mAKAP assembles a protein kinase A/PDE4 phosphodiesterase cAMP signaling module
2001; Springer Nature; Volume: 20; Issue: 8 Linguagem: Inglês
10.1093/emboj/20.8.1921
ISSN1460-2075
AutoresKimberly L. Dodge, Samone Khouangsathiene, Michael S. Kapiloff, R. P. Mouton, Elaine V. Hill, Miles D. Houslay, Lorene K. Langeberg, John D. Scott,
Tópico(s)Cholinesterase and Neurodegenerative Diseases
ResumoArticle17 April 2001free access mAKAP assembles a protein kinase A/PDE4 phosphodiesterase cAMP signaling module Kimberly L. Dodge Kimberly L. Dodge Howard Hughes Medical Institute and Vollum Institute, Oregon Health Sciences University, 3181 SW Sam Jackson Park Road, Portland, OR, 97201-3098 USA Search for more papers by this author Samone Khouangsathiene Samone Khouangsathiene Howard Hughes Medical Institute and Vollum Institute, Oregon Health Sciences University, 3181 SW Sam Jackson Park Road, Portland, OR, 97201-3098 USA Search for more papers by this author Michael S. Kapiloff Michael S. Kapiloff Department of Pediatrics, Oregon Health Sciences University, 3181 SW Sam Jackson Park Road, Portland, OR, 97201-3098 USA Search for more papers by this author Robert Mouton Robert Mouton Howard Hughes Medical Institute and Vollum Institute, Oregon Health Sciences University, 3181 SW Sam Jackson Park Road, Portland, OR, 97201-3098 USA Search for more papers by this author Elaine V. Hill Elaine V. Hill Molecular Pharmacology Group, Division of Biochemistry and Molecular Biology, Davidson Building, Institute of Biomedical and Life Sciences University of Glasgow, Glasgow, G12 8QQ UK Search for more papers by this author Miles D. Houslay Miles D. Houslay Molecular Pharmacology Group, Division of Biochemistry and Molecular Biology, Davidson Building, Institute of Biomedical and Life Sciences University of Glasgow, Glasgow, G12 8QQ UK Search for more papers by this author Lorene K. Langeberg Lorene K. Langeberg Howard Hughes Medical Institute and Vollum Institute, Oregon Health Sciences University, 3181 SW Sam Jackson Park Road, Portland, OR, 97201-3098 USA Search for more papers by this author John D. Scott Corresponding Author John D. Scott Howard Hughes Medical Institute and Vollum Institute, Oregon Health Sciences University, 3181 SW Sam Jackson Park Road, Portland, OR, 97201-3098 USA Search for more papers by this author Kimberly L. Dodge Kimberly L. Dodge Howard Hughes Medical Institute and Vollum Institute, Oregon Health Sciences University, 3181 SW Sam Jackson Park Road, Portland, OR, 97201-3098 USA Search for more papers by this author Samone Khouangsathiene Samone Khouangsathiene Howard Hughes Medical Institute and Vollum Institute, Oregon Health Sciences University, 3181 SW Sam Jackson Park Road, Portland, OR, 97201-3098 USA Search for more papers by this author Michael S. Kapiloff Michael S. Kapiloff Department of Pediatrics, Oregon Health Sciences University, 3181 SW Sam Jackson Park Road, Portland, OR, 97201-3098 USA Search for more papers by this author Robert Mouton Robert Mouton Howard Hughes Medical Institute and Vollum Institute, Oregon Health Sciences University, 3181 SW Sam Jackson Park Road, Portland, OR, 97201-3098 USA Search for more papers by this author Elaine V. Hill Elaine V. Hill Molecular Pharmacology Group, Division of Biochemistry and Molecular Biology, Davidson Building, Institute of Biomedical and Life Sciences University of Glasgow, Glasgow, G12 8QQ UK Search for more papers by this author Miles D. Houslay Miles D. Houslay Molecular Pharmacology Group, Division of Biochemistry and Molecular Biology, Davidson Building, Institute of Biomedical and Life Sciences University of Glasgow, Glasgow, G12 8QQ UK Search for more papers by this author Lorene K. Langeberg Lorene K. Langeberg Howard Hughes Medical Institute and Vollum Institute, Oregon Health Sciences University, 3181 SW Sam Jackson Park Road, Portland, OR, 97201-3098 USA Search for more papers by this author John D. Scott Corresponding Author John D. Scott Howard Hughes Medical Institute and Vollum Institute, Oregon Health Sciences University, 3181 SW Sam Jackson Park Road, Portland, OR, 97201-3098 USA Search for more papers by this author Author Information Kimberly L. Dodge1, Samone Khouangsathiene1, Michael