A dynamic mechanism for AKAP binding to RII isoforms of cAMP-dependent protein kinase

A kinase-anchoring proteins (AKAPs) target PKA to specific microdomains by using an amphipathic helix that docks to N-terminal dimerization and docking (D/D) domains of PKA regulatory (R) subunits. To understand specificity, we solved the crystal structure of the helical motif from D-AKAP2, a dual-specific AKAP, bound to the RIIalpha D/D domain. The 1.6 Angstrom structure reveals how this dynamic, hydrophobic docking site is assembled. A stable, hydrophobic docking groove is formed by the helical interface of two RIIalpha protomers. The flexible N terminus of one protomer is then recruited to the site, anchored to the peptide through two essential isoleucines. The other N terminus is disordered. This asymmetry provides greater possibilities for AKAP docking. Although there is strong discrimination against RIalpha in the N terminus of the AKAP helix, the hydrophobic groove discriminates against RIIalpha. RIalpha, with a cavity in the groove, can accept a bulky tryptophan, whereas RIIalpha requires valine.     

A novel mechanism of PKA anchoring revealed by solution structures of anchoring complexes

The specificity of intracellular signaling events is controlled, in part, by compartmentalization of protein kinases and phosphatases. The subcellular localization of these enzymes is often maintained by protein- protein interactions. A prototypic example is the compartmentalization of the cAMP-dependent protein kinase (PKA) through its association with A-kinase anchoring proteins (AKAPs). A docking and dimerization domain (D/D) located within the first 45 residues of each regulatory (R) subunit protomer forms a high affinity binding site for its anchoring partner. We now report the structures of two D/D-AKAP peptide complexes obtained by solution NMR methods, one with Ht31(493-515) and the other with AKAP79(392-413). We present the first direct structural data demonstrating the helical nature of the peptides. The structures reveal conserved hydrophobic interaction surfaces on the helical AKAP peptides and the PKA R subunit, which are responsible for mediating the high affinity association in the complexes. In a departure from the dimer-dimer interactions seen in other X-type four-helix bundle dimeric proteins, our structures reveal a novel hydrophobic groove that accommodates one AKAP per RIIalpha D/D.     

Analysis of A-kinase anchoring protein (AKAP) interaction with protein kinase A (PKA) regulatory subunits: PKA isoform specificity in AKAP binding

Compartmentalization of cAMP-dependent protein kinase (PKA) is in part mediated by specialized protein motifs in the dimerization domain of the regulatory (R)-subunits of PKA that participate in protein-protein interactions with an amphipathic helix region in A-kinase anchoring proteins (AKAPs). In order to develop a molecular understanding of the subcellular distribution and specific functions of PKA isozymes mediated by association with AKAPs, it is of importance to determine the apparent binding constants of the R-subunit-AKAP interactions. Here, we present a novel approach using surface plasmon resonance (SPR) to examine directly the association and dissociation of AKAPs with all four R-subunit isoforms immobilized on a modified cAMP surface with a high level of accuracy. We show that both AKAP79 and S-AKAP84/D-AKAP1 bind RIIalpha very well (apparent K(D) values of 0.5 and 2 nM, respectively). Both proteins also bind RIIbeta quite well, but with three- to fourfold lower affinities than those observed versus RIIalpha. However, only S-AKAP84/D-AKAP1 interacts with RIalpha at a nanomolar affinity (apparent K(D) of 185 nM). In comparison, AKAP95 binds RIIalpha (apparent K(D) of 5.9 nM) with a tenfold higher affinity than RIIbeta and has no detectable binding to RIalpha. Surface competition assays with increasing concentrations of a competitor peptide covering amino acid residues 493 to 515 of the thyroid anchoring protein Ht31, demonstrated that Ht31, but not a proline-substituted peptide, Ht31-P, competed binding of RIIalpha and RIIbeta to all the AKAPs examined (EC(50)-values from 6 to 360 nM). Furthermore, RIalpha interaction with S-AKAP84/D-AKAP1 was competed (EC(50) 355 nM) with the same peptide. Here we report for the first time an approach to determine apparent rate- and equilibria binding constants for the interaction of all PKA isoforms with any AKAP as well as a novel approach for characterizing peptide competitors that disrupt PKA-AKAP anchoring.     

