Abstract
The stability and subcellular localization of beta-catenin, a protein that plays a major role in cell adhesion and proliferation, is tightly regulated by multiple signaling pathways. While aberrant activation of beta-catenin signaling has been implicated in cancers, the biochemical identity of transcriptionally active beta-catenin (ABC), commonly known as unphosphorylated serine 37 (S37) and threonine 41 (T41) β-catenin, remains elusive. Our current study demonstrates that ABC transcriptional activity is influenced by phosphorylation of T120 by Protein Kinase D1 (PKD1). Whereas the nuclear β-catenin from PKD1-low prostate cancer cell line C4-2 is unphosphorylated S37/T41/T120 with high transcription activity, the nuclear β-catenin from PKD1-overexpressing C4-2 cells is highly phosphorylated at T120, S37 and T41 with low transcription activity, implying that accumulation of nuclear β-catenin alone cannot be simply used as a read-out for Wnt activation. In human normal prostate tissue, the phosphorylated T120 β-catenin is mainly localized to the trans-Golgi network (TGN, 22/30, 73%), and this pattern is significantly altered in prostate cancer (14/197, 7.1%), which is consistent with known down regulation of PKD1 in prostate cancer. These in vitro and in vivo data unveil a previously unrecognized post-translational modification of ABC through T120 phosphorylation by PKD1, which alters subcellular localization and transcriptional activity of β-catenin. Our results support the view that β-catenin signaling activity is regulated by spatial compartmentation and post-translational modifications and protein level of β-catenin alone is insufficient to count signaling activity.
Highlights
Beta-catenin is a dual functional molecule which plays a structural role in cell-cell adhesion and a mediator for cellular signaling pathways [1]. b-catenin bridges the epithelial cell surface to cytoskeletal networks by binding to membranous molecule Ecadherin and a-catenin, which links to F-actin and cytoskeleton [1]
Studies show that b-catenin has two major pools: plasma membrane pool bound to E-cadherin which is relatively stable and cytoplasmic pool in a complex consisting of glycogen synthase kinase-3 (GSK-3), adenomatous polyposis coli (APC) and Axin that is dynamically balanced between degradation and accumulation
Semi-quantitative RT-PCR shows that Wnt/b-catenin target genes were expressed more in C4-2 cells than those in C4-2/Protein Kinase D1 (PKD1) cells (Fig. 1B). These results suggest that Wnt/b-catenin is more active in C4-2 cells than in C4-2/PKD1 cells
Summary
Beta-catenin is a dual functional molecule which plays a structural role in cell-cell adhesion and a mediator for cellular signaling pathways [1]. b-catenin bridges the epithelial cell surface to cytoskeletal networks by binding to membranous molecule Ecadherin and a-catenin, which links to F-actin and cytoskeleton [1]. The S33/S37/ T41/S45 phosphorylated b-catenin localizes at several subcellular sites, including cell-cell contacts where the phosphorylated bcatenin associates with E-cadherin at the adherens junction and with APC at cell protrusions [9,10] These data suggest that Nterminally phosphorylated b-catenin may serve distinct functions in nucleus and cell migration. A key consequence of the Wnt signaling is to generate and accumulate nuclear transcriptionally active b-catenin (ABC), which has been known as unphosphorylated at S37/T41 [11,12] This b-catenin isoform, recognized by a monoclonal antibody 8E7 [11,12], is more readily detected at plasma membrane in complex with E-cadherin [13,14]. Upon Wnt stimulation, this isoform first moves to plasma membrane to form a complex with APC and LRP5 and translocates to nucleus [13]
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