C-terminal binding protein (CtBP) binds to adenovirus early region 1A (AdE1A) through a highly conserved PXDLS motif close to the C terminus. We now have demonstrated that CtBP1 also interacts directly with the transcriptional activation domain (conserved region 3 [CR3]) of adenovirus type 5 E1A (Ad5E1A) and requires the integrity of the entire CR3 region for optimal binding. The interaction appears to be at least partially mediated through a sequence ( 161 RRNTGDP 167 ) very similar to a recently characterized novel CtBP binding motif in ZNF217 as well as other regions of CR3. Using reporter assays, we further demonstrated that CtBP1 represses Ad5E1A CR3-dependent transcriptional activation. Ad5E1A also appears to be recruited to the E-cadherin promoter through its interaction with CtBP. Significantly, Ad5E1A, CtBP1, and ZNF217 form a stable complex which requires CR3 and the PLDLS motif. It has been shown that Ad513SE1A, containing the CR3 region, is able to overcome the transcriptional repressor activity of a ZNF217 polypeptide fragment in a GAL4 reporter assay through recruitment of CtBP1. These results suggest a hitherto-unsuspected complexity in the association of Ad5E1A with CtBP, with the interaction resulting in transcriptional activation by recruitment of CR3-bound factors to CtBP1-containing complexes.
C-terminal binding protein (CtBP) has been shown to bind to a highly conserved five-amino-acid motif (PXDLS) located very close to the C-terminus of adenovirus early region 1A proteins. It has also been demonstrated that amino acids C-terminal and N-terminal to this original proposed binding site contribute to the interaction. However, conflicting evidence has been presented to show that acetylation of an adjacent lysine residue in Ad5E1A may or may not influence binding. It has now been demonstrated here that acetylation of a lysine, equivalent to position 261 in Ad12 E1A and position 285 in Ad5E1A, in a synthetic peptide disrupts the binding to CtBP1 and CtBP2 and alters the K(i) of the peptide, indicative of a reduction in the affinity of the peptide for CtBP1 and CtBP2, but only to a rather limited extent (less than 2-fold). The solution structures of synthetic peptides equivalent to wild-type and acetylated forms of the Ad12 E1A peptide have been determined by proton NMR spectroscopy. The wild-type form of the peptide adopts a series of beta-turns over the region Val(254)-Arg(262). Within the acetylated isoform, the beta-turn conformation is less extensive, Val(260)-Arg(262) adopting a random confirmation. We conclude that secondary structure (beta-turns) and an appropriate series of amino acid side chains over an extended binding site (PXDLSXK) are necessary for recognition by CtBP, acetylation of lysine interfering with both of these features, but not to such an extent as to totally inhibit interaction. Moreover, it is possible that the beta-turn conformation at the C-terminus of AdE1A contributes to binding to alpha importin and nuclear import. Acetylation of lysine (261) could disrupt interaction through structural destabilization as well as charge neutralization and subsequent nuclear localization.
The molecular basis of clinical heterogeneity in paediatric ALL is poorly understood. Defects in cellular responses to DNA double strand breaks (DSBs) have been associated with resistance to DNA damaging agents and poor clinical response in patients with leukaemia. We have previously shown that one third of paediatric ALL tumours exhibit in vitro defect in ionising radiation (IR)-induced apoptosis, despite retained integrity of p53 pathway. In this study we addressed molecular basis of defective (DSB) response in paediatric ALL with the view of identifying novel therapeutic targets.
We analysed differences in transcriptional and posttranscriptional in vitro responses to IR in 22 ALL patients, stratified as either apoptotic resistant or apoptosis sensitive. Analysis of coordinate IR induced expression of functionally related genes, by two independent methods (gene set enrichment analysis, GSEA, and functional module analysis) revealed in resistant cases differential upregulation of multiple pro-survival pathways involved in EGF, PDGF, IGF, PI3 and MAPK signalling. In response to IR resistant ALLs also showed differentially induced phosphorylation of Akt and S6, downstream targets in PI-3 pathway. Array analysis of 42 receptor tyrosine kinases (TRKs), revealed differential phosphorylation of TRKs that act upstream of the PI3 including ErbB3,4, FGF R4,2a, VEGF R1,2 as well as IGF-I R. Furthermore, we were able to demonstrate that in apoptotic resistant leukaemias PI3/Akt upregulation during IR response lead to differential phosphorylation of DNA-PK catalytic subunit, a protein involved in non homologous end joining (NHEJ) repair of DNA DSBs. Consequently, increased repair of DSBs was observed in apoptosis resistant ALL cases and this was documented by faster resolution of IR induced intranuclear foci of γH2AX, a marker of DNA DSBs. Finally, pharmacological inhibition of pro-survival pathways PI3-Akt, IGF and MAPK in resistant leukaemias restored sensitivity to IR.
We suggest that abnormal activation of pro-survival pathways during DSB response may represent one of the mechanisms of resistance to DNA damaging agents in paediatric ALLs and that targeting these pathways should be considered as therapeutic approach in aggressive form of disease.
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