An approximate location for the PKAII tethering site in Akap200 was established by sequencing five cDNAs that directed synthesis of RII-binding, fusion proteins in plaques of a bacteriophage expression library. The sequence of the smallest cDNA (147 bp) encodes a region of the anchor protein bounded by Asp⁴⁹⁰ and Glu⁵³⁸. The same sequence was present in each of the larger (1.1-1.7 kbp) cDNAs. Tethering sites in mammalian AKAPs are composed of ~20 amino acids and contain a precisely spaced group of residues with large, aliphatic side chains (Leu, Val, Ile, and Thr) that cooperatively govern sequestration of RIIa and RIIß subunits. Residues 511-530 in Akap200 can be aligned with tethering regions of mammalian AKAPs, so that Ile⁵¹¹, Ile⁵¹⁸, Val⁵¹⁹, Thr⁵²³, and Val⁵³⁰ are in register with essential hydrophobic residues in previously characterized RII binding sites. A final conserved position is occupied by a smaller hydrophobic residue (Ala⁵²⁷) in the Drosophila anchor protein. RII binding domains of mammalian AKAPs are predicted to fold as an amphipathic a-helix that contains one markedly hydrophobic surface. This feature is also evident in the segment of Akap200 that encompasses residues 511-528. Folding algorithms predict that this partial polypeptide is organized into an a-helix in which 9 of 10 side chains on one surface have hydrophobic character. Thus, amino acids 511-530 constitute a candidate RII binding site in Akap200 (Li, 1999).

To further characterize the tethering domain, a fragment of Akap200 cDNA (nucleotides 1577-2314) that encodes amino acids 475-753 was amplified by polymerase chain reaction. The soluble His tagged fusion protein (named p-DAKAP70) was purified to near homogeneity by affinity chromatography on Ni²⁺-chelate Sepharose 4B resin. Small amounts of immobilized p-DAKAP70 avidly bind ³²P-RIIß (human) in an overlay assay performed with a low concentration of labeled ligand. Similar results were obtained with radiolabeled murine RIIa, thereby indicating that the tethering domain of Akap200 complexes both RII isoforms with high affinity. A caveat is that both binding studies and functional screening of cDNA expression libraries were designed and executed on the basis of a logical but unproved assumption, that mammalian RII isoforms are interchangeable substitutes for authentic Drosophila RII (RII_DR) subunits (Li, 1999).

To verify this assumption and demonstrate more directly the physiological relevance of the novel fly anchor protein, it was essential to examine the ability of Akap200 to bind RII_DR. The gene encoding the 376-residue RII_DR polypeptide has been cloned and sequenced. Structural features that govern subunit dimerization and create a docking surface for AKAPs are located near the amino terminus (residues 1-50) of mammalian RII isoforms. Alignment of the N termini of RII_DR and RIIa reveals that the two sequences are quite divergent (only 44% identity). However, groups of aromatic residues (Phe and Tyr) and amino acids with large aliphatic side chains (Leu, Val, and Ile) contribute the essential functional properties of dimerization and AKAP binding regions in RIIa and RII. Amino acids with these characteristics are conserved at all corresponding positions within residues 1-50 of RII _DR (Li, 1999)

Like mammalian RII subunits, RII_DR contains the PKA phosphorylation site sequence (RRXSX) in a linker region between the dimerization-AKAP binding domain and the cAMP binding sites. Thus, RII_DR was labeled by incubation with Mg-gamma-³²P ATP and the catalytic (C) subunit of PKA. ³²P-Labeled RII_DR binds with low levels of p-DAKAP70 and also forms a stable complex with bovine AKAP75. Thus, structural features that mediate interactions between RII subunits and AKAPs have co-evolved and are conserved from flies to humans. Binding interactions between RIIa, RIIß, or RII_DR and various AKAPs are sufficiently similar to enable their interchangeable use. Since recombinant mammalian RII isoforms are available in plentiful supply and these proteins are more thoroughly characterized than RII_DR, they were employed for most of the studies presented in this study. Repetition of experiments with RII_DR yielded similar results in all instances (Li, 1999).

The physiological relevance of Akap200 was investigated by testing the ability of the anchor protein to bind an endogenous ligand, RII_DR. Several domains in Akap200 may contribute to the targeting of tethered PKAII. Two Pro-rich regions (residues 328-332 and 468-479) are potential binding sites for cytoskeleton/organelle-associated proteins that contain Src homology 3 domains. Amino acids 2-7 constitute an acceptor site for N-myristoyltransferase. Myristoylation of Akap200 would provide a long saturated aliphatic chain that inserts into the hydrocarbon interior of phospholipid bilayers. A segment of Akap200 (residues 118-148) includes a PSD-like cluster of 13 Lys, five Ser, and five large hydrophobic residues. Positive charge in PSD2S promotes electrostatic binding with negatively charged head groups of membrane phospholipids. Intercalation of PSD hydrophobic side chains into the apolar interior of a bilayer further stabilizes association of PSD-containing proteins with membranes. N-terminal myristate and a PSD are critical features of MARCKS proteins, which mediate interactions between plasma membrane and F-actin. The MARCKS PSD is phosphorylated in situ by diacylglycerol-activated protein kinase C isoforms. Phosphorylation inhibits binding with membrane phospholipids, enables translocation of MARCKS from cell surface to cytoplasm, and promotes cytoskeleton remodeling. The nonphosphorylated PSD sequesters calmodulin in a calcium-dependent manner. Binding of Ca²⁺-calmodulin diminishes the ability of MARCKS to cross-link and bundle actin filaments. By analogy, the fly anchor protein may be involved in integrating signals propagated by three critical second messenger molecules: cAMP, diacylglycerol, and calcium. Moreover, the PSD region of Akap200 may enable phosphorylation-controlled shuttling of tethered PKA between two or more intracellular sites (Li, 1999).

Drosophila A kinase anchor protein 200 (Akap200), is predicted to be involved in routing, mediating, and integrating signals carried by cAMP, Ca²⁺, and diacylglycerol. Experiments designed to assess this hypothesis establish (1) the function, boundaries and identity of critical amino acids of the protein kinase AII (PKAII) tethering site of Akap200; (2) demonstrate that residues 119-148 mediate binding with Ca²⁺-calmodulin and F-actin; (3) show that a polybasic region of Akap200 is a substrate for protein kinase C; (4) reveal that phosphorylation of the polybasic domain regulates affinity for F-actin and Ca²⁺-calmodulin, and (5) indicate that Akap200 is myristoylated and that this modification promotes targeting of Akap200 to plasma membrane. DAkap200, a second product of the Akap200 gene, cannot tether PKAII. However, DAkap200 is myristoylated and contains a phosphorylation site domain that binds Ca²⁺-calmodulin and F-actin. An atypical amino acid composition, a high level of negative charge, exceptional thermostability, unusual hydrodynamic properties, properties of the phosphorylation site domain, and a calculated M_r of 38,000 suggest that DAkap200 is a new member of the myristoylated alanine-rich C kinase substrate protein family. Akap200 is a potentially mobile, chimeric A kinase anchor protein-myristoylated alanine-rich C kinase substrate protein that may facilitate localized reception and targeted transmission of signals carried by cAMP, Ca²⁺, and diacylglycerol (Rossi, 1999).

