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ABSTRACT: Excitation-driven entry of Ca(2+) through L-type voltage-gated Ca(2+) channels controls gene expression in neurons and a variety of fundamental activities in other kinds of excitable cells. The probability of opening of Ca(V)1.2 L-type channels is subject to pronounced enhancement by cAMP-dependent protein kinase (PKA), which is scaffolded to Ca(V)1.2 channels by A-kinase anchoring proteins (AKAPs). Ca(V)1.2 channels also undergo negative autoregulation via Ca(2+)-dependent inactivation (CDI), which strongly limits Ca(2+) entry. An abundance of evidence indicates that CDI relies upon binding of Ca(2+)/calmodulin (CaM) to an isoleucine-glutamine motif in the carboxy tail of Ca(V)1.2 L-type channels, a molecular mechanism seemingly unrelated to phosphorylation-mediated channel enhancement. But our work reveals, in cultured hippocampal neurons and a heterologous expression system, that the Ca(2+)/CaM-activated phosphatase calcineurin (CaN) is scaffolded to Ca(V)1.2 channels by the neuronal anchoring protein AKAP79/150, and that overexpression of an AKAP79/150 mutant incapable of binding CaN (ΔPIX; CaN-binding PXIXIT motif deleted) impedes CDI. Interventions that suppress CaN activity-mutation in its catalytic site, antagonism with cyclosporine A or FK506, or intracellular perfusion with a peptide mimicking the sequence of the phosphatase's autoinhibitory domain-interfere with normal CDI. In cultured hippocampal neurons from a ΔPIX knock-in mouse, CDI is absent. Results of experiments with the adenylyl cyclase stimulator forskolin and with the PKA inhibitor PKI suggest that Ca(2+)/CaM-activated CaN promotes CDI by reversing channel enhancement effectuated by kinases such as PKA. Hence, our investigation of AKAP79/150-anchored CaN reconciles the CaM-based model of CDI with an earlier, seemingly contradictory model based on dephosphorylation signaling.
Journal of Neuroscience 10/2012; 32(44):15328-37. · 7.11 Impact Factor
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ABSTRACT: AMPA receptors (AMPARs) are tetrameric ion channels assembled from GluA1-GluA4 subunits that mediate the majority of fast excitatory synaptic transmission in the brain. In the hippocampus, most synaptic AMPARs are composed of GluA1/2 or GluA2/3 with the GluA2 subunit preventing Ca(2+) influx. However, a small number of Ca(2+)-permeable GluA1 homomeric receptors reside in extrasynaptic locations where they can be rapidly recruited to synapses during synaptic plasticity. Phosphorylation of GluA1 S845 by the cAMP-dependent protein kinase (PKA) primes extrasynaptic receptors for synaptic insertion in response to NMDA receptor Ca(2+) signaling during long-term potentiation (LTP), while phosphatases dephosphorylate S845 and remove synaptic and extrasynaptic GluA1 during long-term depression (LTD). PKA and the Ca(2+)-activated phosphatase calcineurin (CaN) are targeted to GluA1 through binding to A-kinase anchoring protein 150 (AKAP150) in a complex with PSD-95, but we do not understand how the opposing activities of these enzymes are balanced to control plasticity. Here, we generated AKAP150ΔPIX knock-in mice to selectively disrupt CaN anchoring in vivo. We found that AKAP150ΔPIX mice lack LTD but express enhanced LTP at CA1 synapses. Accordingly, basal GluA1 S845 phosphorylation is elevated in AKAP150ΔPIX hippocampus, and LTD-induced dephosphorylation and removal of GluA1, AKAP150, and PSD-95 from synapses are impaired. In addition, basal synaptic activity of GluA2-lacking AMPARs is increased in AKAP150ΔPIX mice and pharmacologic antagonism of these receptors restores normal LTD and inhibits the enhanced LTP. Thus, AKAP150-anchored CaN opposes PKA phosphorylation of GluA1 to restrict synaptic incorporation of Ca(2+)-permeable AMPARs both basally and during LTP and LTD.
