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. 2005 Aug;16(8):3574-90.
doi: 10.1091/mbc.e05-02-0134. Epub 2005 Jun 1.

Association of an A-kinase-anchoring protein signaling scaffold with cadherin adhesion molecules in neurons and epithelial cells

Jessica A Gorski  1 , Lisa L Gomez, John D Scott, Mark L Dell'Acqua
Affiliations

Association of an A-kinase-anchoring protein signaling scaffold with cadherin adhesion molecules in neurons and epithelial cells

Jessica A Gorski et al. Mol Biol Cell. 2005 Aug.

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.

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Figures

Figure 1.
Figure 1.
Identification of an interaction between AKAP79/150 and cadherins. (A) Diagram showing the AKAP79 baits used in positive [AKAP79(1-108) and AKAP79WT] and negative [AKAP79(108-427)] yeast two-hybrid screens of a rat brain cDNA library. Locations of three basic regions (A–C) within the targeting domain and PKC-, CaNA-, and PKA-RII-anchoring sites are indicated. (B) Primary structures of Gal4-cDNA PBcad clones identified as interacting with the AKAP79(1-108) bait. The extracellular domain (EC), transmembrane domain (TM), cytoplasmic domain (CD), signal sequence (SS), proteolytic cleavage site (PCS), Ca2+-adhesion repeats (EC1–5), and β-cat are indicated. (C) Representative yeast two-hybrid βgal filter assays for interactions of the indicated baits with PBcad Clone#24. (D) Five hundred nanograms of purified AKAP79(1-153)-His6 tagged fragment or β-cat was assayed for cadherin CD binding in vitro by precipitation with 5 μg of GST (negative control) and 5 μg of GST-CD fusions for human Ecad (hEcad) and Ncad (hNcad) and mouse Ecad (mEcad) followed by immunoblotting (IB:). (E) Diagram showing the primary structures of PBcad and Ecad deletions used to map the AKAP79 binding site (not to scale). Previously delineated (Kaplan et al., 2001) subregions within the cadherin CD that are rich in charged, acidic, serine, or basic residues are indicated. AKAP79(1-153) binding results (shown in F) are summarized by a + or –sign. (F) Representative immunoblots showing AKAP79(1-153) binding activity for the PBcad and Ecad-CD constructs diagramed in E. GST-fusion constructs were present at equal levels (5 μg), except Ecad-Δ2 and Ecad-Δ3 were present at fivefold higher levels, suggesting reduced binding for Ecad-Δ3. (G) PKC phosphorylation or preincubation of 1 μg of AKAP79(1-153) with 1 μM Ca2+-CaM, but not CaM plus EGTA, prevented GST-PBcad pulldown. (H) High salt concentrations (250–1000 mM) inhibit AKAP79(1-153) binding to GST-Ecad. (I) Amino acid sequence alignment of human Ecad, Ncad, and rat PBcad showing regions within the β-cat binding domain that are necessary for AKAP79 binding. Identical residues are shown in red and conservative substitutions in orange. (J) Immunoblots showing saturable binding of AKAP79(1-153) (0.05–2 μM) to a fixed amount of GST-Ecad (80 nM) with equal amounts of β-cat (10 nM). Both AKAP79(1-153) and β-cat are pulled down, even in the presence of a large molar excess of AKAP present (∼200-fold over β-cat).
Figure 2.
Figure 2.
AKAP79/150, cadherins, and β-catenin are present in neuronal postsynaptic complexes. Isolation of endogenous AKAP150 and β-cat from 5 mg of rat brain extracts through binding to 5 μg of GST-PBcadCD (A) and 5 μg of GST-EcadCD (B). (C) Isolation of cadherins and β-cat by immunoprecipitation (IP:) with AKAP150 from 5 mg of rat brain extracts. Five micrograms of anti-AKAP150 or IgG (nonimmune, negative control) IPs was analyzed by immunoblotting (IB:) as indicated. (D) Postsynaptic dendritic spine colocalization (arrows, white in composite panels) of AKAP150 (green) with F-actin (red) and Ncad (blue) or (E) β-cat (blue) in dendrites of hippocampal neurons. (F) Isolation of β-cat in complexes with AKAP150 from rat brain synaptic membranes. AKAP150 or IgG IPs of ∼500 μg of Triton X-100-solubilized crude synaptosomes (P2TX) or DOC-extracted purified synaptosomes (LP1DOC) were analyzed for β-cat by IB. Bar, 5 μm.
Figure 3.
Figure 3.
Polarized localization of endogenous AKAP79 with E-cadherin at the lateral membrane adherens junctions of Caco-2 epithelial cells. Lack of colocalization of AKAP79 (green) with apical membrane ezrin (red) (A and A′) and tight junction ZO-1 (red) (B and B′). Lateral membrane colocalization (yellow) of AKAP79 (green) with Ecad (red) (C and C′) and β-cat (red) (C and C′). (E, E′, and E″) Lateral membrane colocalization (white or yellow) of AKAP79 (green) with cortical F-actin (red, E and E′) and SAP97 (E, blue; E″, red). (A–E) xy, composite images through apical, lateral, and basolateral sections, as labeled. (A′–E″) Individual channels and composite images for a single xy section through apical, lateral, and basolateral membranes as labeled. Bar, 10 μm.