The observation that amino acids 2-7 constitute a potential myristoylation site resulted in identification of a targeting domain. Metabolic labeling with [³H]myristate and immunoprecipitation with anti-Akap200 IgGs has revealed that endogenous Akap200 and DAkap200 are myristoylated in situ in Drosophila S2 cells. Incorporated myristate provides a long alkyl chain that inserts into the interior of phospholipid bilayers, thereby promoting the targeting, anchoring, and enrichment of Akap200 at a membrane surface. Myristoylated anchor protein is also synthesized in mammalian (AV12) cells that contain a Akap200 transgene. A deleted version of Akap200 that lacks residues 1-7 was expressed in AV12 cells, but the protein failed to incorporate [³H]myristate. Thus, the N-terminal region of Akap200 is the sole site of myristoylation and a probable targeting domain for the anchor protein. Several myristoylated signaling proteins contain proximal Cys residues that undergo palmitoylation. Acylation at multiple sites causes extremely stable membrane anchoring. However, Akap200 is not modified by palmitoylation (Rossi, 1999).

N-terminal myristoylation is essential for routing various signaling proteins, including Src and MARCKS, to plasma membrane. Mutations that prevent myristoylation elicit mislocalization and loss of biological function. Myristoylated MARCKS accumulates at focal adhesions or at the junction of cortical cytoskeleton and plasma membrane. MARCKS may simultaneously bind the lipid bilayer (via myristate) and the F-actin-based cytoskeleton. In this configuration, MARCKS mediates interactions between the membrane and actin filaments that stabilize the cytoskeleton. Nonmyristoylated MARCKS is restricted to cytoplasm, and its ability to modulate cytoskeleton organization is abrogated (Rossi, 1999 and references therein).

Akap200 and DAkap200 are specifically enriched in the plasma membrane of Drosophila S2 cells. Epitope-tagged wild type Akap200 is also differentially targeted to plasma membrane in transfected S2 cells. Tagged nonmyristoylated Akap200 is evenly distributed throughout the cell cytoplasm. Thus, N-terminal myristoylation is essential for organelle-specific accumulation of Akap200·RII (PKAII) complexes. Mammalian AKAP18 contains a classical RII tethering site, but docking of this anchor protein with plasma membrane is guided by three acylated amino acids (Rossi, 1999).

Gly¹ is myristoylated, whereas Cys³ and Cys⁴ are palmitoylated. Deletion or mutation of Gly¹ abrogates myristoylation but has no effect on AKAP18 localization. Likewise, mutation of Cys³ or Cys⁴ alone does not alter association of AKAP18 with plasma membrane. However, elimination of all acylation sites yields an AKAP18 variant that accumulates in cytoplasm. Thus, myristoylation of AKAP18 supports, but is not essential for, targeting/anchoring of PKAII (Rossi, 1999 and references therein).

Deletion mutagenesis mapped the Akap200 RII binding domain to a region that includes amino acids 511-531. Activity and selectivity of this PKAII tethering site were not dependent on the context of flanking N- and C-terminal sequences. Conserved hydrophobic residues in the tethering region are predicted to generate an RII binding surface comparable in size and shape with that produced from the corresponding region of mammalian AKAP75 and AKAP79. Substitution of Ile⁵¹⁸ and Val⁵¹⁹ or Thr⁵²³ with Ala sharply diminish RII ligation. This is interpreted as a consequence of the disruption of two parameters (size and shape) in a structure based on cooperative interactions among large, aliphatic side chains. Substitution of Ala⁵¹⁵ with Ser reduced RII binding activity by ~70%, thereby indicating that a hydrophobic side chain at this position is essential for a maximal ligand affinity. This mutation reveals evolutionary divergence in the fly AKAP. The analogous Ala to Ser mutation has no effect on RII sequestration by mammalian AKAP-KL (Rossi, 1999 and references therein).

A PSD (residues 119-148) from Akap200/DAkap200 binds F-actin and Ca²⁺-calmodulin. Ser¹³² and other serines (Ser¹³⁵ and/or Ser¹³⁷) in the PSD region are phosphorylated by PKC. Both calmodulin and actin binding activities are lost as a consequence of PSD-directed phosphorylation. This provides a mechanism for reversibly altering the location and Ca²⁺-calmodulin-related effector functions of Akap200 and DAkap200. The physiological importance of multifunctional PSDs in vertebrate MARCKS and MARCKS-like proteins is paralleled by a high degree of sequence conservation across species. However, alignment of PSD sequences disclosed that the Akap200 PSD is a novel and rather divergent member of this domain family. A distinctive feature of the Drosophila PSD is the compact central cluster of large hydrophobic residues (Trp¹³¹, Phe¹³³, Ile¹³⁶, Phe¹³⁸) that intermingle with three Ser residues that are PKC targets. This arrangement involves a contiguous block of only eight amino acids. Introduction of a high level of negative charge adjacent to sites where Trp, Phe, and Ile side chains are immersed in a lipid bilayer may negate membrane association by electrostatic repulsion from negatively charged phospholipid head groups. This 'electrostatic switch mechanism' may be less efficient and require a higher level of phosphorylation in vertebrate PSDs where serines and hydrophobic amino acids are dispersed among a total of 14 residues. Incorporation of a novel PSD into Akap200 produces a multifunctional anchor protein with enhanced capacities for intracellular targeting, regulation via phosphorylation, and integration of multiple second messenger signals (Rossi, 1999).

MARCKS is a major PKC substrate in many mammalian cells, where it mediates aspects of cytoskeleton dynamics. Incorporation of an RII binding site within a PKC effector protein enables a novel mode of cross-talk between two signal transduction pathways. PKC-catalyzed phosphorylation of the Akap200 PSD could elicit translocation of anchor protein·PKAII complexes from plasma membrane/cortical cytoskeleton to cytoplasm (or elsewhere). Consequently, hormones that stimulate synthesis of lipid second messengers could determine intracellular target sites at which the cAMP/PKA signaling pathway exerts its actions. Phosphorylation-dependent targeting of Akap200·RII complexes could constitute a novel, reversible mode of routing signals carried by cAMP. Previously characterized AKAPs appear to engage in static anchoring of PKAII isoforms (Rossi, 1999 and references therein).

