Abstract
A novel phosphorylation-specific antibody (alphapbeta-catenin) was generated against a peptide corresponding to amino acids 33-45 of human beta-catenin, which contained phosphorylated serines at positions 33 and 37. This antibody is specific to phosphorylated beta-catenin and reacts neither with the non-phosphorylated protein nor with phosphorylated or non-phosphorylated plakoglobin. It weakly interacts with S33Y beta-catenin but not with the S37A mutant. pbeta-catenin is hardly detectable in normal cultured cells and accumulates (up to 55% of total beta-catenin) upon overexpression of the protein or after blocking its degradation by the proteasome. Inhibition of both GSK-3beta and the proteasome resulted in a rapid (t1/2=10 minutes) and reversible reduction in pbeta-catenin levels, suggesting that the protein can undergo dephosphorylation in live cells, at a rate comparable to its phosphorylation by GSK-3beta. pbeta-catenin interacts with LEF-1, but fails to form a ternary complex with DNA, suggesting that it is transcriptionally inactive. Immunofluorescence microscopy indicated that pbeta-catenin accumulates in the nuclei of MDCK and BCAP cells when overexpressed and is transiently associated with adherens junctions shortly after their formation. pbeta-catenin only weakly interacts with co-transfected N-cadherin, although it forms a complex with the ubiquitin ligase component beta-TrCP. SW480 colon cancer cells that express a truncated APC, at position 1338, contain high levels of pbeta-catenin, whereas HT29 cells, expressing APC truncated at position 1555, accumulate non-phosphorylated beta-catenin, suggesting that the 1338-1555 amino acid region of APC is involved in the differential regulation of the dephosphorylation and degradation of pbeta-catenin.
MeSH terms
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Adenomatous Polyposis Coli Protein / genetics
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Adenomatous Polyposis Coli Protein / metabolism
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Amino Acid Sequence / genetics
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Animals
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Cadherins / genetics
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Cadherins / metabolism
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Carcinoma / genetics
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Carcinoma / metabolism*
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Carcinoma / physiopathology
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Cattle
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Cell Division / genetics*
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Cell Line, Transformed
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Cell Transformation, Neoplastic / genetics
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Cell Transformation, Neoplastic / metabolism*
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Colonic Neoplasms / genetics
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Colonic Neoplasms / metabolism*
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Colonic Neoplasms / physiopathology
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Cysteine Endopeptidases / drug effects
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Cysteine Endopeptidases / metabolism
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Cytoskeletal Proteins / genetics
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Cytoskeletal Proteins / metabolism*
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DNA / genetics
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DNA / metabolism
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DNA-Binding Proteins / genetics
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DNA-Binding Proteins / metabolism
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GTP-Binding Proteins / genetics
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GTP-Binding Proteins / metabolism
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Gene Expression Regulation, Neoplastic / genetics
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Glycogen Synthase Kinase 3 / genetics
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Glycogen Synthase Kinase 3 / metabolism
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Glycogen Synthase Kinase 3 beta
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Humans
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Lymphoid Enhancer-Binding Factor 1
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Mice
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Mice, Inbred BALB C
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Multienzyme Complexes / drug effects
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Multienzyme Complexes / metabolism
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Phosphorylation
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Proteasome Endopeptidase Complex
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Rats
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Serine / metabolism
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Trans-Activators / genetics
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Trans-Activators / metabolism*
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Transcription Factors / genetics
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Transcription Factors / metabolism
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Tumor Cells, Cultured
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Up-Regulation / drug effects
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Up-Regulation / genetics
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beta Catenin
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beta-Transducin Repeat-Containing Proteins
Substances
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Adenomatous Polyposis Coli Protein
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BTRC protein, human
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Btrc protein, mouse
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CTNNB1 protein, human
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CTNNB1 protein, mouse
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Cadherins
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Ctnnb1 protein, rat
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Cytoskeletal Proteins
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DNA-Binding Proteins
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LEF1 protein, human
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Lef1 protein, mouse
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Lef1 protein, rat
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Lymphoid Enhancer-Binding Factor 1
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Multienzyme Complexes
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Trans-Activators
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Transcription Factors
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beta Catenin
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beta-Transducin Repeat-Containing Proteins
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Serine
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DNA
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GSK3B protein, human
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Glycogen Synthase Kinase 3 beta
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Gsk3b protein, mouse
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Gsk3b protein, rat
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Glycogen Synthase Kinase 3
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Cysteine Endopeptidases
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Proteasome Endopeptidase Complex
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GTP-Binding Proteins