Conserved Region 2 Research Articles

Binding of PKA-RIIalpha to the Adenovirus E1A12S oncoprotein correlates with its nuclear translocation and an increase in PKA-dependent promoter activity.

The adenovirus type 12 (Ad12) E1A12S oncoprotein utilizes the cAMP/protein kinase A (PKA) signal transduction pathway to activate expression of the viral E2 gene, the products of which are essential for viral replication. A central unsolved question is, however, whether E1A12S interacts directly with PKA in the process of promoter activation. We show here that E1A12S binds to the regulatory subunits (R) of PKA in vitro and in vivo. Interaction depends on the N-terminus and the conserved region 1 (CR1) of E1A12S. Both domains are also essential for the activation of viral E2 gene expression. Infection of cells with Ad12 leads to the cellular redistribution of RIIalpha from the cytoplasm into the nucleus. Furthermore, RIIalpha is also located in the nucleus of cells transformed by E1 of Ad12 and transient expression of E1A12S leads to the redistribution of RIIalpha into the nucleus in a N-terminus- and CR1-dependent manner. Cotransfection of E1A12S with RIIalpha results in strong activation of the E2 promoter. Based on these results we conclude that E1A12S functions as a viral A-kinase anchoring protein redistributing RIIalpha from the cytoplasm into the nucleus where it is involved in E1A12S-mediated activation of the E2 promoter.

Phosphorylation site specificity of the Pak-mediated regulation of Raf-1 and cooperativity with Src

The p21-activated kinase, Pak, has recently been shown to phosphorylate Raf-1 on serine 338 (S338), a critical regulatory residue. The specificity requirements for Pak-mediated phosphorylation of S338 were examined by substitution analysis of Raf-1 peptides and conserved region 3 (CR3) proteins. Phosphorylation was found to be very sensitive to alterations in amino acid side chains proximal to S338. Loss of N-terminal arginines resulted in decreased peptide phosphorylation while loss of these residues, as well as C-terminal glutamates and bulky C-terminal hydrophobic residues, decreased phosphorylation of the CR3 protein. Phosphorylation of Raf-1 on tyrosine 341 is significant in epidermal growth factor- and Src-mediated signaling, suggesting that cooperativity may exist between Pak and Src phosphorylation of Raf-1. Purified Pak and Src were found not to be cooperative in phosphorylating peptides or purified CR3 protein. However, the phosphorylation of Raf-1 S338 by Pak was increased in the presence of Src. The complexity of this signaling module could thus account for the different levels of Raf-1 activation required for fulfillment of different biological roles within the cell.

Vav-2 controls NFAT-dependent transcription inB- but not T-lymphocytes

We show here that Vav-2 is tyrosine phosphorylated following antigen receptor engagement in both B- and T-cells, but potentiates nuclear factor of activated T cells (NFAT)-dependent transcription only in B cells. Vav-2 function requires the N-terminus, as well as functional Dbl homology and SH2 domains. More over, the enhancement of NFAT-dependent transcription by Vav-2 can be inhibited by a number of dominant-negative GTPases. The ability of Vav-2 to potentiate NFAT-dependent transcription correlates with its ability to promote a sustained calcium flux. Thus, Vav-2 augments the calcium signal in B cells but not T cells, and a truncated form of Vav-2 can neither activate NFAT nor augment calcium signaling. The CD19 co-receptor physically interacts with Vav-2 and synergistically enhances Vav-2 phosphorylation induced by the B-cell receptor (BCR). In addition, we found that Vav-2 augments CD19-stimulated NFAT- dependent transcription, as well as transcription from the CD5 enhancer. These data suggest a role for Vav-2 in transducing BCR signals to the transcription factor NFAT and implicate Vav-2 in the integration of BCR and CD19 signaling.

Zinc finger and carboxyl regions of adenovirus E1A 13S CR3 are important for transactivation of the cytomegalovirus major immediate early promoter by adenovirus.