The ubiquitous cAMP-protein kinase A (PKA) signaling pathway exhibits complex temporal requirements during the time course of associative memory processing. This directly raises questions about the molecular mechanisms that provide signaling specificity to this pathway. This study used Drosophila olfactory conditioning to show that divergent cAMP signaling is mediated by functionally distinct pools of PKA. One particular pool is organized via the PKA regulatory type II subunit at the level of A-kinase anchoring proteins (AKAPs), a family of scaffolding proteins that provides focal points of spatiotemporal signal integration. This AKAP-bound pool of PKA is acting within neurons of the mushroom bodies to support a late phase of aversive memory. The requirement for AKAP-bound PKA signaling is limited to aversive memory, but dispensable during appetitive memory. This finding suggests the existence of additional mechanisms to support divergence within the cAMP-PKA signaling pathway during memory processing. Together, these results show that subcellular organization of signaling components plays a key role in memory processing (Schwaerzel, 2007).

This study pioneered the role of AKAPs as organizers of PKA signaling in Drosophila associative olfactory memory processing. First, it was shown that after aversive conditioning ASM can be separated into two phases; whereas an early phase of ASM is AKAP-independent, late ASM requires AKAP-bound PKA-RII signaling within the MBs. Second, it was shown that, after appetitive conditioning, ASM 'bypasses' this AKAP-bound pool of PKA. This shows that PKA signaling at AKAP complexes does not disrupt associative functions per se, but that functionally distinct pools of PKA must exist on a subcellular level to serve defined functions during memory processing. A functional model is suggested in which cAMP signaling is distributed on the subcellular level by involvement of AKAP-bound pools of PKA (Schwaerzel, 2007).

Mutants affecting different steps along the cAMP-PKA cascade have identified distinct anesthesia-sensitive memory phases in Drosophila olfactory conditioning. Mutants directly affecting cAMP (e.g., the adenylyl cyclase rutabaga or the cAMP-specific phosphodiesterase dunce) disturb formation of ASM. Further downstream within the pathway, a conditional allele of the PKA catalytic subunit DC0 X4 impairs memory performance around 20 min to 2 h after training. The amnesiac mutant has revealed a critical time window shortly after conditioning, when olfactory memory requires activity of the dorsal paired medial (DPM) neurons to act onto the mushroom body structure to stabilize ASM. In fact, it has been this temporal requirement for Rutabaga, Dunce, DC0 X4, and Amnesiac that lead to the separation of ASM into STM and MTM. This analysis now reveals that MTM can be further dissected into an early and a late phase. Although the early phase of MTM requires PKA signaling as revealed by the DC0 X4 allele, this study shows that it is independent of AKAP binding. However, a late phase of MTM requires AKAP-bound PKA-RII signaling and supports memory performance from 60 min after conditioning on. As performance of this late MTM becomes completely abolished, it is concluded that no redundancy exists on the molecular level to support this particular late phase. Moreover, early and late MTM must be supported by distinct pools of PKA that serve separable functions during the time course of processing (Schwaerzel, 2007).

The functional dissection of PKA into AKAP-bound and nonbound pools suggested here is derived from the eCOPR2 peptide that constitutes the AKAP binding site of endogenous Drosophila PKA-RII. Based on the conserved mechanism of this interaction across phyla, it is speculated that eCOPR2 disturbs the binding of PKA-RII to all potential AKAPs. Additional experiments are now required to (1) identify the particular AKAP(s) that are involved in late MTM stabilization, and (2) to formally prove that eCOPR2 does indeed disrupt interaction between AKAPs and PKA-RII. However, since it was shown that eCOPR2 expression affects very specific aspects of aversive ASM processing, rather than abolishing associative functions per se, provides strong arguments in favor of distinct PKA pools. A previous study in mammals has shown similar impairment of memory several hours after conditioning when using microinfusion of Ht-31, a specific competitor of PKA-RII-AKAP interaction (Moita, 2002). In Drosophila the effects of AKAP-bound PKA-RII on aversive ASM stabilization could be localized to 700 neurons of the MBs (i.e., to the same neurons that acquire aversive memory via a Rutabaga-dependent process of synaptic plasticity in the first place). It is speculated that processing aversive ASM from STM into early and late MTM takes place within these neurons (Schwaerzel, 2007).

In mammalian cell culture it has been shown that AKAPs are localized to specific subcellular foci of signal integration, including synaptic vesicles, ionotropic and metabotropic receptors, the cytoskeleton, or cellular organelles. Dependent on this localization, AKAPs can organize PKA signaling by selecting specific substrates to the AKAP complex. These findings can now guide research in identifying particular substrates that are involved in Drosophila aversive MTM processing (Schwaerzel, 2007).

Experiencing sugar reward or electric shock punishment during the training procedure gives rise to the formation of either appetitive or aversive olfactory memories. Both are dissociated on the level of catecholamines required, but display numerous similarities with respect to the neural and molecular networks involved in aversive and appetitive memory: both types of memory are acquired within the same subset of 700 MB neurons via a Rutabaga-dependent mechanism of cAMP-mediated plasticity. Moreover, secretion from DPM neurons onto the MBs is required within 30 min after conditioning to support stabilization of aversive and appetitive ASM, respectively. However, this study has shown that AKAP-bound PKA-RII dissociates between both forms, because neither pattern of competitor peptide expression affects appetitive ASM. This argues in favor of the hypothesis that different cAMP-dependent mechanisms support aversive and appetitive ASM; the remarkable stability of appetitive memory within the temporal domain strongly supports this hypothesis. Because the eCOPR2 peptide is believed to specifically block interactions between PKA-RII and appropriate AKAPs, additional pools of PKA might support appetitive ASM processing. Additional experiments can now probe soluble PKA pools or alternative anchoring mechanisms to AKAPs via the PKA regulatory subunit type I (Schwaerzel, 2007).

The finding that cAMP-signaling couples divergently to alternative downstream partners is a theme commonly observed throughout the animal kingdom. In Aplysia neurons, the temporal pattern of receptor activation is sufficient to trigger different cellular responses by threshold-dependent sorting mechanisms. Similar mechanisms might be in place in Drosophila olfactory conditioning, when either catecholamine receptors and/or DPM activity have the potential to trigger different threshold-dependent mechanisms of subcellular signal divergence within the MBs. In Drosophila, aversive olfactory conditioning dopaminergic and DPM signals exhibit complex temporal patterns that sustain the conditioning period. However, additional experiments will be required to substantiate this hypothesis (Schwaerzel, 2007).

