Sam68 as novel CH3-binding protein. Sam68 binds the CH3 domain in part through a conserved FXD/EXXXL motif that is shared among several CH3-binding proteins, including the adenoviral oncoprotein E1A and the tumor suppressor p53. Sam68 and CBP interact in vivo and colocalize in nuclear sub-domains. Sam68 has potent transcriptional repression activity that is independent of its RNA binding activity, which suggests that RNA processing and regulation of gene expression by Sam68 are separable functions. Consistent with this, CBP did not stimulate the ability of Sam68 to promote Rev response element-containing mRNA export. Interestingly, Sam68 can regulate RNA processing in the absence of a Rev response element, suggesting that Sam68 functions through a novel RNA element. Together, these findings reveal a previously unidentified function for Sam68 as a transcriptional repressor and suggest that Sam68 might link cellular signaling pathways with components of the transcriptional machinery.


   Introduction
Top
 Notes
 Abstract
 Introduction
Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
CBP and its close relative p300 are transcriptional adapters that interact with a multitude of nuclear factors (1–3). CBP/p300 have intrinsic and associated acetyltransferase activity that can be directed toward histone and non-histone nuclear proteins (4–6). CBP contains several independent modules that mediate protein contacts, including three cysteine/histidine-rich (CH) domains. The CH3 domain near the COOH terminus of CBP is of particular interest because it is bound by a variety of nuclear proteins that regulate cellular proliferation and differentiation, including p53, E2F, PCAF, and the lineage-restricted transcription factors GATA-1 and MyoD (1). In addition, the CH3 domain is targeted by several viral oncoproteins that promote cell cycle progression and interfere with cell differentiation. The most well studied among these is the adenoviral oncoprotein E1A. E1A makes specific contacts with the CH3 domain leading to inhibition of CBP-dependent transcription activation (7–9). Proposed mechanisms for E1A-mediated transcription inhibition include disruption of protein binding to CH3 [for examples, see Refs. (10, 11)] as well as regulation of CBP's acetyltransferase activity (12–15). Detailed mapping of the E1A-CBP contact sites revealed the presence of a short motif (FXE/DXXXL) near the NH2 terminus of E1A that interacts with a motif called TRAM within the CH3 domain of CBP. The FXE/DXXXL motif is also found in p53 and E2F (11). The importance of a balanced interaction of CBP with critical regulators of cell proliferation and differentiation prompted us to search for additional molecules that bind to the CH3 domain of CBP. Using a yeast two-hybrid screen with CH3 as bait, we identified Sam68 as a CBP-interacting molecule.

Sam68 was originally identified as a substrate of the Src tyrosine kinase which specifically phosphorylates Sam68 during mitosis (16, 17). Sam68 associates with a variety of SH2 and SH3 domain-containing signaling molecules, including members of the Src family of tyrosine kinases and certain adapter molecules (18–20). Sam68 contains a KH domain that mediates RNA binding (21, 22). The KH domain is embedded in a larger conserved domain, alternatively referred to as the STAR (signal transduction and activation of RNA) or GSG (GRP33, Sam68, GLD-1) domain (23, 24). This domain is conserved in a number of proteins shown to have important roles in development [for references, see Ref. (25)]. In addition to mediating RNA binding, the GSG domain is also a protein interaction module (26). Consistent with a role in RNA metabolism, Sam68 has been suggested to function in an analogous fashion to the HIV Rev protein, which regulates RNA export of unspliced Rev response element (RRE)-containing mRNAs (27). We found that Sam68 binds to the CH3 domain in part through a conserved FXE/DXXXL motif. Sam68 by itself has potent transcriptional repressor activity that does not require RNA binding. These results suggest that Sam68 presents a direct link between cell signaling events and the gene regulatory apparatus.


