In resting COS1 cells a dominant negative approach shows that specific, anchored PDE4 cAMP phosphodiesterase isoforms gate the activation, by basal cyclic AMP production, of AKAP-tethered protein kinase A type II located in the centrosomal region

We employ a novel, dominant negative approach to identify a key role for certain tethered cyclic AMP specific phosphodiesterase-4 (PDE4) isoforms in regulating cyclic AMP dependent protein kinase A (PKA) sub-populations in resting COS1 cells. A fraction of PKA is clearly active in resting COS1 cells and this activity increases when cells are treated with the selective PDE4 inhibitor, rolipram. Point mutation of a critical, conserved aspartate residue in the catalytic site of long PDE4A4, PDE4B1, PDE4C2 and PDE4D3 isoforms renders them catalytically inactive. Overexpressed in resting COS1 cells, catalytically inactive forms of PDE4C2 and PDE4D3, but not PDE4A4 and PDE4B1, are constitutively PKA phosphorylated while overexpressed active versions of all these isoforms are not. Inactive and active versions of all these isoforms are PKA phosphorylated in cells where protein kinase A is maximally activated with forskolin and IBMX. By contrast, rolipram challenge of COS1 cells selectively triggers the PKA phosphorylation of recombinant, active PDE4D3 and PDE4C2 but not recombinant, active PDE4A4 and PDE4B1. Purified, recombinant PDE4D3 and PDE4A4 show a similar dose-dependency for in vitro phosphorylation by PKA. Disruption of the tethering of PKA type-II to PKA anchor proteins (AKAPs), achieved using the peptide Ht31, prevents inactive forms of PDE4C2 and PDE4D3 being constitutively PKA phosphorylated in resting cells as does siRNA-mediated knockdown of PKA-RII, but not PKA-RI. PDE4C2 and PDE4D3 co-immunoprecipitate from COS1 cell lysates with 250 kDa and 450 kDa AKAPs that tether PKA type-II and not PKA type-I. PKA type-II co-localises with AKAP450 in the centrosomal region of COS1 cells. The perinuclear distribution of recombinant, inactive PDE4D3, but not inactive PDE4A4, overlaps with AKAP450 and PKA type-II. The distribution of PKA phosphorylated inactive PDE4D3 also overlaps with that of AKAP450 in the centrosomal region of COS1 cells. We propose that a novel role for PDE4D3 and PDE4C2 is to gate the activation of AKAP450-tethered PKA type-II localised in the perinuclear region under conditions of basal cAMP generation in resting cells.     

Phosphodiesterase 4D and protein kinase a type II constitute a signaling unit in the centrosomal area

The mediation of cAMP effects by specific pools of protein kinase A (PKA) targeted to distinct subcellular domains raises the question of how inactivation of the cAMP signal is achieved locally and whether similar targeting of phosphodiesterases (PDEs) to sites of cAMP/PKA action could be observed. Here, we demonstrate that Sertoli cells of the testis contain an insoluble PDE4D3 isoform, which is shown by immunofluorescence to target to centrosomes. Staining of PDE4D and PKA shows co-localization of PDE4D with PKA-RIIalpha and RIIbeta in the centrosomal region. Co-precipitation of RII subunits and PDE4D3 from cytoskeletal extracts indicates a physical association of the two proteins. Distribution of PDE4D overlaps with that of the centrosomal PKA-anchoring protein, AKAP450, and AKAP450, PDE4D3, and PKA-RIIalpha co-immunoprecipitate. Finally, both PDE4D3 and PKA co-precipitate with a soluble fragment of AKAP450 encompassing amino acids 1710 to 2872 when co-expressed in 293T cells. Thus, a centrosomal complex that includes PDE4D and PKA constitutes a novel signaling unit that may provide accurate spatio-temporal modulation of cAMP signals.     

A-kinase anchoring proteins interact with phosphodiesterases in T lymphocyte cell lines