Reactivation of latent cytomegalovirus (CMV) is an important cause of disease in susceptible patients. We previously demonstrated that an adenovirus early gene product can transactivate the CMV major immediate early (IE) promoter in inflammatory cells. This effect was due to the conserved region 3 (CR3) of the adenovirus E1A 13S gene product. There are two domains in the CR3 region, a zinc finger (aa 147-177) and a carboxyl (aa 180-188) domain. Both are crucial for transactivation of downstream promoter elements of adenovirus in E1A 13S. We sought to determine if either or both of these specific domains is also necessary for transactivation of the CMV IE promoter by the adenovirus E1A 13S gene product. We cotransfected T-lymphocyte Jurkat cells and monocyte/macrophage-like THP-1 cells with plasmids expressing wild-type (WT) or CR3 mutant E1A 13S and a CMV IE chloramphenicol acetyltransferase (CAT) reporter construct. With extracts of cells coinfected with E1A WT set to 100%, mutation in the zinc finger domain, the carboxyl domain, or both domains decreased CMV IE CAT activity by >/= 96%. In contrast, a mutation in the region between the zinc finger and carboxyl domains reduced CMV IE CAT activity by only 24 to 26%. Mixing studies in Jurkat cells confirmed the importance of these domains. We also evaluated the active site of the CMV IE promoter involved in transactivation in THP-1 cells using CMV IE promoter deletions and single promoter element constructs. These studies showed that progressive deletion of the 19-bp CMV IE repeats containing cyclic AMP response element binding protein/activating transcription factor (CREB/ATF) sites resulted in progressive loss of activity. The importance of this element was confirmed using single promoter elements containing CMV IE 16-, 18-, 19-, and 21-bp repeats. Finally, using a 19-bp single promoter element construct and the CR3 mutants we demonstrated that mutations in the zinc finger (C171S) carboxyl region (S185N) or both regions (C171S/ S185N) resulted in significant (83, 94, and 85%) loss of activity. We conclude that the zinc finger and carboxyl domains of the CR3 region of E1A 13S are necessary for transactivation of the CMV promoter and that this occurs mainly through activation of the 19-bp CREB/ATF site of the promoter.

Transformation-defective adenovirus 5 E1A mutants exhibit antioncogenic properties in human BLM melanoma cells.

Adenoviral E1 A proteins exhibit a strong tumor-suppressive activity in human tumor cells. However, E1 A is capable of transforming rodent and human cells in cooperation with other oncoproteins, such as activated RAS. Thus, the therapeutic use of wild-type E1A harbors the principal risk of enhancing tumor malignancy. This prompted us to construct E1A 13S cDNA-derived mutants that were unable to transform baby mouse kidney cells in cooperation with E1B and to test their tumor-suppressive activity in BLM human melanoma cells. Anchorage-independent growth in soft agar was reduced for those cell lines expressing the E1AdelCR2 mutant, which lacks the entire conserved region 2 (CR2) sequences, or for cells expressing the E1AcR3Ex2 mutant, which contains CR3 plus exon 2 sequences. In contrast, cell lines expressing the entire E1A wild-type (E1AWT) or only the exon 2 sequences (E1AEx2) grew like the parental BLM cells. Moreover, inoculation of nude mice with BLM cells or cells expressing E1AEx2 revealed large tumors after 2 weeks. In contrast, tumors derived from E1AdelCR2- or E1ACR3Ex2-expressing cells exhibited a substantial delay in tumor growth accompanied by a loss of E1A expression in the outgrown tumors. Cell lines expressing E1AWT showed an intermediate phenotype. Thus, expression of CR3 plus exon 2 sequences is sufficient to enhance both the antioncogenic properties and the therapeutic safety of E1A in our system.