Journal of Neuroscience 10/2012; 32(43):15036-52. · 7.11 Impact Factor
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ABSTRACT: NMDA receptor-dependent long-term potentiation (LTP) and depression (LTD) are forms of synaptic plasticity underlying learning and memory that are expressed through increases and decreases, respectively, in dendritic spine size and AMPA receptor (AMPAR) phosphorylation and postsynaptic localization. The A-kinase anchoring protein 79/150 (AKAP79/150) signaling scaffold regulates AMPAR phosphorylation, channel activity, and endosomal trafficking associated with LTP and LTD. AKAP79/150 is targeted to dendritic spine plasma membranes by an N-terminal polybasic domain that binds phosphoinositide lipids, F-actin, and cadherin cell adhesion molecules. However, we do not understand how regulation of AKAP targeting controls AMPAR endosomal trafficking. Here, we report that palmitoylation of the AKAP N-terminal polybasic domain targets it to postsynaptic lipid rafts and dendritic recycling endosomes. AKAP palmitoylation was regulated by seizure activity in vivo and LTP/LTD plasticity-inducing stimuli in cultured rat hippocampal neurons. With chemical LTP induction, we observed AKAP79 dendritic spine recruitment that required palmityolation and Rab11-regulated endosome recycling coincident with spine enlargement and AMPAR surface delivery. Importantly, a palmitoylation-deficient AKAP79 mutant impaired regulation of spine size, endosome recycling, AMPAR trafficking, and synaptic potentiation. These findings emphasize the emerging importance of palmitoylation in controlling synaptic function and reveal novel roles for the AKAP79/150 signaling complex in dendritic endosomes.
Journal of Neuroscience 05/2012; 32(21):7119-36. · 7.11 Impact Factor
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ABSTRACT: Modulation of phosphorylation states of ion channels is a critical step in the development of hyperalgesia during inflammation. Modulatory enhancement of channel activity may increase neuronal excitability and affect downstream targets such as gene transcription. The specificity required for such regulation of ion channels quickly occurs via targeting of protein kinases and phosphatases by the scaffolding A-kinase anchoring protein 79/150 (AKAP79/150). AKAP79/150 has been implicated in inflammatory pain by targeting protein kinase A (PKA) and protein kinase C (PKC) to the transient receptor potential vanilloid 1 (TRPV1) channel in peripheral sensory neurons, thus lowering threshold for activation of the channel by multiple inflammatory reagents. However, the expression pattern of AKAP150 in peripheral sensory neurons is unknown. Here we identify the peripheral neuron subtypes that express AKAP150, the subcellular distribution of AKAP150, and the potential target ion channels in rat dorsal root ganglion (DRG) slices. We found that AKAP150 is expressed predominantly in a subset of small DRG sensory neurons, where it is localized at the plasma membrane of the soma, axon initial segment, and small fibers. Most of these neurons are peripherin positive and produce C fibers, although a small portion produce Aδ fibers. Furthermore, we demonstrate that AKAP79/150 colocalizes with TRPV1 and Ca(V) 1.2 in the soma and axon initial segment. Thus AKAP150 is expressed in small, nociceptive DRG neurons, where it is targeted to membrane regions and where it may play a role in the modulation of ion channel phosphorylation states required for hyperalgesia.
The Journal of Comparative Neurology 06/2011; 520(1):81-99. · 3.81 Impact Factor
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ABSTRACT: Plasticity at excitatory glutamatergic synapses in the central nervous system is believed to be critical for neuronal circuits to process and encode information, allowing animals to perform complex behaviors such as learning and memory. In addition, alterations in synaptic plasticity are associated with human diseases, including Alzheimer disease, epilepsy, chronic pain, drug addiction, and schizophrenia. Long-term potentiation (LTP) and depression (LTD) in the hippocampal region of the brain are two forms of synaptic plasticity that increase or decrease, respectively, the strength of synaptic transmission by postsynaptic AMPA-type glutamate receptors. Both LTP and LTD are induced by activation of NMDA-type glutamate receptors but differ in the level and duration of Ca(2+) influx through the NMDA receptor and the subsequent engagement of downstream signaling by protein kinases, including PKA, PKC, and CaMKII, and phosphatases, including PP1 and calcineurin-PP2B (CaN). This review addresses the important emerging roles of the A-kinase anchoring protein family of scaffold proteins in regulating localization of PKA and other kinases and phosphatases to postsynaptic multiprotein complexes that control NMDA and AMPA receptor function during LTP and LTD.
