Bax B, Carter P S, Lewis C, Guy A R, Bridges A, Tanner R, Pettman G, Mannix C, Culbert A A, Brown M J, Smith D G, Reith A D
Department of Structural Biology, GlaxoSmithKline Pharmaceuticals, Harlow, Essex CM19 5AD, United Kingdom.
Structure. 2001 Dec;9(12):1143-52. doi: 10.1016/s0969-2126(01)00679-7.
Glycogen synthase kinase-3 (GSK-3) sequentially phosphorylates four serine residues on glycogen synthase (GS), in the sequence SxxxSxxxSxxx-SxxxS(p), by recognizing and phosphorylating the first serine in the sequence motif SxxxS(P) (where S(p) represents a phosphoserine). FRATtide (a peptide derived from a GSK-3 binding protein) binds to GSK-3 and blocks GSK-3 from interacting with Axin. This inhibits the Axin-dependent phosphorylation of beta-catenin by GSK-3.
Structures of uncomplexed Tyr216 phosphorylated GSK-3beta and of its complex with a peptide and a sulfate ion both show the activation loop adopting a conformation similar to that in the phosphorylated and active forms of the related kinases CDK2 and ERK2. The sulfate ion, adjacent to Val214 on the activation loop, represents the binding site for the phosphoserine residue on 'primed' substrates. The peptide FRATtide forms a helix-turn-helix motif in binding to the C-terminal lobe of the kinase domain; the FRATtide binding site is close to, but does not obstruct, the substrate binding channel of GSK-3. FRATtide (and FRAT1) does not inhibit the activity of GSK-3 toward GS.
The Axin binding site on GSK-3 presumably overlaps with that for FRATtide; its proximity to the active site explains how Axin may act as a scaffold protein promoting beta-catenin phosphorylation. Tyrosine 216 phosphorylation can induce an active conformation in the activation loop. Pre-phosphorylated substrate peptides can be modeled into the active site of the enzyme, with the P1 residue occupying a pocket partially formed by phosphotyrosine 216 and the P4 phosphoserine occupying the 'primed' binding site.
糖原合酶激酶-3（GSK-3）通过识别并磷酸化序列基序SxxxS(P)（其中S(p)代表磷酸丝氨酸）中的第一个丝氨酸，依次磷酸化糖原合酶（GS）上的四个丝氨酸残基，顺序为SxxxSxxxSxxx-SxxxS(p)。FRATtide（一种源自GSK-3结合蛋白的肽）与GSK-3结合，并阻止GSK-3与Axin相互作用。这抑制了GSK-3对β-连环蛋白的Axin依赖性磷酸化。
未复合的酪氨酸216磷酸化的GSK-3β及其与一种肽和一个硫酸根离子的复合物的结构均显示，激活环采用的构象类似于相关激酶CDK2和ERK2的磷酸化和活性形式中的构象。激活环上与缬氨酸214相邻的硫酸根离子代表“引发”底物上磷酸丝氨酸残基的结合位点。肽FRATtide在与激酶结构域的C末端叶结合时形成螺旋-转角-螺旋基序；FRATtide结合位点靠近但不阻碍GSK-3的底物结合通道。FRATtide（和FRAT1）不抑制GSK-3对GS的活性。
GSK-3上的Axin结合位点可能与FRATtide的结合位点重叠；其与活性位点的接近程度解释了Axin如何作为促进β-连环蛋白磷酸化的支架蛋白发挥作用。酪氨酸216磷酸化可诱导激活环形成活性构象。预磷酸化的底物肽可模拟进入酶的活性位点，P1残基占据由磷酸酪氨酸216部分形成的口袋，P4磷酸丝氨酸占据“引发”结合位点。
