| The regulated phosphorylation of proteins on specific tyrosyl residues is involved in an increasing number of cellular signaling events including growth, differentiation, migration, and adhesion. In vivo, tyrosine phosphorylation is reversible and dynamic. Phosphorylation states are determined by the balance of opposing activities of protein tyrosine kinases (PTKs), which catalyze phosphate transfer, and protein tyrosine phosphatases (PTPs), which catalyze phosphate hydrolysis. Because many PTKs are oncogenes and growth factors, most studies on tyrosine phosphorylation are focused on PTKs and the importance of PTPs has not been recognized until recent ten years.PCP-2, first identified by Wang HY et al in 1996, is a type lib RPTP characterized by the presence of one Ig-like domain, four FNIII domains and an N-terminal MAM domain. Until now, four types of B phosphotases (PTPu, PTPk , PCP-2 and PTP P) have been reported. The intracellular segment of PCP-2 has two phosphatase domains and a relatively long juxtamembrane segment that is homologous to the conserved cytoplasmic domain of cadherins, which is essential for the complex formation with the intracellular catenins. In adherent junctions, the cytoplasmic domain of E-cadherin binds directly to β-catenin that, in turn, associates with a-catenin, which is thought to link cadherin complex to the actin filament network and mediates stable cell adhesion. In addition to its adhesivefunctions, β-catenin has also been found to serve as a key component in Wnt signaling. Signal transduction via P -catenin involves its posttranslational stabilization and entry into the neucleus, where it interacts with transcription factors of T cell factor/lymphoid enhancer factor family to activate target genes involved in cell growth control and apoptosis such as c-myc and cyclinDl. It has become a hotspot because of its importance in embryonic development and tumorgenesis.There is increasing evidence to suggest that phosphorylation of tyrosyl residues in some components of the cadherin/catenin complex leads to loss of adhesive function and breakdown of adherens junction. But nowadays, there is still few reports on RPTP regulating cadherin/catenin complex and β-catenin transcription activity. Our previous study has proved that PCP-2 colocalized with the cadherin/catenin complex on cellular conjunction. The data suggest that PCP-2 may play a vital role in the regulation of the complex. Phosphorylation dependent release of β-catenin from the cadherin complex not only regulates the integrity and function of the adhesion complex, but may also be an alternative mechanism for activating β-catenin signaling.The wild type β-catenin was cloned by RT-PCR. The truncated 6 catenins were constructed by recombinated PCR and the fragments were inserted into the GFP expression vector. Cotransfection and dual luciferase reporter assay were performed to identify the function of PCP-2, PTPu , and PTPk in regulating the transcriptional activity of β-catenin. Cancer cell line expressing PCP-2 was generated and westernblot, co-immunoprecipitation and dual luciferase reporter assay were performed to detect the change of the transcriptional activity and the expression of target gene modified by β-catenin. The subcellular localization of β-catenin was detected by immunof lurescence staining and its phosphorylation level was measured by coimmunoprecipitation. Western blot and immunoprecipitation were used to reveal the interaction between PCP-2 and cadherin/catenin complex. GST-pulldown, immunoprecipitation and immunoflurescence were used to detect the regulation of PCP-2 to cadherin/catenin complex. We used growth assay, wound assay, colony formation assay and xenograft tumor growth assay to investigate the function of PCP-2 in SW480 cells.In this study, we detected a specific complex formation between PCP-2 and β-catenin through the Oterminus of 3 -catenin using an in vivo binding assay. PCP-2 downregulated the transcriptional activity of wild type and constitutively activated β-catenin... |
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