Figure 2

Groucho binds to LEF-1 via a motif in the CRD and the HMG DNA binding domain. A) Glutathione beads bound with either GST-Groucho (middle panel) or GST (bottom panel) were tested for interaction with in vitro translated ³⁵S LEF-1 deletion constructs (translations shown in the top panel refer to deletions depicted in Fig. 1). B) Additional LEF-1 N-terminal deletions 1–234 and 1–191 (lanes 2 and 3) were not able to interact with GST-Groucho. Again, LEF-1 1–256 (lane 1) strongly interacts with Groucho but a deletion mutant lacking the entire CRD but retaining the HMG (Δ110–295, lane 4) weakly interacts. A GST negative control pull-down shows that LEF-1 does not interact with GST or the beads. Coomassie stained SDS-PAGE gel of purified GST-Groucho (lane 1) or GST alone (lane 2). C) Amino acid alignment of the putative Groucho binding sequence (GBS) and flanking sequences in LEF-1 and other human LEF/TCF family members as well as D. melanogaster and C. elegans worm orthologs, pangolin and POP-1 respectively. The LEF/TCF Groucho binding sequence described in this study, FS/T xxx I/L/V, is similar to the eh1 motif FxIxxIL [20]