Figure 4.
Figure 4.
Polarized localization of AKAP79-GFP and association of AKAP150 with E-cadherin at lateral membranes of MDCK epithelial cells. Lack of colocalization of AKAP79-GFP (green) with apical marker ezrin (79, red) (A) and tight junction marker ZO-1 (M, red) (B). Colocalization (yellow) of AKAP79-GFP (79, green) with lateral membrane Ecad (M, red) (C) and β-cat (M, red) (D). (A–D) Right-hand panels show vertical xz line-scan sections from apical (a) to basolateral (b) for the same cells shown in the left-hand xy panels (y location in xy plane relative to the xz scan is indicated by a black line). (E) Apical, lateral, and basolateral xy sections showing AKAP79-GFP (green) basolateral membrane localization relative to tight junction ZO-1 (red). (F) Lateral membrane (xy) colocalization (yellow) of AKAP150 (green) with Ecad (red). (G) Coimmunoprecipitation of Ecad, β-cat, and SAP97(hDlg) with 5 μg of anti-AKAP150 but not nonimmune IgG from ∼3 mg of transfected MDCK cell lysates. Bars, 10 μm for xy sections, 5 μm for xz sections. Note: We are unable to detect expression of any endogenous canine AKAP protein with our antibodies to human AKAP79 or rat AKAP150.
Figure 5.
Figure 5.
The three basic regions within the AKAP79 N-terminal targeting domain mediate polarized localization to MDCK lateral membranes. (A) AKAP79(1-153)-GFP (green) containing all three (A–C) basic regions, (B) AKAP79(1-108)-GFP (green) containing the first two (A and B) basic regions, (C) AKAP79(75-153)-GFP (green) containing the second two (B and C) basic regions and (D) AKAP79(1-ΔB-153)-GFP (green) containing the first and the third (A and C) basic targeting regions all localize to MDCK lateral membranes (xy2 plane) but not to ZO-1 (red) expressing tight junctions (xy1 plane). The transverse plane (xz) shows a lack of colocalization of AKAP constructs (green) with the tight junction marker ZO-1 (red). (E) AKAP79(108-427)-GFP containing a single (C) basic region fails to show membrane localization. (F) Diagram showing primary structures of AKAP79-GFP fragments used in A–E. Bar, 10 μm.
Figure 6.
Figure 6.
Lateral membrane localization with Ncad and anchoring of PKA and CaN to AKAP79 imaged in living MDCK cells. FRET imaging of PKA-RII-YFP (green) (A) or CaNA-YFP (green) (B) anchoring to AKAP79-CFP (blue) at MDCK lateral membranes with a C-terminal (79ct) but not N-terminal (79nt) CFP chromophore. In A and B, FRETc (see Materials and Methods) is shown both in monochrome (far right) and as a CFP donor-gated image (FRETc/CFP) that represents relative FRET signal intensity on a pseudocolor scale of no FRET, blue; low FRET, green; and high FRET, red. Lateral membrane colocalization of Ncad-YFP with CFP-AKAP79 (C) and AKAP79(1-153)-CFP (D) in living MDCK cells. No FRETc was detected in C and D (our unpublished data). (E) Normalized mean FRETc (FRETNc ± SEM; see Materials and Methods) calculated from multiple FRETc images for AKAP79 anchoring of PKA-RII and CaN (left-hand graph). Apparent mean FRET efficiencies (±SEM) measured from YFP-acceptor photobleaching (see Materials and Methods) for AKAP79 or AKAP79(1-153)-CFP interactions with PKA-RII, CaN, and Ncad-YFP (right-hand graph). Bar, 10 μm.
Figure 7.
Figure 7.
Disruption of the cortical actin cytoskeleton leads to loss of AKAP79 lateral membrane localization in epithelial cells. (A and B) Treatment of MDCK cells with latrunculin A (LtrA, 5 μM, 4 h) causes loss of lateral membrane localization (yellow or white) of AKAP79-GFP (green) with Ecad (red) (A) and F-actin (red), SAP97 (blue) (B). (C and D) Treatment of Caco-2 cells with cytochalasin D (CHD, 5 μM, 4 h) causes loss of lateral membrane localization of endogenous AKAP79 (green) with Ecad (red) (C), F-actin (red) and SAP97 (blue) (D). Although colocalization of the AKAP with Ecad is reduced by LtrA or CHD depolymerization of actin, some intracellular overlap is still present (yellow, see arrows). (E and F) LtrA does not disrupt anti-AKAP coIP of Ecad (E) or β-cat (F) from approximately 5 mg of AKAP150-transfected MDCK cell lysates. Nonimmune IgG is used as a control for IP specificity. Bars, 10 μm.
Figure 8.
Figure 8.
