TI - CDH1 binds to HEF1 C terminal M2 domain containing two D boxes . AB - Upon examining HEF1 amino acids sequence , we found five putative D box [RxxLxxxx(N) ] . These putative D boxes are located within the SH3 domain ( box 1 ) , the SH2 binding sites domain ( box 2 ) , after the Ser rich domain ( box 3 ) and within the M2 domain of HEF1 ( box 4 & 5 ) ( Fig 4B ) . D box and KEN motif are the two motifs recognized by CDH1 on APC SUBstrates [30] . To determine which D box interacts with CDH1 , full-length HEF1 as well as T7-tagged HEF1 deletions , as illustrated in a cartoon in Figure 4C , were co-transfected with myc-tagged CDH1 into 293 cells . Cell lysates were subjected to immunoprecipitation using anti-myc antibody . Full-length HEF1 , HEF1 d113 ( 114-834 ) and HEF1 M2 domain ( 654-834 ) were found to bind to CDH1 ( Fig 4D , top panel , lanes 2 , 4 , & 5 ) . HEF1 1-154 did not show the ability to bind to CDH1 ( Fig 4D , top panel , lane 3 ) . Therefore , we predict that CDH1 binds to a D box located within the M2 (C terminal) domain that contains two putative D box , one located between residues 705 and 714 within the HLH motif , the other between amino acids 826 and 834 . Since CDH1 is a substRATE recognition component for APC ligase complex , the ability of CDH1 to bind to HEF1 suggests that HEF1 is a SUBstrate for the APC ligase . The detected interactions between Smad3 , HEF1 , APC10 and CDH1 is illustrated in a cartoon in Figure 4E .