The cAMP protein kinase A (PKA) pathway in T cells conveys an inhibitory signal to suppress inflammation. This study was performed to understand the mechanisms involved in cAMP-mediated signaling in T lymphocytes. A-kinase anchoring proteins (AKAPs) bind and target PKA to various subcellular locations. AKAPs also bind other signaling molecules such as cyclic nucleotide phosphodiesterases (PDEs) that hydrolyze cAMP in the cell. PDE4 and PDE7 have important roles in T cell activation. Based on this information, we hypothesized that AKAPs associate with PDEs in T lymphocytes. Immunoprecipitation of Jurkat cell lysates with Abs against both the regulatory subunit of PKA (RIIalpha) and specific AKAPs resulted in increased PDE activity associated with RIIalpha and AKAP95, AKAP149, and myeloid translocation gene (MTG) compared with control (IgG). Immunoprecipitation and pull-down analyses demonstrate that PDE4A binds to AKAP149, AKAP95, and MTG, but not AKAP79, whereas PDE7A was found to bind only MTG. Further analysis of MTG/PDE association illustrated that PDE4A and PDE7A bind residues 1-344 of MTG16b. Confocal analysis of HuT 78 cells stained with anti-PDE7A showed overlapping staining patterns with the Golgi marker GM130, suggesting that PDE7A is located in the Golgi. The staining pattern of PDE7A also showed similarity to the staining pattern of MTG, supporting the immunoprecipitation data and suggesting that MTG may interact with PDE7A in the Golgi. In summary, these data suggest that AKAPs interact with both PKA and PDE in T lymphocytes and thus are a key component of the signaling complex regulating T cell activation.     

An anchored PKA and PDE4 complex regulates subplasmalemmal cAMP dynamics

The spatiotemporal regulation of cAMP can generate microdomains just beneath the plasma membrane where cAMP increases are larger and more dynamic than those seen globally. Real-time measurements of cAMP using mutant cyclic nucleotide-gated ion channel biosensors, pharmacological tools and RNA interference (RNAi) were employed to demonstrate a subplasmalemmal cAMP signaling module in living cells. Transient cAMP increases were observed upon stimulation of HEK293 cells with prostaglandin E1. However, pretreatment with selective inhibitors of type 4 phosphodiesterases (PDE4), protein kinase A (PKA) or PKA/A-kinase anchoring protein (AKAP) interaction blocked an immediate return of subplasmalemmal cAMP to basal levels. Knockdown of specific membrane-associated AKAPs using RNAi identified gravin (AKAP250) as the central organizer of the PDE4 complex. Co-immunoprecipitation confirmed that gravin maintains a signaling complex that includes PKA and PDE4D. We propose that gravin-associated PDE4D isoforms provide a means to rapidly terminate subplasmalemmal cAMP signals with concomitant effects on localized ion channels or enzyme activities.     

AKAP3 selectively binds PDE4A isoforms in bovine spermatozoa

Cyclic AMP plays an important role in regulating sperm motility and acrosome reaction through activation of cAMP-dependent protein kinase A (PKA). Phosphodiesterases (PDEs) modulate the levels of cyclic nucleotides by catalyzing their degradation. Although PDE inhibitors specific to PDE1 and PDE4 are known to alter sperm motility and capacitation in humans, little is known about the role or subcellular distribution of PDEs in spermatozoa. The localization of PKA is regulated by A-kinase anchoring proteins (AKAPs), which may also control the intracellular distribution of PDE. The present study was undertaken to investigate the role and localization of PDE4 during sperm capacitation. Addition of Rolipram or RS25344, PDE4-specific inhibitors significantly increased the progressive motility of bovine spermatozoa. Immunolocalization techniques detected both PDE4A and AKAP3 (formerly known as AKAP110) in the principal piece of bovine spermatozoa. The PDE4A5 isoform was detected primarily in the Triton X-100-soluble fraction of caudal epididymal spermatozoa. However, in ejaculated spermatozoa it was seen primarily in the SDS-soluble fraction, indicating a shift in PDE4A5 localization into insoluble organelles during sperm capacitation. AKAP3 was detected only in the SDS-soluble fraction of both caudal and ejaculated sperm. Immunoprecipitation experiments using COS cells cotransfected with AKAP3 and either Pde4a5 or Pde4d provide evidence that PDE4A5 but not PDE4D interacts with AKAP3. Pulldown assays using sperm cell lysates confirm this interaction in vitro. These data suggest that AKAP3 binds both PKA and PDE4A and functions as a scaffolding protein in spermatozoa to regulate local cAMP concentrations and modulate sperm functions.     