The Neuroscientist 06/2011; 17(3):321-36. · 4.57 Impact Factor
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ABSTRACT: Kv4.2, as the primary α-subunit of rapidly inactivating, A-type voltage-gated K(+) (Kv) channels expressed in hippocampal CA1 pyramidal dendrites, plays a critical role in regulating their excitability. Activity-dependent trafficking of Kv4.2 relies on C-terminal protein kinase A (PKA) phosphorylation. A-kinase-anchoring proteins (AKAPs) target PKA to glutamate receptor and ion channel complexes to allow for discrete, local signaling. As part of a previous study, we showed that AKAP79/150 interacts with Kv4.2 complexes and that the two proteins colocalize in hippocampal neurons. However, the nature and functional consequence of their interaction has not been previously explored. Here, we report that the C-terminal domain of Kv4.2 interacts with an internal region of AKAP79/150 that overlaps with its MAGUK (membrane-associated guanylate kinase)-binding domain. We show that AKAP79/150-anchored PKA activity controls Kv4.2 surface expression in heterologous cells and hippocampal neurons. Consistent with these findings, disrupting PKA anchoring led to a decrease in neuronal excitability, while preventing dephosphorylation by the phosphatase calcineurin resulted in increased excitability. These results demonstrate that AKAP79/150 provides a platform for dynamic PKA regulation of Kv4.2 expression, fundamentally impacting CA1 excitability.
Journal of Neuroscience 01/2011; 31(4):1323-32. · 7.11 Impact Factor
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ABSTRACT: Ca(2+)/calmodulin (CaM)-dependent protein kinase II (CaMKII) "autonomy" (T286-autophosphorylation-induced Ca(2+)-independent activity) is required for long-term potentiation (LTP) and for learning and memory, as demonstrated by CaMKII T286A mutant mice. The >20-year-old hypothesis that CaMKII stimulation is required for LTP induction, while CaMKII autonomy is required for LTP maintenance was recently supported using the cell-penetrating fusion-peptide inhibitor antCN27. However, we demonstrate here that ant/penetratin fusion to CN27 compromised CaMKII-selectivity, by enhancing a previously unnoticed direct binding of CaM to ant/penetratin. In contrast to antCN27, the improved cell-penetrating inhibitor tatCN21 (5 mum) showed neither CaM binding nor inhibition of basal synaptic transmission. In vitro, tatCN21 inhibited stimulated and autonomous CaMKII activity with equal potency. In rat hippocampal slices, tatCN21 inhibited LTP induction, but not LTP maintenance. Correspondingly, tatCN21 also inhibited learning, but not memory storage or retrieval in a mouse in vivo model. Thus, CaMKII autonomy provides a short-term molecular memory that is important in the signal computation leading to memory formation, but is not required as long-term memory store.
Journal of Neuroscience 06/2010; 30(24):8214-20. · 7.11 Impact Factor
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ABSTRACT: A hallmark feature of Ca(2+)/calmodulin (CaM)-dependent protein kinase II (CaMKII) regulation is the generation of Ca(2+)-independent autonomous activity by Thr-286 autophosphorylation. CaMKII autonomy has been regarded a form of molecular memory and is indeed important in neuronal plasticity and learning/memory. Thr-286-phosphorylated CaMKII is thought to be essentially fully active ( approximately 70-100%), implicating that it is no longer regulated and that its dramatically increased Ca(2+)/CaM affinity is of minor functional importance. However, this study shows that autonomy greater than 15-25% was the exception, not the rule, and required a special mechanism (T-site binding; by the T-substrates AC2 or NR2B). Autonomous activity toward regular R-substrates (including tyrosine hydroxylase and GluR1) was significantly further stimulated by Ca(2+)/CaM, both in vitro and within cells. Altered K(m) and V(max) made autonomy also substrate- (and ATP) concentration-dependent, but only over a narrow range, with remarkable stability at physiological concentrations. Such regulation still allows molecular memory of previous Ca(2+) signals, but prevents complete uncoupling from subsequent cellular stimulation.