   Results
Top
 Notes
 Abstract
 Introduction
 Results
Discussion
 Materials and Methods
 Acknowledgements
 References
 
Sam68 Binds to the E1A-binding Domain of CBP in a Yeast Two-hybrid Assay
To identify cellular proteins that interact with the E1A-BD of CBP, a yeast two-hybrid screen was performed. The bait plasmid contained the GAL4-DNA-binding domain (DBD) fused in-frame to amino acids 1805–1892 of CBP. This portion of CBP contains the TRAM motif (amino acids 1811–1822) that mediates interaction with the FXE/DXXXL motif of E1A (11) (Fig. 1E ). We screened 1 x 106 primary transformants of a cDNA library derived from murine erythroleukemia (MEL) cells (28) and obtained 23 positive colonies. One colony contained a cDNA encoding amino acids 234–443 of Sam68. Retransformation of the Sam68-containing plasmid into yeast cells expressing the bait yielded positive clones, thus verifying the interaction between Sam68 and CBP. Sam68 contains a FXE/DXXXL motif at positions 268–274 that might mediate binding to CBP. This was directly examined through in vitro binding studies.



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  		FIGURE 1. Mapping of the CBP and Sam68 binding sites. A. Indicated Sam68 constructs were examined for binding to GST-E1A-BD. KH, KH domain; FLELSYL, amino acids 268–274; YR, tyrosine-rich region; black bars, proline-rich sites. B. GST fused to Sam68 was incubated with in vitro translated CBP in the presence or absence of 0.15 mg/ml of RNase A. C. Indicated CBP fragments were tested for binding to full-length Sam68 fused to GST. {Delta}E1ABD lacks amino acids 1805–1892. D. Sam68 form contacts outside the E1A-BD. Binding of in vitro translated Sam68 to GST fused to the CH3 domain (amino acids 1680–1915), acetyltransferase (AT) domain (amino acids 1196–1718), or E1A-BD (amino acids 1805–1892). E. Conservation of the CH3-binding site among human (h), mouse (m), rat (r), and chicken (c) Sam68 and select cellular and viral molecules. X, any amino acid.

 
A Conserved FXE/DXXXL Motif in Sam68 Mediates Direct Interaction with CBP
To determine whether Sam68 and CBP interact directly and to map the sites of interaction, GST-pull-down experiments were performed. We began by examining Sam68 binding to GST fused to the E1A-BD of CBP (amino acids 1805–1892). The results show that in vitro translated full-length Sam68 bound to GST-E1A-BD with high efficiency, but not to GST alone (Fig. 1A). To map the sites of interaction, a series of Sam68 deletion constructs (Fig. 1A) was analyzed for binding to GST-E1A-BD. The results show that the COOH terminus of Sam68 (amino acids 234–443) is necessary and sufficient for binding (Fig. 1A). This region of Sam68 contains a FXE/DXXXL motif that is conserved among mouse, human, rat, and chicken Sam68, and is present in other molecules that bind to the CH3 domain of CBP, including E1A, p53, and E2F (11) (Fig. 1E). Deletion of this motif in full-length Sam68 dramatically reduced binding to GST-E1A-BD, suggesting that it mediates binding to the E1A-BD (Fig. 1A). However, it appears that additional contact sites are formed between Sam68 and CBP because a construct that retains the FXEXXXL motif but lacks the C-terminal 80 amino acids also showed diminished binding (Fig. 1A). The proline-rich domain (amino acids 295–363) is dispensable for E1A-BD binding (Fig. 1A).

To determine whether mRNA present in the in vitro translation reaction might mediate binding between Sam68 and CBP, we performed a GST-pull-down experiment using full-length GST-Sam68 and full-length in vitro translated CBP in the presence or absence of 0.15 mg/ml RNase A. The results in Fig. 1B show that Sam68 and CBP interacted strongly under both conditions, confirming the results obtained in the converse experiment and indicating that their interaction is independent of the presence of mRNA.

