Induction of flexibility through protein-protein interactions

The dimerization/docking (D/D) domain of the cyclic AMP-dependent protein kinase (PKA) holoenzyme mediates important protein-protein interactions that direct the subcellular localization of the enzyme. A kinase anchoring proteins (AKAPs) provide the molecular scaffold for the localization of PKA. The recent solution structures of two D/D AKAP complexes revealed that the AKAP binds to a surface-exposed, hydrophobic groove on the D/D. In the present study, we present an analysis of the changes in hydrogen/deuterium exchange protection and internal motions of the backbone of the D/D when free and bound to the prototype anchoring protein, Ht31(pep). We observe that formation of the complex results in significant, but small, increases in H/D exchange protection factors as well as increases in backbone flexibility, throughout the D/D, and in particular, in the hydrophobic binding groove. This unusual observation of increased backbone flexibility and marginal H/D exchange protection, despite high affinity protein-ligand interactions, may be a general effect observed for the stabilization of hydrophobic ligand/hydrophobic pocket interactions.     

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.     

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.     

Alternative splicing regulates the subcellular localization of A-kinase anchoring protein 18 isoforms

The cAMP-dependent protein kinase (PKA) is localized to specific subcellular compartments by association with A-kinase anchoring proteins (AKAPs). AKAPs are a family of functionally related proteins that bind the regulatory (R) subunit of PKA with high affinity and target the kinase to specific subcellular organelles. Recently, AKAP18, a low molecular weight plasma membrane AKAP that facilitates PKA-mediated phosphorylation of the L-type Ca(2+) channel, was cloned. We now report the cloning of two additional isoforms of AKAP18, which we have designated AKAP18beta and AKAP18gamma, that arise from alternative mRNA splicing. The AKAP18 isoforms share a common R subunit binding site, but have distinct targeting domains. The original AKAP18 (renamed AKAP18alpha) and AKAP18beta target the plasma membrane when expressed in HEK-293 cells, while AKAP18gamma is cytosolic. When expressed in epithelial cells, AKAP18alpha is targeted to lateral membranes, whereas AKAP18beta is accumulated at the apical membrane. A 23-amino acid insert, following the plasma membrane targeting domain, facilitates the association of AKAP18beta with the apical membrane. The data suggest that AKAP18 isoforms are differentially targeted to modulate distinct intracellular signaling events. Furthermore, the data suggest that plasma membrane AKAPs may be targeted to subdomains of the cell surface, adding additional specificity in intracellular signaling.     

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.     

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.     

Association of the type 1 protein phosphatase PP1 with the A-kinase anchoring protein AKAP220

The cyclic AMP (cAMP)-dependent protein kinase (PKA) and the type 1 protein phosphatase (PP1) are broad-specificity signaling enzymes with opposing actions that catalyze changes in the phosphorylation state of cellular proteins. Subcellular targeting to the vicinity of preferred substrates is a means of restricting the specificity of each enzyme [1] [2]. Compartmentalization of the PKA holoenzyme is mediated through association of the regulatory subunits with A-kinase anchoring proteins (AKAPs), whereas a diverse family of phosphatase-targeting subunits directs the location of the PP1 catalytic subunit (PP1c) [3] [4]. Here, we demonstrate that the PKA-anchoring protein, AKAP220, binds PP1c with a dissociation constant (KD) of 12.1 +/- 4 nM in vitro. Immunoprecipitation of PP1 from cell extracts resulted in a 10.4 +/- 3.8-fold enrichment of PKA activity. AKAP220 co-purified with PP1c by affinity chromatography on microcystin sepharos Immunocytochemical analysis demonstrated that the kinase, the phosphatase and the anchoring protein had distinct but overlapping staining patterns in rat hippocampal neurons. Collectively, these results provide the first evidence that AKAP220 is a multivalent anchoring protein that maintains a signaling scaffold of PP1 and the PKA holoenzyme.     

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.     

Pericentrin anchors protein kinase A at the centrosome through a newly identified RII-binding domain

Centrosomes orchestrate microtubule nucleation and spindle assembly during cell division [1,2] and have long been recognized as major anchoring sites for cAMP-dependent protein kinase (PKA) [3,4]. Subcellular compartmentalization of PKA is achieved through the association of the PKA holoenzyme with A-kinase anchoring proteins (AKAPs) [5,6]. AKAPs have been shown to contain a conserved helical motif, responsible for binding to the type II regulatory subunit (RII) of PKA, and a specific targeting motif unique to each anchoring protein that directs the kinase to specific intracellular locations. Here, we show that pericentrin, an integral component of the pericentriolar matrix of the centrosome that has been shown to regulate centrosome assembly and organization, directly interacts with PKA through a newly identified binding domain. We demonstrate that both RII and the catalytic subunit of PKA coimmunoprecipitate with pericentrin isolated from HEK-293 cell extracts and that PKA catalytic activity is enriched in pericentrin immunoprecipitates. The interaction of pericentrin with RII is mediated through a binding domain of 100 amino acids which does not exhibit the structural characteristics of similar regions on conventional AKAPs. Collectively, these results provide strong evidence that pericentrin is an AKAP in vivo.     