Journal of Biological Chemistry 03/2010; 285(23):17930-7. · 4.77 Impact Factor
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ABSTRACT: Alterations in N-methyl-d-aspartate receptor (NMDAR) protein levels or subcellular localization in brain after chronic ethanol exposure may contribute to withdrawal-associated seizures and neurotoxicity. We have investigated synaptic localization of NMDARs in cultured hippocampal pyramidal neurons after prolonged (7 days) exposure to, and acute withdrawal from, 80 mM ethanol using fluorescence immunocytochemistry techniques. After chronic ethanol exposure, there was a significant increase in the clustering of NR1 and NR2B subunits and their colocalization with the synaptic proteins synaptophysin and postsynaptic density protein 95, respectively. There was also increased expression of NR1 variants containing the C2' cassette after chronic ethanol exposure. The ethanol-induced synaptic clustering and colocalization were rapidly reversed within 4 h after ethanol withdrawal. Surface labeling of NR2B subunits suggested that this rapid reversal involved lateral receptor movement to extrasynaptic sites rather than internalization of receptors. Receptor removal from the synapse during ethanol withdrawal was associated with changes in the phosphorylation state of NR2B Ser1480, controlled by the protein kinase CK2. The redistribution of NMDAR to synapses produced by long-term ethanol exposure, as well as the rapid removal during withdrawal, may not only affect neuronal withdrawal hyperexcitability but also may sensitize the system to subsequent synaptic plasticity.
Journal of Pharmacology and Experimental Therapeutics 12/2009; 332(3):720-9. · 3.83 Impact Factor
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ABSTRACT: A-kinase anchoring protein (AKAP) 79/150 is a scaffold protein found in dendritic spines that recruits the cAMP-dependent protein kinase (PKA) and protein phosphatase 2B-calcineurin (CaN) to membrane-associated guanylate kinase (MAGUK)-linked AMPA receptors (AMPARs) to control receptor phosphorylation and synaptic plasticity. However, AKAP79/150 may also coordinate regulation of AMPAR activity with spine structure directly through MAGUK binding and membrane-cytoskeletal interactions of its N-terminal targeting domain. In cultured hippocampal neurons, we observed that rat AKAP150 expression was low early in development but then increased coincident with spine formation and maturation. Overexpression of human AKAP79 in immature or mature neurons increased the number of dendritic filopodia and spines and enlarged spine area. However, RNA interference knockdown of AKAP150 decreased dendritic spine area only in mature neurons. Importantly, AKAP79 overexpression in immature neurons increased AMPAR postsynaptic localization and activity. Neither the AKAP79 PKA nor CaN anchoring domain was required for increasing dendritic protrusion numbers, spine area, or AMPAR synaptic localization; however, an internal region identified as the MAGUK binding domain was found to be essential as shown by expression of a MAGUK binding mutant that formed mainly filopodia and decreased AMPAR synaptic localization and activity. Expression of the AKAP79 N-terminal targeting domain alone also increased filopodia numbers but not spine area. Overall, these results demonstrate a novel structural role for AKAP79/150 in which the N-terminal targeting domain induces dendritic filopodia and binding to MAGUKs promotes spine enlargement and AMPAR recruitment.