S. Kapiloff2, Robert Mouton1, Elaine V. Hill3, Miles D. Houslay3, Lorene K. Langeberg1 and John D. Scott 1 1Howard Hughes Medical Institute and Vollum Institute, Oregon Health Sciences University, 3181 SW Sam Jackson Park Road, Portland, OR, 97201-3098 USA 2Department of Pediatrics, Oregon Health Sciences University, 3181 SW Sam Jackson Park Road, Portland, OR, 97201-3098 USA 3Molecular Pharmacology Group, Division of Biochemistry and Molecular Biology, Davidson Building, Institute of Biomedical and Life Sciences University of Glasgow, Glasgow, G12 8QQ UK *Corresponding author. E-mail: [email protected] The EMBO Journal (2001)20:1921-1930https://doi.org/10.1093/emboj/20.8.1921 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Spatiotemporal regulation of protein kinase A (PKA) activity involves the manipulation of compartmentalized cAMP pools. Now we demonstrate that the muscle-selective A-kinase anchoring protein, mAKAP, maintains a cAMP signaling module, including PKA and the rolipram-inhibited cAMP-specific phosphodiesterase (PDE4D3) in heart tissues. Functional analyses indicate that tonic PDE4D3 activity reduces the activity of the anchored PKA holoenzyme, whereas kinase activation stimulates mAKAP-associated phosphodiesterase activity. Disruption of PKA–mAKAP interaction prevents this enhancement of PDE4D3 activity, suggesting that the proximity of both enzymes in the mAKAP signaling complex forms a negative feedback loop to restore basal cAMP levels. Introduction Understanding the molecular organization of intracellular signaling pathways has recently become a topic of considerable research interest (Hunter, 2000). It is now appreciated that the assembly of multiprotein signaling complexes creates focal points of enzyme activity that transduce the intracellular action of many hormones, neurotransmitters and cardiotonic agonists (Jordan et al., 2000). Accordingly, the spatiotemporal activation of protein kinases and/or phosphatases is important in controlling where and when phosphorylation events occur. Anchoring proteins and targeting subunits provide a molecular framework that orients protein kinases and phosphatases towards selected substrates (Pawson and Scott, 1997; Hunter, 2000). Prototypic examples of these 'signal-directing molecules’ are A-kinase anchoring proteins (AKAPs), which sustain multi-component signaling complexes of protein kinase A (PKA) and other enzymes (Colledge and Scott, 1999). These protein–protein interactions not only focus PKA toward the phosphorylation of certain ion channels, cytoskeletal elements and transcription factors, but also spatially segregate parallel cAMP signaling pathways (Johnson et al., 1994; Rosenmund et al., 1994; Feliciello et al., 1997; Fraser et al., 1998; Colledge and Scott, 1999; Marx et al., 2000; Westphal et al., 2000). However, activation of each intracellular PKA pool is ultimately determined by the availability of cAMP. Accordingly, the accumulation of cAMP at sites of PKA anchoring must be tightly and dynamically regulated. Phosphodiesterases (PDEs) are critical regulators of cyclic nucleotide homeostasis (Beavo, 1995; Conti, 2000). Together with phosphatases, they control the signal termination processes by counteracting the actions of second messenger-dependent enzymes. This is achieved by limiting the diffusion of second messengers, thereby influencing the activity state of cyclic nucleotide-gated ion channels, cAMP-GEFs, and enzymes such as PKA or PKG (Conti, 2000). Given the broad role that these enzymes play in all aspects of cellular regulation, it is not surprising that control of PDE activity is of considerable clinical importance. In fact, PDE inhibitor drugs are used to treat clinical depression, asthma and erectile dysfunction (Corbin and Francis, 1999; Soderling and Beavo, 2000). Inhibition of PDE activity is particularly relevant in the cardiovascular system where certain PDE families have been implicated in promoting vasodilatation of pulmonary arteries and aortic endothelial cells (Kessler and Lugnier, 1995; Maclean et al., 1997). Consequently, PDE inhibitors such as indolidan, enoximone and cilostazol have been developed as cardiotonic agents (reviewed by Beavo, 1995). The PDE superfamily currently consists