Bioinformatic design of A-kinase anchoring protein-in silico: a potent and selective peptide antagonist of type II protein kinase A anchoring.
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 RII-selective peptide, called AKAP-in silico (AKAP-IS), that binds RII with a K(d) of 0.4 nM and binds RI with a K(d) 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 alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor currents than previously described anchoring inhibitor peptides. Thus, computer-based and peptide array screening approaches have generated a reagent that binds PKA with higher affinity than previously described AKAPs.
Alto
Neal M
NM
Howard Hughes Medical Institute, Vollum Institute, Oregon Health and Science University, Portland OR 97239, USA.
Soderling
Scott H
SH
Hoshi
Naoto
N
Langeberg
Lorene K
LK
Fayos
Rosa
R
Jennings
Patricia A
PA
Scott
John D
JD
D000595:Amino Acid Sequence; D001483:Base Sequence; D002460:Cell Line; D019295:Computational Biology; D016384:Consensus Sequence; D054746:Cyclic AMP-Dependent Protein Kinase Type II; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics; D015195:Drug Design; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D015202:Protein Engineering; D011506:Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology; D015398:Signal Transduction
D018076:DNA, Complementary; D011506:Proteins; D011994:Recombinant Proteins; D054746:Cyclic AMP-Dependent Protein Kinase Type II; D017868:Cyclic AMP-Dependent Protein Kinases