Induction of S phase and apoptosis by the human papillomavirus type 16 E7 protein are separable events in immortalized rodent fibroblasts

The HPV16 E7 oncoprotein neutralizes several cell cycle checkpoints, favouring the entry of quiescent cells into S phase. This activity is mediated in part by association of E7 with the pocket proteins and consequent activation of E2F transcription factors. In addition, HPV16 E7 protein is able to promote apoptosis. In this study we demonstrate that the ability to induce apoptosis is a common property of E7s belonging to both benign and malignant HPV types. The E7-induced apoptosis is mediated by inactivation of pRb, whilst neutralization of the other two pRB-related proteins, p107 and 130, is not sufficient to trigger apoptosis. Moreover, we show that certain point mutations in the conserved region 1 (CR1) of HPV16 E7 abolish the induction of apoptosis without altering the ability to stimulate S phase. Thus, these two E7-mediated cellular events, apoptosis and S phase entry, can be separated in immortalized rodent fibroblasts. Our findings demonstrate that the E7-mediated pRb destabilization is not required for its ability to drive quiescent cells into S phase and to induce apoptosis. Finally, expression of E7 proteins in NIH3T3, which lack a functional p19ARF, does not lead to p53 accumulation, indicating that the E7 impacts upon additional cellular pathways to promote apoptosis.

The Structure of the Site on Adenovirus Early Region 1A Responsible for Binding to TATA-binding Protein Determined by NMR Spectroscopy

Previous detailed mutational analysis has shown that the binding site on adenovirus (Ad) early region 1A (E1A) for TATA-binding protein (TBP) is located toward the N terminus of conserved region 3 (CR3). Here we demonstrate that synthetic peptides of between 15 and 22 amino acids, identical to amino acid sequences of CR3 present in the larger Ad5 E1A (13 S product) and in both the Ad12 E1A (13 and 12 S products) proteins that lie N-terminal to the zinc finger motif, can disrupt binding of E1A to TBP. These findings suggest that the peptides are biologically active in terms of interacting with TBP and must therefore comprise some, if not all, of the TBP binding site on E1A. The interaction between Ad12 E1A and TBP was confirmed by direct co-precipitation experiments. In 1H NMR studies of CR3 peptides, regular patterns of NOEs were observed from which their conformational preferences in aqueous solution were determined. Both Ad5 and Ad12 peptides were shown to contain regions of helical backbone structure in 50% trifluoroethanol. In each case, the type and intensities of NOE cross-peaks observed correlated best to alpha-helical turns. These helices are more extensive in larger peptides and extend from Glu141 to Val147 and from Arg144 to Pro152 in the full-length Ad5 and Ad12 13S E1A proteins, respectively. The structure of a 19-residue Ad5 CR3 peptide carrying the V147L mutation in the full-length protein that abolishes TBP binding was examined. No significant differences between the substituted and wild type peptides were observed, suggesting that this substitution in the intact protein may cause disruption of global rather than local structures.

The CXXC Zn binding motifs of the human papillomavirus type 16 E7 oncoprotein are not required for its in vitro transforming activity in rodent cells.

The conserved region 3 (CR3) of the E7 protein of human papillomaviruses contains two CXXC motifs involved in zinc binding and in the homodimerization of the molecule. Studies have suggested that the intact CXXC motifs in the CR3 of HPV16 and HPV18 E7 are required for the in vitro transforming activity of these proteins. CR3 also contains a low affinity pRb binding site and is involved in the disruption of the E2F/Rb1 complex. E7 is structurally and functionally related to Adenovirus E1A protein, which also has two CXXC motifs in CR3. However, the Ad E1A transforming activity appears to be independent of the presence of such domains. In fact, this viral protein exists in vivo as two different forms of 289 and 243 amino acids. The shorter Ad E1A form (Ad E1A243), where both CXXC motifs are deleted by internal splicing, retains its in vitro transforming activity. We have investigated if the HPV16 E7 CR3 can be functionally replaced by the Ad E1A243 CR3, which lacks both CXXC motifs. A chimeric protein (E7/E1A243) containing the CR1 and CR2 of HPV16 E7 fused to the CR3 of Ad E1A 243 was constructed. The E7/E1A243 while not able to homodimerize in the S. cerevisiae two-hybrid system retains several of the properties of the parental proteins, HPV16 E7 and Ad E1A. It associates with the 'pocket' proteins, induces growth in soft agar of NIH3T3 cells and immortalizes rat embryo fibroblasts. These data suggest that the CXXC motifs in CR3 of E7 do not play a direct role in the transforming properties of this viral protein but probably are important for maintaining the correct protein configuration.