Distinct interaction modes of an AKAP bound to two regulatory subunit isoforms of protein kinase A revealed by amide hydrogen/deuterium exchange

The structure of an AKAP docked to the dimerization/docking (D/D) domain of the type II (RIIalpha) isoform of protein kinase A (PKA) has been well characterized, but there currently is no detailed structural information of an AKAP docked to the type I (RIalpha) isoform. Dual-specific AKAP2 (D-AKAP2) binds in the nanomolar range to both isoforms and provided us with an opportunity to characterize the isoform-selective nature of AKAP binding using a common docked ligand. Hydrogen/deuterium (H/D) exchange combined with mass spectrometry (DXMS) was used to probe backbone structural changes of an alpha-helical A-kinase binding (AKB) motif from D-AKAP2 docked to both RIalpha and RIIalpha D/D domains. The region of protection upon complex formation and the magnitude of protection from H/D exchange were determined for both interacting partners in each complex. The backbone of the AKB ligand was more protected when bound to RIalpha compared to RIIalpha, suggesting an increased helical stabilization of the docked AKB ligand. This combined with a broader region of backbone protection induced by the AKAP on the docking surface of RIalpha indicated that there were more binding constraints for the AKB ligand when bound to RIalpha. This was in contrast to RIIalpha, which has a preformed, localized binding surface. These distinct modes of AKAP binding may contribute to the more discriminating nature of the RIalpha AKAP-docking surface. DXMS provides valuable structural information for understanding binding specificity in the absence of a high-resolution structure, and can readily be applied to other protein-ligand and protein-protein interactions.     

Molecular basis of AKAP specificity for PKA regulatory subunits

Localization of cyclic AMP (cAMP)-dependent protein kinase (PKA) by A kinase-anchoring proteins (AKAPs) restricts the action of this broad specificity kinase. The high-resolution crystal structures of the docking and dimerization (D/D) domain of the RIIalpha regulatory subunit of PKA both in the apo state and in complex with the high-affinity anchoring peptide AKAP-IS explain the molecular basis for AKAP-regulatory subunit recognition. AKAP-IS folds into an amphipathic alpha helix that engages an essentially preformed shallow groove on the surface of the RII dimer D/D domains. Conserved AKAP aliphatic residues dominate interactions to RII at the predominantly hydrophobic interface, whereas polar residues are important in conferring R subunit isoform specificity. Using a peptide screening approach, we have developed SuperAKAP-IS, a peptide that is 10,000-fold more selective for the RII isoform relative to RI and can be used to assess the impact of PKA isoform-selective anchoring on cAMP-responsive events inside cells.     

The molecular basis for protein kinase A anchoring revealed by solution NMR

Compartmentalization of signal transduction enzymes into signaling complexes is an important mechanism to ensure the specificity of intracellular events. Formation of these complexes is mediated by specialized protein motifs that participate in protein-protein interactions. The adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase (PKA) is localized through interaction of the regulatory (R) subunit dimer with A-kinase-anchoring proteins (AKAPs). We now report the solution structure of the type II PKA R-subunit fragment RIIalpha(1-44), which encompasses both the AKAP-binding and dimerization interfaces. This structure incorporates an X-type four-helix bundle dimerization motif with an extended hydrophobic face that is necessary for high-affinity AKAP binding. NMR data on the complex between RIIalpha(1-44) and an AKAP fragment reveals extensive contacts between the two proteins. Interestingly, this same dimerization motif is present in other signaling molecules, the S100 family. Therefore, the X-type four-helix bundle may represent a conserved fold for protein-protein interactions in signal transduction.     

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.     

