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High-affinity AKAP7delta-protein kinase A interaction yields novel protein kinase A-anchoring disruptor peptides.
- Source :
-
The Biochemical journal [Biochem J] 2006 Jun 01; Vol. 396 (2), pp. 297-306. - Publication Year :
- 2006
-
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.
- Subjects :
- A Kinase Anchor Proteins
Adaptor Proteins, Signal Transducing chemistry
Amino Acid Sequence
Amino Acids chemistry
Amino Acids metabolism
Animals
Binding Sites
Cells, Cultured
Cyclic AMP-Dependent Protein Kinases chemistry
Electrophysiology
Hydrogen Bonding
Hydrophobic and Hydrophilic Interactions
Membrane Proteins chemistry
Models, Molecular
Molecular Sequence Data
Peptides metabolism
Peptides pharmacology
Protein Structure, Tertiary
Protein Subunits
Rats
Sequence Alignment
Time Factors
Adaptor Proteins, Signal Transducing metabolism
Cyclic AMP-Dependent Protein Kinases metabolism
Membrane Proteins metabolism
Peptides chemistry
Protein Binding
Subjects
Details
- Language :
- English
- ISSN :
- 1470-8728
- Volume :
- 396
- Issue :
- 2
- Database :
- MEDLINE
- Journal :
- The Biochemical journal
- Publication Type :
- Academic Journal
- Accession number :
- 16483255
- Full Text :
- https://doi.org/10.1042/BJ20051970