To map the Sam68-binding sites in CBP, full-length GST-Sam68 was used as affinity reagent to examine binding of various in vitro translated CBP fragments. Of all CBP fragments examined, only one that contained the CH3 domain bound to Sam68 (Fig. 1C). Deletion of the E1A-BD in full-length CBP significantly reduced but did not eliminate Sam68 binding, indicating that contacts outside this domain contribute to the Sam68-CBP interaction (Fig. 1C). Consistent with this finding, in vitro translated Sam68 bound to the entire CH3 domain of CBP (amino acids 1680–1915) more efficiently than the E1A-BD alone (Fig. 1D). The acetyltransferase domain of CBP (amino acids 1196–1718) did not show substantial Sam68 binding (Fig. 1D) which might explain the inability of Sam68 to regulate CBP acetyltransferase activity (see below).

In summary, the FXEXXXL motif of Sam68 and the E1A-BD of CBP are required for interaction, but additional contacts are formed that might involve the tyrosine-rich domain Sam68.

In Vivo Interaction Between Sam68 and CBP
To determine whether Sam68 and CBP associate in mammalian cells in vivo, immunoprecipitation experiments were performed with nuclear extracts from MEL cells. Antibodies against CBP but not control serum coprecipitated Sam68, whereas, anti-Sam68 coprecipitated CBP (Fig. 2A ). Similar results were obtained when NIH3T3 (Fig. 2B) and HeLa cells (data not shown) were used, but coprecipitation was less efficient, suggesting that cell-type specific modifications might stabilize the CBP-Sam68 interaction.



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  		FIGURE 2. Interaction between CBP and Sam68 in vivo. Coimmunoprecipitation of Sam68 and CBP in MEL (A) and NIH 3T3 cells (B). i.p., antibodies used for immunoprecipitation; n.i., nonimmune serum. Ten percent of nonprecipitated extract was analyzed directly. C. Mammalian two-hybrid assay. CBP enhances Sam68 transcriptional activity in a manner dependent on the FLELSYL motif. Reporter vector pGL2-GAL4-Luc, GAL4-Sam68, or GAL4-Sam68-{Delta}268-274 were transfected into 3T3 cells alone or together with CBP.

 
To further examine whether CBP and Sam68 interact in vivo, a mammalian two-hybrid assay was performed. A GAL4-dependent reporter construct was transfected into 3T3 cells alone or together with GAL4-Sam68 and CBP. Since CBP activates transcription, an interaction between the two molecules would be expected to activate reporter gene activity. When GAL4-Sam68 was expressed alone, we observed a surprisingly strong inhibition of reporter gene activation (Fig. 5, see below). The reporter gene activity observed in the presence of GAL4-Sam68 was enhanced 2-fold in the presence of coexpressed CBP (Fig. 2C). To examine whether this stimulation required direct interaction with CBP, we generated a GAL4-Sam68 construct (GAL4-Sam68-{Delta}268-274) that carries a deletion of the FXE/DXXXL motif and is impaired in CBP binding (Fig. 1A). Although GAL4-Sam68-{Delta}268-274 repressed transcription with the same efficiency as wild-type GAL4-Sam68 (see Fig. 5A), coexpression of CBP failed to enhance reporter gene activity (Fig. 2C). These results suggest that Sam68 and CBP interact in vivo in a manner dependent on the FXE/DXXXL motif. Additional mammalian two-hybrid experiments (see below and Fig. 5B) further confirm the Sam68/CBP interaction in vivo.



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  		FIGURE 5. A. Sam68 represses transcription in the absence of RNA binding. GAL4-Sam68 constructs and reporter constructs were transfected into 3T3 cells. GAL4-DBD alone served as reference. Western blot with anti-GAL4 antibodies showed comparable amounts of GAL4-Sam68 proteins. B. Sam68 inhibits CBP-dependent transcriptional activation. GAL4-CBP(1099–1877) containing the E1A-BD and GAL4-CBP(1099–1758) lacking the E1A-BD were expressed together with a GAL4-binding site-containing reporter plasmid (G5AdML-CAT) in U2OS cells. C. Sam68 blocks the interaction between CBP and GATA-1. The CH3 domain of CBP (amino acids 1680–1910) fused to a histidine tag (His-CH3) was immobilized and examined for binding to GST-GATA-1 and GST-Sam68. GST alone served as negative control. Bound proteins were analyzed by Western blot with antibodies to GATA-1 and GST, respectively.