Defining the molecular and neuronal basis of associative memories is based upon behavioral preparations that yield high performance due to selection of salient stimuli, strong reinforcement, and repeated conditioning trials. One of those preparations is the Drosophila aversive olfactory conditioning procedure where animals initiate multiple memory components after experience of a single cycle training procedure. This study explored the analysis of acquisition dynamics as a means to define memory components and revealed strong correlations between particular chronologies of shock impact and number experienced during the associative training situation and subsequent performance of conditioned avoidance. Analyzing acquisition dynamics in Drosophila memory mutants revealed that rutabaga (rut)-dependent cAMP signals couple in a divergent fashion for support of different memory components. In case of anesthesia-sensitive memory (ASM) this study identified a characteristic two-step mechanism that links rut-AC1 to A-kinase anchoring proteins (AKAP)-sequestered protein kinase A at the level of Kenyon cells, a recognized center of olfactory learning within the fly brain. It is proposed that integration of rut-derived cAMP signals at level of AKAPs might serve as counting register that accounts for the two-step mechanism of ASM acquisition (Scheunemann, 2013).

Conditioned odor avoidance is subject to a general dichotomy since multiple memory components are engaged in control of behavior. This is usually analyzed at two time points, i.e., 3 min and 3 h after training. At 3 min, basal and dynamic STM are separable by genetic means as revealed by opposing phenotypes of rut¹ and dnc¹ mutants. Moreover, those components are also separable due to characteristic differences in their acquisition dynamics as revealed by different effects of shock number. A similar dichotomy applied to 3 h memory when ASM and ARM were separable by means of amnestic treatment. It was striking that basal STM and consolidated ARM were instantaneously acquired, resulting in a front line of protection by eliciting conditioned avoidance after a singular experience of a CS/US pairing. Interestingly, consolidated ARM (as defined by means of resisting amnestic treatment) was installed quickly after training. However, it remains to be addressed at the genetic and molecular level, whether 3 h ARM linearly results from the functionally similar basal SMT component (Scheunemann, 2013).

In contrast, the two components of dynamic STM and labile ASM acquire in a dynamic fashion but are clearly dissociated from each other by characteristic chronologies of CS/US pairings required for their acquisition. However, either component contributes to behavioral performance in addition to the appropriate instantaneous component, and hence, increases avoidance probability during the test situation. Considering a potential benefit from avoiding aversive situations this overall dichotomy of behavioral control seems plausible and is also reflected at the genetic level since rut-dependent cAMP signals are limited to support of dynamic but not instantaneous memory components. Rut-dependent STM and ASM, however, are dissociated by means of shock impact and discontinuous formation of ASM is limited to situations where animals repeatedly experience high-impact CS/US pairings within a predefined time window. Experience that does not meet this criterion, however, is not discounted but adds to continuously acquired dynamic STM. By functional means these two components are thus clearly separated but commonly dependent on rut-derived cAMP signals within the KC layer, forming ties between genetically and functionally defined memory components (Scheunemann, 2013).

Genetic dissection of memory has a long-standing history in Drosophila and provided a powerful means to define molecular, cellular, and neuronal networks involved in regulation of conditioned odor avoidance. Among others, the cAMP-signaling cascade has been identified as one central tenet of aversive odor memory foremost by means of two single-gene mutants affecting either a Ca²⁺-sensitive type 1 adenylyl cyclase (AC1) and/or a cAMP-specific type 4 phosphodiesterase (PDE4) affected in the Drosophila learning mutants rutabaga (rut-AC1) and dunce (dnc-PDE4). Although originally isolated due to poor performance in the aversive odor learning paradigm, a general dichotomy has been recently revealed that separates memory components by their dependency on either rut-AC1 or dnc-PDE4 function, and the view was established that two different types of cAMP signals are engaged during the single-cycle training procedure (Scheunemann, 2012). A similar dichotomy is observed at level of acquisition dynamics and suggests that rut-dependent cAMP signals are limited to formation of dynamically acquired memory components, i.e., dynamic STM and ASM. Interestingly, rut-dependent cAMP is also required for long-term memory (LTM), which acquires after spaced and repeated training sessions. Downstream the signaling cascade, however, appropriate cAMP signals are differently channeled to either support LTM in a CREB-dependent manner, ASM via tomosyn-dependent plasticity, or basal STM via synapsin-dependent regulation of synaptic efficacy. It appears that the chronology of CS/US pairings is an important determinant of which downstream effect is triggered and hence molecular mechanisms must be installed that are sensitive to the temporal order of training (Scheunemann, 2013).

At the level of molecular scaffolds, literature suggests that AKAPs serve the integration of cAMP with other signaling processes and are crucially involved in the control of a plethora of cellular functions in any organ. For example, AKAP79 coordinates cAMP and Ca²⁺ signaling in neurons to control ion channel activities. The recognized design principle of AKAPs to serve as molecular switch is well in line with the recognized two-step register mechanism involved in ASM formation. An increasing body of evidence shows that AKAPs are involved in memory processing across phyla and accordantly those studies revealed a contribution for support of matured, but not immediate memories. Communality among all those AKAP-dependent memories is the need for repeated and temporally organized training sessions, i.e., only spaced training sessions are effective to induce protein synthesis-dependent LTM in flies and mammals. Similarly, ASM requires the precise timing of two training sessions and mechanistically acts via an 'activated' state generated by the initial CS/US pairing that persists within the brain for ~5 min. Such temporal integration might well take place at level of AKAPs within the KC layer to operate rut-AC1-dependent cAMP signals finally onto phosphorylation of tomosyn. Identification of the particular AKAPs involved in two-step ASM formation will require further analysis of appropriate mutants. To date, only four Drosophila AKAPs are characterized, i.e., rugose, a 550 kDa protein that impacts on STM performance probably via molecular domains other than its AKAP function; yu/spoonbill that supports LTM; and Nervy and AKAP²⁰⁰ have not been tested for their impact on aversive odor memory (Scheunemann, 2013).

Together, the benchmarking of Drosophila aversive odor memory performance by means of acquisition dynamics that were demonstrated in this study will provide a valuable tool since dynamic aspects of acquisition are obviously informative and add to the steady-state condition determined by the single-cycle training procedure (Scheunemann, 2013).