Isoform-specific differences between the type Ialpha and IIalpha cyclic AMP-dependent protein kinase anchoring domains revealed by solution NMR

Cyclic AMP dependent protein kinase (PKA) is controlled, in part, by the subcellular localization of the enzyme (). Discovery of dual specificity anchoring proteins (d-AKAPs) indicates that not only is the type II, but also the type I, enzyme localized (). It appears that the type I enzyme is localized in a novel, dynamic fashion as opposed to the apparent static localization of the type II enzyme. Recently, the structure of the dimerization/docking (D/D) domain from the type II enzyme was solved (). This work revealed an X-type four-helix bundle motif with a hydrophobic patch that modulates AKAP interactions. To understand the dynamic versus static localization of PKA, multidimensional NMR techniques were used to investigate the structural features of the type I D/D domain. Our results indicate a conserved helix-turn-helix motif in the type I and type II D/D domains. However, important differences between the two domains are evident in the extreme NH(2) terminus: this region is extended in the type II domain, whereas it is helical in the type I protein. The NH(2)-terminal residues in RIIalpha contain determinants for anchoring, and the orientation and packing of this helical element in the RIalpha structure may have profound consequences in the recognition surface presented to the AKAPs.     

Single amino acids determine specificity of binding of protein kinase A regulatory subunits by protein kinase A anchoring proteins.

Cyclic AMP-dependent protein kinase is tethered to protein kinase A anchoring proteins (AKAPs) through regulatory subunits (R) by RIalpha-specific, RIIalpha-specific, or RIalpha/RIIalpha dual-specific binding. Ala- and Val-scanning mutagenesis determined that hydrophobic amino acids at three homologous positions are required for binding of RIalpha to FSC1/AKAP82 domain B and RIIalpha to AKAP Ht31. A mutation at the middle position reversed the binding specificity of both AKAPs, and mutations at this same position of the dual-specific domain A of FSC1/AKAP82 converted it into either an RIalpha or RIIalpha binding domain. This suggests that hydrophobic amino acids at three conserved positions within the primary sequence and an amphipathic helix of AKAPs are required for cyclic AMP-dependent protein kinase binding, with the size of the aliphatic side chain at the middle position determining RIalpha or RIIalpha binding specificity.     

Conformational differences among solution structures of the type Ialpha, IIalpha and IIbeta protein kinase A regulatory subunit homodimers: role of the linker regions

The regulatory (R) subunits of the cAMP-dependent protein kinase (protein kinase A or PKA) are multi-domain proteins responsible for conferring cAMP-dependence and localizing PKA to specific subcellular locations. There are four isoforms of the R subunit in mammals that are similar in molecular mass and domain organization, but clearly serve different biological functions. Although high-resolution structures are available for the cAMP-binding domains and dimerization/docking domains of two isoforms, there are no high-resolution structures of any of the intact R subunit homodimer isoforms. The results of small-angle X-ray scattering studies presented here indicate that the RIalpha, RIIalpha, and RIIbeta homodimers differ markedly in overall shape, despite extensive sequence homology and similar molecular masses. The RIIalpha and RIIbeta homodimers have very extended, rod-like shapes, whereas the RIalpha homodimer likely has a compact Y-shape. Based on a comparison of the R subunit sequences, we predict that the linker regions are the likely cause of these large differences in shape among the isoforms. In addition, we show that cAMP binding does not cause large conformational changes in type Ialpha or IIalpha R subunit homodimers, suggesting that the activation of PKA by cAMP involves only local conformational changes in the R subunits.     