The Relative Ability of Human Papillomavirus Type 6 and Human Papillomavirus Type 16 E7 Proteins to Transactivate E2F-Responsive Elements Is Promoter- and Cell-Dependent

The human papillomavirus 16 E7 protein (HPV 16 E7) transactivates the adenovirus E2 promoter (AdE2) by altering interactions between retinoblastoma (pRb) family members and the transcription factor E2F. To understand factors limiting the oncogenic potential of HPV 6, the relative ability of HPV 6 E7 as compared to HPV 16 E7 to transactivate the AdE2 promoter was determined. In primary baby rat kidney cells and human foreskin keratinocytes, HPV 16 E7 transactivated the AdE2 promoter to a greater extent than HPV 6 E7, consistent with the observation that HPV 16 E7 binds pRb with greater affinity. HPV 6 E7 gain of function correlated with increasing the affinity of the HPV 6 E7 pRb binding site of conserved region 2 (CR2). In keratinocytes, in contrast to the AdE2 promoter, the abilities of the two E7 proteins to transactivate the B-mybpromoter, a promoter regulated by E2F bound to p107/p130, were comparable. Introducing a negative charge into the N-terminus (CR1) and a high affinity pRb binding site into CR2 of HPV 6 E7 resulted in a transactivator with greater activity than HPV 16 E7 for both the AdE2 and B-mybpromoters. Both of the promoters were negatively regulated by E2F and transactivation by the E7 proteins required an intact E2F site. In C33-A cells, which contain a mutated pRb, the two E7 proteins had comparable transactivating activity on both the AdE2 and B-mybpromoters. The data are consistent with the interpretation that HPV 16 E7 affects interactions of pRb and p107/p130 with the E2F transcription factor, whereas HPV 6 E7 only affects interactions of p107/p130.

Both conserved region 1 (CR1) and CR2 of the human papillomavirus type 16 E7 oncogene are required for induction of epidermal hyperplasia and tumor formation in transgenic mice.

High-risk human papillomavirus type 16 (HPV-16) and HPV-18 are associated with the majority of human cervical carcinomas, and two viral genes, HPV E6 and E7, are commonly found to be expressed in these cancers. The presence of HPV-16 E7 is sufficient to induce epidermal hyperplasia and epithelial tumors in transgenic mice. In this study, we have performed experiments in transgenic mice to determine which domains of E7 contribute to these in vivo properties. The human keratin 14 promoter was used to direct expression of mutant E7 genes to stratified squamous epithelia in mice. The E7 mutants chosen had either an in-frame deletion in the conserved region 2 (CR2) domain, which is required for binding of the retinoblastoma tumor suppressor protein (pRb) and pRb-like proteins, or an in-frame deletion in the E7 CR1 domain. The CR1 domain contributes to cellular transformation at a level other than pRb binding. Four lines of animals transgenic for an HPV-16 E7 harboring a CR1 deletion and five lines harboring a CR2 deletion were generated and were observed for overt and histological phenotypes. A detailed time course analysis was performed to monitor acute effects of wild-type versus mutant E7 on the epidermis, a site of high-level expression. In the transgenic mice with the wild-type E7 gene, age-dependent expression of HPV-16 E7 correlated with the severity of epidermal hyperplasia. Similar age-dependent patterns of expression of the mutant E7 genes failed to result in any phenotypes. In addition, the transgenic mice with a mutant E7 gene did not develop tumors. These experiments indicate that binding and inactivation of pRb and pRb-like proteins through the CR2 domain of E7 are necessary for induction of epidermal hyperplasia and carcinogenesis in mouse skin and also suggest a role for the CR1 domain in the induction of these phenotypes through as-yet-uncharacterized mechanisms.

