Solubilisation of the armadillo‐repeat protein β‐catenin using a zwitterionic detergent allows resolution of phosphorylated forms by 2DE

The Ludwig Institute for Cancer Research, Royal Melbourne Hospital, Parkville, Australia.
Electrophoresis (Impact Factor: 3.03). 07/2012; 33(12):1804-13. DOI: 10.1002/elps.201100671
Source: PubMed


β-catenin is a member of the armadillo repeat family of proteins and has important functions in cell-cell adhesion and Wnt signalling. Different protein species of β-catenin have been shown to exist in the cell and the relative proportions of these species are altered upon stimulation of cells with Wnt-3a (Gottardi and Gumbiner, 2004). In order to determine whether posttranslational modifications (PTMs) of β-catenin underlie these different protein species, we have used 2DE separation and immunoblotting with an antibody specific for β-catenin. High-resolution separation of differentially modified species of β-catenin in 2DE required the addition of ASB-16, a zwitterionic detergent that can solubilise integral membrane proteins. ASB-16 was also necessary for focussing of other armadillo repeat proteins, such as γ-catenin and p120-catenin. 2DE using ASB-16 allowed detection of a previously unreported phosphorylation site in the transcriptionally active form of β-catenin that binds to GST-Tcf in response to Wnt signalling.

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    • "The use of surfactants to re-solubilise membrane related proteins has been investigated by various authors (Di Ciero et al., 2004; Zhang et al., 2005; Zuobi-Hasona et al., 2005; Layton et al., 2012). SDS is traditionally used for protein solubilisation, but produces poor solubilisation for membrane associated proteins and results in a loss of electrostatic repulsive effect brought about by the polar head group (Luche et al., 2003). "
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    ABSTRACT: The mechanisms involved in anthelmintic resistance (AR) are complex but a greater understanding of AR management is essential for effective and sustainable control of parasitic helminth worms in livestock. Current tests to measure AR are time consuming and can be technically problematic, gold standard diagnostics are therefore urgently required to assist in combatting the threat from drug resistant parasites. For anthelmintics such as ivermectin (IVM), target proteins may be present in the cellular membrane. As proteins usually act in complexes and not in isolation, AR may develop and be measurable in the target associated proteins present in the parasite membrane. The model nematode Caenorhabditis elegans was used to develop a sub-proteomic assay to measure protein expression differences, between IVM resistant and IVM susceptible isolates in the presence and absence of drug challenge. Evaluation of detergents including CHAPS, ASB-14, C7BzO, Triton ×100 and TBP (tributyl phosphine) determined optimal conditions for the resolution of membrane proteins in Two Dimensional Gel Electrophoresis (2DE). These sub-proteomic methodologies were then translated and evaluated using IVM-susceptible and IVM-resistant Haemonchus contortus; a pathogenic blood feeding parasitic nematode which is of global importance in livestock health, welfare and productivity. We have demonstrated the successful resolution of membrane associated proteins from both C. elegans and H. contortus isolates, using a combination of CHAPS and the zwitterionic amphiphilic surfactant ASB-14 to further support the detection of markers for AR. Copyright © 2014. Published by Elsevier B.V.
    Veterinary Parasitology 12/2014; DOI:10.1016/j.vetpar.2014.12.003 · 2.46 Impact Factor
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    • "We compared three different protein phosphatases (Figure 2A). We have previously used Antarctic Phosphatase to dephosphorylate affinity-precipitated proteins for characterisation on 2D gels [21], however Antarctic Phosphatase was inactive at temperatures >4°C that are required for significant lipid and protein kinase activity of PI3Kα, and so had no effect on levels of phosphorylation of p85α or PI at room temperature (Figure 2A). Alkaline phosphatase is a broad spectrum phosphatase that was able to dephosphorylate both S608 and PI-3-P. "
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    ABSTRACT: Background The α-isoform of the Type 1A Phosphoinositide 3-kinases (PI3Kα) has protein kinase activity as well as phosphoinositide lipid kinase activity. The best described substrate for its protein kinase activity is its regulatory subunit, p85α, which becomes phosphorylated on Serine 608. Phosphorylation of Serine 608 has been reported to down-regulate its lipid kinase activity. Results We have assessed whether oncogenic mutants of PI3Kα, which have up-regulated lipid kinase activity, have altered levels of Serine 608 phosphorylation compared to wild type PI3Kα, and whether differential phosphorylation of Serine 608 contributes to increased activity of oncogenic forms of PI3Kα with point mutations in the helical or the kinase domains. Despite markedly increased lipid kinase activity, protein kinase activity was not altered in oncogenic compared to wild type forms of PI3Kα. By manipulating levels of phosphorylation of Serine 608 in vitro, we found no evidence that the protein kinase activity of PI3Kα affects its phosphoinositide lipid kinase activity in either wild-type or oncogenic mutants of PI3Kα. Conclusions Phosphorylation of p85α S608 is not a significant regulator of wild-type or oncogenic PI3Kα lipid kinase activity.
    BMC Biochemistry 12/2012; 13(1):30. DOI:10.1186/1471-2091-13-30 · 1.44 Impact Factor