 
Colocalization of Sam68 and CBP
If Sam68 and CBP can physically interact, they might be expected to localize to the same nuclear compartments. Both Sam68 and CBP have been shown to localize to discrete nuclear domains in certain cell types (25, 29). Specifically, previous work showed that Sam68 exhibits punctate nuclear staining in MCF7 breast carcinoma cells (25). Therefore, to examine whether Sam68 and CBP might colocalize, MCF7 cells were fixed and reacted with antibodies against CBP and Sam68. When examined by immunofluorescence microscopy, both Sam68 and CBP antibodies revealed a punctate nuclear staining pattern (Fig. 3 ). When the images were merged, it became apparent that CBP and Sam68 colocalize in a large proportion, although not all, of the nuclear spots (Fig. 3). These results are consistent with the observed physical interaction of Sam68 and CBP.



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  		FIGURE 3. Colocalization of Sam68 and CBP in MCF7 cells. MCF7 cells were immunostained for both CBP and Sam68 and analyzed by indirect immunofluorescence as described in "Materials and Methods." A. DAPI (blue); B. anti-CBP (red); C. anti-Sam68 (green); D. merge of B and C.

 
CBP Does Not Stimulate Sam68-mediated Messenger RNA Export
In an effort to understand the functional consequences of the Sam68-CBP interaction, we examined whether CBP can stimulate the ability of Sam68 to regulate RNA processing. Sam68 has previously been suggested to function in a fashion similar to HIV Rev by stimulating RNA export through direct interaction with a RRE (27). To assay RNA export, Sam68 was transfected into COS cells together with a reporter plasmid (pDM128, Fig. 4A ) that allows monitoring of export of an unspliced CAT-containing mRNA. pDM128 contains the SV40 promoter driving the expression of the CAT gene (30). Downstream of the CAT open reading frame is a RRE. Near the 5' and 3' ends of the transcribed regions are a splice donor and an acceptor site, respectively (Fig. 4A). Expression of Sam68 led to a 3- to 5-fold increase in CAT activity, consistent with published results (27) (Fig. 4B). Coexpression of CBP led to a further 3-fold increase in reporter activity (Fig. 4B, left panel). However, expression of CBP also enhanced by 3-fold the expression of the reporter gene in the absence of coexpressed Sam68 (Fig. 4B, left panel). In addition, CBP stimulated other reporter gene constructs containing an SV40 promoter (Fig. 4B, right panel), indicating that the 3-fold increase in CAT activity is the result of enhanced transcription from the SV40 promoter rather than increased Sam68-mediated mRNA export. Thus, CBP appears to have little or no effect on Sam68's ability to stimulate protein expression from unspliced RNAs containing a RRE.



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  		FIGURE 4. CBP does not stimulate Sam68-mediated mRNA export. A. Reporter plasmid pDM128. SD, splice donor site; SA, splice acceptor site; SV40, SV40 promoter; RRE, Rev response element; CAT, chloramphenicol acetyltransferase. B. Left panel, pDM128 was cotransfected with pcDNA3-Sam68 and pCMV5-CBP into COS cells. Right panel, pGL2-luciferase containing the SV40 promoter was transfected into COS cells alone or together with indicated amounts of pCMV5-CBP. C. Sam68 stimulates RNA export of pDM128 in the absence of a RRE. Activity of reporter plasmid alone served as control (ctr.).

 
In an attempt to separate potential effects of CBP on promoter activity of the reporter gene from those that might regulate Sam68 activity, we deleted the RRE from pDM128 (pDM128-{Delta}RRE). To our surprise, we found that Sam68 enhanced CAT reporter expression from pDM128-{Delta}RRE with the same efficiency as from pDM128 (Fig. 4C). In contrast, mRNA export mediated by HIV Rev was strictly dependent on the presence of the RRE (Fig. 4C). These results show that Sam68 can work by a RRE-independent mechanism to promote mRNA export.

