High-affinity AKAP7δ–protein kinase A interaction yields novel protein kinase A-anchoring disruptor peptides

Abstract

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 RIIalpha subunits with high affinity is AKAP7delta [AKAP18delta; K(d) (equilibrium dissociation constant) value of 31 nM]. An N-terminally truncated AKAP7delta mutant binds RIIalpha 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 AKAP7delta bind RIIalpha subunits with higher affinity (K(d)=0.4+/-0.3 nM) than either full-length or N-terminally truncated AKAP7delta, or peptides derived from other RII binding domains. The AKAP7delta-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 AKAP7delta, 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.

Compartmentalization of cAMP-Dependent Signaling by Phosphodiesterase-4D Is Involved in the Regulation of Vasopressin-Mediated Water Reabsorption in Renal Principal Cells

“…Furthermore, AVP induces focal activation of PKA in the perinuclear region, where AQP2 and PKA predominantly are located ( Figures 1B, 2, and 3) (34). PDE4D3/9 and PKA both reside on AQP2-bearing vesicles, which also contain AKAP ( Figure 6A) (17,41) that tether PKA to the vesicles ( Figure 5C) (41) and underpin activation of the AQP2 shuttle (2). Collectively, our data provide strong evidence for the presence of a compartmentalized cAMP-dependent signaling system on AQP2-bearing vesicles that regulates the AQP2 shuttle and thereby the Pf in renal principal cells (Figure 7).…”

Pharmacological Interference With Protein-protein Interactions of Akinase Anchoring Proteins as a Strategy for the Treatment of Disease

“…Small molecules, which inhibit AKAP--PKA interactions but at the same time activate PKA also increase contractility and may thus be beneficial for the treatment of hypertrophy and heart failure [36]. However, in another study peptides for the disruption of AKAP--PKA interactions had negative effects on chronotropy (frequency), inotropy (contraction), and lusitropy (relaxation) on cultured cardiac myocytes and isolated hearts [35], and, in line, prevented β--adrenoceptor--induced increases in L--type Ca 2+ channel currents in isolated cardiac myocytes [53]. Targeting the interaction of AKAP18 and PLN may lower the energy expenditure by SERCA2, which is favorable in the failing heart when energy for contraction is limited [54].…”

Hoch funktionalisierte Terpyridine als kompetitive Inhibitoren von AKAP‐PKA‐Wechselwirkungen

“…B. AKAP18d-L314E aus AKAP18d (K D = 4 nm ; Abbildung S1 und S2 sowie Tabelle S2 in den Hintergrundinformationen). [6] Diese Peptide hemmen effektiv AKAP-PKA-Wechselwirkungen. [1] In kultivierten renalen Hauptzellen verhindern z.…”

“…[10] Ausgangspunkt war ein Modell, bei dem zum einen die hydrophoben Reste (L301, L304, L308) des Peptids mit dem hydrophoben Boden der Tasche des D/ D-Domänen-Dimers von RIIa und zum anderen die hydrophilen Seitenketten des Peptids, wie E300, mit hydrophilen Resten, wie Q4 am peripheren Rand der durch das D/DDimer gebildeten Tasche, wechselwirken. [6] Verschiedene Terpyridine, 1 a-f (Schema 1 und 4), wurden in silico durch Docking-Studien entworfen. Der hydrophobe Bereich der AKAP18d-L314E-Helix entspricht der meta-Methylgruppe des zweiten Pyridins, dem kondensierten Cyclopentylring des dritten Pyridins und den folgenden zwei Benzylringen in 1 b. Damit kçnnen sie mit dem hydrophoben Kern (gelb in Abbildung 4 und Abbildung S1) der Bindungstasche wechselwirken.…”

“…138 mm ; Abbildung 2). Wie zu erwarten war, hemmt das Peptid AKAP18d-L314E die Wechselwirkung im nanomolaren Konzentrationsbereich (12.7 nm), [6] und das inaktive Kontrollpeptid AKAP18d-PP hat keinen Einfluss auf die Wechselwirkung (Abbildung 2). Eine hemmende Wirkung von 1 a und 1 f war zu gering, um IC 50 -Werte zu bestimmen.…”