Structure of smAKAP and its regulation by PKA-mediated phosphorylation

The A-kinase anchoring protein (AKAP) smAKAP has three extraordinary features; it is very small, it is anchored directly to membranes by acyl motifs, and it interacts almost exclusively with the type I regulatory subunits (RI) of cAMP-dependent kinase (PKA). Here, we determined the crystal structure of smAKAP's A-kinase binding domain (smAKAP-AKB) in complex with the dimerization/docking (D/D) domain of RIα which reveals an extended hydrophobic interface with unique interaction pockets that drive smAKAP's high specificity for RI-subunits. We also identify a conserved PKA phosphorylation site at Ser66 in the AKB domain which we predict would cause steric clashes and disrupt binding. This correlates with in vivo co-localization and fluorescence polarization studies where Ser66 AKB phosphorylation ablates RI-binding. Hydrogen/deuterium exchange studies confirm that the AKB helix is accessible and dynamic. Furthermore, full-length smAKAP as well as the unbound AKB is predicted to contain a break at the phosphorylation site, and circular dichroism measurements confirm that the AKB domain loses its helicity following phosphorylation. Since the active site of PKA's catalytic subunit does not accommodate α-helices, we predict that the inherent flexibility of the AKB domain enables its phosphorylation by PKA. This represents a novel mechanism, whereby activation of anchored PKA can terminate its binding to smAKAP affecting the regulation of localized cAMP-signaling events. This article is protected by copyright. All rights reserved

Спроби розв’язання Радою Народних Міністрів Української Народної Республіки земельного питання у березні-квітні 1918 року

У статті розглядається діяльність РНМ УНР із реформування земельних відносин в Україні, реалізації тимчасового земельного закону, вирішення питання засіву землі весною 1918 р.В статье рассматривается деятельность СНМ УНР по реформированию земельных отношений в Украине, реализации временного земельного закона, решение вопроса засева земли весной 1918 г.In the article activity of CFМ of UNR is examined from reformation of the landed relations in Ukraine, realization of the temporal landed law, decision of sowing of the land in spring 1918

A systematic evaluation of protein kinase A-A-kinase anchoring protein interaction motifs

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

A systematic evaluation of protein kinase a-a-kinase anchoring protein interaction motifs

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

A Systematic Evaluation of Protein Kinase A–A-Kinase Anchoring Protein Interaction Motifs

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

A Systematic Evaluation of Protein Kinase A–A-Kinase Anchoring Protein Interaction Motifs

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