Role of the C-Terminal Binding Protein PXDLS Motif Binding Cleft in Protein Interactions and Transcriptional Repression

Abstract

C-terminal binding proteins (CtBPs) are multifunctional proteins that can mediate gene repression. CtBPs contain a cleft that binds Pro-X-Asp-Leu-Ser (PXDLS) motifs. PXDLS motifs occur in numerous transcription factors and in effectors of gene repression, such as certain histone deacetylases. CtBPs have been depicted as bridging proteins that self-associate and link PXDLS-containing transcription factors to PXDLS-containing chromatin-modifying enzymes. CtBPs also recruit effectors that do not contain recognizable PXDLS motifs. We have investigated the importance of the PXDLS binding cleft to CtBP's interactions with various partner proteins and to its ability to repress transcription. We used CtBP cleft mutant and cleft-filled fusion derivatives to distinguish between partner proteins that bind in the cleft and elsewhere on the CtBP surface. Functional assays demonstrate that CtBP mutants that carry defective clefts retain repression activity when fused to heterologous DNA-binding domains. This result suggests that the cleft is not essential for recruiting effectors. In contrast, when tested in the absence of a fused DNA-binding domain, disruption of the cleft abrogates repression activity. These results demonstrate that the PXDLS binding cleft is functionally important but suggest that it is primarily required for localization of the CtBP complex to promoter-bound transcription factors.The C-terminal binding proteins (CtBPs) function as transcriptional corepressors but also play roles in Golgi maintenance and in synaptic ribbon formation (6,8,49,55,58). They mediate transcriptional repression by associating with DNAbound transcription factors and recruiting a complex of chromatin-modifying enzymes (46)(47)(48)64). CtBPs have been shown to regulate gene expression in organisms from Drosophila to mammals. In Drosophila, CtBPs associate with developmental transcription factors such as Krüppel, Hairy,. In mammals, CtBPs associate with a number (about 30) of different sequence-specific transcription factors (9,25,31,57,61). Knockout studies with mice, in combination with microarray analyses, have implicated the mammalian CtBPs in the repression of genes involved in apoptosis and in the epithelialto-mesenchymal transition (13,15,16).Structural analyses have revealed that CtBP is composed of three domains, the substrate-binding domain that primarily comprises the PXDLS binding cleft, the central nucleotidebinding domain that is responsible for NAD(H) binding and dimerization, and an intrinsically unstructured C-terminal region (32, 33). Details of the PXDLS cleft (hereafter referred to as the PIDLS cleft, as X is often Ile) have come from studies of one member of the CtBP family, rat CtBP3/BARS (recently renamed short-CtBP1 or CtBP1-S) (5, 32). Mutagenesis has confirmed that two residues in the CtBP cleft, A41 and V55, are critical to PIDLS recognition (32).Although PIDLS motifs in transcription factors have been implicated in CtBP binding, the mechanisms through which CtBP then recruits chromatin-modifying enzymes ar...

The CACCC-Binding Protein KLF3/BKLF Represses a Subset of KLF1/EKLF Target Genes and Is Required for Proper Erythroid MaturationIn Vivo

“…Interestingly, only three genes are significantly downregulated in the absence of KLF3. These results suggest that KLF3 primarily acts as a transcriptional repressor in vivo, consistent with previous functional studies in vitro (17,22,29).…”

CtBPs promote mitotic fidelity through their activities in the cell nucleus

“…Structural and mutational analysis of CtBPs have identified discreet functional domains within the proteins; the N terminus forms a hydrophobic cleft that contacts the PxDLS motif in many of its partner transcriptional regulators, and the core central domain undergoes NAD þ /NADH-dependent dimerization, which is required for the assembly of the chromatinmodifying complex (Nardini et al, 2003;Quinlan et al, 2006;Chinnadurai, 2007;Kuppuswamy et al, 2008). CtBP chromatin-modifying complexes are readily detectable in proliferating cancer cells in culture (Shi et al, 2003), and there is evidence that their formation is increased under conditions of increased glycolysis, for example, in response to hypoxia (Zhang et al, 2002).…”

“…To test this assumption, we generated GST-CtBP DN V72R. V72 in CtBP2 is a critical residue in the PxDLS-binding domain, and its mutation to arginine (or mutation of the equivalent residue in CtBP1) has been clearly demonstrated by independent research groups to abrogate binding to PxDLS-containing proteins, including both chromatin-modifying enzymes and DNA-binding transcription factors (Nardini et al, 2003;Quinlan et al, 2006;Kuppuswamy et al, 2008). Thus, this mutant of CtBP DN would be unable to effectively compete with CtBPs for binding to PxDLS-motif-containing proteins.…”

Role of the PLDLS-Binding Cleft Region of CtBP1 in Recruitment of Core and Auxiliary Components of the Corepressor Complex

“…It is widely believed that the primary function of the PLDLS cleft is to link CtBP with DNA-binding factors. The second is a surface groove (located on the dinucleotide-binding domain) with which the RRT-motifcontaining proteins interact (43). The RRT binding groove is functionally redundant with the PLDLS cleft, and all RRTmotif-containing proteins also contain the PLDLS-like motifs.…”