Phosphorylation within the transactivation domain of adenovirus E1A protein by mitogen-activated protein kinase regulates expression of early region 4.

A critical role of the 289-residue (289R) E1A protein of human adenovirus type 5 during productive infection is to transactivate expression of all early viral transcription. Sequences within and proximal to conserved region 3 (CR3) promote expression of these viral genes through interactions with a variety of transcription factors requiring the zinc binding motif in CR3 and in some cases a region at the carboxy-terminal end of CR3, including residues 183 to 188. It is known that 3',5' cyclic AMP (cAMP) reduces the level of phosphorylation of the 289R E1A protein through the activation of protein phosphatase 2A by the E4orf4 protein. This study was designed to identify the E1A phosphorylation sites affected by E4orf4 expression and to determine their importance in regulation of E1A activity. We report here that two previously unidentified sites at Ser-185 and Ser-188 are the targets for decreased phosphorylation in response to cAMP. At least one of these sites, presumably Ser-185, is phosphorylated in vitro by purified mitogen-activated protein kinase (MAPK), and both are hyperphosphorylated in cells which express a constitutively active form of MAPK kinase. Analysis of E1A-mediated transactivation activity indicated that elevated phosphorylation at these sites increased expression of the E4 promoter but not that of E3. We have recently shown that one or more E4 products induce cell death due to p53-independent apoptosis, and thus it seems likely that one role of the E4orf4 protein is to limit production of toxic E4 products by limiting expression of the E4 promoter.

Accumulation of p53 induced by the adenovirus E1A protein requires regions involved in the stimulation of DNA synthesis.

It has been known for some time that expression of the 243-residue (243R) human adenovirus type 5 (Ad5) early region 1A (E1A) protein causes an increase in the level of the cellular tumor suppressor p53 and induction of p53-dependent apoptosis. Deletion of a portion of conserved region 1 (CR1) had been shown to prevent apoptosis, suggesting that binding of p300 and/or the pRB retinoblastoma tumor suppressor and related proteins might be implicated. To examine the mechanism of the E1A-induced accumulation of p53, cells were infected with viruses expressing E1A-243R containing various deletions which have well-characterized effects on p300 and pRB binding. It was found that in human HeLa cells and rodent cells, complex formation with p300 but not pRB was required for the rise in p53 levels. However, in other human cell lines, including MRC-5 cells, E1A proteins which were able to form complexes with either p300 or pRB induced a significant increase in p53 levels. Only E1A mutants defective in binding both classes of proteins were unable to stimulate p53 accumulation. This same pattern was also apparent in p53-null mouse cells coinfected by Ad5 mutants and an adenovirus vector expressing either wild-type or mutant human p53 under a cytomegalovirus promoter, indicating that the difference in importance of pRB binding may relate to differences between rodent and human p53 expression. The increase in p53 levels correlated well with the induction of apoptosis and, as shown previously, with the stimulation of cellular DNA synthesis. Thus, it is possible that the accumulation of p53 is induced by the induction of unscheduled DNA synthesis by E1A proteins and that increased levels of p53 then activate cell death pathways.

Role of p300-family proteins in E1A oncogene induction of cytolytic susceptibility and tumor cell rejection.