of 21 different genes encoding 11 families of distinct PDE subtypes (Conti, 2000). The PDE4 cAMP-specific, rolipram-inhibited PDEs are derived from four related genes that encode numerous isoforms through alternate splicing of RNA (PDE4, A–D) (Houslay et al., 1998). Each isoenzyme contains a conserved catalytic core (Xu et al., 2000), but has divergent N- and C-terminal sequences, which have been proposed to regulate enzyme activity and direct the subcellular location of individual PDE4D isoforms (Jin et al., 1998; McPhee et al., 1999; Beard et al., 2000). On the basis of these findings, we reasoned that certain PDE4 cAMP-specific PDE isoforms may be compartmentalized with the cAMP-dependent protein kinase in heart tissue. In this report, we provide evidence that the PDE4D3 isoform and the PKA holoenzyme exist in the same signaling complex through their association with the muscle-selective A-kinase anchoring protein, mAKAP. Functional studies indicate that anchored PKA enhances cAMP degradation 2-fold, indicating that mAKAP provides the molecular framework for the assembly of a PKA/PDE negative feedback loop. Results mAKAP associates with a type 4 PDE We focused initially on measuring PDE activity that co-purified with the PKA holoenzyme from rat heart extracts (Figure 1A–C). Immunoprecipitation of the type II regulatory (RII) subunit of PKA resulted in a modest increase of 1.86 ± 0.4-fold (n = 3) in PDE activity over an IgG control using [3H]cAMP as a substrate (Figure 1A). This implied that both enzymes were recruited to the same signaling complex, but did not indicate whether an anchoring protein maintained these interactions. To test this, two AKAP signaling complexes known to be present in heart were isolated from tissue extract (Fraser et al., 1998; Kapiloff et al., 1999). Immunoprecipitation of mAKAP resulted in a 5.1 ± 0.2-fold (n = 5) increase in PDE activity over an IgG control, whereas immunoprecipitation of AKAP 15/18 only elicited a 1.7 ± 0.3-fold (n = 3) increase in enzyme activity (Figure 1B). In addition, AKAP150 immune complexes isolated from brain extracts displayed little PDE activity [1.45 ± 0.7-fold (n = 3); Figure 1B]. Owing to the significant amount of PDE activity associated with mAKAP, further experiments focused upon characterizing this interaction. Figure 1.Type 4 PDE activity co-purifies with mAKAP. (A) Immune complexes were isolated from rat heart extracts using antibodies against the RII subunit of cAMP-dependent protein kinase or control IgG serum. Co-precipitating PDE activity [pmol/min/immunoprecipitation (IP)] was measured using [3H]cAMP as a substrate. Data are presented as the average of three independent experiments. (B) Immune complexes were isolated from rat heart extracts using antibodies against mAKAP, AKAP18 or control IgG and from brain extracts using antibodies against AKAP150. The source of the antibody is indicated below each column. Co-precipitating PDE activity (pmol/min/IP) was measured as described above. Data presented are the average of three independent experiments. (C) mAKAP immune complexes were treated with PDE inhibitors (indicated below each column) and PDE activity was measured as described above. Data presented are the average of three independent experiments. (D) Immunoprecipitations were performed from rat heart extracts using antibodies against the RII subunit of PKA or IgG control. The resulting immune complexes were separated by electrophoresis on a 7.5% SDS gradient polyacrylamide gel and electrotransferred to nitrocellulose membranes. Detection of PDE4D in the extract (lane 1), IgG control (lane 2) or the RII immunoprecipitation (lane 3) was by immunoblot analysis using a monoclonal antibody against the PDE4D family. Detection of signals was by chemiluminescence. Molecular weight markers are indicated. The migration of PDE4D is indicated. Download figure Download PowerPoint PDE inhibitors were added to mAKAP immune complexes to establish which family of PDE associated with the anchoring protein (Figure 1C). The general PDE inhibitor 3-isobutyl-1-methylxanthine (IBMX; 15 μM) reduced PDE activity by 74 ± 8% (n = 3). In contrast, application of milrinone (1 μM), a selective PDE3 inhibitor, had no effect on the