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Structural basis for recruitment of glycogen synthase kinase 3beta to the axin-APC scaffold complex - PubMed

  • ️Wed Jan 01 2003

Structural basis for recruitment of glycogen synthase kinase 3beta to the axin-APC scaffold complex

Rana Dajani et al. EMBO J. 2003.

Abstract

Glycogen synthase kinase 3beta (GSK3beta) is a serine/threonine kinase involved in insulin, growth factor and Wnt signalling. In Wnt signalling, GSK3beta is recruited to a multiprotein complex via interaction with axin, where it hyperphosphorylates beta-catenin, marking it for ubiquitylation and destruction. We have now determined the crystal structure of GSK3beta in complex with a minimal GSK3beta-binding segment of axin, at 2.4 A resolution. The structure confirms the co-localization of the binding sites for axin and FRAT in the C-terminal domain of GSK3beta, but reveals significant differences in the interactions made by axin and FRAT, mediated by conformational plasticity of the 285-299 loop in GSK3beta. Detailed comparison of the axin and FRAT GSK3beta complexes allows the generation of highly specific mutations, which abrogate binding of one or the other. Quantitative analysis suggests that the interaction of GSK3beta with the axin scaffold enhances phosphorylation of beta-catenin by >20 000-fold.

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Figures

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Fig. 1. Structural and biochemical consequences of Tyr216 phosphorylation. (A) Secondary structure cartoon of GSK3β showing the activation segment (thickened) and the conformations of Tyr216 in the unphosphorylated protein (orange and red) and pTyr216 in the present structure (pink and red). (B) Comparison of the activation segment in the Tyr216 phosphorylated (buff and pink) and unphosphorylated conformations (yellow and orange). Phosphorylation causes an ∼120° rotation of the tyrosine side chain and repositioning of the side chains of Arg220 and Arg223, to hydrogen bond to the phosphate group of pTyr 216. (C) Kinase activity of unphosphorylated (diamonds) and pTyr 216 (squares) GSK3β on a phospho-primed peptide substrate as a function of peptide substrate concentration. All points are the means of three measurements. (D) Double reciprocal plot of the data from (C). For the unphosphorylated GSK3β, Km = 60 µM, kcat = 0.7; for pTyr216 phosphorylated GSK3β, Km = 85 µM, kcat = 3.7. pTyr216 phosphorylation causes an ∼5-fold increase in activity.

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Fig. 2. C-terminal axin-binding site. (A) Secondary structure cartoon of GSK3β (colour-ramped blue to red from the N- to the C-terminus) with the bound axin(383–401) peptide (magenta). (B) As (A), but rotated 90° around the vertical.

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Fig. 3. Axin–GSK3β interactions. (A) Axin residues Phe388, Leu392, Leu396 and Val399 form a hydrophobic helical ‘ridge’ that packs into a hydrophobic groove formed between helix 262–273 (green surface) and the extended loop from 285–299 (yellow surface) in GSK3β. (B) The axin(383–401) peptide (magenta and white) makes only a single side chain hydrogen bond to GSK3β (green-yellow), from Arg395 to Asp264.

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Fig. 4. Comparison of axin and FRAT binding to GSK3β. (A) The binding sites for the axin(383–401) peptide and FRAT(197–222) peptides are co- localized in the C-terminal domain of GSK3β. However, the two peptide have no sequence homology, and bind with different conformations and interactions. (B) The extended loop formed by residues 285–299 of GSK3β (yellow) adopts different conformations in binding axin and FRAT. In particular, residues Tyr288, Glu290 (orange), Phe291 and Phe293 adopt radically different conformations and interactions in the two complexes.

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Fig. 5. Templating factor for axin phosphorylation of β-catenin. (A) Estimation of levels of axin-bound, β-catenin levels. A 3 µg aliquot of His-tagged β-catenin was incubated with the molar equivalent of each GST protein and precipitated with glutathione–Sepharose beads. A one-fifth equivalent of the washed beads was analysed by SDS–PAGE and silver stained. GSTAx-(351–701) is the central region of axin which encompasses both the β-catenin- and GSK3β-binding sites. The L521P mutation prevents GSK3β binding (Smalley et al., 1999). GSTAx-(501–560) is the GSK3β-binding domain alone. (B) Kinase assay. GSK3β (10 µM final concentration) was added to beads with bound GST proteins as shown in (A) or to increasing concentrations of free β-catenin under kinase assay conditions. Samples were analysed by SDS–PAGE and immunoblotting. GSTAx-(351–701) and GSTAx-(351–701)-L521P were normalized for β-catenin levels (2-fold increase in loading of the latter). Comparison of the levels of N-terminal phosphorylation of the β-catenin were made using an anti-phospho β-catenin Ser33/37 Thr41 antibody by timed autoradiographic exposures. Calculation: comparisons of phospho-β-catenin levels in lane 2 were made with comparable levels in the β-catenin titration. These were then normalized to the total level of β-catenin present, resulting in an estimate of a 40-fold molar excess of phospho-β-catenin S33/37/T41 in the presence of axin compared with that in free solution. To derive the scaffolding factor, this figure was multiplied by 3 (since only 1 in 3 axin molecules was estimated to bind a β-catenin; see A, track 2) and by 200 to compensate for the proportion of templates containing GSK3β [since GSK3β was added at a final concentration of 10 µM while GST–axin(368–701) was present at 2 µM]. The calculated scaffolding factor was consistent with additional assays in which the axin–β-catenin complex was double purified to remove unbound axin and β-catenin (data not shown).

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