The mechanism by which the adenoviral (Ad) E1A oncogene induces cellular susceptibility to lysis by killer lymphocytes involves interactions between its first exon and different second-exon accessory regions. Mutational analysis showed that two first-exon regions--one in the N terminus and one in the conserved region 1 (CR1) domain--are necessary for this activity. E1A complex formation with cellular p300 protein through these first-exon-encoded regions correlated with induction of the cytolytic susceptible phenotype but was only effective in the context of E1A second-exon expression. An E1A first-exon deletion that prevented p300 binding eliminated both oncoprotein-induced cytolytic susceptibility and rejection of transfected sarcoma cells by immunocompetent animals. These results suggest that the E1A oncogene induces cytolytic susceptibility and tumor rejection by interactions with cellular proteins of the p300 family that affect transcription of genes involved in the cellular response to injury inflicted by host killer cells.

The CR1 and CR3 domains of the adenovirus type 5 E1A proteins can independently mediate activation of ATF-2.

The adenovirus 12S E1A protein can stimulate the activity of the c-jun promoter through a conserved region 1 (CR1)-dependent mechanism. The effect is mediated by two AP-1/ATF-like elements, jun1 and jun2, that preferentially bind c-Jun-ATF-2 heterodimers. In this study, we show that the ATF-2 component of the c-Jun-ATF-2 heterodimer is the primary target for 12S E1A: 12S E1A can enhance the transactivating activity of the N terminus of ATF-2 when fused to a heterologous DNA-binding domain, whereas the transactivating activity of the c-Jun N terminus is not significantly affected. Activation of the ATF-2 N terminus by 12S E1A is dependent on CR1. In the context of the 13S E1A protein, CR1 and CR3 can both contribute to activation of ATF-2, and their relative contributions are dependent on the cell type. In contrast to activation of ATF-2 by stress-inducing agents, CR1-dependent activation of ATF-2 was found not to depend strictly on the presence of threonines 69 and 71 in the N terminus of ATF-2, which are targets for phosphorylation by stress-activated protein kinases (SAPKs). In agreement with this observation, we did not observe phosphorylation of threonines 69 and 71 or constitutively enhanced SAPK activity in E1A- plus E1B-transformed cell lines. These data suggest that CR1-dependent activation of ATF-2 by 12S E1A does not require phosphorylation of threonines 69 and 71 by SAPK.

The CtBP binding domain in the adenovirus E1A protein controls CR1- dependent transactivation

The adenovirus E1A-243R protein has the ability to force a resting cell into uncontrolled proliferation by modulating the activity of key targets in cell cycle control. Most of these regulatory mechanisms are dependent on activities mapping to conserved region 1 (CR1) and the non-conserved N-terminal region of E1A. We have previously shown that CR1 functions as a very patent transactivator when it is tethered to a promoter through a heterologous DNA binding domain. However, artificial DNA binding was not sufficient to convert full-length E1A-243R to a transactivator. Thus, an additional function(s) of the E1A-243R protein modulates the effect of CR1 in transcription regulation. Here we demonstrate that a 44 amino acid region at the extreme C-terminus of ElA inhibited transactivation by a Gal4-CR1 fusion protein. Inhibition correlated with binding of the nuclear 48 kDa C-terminal binding protein (CtBP), which has been implicated in E1A-mediated suppression of the metastazing potential of tumour cells. This might suggest that CtBP binding can regulate E1A-mediated transformation by modulating CR1-dependent control of transcription.

Adenovirus E1A Proteins Regulate Phosphoenolpyruvate Carboxykinase Gene Transcription through Multiple Mechanisms

