Phosphorylation of O6-alkylguanine-DNA alkyltransferase: experience with a GST-fusion protein and a new pull-down assay.
We showed recently that human O6-alkylguanine-DNA alkyltransferase (AGT), a key target for enhancing the efficacy of anticancer alkylating agents, is regulated by phosphorylation in brain tumor cells. This report describes the problems we encountered in using a glutathione S-transferase (GST)-tagged AGT as the substrate in our search for cellular AGT kinases, validation of a new pull-down assay for AGT phosphorylation, and its wide applicability for quantitating protein kinases in crude extracts and purified fractions. The GST-tag present in the fusion protein, by itself, was found to undergo significant phosphorylation by tumor cell extracts and contribute to spurious results. Instead, we used a histidine-tagged AGT protein, and its micro-scale purification with Talon resin as the basis for a quantitative pull-down assay, and applied it for measuring AGT phosphorylation by protein kinase C (PKC) and other cellular kinases. The pull-down procedure can be easily adopted for quantitating protein kinases in a variety of settings, as it overcomes the need for substrate immunoprecipitation when whole cell extracts are used, and eliminates the autophosphorylated kinase proteins, when purified kinases are used. Our observations call for caution in interpreting the results with GST-fusion proteins in phosphorylation studies.
Duke Scholars
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- Tumor Cells, Cultured
- Sequence Tagged Sites
- Reproducibility of Results
- Recombinant Fusion Proteins
- Phosphorylation
- Oncology & Carcinogenesis
- O(6)-Methylguanine-DNA Methyltransferase
- Molecular Sequence Data
- Humans
- Glutathione Transferase
Citation
Published In
DOI
ISSN
Publication Date
Volume
Issue
Start / End Page
Location
Related Subject Headings
- Tumor Cells, Cultured
- Sequence Tagged Sites
- Reproducibility of Results
- Recombinant Fusion Proteins
- Phosphorylation
- Oncology & Carcinogenesis
- O(6)-Methylguanine-DNA Methyltransferase
- Molecular Sequence Data
- Humans
- Glutathione Transferase