Sam68 has Transcriptional Repressor Activity
During our transfection studies, we noticed that expression of Sam68 often resulted in transcriptional repression of various mammalian and viral promoter constructs, similar to what has been frequently observed with expression of E1A [for review, see Ref. (1)]. The inhibitory effects of E1A have been correlated with its ability to bind to CBP. Therefore, we speculated that Sam68 might act as a transcriptional repressor by virtue of its interaction with CBP. In support of this hypothesis, we observed that GAL4-Sam68 potently repressed a GAL4-dependent reporter gene in our mammalian two-hybrid experiments (see above). Fig. 5A shows that GAL4-Sam68 strongly repressed transcription that depended on the presence of intact GAL4-binding sites. To determine which domain of Sam68 mediates transcriptional repression, deletion analysis was performed. The results of these experiments show that a truncated form of Sam68 lacking the NH2-terminal 233 amino acids that include the RNA-binding KH domain repressed transcription with the same efficiency as wild-type Sam68 (Fig. 5A). This suggests that transcriptional repression is independent of RNA binding. Deletion of the FXEXXXL motif that contributes to CBP binding had little effect on repression, indicating that Sam68 might possess repressor function independent of its ability to bind to CBP. Deletion of the C-terminal amino acids 364–443 abrogated its repressor activity. Together, these results identify a novel role for Sam68 in transcriptional repression.

Sam68 Represses CBP-dependent Transcriptional Activation
Our observation that Sam68 possesses a transcriptional repression activity and binds to CBP raised the possibility that Sam68 might directly inhibit transcriptional activation by CBP. Therefore, we measured CBP activity in the presence and absence of coexpressed Sam68. Since CBP regulates the activity of numerous cellular transcription factors, which complicates the analysis of conventional reporter plasmids, we measured CBP activity by analyzing GAL4-CBP fusion constructs. GAL4 fused to amino acids 1099–1877 activates transcription of a GAL4-containing reporter construct (G5AdML-CAT) (31). Coexpression of Sam68 inhibited GAL4-CBP(1099–1877) activity in a dose-dependent manner (Fig. 5B), consistent with a model in which Sam68 links CBP with a repression function. To determine whether inhibition of CBP by Sam68 requires physical interaction between them, we analyzed the effects of Sam68 on the activity of GAL4-CBP(1099–1758) that lacks the E1A-BD. GAL4-CBP(1099–1758) activated transcription at a level somewhat below that found in GAL4-CBP(1099–1877), consistent with previous studies (31). However, Sam68 was substantially less potent in inhibiting GAL4-CBP(1099–1758). Thus, Sam68 and CBP interact in vivo in a manner dependent on the E1A-BD. Furthermore, these results demonstrate a direct effect of Sam68 on the transcriptional activity of CBP.

Sam68 Blocks the Interaction Between CBP and the CH3-binding Protein GATA-1
E1A interferes with the activity of virtually all CBP-dependent transcription factors [for review, see Ref. (1)]. In certain cases, this inhibition has been proposed to result from inhibition of CBP acetyltransferase activity (12–14). In addition, E1A has been shown to compete with transcriptional regulators for physical interaction with CBP, including p53, E2F, and the acetyltransferase PCAF [for examples, see Refs. (10, 11)]. To test whether Sam68 might inhibit acetyltransferase activity of CBP, in vitro acetylation reactions were carried out using recombinant CBP and histones as substrate. The results showed that Sam68 did not significantly alter acetyltransferase activity of CBP (data not shown). We next examined whether Sam68 might compete with other transcription factors for CBP binding. The hematopoietic transcription factor GATA-1 binds to the CH3 domain of CBP and is inhibited by E1A (32). Thus, we examined whether Sam68 might inhibit the interaction between CBP and GATA-1. His-tagged CH3 domain of CBP was immobilized on nickel agarose beads and bound by recombinant purified GST-GATA-1. Consistent with previous results, GATA-1 bound the CH3 domain very efficiently (Fig. 5C). When increasing amounts of Sam68 were added to the binding reaction, a dose-dependent loss of GATA-1 binding was observed (Fig. 5C). These results show that Sam68 can compete with other nuclear factors for CH3 binding, thus providing a potential mechanism for transcriptional repression by Sam68.


