Beta-catenin Forms Protein Aggregation at High Concentrations in HEK293TCells

Marie Saghaeian Jazi, Seyed Mahmoud Arab Najafi

Abstract


Background: The canonical Wnt signal transduction (or the Wnt/β-catenin pathway) plays a crucial role in the development of animals and in carcinogenesis. Beta-catenin is the central component of this signaling pathway. The activation of Wnt/β-catenin signaling results in the cytoplasmic and nuclear accumulation of β-catenin. In the nucleus, β-catenin interacts with the TCF/LEF transcription factors and, therefore, participates in the upregulation or downregulation of some important genes involved in diverse cellular activities. In addition, β-catenin is a critical component of the cadherin-mediated cell adherens junction. We had previously noticed that very high cellular concentrations of β-catenin had a negative effect on the transcriptional activity of this protein and, therefore, the aim of this study was to find a mechanism for this negative interaction. Methods: Cell fractionation, western blotting, and immunofluorescence microscopy experiments were performed to measure β-catenin protein levels and β-catenin cellular localization in HEK293Tcells transfected with various amounts of a β-catenin-encoding plasmid. Also, total RNA was extracted from the cells and used for reverse transcriptase-PCR experiments to measure the expression of the β-catenin target genes. SPSS, version 16, was used to analyze the results statistically. Results: We demonstrated that overexpression of β-catenin led to the formation of rod-shaped protein aggregates. The aggregate structures were mainly formed in the cell nucleus and were heavy enough to be isolated by centrifugation. Beta-catenin aggregate formation was accompanied by a decrease in the expression of theβ-catenin target genes used in this study.
Conclusion: Since deregulation of β-catenin function occurs in several human diseases, including cancer and neurological disorders, the results of this paper further support the possible biological and clinical significance of β-catenin aggregate formation.


Keywords


HEK293Tcells, Beta catenin, Cell aggregation factors, Nuclear localization signals

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pISSN: 0253-0716         eISSN: 1735-3688