quantitative RT-PCR shows that Wnt/b-catenin target genes were expressed more in C4-2 cells than those in C4-2/PKD1 cells. These results suggest that Wnt/b-catenin is more active in C4-2 cells than in C4-2/PKD1 cells. Since PKD1 directly phosphorylates b-catenin at ” threonine-112 and 120 residues, we examined the subcellular locations of bcatenin in the presence and absence of PKD1 activity. The endogenous b-catenin localizes to plasma membrane and a 2 Beta-Catenin T120 Phosphorylation portion of cytoplasm b-catenin accumulates in transGolgi Network, where it co-localizes with TGN marker P230. Pre-incubation with a PKD1 small molecule inhibitor CID755673, the accumulation of b-catenin in TGN becomes (-)-Blebbistatin diminished. Development and characterization of pT120 antibody Among the two PKD1 phosphorylation sites T112 and T120, T112 is also a site for casein kinase II phosphorylation, PKD1 is the only known kinase to phosphorylate T120. Therefore, we chose the pT120 for developing phosphothreonine specific antibody. In order to confirm specificity of pT120 
antibody, we first transfected HA-tagged wild type, T120I and T102I/T112R/ T120I mutants of b-catenin into mouse 3T3 cell, a PKD1 positive cell line. Cell lysates were used for Western blot. The pT120 antibody recognizes wild type b-catenin presumably phosphorylated, but not the mutants that lack T120 phosphorylation, suggesting the pT120 antibody specifically recognizes T120 phosphorylation. Next, we compared the pT120 b-catenin in C4-2 and C4-2/ PKD1 cells. Cell lysates from C4-2 cells and C4-2/PKD1 were blotted with either a conventional b-catenin H102 antibody or pT120 antibody. The H102 antibody detects a major b-catenin band around 90 kDa in both C4-2 and C4-2/PKD1 cells and a minor band below 90 kDa in C4-2/PKD1 cell lysate only. The pT120 antibody strongly reacts to the lower band in C4-2/PKD1 cell and weakly to “
10411478“the band at 90 kDa in C4-2 cells. The results are consistent with our previous observation that the PKD1 phosphorylated b-catenin abnormally appeared at lower molecular weight, although phosphorylated proteins usually appear at higher molecular weights in most cases. To further address the specificity of this pT120 antibody, we performed peptide competition assay. The antigenic phosphopeptide and non-phospho peptide were pre-mixed with the pT120 antibody individually and used to blot C4-2/PKD1 cell lysate. The phosphopeptide can compete with pT120 antibody binding, but not the normal peptide. Finally, knockdown PKD1 protein levels in LNCaP cells by shRNA ” resulted in lower pT120 antibody signal, suggesting that the T120 residue is a phosphorylation site for PKD1 in vitro. The data suggests that that pT120 antibody specifically recognizes the T120 phosphorylation of b-catenin. Overexpression of PKD1 blocks nuclear accumulation of active b-catenin To study the influence of PKD1 activity on b-catenin, we compared b-catenin subcellular distribution in C4-2 and C4-2/ PKD1 cells. Subcellular fractions enriched with soluble cytosolic, nuclear or total membrane fractions were prepared and blotted with pT120, 8E7, pS33/S37/T41, and H102 antibodies. In C42 cells, the pT120 b-catenin is predominately localized to total membrane fraction, with minor amounts in soluble cytosol and nucleus. In contrast, the pT120 b-catenin is readily detected in every subcellular fraction of C4-2/PKD1 cells. The active b-catenin detected by 8E7 antibody generally demonstrates an inverse relationship to pT120 b-ca
