Hagen Thilo, Di Daniel Elena, Culbert Ainsley A, Reith Alastair D
Systems Research-Kinase Biology Discovery Research, GlaxoSmithKline Pharmaceuticals, Harlow, Essex, CM19 5AD, United Kingdom.
J Biol Chem. 2002 Jun 28;277(26):23330-5. doi: 10.1074/jbc.M201364200. Epub 2002 Apr 19.
Glycogen synthase kinase-3 (GSK-3) is a serine-threonine 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(45) in intact cells. We have shown that the Arg(96) 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 Arg(96) 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).
糖原合酶激酶-3（GSK-3）是一种丝氨酸-苏氨酸激酶，参与多种细胞信号通路，包括Wnt信号级联反应，在该反应中它使β-连环蛋白磷酸化，从而使其靶向蛋白酶体介导的降解。与糖原合酶的磷酸化不同，GSK-3对β-连环蛋白的磷酸化在体外不需要引发，即它不依赖于在GSK-3磷酸化位点C端四个残基处存在磷酸丝氨酸。最近，通过鉴定GSK-3β的R96A突变体，使得剖析GSK-3对引发和未引发底物的活性成为可能。该突变体无法使引发的底物磷酸化，但仍能使未引发的底物磷酸化（弗雷姆，S.，科恩，P.，以及比昂迪，R. M.（2001年）《分子细胞》7卷，1321 - 1327页）。在此，我们研究了在完整细胞中β-连环蛋白中Ser(33)、Ser(37)和Thr(41)的磷酸化是否需要通过Ser(45)处的预先磷酸化来引发。我们已经表明，Arg(96)突变体不会诱导β-连环蛋白降解，反而会使其稳定，这表明它在完整细胞中无法使β-连环蛋白磷酸化。此外，如果β-连环蛋白中的Ser(45)突变为Ala，β-连环蛋白会明显稳定，并且在完整细胞中野生型GSK-3β对β-连环蛋白中Ser(33)、Ser(37)和Thr(41)的磷酸化会受到抑制。另外，我们已经表明，在体外缺乏使Axin磷酸化能力的L128A突变体，尽管其活性降低，但在完整细胞中仍能使β-连环蛋白磷酸化。将Tyr(216)突变为Phe会显著降低GSK-3β使β-连环蛋白磷酸化并下调β-连环蛋白的能力。总之，我们发现Arg(96)突变体对GSK-3β依赖的β-连环蛋白磷酸化具有显性负效应，并且β-连环蛋白靶向降解需要通过Ser(45)的磷酸化进行预先引发。
