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.
Key words
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Bar, H. P., and Hechter, O. (1969) Adenyl cyclase and hormone action. I. Effects of adrenocorticotropic hormone, glucagon, and epinephrine on the plasma membrane of rat fat cells, Proc Natl Acad Sci USA 63, 350–356.
Wong, W., and Scott, J. D. (2004) AKAP signalling complexes: focal points in space and time, Nat Rev Mol Cell Biol 5, 959–970.
Huang, L. J., Durick, K., Weiner, J. A., Chun, J., and Taylor, S. S. (1997) Identification of a novel protein kinase A anchoring protein that binds both type I and type II regulatory subunits, J Biol Chem 272, 8057–8064.
Wang, L., Sunahara, R. K., Krumins, A., Perkins, G., Crochiere, M. L., Mackey, M., Bell, S., Ellisman, M. H., and Taylor, S. S. (2001) Cloning and mitochondrial localization of full-length D-AKAP2, a protein kinase A anchoring protein, Proc Natl Acad Sci USA 98, 3220–3225.
Angelo, R., and Rubin, C. S. (1998) Molecular characterization of an anchor protein (AKAPCE) that binds the RI subunit (RCE) of type I protein kinase A from Caenorhabditis elegans, J Biol Chem 273, 14633–14643.
Kovanich, D., van der Heyden, M. A., Aye, T. T., van Veen, T. A., Heck, A. J., and Scholten, A. Sphingosine kinase interacting protein is an A-kinase anchoring protein specific for type I cAMP-dependent protein kinase, Chembiochem 11, 963–971.
Scholten, A., Poh, M. K., van Veen, T. A., van Breukelen, B., Vos, M. A., and Heck, A. J. (2006) Analysis of the cGMP/cAMP interactome using a chemical proteomics approach in mammalian heart tissue validates sphingosine kinase type 1-interacting protein as a genuine and highly abundant AKAP, J Proteome Res 5, 1435–1447.
Scholten, A., van Veen, T. A., Vos, M. A., and Heck, A. J. (2007) Diversity of cAMP-dependent protein kinase isoforms and their anchoring proteins in mouse ventricular tissue, J Proteome Res 6, 1705–1717.
Poppe, H., Rybalkin, S. D., Rehmann, H., Hinds, T. R., Tang, X. B., Christensen, A. E., Schwede, F., Genieser, H. G., Bos, J. L., Doskeland, S. O., Beavo, J. A., and Butt, E. (2008) Cyclic nucleotide analogs as probes of signaling pathways, Nat Methods 5, 277–278.
Vliem, M. J., Ponsioen, B., Schwede, F., Pannekoek, W. J., Riedl, J., Kooistra, M. R., Jalink, K., Genieser, H. G., Bos, J. L., and Rehmann, H. (2008) 8-pCPT-2′-O-Me-cAMP-AM: an improved Epac-selective cAMP analogue, Chembiochem 9, 2052–2054.
Aye, T. T., Mohammed, S., van den Toorn, H. W., van Veen, T. A., van der Heyden, M. A., Scholten, A., and Heck, A. J. (2009) Selectivity in enrichment of cAMP-dependent protein kinase regulatory subunits type I and type II and their interactors using modified cAMP affinity resins, Mol Cell Proteomics 8, 1016–1028.
Mortensen, P., Gouw, J. W., Olsen, J. V., Ong, S. E., Rigbolt, K. T., Bunkenborg, J., Cox, J., Foster, L. J., Heck, A. J., Blagoev, B., Andersen, J. S., and Mann, M. MSQuant, an open source platform for mass spectrometry-based quantitative proteomics, J Proteome Res 9, 393–403.
Boersema, P. J., Raijmakers, R., Lemeer, S., Mohammed, S., and Heck, A. J. (2009) Multiplex peptide stable isotope dimethyl labeling for quantitative proteomics, Nat Protoc 4, 484–494.
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.).
Author information
Authors and Affiliations
Corresponding authors
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2012 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
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
Download citation
DOI: https://doi.org/10.1007/978-1-61779-364-6_12
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-61779-363-9
Online ISBN: 978-1-61779-364-6
eBook Packages: Springer Protocols