Beyond PKA: Evolutionary and structural insights that define a docking and dimerization domain superfamily

Protein-interaction domains can create unique macromolecular complexes that drive evolutionary innovation. By combining bioinformatic and phylogenetic analyses with structural approaches, we have discovered that the docking and dimerization (D/D) domain of the PKA regulatory subunit is an ancient and conserved protein fold. An archetypal function of this module is to interact with A-kinase-anchoring proteins (AKAPs) that facilitate compartmentalization of this key cell-signaling enzyme. Homology searching reveals that D/D domain proteins comprise a superfamily with 18 members that function in a variety of molecular and cellular contexts. Further in silico analyses indicate that D/D domains segregate into subgroups on the basis of their similarity to type I or type II PKA regulatory subunits. The sperm autoantigenic protein 17 (SPA17) is a prototype of the type II or R2D2 subgroup that is conserved across metazoan phyla. We determined the crystal structure of an extended D/D domain from SPA17 (amino acids 1–75) at 1.72 Å resolution. This revealed a four-helix bundle-like configuration featuring terminal β-strands that can mediate higher order oligomerization. In solution, SPA17 forms both homodimers and tetramers and displays a weak affinity for AKAP18. Quantitative approaches reveal that AKAP18 binding occurs at nanomolar affinity when SPA17 heterodimerizes with the ropporin-1-like D/D protein. These findings expand the role of the D/D fold as a versatile protein-interaction element that maintains the integrity of macromolecular architectures within organelles such as motile cilia.     

Electrostatic properties of the structure of the docking and dimerization domain of protein kinase A IIalpha.

The structure of the N-terminal docking and dimerization domain of the type IIalpha regulatory subunit (RIIalpha D/D) of protein kinase A (PKA) forms a noncovalent stand-alone X-type four-helix bundle structural motif, consisting of two helix-loop-helix monomers. RIIalpha D/D possesses a strong hydrophobic core and two distinct, exposed faces. A hydrophobic face with a groove is the site of protein-protein interactions necessary for subcellular localization. A highly charged face, opposite to the former, may be involved in regulation of protein-protein interactions as a result of changes in phosphorylation state of the regulatory subunit. Although recent studies have addressed the hydrophobic character of packing of RIIalpha D/D and revealed the function of the hydrophobic face as the binding site to A-kinase anchoring proteins (AKAPs), little attention has been paid to the charges involved in structure and function. To examine the electrostatic character of the structure of RIIalpha D/D we have predicted mean apparent pKa values, based on Poisson-Boltzmann electrostatic calculations, using an ensemble of calculated dimer structures. We propose that the helix promoting sequence Glu34-X-X-X-Arg38 stabilizes the second helix of each monomer, through the formation of a (i, i +4) side chain salt bridge. We show that a weak inter-helical hydrogen bond between Tyr35-Glu19 of each monomer contributes to tertiary packing and may be responsible for discriminating from alternative quaternary packing of the two monomers. We also show that an inter-monomer hydrogen bond between Asp30-Arg40 contributes to quaternary packing. We propose that the charged face comprising of Asp27-Asp30-Glu34-Arg38-Arg40-Glu41-Arg43-Arg44 may be necessary to provide flexibility or stability in the region between the C-terminus and the interdomain/autoinhibitory sequence of RIIalpha, depending on the activation state of PKA. We also discuss the structural requirements necessary for the formation of a stacked (rather than intertwined) dimer, which has consequences for the orientation of the functionally important and distinct faces.     

High-affinity AKAP7delta-protein kinase A interaction yields novel protein kinase A-anchoring disruptor peptides

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.     

Identification of sperm-specific proteins that interact with A-kinase anchoring proteins in a manner similar to the type II regulatory subunit of PKA