Overall patterns of in vivo expression of Akap200 and DAkap200 were established by performing Western blot analysis on protein samples isolated from flies at various developmental stages. Results obtained for cytosolic and detergent-solubilized, particulate proteins were similar. The Akap200 protein cluster was enriched in pupae (3-4-fold higher than other stages), but substantial levels of these anchor proteins were also evident in embryos, adults, and larvae. DAkap200 is also detected at all phases of the Drosophila life cycle. This indicates that alternative splicing of Akap200 gene transcripts is operative during the progression of embryonic and postembryonic development. The concentration of DAkap200 in the adult head, which is enriched in neurons, is ~7-fold higher than that observed in body parts. Therefore, alternative excision of exon 5 from Akap200 mRNA and/or stability of DAkap200 protein may be differentially regulated in a cell/tissue specific fashion in mature Drosophila (Li, 1999).

Analyses of abnormal nurse cell fusions during Drosophila oogenesis have revealed that genes regulating cytoskeleton function and genes involved in cAMP metabolism are required to preserve the structural integrity of the nurse cells. It was hypothesized that the anchoring of PKA holoenzyme near the cortex of cells may regulate cytoskeletal function. To test this hypothesis, the distribution of AKAPs was examined in wild-type ovaries. One of these, Akap200, can be detected near the membranes of the germline cells throughout oogenesis and is therefore a candidate for regulating PKA activity near the subcortical cytoskeleton. In somatic follicle cells, Akap200 is also associated with membranes during the early stages of oogenesis. Expression decreases in the oocyte-associated follicle cells as they begin their migration over the oocyte at approximately stage nine of oogenesis. Akap200 expression remains in the follicle cells associated with the nurse cells and in the nurse cells themselves. Akap200 is enriched on the ring canals of the nurse cells. Ring canals have an inner and outer rim, and although some proteins associate only with inner rims, specific functions have not yet been attributed to either the inner or the outer rim. Nevertheless, costaining with Hts-RC antibodies, which recognize inner-rim-specific epitopes of the hu-li tai shao gene product, suggests that Akap200 localizes to the outer rim of the ring canals. Finally, in addition to the membrane and ring canal association, Akap200 immunoreactivity is also associated with cytoplasmic puncta at all observable stages (Jackson, 2002).

The catalytic subunit of Pka-C is localized to the membranes of germline cells in wild-type ovaries. The subcellular localization of the regulatory subunits has not yet been described however, and because the catalytic subunits dissociate from the regulatory subunits in the presence of cAMP, the subcellular localization of the regulatory and catalytic subunits may not necessarily overlap. Therefore wild-type ovaries were stained with antibodies prepared against the Drosophila Pka-RII subunit. In the ovary, the localization of Pka-RII resembles Akap200. A majority of Pka-RII is associated with membranes of both the follicle cells and the germline-derived cells throughout oogenesis. At the anteriormost region of the germarium (region 1), Pka-RII is found on the membranes of the germline stem cells. Pka-RII levels appeared to be higher in the follicle cells than in the germline cells. In the middle stages of oogenesis, Pka-RII expression increases in the anteriormost group of follicle cells that will become border cells. Pka-RII is also found on the outer rim of ring canals of nurse cells. It persists on the ring canals until oogenesis is completed. Pka-RI localization is diffuse throughout the cytoplasm in all the cells in the ovary, and does not appear to be localized to particular structures in wild-type egg chambers (Jackson, 2002).

To evaluate the significance of Akap200 localization and to test its function in PKA anchoring, a P element insertion in the Akap200 locus (l(2)k07118^k07118), identified and mapped by the Berkeley Drosophila Genome Project, was characterized. This P element is located in the large first intron of the Akap200 gene, and although the mutation was originally described as lethal, an unrelated background mutation found on the chromosome accounted for a majority of the lethality. Once this background lethal mutation was removed, Akap200 homozygotes could be recovered at slightly less than Mendelian-predicted frequencies; this allele is therefore referred to as Akap200^k07118. Nevertheless, in all experiments, the phenotypes of hemizygous Akap200 mutants (Akap200/Df(2L)N22-14) were examined to reduce the contribution of other recessive mutations on the original P element chromosome. To verify that the P element is responsible for Akap200 phenotypes, and to generate other alleles, the P element was mobilized and multiple, independent excision lines were established. Precise excision reverted all the examined phenotypes, demonstrating that the P element is responsible for the observed abnormalities. Several lines exhibited decreased viability and/or the presence of extra notal bristles when trans to the Df(2L)N22-14 chromosome; four of these lines were chosen for further analysis. Although all of these lines produce extra notal bristles, neither the original insertion allele nor any of the excision lines demonstrated a complete penetrance in the reduced viability or bristle phenotype (Jackson, 2002).

Cuticles and other adult structures from Akap200^k07118/Df(2L)N22-14 hemizygotes have a mostly wild-type morphology. In approximately a fifth of the adult flies, however, extra notal macrochaetae were present. These extra macrochaetae were accompanied by a socket cell and were either directly adjacent to the normal macrochaete or midway between the proximal and distal macrochaetae. Extra bristles were not observed in other locations, however, and the presence of more than one extra bristle on a single notum was rare. These observations suggest that Akap200 could be involved in the choice between the alternate cell fates of epidermis or sensory organ (Jackson, 2002).

Based on the similar staining patterns, it was hypothesized that Akap200 is responsible for the Pka-RII membrane sequestration and therefore, disrupting Akap200 function would redistribute Pka-RII to other areas. To test this hypothesis, Pka-RII localization was examined in Akap200 mutants. In control germaria, Akap200 and Pka-RII are associated with membranes of both the germline and follicle cells. In Akap200^k07118 mutant ovaries, almost all the Akap200 immunoreactivity was abolished. This low but detectable residual staining suggested that the Akap200^k07118 allele is not null. The effects of loss of Akap200 function are most evident in the germline cells, in which Pka-RII membrane staining decreases and cytoplasmic staining increases. Membrane association persists in the follicle cells however, and to a lesser degree in later-staged nurse cells. This persistent membrane staining may result from partial Akap200 activity or because other AKAPs anchor Pka-RII to the membranes of these cells. In the excision allele Akap200^ix4, a partial restoration of Akap200 immunoreactivity correlates with a partial restoration of Pka-RII membrane localization, with a concomitant decrease in cytoplasmic staining. In Akap200^D7 flies, Akap200 immunoreactivity is undetectable and is indistinguishable from controls with secondary antibody only. As a result, Pka-RII membrane association is reduced in the germline stem cells and in other cells of the ovary. Notably, the total amount of Pka-RII protein is unchanged in each of the Akap200 mutants. Akap200 is therefore responsible for sequestering a majority of Pka-RII to the membranes of germline cells during the early stages of oogenesis (Jackson, 2002).

