Membrane fractions were either resuspended in PBS and analyzed by Western blot, or were subjected to sucrose density gradient centrifugation, performed according to [22]

Membrane fractions were either resuspended in PBS and analyzed by Western blot, or were subjected to sucrose density gradient centrifugation, performed according to [22]. [35S]-methionine/cysteine metabolic labeling Steady state metabolic labeling of proteins with [35S]-methionine/cysteine was done according to [25]. 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 [22]. 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 [47]. Staining patterns observed for all phospho–catenin antibodies are (R)-ADX-47273 diminished by -catenin silencing by siRNA (not shown) or genetic ablation [22].(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 [39].(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 [1], [2], [3], [4]. 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 [6]. 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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