"Shaping" of cell signaling via AKAP-tethered PDE4D: Probing with AKAR2-AKAP5 biosensor

ABSTRACT: BACKGROUND: PKA, a key regulator of cell signaling, phosphorylates a diverse and important array of target molecules and is spatially docked to members of the A-kinase Anchoring Protein (AKAP) family. AKAR2 is a biosensor which yields a FRET signal in vivo, when phosphorylated by PKA. AKAP5, a prominent member of the AKAP family, docks several signaling molecules including PKA, PDE4D, as well as GPCRs, and is obligate for the propagation of the activation of the mitogen-activated protein kinase cascade from GPCRs to ERK1,2. RESULTS: Using an AKAR2-AKAP5 fusion "biosensor", we investigated the spatial-temporal activation of AKAP5 undergoing phosphorylation by PKA in response to beta-adrenergic stimulation. The pattern of PKA activation reported by AKAR2-AKAP5 is a more rapid and spatially distinct from those "sensed" by AKAR2-AKAP12. Spatial-temporal restriction of activated PKA by AKAP5 was found to "shape" the signaling response. Phosphatase PDE4D tethered to AKAP5 also later reverses within 60 s elevated intracellular cyclic AMP levels stimulated by beta-adrenergic agonist. AKAP12, however, fails to attenuate the rise in cyclic AMP over this time. Fusion of the AKAP5 PDE4D-binding-domain to AKAP12 was found to accelerate a reversal of accumulation of intracellular cyclic AMP. CONCLUSION: AKAPs, which are scaffolds with tethered enzymes, can "shape" the temporal and spatial aspects of cell signaling.     

“Shaping” of cell signaling via AKAP-tethered PDE4D: Probing with AKAR2-AKAP5 biosensor

Background

PKA, a key regulator of cell signaling, phosphorylates a diverse and important array of target molecules and is spatially docked to members of the A-kinase Anchoring Protein (AKAP) family. AKAR2 is a biosensor which yields a FRET signal in vivo, when phosphorylated by PKA. AKAP5, a prominent member of the AKAP family, docks several signaling molecules including PKA, PDE4D, as well as GPCRs, and is obligate for the propagation of the activation of the mitogen-activated protein kinase cascade from GPCRs to ERK1,2.

Results

Using an AKAR2-AKAP5 fusion ??biosensor??, we investigated the spatial-temporal activation of AKAP5 undergoing phosphorylation by PKA in response to ??-adrenergic stimulation. The pattern of PKA activation reported by AKAR2-AKAP5 is a more rapid and spatially distinct from those ??sensed?? by AKAR2-AKAP12. Spatial-temporal restriction of activated PKA by AKAP5 was found to ??shape?? the signaling response. Phosphatase PDE4D tethered to AKAP5 also later reverses within 60?s elevated intracellular cyclic AMP levels stimulated by ??-adrenergic agonist. AKAP12, however, fails to attenuate the rise in cyclic AMP over this time. Fusion of the AKAP5 PDE4D-binding-domain to AKAP12 was found to accelerate a reversal of accumulation of intracellular cyclic AMP.

Conclusion

AKAPs, which are scaffolds with tethered enzymes, can ??shape?? the temporal and spatial aspects of cell signaling.     

Identification and characterization of myeloid translocation gene 16b as a novel a kinase anchoring protein in T lymphocytes

Increased levels of intracellular cAMP inhibit T cell activation and proliferation. One mechanism is via activation of the cAMP-dependent protein kinase (PKA). PKA is a broad specificity serine/threonine kinase whose fidelity in signaling is maintained through interactions with A kinase anchoring proteins (AKAPs). AKAPs are adaptor/scaffolding molecules that convey spatial and temporal localization to PKA and other signaling molecules. To determine whether T lymphocytes contain AKAPs that could influence the inflammatory response, PBMCs and Jurkat cells were analyzed for the presence of AKAPs. RII overlay and cAMP pull down assays detected at least six AKAPs. Western blot analyses identified four known AKAPs: AKAP79, AKAP95, AKAP149, and WAVE. Screening of a PMA-stimulated Jurkat cell library identified two additional known AKAPs, AKAP220 and AKAP-KL, and one novel AKAP, myeloid translocation gene 16 (MTG16b). Mutational analysis identified the RII binding domain in MTG16b as residues 399-420, and coimmunoprecipitation assays provide strong evidence that MTG16b is an AKAP in vivo. Immunofluorescence and confocal microscopy illustrate distinct subcellular locations of AKAP79, AKAP95, and AKAP149 and suggest colocalization of MTG and RII in the Golgi. These experiments represent the first report of AKAPs in T cells and suggest that MTG16b is a novel AKAP that targets PKA to the Golgi of T lymphocytes.     

