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, 8 (10), e77257
eCollection

Large Extent of Disorder in Adenomatous Polyposis Coli Offers a Strategy to Guard Wnt Signalling Against Point Mutations

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Large Extent of Disorder in Adenomatous Polyposis Coli Offers a Strategy to Guard Wnt Signalling Against Point Mutations

David P Minde et al. PLoS One.

Abstract

Mutations in the central region of the signalling hub Adenomatous Polyposis Coli (APC) cause colorectal tumourigenesis. The structure of this region remained unknown. Here, we characterise the Mutation Cluster Region in APC (APC-MCR) as intrinsically disordered and propose a model how this structural feature may contribute to regulation of Wnt signalling by phosphorylation. APC-MCR was susceptible to proteolysis, lacked α-helical secondary structure and did not display thermal unfolding transition. It displayed an extended conformation in size exclusion chromatography and was accessible for phosphorylation by CK1ε in vitro. The length of disordered regions in APC increases with species complexity, from C. elegans to H. sapiens. We speculate that the large disordered region harbouring phosphorylation sites could be a successful strategy to stabilise tight regulation of Wnt signalling against single missense mutations.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Charge distribution of APC-MCR.
Charges present in the primary structure, putative phosphorylation sites and regulatory important motifs are indicated. A large number of putative phosphorylation sites scatter over the entire MCR (Phosida predictor for S/T phosphorylation). Two of the three 20 amino acid repeats (blue), bind to β-catenin (β). A short peptide stretch called “CID” (yellow) was recently implicated in Wnt/β-catenin downregulation [4,70].
Figure 2
Figure 2. MCR is susceptible to TL digestion.
Resistance of well-characterised folded and unfolded proteins is compared with MCR by using TL concentrations of 0 g/L, 0.001 g/L and 0.1 g/L. Folded protein MBP (diamond), is resistant against the highest protease concentration while β-caseine and Axin CR are already cleaved at low protease concentration (0.001g/L). A fusion construct of NusA and β-catenin gives several high molecular weight bands, likely due to the presence of at least one large protease susceptible internal linker segment [65].
Figure 3
Figure 3. MCR lacks secondary structure.
Far-UV CD spectra of APC-MCR in the absence (black) and presence of TFE (dark grey, 20%; light grey, 80%) in 10 mM Na-phosphate buffer (pH 7.2), 50 mM NaF, and 0.5 mM TCEP.
Figure 4
Figure 4. APC-MCR lacks a cooperative unfolding transition.
A, Intrinsic protein fluorescence spectra of APC-MCR measured in 1°C steps from 20°C to 70°C, indicated by a greyscale gradient from black to light grey. B, Comparison of intrinsic tyrosine fluorescence spectra normalised to the maxima. Experiment and colour code as in A. C Temperature dependence of intrinsic tyrosine fluorescence emission of MCR upon stepwise increase of temperature at 340 nm (black) and 304 nm (grey) normalised on maxima of emission.
Figure 5
Figure 5. APC-MCR runs at a higher apparent molecular weight upon size exclusion chromatography.
A, APC-MCR was applied to analytical size exclusion chromatography. MCR eluted after 8.36 min between the indicated globular size standards. By semilogarithmic fitting to the molecular weights of the standards, an apparent molecular weight of 220 kDa has been determined for APC-MCR. B, The straight line indicates a semilogarithmic fit to the known stokes radii of the indicated standard proteins. According to this analysis, MCR has a stokes radius of 5.9 nm. C, SEC-MALLS reveals that the ySUMO-APC-MCR fusion protein is monomeric. ySUMO-APC-MCR eluted as a single peak with a mass of 44.4 ± 4.0 kDa (UV absorption at 220 nm, black, arbitrary units; differential refractive index, red, arbitrary units; Molecular mass, blue, kDa).
Figure 6
Figure 6. MCR can be phosphorylated by CK1ε.
ySUMO-MCR was incubated for the times indicated with CK1ε. The bandshift of phosphorylated ySUMO-MCR after 16 h phosphorylation was fully reversed by heat inactivation of CK1ε followed by dephosphorylation with CIP for 30 min (last lane).
Figure 7
Figure 7. The three Wnt signalling hubs APC, Axin, WTX contain large intrinsically disordered regions.
A, Meta-predictions of disorder using the PONDR-FIT algorithm are displayed. (black for scores >= 0.5 and white for scores < 0.5). B, Same as A, for C. elegans homologues of APC, Axin, Hsp90, CBP.

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Grant support

SGDR was supported by the European Union's Sixth Framework Programme (FP6) by a Marie-Curie Excellence Grant (MEXT-CT-2005-025651) and by the Seventh Framework Programme (FP7) under grant agreement ManiFold n°317371, by a VIDI career development grant (700.55.421) by the Netherlands Organization for Scientific Research (NWO) and by a High Potential Grant of Utrecht University. MMM was supported by the European Research Council (ERC-StG no.242958) and by a High Potential Grant of Utrecht University. URLs of funders: NWO: http://www.nwo.nl/; EU: http://europa.eu/index_en.htm; Utrecht University: http://www.uu.nl/; ERC: http://erc.europa.eu/. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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