It was predicted that if PKA anchoring is important for preserving nurse cell membrane and actin integrity, then disrupting anchoring should produce binucleate nurse cells. Both loss-of-function and gain-of-function approaches were used to test this prediction. The effects of Akap200 mutations on the morphology of the egg chamber were analyzed by staining mutant egg chambers with fluorescent-phalloidin conjugates, to examine actin-rich ring canals and subcortical actin, and DAPI, to visualize the nuclei. Females mutant for Akap200 produce egg chambers that have multinucleate cells with ring canal remnants. As is the case with most other mutations that produce multinucleate cells, these remnants stain with Hts-RC antibodies, suggesting that cytokineses and ring canal formation is initially normal in these mutants (Jackson, 2002).

It was reasoned that loss of Akap200 activity and the resulting failure to sequester PKA holoenzyme would produce a change in the local concentration of available Pka-C. This hypothesis was tested directly by expressing a constitutively active Pka-C mutant subunit (Pka-C*) throughout the germline cells. This transgene, driven by a UAS promoter, has a point mutation that diminishes its ability to bind to PKA regulatory subunits; it therefore cannot be sequestered. Two lines that express GAL4 at different levels in the germline [nos-GAL4-VP16 and NGT40, were used to determine the relative dosage effects of active Pka-C. When Pka-C* was expressed in the germline, binucleate cells were produced at a frequency that correlated with the levels of GAL4 expression (NGT40/+ < nos-GAL4-VP16/+). Although both NGT40 and nos-GAL4-VP16 direct expression during the earliest stages of oogenesis, binucleate cells were only observed after approximately stage five or six. A similar phenotype was observed when NGT40 was used to drive wild-type Pka-C instead of Pka-C*, although fewer affected egg chambers resulted. Nevertheless, to ensure the analysis of all potential mutant egg chambers, the frequency of binucleate cells was counted at all stages between 2 and 11, resulting in binucleate cell frequencies of less than 10% for all egg chambers. No binucleate cells were observed with a peptide inhibitor of Pka-C (UAS-PKIF) or a mutant that fails to bind to Pka-C (UAS-PKIG^19,20), demonstrating that the binucleate cell phenotype is not due to GAL4 expression in the germline. Therefore, ectopic Pka-C activity disrupts the morphology of the germline cells (Jackson, 2002).

Models of AKAP function have suggested two distinct roles for AKAPs: they either control the local activity of Pka-C by sequestering inhibitory subunits to specific subcellular locales, or they act as a scaffold, bringing PKA signaling components together. The first model predicts that increased Pka-C activity would either have no effect or enhance AKAP loss of function phenotypes, whereas in the scaffold model, increased Pka-C activity would overcome the loss of AKAP function. To distinguish between these roles of Akap200 in regulating actin morphology, Pka-C* was expressed in Akap200 loss of function ovaries. The frequency of multinucleate cells increased when Pka-C* was expressed in an Akap200/Df(2L)N22-14 mutant background. These results are consistent with the hypothesis that Akap200 regulates the activity of the Pka-C subunit in the vicinity of subcortical actin in the nurse cells by localizing Pka-RII to these regions (Jackson, 2002).

Interestingly, it was noticed during these studies that Akap200 mutations also affect the size of the ring canals in the intact, mononucleate nurse cells. Ring canal size has been measured throughout oogenesis, and although variability is found because of the age of the ring canal, the size falls within a stereotypical range for a particular stage. In the strong Akap200 alleles, the ring canals appear to be larger and thinner than wild type. Ring canals were stained with phalloidin, anti-Hts-RC and anti-phosphotyrosine, then the inner and outer ring canal diameter was measured between stages seven and ten in mutant and wild-type egg chambers. At each stage and with each epitope, both the inner and outer rims are larger in the mutants. Although the ring canals are large and thin, phosphotyrosine, Hts-RC, Kelch and Filamin all appeared to localize normally to the mutant ring canals. The Akap200^D7 allele also produces large, thin ring canals that are similar in size to Akap200^k07118 ring canals. By contrast, the weaker Akap200^ix4 allele, which retains some Akap200 function, has an approximately normal ring canal size, even though it produces multinucleate nurse cells (Jackson, 2002).

Because loss of Akap200 function produces large ring canals, it was hypothesized that the gain of Akap200 function might result in smaller ring canals. Therefore Akap200 was overexpressed in the nurse cells by crossing a GAL4-dependent 'enhancer piracy' line, EP2254, which maps to the Akap200 locus, to flies containing a germline-expressing GAL4 transgene (nos-GAL4-VP16). Ovaries from females carrying both nos-GAL4-VP16 and UAS-Akap200 produce egg chambers in which ring canals are smaller and thicker than wild type. These females are fertile however, and the small ring canals do not interfere with dumping (the transfer of nurse cell contents to the oocyte). Although nos-GAL4-VP16 is expressed at the earliest stages of oogenesis, no differences were detected in ring canal morphology prior to stage four, nor were other defects observed that could be correlated with altered germline stem cell function (i.e. extra or fewer divisions, changes in fate). Overexpressing Akap200 in the follicle cells results in ovaries that are indistinguishable from wild-type (Jackson, 2002).

Although many genes have been identified that alter ring canal function, altered ring canal size has only been observed in mutants of the non-receptor tyrosine kinases Src64B and Tec29. In these mutants, ring canals are smaller, a phenotype opposite that of the Akap200 mutants. Ring canals were examined in Src64B mutant ovaries when Akap200 gene dose is reduced, to determine whether Akap200 could antagonize Src64B in regulating ring canal size. The hypomorphic Src64B^PI allele is homozygous viable and produces ring canals that are smaller than wild type. Akap200^k07118 acted as a dominant suppressor of the small ring canal size phenotype produced by these mutants. Females of genotype Akap200^k07118/+; Src64B^PI/Src64B^PI produce ring canals that are almost wild type in size and have near normal amounts of phosphotyrosine. Reducing Src64B gene dose by half, however, fails to affect the size of the ring canals produced by Akap200^k07118 mutants. Ring canals produced by Akap200^k07118/Akap200^k07118; Src64B^PI/+ are slightly smaller than those produced by Akap200^k07118 mutants. These results suggest that Src64B and Akap200 act antagonistically to regulate ring canal growth. Akap200 protein localization is not altered detectably in Src64B^PI mutant egg chambers, nor is Src64B localization changed visibly in Akap200 mutant ovaries. These observations suggest that post-translational mechanisms may be responsible for the genetic interaction (Jackson, 2002).

