DIPA-family coiled-coils bind conserved isoform-specific head domain of p120-catenin family: potential roles in hydrocephalus and heterotopia

... exact role is unknown, the head domain likely mediates developmental and cancer-associated events linked to p120-1A expression (e.g., motility, invasion, metastasis). Here we identified delta-interacting protein A (DIPA) as the first head domainspecific binding partner and candidate mediator of isoform 1A activity. DIPA colocalizes with AJs in a p120-1A- but not 3A-dependent manner. Moreover, all DIPA family members (Ccdc85a, Ccdc85b/DIPA, and Ccdc85c) interact reciprocally with p120 family members

... and AP-1 signaling ( Du etal . , 2006 ). DIPA belongs to a family comprised of Ccdc85a, Ccdc85b (DIPA), and Ccdc85c. Each contains a pair of conserved coiled-coil motifs that are believed to participate in DNA transcriptional regulation. The C-terminal sequences following the coiled-coil domains are not conserved and do not contain known functional domains. Of interest, a mouse Ccdc85c mutation was identified recently as the causal factor for the hemorrhagic hydrocephalus ( hhy ) mutant mouse

...-binding domain. Y2H experiments using -catenin required the addition of 12.5 mM 3-amino-1,2,4-triazole (3-AT) to the medium to control for autoactivation. (B) Immunofluorescence detection of p120-knockdown MDCK cells with endogenous DIPA (3E3 anti-DIPA mAb) and one of five exogenous GFP fusion proteins: GFP alone, p120-3A, p120-1A, -catenin, or p0071. Scale bars, 25 m. Images are representative of at least three independent experiments. DIPA family members Ccdc85a and Ccdc85c share high homology in their N-terminal sequences, each of which contains two coiled-coil motifs ( Figure 4, A and B ). Thus we performed Y2H assays with full-length Ccdc85a or Ccdc85c versus p120-1A, p120-3A, -catenin, p0071, or ARVCF. Both DIPA family members exhibited specific interactions with p120-1A and not p120-3A ( Figure 4C ). They also interacted with all three p120 family members ( Figure 4C ). To test for interaction in cells, we stably introduced Flag-tagged Ccdc85a or Ccdc85c into p120-knockdown MDCK cells. p120-1A, p120-3A, -catenin, or p0071 tagged with GFP were then introduced and analyzed as before for colocalization ( Figure 4, D and E ). Exogenous Ccdc85a and Ccdc85c were expressed at similar levels and were effectively recruited to junctions by p120-1A-GPF, -catenin-GFP, and p0071-GFP. GFP alone or GFP-p120-3A, on the other hand, had no effect. In wild-type cells, both overexpressed Ccdc85a and Ccdc85c localized to AJs ( Figure 4, D and E , bottom). ARVCF-GFP vectors were generated several times and directly validated by sequencing but could not be expressed. Considering the positive Y2H data and the similarity between the ARVCF N-terminal region and the other p120 family members, however, it is likely that DIPA, Ccdc85a, and Ccdc85c interact with ARVCF as well. FIGURE 4: (AC) DIPA family members bind to p120-1A and its family members. (A) Schematic of DIPA, Ccdc85a, and Ccdc85c proteins with structured domains and relative sizes. CC1, coiled-coil 1. CC2, coiled-coil 2; Gly-rich, glycine-rich domain. Numbers indicate amino acids. (B) ClustalW alignment of approximately the first 250 amino acids of the human DIPA family members shows at least 54% similarity and 73% identity in the coiled-coil regions. Shaded boxed areas indicate identical amino acids for at least two of the proteins. Blue and red brackets enclose the DIPA CC1 and CC2, respectively. National Center for Biotechnology Information accession numbers: Ccdc85b (DIPA), AAB05928.1; Ccdc85a, AAI31558.1; Ccdc85c

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Gene: CCDC85A - coiled-coil domain containing 85A - (NCBI)

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