mAKAP-associated PDE activity (Figure 1C). However, rolipram (10 μM), a specific PDE4 inhibitor, blocked all mAKAP-associated PDE activity (Figure 1C), indicating that PDE4 activity associates with mAKAP in heart extracts. We obtained independent confirmation of this result when RII antibodies co-precipitated a 100 kDa protein recognized by monoclonal antibodies against the PDE4D gene family (Figure 1D, lane 3). On the basis of molecular weight, this protein was likely to be either PDE4D3 (97 kDa) or PDE4D5 (105 kDa). Both enzymes are expressed in cardiac tissues and PKA phosphorylation stimulates their PDE activity (Kostic et al., 1997). PDE4D associates with mAKAP inside cells To characterize the mAKAP signaling complex biochemically, additional co-precipitation experiments and PKA activity measurements were performed (Figure 2). Immunoprecipitation of mAKAP from rat heart extracts using polyclonal antisera against the rat anchoring protein resulted in co-purification of a 100 kDa PDE4D isoform, as detected by western blotting (Figure 2B, lane 3). Identical results were obtained when experiments were repeated using antisera raised against the human mAKAP protein (data not shown). PDE immunoreactivity was not co-precipitated with a control rabbit IgG control (Figure 2B, lane 2). It was estimated that ∼5% of the total cardiac PDE4 pool was associated with mAKAP. In reciprocal experiments, immunoprecipitation of PDE4D family members resulted in the co-purification of mAKAP, as detected by western blotting (Figure 2D, top panel, lane 3). An RII binding protein corresponding in size to mAKAP was detected when the same filter was probed for AKAPs by the overlay assay (data not shown). The anchoring protein was not detected when immunoprecipitations were performed with control IgG or pre-immune serum (Figure 2D, lane 2). Further analysis confirmed that the PKA holoenzyme was co-purified with the signaling complex, as RII (Figure 2D, middle panel, lane 3) and the C subunit of PKA (Figure 2D, bottom panel, lane 3) were detected by immunoblotting. Figure 2.Biochemical characterization of the mAKAP signaling complex. The mAKAP signaling complex was analyzed by a series of complementary biochemical approaches. (A) A schematic diagram depicting the isolation of the mAKAP immune complexes. (B) Immunoprecipitations were performed from rat heart extracts using antibodies against rat mAKAP or control IgG. The resulting immune complexes were separated by electrophoresis on a 7.5% SDS–polyacrylamide gel and electrotransferred to nitrocellulose membranes. Immunoblot analyses using monoclonal antibodies against the PDE4D family were used to identify the PDE in heart extracts (lane 1) and immunoprecipitations with the IgG control (lane 2) or anti-mAKAP antisera (lane 3). Detection of signals was by chemiluminescence. Molecular weight markers are indicated. The migration of PDE4D is indicated. (C) A schematic diagram depicting immunoprecipitation of PDE4D and associated proteins. (D) Immune complexes were isolated from rat heart extracts (lane 1) using goat polyclonal antibodies against PDE4D isoforms (lane 3) or control goat IgG (lane 2). Immune complexes were separated by electrophoresis on a 7.5% SDS–polyacrylamide gel and electrotransferred to nitrocellulose membranes. The filter was subjected to immunoblot analysis using rabbit polyclonal antibodies against rat mAKAP (top panel), RII regulatory subunit (middle panel) and catalytic subunit of PKA (bottom panel). Detection of signals was by chemiluminescence. Molecular weight markers and the migration position of mAKAP are indicated. (E) PKA activity in PDE4D immune complexes was measured using the heptapeptide Kemptide as a substrate. PKA specific activity (pmol/min/IP) was measured from PDE4D immune complexes, the IgG control and in the presence of the PKI 5–24 peptide, a specific inhibitor of PKA. The source of the sample is indicated below each lane. The accumulated data from three experiments are presented. Download figure Download PowerPoint Additional experiments were performed to establish whether PDE4D could immunoprecipitate the PKA holoenzyme through the association with mAKAP (Figure 2C). The presence of the PKA catalytic subunit was detected by