The catenin p120(ctn) interacts with Kaiso, a novel BTB/POZ domain zinc finger transcription factor

Abstract

p120(ctn) is an Armadillo repeat domain protein with structural similarity to the cell adhesion cofactors beta-catenin and plakoglobin. All three proteins interact directly with the cytoplasmic domain of the transmembrane cell adhesion molecule E-cadherin; beta-catenin and plakoglobin bind a carboxy-terminal region in a mutually exclusive manner, while p120 binds the juxtamembrane region. Unlike beta-catenin and plakoglobin, p120 does not interact with alpha-catenin, the tumor suppressor adenomatous polyposis coli (APC), or the transcription factor Lef-1, suggesting that it has unique binding partners and plays a distinct role in the cadherin-catenin complex. Using p120 as bait, we conducted a yeast two-hybrid screen and identified a novel transcription factor which we named Kaiso. Kaiso's deduced amino acid sequence revealed an amino-terminal BTB/POZ protein-protein interaction domain and three carboxy-terminal zinc fingers of the C2H2 DNA-binding type. Kaiso thus belongs to a rapidly growing family of POZ-ZF transcription factors that include the Drosophila developmental regulators Tramtrak and Bric à brac, and the human oncoproteins BCL-6 and PLZF, which are causally linked to non-Hodgkins' lymphoma and acute promyelocytic leukemia, respectively. Monoclonal antibodies to Kaiso were generated and used to immunolocalize the protein and confirm the specificity of the p120-Kaiso interaction in mammalian cells. Kaiso specifically coprecipitated with a variety of p120-specific monoclonal antibodies but not with antibodies to alpha- or beta-catenin, E-cadherin, or APC. Like other POZ-ZF proteins, Kaiso localized to the nucleus and was associated with specific nuclear dots. Yeast two-hybrid interaction assays mapped the binding domains to Arm repeats 1 to 7 of p120 and the carboxy-terminal 200 amino acids of Kaiso. In addition, Kaiso homodimerized via its POZ domain but it did not heterodimerize with BCL-6, which heterodimerizes with PLZF. The involvement of POZ-ZF proteins in development and cancer makes Kaiso an interesting candidate for a downstream effector of cadherin and/or p120 signaling.

FIG. 1

(A) Schematic representation of Kaiso structure. The POZ domain and three C₂H₂ ZFs are indicated. The longest cDNA clone (S11) is shown aligned with clone F-38, which was isolated from the yeast two-hybrid screen. (B) Kaiso’s deduced amino acid sequence. The POZ domain residues (amino acids 12 to 117) are highlighted by a hatched line above the sequence, while the residues of the three ZFs are boxed and shaded. Eight proline-dependent serine or threonine phosphorylation sites are boxed, and the two highly acidic regions are highlighted by a gray line above the sequence. (C) Kaiso alignment with its human homolog. Kaiso has 87% identity with the deduced amino acid sequence of a human EST which localizes to chromosome Xq23. Most amino acid differences are conservative changes and occur outside the highly conserved POZ and ZF domains. This human EST, coupled with comigration of overexpressed and endogenous Kaiso, confirms the designation of Kaiso’s initiation and termination codons.

FIG. 1

(A) Schematic representation of Kaiso structure. The POZ domain and three C₂H₂ ZFs are indicated. The longest cDNA clone (S11) is shown aligned with clone F-38, which was isolated from the yeast two-hybrid screen. (B) Kaiso’s deduced amino acid sequence. The POZ domain residues (amino acids 12 to 117) are highlighted by a hatched line above the sequence, while the residues of the three ZFs are boxed and shaded. Eight proline-dependent serine or threonine phosphorylation sites are boxed, and the two highly acidic regions are highlighted by a gray line above the sequence. (C) Kaiso alignment with its human homolog. Kaiso has 87% identity with the deduced amino acid sequence of a human EST which localizes to chromosome Xq23. Most amino acid differences are conservative changes and occur outside the highly conserved POZ and ZF domains. This human EST, coupled with comigration of overexpressed and endogenous Kaiso, confirms the designation of Kaiso’s initiation and termination codons.

