Bioinformatic design of A-kinase anchoring protein- in silico : A potent and selective peptide antagonist of type II protein kinase A anchoring

Abstract

Compartmentalization of the cAMP-dependent protein kinase (PKA) is coordinated through association with A-kinase anchoring proteins (AKAPs). A defining characteristic of most AKAPs is a 14-to 18-aa sequence that binds to the regulatory subunits (RI or RII) of the kinase. Cellular delivery of peptides to these regions disrupts PKA anchoring and has been used to delineate a physiological role for AKAPs in the facilitation of certain cAMP-responsive events. Here, we describe a bioinformatic approach that yields an RIIselective peptide, called AKAP-in silico (AKAP-IS), that binds RII with a Kd of 0.4 nM and binds RI with a Kd of 277 nM. AKAP-IS associates with the type II PKA holoenzyme inside cells and displaces the kinase from natural anchoring sites. Electrophysiological recordings indicate that perfusion of AKAP-IS evokes a more rapid and complete attenuation of ␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor currents than previously described anchoring inhibitor peptides. Thus, computerbased and peptide array screening approaches have generated a reagent that binds PKA with higher affinity than previously described AKAPs.

Stably tethered multifunctional structures of defined composition made by the dock and lock method for use in cancer targeting

“…The AD of AKAPs for PKA is an amphipathic helix of 14-18 residues (12). The amino acid sequences of the AD are quite varied among AKAPs, and the binding affinities for RII dimers range from 2 ϫ 10 Ϫ9 M to 9 ϫ 10 Ϫ8 M, whereas the binding affinities for RI dimers are Ϸ100-fold weaker (13). AKAPs will bind only to dimeric R subunits.…”

Anchoring of Protein Kinase A-Regulatory Subunit IIα to Subapically Positioned Centrosomes Mediates Apical Bile Canalicular Lumen Development in Response to Oncostatin M but Not cAMP

“…This increases to ϳ65% (p Ͻ 0.01) in oncostatin M-treated cells, but not in db-cAMP-treated cells. The enhanced association of PKA-RII␣ with centrosomes is a result of anchoring via AKAPs as expression of the PKA-RII-displacing peptide AKAP-IS (Alto et al, 2003), which reduced the anchoring of PKA-RII␣ to centrosomes, prevents the stimulatory effect of OSM. A scrambled version of the peptide was without effect.…”

“…To confirm that the OSM-stimulated association of PKA-RII␣ with centrosomes occurs via an AKAP, and to investigate whether the OSM-stimulated association of PKA-RII␣ with centrosomes plays a role in OSM-stimulated bile canalicular lumen development (van der Wouden et al, 2002;van IJzendoorn et al, 2004a), HepG2 cells were stably transfected with epitope-tagged AKAP-IS, a peptide that specifically binds to PKA-RII with high affinity and displaces it from its natural anchoring sites, or, as a control, stably transfected with an epitope-tagged nonfunctional scrambled peptide (details with regard to these peptides are described in Alto et al, 2003). Stable transfectants were selected, and they were shown to express AKAP-IS or the scrambled peptide and displace PKA-RII␣ from natural anchoring sites or not, respectively (cf.…”

“…HepG2 cells stably expressing the GFP/V5/His-tagged AKAP-IS peptide or scrambled peptide (GFP-QDVEIQLKAAYNQKLIAI-V5/His) (provided by John Scott (Howard Hughes Medical Institute, Vollum Institute, Oregon Health & Science University, Portland, OR 97239) and described in Alto et al (2003)) were created as described previously (Wojtal et al, 2006). Stable transfectants were cultured in medium supplemented with 800 g/ml Geneticin (G-418; Invitrogen, Carlsbad, CA).…”

Synthetic modular systems – reverse engineering of signal transduction

“…Interactions in which a folded domain binds a short peptide sequence can be of high affinity (low nM), and consequently stable and longlived, exemplified by interactions between the regulatory subunits of cAMP-dependent protein kinase (PKA) and their binding motifs on Akinase anchoring proteins [19]. This particular protein-protein interaction functions to hold PKA in an inactive state close to specific targets, in readiness for a local increase in the concentration of cAMP [20].…”