Phosphodiesterase 4 interacts with the 5-HT4(b) receptor to regulate cAMP signaling

Phosphodiesterase (PDE) 3 and PDE4, which degrade cyclic adenosine monophosphate (cAMP), are important regulators of 5-hydroxytryptamine (5-HT) 4 receptor signaling in cardiac tissue. Therefore, we investigated whether they interact with the 5-HT_4(b) receptor, and whether A-kinase anchoring proteins (AKAPs), scaffolding proteins that bind to the regulatory subunit of protein kinase A (PKA) and contribute to the spacial-temporal control of cAMP signaling, are involved in the regulation of 5-HT_4(b) receptor signaling. By measuring PKA activity in the absence and presence of PDE3 and PDE4 inhibitiors, we found that constitutive signaling of the overexpressed HA-tagged 5-HT_4(b) receptor in HEK293 cells is regulated predominantly by PDE4, with a secondary role for PDE3 that is unmasked in the presence of PDE4 inhibition. Overexpressed PDE4D3 and PDE3A1, and to a smaller extent PDE4D5 co-immunoprecipitate constitutively with the 5-HT_4(b) receptor. PDE activity measurements in immunoprecipitates of the 5-HT_4(b) receptor confirm the association of PDE4D3 with the receptor and provide evidence that the activity of this PDE may be increased upon receptor stimulation with 5-HT. A possible involvement of AKAPs in 5-HT_4(b) receptor signaling was uncovered in experiments using the St-Ht31 inhibitor peptide, which disrupts the interaction of AKAPs with PKA. However, St-Ht31 did not influence 5-HT_4(b) receptor-stimulated PKA activity, and endogenous AKAP79 and gravin were not found in immunoprecipitates of the 5-HT_4(b) receptor. In conclusion, we found that both PDE3A1 and PDE4D3 are integrated into complexes that contain the 5-HT_4(b) receptor and may thereby regulate 5-HT_4(b) receptor-mediated signaling.     

Role of AKAP 149–PKA–PDE4A complex in cell survival and cell differentiation processes

The cellular localization of A-kinase anchoring proteins (AKAPs), protein kinase A (PKAs) and phosphodiesterases (PDEs) is a key step to the spatiotemporal regulation of the second messenger adenosine 3′,5′-cyclic monophosphate (cAMP). In this paper the cellular distribution of the mitochondrial AKAP 149–PKA–PDE4A complex and its implications in the cell death induced by YTX treatment, a known PDE modulator, was studied. K-562 cell line was incubated with YTX for 24 or 48 h. Under these conditions AKAP 149, PKA and type-4A PDE (PDE4A) levels were measured in the cytosol, in the plasma membrane and in the nucleus. Apoptotic hallmarks were also measured after the same conditions. In addition, YTX effect on cell viability was checked after AKAP 149 and PDE4A silencing. The results obtained show a decrease in AKAP 149–PKA–PDE4A levels in cytosol after YTX exposure. 24 h after the toxin addition, the complex expression increased in the plasma membrane and after 48 h in the nucleus domain. Furthermore Bcl-2 levels were decreased and the expression of caspase 3 together with caspase 8 activity were increased after 24 h of toxin incubation but not after 48 h. These results suggest apoptotic cell death at 24 h and a non-apoptotic cell death after 48 h. When AKAP 149 and PDE4A were silenced YTX did not induce cellular death. In summary, AKAP 149–PKA–PDE4A complex localization is related with YTX effect in K-562 cell line. When this complex is mainly located in the plasma membrane apoptosis is activated while when the complex is in the nuclear domain non-apoptotic cellular death or cellular differentiation is activated. Therefore AKAP 149–PKA–PDE4A distribution and integrity have a key role in cellular survival.     

Anchoring of protein kinase A-regulatory subunit IIalpha to subapically positioned centrosomes mediates apical bile canalicular lumen development in response to oncostatin M but not cAMP

Oncostatin M and cAMP signaling stimulate apical surface-directed membrane trafficking and apical lumen development in hepatocytes, both in a protein kinase A (PKA)-dependent manner. Here, we show that oncostatin M, but not cAMP, promotes the A-kinase anchoring protein (AKAP)-dependent anchoring of the PKA regulatory subunit (R)IIalpha to subapical centrosomes and that this requires extracellular signal-regulated kinase 2 activation. Stable expression of the RII-displacing peptide AKAP-IS, but not a scrambled peptide, inhibits the association of RIIalpha with centrosomal AKAPs and results in the repositioning of the centrosome from a subapical to a perinuclear location. Concomitantly, common endosomes, but not apical recycling endosomes, are repositioned from a subapical to a perinuclear location, without significant effects on constitutive or oncostatin M-stimulated basolateral-to-apical transcytosis. Importantly, however, the expression of the AKAP-IS peptide completely blocks oncostatin M-, but not cAMP-stimulated apical lumen development. Together, the data suggest that centrosomal anchoring of RIIalpha and the interrelated subapical positioning of these centrosomes is required for oncostatin M-, but not cAMP-mediated, bile canalicular lumen development in a manner that is uncoupled from oncostatin M-stimulated apical lumen-directed membrane trafficking. The results also imply that multiple PKA-mediated signaling pathways control apical lumen development and that subapical centrosome positioning is important in some of these pathways.     