FIG. 2

Functional Kaiso motifs. (A) POZ domain comparison. Alignment of amino acid sequences of POZ domains of the most closely related POZ family proteins, murine PLZF and ZF-5 (Zn-5), is shown. A consensus sequence (Consen) (2) is shown for comparison. Kaiso has all 37 highly conserved residues found in the majority of POZ proteins as well as the 6 invariant residues (underlined). (B) Alignment of Kaiso’s three ZFs. Kaiso has three conserved ZFs of the C₂H₂ type that is commonly associated with DNA-binding transcription factors. All three fingers fit the consensus sequence [(F/Y)XCX₂CX₁₂HX_3-4H]. While the HX₃H (ZF-1 and ZF-2) spacing is more common than HX₄H (ZF-3), it is believed that HX₄H gives more flexibility for DNA binding.

FIG. 3

p120 coprecipitates Kaiso from MDCK and HCT116 cells. Whole-cell lysates were immunoprecipitated with the indicated antibodies, separated by SDS-PAGE, transferred to nitrocellulose, and then Western blotted with Kaiso-specific polyclonal antibody (pAb). (A) Kaiso was coprecipitated by p120 MAbs 15D2 and 12F4 from both MDCK and HCT116 cells (lanes 4, 6, 11, and 13). The p120 MAb 8D11, which does not cross-react with human p120, coprecipitated Kaiso from canine MDCK cells but not from human HCT116 cells, highlighting the requirement for p120 in the immunoprecipitate (compare lanes 5 and 12). In addition, p120 MAbs 6H11 and 5A7, which recognize only the p120 type 1 isoforms, did not coprecipitate Kaiso, consistent with the poor expression of this splice form in epithelial cells. (B) The reciprocal Western blot (WB) indicates the levels of p120 immunoprecipitated by the p120 MAbs and illustrates the specificity of MAb 8D11 for canine (MDCK) but not human (HCT116) p120.

FIG. 4

Kaiso interacts specifically with p120. Whole-cell lysates from MDCK and HCT116 cells were immunoprecipitated with antibodies to different components of the cadherin-catenin complex (indicated across the top of the panel), separated by SDS-PAGE, and Western blotted with Kaiso-specific polyclonal antibody (pAb). Kaiso coprecipitated efficiently with p120 MAb (lane 5) but not with antibodies to α-catenin (α-cat), β-catenin (β-cat), E-cadherin (E-cad), APC, BCL-6, or the control antibody, KT3.

FIG. 5

Subcellular localization of Kaiso in MDCK and NIH 3T3 cells by immunofluorescence. Endogenous Kaiso was primarily concentrated in the nucleus (A and C) as detected by immunofluorescence with Kaiso polyclonal antibodies. The staining was mostly diffuse (panel A) but was also associated with small punctate dot structures (panel C). Overexpressed Kaiso was efficiently localized to the nucleus (B) as detected by the Kaiso-specific MAb 12H9. However, in some cells, nuclear and cytosolic Kaiso was detected (D).

FIG. 6

Kaiso is ubiquitously expressed. (A) Northern blot analysis of Kaiso expression in tissues. A murine multiple tissue Northern blot (Clontech) was probed with a Kaiso cDNA probe (nt 870 to 2583) and washed under high stringency. A Kaiso mRNA of approximately 5 to 6 kb was ubiquitously expressed, and the lowest levels were found in the brain and testis. (B) Western blot analysis of Kaiso protein expression in various cell types. Whole-cell lysates of each cell line were normalized for equal protein amounts, immunoprecipitated with Kaiso MAb 6F8, and detected by Western blotting with a Kaiso polyclonal antibody (pAb). Cell lines are indicated at the top of the panel. Kaiso migrates as an ∼95-kDa doublet in most cells, but interestingly rodent Kaiso (NIH 3T3, CHO, MLL, AT2, and MC26 cell lines) migrated as a slightly larger doublet (∼110 kDa).

FIG. 7

Yeast two-hybrid interactions. Schematic representation of p120 and Kaiso mutant constructs. (A) The 10 Arm repeats which constitute p120’s Arm domain are indicated by the hatched boxes. Clones F-15 (column 15) and F-38 (column 38) are representative short and long Kaiso clones isolated in the yeast two-hybrid screen. A summary of the binding interactions is shown on the right. NA, not addressed because of autoactivation. (B) Kaiso’s POZ and ZF domains are indicated, and a summary of the binding results is shown on the right.