Disruption of cadherin homophilic interactions by low extracellular Ca2+ leads to loss of AKAP79 lateral membrane localization in epithelial cells. (A–C) MDCK cells transfected with AKAP79-CFP and Ncad-YFP were imaged under control conditions (top row, 2 mM Ca2+), in reduced calcium (middle row, 2 μM Ca2+, 1 h), and after recovery (bottom row, 2 mM Ca2+, 1 h). (A) F-actin (monochrome, not in composite) lateral membrane localization (control, top row) is lost in low calcium media (middle row) that is restored to pretreatment conditions after 1-h recovery in normal calcium (bottom row). (B) Under control conditions (top row), AKAP79-CFP (green) colocalizes (yellow) with endogenous Ecad (red) at lateral membranes. Despite reduced colocalization of AKAP79-CFP with endogenous Ecad at lateral membranes in low calcium conditions (middle row), some intracellular colocalization is still seen (arrow). AKAP and Ecad lateral membrane localization is restored after 1-h recovery (bottom row). (C) Ncad-YFP (monochrome, not in composite) shows a similar distribution to endogenous Ecad in all experimental conditions. (D) Despite loss of membrane colocalization, inhibition of cadherin adhesion by low Ca2+ does not disrupt AKAP coIP of Ecad or β-cat in approximately 5 mg of AKAP150-transfected MDCK cell lysates. Nonimmune IgG is used as a control for IP specificity. (E) AKAP79-CFP (blue) and Ncad-YFP (green) are lost from lateral membranes and occur in common intracellular structures (arrows) over the same time course in living MDCK cells in low Ca2+ (t = 60–75 min, 33°C). Bar, 10 μm.
Figure 9.
Figure 9.
Regulation of AKAP79/150–cadherin complexes by NMDA receptor activation. (A and B) Loss of AKAP150 (red) dendritic spine colocalization (yellow) with Ncad (green) (A) and β-cat (green) (B) in response to NMDA (50 μM, 37°C, 10 min) in hippocampal neurons. Smaller panels show magnification of dendrites, arrows point to colocalized signal in control spines and loss of spine colocalization after NMDA treatment. (C) Quantitation (n = 10 cells) showing a significant decrease in AKAP150, but not Ncad or β-cat, localization to dendrites relative to the soma (dendrite/soma ratio, left-hand graph) and a significant loss of AKAP150 punctate colocalization with Ncad and β-cat (colocalization, left-hand graph) with NMDA. Colocalization of Ncad and β-cat with the presynaptic marker Syp was unchanged by NMDA (n = 7 cells). (D) Disruption of AKAP79/150 association with cadherins, catenins, and PSD-95 MAGUKs in response to NMDA. Approximately 7 mg of Triton-soluble extracts from control untreated (–) and NMDA treated (+) neurons were immunoprecipitated (IP:) with either 5 μg of anti-AKAP150 or nonimmune IgG as indicated. IPs and cell extracts were probed (IB:) as indicated. Bars, 10 μm.
Figure 10.
Figure 10.
NMDA receptor regulation of AKAP79 postsynaptic localization with PSD-95 and Ncad imaged in living neurons. (A) AKAP79-YFP (green) colocalizes (arrow, white) on dendritic spines with endogenous rat AKAP150 (red) and PSD-95 (blue) in hippocampal neurons. (B) Time-lapse imaging in living hippocampal neurons of AKAP79-YFP (green) redistribution (arrows) away from PSD-95-CFP (blue) on dendritic spines in response to NMDA (50 μM, 33°C, 5–40 min). (C) Postsynaptic localization of AKAP79(1-153)-YFP (green) with PSD-95-CFP (blue) on dendritic spines (arrows) seen in control conditions is lost in response to NMDA. (D) Postsynaptic localization of Ncad-YFP (green) with PSD-95-CFP on dendritic spines (arrows) is unchanged in response to NMDA. (E) AKAP79-CFP (blue) redistributes away from Ncad-YFP (green) on dendritic spines in response to NMDA (arrows). (C–E) Post-NMDA 30–40 min; smaller panels in A–E show magnifications of dendrites, arrows mark spines where CFP/YFP colocalization is seen in control neurons but is not seen after NMDA treatment). (F) Quantitation (n = 10 cells) showing significant decreases in NMDA-treated relative to untreated controls for AKAP79-YFP and (1-153)-YFP but not Ncad-YFP localization to dendrites relative to the soma (dend/soma ratio), dendritic spines relative to the cytoplasm of dendrites shafts (spine/shaft ratio) and colocalization with PSD-95-CFP (PS-Dcolo). Bars, 10 μm.
Figure 11.
Figure 11.
Model for NMDAR regulation of MAGUK–AKAP–cadherin signaling complexes at neuronal synapses. Under basal conditions, scaffold and anchoring proteins tether NMDAR and AMPAR to the actin cytoskeleton at the PSD. On NMDA stimulation and under LTD conditions, AKAP moves away from the PSD. Actin depolymerization and PKC or Ca2+–CaM interactions with the targeting domain might participate in the disruption of AKAP targeting. AKAP takes with it PKA, leaving some CaN behind (Gomez et al., 2002). This AKAP-mediated delocalization of PKA shifts the balance of AMPAR phosphorylation in favor of dephosphorylation. After dephosphorylation by CaN and PP1, AMPARs diffuse laterally out of the PSD, where they undergo CaN-dependent internalization and endocytosis. Actin depolymerization also is regulated by CaN and may participate in AMPAR untethering (Halpain et al., 1998; Shen et al., 2000). In contrast, cadherin–catenin complexes do not seem to be displaced in response to NMDA stimulation associated with LTD, despite reduction in spine F-actin (Okamoto et al., 2004; Zhou et al., 2004).
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References

