Abstract
CtBP (carboxyl-terminal binding protein) has been shown to be a highly conserved co-repressor of transcription that is important in development, cell cycle regulation, and transformation. Viral proteins E1A and EBNA3C and all the various Drosophila and vertebrate transcription factors to which CtBP has been reported to bind contain a conserved "PXDLS" CtBP-interaction domain. Here we show that EBNA3A binds CtBP both in vitro and in vivo but that this interaction does not require a near consensus (98)PLDLR(102) motif in the NH(2) terminus of EBNA3A. However, further deletion and mutation analysis revealed that CtBP interacts with this viral protein through a cryptic, bipartite motif located in the COOH terminus of EBNA3A. The two components of this binding domain are similar to the canonical PXDLS motif but do not include the highly conserved, and normally critical, first proline residue. These nonconsensus sites, (857)ALDLS(861) and (886)VLDLS(890), synergize to produce very efficient binding to CtBP. Interaction with CtBP was shown to be important in the repression of transcription by EBNA3A and in the ability of EBNA3A to cooperate with activated Ras to immortalize and transform primary rat embryo fibroblasts. Similar bipartite sequences can be found in other viral and cellular proteins that can interact with CtBP, including the retinoblastoma-interacting protein-methyltransferase RIZ, the oncoprotein EVI1, and Marek's disease virus transforming protein Meq.
Highlights
E1A COOH-terminal binding protein (CtBP1)1 was initially identified as a cellular factor interacting with the COOH terminus of adenovirus E1A oncoproteins
EBNA3A Binds to GST-CtBP through a Sequence(s) Located in the COOH Terminus, Not Through 98PLDLR102—An analysis of the predicted amino acid sequence of EBNA3A had revealed a sequence in the NH2 terminus similar to the PLDLR CtBP binding motif in HDAC4, HPC2, and xPC [18]
Because CtBP is a co-repressor of transcription and EBNA3A can repress transcription and is a close relative of EBNA3C, the ability of in vitro translated EBNA3A to bind to a GST fusion with CtBP was tested
Summary
E1A COOH-terminal binding protein (CtBP1) was initially identified as a cellular factor interacting with the COOH terminus (amino acids 225–238) of adenovirus E1A oncoproteins. On some promoters CtBP-mediated repression is independent of HDAC activity [15], so presently the precise molecular mechanism by which CtBP inhibits transcription is unknown and may turn out to be quite different in different situations [11, 12, 15,16,17] All these various factors that regulate transcription and bind to CtBP contain a conserved Pro-X-Asp-Leu-Ser (“PXDLS”) CtBPinteraction domain that is necessary and probably sufficient for the interaction. Because Cp is the promoter for all EBNA mRNA initiation in lymphoblastoid cells immortalized by EBV (LCLs), the EBNA3 proteins probably contribute to a negative autoregulatory control loop Both EBNA3A and EBNA3C exhibit robust repressor activity when targeted to DNA by fusion with the DNA-binding domain of Gal4 [33,34,35]. The repression by EBNA3C is partly mediated by interactions with cellular histone deacetylase and possibly CtBP, but the mechanism of EBNA3A-mediated repression is unknown
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