The cAMP-dependent protein kinase (PKA) is targeted to specific subcellular compartments through its interaction with A-kinase anchoring proteins (AKAPs). AKAPs contain an amphipathic helix domain that binds to the type II regulatory subunit of PKA (RII). Synthetic peptides containing this amphipathic helix domain bind to RII with high affinity and competitively inhibit the binding of PKA with AKAPs. Addition of these anchoring inhibitor peptides to spermatozoa inhibits motility (Vijayaraghavan, S., Goueli, S. A., Davey, M. P., and Carr, D. W. (1997) J. Biol. Chem. 272, 4747-4752). However, inhibition of the PKA catalytic activity does not mimic these peptides, suggesting that the peptides are disrupting the interaction of AKAP(s) with proteins other than PKA. Using the yeast two-hybrid system, we have now identified two sperm-specific human proteins that interact with the amphipathic helix region of AKAP110. These proteins, ropporin (a protein previously shown to interact with the Rho signaling pathway) and AKAP-associated sperm protein, are 39% identical to each other and share a strong sequence similarity with the conserved domain on the N terminus of RII that is involved in dimerization and AKAP binding. Mutation of conserved residues in ropporin or RII prevents binding to AKAP110. These data suggest that sperm contains several proteins that bind to AKAPs in a manner similar to RII and imply that AKAPs may have additional and perhaps unique functions in spermatozoa.     

A kinase anchoring protein (AKAP) interaction and dimerization of the RIalpha and RIbeta regulatory subunits of protein kinase a in vivo by the yeast two hybrid system

Protein kinase A (PKA) regulatory (R) subunits dimerize through an N-terminal motif. Such dimerization is necessary for binding to PKA anchoring proteins (AKAPs) and targeting of PKA to its site of action. In the present study, we used the yeast two-hybrid system as an in vivo bio-reporter assay and analyzed the formation of homo- and heterodimeric complexes of RIalpha and RIbeta as well as AKAP binding of RI dimers. Native polyacrylamide gel electrophoresis (PAGE) of yeast extracts confirmed the two-hybrid data. Both RIalpha- and RIbeta homodimers as well as an RIalpha:RIbeta heterodimer were observed. Single, double and one triple mutation were introduced into the RIalpha and RIbeta subunits and dimerization properties of the mutants were analyzed. Consistent with previous reports, RIalpha(C37H) dimerized, although the disulfide bridges were disrupted, whereas the additional mutation of F47 or F52 abolished the dimerization. Corresponding mutations (C38H, F48A, F53A) in RIbeta were not sufficient to abolish the RIbeta dimerization, indicating that additional or other amino acids are important. RIalpha:RIbeta heterodimers of the mutants were formed at intermediate stringency. Analysis of ternary complexes by the yeast two-hybrid system revealed that RIalpha and RIbeta homodimers as well as an RIalpha:RIbeta heterodimer and several of the mutants were able to bind to the R-binding domain of AKAP149/D-AKAP1. Furthermore, an RIbeta:AKAP149 complex was identified following introduction of RIbeta into HEK293 cells. Importantly, RIbeta revealed AKAP binding properties similar to those of RIalpha, indicating that RIbeta holoenzymes may be anchored.     

Protein kinase A RII-like (R2D2) proteins exhibit differential localization and AKAP interaction

A-kinase anchoring proteins (AKAPs) bind to protein kinase A (PKA) via an amphipathic helix domain that interacts with a dimerization/docking domain on the regulatory (R) subunit of PKA. Four other mammalian proteins (ROPN1, ASP, SP17, and CABYR) also contain a highly conserved RII dimerization/docking (R2D2) domain, suggesting all four proteins may interact with all AKAPs in a manner similar to RII. All four of these proteins were originally detected in the flagellum of mammalian sperm. In this report, we demonstrate that all four R2D2 proteins are expressed in a wide variety of tissues and three of the proteins SP17, CABYR, and ASP are located in motile cilia of human bronchus and fallopian tubes. In addition, we detect SP17 in primary cilia. We also provide evidence that ROPN1 and ASP bind to a variety of AKAPs and this interaction can be disrupted with anchoring inhibitor peptides. The interaction of SP17 and CABYR with AKAPs appears to be much more limited. None of the R2D2 proteins appears to bind cAMP, a fundamental characteristic of the regulatory subunits of PKA. These observations suggest that R2D2 proteins utilize docking interactions with AKAPs to accomplish their function of regulating cilia and flagella. Based on location, affinity for AKAPs and lack of affinity for cAMP, it appears that each R2D2 protein has a unique role in this process.     