Differentiation of the R7 photoreceptor cell is dependent on the Sevenless receptor tyrosine kinase, which activates the Ras1/mitogen-activated protein kinase signaling cascade. Kinase suppressor of ras (Ksr) functions genetically downstream of Ras1 in this signal transduction cascade. Expression of dominant-negative Ksr (KDN) in the developing eye blocks Ras pathway signaling, prevents R7 cell differentiation, and causes a rough eye phenotype. To identify genes that modulate Ras signaling, a screened was carried out for genes that alter Ras1/Ksr signaling efficiency when misexpressed. In this screen, three known genes, Lk6, misshapen, and Akap200, were recovered. Seven previously undescribed genes were recovered; one encodes a novel rel domain member of the NFAT family, and six encode novel proteins. These genes may represent new components of the RAS pathway or components of other signaling pathways that can modulate signaling by RAS (Huang, 2000).

One of the misexpression interactors, MESR2, was an insertion upstream of the Akap200 locus. Akap200 refers to Drosophila A kinase anchor protein of molecular weight 200 kd, and binds the regulatory II (rII) subunit of cyclic AMP-dependent protein kinase (PKA). The Akap200 gene produces two different transcripts, one that contains the binding site for RII and one where the exon encoding for the RII binding site is spliced out to generate a protein that does not interact directly with PKA. Both isoforms of AKAP200 are expressed at relatively similar levels throughout development as well as in adult heads (Huang, 2000).

PKA is the principal mediator of signals that activate adenylate cyclase. cAMP signals are often targeted to effectors that accumulate to discrete intracellular locations. This targeting is due to a nonuniform distribution of PKA molecules within cells. In Drosophila, PKA has been implicated in normal developmental events in all imaginal tissues through the Hedgehog signaling pathway and is involved in signaling pathways that generate cell polarity: this requires that Hh be localized to distinct intracellular locations. Subcellular localization of PKA occurs through association with AKAPs. AKAPs are a functionally related family of proteins, defined by their ability to associate with PKA. Each AKAP contains a unique targeting domain that directs the complex to a defined intracellular location where PKA is placed proximal to both a signal generator (adenylate cyclase) as well as to potential PKA effector molecules. Coordinate binding of specific combinations of enzymes can allow such complexes to respond to distinct second messenger-mediated signals (Huang, 2000).

Studies in mammalian cells have suggested that PKA signaling via Rap1, another small molecular weight GTP-binding protein, antagonizes RAS1 signaling by competing for RAS pathway components such as B-Raf and MAPK. However, more recent studies suggest no genetic interaction between Drosophila Rap1 and RAS1. In Drosophila, overexpression of Rap1 in a heterozygous RAS1 mutant background has no effect on photoreceptor determination, suggesting no interaction between the two gene functions. A heterozygous Rap1 mutation does not reduce the number of R7 cells in a sev-RAS1^V12 rough eye, also suggesting that the two pathways do not interact. Although there is no direct evidence linking PKA activation to MAPK activation via Rap1, there may be a still unknown pathway by which these molecules can signal (Huang, 2000).

The screen isolated Akap200 as a misexpression enhancer of KDN and suppressor of RAS1^V12. This suggests that overexpression of this AKAP decreases signaling through RAS1. Overexpression of an AKAP might cause mislocalization of PKA molecules to the plasma membrane. This could activate a signaling pathway that normally is not utilized in this cell or at this time in development. If PKA and Rap1 are involved in RAS signaling, why were they not uncovered in previous loss-of-function screens? One possibility is that mutations in either gene may not be dose sensitive and therefore be unable to dominantly modify a rough eye phenotype. Another is the possibility that overexpression of an AKAP causes abnormal targets of PKA to become activated. Whether PKA signals through Rap1 is still unclear; however, the reported effects of attenuating RAS1/MAPK signaling are supported by this study. The enhancement of the KDN rough eye phenotype could be due to the additive effects of inefficient signaling due to KDN as well as the attenuation of MAPK by mislocalized PKA. In the activated RAS1^V12 background, the attenuating effects of activated PKA due to mislocalization to the plasma membrane might reduce the amount of signaling through the pathway to suppress the RAS1-dependent rough eye phenotype (Huang, 2000).

The Drosophila orthologue of the c-Cbl proto-oncogene acts to downregulate signalling from receptor tyrosine kinases by enhancing endocytosis of activated receptors. Expression of an analogue of the C-terminally truncated v-Cbl oncogene, Dv-cbl, in the developing Drosophila eye conversely leads to excess signalling and disruption to the well-ordered adult compound eye. Co-expression of activated Ras with Dv-cbl leads to a severe disruption of eye development. A transposon-based inducible expression system was used to screen for molecules that can suppress the Dv-cbl phenotype and an allele was identified that upregulates the A-kinase anchoring protein, Akap200. Overexpression of Akap200 not only suppresses the phenotype caused by Dv-cbl expression, but also the severe disruption to eye development caused by the combined expression of Dv-cbl and activated Ras. Akap200 is also endogenously expressed in the developing Drosophila eye at a level that modulates the effects of excessive signalling caused by expression of Dv-cbl (Sannang, 2012).

Preferential phosphorylation of specific proteins by cAMP-dependent protein kinase (PKA) may be mediated in part by the anchoring of PKA to a family of A-kinase anchor proteins (AKAPs) positioned in close proximity to target proteins. This interaction is thought to depend on binding of the type II regulatory (RII) subunits to AKAPs and is essential for PKA-dependent modulation of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/kainate receptor, the L-type Ca2+ channel, and the KCa channel. It was hypothesized that the targeted disruption of the gene for the ubiquitously expressed RIIalpha subunit would reveal those tissues and signaling events that require anchored PKA. RIIalpha knockout mice appear normal and healthy. In adult skeletal muscle, RIalpha protein levels increase to partially compensate for the loss of RIIalpha. Nonetheless, a reduction in both catalytic (C) subunit protein levels and total kinase activity is observed. Surprisingly, the anchored PKA-dependent potentiation of the L-type Ca2+ channel in RIIalpha knockout skeletal muscle is also unchanged, as compared with wild type, although it is more sensitive to inhibitors of PKA-AKAP interactions. The C subunit colocalizes with the L-type Ca2+ channel in transverse tubules in wild-type skeletal muscle and retain this localization in knockout muscle. The RIalpha subunit binds AKAPs, although with a 500-fold lower affinity than the RIIalpha subunit. The potentiation of the L-type Ca2+ channel in RIIalpha knockout mouse skeletal muscle suggests that, despite a lower affinity for AKAP binding, RIalpha is capable of physiologically relevant anchoring interactions (Burton, 1997).