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.     

G-Protein-coupled receptor-associated A-kinase anchoring proteins: AKAP79 and AKAP250 (gravin)

A-kinase anchoring proteins (AKAPs) define an expanding group of scaffold proteins that display a signature binding site for the RI/RII subunit of protein kinase A. AKAPs are multivalent and a subset of these scaffold proteins also display the ability to associate with the prototypic member of G-protein-coupled receptors, the beta(2)-adrenergic receptor. Both AKAP79 (also known as AKAP5) and AKAP250 (also known as gravin or AKAP12) have been shown to associate with the beta(2)-adrenergic receptor, but each directs downstream signaling events in decidedly different manners. The primary structures, common and unique protein motifs are of interest. Both proteins display largely natively unfolded primary sequences that provide a necklace on which short, structured regions of sequence are found. Membrane association appears to involve both interactions with the lipid bilayer via docking to a G-protein-coupled receptor as well as interactions of short positively charged domains with the inner leaflet of the cell membrane. Gravin, unlike AKAP79, displays a canonical site at its N-terminus that is subject to N-myristoylation. AKAP79 appears to function in switching signaling pathways of the receptor from adenylylcyclase to activation of the mitogen-activated protein kinase cascade. Gravin, in contrast, is essential for the resensitization and recycling of the receptors following agonist-induced activation, desensitization, and internalization. Each AKAP provides a template that enables space-time continuum features to G-protein-coupled signaling pathways as well as a paradigm for explaining apparent compartmentalization of cell signaling.     

Delineation of type I protein kinase A-selective signaling events using an RI anchoring disruptor

Control of specificity in cAMP signaling is achieved by A-kinase anchoring proteins (AKAPs), which assemble cAMP effectors such as protein kinase A (PKA) into multiprotein signaling complexes in the cell. AKAPs tether the PKA holoenzymes at subcellular locations to favor the phosphorylation of selected substrates. PKA anchoring is mediated by an amphipathic helix of 14-18 residues on each AKAP that binds to the R subunit dimer of the PKA holoenzymes. Using a combination of bioinformatics and peptide array screening, we have developed a high affinity-binding peptide called RIAD (RI anchoring disruptor) with >1000-fold selectivity for type I PKA over type II PKA. Cell-soluble RIAD selectively uncouples cAMP-mediated inhibition of T cell function and inhibits progesterone synthesis at the mitochondria in steroid-producing cells. This study suggests that these processes are controlled by the type I PKA holoenzyme and that RIAD can be used as a tool to define anchored type I PKA signaling events.     

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.     

Role of A-kinase anchoring proteins in cyclic-AMP-mediated Schwann cell proliferation

Proliferation of Schwann cells during peripheral nerve development is stimulated by the heregulin/neuregulin family of growth factors expressed by neurons. However, for neonatal rat Schwann cells growing in culture, heregulins produce only a weak mitogenic response. Supplementing heregulin with forskolin, an agent that elevates cyclic AMP levels, produces a dramatic increase in the proliferation of cultured Schwann cells. The mechanisms underlying this synergistic effect required for Schwann cell proliferation in vivo is not well established. Characterizing the A-kinase anchoring proteins (AKAPs) in Schwann cells might help identify substrates tethered to and phosphorylated by the cAMP-dependent protein kinase A (PKA). Using an RII overlay assay that detects AKAPs that are bound to the type II regulatory subunits of PKA, we identified AKAP150 in Schwann cells. Western blot analysis revealed that additional AKAPs, specifically AKAP95, and yotiao were also present. Disruption of PKA/AKAP interaction with Ht-31 peptide resulted in an increase in luciferase-conjugated cyclin D3 promoter activity. Transfection with sequence-specific AKAP siRNAs for AKAP150 and AKAP95 produced a marked reduction in cell proliferation. Immunoblot analysis revealed that knock down of AKAP95 protein caused a significant decrease in expression of the cell cycle regulatory proteins cyclin D2, cyclin D3 and the cell survival signal Akt/Protein Kinase B (Akt/PKB). Morphological characterization of Schwann cell AKAPs indicated the presence of nuclear (AKAP95), cytoplasm-associated (AKAP150) and perinuclear (yotiao) A-kinase anchoring proteins. These results indicate a role for AKAP95 and AKAP150 in the synergistic response of Schwann cells to treatment with heregulin and forskolin.     

