cAMP Signaling pp 191-201 | Cite as

Separation of PKA and PKG Signaling Nodes by Chemical Proteomics

  • Eleonora Corradini
  • Albert J. R. Heck
  • Arjen Scholten
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1294)

Abstract

The chemically quite similar cyclic nucleotides cAMP and cGMP are two second messengers that activate the homologous cAMP- and cGMP-dependent protein kinases (PKA and PKG, respectively). To gain specificity in space and time in vivo, PKA is compartmentalized by the interaction of its regulatory subunits with A-kinase-anchoring proteins (AKAPs), which often form the core of larger signaling protein machineries. In a similar manner, PKG is also found to be compartmentalized close to specific, local pools of cGMP through interaction with G-kinase-anchoring proteins (GKAPs), although the extent and mechanisms mediating these interactions are only marginally understood.

In affinity-based chemical proteomics strategies, small molecules are immobilized on solid supports in order to enrich for specific target proteins. We have shown the utility of immobilized cAMP and cGMP to enrich for PKA and PKG and their associated proteins. Unfortunately, both PKA and PKG are enriched in the pull downs with both immobilized compounds. Although this proved sufficient to identify novel AKAPs, the lower abundance of PKG has seriously hampered the enrichment and identification of novel GKAPs. Here we present an improved chemical proteomics method involving in-solution competition with low doses of different free cyclic nucleotides to segregate the cAMP/PKA- and cGMP/PKG-based signaling nodes, allowing the purification and subsequent identification of new scaffold proteins for PKG.

Keywords

Chemical proteomics cAMP cGMP PKA AKAP PKG GKAP Mass spectrometry 

Notes

Acknowledgments

This work was supported by the Netherlands Proteomics Center, by the PRIME-XS project, grant agreement number 262067, funded by the European Union 7th Framework Programme.

References

  1. 1.
    Hofmann F, Bernhard D, Lukowski R et al (2009) cGMP regulated protein kinases (cGK). Handb Exp Pharmacol 191:137–162CrossRefPubMedGoogle Scholar
  2. 2.
    Skalhegg BS, Tasken K (2000) Specificity in the cAMP/PKA signaling pathway. Differential expression, regulation, and subcellular localization of subunits of PKA. Front Biosci 5:D678–D693CrossRefPubMedGoogle Scholar
  3. 3.
    Zagotta WN, Siegelbaum SA (1996) Structure and function of cyclic nucleotide-gated channels. Annu Rev Neurosci 19:235–263CrossRefPubMedGoogle Scholar
  4. 4.
    Wegener JW, Nawrath H, Wolfsgruber W et al (2002) cGMP-dependent protein kinase I mediates the negative inotropic effect of cGMP in the murine myocardium. Circ Res 90:18–20CrossRefPubMedGoogle Scholar
  5. 5.
    Moncada S, Gryglewski R, Bunting S et al (1976) An enzyme isolated from arteries transforms prostaglandin endoperoxides to an unstable substance that inhibits platelet aggregation. Nature 263:663–665CrossRefPubMedGoogle Scholar
  6. 6.
    Taylor SS, Kim C, Vigil D et al (2005) Dynamics of signaling by PKA. Biochim Biophys Acta 1754:25–37CrossRefPubMedGoogle Scholar
  7. 7.
    Wong W, Scott JD (2004) AKAP signalling complexes: focal points in space and time. Nat Rev Mol Cell Biol 5:959–970CrossRefPubMedGoogle Scholar
  8. 8.
    Scholten A, Aye TT, Heck AJ (2008) A multi-angular mass spectrometric view at cyclic nucleotide dependent protein kinases: in vivo characterization and structure/function relationships. Mass Spectrom Rev 27:331–353CrossRefPubMedGoogle Scholar
  9. 9.
    Corbin JD, Francis SH (1999) Cyclic GMP phosphodiesterase-5: target of sildenafil. J Biol Chem 274:13729–13732CrossRefPubMedGoogle Scholar
  10. 10.
    Schlossmann J, Ammendola A, Ashman K et al (2000) Regulation of intracellular calcium by a signalling complex of IRAG, IP3 receptor and cGMP kinase Ibeta. Nature 404:197–201CrossRefPubMedGoogle Scholar
  11. 11.
    Sharma AK, Zhou GP, Kupferman J et al (2008) Probing the interaction between the coiled coil leucine zipper of cGMP-dependent protein kinase Ialpha and the C terminus of the myosin binding subunit of the myosin light chain phosphatase. J Biol Chem 283:32860–32869CrossRefPubMedCentralPubMedGoogle Scholar
  12. 12.
    Scholten A, Poh MK, van Veen TA et al (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 5:1435–1447CrossRefPubMedGoogle Scholar
  13. 13.
    Kovanich D, van der Heyden MA, Aye TT et al (2010) Sphingosine kinase interacting protein is an A-kinase anchoring protein specific for type I cAMP-dependent protein kinase. Chembiochem 11:963–971CrossRefPubMedGoogle Scholar
  14. 14.
    Burgers PP, Ma Y, Margarucci L et al (2012) A small novel A-kinase anchoring protein (AKAP) that localizes specifically protein kinase A-regulatory subunit I (PKA-RI) to the plasma membrane. J Biol Chem 287:43789–43797CrossRefPubMedCentralPubMedGoogle Scholar
  15. 15.
    Poppe H, Rybalkin SD, Rehmann H et al (2008) Cyclic nucleotide analogs as probes of signaling pathways. Nat Methods 5:277–278CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Eleonora Corradini
    • 1
    • 2
  • Albert J. R. Heck
    • 1
    • 2
  • Arjen Scholten
    • 1
    • 2
  1. 1.Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical SciencesUtrecht UniversityUtrechtThe Netherlands
  2. 2.Netherlands Proteomics CentreUtrecht UniversityUtrechtThe Netherlands

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