Wild type PKD1 was able to suppress the b-catenin/TCF transcription activity

lly demonstrates an inverse relationship to pT120 b-catenin, i.e. high levels of ABC in C4-2, especially in nuclei. The 17328890 nuclear 3 Beta-Catenin T120 Phosphorylation ABC is not detectable in the C4-2/PKD1 cells, although relative amount of nuclear b-catenin is comparable to C4-2 cells. Furthermore, phosphorylated S33/S37/T41 primarily present in C4-2/PKD1 correlates positively with pT120 distribution. Since the expression of Wnt target genes in C4-2/PKD1 cells is less than those in C4-2 cells, the nuclear b-catenin in C4-2/PKD1 cells, which consists of phosphorylated T120 b-catenin, is less transcriptionally active, suggesting that b-catenin protein level alone is insufficient to count signaling activity. Large differences in total b-catenin in cytoplasm and membrane fractions are also observed. Since the C4-2 is an E-cadherin negative cell and C4-2/PKD1 is an E-cadherin positive cell, it is reasoned that the b-catenin in C42/PKD1 cell is more membrane bound due to binding to Ecadherin and less likely to be found as soluble cytoplasm protein. In addition, immunofluorescence image demonstrate that the pT120 b-catenin in C4-2/PKD1 distributes in cytosol, nucleus and plasma membrane and co-localizes with TGN marker p230 in cytosol, which is consistent with H102 staining pattern. remains nearly unchanged after 6 hours treatment, prolonged treatment actually decreases the amount of ABC. Hendriksen et al. reported similar findings in mammary epithelial 9600591 cell cultures and suggested that the E-cadherin/bcatenin membranous complex resists Wnt stimulation. Interestingly, the known patterns of pT120 in un-stimulated C42 and C4-2/PKD1 cells, i.e., a high molecular weight band in C42 and a low molecular weight band in C4-2 PKD1 remain relatively unchanged throughout Wnt treatment. However, a new band recognized by pT120 antibody appears and accumulates in C4-2 cells after Wnt treatment up to 24 hours. The pT120 b-catenin accumulates at TGN of prostate epithelial cells We previously observed that PKD1 activity is associated with bcatenin membrane trafficking and the two proteins co-localize with multiple TGN markers, suggesting that pT120 b-catenin may involve in b-catenin membrane trafficking. Immunofluorescence staining with pT120 antibody in various prostate epithelial cells BAY-41-2272 reveals that pT120 subcellular localization has different patterns from H102 antibody. In C4-2 cells, which are E-cadherin negative, total b-catenin level is low and cytosol, pT120 b-catenin mainly localizes to paranuclear area. In E-cadherin positive cells, including C4-2/PKD1, LNCaP, RWPE1 and BPH-1 cells, the H102 antibody staining reveals bcatenin at plasma membrane. In addition, the pT120 antibody shows different localization patterns:, In C4-2/PKD1 cells, the pT120 b-catenin distributes to cytosol, nucleus and plasma membrane and the Overexpression of PKD1 prevents Wnt-induced b-catenin accumulation Although C4-2 cells do not express E-cadherin, C4-2/PKD1 is E-cadherin positive due to inhibition of transcription factor Snail by PKD1. Upon Wnt3a treatment, both total and active b-catenin increasingly accumulates in C4-2 cells after 6 and 24-hour stimulation. In contrast, total and pT120 b-catenin in C4-2/PKD1 cells remains unchanged before and after Wnt stimulation, active b-catenin 4 Beta-Catenin T120 Phosphorylation cytosol pT120 b-catenin co-localizes with TGN. In LNCaP and RWPE1 cells, the pT120 b-catenin distributes predominately as diffused cytosol protein. However, in