Isoform specific differences in binding of a dual-specificity A-kinase anchoring protein to type I and type II regulatory subunits of PKA.
Dual-specificity AKAPs bind to type I (RI) and type II (RII) regulatory subunits of cAMP-dependent protein kinase A (PKA), potentially recruiting distinct cAMP responsive holoenzymes to a given intracellular location. To understand the molecular basis for this "dual" functionality, we have examined the pH-dependence, the salt-dependence, and the kinetics of binding of the A-kinase binding (AKB) domain of D-AKAP2 to the regulatory subunit isoforms of PKA. Using fluorescence anisotropy, we have found that a 27-residue peptide corresponding to the AKB domain of D-AKAP2 bound 25-fold more tightly to RIIalpha than to RIalpha. The higher affinity for RIIalpha was the result of a slower off-rate as determined by surface plasmon resonance. The high-affinity interaction for RIalpha and RIIalpha was pH-independent from pH 7.4 to 5.0. At pH 4.0, both isoforms had a reduction in binding affinity. Additionally, binding of the AKB domain to RIalpha was independent of solution ionic strength, whereas RIIalpha had an increased binding affinity at higher ionic strength. This suggests that the relative energetic contribution of the charge stabilization is different for the two isoforms. This prediction was confirmed by mutagenesis in which acidic mutations, primarily of E10 and D23, in the AKB domain affected binding to RIalpha but not to RIIalpha. These isoform-specific differences provide a foundation for developing isoform-specific peptide inhibitors of PKA anchoring by dual-specificity AKAPs, which can be used to evaluate the physiological significance of dual-specificity modes of PKA anchoring.
Howard Hughes Medical Institute, Department of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman Drive, La Jolla, California 92093-0654, USA.
Lora L
LL
Burns
Jaume M
JM
Canaves
Juniper K
JK
Pennypacker
Donald K
DK
Blumenthal
Susan S
SS
Taylor
D054758:A Kinase Anchor Proteins; D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002417:Cattle; D002942:Circular Dichroism; D000242:Cyclic AMP / Q000378:metabolism; D054746:Cyclic AMP-Dependent Protein Kinase Type II; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005454:Fluorescence Polarization; D006801:Humans; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021122:Protein Subunits; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D055672:Static Electricity; D020349:Surface Plasmon Resonance
D054758:A Kinase Anchor Proteins; C484639:AKAP10 protein, human; D048868:Adaptor Proteins, Signal Transducing; C108772:Akap10 protein, mouse; D002352:Carrier Proteins; D007527:Isoenzymes; D010446:Peptide Fragments; D021122:Protein Subunits; D011994:Recombinant Proteins; D000242:Cyclic AMP; D054746:Cyclic AMP-Dependent Protein Kinase Type II; D017868:Cyclic AMP-Dependent Protein Kinases