Pontin52, an interaction partner of beta-catenin, binds to the TATA box binding protein - PubMed (original) (raw)
Pontin52, an interaction partner of beta-catenin, binds to the TATA box binding protein
A Bauer et al. Proc Natl Acad Sci U S A. 1998.
Abstract
beta-catenin, the vertebrate homolog of the Drosophila Armadillo protein, has been shown to have dual cellular functions, as a component of both the cadherin-catenin cell adhesion complex and the Wnt signaling pathway. At Wnt signaling, beta-catenin becomes stabilized in the cytoplasm and subsequently available for interaction with transcription factors of the lymphocyte enhancer factor-1/T-cell factor family, resulting in a nuclear localization of beta-catenin. Although beta-catenin does not bind DNA directly, its carboxyl- and amino-terminal regions exhibit a transactivating activity still not well understood molecularly. Here we report the identification of an interaction partner of beta-catenin, a nuclear protein designated Pontin52. Pontin52 binds beta-catenin in the region of Armadillo repeats 2-5 and, more importantly, also binds the TATA box binding protein. We provide evidence for an in vivo multiprotein complex composed of Pontin52, beta-catenin, and lymphocyte enhancer factor-1/T-cell factor. Our results suggest involvement of Pontin52 in the nuclear function of beta-catenin.
Figures
Figure 1
Affinity binding experiments with recombinant GST-β-catenin284 (GST-β284). Cell lysates from 1 × 107 metabolically labeled and 1.5 × 109 nonlabeled SW480 cells were affinity precipitated with a recombinant GST-β284 fusion protein. Bound proteins were eluted, separated by SDS/PAGE and analyzed by autoradiography (A) and Coomassie blue staining (CB).
Figure 2
Sequence-alignment of human Pontin52 with homologous proteins from rat (rTIP49) and S. cerevisiae (YDR190c). The boxed sequences contain WalkerA and WalkerB motifs responsible for ATP binding and ATP hydrolysis. Sequences derived from peptide sequencing analysis are underlined. Protein regions used for antibody production are indicated in filled boxes.
Figure 3
Association of Pontin52 and β-catenin in vivo. (A) Two independently raised anti-Pontin52 antibodies, anti-23 and anti-24, were used for immunoblots against cell lysates of SW480 cells. Immunoprecipitates (IP) collected from SW480 cell lysates with the same antibodies were subjected to Western blot analysis with anti-β-catenin antibodies, demonstrating an association of Pontin52 and β-catenin in living cells. Corresponding nonimmune sera were used in controls. Arrowhead indicates β-catenin. (B) For the reciprocal experiment, myc-tagged Pontin52 was transfected into SW480 cells, and the expression of Pontin52-MYC was monitored by immunoblotting with anti-myc antibodies (lysate). Transfected cells were immunoprecipitated with antibodies as indicated, and the immunoprecipitates were blotted and probed with anti-myc antibodies. Only immunoprecipitates collected with anti-β-catenin from transfected (t) cells contained Pontin52-MYC. Immunoprecipitations with anti-β-catenin from nontransfected SW480 cells (n) were included as controls. Arrowhead indicates Pontin52-MYC; ∗ indicates unreduced IgG heavy chains.
Figure 4
Pontin52 interacts directly with β-catenin and TBP. (A) A direct interaction of Pontin52 with β-catenin and TBP was demonstrated by affinity precipitation of recombinant His6-tagged β-catenin or His6-tagged TBP with the recombinant GST-Pontin52 fusion protein. The precipitate was analyzed by SDS/PAGE and Western blotting with anti-β-catenin antibodies (Left) or anti-TBP antibodies (Right). Filled arrowhead indicates His6-tagged TBP; open arrowhead indicates His6-tagged β-catenin. (B) To map the binding site(s) of Pontin52 in β-catenin several recombinant GST-β-catenin fusion proteins were used to affinity precipitate _in vitro-_translated Pontin52. The precipitates were analyzed by autoradiography. Arrowhead indicates Pontin52.
Figure 5
Pontin52 is a nuclear protein. Indirect immunofluorescence tests with the Pontin52-specific anti-25 antibody were performed on COS cells (A and _A_′). Pontin52 was found predominantly localized in the nucleus in dot-like structures. The dotted structures throughout the cytoplasm represent unspecific staining, because they also can be seen with the nonimmune sera (B and _B_′) or with anti-25 preincubated with recombinant GST-Pontin52 fusion protein (C and _C_′). (_A_′, _B_′, and _C_′) Hoechst 33342 staining of the same nuclei. (Bar: 20 μm.)
Figure 6
Pontin52 exhibits a widespread expression pattern. (A) Western blot analysis of cell lysates from human and mouse cell lines with Pontin52-specific antibody (anti-24). An equal number of cells for each cell line was taken for comparing the relative amount of Pontin52 protein. Arrowhead indicates Pontin52. (B) Northern blot analysis on human tissues probed with a 0.69-kb _Bgl_I–_Eco_RV fragment of the Pontin52 cDNA. A REAL blot was purchased from Invitrogen, which as indicated by the manufacturer had been loaded with 2 μg of total RNA per lane. A band expected for the size of the Pontin52 mRNA was detected in each of the tissues. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
Figure 7
In vivo association of Pontin52 with the LEF-1-β-catenin complex. Several combinations of plasmids were transfected into N2A cells as indicated. Pontin52-myc was precipitated from cell lysates with myc-specific antibodies, and the immunoprecipitates were analyzed by Western blotting of HA-tagged LEF-1 with anti-HA specific antibodies. The expression of transfected β-catenin and Pontin52-MYC was monitored by Western blotting of cell lysates with anti-β-catenin and anti-myc antibodies. Arrowhead indicates LEF-1-HA; ∗ indicates degradation products of LEF-1-HA as monitored in parallel experiments with anti-LEF-1 antibodies (not shown).
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