   Discussion
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
Materials and Methods
 Acknowledgements
 References
 
The CH3 domain is a central module linking CBP to nuclear proteins that play critical roles in cell differentiation and proliferation. The CH3 domain is also the target for viral oncoproteins that interfere with the tumor suppressor function of CBP. Here we identified Sam68 as a new CH3-binding protein. Binding of Sam68 to CBP occurred in vivo in yeast and mammalian cells. Sam68 contains a conserved FXE/DXXXL motif that is required for CBP binding similar to certain other CH3-binding proteins. Immunofluorescence experiments showed that CBP and Sam68 colocalize in nuclear subdomains in human cancer cells. These findings led us to examine whether these molecules might regulate each other's activities. We initially observed that Sam68 has strong inhibitory activity toward a variety of promoters in transient transfections reminiscent of the actions of the E1A oncoprotein (data not shown). Further studies revealed that Sam68 inhibits transcription by at least two separable mechanisms. First, Sam68 can compete with other nuclear factors for CBP binding, as demonstrated by its ability to inhibit GATA-1 binding to the CH3 domain. This mechanism of inhibition has also been invoked for E1A (10, 11). However, in contrast to E1A (12–14), Sam68 did not substantially alter CBP acetyltransferase activity (data not shown). Second, Sam68 also has strong transcriptional repressor activity when recruited to a promoter via the GAL4-DBD. Repression by GAL4-Sam68 occurred in the absence of the FXEXXXL motif, indicating that CBP binding is not required for the repressive function in this context. Transcriptional activation by GAL4 fused to a portion of CBP was inhibited by Sam68, in a manner dependent on the E1A-BD of CBP. The simplest interpretation of these results is that Sam68 binds to CBP, thereby linking it with a transcriptional repression domain. In addition, Sam68 might also interfere with CBP function by displacing other factors bound to the CH3 domain. We also found that repression by GAL4-Sam68 does not require RNA binding. Overexpression of wild-type and RNA binding-defective Sam68 in mouse fibroblasts leads to cell cycle arrest.2 The arrest in G1 phase seen after Sam68 overexpression is accompanied by, and perhaps caused by, decreased RNA and protein levels of cyclins D1 and E. These results raise the possibility that Sam68 might play a role in repression of cyclin gene expression. Moreover, because our results suggest that Sam68 interaction with CBP is not required for repressor potential, it may be that cell cycle control by Sam68 is independent of CBP. Because Sam68 levels do not change with respect to cell cycle status, it will be important to determine how its repressor activity might be regulated and how the interaction with CBP might be modified. Because Sam68 is regulated by phosphorylation in response to a variety of signaling events, it is possible that phosphorylation affects its interaction with CBP and/or its repressor activity. The former possibility is supported by our observation that the Sam68-CBP interaction varies depending on cell type. In addition, a mutant form of Sam68 lacking the tyrosine-rich domain binds CBP less avidly than wild-type Sam68, which raises the possibility that tyrosine phosphorylation might regulate the Sam68-CBP interaction.

