Sadot Einat, Conacci-Sorrell Maralice, Zhurinsky Jacob, Shnizer Dalia, Lando Zeev, Zharhary Dorit, Kam Zvi, Ben-Ze'ev Avri, Geiger Benjamin
Department of Molecular Cell Biology, Weizmann Institute of Science Rehovot 76100 Israel.
J Cell Sci. 2002 Jul 1;115(Pt 13):2771-80. doi: 10.1242/jcs.115.13.2771.
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.
针对一段对应于人β-连环蛋白33至45位氨基酸的肽段制备了一种新型的磷酸化特异性抗体（αβ-连环蛋白），该肽段在33位和37位含有磷酸化丝氨酸。这种抗体对磷酸化的β-连环蛋白具有特异性，既不与非磷酸化蛋白反应，也不与磷酸化或非磷酸化的桥粒芯蛋白反应。它与S33Y β-连环蛋白有微弱相互作用，但与S37A突变体不相互作用。在正常培养细胞中几乎检测不到磷酸化β-连环蛋白（pβ-连环蛋白），在该蛋白过表达或通过蛋白酶体阻断其降解后会积累（高达总β-连环蛋白的55%）。抑制糖原合成酶激酶-3β（GSK-3β）和蛋白酶体都会导致pβ-连环蛋白水平迅速（半衰期=10分钟）且可逆地降低，这表明该蛋白在活细胞中可发生去磷酸化，其速率与被GSK-3β磷酸化的速率相当。pβ-连环蛋白与淋巴样增强因子1（LEF-1）相互作用，但无法与DNA形成三元复合物，这表明它在转录上无活性。免疫荧光显微镜检查表明，pβ-连环蛋白过表达时会在MDCK细胞和BCAP细胞的细胞核中积累，并在紧密连接形成后不久短暂地与紧密连接相关联。pβ-连环蛋白仅与共转染的N-钙黏蛋白有微弱相互作用，尽管它与泛素连接酶成分β-转导素重复序列包含蛋白（β-TrCP）形成复合物。表达1338位截短型结肠腺瘤性息肉病（APC）蛋白的SW480结肠癌细胞含有高水平的pβ-连环蛋白，而表达1555位截短型APC蛋白的HT29细胞积累非磷酸化的β-连环蛋白，这表明APC蛋白的1338 - 1555位氨基酸区域参与了pβ-连环蛋白去磷酸化和降解的差异调节。
