Regulation of S33/S37 phosphorylated beta-catenin in normal and transformed cells.
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.
Sadot
Einat
E
Department of Molecular Cell Biology, Weizmann Institute of Science Rehovot 76100 Israel.
Conacci-Sorrell
Maralice
M
Zhurinsky
Jacob
J
Shnizer
Dalia
D
Lando
Zeev
Z
Zharhary
Dorit
D
Kam
Zvi
Z
Ben-Ze'ev
Avri
A
Geiger
Benjamin
B
D025601:Adenomatous Polyposis Coli Protein / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence / Q000235:genetics; D000818:Animals; D015820:Cadherins / Q000235:genetics / Q000378:metabolism; D002277:Carcinoma / Q000235:genetics / Q000378:metabolism* / Q000503:physiopathology; D002417:Cattle; D002455:Cell Division / Q000235:genetics*; D002461:Cell Line, Transformed; D002471:Cell Transformation, Neoplastic / Q000235:genetics / Q000378:metabolism*; D003110:Colonic Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000503:physiopathology; D003546:Cysteine Endopeptidases / Q000187:drug effects / Q000378:metabolism; D003598:Cytoskeletal Proteins / Q000235:genetics / Q000378:metabolism*; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism; D015972:Gene Expression Regulation, Neoplastic / Q000235:genetics; D038362:Glycogen Synthase Kinase 3 / Q000235:genetics / Q000378:metabolism; D000071679:Glycogen Synthase Kinase 3 beta; D006801:Humans; D051821:Lymphoid Enhancer-Binding Factor 1; D051379:Mice; D008807:Mice, Inbred BALB C; D009097:Multienzyme Complexes / Q000187:drug effects / Q000378:metabolism; D010766:Phosphorylation; D046988:Proteasome Endopeptidase Complex; D051381:Rats; D012694:Serine / Q000378:metabolism; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D014407:Tumor Cells, Cultured; D015854:Up-Regulation / Q000187:drug effects / Q000235:genetics; D051176:beta Catenin; D044844:beta-Transducin Repeat-Containing Proteins
D025601:Adenomatous Polyposis Coli Protein; C471644:BTRC protein, human; C471643:Btrc protein, mouse; C495270:CTNNB1 protein, human; C495267:CTNNB1 protein, mouse; D015820:Cadherins; C495268:Ctnnb1 protein, rat; D003598:Cytoskeletal Proteins; D004268:DNA-Binding Proteins; C497573:LEF1 protein, human; C497574:Lef1 protein, mouse; C497575:Lef1 protein, rat; D051821:Lymphoid Enhancer-Binding Factor 1; D009097:Multienzyme Complexes; D015534:Trans-Activators; D014157:Transcription Factors; D051176:beta Catenin; D044844:beta-Transducin Repeat-Containing Proteins; D012694:Serine; D004247:DNA; C000605506:GSK3B protein, human; D000071679:Glycogen Synthase Kinase 3 beta; C000605507:Gsk3b protein, mouse; C000605508:Gsk3b protein, rat; D038362:Glycogen Synthase Kinase 3; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D019204:GTP-Binding Proteins