Selectivity and regulation of A-kinase anchoring proteins in the heart - The role of autophosphorylation of the type II regulatory subunit of cAMP-dependent protein kinase

Downstream regulation of the cAMP-dependent protein kinase (PKA) pathway is mediated by anchoring proteins (AKAPs) that sequester PKA to specific subc ellular locations through binding to PKA regulatory subunits (RI or RII). T he RII-binding domain of all AKAPs forms an amphipathic alpha -helix with s imilar secondary structure. However, the importance of sequence differences in the RII-binding domains of different AKAPs is unknown, and mechanisms t hat regulate AKAP-PKA affinity are not clearly defined. Using surface plasm on resonance (SPR) spectroscopy, we measured real-time kinetics of RII inte raction with various AKAPs, Baseline equilibrium binding constants (K-d) fo r RII binding to Ht31, mAKAP, and AKAP15/18 were 10 nM, 119 nM, and 6.6 muM , respectively, PKA stimulation of intact Chinese hamster ovary cells incre ased RII alpha binding to AKAP100/mAKAP and AKAP15/18 by similar to7- and 8 2-fold, respectively. These results suggest that differences in primary seq uence of the RII-binding domain may be responsible for the selective affini ty of RII for different AKAPs. Furthermore, RII autophosphorylation may pro vide additional localized regulation of kinase anchoring. In cardiac myocyt es, disruption of RII-AKAP interaction decreased PKA phosphorylation of the PKA substrate, myosin-binding protein C. Thus, these mechanisms may be inv olved in adding additional specificity in intracellular signaling in divers e cell types and under conditions of cAMP/PKA activation.