Expression and characterization of GSK-3 mutants and their effect on β-catenin phosphorylation in intact cells
Expression and characterization of GSK-3 mutants and their effect on β-catenin phosphorylation in intact cells
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DOI：
10.1074/jbc.m201364200
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发表时间：
2002-06-28
影响因子：
4.8
通讯作者：
Reith, AD
中科院分区：
文献类型：
--
作者：
Hagen, T;Di Daniel, E;Reith, AD
糖原合成酶激酶3 （GSK-3）是一种丝氨酸-3。9激酶参与多种细胞信号通路，包括Wnt信号级联，其中它磷酸化-连环蛋白，从而靶向蛋白酶体介导的降解。与糖原合成酶的磷酸化不同，GSK-3对-连环蛋白的磷酸化不需要体外引物，也就是说，它不依赖于磷酸化丝氨酸的存在，在GSK-3磷酸化位点的c端有四个残基。最近，通过鉴定GSK-3beta的R96A突变体，可以分析GSK-3对引物和非引物的活性。该突变体不能磷酸化引物，但仍能磷酸化未引物的底物（Frame， S., Cohen， P., and Biondi R. M. (2001) Mol. Cell 7,1321-1327）。在这里，我们研究了β -连环蛋白中Ser（33）、Ser（37）和Thr（41）的磷酸化是否需要通过完整细胞中Ser（41）的先前磷酸化来启动。我们已经证明，突变体不会诱导β -连环蛋白降解，而是稳定β -连环蛋白，这表明它不能在完整细胞中磷酸化β -连环蛋白。此外，如果β -catenin中的Ser（45）突变为Ala，则β -catenin显着稳定，并且野生型gsk -3 β在完整细胞中阻止了β -catenin中Ser(33), Ser（37）和Thr（41）的磷酸化。此外，我们已经证明，在体外缺乏磷酸化轴蛋白的L128A突变体，尽管活性降低，但在完整细胞中仍然能够磷酸化β -连环蛋白。Tyr（216）突变为Phe显著降低gsk -3 β磷酸化和下调β -连环蛋白的能力。总之，我们发现Are突变体对gsk -3beta依赖性的β -catenin磷酸化具有显性负向影响，并且靶向β -catenin降解需要通过Ser磷酸化预先启动（45）。
Glycogen synthase kinase-3 (GSK-3) is a serine-threo. nine kinase that is involved in multiple cellular signaling pathways, including the Wnt signaling cascade where it phosphorylates beta-catenin, thus targeting it for proteasome-mediated degradation. Unlike phosphorylation of glycogen synthase, phosphorylation of beta-catenin by GSK-3 does not require priming in vitro, i.e. it is not dependent on the presence of a phosphoserine, four residues C-terminal to the GSK-3 phosphorylation site. Recently, a means of dissecting GSK-3 activity toward primed and non-primed substrates has been made possible by identification of the R96A mutant of GSK-3beta. This mutant is unable to phosphorylate primed but can still phosphorylate unprimed substrates (Frame, S., Cohen, P., and Biondi R. M. (2001) Mol. Cell 7,1321-1327). Here we have investigated whether phosphorylation of Ser(33), Ser(37), and Thr(41) in beta-catenin requires priming through prior phosphorylation at Ser(41) in intact cells. We have shown that the Are mutant does not induce beta-catenin degradation but instead stabilizes beta-catenin, indicating that it is unable to phosphorylate beta-catenin in intact cells. Furthermore, if Ser(45) in beta-catenin is mutated to Ala, beta-catenin is markedly stabilized, and phosphorylation of Ser(33), Ser(37), and Thr(41) in beta-catenin by wild type GSK-3beta is prevented in intact cells. In addition, we have shown that the L128A mutant, which is deficient in phosphorylating Axin in vitro, is still able to phosphorylate beta-catenin in intact cells although it has reduced activity. Mutation of Tyr(216) to Phe markedly reduces the ability of GSK-3beta to phosphorylate and down-regulate beta-catenin. In conclusion, we have found that the Are mutant has a dominant-negative effect on GSK-3beta-dependent phosphorylation of beta-catenin and that targeting of beta-catenin for degradation requires prior priming through phosphorylation of Ser(45).