We also examined a possible role for CBP in regulating Sam68 activity. Because the CBP-binding motif (FXEXXXL) lies in the CK (COOH-terminal of KH) region of the GSG domain (25), we considered the possibility that the interaction with CBP might regulate RNA binding by Sam68. However, CBP did not significantly influence Sam68-mediated export of a RRE-containing CAT reporter gene, an effect that has been shown to require RNA binding, when compared to control reporter genes. To distinguish between possible transcriptional effects of CBP on the promoter of the reporter gene and effects on RNA export, we generated a reporter gene lacking the RRE. Unexpectedly, we found that although Rev-mediated RNA export was lost in the absence of the RRE, Sam68 increased CAT levels to the same extent as the RRE-containing reporter gene. This suggests that Sam68 might function by a mechanism that is distinct from Rev. Because the KH domain of Sam68 is required for stimulation of reporter gene activity (27), this raises the interesting possibility that Sam68 might stimulate RNA processing through interaction with a novel RNA element. Future work is directed at defining this element. However, because the KH domain also mediates protein contacts (26), it remains possible that Sam68 works indirectly via a yet to be identified RNA-binding protein.

As an additional way of regulating Sam68 activity, we examined whether CBP might acetylate Sam68. In vitro acetylation reactions using recombinant purified CBP and Sam68 proteins showed little or no Sam68 acetylation (data not shown). Furthermore, Sam68 had no effect on CBP acetyltransferase activity. Thus, although it remains formally possible that additional factors might influence the ability of Sam68 to either act as a substrate or regulator of CBP acetyltransferase activity in vivo, it appears that despite its association with CBP, protein acetylation is unlikely to be an important determinant in Sam68 function.

Sam68 displays many of the hallmarks of a tumor suppressor gene in that overexpression can cause both cell cycle arrest and apoptosis3 and reduced expression by antisense targeting results in cell transformation (33). It is therefore possible that Sam68 acts in concert with CBP to regulate proliferation and apoptosis. Although we have not formally demonstrated that E1A can compete with Sam68 for binding to CBP, both proteins contain a FXEXXXL motif that contributes to CBP binding. It is therefore likely that E1A is able to displace Sam68 from CBP in adenovirus-infected cells; this may neutralize the ability of Sam68 to promote growth arrest or apoptosis.

CBP is a widely expressed cofactor that interacts with a multitude of nuclear factors. In addition, CBP interacts with basal transcription factors, including TFIIB, TBP, and RNA polymerase II [for review, see Refs. (1, 2)]. As such, CBP can be viewed as general transcription factor. The finding that CBP interacts with Sam68 raises the interesting possibility that Sam68 might link the transcriptional apparatus with the RNA processing machinery. Such a link has been proposed to exist on several levels where RNA polymerase and associated factors are physically coupled to proteins that mediate mRNA capping, splicing, and polyadenylation to form what is referred to as transcriptosome [for review, see Ref. (34)]. If Sam68 were indeed such a link, this function would be separable from its inhibitory effects on transcription initiation. Finally, both Sam68 and CBP are phosphoproteins and phosphorylation might affect their interaction and respective activities. Future work will address these important questions.


   Materials and Methods
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
Acknowledgements
 References
 
Yeast Two-Hybrid System
The E1A-BD of murine CBP (amino acids 1805–1892) was cloned in-frame by PCR downstream the GAL4-DBD of plasmid pGBT9 (Clontech Laboratories, Inc., Palo Alto, CA). A MEL cDNA expression library in pGAD10 (28) was transformed into the yeast strain PJ69-4{alpha} (gift from Erfei Bi). PJ69-4{alpha} and the two-hybrid screening procedure have been described (35).

Plasmids
CBP constructs and GST-GATA-1 fusion constructs have been described (36, 37); pcDNA3-Sam68 and GST-Sam68 fusion constructs have been described by Lin et al. (22). Truncated variants of Sam68 constructs were generated by cloning PCR fragments into pcDNA3 vectors (Invitrogen, Frederick, MD). G5AdML reporter plasmid, GAL4-CBP(1099–1758), and GAL4-CBP(1099–1877) were gifts from Tony Kouzarides (31). GAL4-Sam68 constructs were prepared by transferring corresponding Sam68 fragments from pcDNA3-Sam68 constructs to pcMX-GAL4 vectors. The reporter vectors used in transient transfections were pGL2-Luc (Promega, Madison, WI) and pGL2-GAL4-Luc, which contains five GAL4-binding sites (gift from Mitch Lazar). COOH-terminally His-tagged CBP-CH3 (amino acids 1680–1910) was generated by PCR and subcloned between the BamHI and Hind III sites of pET-21a(+) (Novagen, Madison, WI). pDM128 has been described (30). pDM128-{Delta}RRE was made by deleting the RRE (nucleotides 7787–7997 of HIVSF2) with overlapping PCR. All constructs were verified by sequencing.