    1. Abe, K., Chisaka, O., Van Roy, F., and Takeichi, M. (2004). Stability of dendritic spines and synaptic contacts is controlled by αN-catenin. Nat. Neurosci. 7, 357–363. - PubMed
    1. Banke, T. G., Bowie, D., Lee, H., Huganir, R. L., Schousboe, A., and Traynelis, S. F. (2000). Control of GluR1 AMPA receptor function by cAMP-dependent protein kinase. J. Neurosci. 20, 89–102. - PMC - PubMed
    1. Bauman, A. L., Goehring, A. S., and Scott, J. D. (2004). Orchestration of synaptic plasticity through AKAP signaling complexes. Neuropharmacology 46, 299–310. - PubMed
    1. Beattie, E. C., Carroll, R. C., Yu, X., Morishita, W., Yasuda, H., von Zastrow, M., and Malenka, R. C. (2000). Regulation of AMPA receptor endocytosis by a signaling mechanism shared with LTD. Nat. Neurosci. 3, 1291–1300. - PubMed
    1. Bozdagi, O., Shan, W., Tanaka, H., Benson, D. L., and Huntley, G. W. (2000). Increasing numbers of synaptic puncta during late-phase LTP: N-cadherin is synthesized, recruited to synaptic sites, and required for potentiation. Neuron 28, 245–259. - PubMed

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