The PDZ Protein Tax-interacting Protein-1 Inhibits β-Catenin Transcriptional Activity and Growth of Colorectal Cancer Cells

Abstract

Wnt signaling is essential during development while deregulation of this pathway frequently leads to the formation of various tumors including colorectal carcinomas. A key component of the pathway is ␤-catenin that, in association with TCF-4, directly regulates the expression of Wnt-responsive genes. To identify novel binding partners of ␤-catenin that may control its transcriptional activity, we performed a mammalian twohybrid screen and isolated the Tax-interacting protein (TIP-1). The in vivo complex formation between ␤-catenin and TIP-1 was verified by coimmunoprecipitation, and a direct physical association was revealed by glutathione S-transferase pull-down experiments in vitro. By using a panel of deletion mutants of both proteins, we demonstrate that the interaction is mediated by the PDZ (PSD-95/DLG/ZO-1 homology) domain of TIP-1 and requires primarily the last four amino acids of ␤-catenin. TIP-1 overexpression resulted in a dose-dependent decrease in the transcriptional activity of ␤-catenin when tested on the TOP/FOPFLASH reporter system. Conversely, siRNA-mediated knock-down of endogenous TIP-1 slightly increased endogenous ␤-catenin transactivation function. Moreover, we show that overexpression of TIP-1 reduced the proliferation and anchorageindependent growth of colorectal cancer cells. These data suggest that TIP-1 may represent a novel regulatory element in the Wnt/␤-catenin signaling pathway.

The Terminal Region of β-Catenin Promotes Stability by Shielding the Armadillo Repeats from the Axin-scaffold Destruction Complex

“…1, B and C). This suggests that the PDZ binding domain motif and its various potential binding partners (32)(33)(34)(35) are dispensable for ␤-catenin stabilization. To rule out the possibility that other regions of the C terminus interact with a factor required for stabilization (Fig.…”

Cell polarity and cancer – cell and tissue polarity as a non-canonical tumor suppressor

“…The interaction between an oncoprotein and Dlg is not unique to E6. Dlg also binds to the Tax1 oncoprotein from human Tlymphotropic virus type 1 (HTLV-1) and the adenovirus oncoprotein 9ORF1 (Hirata et al, 2004;Kanamori et al, 2003;Lee et al, 1997). As viral proteins are under selective evolutionary pressure to keep only the most essential functions, these findings suggest that degradation of Dlg and Scrib is necessary for malignant transformation.…”

PDZ Protein Interactions Underlying NMDA Receptor-Mediated Excitotoxicity and Neuroprotection by PSD-95 Inhibitors

“…The interactions of these four MAGUKs with NMDARs have already been established (Kornau et al, 1995;Kim et al, 1996;Muller et al, 1996;Christopherson et al, 1999). The fifth PDZ interacting strongly at 7 nM Tat-NR2B9c was TIP1, a protein originally identified on the basis of binding to the TAX oncoprotein from human T-cell leukemia virus that has subsequently been demonstrated to interact with ␤-catenin in oncogenic pathways (Kanamori et al, 2003). It is plausible that TIP1 could act as a tumor suppressor and that, when bound by a virus or TatNR2B9c, it allows derepression of oncogenic or anti-apoptotic pathways.…”

“…Examples include stroke (Sattler et al, 1999; Aarts et al, 2002), cancers (Kanamori et al, 2003;Hirai et al, 2004;Doorbar, 2006), infection (Excoffon et al, 2004;Xie et al, 2006), and disorders of lipid metabolism (Yesilaltay et al, 2005). Although we focused on relevant PDZ interactions of NMDAR subunits and of Tat-NR2B9c, the broader utility of this interaction assay as a drug discovery platform is evident.…”

“…A presynthesized small interfering RNA (siRNA; Invitrogen) mix was prepared in DEPC water. The following mRNA target sequences were used in designing the siRNA: SAP97, 5Ј-AAAC-CCAAATCCATGGAAAAT-3Ј; PSD-93, 5Ј-CCCGATTGAAGACAGT-GAAGTTCAA-3Ј; SAP102, 5Ј-GGCCAGACATACGAACAAG-CAAATAA-3Ј; PSD-95, 5Ј-AAAGAGGCAGGTTCCATCGTT-3Ј (Futai et al, 2007); nNOS, 5Ј-AAGAACAAGGGCGTCTTCAGA-3Ј; and TIP1, 5Ј-AAGTTGCGTCAAGGTGAGAAC-3Ј (Kanamori et al, 2003).…”