Journal of Neuroscience 07/2009; 29(24):7929-43. · 7.11 Impact Factor
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ABSTRACT: Neuronal L-type calcium channels contribute to dendritic excitability and activity-dependent changes in gene expression that influence synaptic strength. Phosphorylation-mediated enhancement of L-type channels containing the CaV1.2 pore-forming subunit is promoted by A-kinase anchoring proteins (AKAPs) that target cAMP-dependent protein kinase (PKA) to the channel. Although PKA increases L-type channel activity in dendrites and dendritic spines, the mechanism of enhancement in neurons remains poorly understood. Here, we show that CaV1.2 interacts directly with AKAP79/150, which binds both PKA and the Ca2+/calmodulin-activated phosphatase calcineurin (CaN). Cotargeting of PKA and CaN by AKAP79/150 confers bidirectional regulation of L-type current amplitude in transfected HEK293 cells and hippocampal neurons. However, anchored CaN dominantly suppresses PKA enhancement of the channel. Additionally, activation of the transcription factor NFATc4 via local Ca2+ influx through L-type channels requires AKAP79/150, suggesting that this signaling complex promotes neuronal L channel signaling to the nucleus through NFATc4.
Neuron 08/2007; 55(2):261-75. · 14.74 Impact Factor
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ABSTRACT: NMDA receptor (NMDAR)-dependent hippocampal synaptic plasticity underlying learning and memory coordinately regulates dendritic spine structure and AMPA receptor (AMPAR) postsynaptic strength through poorly understood mechanisms. Induction of long-term depression (LTD) activates protein phosphatase 2B/calcineurin (CaN), leading to dendritic spine shrinkage through actin depolymerization and AMPAR depression through receptor dephosphorylation and internalization. The scaffold proteins A-kinase-anchoring protein 79/150 (AKAP79/150) and postsynaptic density 95 (PSD95) form a complex that controls the opposing actions of the cAMP-dependent protein kinase (PKA) and CaN in regulation of AMPAR phosphorylation. The AKAP79/150-PSD95 complex is disrupted in hippocampal neurons during LTD coincident with internalization of AMPARs, decreases in PSD95 levels, and loss of AKAP79/150 and PKA from spines. AKAP79/150 is targeted to spines through binding F-actin and the phospholipid phosphatidylinositol-(4,5)-bisphosphate (PIP2). Previous electrophysiological studies have demonstrated that inhibition of phospholipase C (PLC)-catalyzed hydrolysis of PIP2 inhibits NMDAR-dependent LTD; however, the signaling mechanisms that link PLC activation to alterations in dendritic spine structure and AMPAR function in LTD are unknown. We show here that NMDAR stimulation of PLC in cultured hippocampal neurons is necessary for AKAP79/150 loss from spines and depolymerization of spine actin. Importantly, we demonstrate that NMDAR activation of PLC is also necessary for decreases in spine PSD95 levels and AMPAR internalization. Thus, PLC signaling is required for structural and functional changes in spine actin, PSD scaffolding, and AMPAR trafficking underlying postsynaptic expression of LTD.