Recently, Kalvakolanu et al. (Kalvakolanu, D. V. R., Liu, J., Hanson, R. W., Harter, M. L., and Sen, G. C. (1992) J. Biol. Chem. 267, 2530-2536) showed that E1A inhibited the basal and cAMP-stimulated transcription of the gene for phosphoenolpyruvate carboxykinase (PEPCK). This inhibition was mediated by the conserved region 1 (CR1) domain of E1A, which has been shown by other laboratories to bind to the cellular transcriptional adaptor proteins, p300 and cAMP response element binding protein (CREB)-binding protein (CBP). The PEPCK gene promoter contains a functional cAMP-response element, through which CREB and, therefore, CBP modulate transcription, and a consensus p300 DNA binding sequence is also present in a distal protein binding site of the promoter. We hypothesized that E1A might inhibit PEPCK gene transcription by binding to p300 and/or CBP. Surprisingly, we found that E1A consistently stimulated basal transcription from the PEPCK promoter in transfection assays in adenovirus (Ad)-infected HepG2 hepatoma cells or E1A-expressing, stably transfected 3T3 fibroblasts and nuclear run-on assays in Ad-infected H4IIE hepatoma cells. E1A also enhanced the stimulation of PEPCK gene transcription by Bt2cAMP. In transfection assays, wild type Ad5 expressing both 243R and 289R forms of E1A or a mutant virus expressing the 289R form alone stimulated transcription from the PEPCK promoter by approximately 5-fold 20 h postinfection. However, no stimulation was observed in cells infected with a virus expressing either the 243R protein alone or a 289R protein from which conserved region 3 (CR3) was mutated. Mutation or deletion of CR1 of E1A had no significant effect on transcription from the PEPCK promoter. Mutations within conserved region 2 (CR2) of E1A that inhibit the binding of E1A to the retinoblastoma gene product (pRb) further enhanced the stimulation of transcription from the PEPCK promoter by 2 3-fold compared with wild type E1A. These findings suggested that the normal function of pRb is to stimulate PEPCK gene transcription, and that this process is inhibited by the binding of E1A to pRb. This hypothesis was confirmed by overexpressing pRb in HepG2 cells, which stimulated transcription from the PEPCK promoter. Our findings indicate that Ad E1A regulates PEPCK gene transcription through a stimulatory mechanism involving CR3, and by attenuating a stimulatory effect of pRb through CR2.

Adenovirus E1A activates cyclin A gene transcription in the absence of growth factors through interaction with p107.

Using the infection of quiescent human fibroblasts with adenovirus type 5 and various deletion mutants, we show that E1A can stimulate transcription of the cyclin A gene in the absence of exogenous growth factors. Required for this activity is conserved region 2 (CR2), while both the N-terminal part of E1A and CR1 are dispensable. This indicates that activation of cyclin A gene expression requires the binding of E1A to p107, while binding to either pRB or p300 is not involved in transcriptional activation. We demonstrate that p107 represses the cyclin A promoter through its cell cycle-regulatory E2F binding site and that 12S E1A can activate the cyclin A promoter, essentially by counteracting its repression by p107. Since Cr2 is required for cell transformation, transcriptional activation of the cyclin A gene by E1A appears to be important for its capacity to override control of cellular growth.

The human papillomavirus type 16 E7 protein complements adenovirus type 5 E1A amino-terminus-dependent transactivation of adenovirus type 5 early genes and increases ATF and Oct-1 DNA binding activity

We have previously shown that conserved region 1 (CR1) of the adenovirus type 5 (Ad5) E1A protein synergizes with CR3 in the transactivation of Ad5 early genes (H.K. Wong and E. B. Ziff, J. Virol. 68:4910-4920, 1994). CR1 lies within the E1A amino terminus and binds host regulatory proteins such as the RB protein, p107, p130, and p300. Since simian virus 40 (SV40) large T antigen and human papillomavirus type 16 (HPV16) E7 protein also bind host regulatory factors, we investigated whether these viral proteins can complement E1A mutants which are defective in early gene activation. We show that the HPV16 E7 protein but not SV40 T antigen can complement mutations in the Ad5 E1A CR1 in the transactivation of viral early promoters. The inability of SV40 T antigen to complement suggests that RB binding on its own is not sufficient for early promoter transactivation by the E1A amino terminus. Nuclear runoff assays show that complementation by HPV16 E7 restores the ability of the E1A mutants to stimulate early gene expression at the level of transcription. Furthermore, nuclear extracts from the E7-transformed cells show increased binding activity of ATF and Oct-1, factors that can recognize the elements of Ad5 early genes, consistent with gene activation by E1A and E7 at the transcriptional level.