activity measurements using the heptapeptide Kemptide as a substrate (Corbin and Reimann, 1974). The specific activity of PKA was enriched 3.3 ± 0.6-fold (n = 3) over the IgG control in PDE4D immunoprecipitates from rat heart extracts (Figure 2E). Furthermore, PKA activity was reduced to baseline levels in the presence of the PKI 5–24 peptide, a specific inhibitor of PKA (Scott et al., 1985) (Figure 2E). Collectively, these results demonstrate that mAKAP maintains a signaling complex of a cAMP-selective type 4D PDE and the PKA holoenzyme in vivo. PDE4D3 binds directly to mAKAP The PDE4D3 and PDE4D5 isoforms are splice variants of the same gene that differ only in their extreme N-terminal sequences (Beavo et al., 1994) (Figure 3A). In order to establish which PDE4D isoform associated with the anchoring protein, HEK 293 cells were transfected with pcDNA3 expression vectors encoding myc-tagged mAKAP and Flag-tagged versions of either PDE4D3 or PDE4D5. The epitope-tagged PDEs were immunoprecipitated from cell extracts with anti-Flag-tag antibodies, and co-purification of the anchoring protein was assessed by western blotting using anti-Myc antibodies (Figure 3B and C). Co-precipitation of mAKAP was only seen with co-expression of PDE4D3 (Figure 3B, lanes 1–3). In contrast, the anchoring protein did not co-precipitate with PDE4D5 (Figure 3B, lanes 4–6). Thus, PDE4D3 appears to associate with mAKAP selectively. Figure 3.PDE4D3 directly binds a central region of mAKAP. Mapping studies were performed to determine which PDE4D isoform interacts with mAKAP. (A) A schematic diagram of the PDE4D gene family highlights the conserved catalytic core and upstream conserved regions (UCR). The locations of divergent sequences in each isoform are indicated. The unique first 15 residues of PDE4D3 are presented using the one letter amino acid code. (B) Recombinant Flag-tagged PDE4D3 or Flag-tagged PDE4D5 was expressed in HEK293 cells. Anti-Flag antibodies or control IgG were used to isolate immune complexes. The precipitates were separated by gel electrophoresis on a 7.5% SDS–polyacrylamide gel and electrotransferred to nitrocellulose membranes. Co-precipitation of epitope-tagged mAKAP from cell extracts expressing PDE4D3 (lanes 1–3) or PDE4D5 (lanes 4–6) was assessed by immunoblotting (I.B.) using monoclonal antibodies against the myc tag. Detection of the signal was by chemiluminescence. Molecular weight markers and the migration position of Myc-mAKAP are indicated. (C) A His-tagged fusion protein encompassing the unique region of PDE4D3 or a His-tagged control fragment was co-expressed with mAKAP in HEK293 cells. Anti-His-tag antisera were used to isolate immune complexes. The presence of mAKAP was assessed by immunoblotting using anti-Myc monoclonal antibodies in HEK 293 cell extracts (lane 1) and in immune complexes from cells expressing His-PDE4D3 fragment (lane 3) or control fragment (lane 2). Detection of the signal was by chemiluminescence. Molecular weight markers and the migration position of Myc-mAKAP are indicated. A family of His-tagged-mAKAP fragments encompassing distinct regions of the anchoring protein were expressed in Escherichia coli. (D) A schematic diagram shows the size of each mAKAP fragment and its location within the linear sequence of the anchoring protein. The first and last amino acids of each fragment are indicated. The region of the anchoring protein that interacts with the PDE (closed box) and the binding site for PKA are highlighted. (E) A GST fusion protein including the unique N-terminal sequence of PDE4D3 was used to screen the purified mAKAP fragments by in vitro pull-down assay (lanes 2, 4, 6 and 8). Control experiments were performed with GST alone (lanes 1, 3, 5 and 7). The isolated materials were separated by gel electrophoresis on a 7.5% SDS–polyacrylamide gel and electrotransferred to nitrocellulose membranes. Isolation of mAKAP fragments was assessed by immunoblotting (I.B.) using monoclonal antibodies against the His tag. Detection of the signal was by chemiluminescence. Molecular weight markers are indicated. Download figure Download PowerPoint Given that PDE4D3 and PDE4D5 differ only in their extreme N-termini, we hypothesized that association between mAKAP