Abstract
Chemical proteomics is a versatile tool to investigate protein–small molecule interactions, but can be extended to probe also secondary binding investigating small molecule–protein 1–protein 2 interactions, providing insight into protein scaffolds. This application of chemical proteomics has in particular been applied extensively to cyclic nucleotide (cAMP, cGMP) signaling. cAMP regulates cellular functions primarily by activating cAMP-dependent protein kinase (PKA). Compartmentalization of PKA plays an important role in the specificity of cAMP signaling events and is mediated by interaction of the regulatory subunit (PKA-R) with A-kinase anchoring proteins (AKAPs), which often form the core of even larger protein machineries. The selective binding of AKAPs to one of the major isoforms PKA-R type I (PKA-RI) and PKA-R type II (PKA-RII) is an important feature of cAMP/PKA signaling. However, this specificity is not well established for most AKAPs. Here, we describe a chemical proteomics approach that combines cAMP-based affinity chromatography with quantitative mass spectrometry to investigate PKA-R isoform/AKAP specificity directly in lysates of cells and tissues of any origin. With this tool, several novel PKA-R/AKAP specificities can be easily resolved.
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Acknowledgments
The authors would like to thank Toon A.B. van Veen for lysate preparation. This work was supported by the Netherlands Proteomics Centre (D.K., T.A.A., A.J.R.H., and A.S.), the Utrecht Institute of Pharmaceutical Sciences (D.K. and A.S.), and a Focus and Massa grant from Utrecht University (A.S.).
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Kovanich, D., Aye, T.T., Heck, A.J.R., Scholten, A. (2012). Probing the Specificity of Protein–Protein Interactions by Quantitative Chemical Proteomics. In: Drewes, G., Bantscheff, M. (eds) Chemical Proteomics. Methods in Molecular Biology, vol 803. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-364-6_12
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DOI: https://doi.org/10.1007/978-1-61779-364-6_12
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