High-affinity AKAP7delta-protein kinase A interaction yields novel protein kinase A-anchoring disruptor peptides.
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.
Leibniz-Institut für Molekulare Pharmakologie, Campus Berlin-Buch, Robert-Rössle-Str. 10, 13125 Berlin, Germany.
Christian
C
Hundsrucker
Gerd
G
Krause
Michael
M
Beyermann
Anke
A
Prinz
Bastian
B
Zimmermann
Oliver
O
Diekmann
Dorothea
D
Lorenz
Eduard
E
Stefan
Pavel
P
Nedvetsky
Margitta
M
Dathe
Frank
F
Christian
Theresa
T
McSorley
Eberhard
E
Krause
George
G
McConnachie
Friedrich W
FW
Herberg
John D
JD
Scott
Walter
W
Rosenthal
Enno
E
Klussmann
D054758:A Kinase Anchor Proteins; D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D002478:Cells, Cultured; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry / Q000378:metabolism*; D004594:Electrophysiology; D006860:Hydrogen Bonding; D057927:Hydrophobic and Hydrophilic Interactions; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021122:Protein Subunits; D051381:Rats; D016415:Sequence Alignment; D013997:Time Factors
D054758:A Kinase Anchor Proteins; C112596:AKAP7 protein, human; D048868:Adaptor Proteins, Signal Transducing; D000596:Amino Acids; D008565:Membrane Proteins; D010455:Peptides; D021122:Protein Subunits; D017868:Cyclic AMP-Dependent Protein Kinases