Membrane fractions were either resuspended in PBS and analyzed by Western blot, or were subjected to sucrose density gradient centrifugation, performed according to . [35S]-methionine/cysteine metabolic labeling Steady state metabolic labeling of proteins with [35S]-methionine/cysteine was done according to . myc epitope tags, which retard -catenin mobility in SDS-PAGE by 20 kDa.(0.43 MB TIF) pone.0010184.s001.tif (417K) GUID:?260B3A94-3D26-45F6-A2E8-E8CF73092A64 Figure S2: N-terminally phosphorylated -catenin does not associate with a GST-cadherin cytoplasmic domain by affinity precipitation. A detergent-free cytosolic fraction from SW480 cells was sequentially incubated with GST-cadherin cytoplasmic domain coupled-glutathione sepharose beads. nonbinding lane contains the total unbound fraction precipitated with Trichloroacetic Acid. Note that while a significant fraction of total -catenin can be affinity precipitated by GST-cadherin, -catenin phosphorylated at S33/37/T41 does not associate.(0.38 MB TIF) pone.0010184.s002.tif (372K) GUID:?CB105C9A-0691-40E1-812B-3FBBE4BC9DEE Figure S3: Specificity of phospho–catenin antibodies in SW480 cells. SW480 cells were transfected with siRNAs against human -catenin (L-003482-00-0005) ON-TARGETplus SMARTpool siRNA (Thermo Scientific Dharmacon) or non-targeting control sequences (D-001810-10-05) using DharmaFECT reagent (Thermo). After 48 (R)-ADX-47273 hours, cells were solubilized and subjected to SDS-PAGE immunoblot analysis with the antibodies specified. GAPDH protein levels (control) do not change upon -catenin silencing (not shown). Note that the phospho–catenin antibodies almost exclusively recognize a single band over a 15C200 kDa range, and this band disappears upon -catenin silencing. The 120 kDa band detected with the P33/37/41 antibody is typically much less abundant than phospho–catenin . We have previously determined that the 160 kDa band detected with the ABC antibody (*) does not account for the nuclear staining in SW480s, although may be an issue in other cell types . Staining patterns observed for all phospho–catenin antibodies are (R)-ADX-47273 diminished by -catenin silencing by siRNA (not shown) or genetic ablation .(2.33 MB TIF) pone.0010184.s003.tif (2.2M) GUID:?1336EDDB-1C36-44B6-8D62-77E28E352BBC Figure S4: Phosphatase treatment of total -catenin (R)-ADX-47273 removes N-terminal phosphorylations but does not unmask the ABC epitope. SW480 cells were solubilized in 1% TX-100 lysis buffer and total -catenin was immunoprecipitated. Reactions were divided and treated with and without lambda phosphatase or phosphatase inhibitors for 30 minutes. Reaction was quenched with sample buffer prior to SDS-PAGE. Phosphatase treatment does not appear to unmask the ABC epitope, but does remove N-terminal phosphates. Note that longer incubation times (up to 18 hours) were still unable to unmask the ABC epitope, despite evidence to the contrary by Hendriksen et al .(0.72 MB TIF) pone.0010184.s004.tif (701K) GUID:?F63091F8-187F-4F79-94C4-721C1E97576B Abstract and generate distinct signaling and adhesive forms of -catenin at the level of gene expression. Whether vertebrates, which rely on a single -catenin gene, generate unique adhesive and signaling forms at the level of protein modification remains unresolved. We show that -catenin unphosphorylated at serine 37 (S37) and threonine 41 (T41), commonly referred to as transcriptionally Active -Catenin (ABC), is a minor nuclear-enriched monomeric form of -catenin in SW480 cells, which express low levels of E-cadherin. Despite earlier indications, the superior signaling activity of ABC is not due to reduced cadherin binding, as ABC is readily FABP5 incorporated into cadherin contacts in E-cadherin-restored cells. -catenin phosphorylated at serine 45 (S45) or threonine 41 (T41) (T41/S45) or along the GSK3 regulatory cassette S33, S37 or T41 (S33/37/T41), however, is largely unable to associate with cadherins. -catenin phosphorylated at T41/S45 and unphosphorylated at S37 and T41 is predominantly nuclear, while -catenin phosphorylated at S33/37/T41 is mostly cytoplasmic, suggesting that -catenin hypophosphorylated at S37 and T41 may be more active in transcription due to its enhanced nuclear accumulation. Evidence that phosphorylation at T41/S45 can be spatially separated from phosphorylations at S33/37/T41 suggests that these phosphorylations may not always be coupled, raising the possibility that phosphorylation at S45 serves a distinct nuclear function. Introduction -catenin is a prototypic example of a dual-function adhesion signaling protein. At the cell surface, -catenin binds the cytoplasmic domain of cadherin-type adhesion receptors which, together with the actin-binding protein, -catenin, allows cells to link their cytoskeletal networks through robust intercellular adhering junctions , , , . In the cytoplasm and nucleus, a cadherin-independent pool of -catenin transduces extracellular Wnt signals by interacting with TCF-type transcription factors to activate target genes that control cellular differentiation. In make a neural splice form of -catenin, which lacks the C-terminus and is used exclusively for neural cell adhesion . Since vertebrates only rely on a single -catenin gene, it has been speculated that vertebrates generate distinct signaling and adhesive forms of -catenin through post-translational modification. The best-known modifications of -catenin are a series of phosphorylations that continually promote degradation of the cadherin-free pool of -catenin. Specifically, CK1 phosphorylates.
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