and PDE4D3 was due to the unique sequence at the extreme N-terminus of PDE4D3 (Figure 3A). A His-tagged fusion protein including this region was expressed in HEK 293 cells and used as a ligand to isolate recombinant mAKAP (Figure 3C). Immunoprecipitation of the His-tagged 4D3 fragment resulted in co-purification of recombinant mAKAP (Figure 3C, lane 3). The anchoring protein was not detected upon immunoprecipitation of a control His-tagged fragment (Figure 3C, lane 2). To confirm these results, a glutathione S-transferase (GST) fusion protein encompassing the 15-amino-acid unique region of PDE4D3 was used to screen a family of mAKAP fragments in an in vitro pull-down assay (Figure 3D). The GST–PDE4D3 fragment bound to a central portion of mAKAP encompassing residues 1286–1831 (Figure 3E, lane 6), but did not bind adjacent regions of the anchoring protein including a fragment encompassing residues 1401–2301 (Figure 3E, lane 8). GST alone did not bind to any of the mAKAP fragments (Figure 3E). Control experiments confirmed that approximately equal amounts of each bacterially purified mAKAP-His-tag fragment were used in the pull-downs (data not shown). Taken together, these mapping studies indicate that there is a direct interaction between the N-terminus of PDE4D3 and a site located between residues 1286 and 1401 of mAKAP. Redistribution of PDE4 upon induction of mAKAP expression in cultured rat neonatal ventriculocytes We have demonstrated previously that mAKAP expression is induced in rat neonatal ventriculocytes (RNVs) by treatment with hypertrophic stimuli. This promotes accumulation of the anchoring protein at perinuclear sites and movement of the PKA holoenzyme to this region (Kapiloff et al., 1999). It seemed likely that PDE4D3 localization would be altered if mAKAP expression levels were manipulated. Immunofluorescence techniques were used to visualize PDE4 location in primary cultures of RNV before and after induction of mAKAP expression following treatment with phenylephrine (100 μM) for 24 h (Figure 4). Since the antibodies used in our biochemical analyses did not work for immunocytochemical detection of the enzyme, it was necessary to use a monoclonal antibody that recognized all PDE4 subtypes for these experiments. Control experiments confirmed that this antibody detected a 100 kDa PDE4 species upon immunoprecipitation of mAKAP from RNV (Figure 4A). Figure 4.Recruitment of mAKAP and PDE4D3 to the perinuclear regions of RNVs upon induction of hypertrophy. Expression of mAKAP is upregulated upon the induction of hypertrophy in RNV, resulting in accumulation of the anchoring protein at perinuclear regions (Kapiloff et al., 1999). (A) As a prelude to immunocytochemical analysis, control experiments confirmed that a monoclonal antibody against PDE4 family members recognized the PDE that co-precipitated with mAKAP from cardiomyocyte extracts. Immunoprecipitations were performed using antibodies against rat mAKAP or control IgG. The resulting immune complexes were separated by electrophoresis on a 7.5% SDS–polyacrylamide gel and electrotransferred to nitrocellulose membranes. Immunoblot analysis using a monoclonal antibody that recognizes the PDE4 family was used to identify the PDE in immunoprecipitations with anti-mAKAP antisera or the IgG control (indicated above each lane). This antibody was subsequently used in immunofluorescence experiments. Dissociated RNVs were plated on coverslips and grown in the absence (upper panels) or presence (lower panels) of phenylephrine (100 μM) for 24 h at 37°C. Ventriculocytes were fixed by incubation in 3.7% formaldehyde for 10 min at room temperature, permeablized with 0.3% Triton X-100, and subjected to labeling with primary antibodies against mAKAP (1:500 dilution) and PDE4 family(1:500 dilution) for 16 h at 4°C. The intracellular location of mAKAP (blue; B and F) was detected with Cy5-labeled secondary antibodies (1:100 dilution), PDE4 (green; C and G) was detected with fluorescein-labeled secondary antibodies (1:100 dilution) and the actin cytoskeleton (red; D and H) was detected with Texas red–phalloidin. Immunofluorescent detection of each signal was performed by confocal microscopy on a BioRad 1024 UV/Vis confocal microscope. Composite