title: Separation of PKA and PKG signaling nodes by chemical proteomics.
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.
authors.affiliation: Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Padualaan 8, 3584 CH, Utrecht, The Netherlands.
authors.forename: Eleonora
authors.initials: E
authors.lastname: Corradini
authors.forename: Albert J R
authors.initials: AJ
authors.lastname: Heck
authors.forename: Arjen
authors.initials: A
authors.lastname: Scholten
mesh_terms: D000818:Animals; D002460:Cell Line; D002846:Chromatography, Affinity; D000242:Cyclic AMP / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry / Q000378:metabolism*; D006152:Cyclic GMP / Q000378:metabolism*; D017869:Cyclic GMP-Dependent Protein Kinases / Q000737:chemistry / Q000378:metabolism*; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D040901:Proteomics / Q000379:methods*; D051381:Rats; D015398:Signal Transduction; D054852:Small Molecule Libraries / Q000737:chemistry / Q000302:isolation & purification*
chemical_list: D054852:Small Molecule Libraries; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D017869:Cyclic GMP-Dependent Protein Kinases; D006152:Cyclic GMP