Using a combination of protein kinase A type II overlay screening, rapid amplification of cDNA ends, and database searches, a contig of 9923 bp was assembled and characterized in which the open reading frame encodes a 1901-amino-acid A-kinase-anchoring protein (AKAP) with an apparent SDS-PAGE mobility of 220 kDa. It has been named human AKAP220 (hAKAP220). The hAKAP220 amino acid sequence reveals high similarity to rat AKAP220 in the 1167 C-terminal residues, but contains 727 residues in the N-terminus not present in the reported rat AKAP220 sequence. The hAKAP220 mRNA is expressed at high levels in testis and in isolated pachytene spermatocytes and round spermatids. The hAKAP220 protein is present in male germ cells and mature sperm. Immunofluorescent labeling with specific antibodies indicates that hAKAP220 is localized in the cytoplasm of premeiotic pachytene spermatocytes and in the centrosome of developing postmeiotic germ cells, while a midpiece/centrosome localization is found in elongating spermatocytes and mature sperm. The hAKAP220 protein together with a fraction of PKA types I and II and protein phosphatase I is resistant to detergent extraction of sperm tails, suggesting an association with cytoskeletal structures. In contrast, S-AKAP84/D-AKAP1, which is also present in the midpiece, is extracted under the same conditions. Anti-hAKAP220 antisera coimmunoprecipitates both type I and type II regulatory subunits of PKA in human testis lysates, indicating that hAKAP220 interacts with both classes of R subunits, either through separate or through a common binding motif(s) (Reinton, 2000).

WAVE proteins (see Drosophila Scar/Wave)are members of the Wiskott-Aldrich syndrome protein (WASP) family of scaffolding proteins that coordinate actin reorganization by coupling Rho-related small molecular weight GTPases to the mobilization of the Arp2/3 complex. WAVE-1 has been identified in a screen for rat brain A kinase-anchoring proteins (AKAPs), which bind to the SH3 domain of the Abelson tyrosine kinase (Abl). Recombinant WAVE-1 interacts with cAMP-dependent protein kinase (PKA) and Abl kinases when expressed in HEK-293 cells, and both enzymes co-purify with endogenous WAVE from brain extracts. Mapping studies have defined binding sites for each kinase. Competition experiments suggest that the PKA-WAVE-1 interaction may be regulated by actin because the kinase binds to a site overlapping a verprolin homology region, which has been shown to interact with actin. Immunocytochemical analyses in Swiss 3T3 fibroblasts suggest that the WAVE-1 kinase scaffold is assembled dynamically as WAVE, PKA and Abl translocate to sites of actin reorganization in response to platelet-derived growth factor treatment. Thus, a previously unrecognized function is proposed for WAVE-1 as an actin-associated scaffolding protein that recruits PKA and Abl (Westphal, 2000).

A-kinase-anchoring proteins (AKAP) help regulate the intracellular organization of cyclic AMP-dependent kinase (PKA) and actin within somatic cells. Elevated levels of cAMP also help maintain meiotic arrest in immature oocytes, with AKAPs implicated as critical mediators but poorly understood during this process. This study tests the hypothesis that the AKAP WAVE1 is required during mammalian fertilization, and identify a nuclear localization of WAVE1 that is independent of actin and actin-related proteins (Arp). Immunofluorescence and immunoprecipitation experiments show a redistribution of WAVE1 from the cortex in germinal vesicle (GV) oocytes to cytoplasmic foci in oocytes arrested in second meiosis (Met II). Following sperm entry, WAVE1 relocalizes to the developing male and female pronuclei. Association of WAVE1 with a regulatory subunit of PKA is detected in both Met II oocytes and pronucleate zygotes, but interaction with Arp 2/3 is observed only in Met II oocytes. WAVE1 redistributes to the cytoplasm upon nuclear envelope breakdown at mitosis, and concentrates at the cleavage furrow during embryonic cell division. Blocking nuclear pore formation with microinjected wheat germ agglutinin does not inhibit the nuclear localization of WAVE1, suggesting that this event precedes nuclear envelope formation. Neither depolymerization nor stabilization of actin affects WAVE1 distribution. Microtubule stabilization with Taxol, however, redistributes WAVE1 to the centrosome, and anti-WAVE1 antibodies prevent both the nuclear distribution of WAVE1 and the migration and apposition of pronuclei. These findings show that WAVE1 sequestration to the nucleus is required during fertilization, and is an actin-independent event that relies on dynamic microtubules but not nuclear pores (Rawe, 2004).

Within neurons, Ca(2+)-dependent inactivation (CDI) of voltage-gated L-type Ca(2+) channels shapes cytoplasmic Ca(2+) signals. CDI is initiated by Ca(2+) binding to channel-associated calmodulin and subsequent Ca(2+)/calmodulin activation of the Ca(2+)-dependent phosphatase, calcineurin (CaN), which is targeted to L channels by the A-kinase-anchoring protein AKAP79/150. This study reports that CDI of neuronal L channels is abolished by inhibition of PKA activity or PKA anchoring to AKAP79/150 and that CDI is also suppressed by stimulation of PKA activity. Although CDI was reduced by positive or negative manipulation of PKA, interference with PKA anchoring or activity lowered Ca(2+) current density whereas stimulation of PKA activity elevated it. In contrast, inhibition of CaN reduced CDI but had no effect on current density. These results suggest a model wherein PKA-dependent phosphorylation enhances neuronal L current, thereby priming channels to undergo CDI, and Ca(2+)/calmodulin-activated CaN actuates CDI by reversing PKA-mediated enhancement of channel activity (Dittmer, 2014).

L-type voltage-gated Ca²⁺ channels (LTCC) couple neuronal excitation to gene transcription. LTCC activity is elevated by the cyclic AMP (cAMP)-dependent protein kinase (PKA) and depressed by the Ca²⁺-dependent phosphatase calcineurin (CaN), and both enzymes are localized to the channel by A-kinase anchoring protein 79/150 (AKAP79/150). AKAP79/150 anchoring of CaN also promotes LTCC activation of transcription through dephosphorylation of the nuclear factor of activated T cells (NFAT). This study reports that the basal activity of AKAP79/150-anchored PKA maintains neuronal LTCC coupling to CaN-NFAT signaling by preserving LTCC phosphorylation in opposition to anchored CaN. Genetic disruption of AKAP-PKA anchoring promoted redistribution of the kinase out of postsynaptic dendritic spines, profound decreases in LTCC phosphorylation and Ca²⁺ influx, and impaired NFAT movement to the nucleus and activation of transcription. Thus, LTCC-NFAT transcriptional signaling in neurons requires precise organization and balancing of PKA and CaN activities in the channel nanoenvironment, which is only made possible by AKAP79/150 scaffolding (Murphy, 2014).

date revised: 23 August 2014

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