Int J Stem Cells.  2018 Nov;11(2):242-247. 10.15283/ijsc18032.

Pja2 Inhibits Wnt/β-catenin Signaling by Reducing the Level of TCF/LEF1

Affiliations
  • 1Department of Life Science, University of Seoul, Seoul, Korea. ej70@uos.ac.kr
  • 2Department of Mathematics, University of Seoul, Seoul, Korea.

Abstract

Ubiquitination of proteins plays an essential role in various cellular processes, including protein degradation, DNA repair, and cell signaling pathways. Previous studies have shown that protein ubiquitination is implicated in regulating pluripotency as well as fate determination of stem cells. To identify how protein ubiquitination affects differentiation of embryonic stem cells, we analyzed microarray data, which are available in the public domain, of E3 ligases and deubiquitinases whose levels changed during stem cell differentiation. Expression of pja2, a member of the RING-type E3 ligase family, was up-regulated during differentiation of stem cells. Wnt/β-catenin signaling is one of the most important signaling pathways for regulation of the self-renewal and differentiation of embryonic stem cells. Pja2 was shown to bind to TCF/LEF1, which are transcriptional factors for Wnt/β-catenin signaling, and regulate protein levels by ubiquitination, leading to down-regulation of Wnt signaling activity. Based on these results, we suggest that E3 ligase Pja2 regulates stem cell differentiation by controlling the level of TCF/LEF1 by ubiquitination.

Keyword

Pja2; E3 ligase; Wnt; TCF/LEF1; Embryonic stem cell; Differentiation

MeSH Terms

DNA Repair
Down-Regulation
Embryonic Stem Cells
Humans
Ligases
Proteolysis
Public Sector
Stem Cells
Ubiquitin
Ubiquitin-Protein Ligases
Ubiquitin-Specific Proteases
Ubiquitination
Ligases
Ubiquitin
Ubiquitin-Protein Ligases

Figure

  • Fig. 1 Levels of pja2 mRNA and protein increase during mESC differentiation. (A) qPCR analyses for pja2 mRNA expression during mESC differentiation. Total RNAs were isolated at the indicated times. (B) Lysates were obtained at the indicated times, and the levels of proteins were detected by Western blotting using the indicated antibodies. Differentiation of mESCs was induced by withdrawal of LIF from culture media.

  • Fig. 2 Pja2 inhibits Wnt/β-catenin signaling. (A) Pja2 inhibits Wnt 3a-mediated reporter activity. TOP-Flash Wnt reporter construct was transfected with HA-pja2. Reporter activity was analyzed upon treatment with control and Wnt 3a-conditioned media. (B) Pja2 inhibits activator of Wnt signaling-mediated reporter activity. Reporter activity was measured after transfection with the indicated plasmids into HEK293T cells. **p<0.01.

  • Fig. 3 Inverse correlation between expression of pja2 and TCF/LEF1. Samples were collected from undifferentiated and differentiated mESCs on the indicated days in the figure, and Western blotting was performed with the indicated antibodies. Differentiation of mESCs was induced by removal of LIF from culture media.

  • Fig. 4 Pja2 interacts and down-regulates TCF/LEF1 by enhancing ubiquitination. (A) Pja2 interacts with TCF/LEF1. MYC-pja2- and HA-tagged LEF1, TCF1, and TCF3 were co-transfected into HEK293T cells. Lysates were immunoprecipitated with anti-MYC and immunoblotted by HA. (B) Pja2 reduces levels of TCF1 and LEF1. HEK293T cells were transiently transfected with siRNAs for pja2 and HA-pja2, which is resistant to siRNA, as indicated in the figure. Expression levels of proteins were measured by immunoblotting with the antibodies indicated in the figure. (C, D) Pja2 ubiquitinates TCF/LEF1. Flag-UB, HA-TCF1/LEF1, and MYC-pja2 were transfected into HEK293T cells. Lysates were immunoprecipitated with anti-HA antibody and blotted with anti-Flag antibody to measure ubiquitination of TCF (C) and LEF1 (D). Western blotting was performed with the antibodies indicated in the figure.


Reference

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