Cell Culture, Transfections, Immunoprecipitations, and Western Blots
MEL and COS cells were maintained in DMEM (Life Technologies, Inc., Carlsbad, CA) supplemented with 10% FCS. NIH 3T3 cells were grown in 10% calf serum. Cells were transiently transfected with Lipofectamine (Life Technologies, Inc.) or FuGene 6 (Roche, Basel, Switzerland) according to the manufacturer's instructions. Nuclear extracts were prepared as described (38). Immunoprecipitations were performed with anti-CBP serum (1–100, gift from T. Nakajima), anti-Sam68 serum (19). Nonimmune serum was used as negative control. Anti-CBP antibodies (A22, Santa Cruz Biotechnology, Santa Cruz, CA) and anti-Sam68 serum were used for Western blots. Commercially available antibodies against CBP were significantly less efficient in precipitating both CBP and Sam68 (data not shown). Both anti-GAL4-DBD and anti-GATA-1 (N6) antibodies were from Santa Cruz Biotechnology. In the mammalian two-hybrid assays, 0.5 µg of reporter vector pGL2-GAL4-Luc, 0.5 µg of GAL4-Sam68 constructs, and 1 µg of CBP expression plasmid were transfected into 3T3 cells using Lipofectamine. For Sam68 reporter assays, 0.5 µg of GAL4-Sam68 constructs and 0.5 µg of reporter vectors were used.

In Vitro Protein Binding Assays
GST fusion protein preparations and interaction assays were performed as described (39). His-tagged proteins were expressed in Escherichia coli BL21 (DE3, Novagen) and purified with Ni-NTA agarose beads (Qiagen, Valencia, CA), according to the manufacturer's instruction. Equal amounts of His-CH3 bound to Ni-NTA beads were incubated with 4 µg GST-GATA-1 and increasing amounts of GST-Sam68 (1.5–15 µg) for 2 h in buffer containing 50 mM NaH2PO4 (pH 8.0), 300 mM NaCl, 20 mM imidazole, 0.1% Triton X-100, 0.5 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, 1 µg/ml pepstatin, and 1 µg/ml aprotinin. Beads were washed four times with the above buffer. His-CH3, GST-Sam68, and GST-GATA-1 were separated by SDS-PAGE and detected by Western blotting.

Immunofluorescence Microscopy
MCF7 cells were grown on glass coverslips for 2 days, fixed with ethanol/glycine, pH 2.0, at -20°C for 30 min, washed three times with PBS, and incubated with primary antibodies in PBS plus 3% BSA for 1 h at room temperature. Immunoaffinity purified rabbit anti-Sam68 serum and mouse monoclonal anti-CBP antibody NM11 (Neomarker, Fremont, CA) served as primary antibodies. Cells were washed three times with PBS and incubated for 1 h with goat anti-rabbit or goat anti-mouse antibodies coupled to FITC or Texas Red, respectively (Jackson ImmunoResearch Laboratories, Westgrove, PA). Cells were washed three times with PBS, drained, and mounted on glass slides using Vectashield containing DAPI.


   Acknowledgements
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
References
 
We thank Margaret Chou for critical reading of the manuscript and Helen Bayes for help with the immunoprecipitation experiments.


   Notes
Top
 Notes
Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
1 NIH Grant 1RO1 DK54937-01 (to G.A.B.). Back

2 S. J. Taylor, R. J. Resnick, and D. Shalloway, unpublished observations. Back

3 S. J. Taylor, unpublished observations. Back

Received May 7, 2002; revised July 3, 2002; accepted August 20, 2002.


   References
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 

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