Journal of Neuroscience 03/2007; 27(13):3523-34. · 7.11 Impact Factor
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ABSTRACT: Central to organization of signaling pathways are scaffolding, anchoring and adaptor proteins that mediate localized assembly of multi-protein complexes containing receptors, second messenger-generating enzymes, kinases, phosphatases, and substrates. At the postsynaptic density (PSD) of excitatory synapses, AMPA (AMPAR) and NMDA (NMDAR) glutamate receptors are linked to signaling proteins, the actin cytoskeleton, and synaptic adhesion molecules on dendritic spines through a network of scaffolding proteins that may play important roles regulating synaptic structure and receptor functions in synaptic plasticity underlying learning and memory. AMPARs are rapidly recruited to dendritic spines through NMDAR activation during induction of long-term potentiation (LTP) through pathways that also increase the size and F-actin content of spines. Phosphorylation of AMPAR-GluR1 subunits by the cAMP-dependent protein kinase (PKA) helps stabilize AMPARs recruited during LTP. In contrast, induction of long-term depression (LTD) leads to rapid calcineurin-protein phosphatase 2B (CaN) mediated dephosphorylation of PKA-phosphorylated GluR1 receptors, endocytic removal of AMPAR from synapses, and a reduction in spine size. However, mechanisms for coordinately regulating AMPAR localization, phosphorylation, and synaptic structure by PKA and CaN are not well understood. A kinase-anchoring protein (AKAP) 79/150 is a PKA- and CaN-anchoring protein that is linked to NMDARs and AMPARs through PSD-95 and SAP97 membrane-associated guanylate kinase (MAGUK) scaffolds. Importantly, disruption of PKA-anchoring in neurons and functional analysis of GluR1-MAGUK-AKAP79 complexes in heterologous cells suggests that AKAP79/150-anchored PKA and CaN may regulate AMPARs in LTD. In the work presented at the "First International Meeting on Anchored cAMP Signaling Pathways" (Berlin-Buch, Germany, October 15-16, 2005), we demonstrate that AKAP79/150 is targeted to dendritic spines by an N-terminal basic region that binds phosphatidylinositol-4,5-bisphosphate (PIP(2)), F-actin, and actin-linked cadherin adhesion molecules. Thus, anchoring of PKA and CaN as well as physical linkage of the AKAP to both cadherin-cytoskeletal and MAGUK-receptor complexes could play roles in coordinating changes in synaptic structure and receptor signaling functions underlying plasticity. Importantly, we provide evidence showing that NMDAR-CaN signaling pathways implicated in AMPAR regulation during LTD lead to a disruption of AKAP79/150 interactions with actin, MAGUKs, and cadherins and lead to a loss of the AKAP and anchored PKA from postsynapses. Our studies thus far indicate that this AKAP79/150 translocation depends on activation of CaN, F-actin reorganization, and possibly Ca(2+)-CaM binding to the N-terminal basic regions. Importantly, this tranlocation of the AKAP79/150-PKA complex from spines may shift the balance of PKA kinase and CaN/PP1 phosphatase activity at the postsynapse in favor of the phosphatases. This loss of PKA could then promote actions of CaN and PP1 during induction of LTD including maintaining AMPAR dephosphorylation, promoting AMPAR endocytosis, and preventing AMPAR recycling. Overall, these findings challenge the accepted notion that AKAPs are static anchors that position signaling proteins near fixed target substrates and instead suggest that AKAPs can function in more dynamic manners to regulate local signaling events.
European Journal of Cell Biology 08/2006; 85(7):627-33. · 2.81 Impact Factor
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ABSTRACT: NMDA receptor-dependent long-term potentiation and long-term depression (LTD) involve changes in AMPA receptor activity and postsynaptic localization that are in part controlled by glutamate receptor 1 (GluR1) subunit phosphorylation. The scaffolding molecule A-kinase anchoring protein (AKAP)79/150 targets both the cAMP-dependent protein kinase (PKA) and protein phosphatase 2B/calcineurin (PP2B/CaN) to AMPA receptors to regulate GluR1 phosphorylation. Here, we report that brief NMDA receptor activation leads to persistent redistribution of AKAP79/150 and PKA-RII, but not PP2B/CaN, from postsynaptic membranes to the cytoplasm in hippocampal slices. Similar to LTD, AKAP79/150 redistribution requires PP2B/CaN activation and is accompanied by GluR1 dephosphorylation and internalization. Using fluorescence resonance energy transfer microscopy in hippocampal neurons, we demonstrate that PKA anchoring to AKAP79/150 is required for NMDA receptor regulation of PKA-RII localization and that movement of AKAP-PKA complexes underlies PKA redistribution. These findings suggest that LTD involves removal of AKAP79/150 and PKA from synapses in addition to activation of PP2B/CaN. Movement of AKAP79/150-PKA complexes from the synapse could further favor the actions of phosphatases in maintaining dephosphorylation of postsynaptic substrates, such as GluR1, that are important for LTD induction and expression. In addition, our observations demonstrate that AKAPs serve not solely as stationary anchors in cells but also as dynamic signaling components.