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.     

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.     

A型激酶锚定蛋白及其复合物结构与功能

A型激酶锚定蛋白(A-kinase anchoring proteins,AKAPs)是一类结构不同而功能相关的蛋白家族,其主要功能是将cAMP依赖性蛋白激酶A（PKA）锚定于特定的亚细胞结构.PKA是第二信使cAMP的主要效应器,而AKAPs在靶向定位和调节PKA介导的磷酸化事件方面扮演重要角色. AKAPs更为重要的功能是与多种信号分子形成信号复合物,从时间和空间上整合cAMP-PKA和其他信号途径.本文将对AKAPs及其信号复合物的结构特点和参与细胞信号转导的功能机制及其研究现状进行概述.     

RNA silencing identifies PDE4D5 as the functionally relevant cAMP phosphodiesterase interacting with beta arrestin to control the protein kinase A/AKAP79-mediated switching of the beta2-adrenergic receptor to activation of ERK in HEK293B2 cells

PDE4B and PDE4D provide >90% of PDE4 cAMP phosphodiesterase activity in human embryonic kidney (HEK293B2) cells. Their selective small interference RNA (siRNA)-mediated knockdown potentiates isoprenaline-stimulated protein kinase A (PKA) activation. Whereas endogenous PDE4D co-immunoprecipitates with beta arrestin, endogenous PDE4B does not, even upon PDE4D knockdown. Ectopic overexpression of PDE4B2 confers co-immunoprecipitation with beta arrestin. Knockdown of PDE4D, but not PDE4B, amplifies isoprenaline-stimulated phosphorylation of the beta2-adrenergic receptor (beta2-AR) by PKA and activation of extracellular signal-regulated kinase (ERK) through G(i). Isoform-selective knockdown identifies PDE4D5 as the functionally important species regulating isoprenaline stimulation of both these processes. Ht31-mediated disruption of the tethering of PKA to AKAP scaffold proteins attenuates isoprenaline activation of ERK, even upon PDE4D knockdown. Selective siRNA-mediated knockdown identifies AKAP79, which is constitutively associated with the beta2-AR, rather than isoprenaline-recruited gravin, as being the functionally relevant AKAP in this process. Isoprenaline-stimulated membrane recruitment of PDE4D is ablated upon beta arrestin knockdown. A mutation that compromises interactions with beta arrestin prevents catalytically inactive PDE4D5 from performing a dominant negative role in potentiating isoprenaline-stimulated ERK activation. Beta arrestin-recruited PDE4D5 desensitizes isoprenaline-stimulated PKA phosphorylation of the beta2-AR and the consequential switching of its signaling to ERK. The ability to observe a cellular phenotype upon PDE4D5 knockdown demonstrates that other PDE4 isoforms, expressed at endogenous levels, are unable to afford rescue in HEK293B2 cells.     

The Role of Type 4 Phosphodiesterases in Generating Microdomains of cAMP: Large Scale Stochastic Simulations

Cyclic AMP (cAMP) and its main effector Protein Kinase A (PKA) are critical for several aspects of neuronal function including synaptic plasticity. Specificity of synaptic plasticity requires that cAMP activates PKA in a highly localized manner despite the speed with which cAMP diffuses. Two mechanisms have been proposed to produce localized elevations in cAMP, known as microdomains: impeded diffusion, and high phosphodiesterase (PDE) activity. This paper investigates the mechanism of localized cAMP signaling using a computational model of the biochemical network in the HEK293 cell, which is a subset of pathways involved in PKA-dependent synaptic plasticity. This biochemical network includes cAMP production, PKA activation, and cAMP degradation by PDE activity. The model is implemented in NeuroRD: novel, computationally efficient, stochastic reaction-diffusion software, and is constrained by intracellular cAMP dynamics that were determined experimentally by real-time imaging using an Epac-based FRET sensor (H30). The model reproduces the high concentration cAMP microdomain in the submembrane region, distinct from the lower concentration of cAMP in the cytosol. Simulations further demonstrate that generation of the cAMP microdomain requires a pool of PDE4D anchored in the cytosol and also requires PKA-mediated phosphorylation of PDE4D which increases its activity. The microdomain does not require impeded diffusion of cAMP, confirming that barriers are not required for microdomains. The simulations reported here further demonstrate the utility of the new stochastic reaction-diffusion algorithm for exploring signaling pathways in spatially complex structures such as neurons.     

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.