The Mouse B-raf Gene Encodes Multiple Protein Isoforms with Tissue-specific Expression

The c-Rmil/B-raf proto-oncogene is a member of the mil/raf family encoding serine/threonine protein kinases shown to be involved in signal transduction from the membrane to the nucleus. We isolated from a mouse brain library B-raf cDNAs containing a previously unidentified 36-base pair alternatively spliced exon located between exons 8 and 9 and, therefore, designated exon 8b. Human and mouse B-raf mRNAs also contain the 120-base pair alternatively spliced exon 10 previously described in the avian c-Rmil gene. Independent splicing of these two exons, located between the conserved region 2 (CR2) and the catalytic domain (CR3) gives rise to mRNAs potentially encoding four distinct proteins. By using specific sera generated against different portions of B-Raf, we identified at least 10 protein isoforms in adult mouse tissues. Some isoforms, in the range of 69-72 kDa, are not recognized by antisera directed against peptides encoded by exons 1 and 2, indicating the existence of B-Raf proteins with two different NH2 extremities. The other isoforms, in the range of 79-99 kDa, contain the amino acids encoded by exons 1 and 2, by either or both of the alternatively spliced exons, and, possibly, by another of the unidentified exon. Analysis of B-raf mRNA expression by reverse transcriptase-polymerase chain reaction and immunocharacterization of B-Raf proteins in different tissues of the adult mouse showed a tissue-specific pattern of B-Raf isoforms expression. Interestingly, isoforms containing amino acids encoded by exon 10 are specifically expressed in neural tissues. Taken together, these results suggest that distinct B-Raf proteins could be involved, in a tissue-specific manner, in signal transduction pathways.

Suppression of Mutations in Two Saccharomyces cerevisiae Genes by the Adenovirus E1A Protein

The protein products of the adenoviral E1A gene are implicated in a variety of transcriptional and cell cycle events, involving interactions with several proteins present in human cells, including parts of the transcriptional machinery and negative regulators of cell division such as the Rb gene product and p107. To determine if there are functional homologs of E1A in Saccharomyces cerevisiae, we have developed a genetic screen for mutants that depend on E1A for growth. The screen is based on a colony color sectoring assay which allows the identification of mutants dependent on the maintenance and expression of an E1A-containing plasmid. Using this screen, we have isolated five mutants that depend on expression of the 12S or 13S cDNA of E1A for growth. A plasmid shuffle assay confirms that the plasmid-dependent phenotype is due to the presence of either the 12S or the 13S E1A cDNA and that both forms of E1A rescue growth of all mutants equally well. The five mutants fall into two classes that were named web1 and web2 (for "wants E1A badly"). Plasmid shuffle assays with mutant forms of E1A show that conserved region 1 (CR1) is required for rescue of the growth of the web1 and web2 E1A-dependent yeast mutants, while the N-terminal 22 amino acids are only partially required; conserved region 2 (CR2) and the C terminus are dispensable. The phenotypes of mutants in both the web1 and the web2 groups are due to a single gene defect, and the yeast genes that fully complement the mutant phenotypes of both groups were cloned. The WEB1 gene sequence encodes a 1,273-amino-acid protein that is identical to SEC31, a protein involved in the budding of transport vesicles from the endoplasmic reticulum. The WEB2 gene encodes a 1,522-amino-acid protein with homology to nucleic acid-dependent ATPases. Deletion of either WEB1 or WEB2 is lethal. Expression of E1A is not able to rescue the lethality of either the web1 or the web2 null allele, implying allele-specific mutations that lead to E1A dependence.