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.

E1A12S-mediated activation of the adenovirus type 12 E2 promoter depends on the histone acetyltransferase activity of p300/CBP.

Activation of the transcription unit early region 2 (E2) promoter of the oncogenic adenovirus serotype 12 (Ad12), which regulates the expression of proteins essential for viral replication, requires the assembly of a ternary complex consisting of cAMP response element-binding protein (CREB)-1/activating transcription factor (ATF)-1, the Ad12 12S oncogene product of early region 1A (E1A(12S)), and the co-activator p300/CBP on the E2(Ad12) cAMP response element (E2-CRE). Here we show that the active E2(Ad12) promoter is associated with acetylated histone H4 whereas an E2-CRE point-mutated promoter which is transcriptionally inactive due to its inability to assemble this ternary complex is not bound by acetylated histone H4. The histone deacetylase 1 as well as Roscovitine, which blocks the activation of the histone acetyltransferase (HAT) activity of CBP by cyclin E-Cdk2, prevents E2(Ad12) promoter activation through E1A(12S). p300/CBP counteracts the repressive function of histone deacetylase 1 in a HAT domain-dependent manner whereas the p300/CBP-associated factor PCAF failed to rescue E2(Ad12) promoter activity. E1A(12S) bound p300/CBP displays strong HAT activity. Most interestingly, E1A(12S)-mediated activation of the E2(Ad12) promoter correlates well with the ability of the viral protein to associate with the HAT activity of p300/CBP in vivo. Taken together these data indicate that the recruitment of the HAT activity of p300/CBP by E1A(12S) plays an important role in E2(Ad12) promoter activation.

cAMP-independent activation of the adenovirus type 12 E2 promoter correlates with the recruitment of CREB-1/ATF-1, E1A(12S), and CBP to the E2-CRE.

Expression of the transcription unit early region 2 (E2) is of crucial importance for adenoviruses because this region encodes proteins essential for viral replication. Here, we demonstrate that the E1A(12S) protein of the oncogenic adenovirus serotype 12 activates the E2 promoter in dependence of the N terminus and the conserved region 1. Activation is mediated through a cAMP-response element that is bound by CREB-1 and ATF-1. Moreover, the Ad12 E2 promoter is inducible by protein kinase A and repressed by either a dominant-negative cAMP-response element-binding protein (CREB) mutant or the highly specific protein kinase A inhibitor protein underscoring the participation of CREB-1/ATF-1 in promoter activation. E1A(12S) binds to CREB-1 and ATF-1 in dependence of the N terminus and CR1 and is recruited to the E2 cAMP-response element through both cellular transcription factors. Most interestingly, point mutations revealed that E1A(12S) domains essential for binding to CREB-1/ATF-1 and for activation of the Ad12 E2 promoter are also essential for binding to the CREB-binding protein. Due to these data and results obtained in DNA-dependent protein-protein interaction assays, we propose a model in which the cAMP-independent activation of the Ad12 E2 promoter is mediated through a ternary complex consisting of CREB-1/ATF-1, E1A(12S), and CREB-binding protein, which assembles on the E2 cAMP-response element.

Differences in the interactions of oncogenic adenovirus 12 early region 1A and nononcogenic adenovirus 2 early region 1A with the cellular coactivators p300 and CBP.

Association with the cellular coactivators p300 and CBP is required for the growth-regulatory function of adenoviral (Ad) early region 1A (E1A) proteins. E1A regions necessary for these interactions overlap with domains involved in the induction of tumours in immunocompetent rodents through highly oncogenic Ad12. Differences in the association of cellular factors with the respective E1A domains of Ad12 and nononcogenic Ad2 might therefore be involved in serotype-specific oncogenicity. We analyzed the interaction of the Ad12 E1A 235R protein with p300 and CBP. Here we demonstrate that in the case of Ad12, but not Ad2/5, amino acids (aa) 1-29 of E1A proteins are sufficient to bind the p300-C/H3 domain in vivo and wild-type p300 in vitro. The conserved arginine-2, which is essential for the interaction between Ad2 E1A and p300, was dispensable for the Ad12 E1A 235R-p300 interaction in vitro. In addition to the p300-C/H3 region, we identified a second domain within p300 (aa 1999-2200) binding to the 235R protein. Contrary to p300, the amino-terminus and CR1 are necessary to associate with CBP. The aa 1-29 of the 235R protein but not CR1 are essential for the repression of colTRE-driven gene expression. This repression function is strictly dependent on p300 but not on CBP.