Journal of Neuroscience 04/2006; 26(9):2391-402. · 7.11 Impact Factor
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ABSTRACT: A-kinase-anchoring protein (AKAP) 79/150 organizes a scaffold of cAMP-dependent protein kinase (PKA), protein kinase C (PKC), and protein phosphatase 2B/calcineurin that regulates phosphorylation pathways underlying neuronal long-term potentiation and long-term depression (LTD) synaptic plasticity. AKAP79/150 postsynaptic targeting requires three N-terminal basic domains that bind F-actin and acidic phospholipids. Here, we report a novel interaction of these domains with cadherin adhesion molecules that are linked to actin through beta-catenin (beta-cat) at neuronal synapses and epithelial adherens junctions. Mapping the AKAP binding site in cadherins identified overlap with beta-cat binding; however, no competition between AKAP and beta-cat binding to cadherins was detected in vitro. Accordingly, AKAP79/150 exhibited polarized localization with beta-cat and cadherins in epithelial cell lateral membranes, and beta-cat was present in AKAP-cadherin complexes isolated from epithelial cells, cultured neurons, and rat brain synaptic membranes. Inhibition of epithelial cell cadherin adhesion and actin polymerization redistributed intact AKAP-cadherin complexes from lateral membranes to intracellular compartments. In contrast, stimulation of neuronal pathways implicated in LTD that depolymerize postsynaptic F-actin disrupted AKAP-cadherin interactions and resulted in loss of the AKAP, but not cadherins, from synapses. This neuronal regulation of AKAP79/150 targeting to cadherins may be important in functional and structural synaptic modifications underlying plasticity.
Molecular Biology of the Cell 09/2005; 16(8):3574-90. · 4.94 Impact Factor
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ABSTRACT: Sensing the osmolarity of the environment is a critical response for all organisms. Whereas bacteria will migrate away from high osmotic conditions, most eukaryotic cells are not motile and use adaptive metabolic responses for survival. The p38 MAPK pathway is a crucial mediator of survival during cellular stress. We have discovered a novel scaffold protein that binds to actin, the GTPase Rac, and the upstream kinases MEKK3 and MKK3 in the p38 MAPK phospho-relay module. RNA interference (RNAi) demonstrates that MEKK3 and the scaffold protein are required for p38 activation in response to sorbitol-induced hyperosmolarity. FRET identifies a cytoplasmic complex of the MEKK3 scaffold protein that is recruited to dynamic actin structures in response to sorbitol treatment. Through its ability to bind actin, relocalize to Rac-containing membrane ruffles and its obligate requirement for p38 activation in response to sorbitol, we have termed this protein osmosensing scaffold for MEKK3 (OSM). The Rac-OSM-MEKK3-MKK3 complex is the mammalian counterpart of the CDC42-STE50-STE11-Pbs2 complex in Saccharomyces cerevisiae that is required for the regulation of p38 activity.
Nature Cell Biology 01/2004; 5(12):1104-10. · 19.49 Impact Factor
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ABSTRACT: 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.
The Journal of Cell Biology 02/2003; 160(1):101-12. · 10.26 Impact Factor
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ABSTRACT: Compartmentalization of protein kinases and phosphatases with substrates is a means to increase the efficacy of signal transduction events. The A-kinase anchoring protein, AKAP79, is a multivalent anchoring protein that maintains the cAMP-dependent protein kinase, protein kinase C, and protein phosphatase-2B (PP2B/calcineurin) at the postsynaptic membrane of excitatory synapses where it is recruited into complexes with N-methyl-d-aspartic acid or alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA)-subtype glutamate receptors. We have used cellular targeting of AKAP79 truncation and deletion mutants as an assay to map the PP2B-binding site on AKAP79. We demonstrate that residues 315-360 are necessary and sufficient for AKAP79-PP2B anchoring in cells. Multiple determinants contained within this region bind directly to the A subunit of PP2B and inhibit phosphatase activity. Peptides spanning the 315-360 region of AKAP79 can antagonize PP2B anchoring in vitro and targeting in transfected cells. Electrophysiological experiments further emphasize this point by demonstrating that a peptide encompassing residues 330-357 of AKAP79 attenuates PP2B-dependent down-regulation of GluR1 receptor currents when perfused into HEK293 cells. We propose that the structural features of this AKAP79-PP2B-binding domain may share similarities with other proteins that serve to coordinate PP2B localization and activity.