Glycogen Synthase Kinase 3�� Interaction Protein Functions as an A-kinase Anchoring Protein

A-kinase anchoring proteins (AKAPs) include a family of scaffolding proteins that target protein kinase A (PKA) and other signaling proteins to cellular compartments and thereby confine the activities of the associated proteins to distinct regions within cells. AKAPs bind PKA directly. The interaction is mediated by the dimerization and docking domain of regulatory subunits of PKA and the PKA-binding domain of AKAPs. Analysis of the interactions between the dimerization and docking domain and various PKA-binding domains yielded a generalized motif allowing the identification of AKAPs. Our bioinformatics and peptide array screening approaches based on this signature motif identified GSKIP (glycogen synthase kinase 3β interaction protein) as an AKAP. GSKIP directly interacts with PKA and GSK3β (glycogen synthase kinase 3β). It is widely expressed and facilitates phosphorylation and thus inactivation of GSK3β by PKA. GSKIP contains the evolutionarily conserved domain of unknown function 727. We show here that this domain of GSKIP and its vertebrate orthologues binds both PKA and GSK3β and thereby provides a mechanism for the integration of PKA and GSK3β signaling pathways.     

D‐AKAP2:PKA RII:PDZK1 ternary complex structure: Insights from the nucleation of a polyvalent scaffold

A‐kinase anchoring proteins (AKAPs) regulate cAMP‐dependent protein kinase (PKA) signaling in space and time. Dual‐specific AKAP2 (D‐AKAP2/AKAP10) binds with high affinity to both RI and RII regulatory subunits of PKA and is anchored to transporters through PDZ domain proteins. Here, we describe a structure of D‐AKAP2 in complex with two interacting partners and the exact mechanism by which a segment that on its own is disordered presents an α‐helix to PKA and a β‐strand to PDZK1. These two motifs nucleate a polyvalent scaffold and show how PKA signaling is linked to the regulation of transporters. Formation of the D‐AKAP2: PKA binary complex is an important first step for high affinity interaction with PDZK1, and the structure reveals important clues toward understanding this phenomenon. In contrast to many other AKAPs, D‐AKAP2 does not interact directly with the membrane protein. Instead, the interaction is facilitated by the C‐terminus of D‐AKAP2, which contains two binding motifs—the D‐AKAP2_AKB and the PDZ motif—that are joined by a short linker and only become ordered upon binding to their respective partner signaling proteins. The D‐AKAP2_AKB binds to the D/D domain of the R‐subunit and the C‐terminal PDZ motif binds to a PDZ domain (from PDZK1) that serves as a bridging protein to the transporter. This structure also provides insights into the fundamental question of why D‐AKAP2 would exhibit a differential mode of binding to the two PKA isoforms.     

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 subcellular locations through binding to PKA regulatory subunits (RI or RII). The RII-binding domain of all AKAPs forms an amphipathic alpha-helix with similar secondary structure. However, the importance of sequence differences in the RII-binding domains of different AKAPs is unknown, and mechanisms that regulate AKAP-PKA affinity are not clearly defined. Using surface plasmon resonance (SPR) spectroscopy, we measured real-time kinetics of RII interaction with various AKAPs. Base-line equilibrium binding constants (K(d)) for RII binding to Ht31, mAKAP, and AKAP15/18 were 10 nm, 119 nm, and 6.6 microm, respectively. PKA stimulation of intact Chinese hamster ovary cells increased RIIalpha binding to AKAP100/mAKAP and AKAP15/18 by approximately 7- and 82-fold, respectively. These results suggest that differences in primary sequence of the RII-binding domain may be responsible for the selective affinity of RII for different AKAPs. Furthermore, RII autophosphorylation may provide additional localized regulation of kinase anchoring. In cardiac myocytes, disruption of RII-AKAP interaction decreased PKA phosphorylation of the PKA substrate, myosin-binding protein C. Thus, these mechanisms may be involved in adding additional specificity in intracellular signaling in diverse cell types and under conditions of cAMP/PKA activation.