title: Probing the specificity of protein-protein interactions by quantitative chemical proteomics.
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.
authors: Biomolecular Mass Spectrometry and Proteomics Group, Utrecht University and Netherlands Proteomics Centre, Utrecht, The Netherlands.
authors: Duangnapa
authors: D
authors: Kovanich
authors: Thin Thin
authors: TT
authors: Aye
authors: Albert J R
authors: AJ
authors: Heck
authors: Arjen
authors: A
authors: Scholten
mesh_terms: D000595:Amino Acid Sequence; D002457:Cell Extracts; D005591:Chemical Fractionation; D002853:Chromatography, Liquid; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D007474:Ion Exchange; D007527:Isoenzymes / Q000378:metabolism; D007553:Isotope Labeling; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010447:Peptide Hydrolases / Q000378:metabolism; D011485:Protein Binding; D060066:Protein Interaction Maps; D040901:Proteomics / Q000379:methods*; D012984:Software; D013223:Statistics as Topic
chemical_list: D002457:Cell Extracts; D007527:Isoenzymes; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D010447:Peptide Hydrolases