Imaging kinase--AKAP79--phosphatase scaffold complexes at the plasma membrane in living cells using FRET microscopy

Scaffold, anchoring, and adaptor proteins coordinate the assembly and localization of signaling complexes providing efficiency and specificity in signal transduction. The PKA, PKC, and protein phosphatase-2B/calcineurin (CaN) scaffold protein A-kinase anchoring protein (AKAP) 79 is localized to excitatory neuronal synapses where it is recruited to glutamate receptors by interactions with membrane-associated guanylate kinase (MAGUK) scaffold proteins. Anchored PKA and CaN in these complexes could have important functions in regulating glutamate receptors in synaptic plasticity. However, direct evidence for the assembly of complexes containing PKA, CaN, AKAP79, and MAGUKs in intact cells has not been available. In this report, we use immunofluorescence and fluorescence resonance energy transfer (FRET) microscopy to demonstrate membrane cytoskeleton-localized assembly of this complex. Using FRET, we directly observed binding of CaN catalytic A subunit (CaNA) and PKA-RII subunits to membrane-targeted AKAP79. We also detected FRET between CaNA and PKA-RII bound simultaneously to AKAP79 within 50 A of each other, thus providing the first direct evidence of a ternary kinase-scaffold-phosphatase complex in living cells. This finding of AKAP-mediated PKA and CaN colocalization on a nanometer scale gives new appreciation to the level of compartmentalized signal transduction possible within scaffolds. Finally, we demonstrated AKAP79-regulated membrane localization of the MAGUK synapse-associated protein 97 (SAP97), suggesting that AKAP79 functions to organize even larger signaling complexes.     

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.     

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.     

Multiple interactions within the AKAP220 signaling complex contribute to protein phosphatase 1 regulation

The phosphorylation status of cellular proteins is controlled by the opposing actions of protein kinases and phosphatases. Compartmentalization of these enzymes is critical for spatial and temporal control of these phosphorylation/dephosphorylation events. We previously reported that a 220-kDa A-kinase anchoring protein (AKAP220) coordinates the location of the cAMP-dependent protein kinase (PKA) and the type 1 protein phosphatase catalytic subunit (PP1c) (Schillace, R. V., and Scott, J. D. (1999) Curr. Biol. 9, 321-324). We now demonstrate that an AKAP220 fragment is a competitive inhibitor of PP1c activity (K(i) = 2.9 +/- 0.7 micrometer). Mapping studies and activity measurements indicate that several protein-protein interactions act synergistically to inhibit PP1. A consensus targeting motif, between residues 1195 and 1198 (Lys-Val-Gln-Phe), binds but does not affect enzyme activity, whereas determinants between residues 1711 and 1901 inhibit the phosphatase. Analysis of truncated PP1c and chimeric PP1/2A catalytic subunits suggests that AKAP220 inhibits the phosphatase in a manner distinct from all known PP1 inhibitors and toxins. Intermolecular interactions within the AKAP220 signaling complex further contribute to PP1 inhibition as addition of the PKA regulatory subunit (RII) enhances phosphatase inhibition. These experiments indicate that regulation of PP1 activity by AKAP220 involves a complex network of intra- and intermolecular interactions.     

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.     

PKA and PDE4D3 anchoring to AKAP9 provides distinct regulation of cAMP signals at the centrosome

Previous work has shown that the protein kinase A (PKA)-regulated phosphodiesterase (PDE) 4D3 binds to A kinase-anchoring proteins (AKAPs). One such protein, AKAP9, localizes to the centrosome. In this paper, we investigate whether a PKA-PDE4D3-AKAP9 complex can generate spatial compartmentalization of cyclic adenosine monophosphate (cAMP) signaling at the centrosome. Real-time imaging of fluorescence resonance energy transfer reporters shows that centrosomal PDE4D3 modulated a dynamic microdomain within which cAMP concentration selectively changed over the cell cycle. AKAP9-anchored, centrosomal PKA showed a reduced activation threshold as a consequence of increased autophosphorylation of its regulatory subunit at S114. Finally, disruption of the centrosomal cAMP microdomain by local displacement of PDE4D3 impaired cell cycle progression as a result of accumulation of cells in prophase. Our findings describe a novel mechanism of PKA activity regulation that relies on binding to AKAPs and consequent modulation of the enzyme activation threshold rather than on overall changes in cAMP levels. Further, we provide for the first time direct evidence that control of cell cycle progression relies on unique regulation of centrosomal cAMP/PKA signals.