PKA (protein kinase A) is tethered to subcellular compartments by direct interaction of its regulatory subunits (RI or RII) with AKAPs (A kinase-anchoring proteins). AKAPs preferentially bind RII subunits via their RII-binding domains. RII-binding domains form structurally conserved amphipathic helices with unrelated sequences. Their binding affinities for RII subunits differ greatly within the AKAP family. Amongst the AKAPs that bind RIIα subunits with high affinity is AKAP7δ [AKAP18δ; Kd (equilibrium dissociation constant) value of 31 nM]. An N-terminally truncated AKAP7δ mutant binds RIIα subunits with higher affinity than the full-length protein presumably due to loss of an inhibitory region [Henn, Edemir, Stefan, Wiesner, Lorenz, Theilig, Schmidtt, Vossebein, Tamma, Beyermann et al. (2004) J. Biol. Chem. 279, 26654–26665]. In the present study, we demonstrate that peptides (25 amino acid residues) derived from the RII-binding domain of AKAP7δ bind RIIα subunits with higher affinity (Kd=0.4±0.3 nM) than either full-length or N-terminally truncated AKAP7δ, or peptides derived from other RII binding domains. The AKAP7δ-derived peptides and stearate-coupled membrane-permeable mutants effectively disrupt AKAP–RII subunit interactions in vitro and in cell-based assays. Thus they are valuable novel tools for studying anchored PKA signalling. Molecular modelling indicated that the high affinity binding of the amphipathic helix, which forms the RII-binding domain of AKAP7δ, with RII subunits involves both the hydrophobic and the hydrophilic faces of the helix. Alanine scanning (25 amino acid peptides, SPOT technology, combined with RII overlay assays) of the RII binding domain revealed that hydrophobic amino acid residues form the backbone of the interaction and that hydrogen bond- and salt-bridge-forming amino acid residues increase the affinity of the interaction.
High-affinity AKAP7δ–protein kinase A interaction yields novel protein kinase A-anchoring disruptor peptides
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Christian Hundsrucker, Gerd Krause, Michael Beyermann, Anke Prinz, Bastian Zimmermann, Oliver Diekmann, Dorothea Lorenz, Eduard Stefan, Pavel Nedvetsky, Margitta Dathe, Frank Christian, Theresa Mcsorley, Eberhard Krause, George Mcconnachie, Friedrich W. Herberg, John D. Scott, Walter Rosenthal, Enno Klussmann; High-affinity AKAP7δ–protein kinase A interaction yields novel protein kinase A-anchoring disruptor peptides. Biochem J 1 June 2006; 396 (2): 297–306. doi: https://doi.org/10.1042/BJ20051970
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