title: A systematic evaluation of protein kinase A-A-kinase anchoring protein interaction motifs.
abstract: Protein kinase A (PKA) in vertebrates is localized to specific locations in the cell via A-kinase anchoring proteins (AKAPs). The regulatory subunits of the four PKA isoforms (RIα, RIβ, RIIα, and RIIβ) each form a homodimer, and their dimerization domain interacts with a small helical region present in each of the more than 40 AKAPs reported so far. This allows for tight anchoring of PKA and efficient communication with other signaling proteins that interact with the AKAP scaffold in a spatial and temporal manner. The hydrophobic interaction surfaces of the PKA-R dimer and several AKAP helices have been investigated in great detail. Despite this knowledge, not every suggested AKAP has had its binding motif specified. Here we created an efficient bioinformatic tool, termed THAHIT, to accurately map the PKA binding motif and/or additional motifs of all previously reported AKAPs. Moreover, THAHIT predicts its specificity toward PKA-RIα and/or PKA-RIIα binding. To verify the validity of these newly predicted anchoring sites and their putative specificities, we used computational modeling approaches (HADDOCK), biochemical affinity studies (fluorescence anisotropy), and cellular colocalization studies. We further demonstrate the potential of THAHIT to identify novel AKAPs in cAMP-based chemical proteomics discovery data sets, and the human proteome. We retrieved numerous novel AKAP candidates, including a never reported 330 kDa AKAP observed in heart tissue, which we further characterized biochemically as a PKA-RIIα binder. Altogether, THAHIT provides a comprehensive overview of known and novel PKA-AKAP interaction domains and their PKA-R specificities.
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: Pepijn P
authors.initials: PP
authors.lastname: Burgers
authors.forename: Marcel A G
authors.initials: MA
authors.lastname: van der Heyden
authors.forename: Bart
authors.initials: B
authors.lastname: Kok
authors.forename: Albert J R
authors.initials: AJ
authors.lastname: Heck
authors.forename: Arjen
authors.initials: A
authors.lastname: Scholten
mesh_terms: D054758:A Kinase Anchor Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D008969:Molecular Sequence Data; D011485:Protein Binding / Q000502:physiology; D054730:Protein Interaction Domains and Motifs / Q000502:physiology*; D017433:Protein Structure, Secondary
chemical_list: D054758:A Kinase Anchor Proteins; D017868:Cyclic AMP-Dependent Protein Kinases