PXDLS Motif Research Articles

Two Nonconsensus Sites in the Epstein-Barr Virus Oncoprotein EBNA3A Cooperate to Bind the Co-repressor Carboxyl-terminal-binding Protein (CtBP)

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.

Structural determinants outside the PXDLS sequence affect the interaction of adenovirus E1A, C-terminal interacting protein and Drosophila repressors with C-terminal binding protein

C-Terminal binding protein (CtBP) interacts with a highly conserved amino acid motif (PXDLS) at the C terminus of adenovirus early region 1A (AdE1A) protein. This amino acid sequence has recently been demonstrated in the mammalian protein C-terminal interacting protein (CtIP) and a number of Drosophila repressors including Snail, Knirps and Hairy. In the study described here we have examined the structures of synthetic peptides identical to the CtBP binding sites on these proteins using NMR spectroscopy. It has been shown that peptides identical to the CtBP binding site in CtIP and at the N terminus of Snail form a series of β-turns similar to those seen in AdE1A. The PXDLS motif towards the C terminus of Snail forms an α-helix. However, the motifs in Knirps and Hairy did not adopt well-defined structures in TFE/water mixtures as shown by the absence of medium range NOEs and a high proportion of signal overlap. The affinities of peptides for Drosophila and mammalian CtBP were compared using enzyme-linked immunosorbent assay. CtIP, Snail (N-terminal peptide) and Knirps peptides all bind to mammalian CtBP with high affinity ( K i of 1.04, 1.34 and 0.52 μM, respectively). However, different effects were observed with dCtBP, most notably the affinity for the Snail (N-terminal peptide) and Knirps peptides were markedly reduced ( K i of 332 and 56 μM, respectively) whilst the Hairy peptide bound much more strongly ( K i for dCtBP of 6.22 compared to 133 μM for hCtBP). In addition we have shown that peptides containing identical PXDLS motifs but with different N and C terminal sequences have appreciably different affinities for mammalian CtBP and different structures in solution. We conclude that the factors governing the interactions of CtBPs with partner proteins are more complex than simple possession of the PXDLS motif. In particular the overall secondary structures and amino acid side chains in the binding sites of partner proteins are of importance as well as possible global structural effects in both members of the complex. These data are considered evidence for a multiplicity of CtBPs and partner proteins in the cell.

Acetylation of adenovirus E1A regulates binding of the transcriptional corepressor CtBP.

Adenovirus E1A mediates its effects on cellular transformation and transcription by interacting with critical cellular proteins involved in cell growth and differentiation. The amino terminus of E1A binds to CBP/p300 and associated histone acetyltransferases such as P/CAF. The carboxyl terminus binds to the carboxyl-terminal binding protein (CtBP), which associates with histone deacetylases. We show that 12S E1A can be acetylated by p300 and P/CAF and map one of the acetylation sites to Lys-239. This Lys residue is adjacent to the consensus CtBP binding motif, PXDLS. Mutation of Lys-239 to Gln or Ala blocks CtBP binding in vitro and disrupts the E1A-CtBP interaction in vivo. Peptide competition assays demonstrated that the interaction of E1A with CtBP is also blocked by Lys-239 acetylation. Supporting a functional role for Lys-239 in CtBP binding, mutation of this residue to Ala decreases the ability of E1A to block cAMP-regulated enhancer (CRE)-binding protein (CREB)-stimulated gene expression. Finally, we demonstrate that Lys-239 is acetylated in cells by using an antibody directed against an acetyl-Lys-239 E1A peptide. CtBP interacts with a wide variety of other transcriptional repressors through the PXDLS motif, and, in many instances, this motif is followed by a Lys residue. We suggest that acetylation of this residue by histone acetyltransferases, and the consequent disruption of repressor complexes, might be a general mechanism for gene activation.