Wnt信号通路.ppt

Wnt信号通路 Wnt 信号传导途径 Abundance, complexation, and trafficking of Wnt/ -catenin signalingelements in response to Wnt3a Wnt3a regulates a canonical signaling pathway in early development that controls the nuclear accumulation of β-catenin and its activation of Lef/Tcf-sensitive transcription of developmentally important genes. Wnt3a stimulates changes in cellular abundance of several key signaling molecules in the canonical Wnt/ β-catenin pathway. The mouse F9 teratocarcinoma (F9) cells are embryonal and totipotent cells that can be stimulated to form primitive endoderm. Primitive endoderm formation was observed within 3-6 days of treatment with Wnt3a, established by immunoblotting of the PE-specific marker, cytokeratin Endo-A. Staining of blots of whole-cell lysates of Wnt3a-stimulated F9 cells with the TROMA-1 monoclonal antibody to Endo-A reveals increased expression of Endo-A. M50 construct, which harbors multiple ??-catenin binding sites in its promoter F9 cell lysates were subjected to SDSanalysis and the resolved proteins to immunoblotting. The blots were stained with antibodies against Axin, ????β-catenin, Dvl2, GSK3???? (phospho- and dephospho-forms), the phosphoprotein phosphatase 2A (i.e., the conserved catalytic or “C”-subunit) as well as for GAPDH (as a protein loading control). The F9 cells were either untreated (time = 0) or treated with purified Wnt3a for 5 to 90 min and then subjected to disruption, SDS, and quantification of cellular protein (via immunoblotting), setting the abundance of each protein established at zero time as “1” (fig. 1C). Axin and ??β-catenin display Wnt3a-induced accumulation over the 90 min stimulation. At 60-90 min post stimulation by Wnt3a, the abundance of ?? β -catenin is increased 3-fold, the abundance of Axin is increased 4- to 5-fold. The abundance of the phospho-GSK3?? also increases, but much more modestly (~ 50%), in response to Wnt3a stimulation. The abundance of Dvl2, total GSK3????????the protein phosphatase??2A (PP