Journal of Biological Chemistry 01/2003; 277(50):48796-802. · 4.77 Impact Factor
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ABSTRACT: Compartmentalization of protein kinases and phosphatases with substrates is a means to increase the efficacy of signal transduction
events. The A-kinaseanchoring protein, AKAP79, is a multivalent anchoring protein that maintains the cAMP-dependent protein kinase, protein kinase C, and
protein phosphatase-2B (PP2B/calcineurin) at the postsynaptic membrane of excitatory synapses where it is recruited into complexes
withN-methyl-d-aspartic acid or α-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA)-subtype glutamate receptors. We have used cellular
targeting of AKAP79 truncation and deletion mutants as an assay to map the PP2B-binding site on AKAP79. We demonstrate that
residues 315–360 are necessary and sufficient for AKAP79-PP2B anchoring in cells. Multiple determinants contained within this
region bind directly to the A subunit of PP2B and inhibit phosphatase activity. Peptides spanning the 315–360 region of AKAP79
can antagonize PP2B anchoring in vitro and targeting in transfected cells. Electrophysiological experiments further emphasize this point by demonstrating that a
peptide encompassing residues 330–357 of AKAP79 attenuates PP2B-dependent down-regulation of GluR1 receptor currents when
perfused into HEK293 cells. We propose that the structural features of this AKAP79-PP2B-binding domain may share similarities
with other proteins that serve to coordinate PP2B localization and activity.
Journal of Biological Chemistry 12/2002; 277(50):48796-48802. · 4.77 Impact Factor
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ABSTRACT: At the postsynaptic membrane of glutamatergic synapses, the cAMP-dependent protein kinase (PKA), protein kinase C (PKC), and calcineurin (CaN) anchoring protein AKAP79/150 is recruited to NMDA and AMPA glutamate receptors by postsynaptic density (PSD)-95 family membrane-associated guanylate kinase (MAGUK) scaffold proteins. These signaling scaffold complexes may function to regulate receptor phosphorylation in synaptic plasticity. Thus, it is important to understand regulation of AKAP79/150 targeting to synapses and recruitment to PSD-MAGUK complexes. AKAP79 is targeted to the plasma membrane by an N-terminal basic domain that binds phosphatidylinositol-4,5-bisphosphate (PI-4,5-P(2)) and is regulated by PKC phosphorylation and calmodulin binding. Here we demonstrate that this same domain also binds F-actin in a calmodulin- and PKC-regulated manner, targets to membrane ruffles enriched in F-actin and PI-4,5-P(2) in COS7 cells, and localizes to dendritic spines with F-actin and PSD-MAGUKs in hippocampal neurons. Inhibition of actin polymerization disrupted AKAP79 targeting of PKA and CaN to ruffles in COS7 cells and endogenous AKAP79/150 dendritic spine localization with PKA, CaN, and PSD-MAGUKs in neurons. AKAP79/150 postsynaptic localization was rapidly regulated by NMDA receptors through CaN activation and F-actin remodeling, further suggesting that AKAP79/150 signaling scaffold targeting depends on actin dynamics. NMDA receptor activation also regulated dendritic spine localization of PKA and CaN and association of the AKAP79/150-PKA complex with PSD-MAGUKs. Because AMPA receptor PKA phosphorylation and synaptic localization are regulated by similar NMDA receptor-CaN signaling pathways linked to hippocampal long-term depression, this regulation of AKAP79/150 postsynaptic targeting might be important for synaptic plasticity.
Journal of Neuroscience 09/2002; 22(16):7027-44. · 7.11 Impact Factor