images are presented in (E) and (I). Download figure Download PowerPoint mAKAP was barely detected in undifferentiated RNV (Figure 4B), and PDE4 exhibited a general cytoplasmic staining pattern (Figure 4C). Actin staining defined cell morphology and indicated a moderate level of stress fiber formation (Figure 4D). Prominent mAKAP staining was detected at the perinuclear regions upon the induction of cardiomyocyte hypertrophy with phenylephrine (Figure 4F). Significantly, a proportion of the PDE4 signal was now detected at these sites (Figure 4G). In addition, stress fiber formation was more pronounced (Figure 4H), which is a recognized hallmark of RNV hypertrophy (Thorburn et al., 1995). These results suggest that induction of mAKAP promotes the recruitment of PDE4 to the perinuclear membranes of RNV. This is best illustrated in a composite image showing that a proportion of the mAKAP and PDE4 signals overlap (Figure 4I). This finding is consistent with subcellular fractionation data indicating that PDE4D subtypes are present in the outer perinuclear membranes of isolated cardiac nuclei (Lugnier et al., 1999). Regulation of the mAKAP signaling complex Our combined biochemical, enzymological and immunofluorescence data suggest that mAKAP maintains a signaling complex of active PDE4D3 and the PKA holoenzyme. A functional consequence of these interactions could be to control the activity status of the anchored kinase through regulation of local cAMP concentrations. To test this hypothesis, the dose dependency of PKA activation within the mAKAP signaling complex was measured over a range of cAMP concentrations. Untreated mAKAP immunoprecipitates from heart extracts displayed a dose-dependent increase in PKA activity (Figure 5A, squares) that resulted in a 7.8 ± 0.9-fold (n = 3) maximal stimulation of the kinase (Figure 5A). Addition of the PDE4 inhibitor rolipram enhanced sensitivity to cAMP (Figure 5A, diamonds), resulting in a 15.4 ± 2-fold (n = 3) stimulation of kinase activity at the highest concentration of cAMP (Figure 5A). Control experiments using the specific PKA inhibitor peptide PKI 5–24 confirmed that all of the anchored kinase activity was PKA (Figure 5A, circles). These data indicate that the pool of PDE4D3 associated with mAKAP influences the activity status of the anchored PKA. This result was confirmed by assembly of the signaling complex with recombinant components in HEK 293 cells (Figure 5B). Following immunoprecipitation of the mAKAP signaling complex, PKA activity was decreased 40 ± 10% (n = 3) in cells co-transfected with PDE4D3 (Figure 5B). Upon application of rolipram, the mAKAP-associated PKA activity was restored to a level equivalent to that measured from cells expressing mAKAP alone (Figure 5B). Thus, recruitment of the PDE into the mAKAP signaling complex attenuates PKA activation. Furthermore, these experiments indicate that mAKAP organizes two functionally coupled enzymes in a cAMP-responsive signaling scaffold to locally regulate PKA activity. Figure 5.mAKAP assembles a PDE/PKA negative feedback loop. The functional consequences of PDE4D3 localization through association with mAKAP were assessed on PKA activity. (A) The mAKAP signaling complex was immunoprecipitated from rat heart extracts using antibodies against the mouse anchoring protein. PKA activity (squares) was measured over a range of cAMP concentrations (0.1–10 mM) as described in Materials and methods, upon inhibition of PDE4D3 by rolipram (diamonds) and in the presence of the PKI 5–24 peptide (circles). The specific activity of the PKA catalytic subunit is presented as pmol/min/IP. The averaged data from three experiments are presented. (B) Recombinant myc-tagged mAKAP was expressed in HEK293 cells in the absence or presence of PDE-4D3 (indicated below each lane). Anti-Myc antibodies were used to isolate immune complexes and PKA activity was measured in the presence of 1 mM cAMP. The specific activity of the PKA catalytic subunit is presented as pmol/min/IP. The averaged data from three experiments are presented. Rolipram (10 μM) was used to selectively inhibit PDE activity. (C) Expression vectors encoding VSV-tagged PDE4D3 (lanes 1–4) or the phosphorylation site mutant VSV-tagged PDE4D3 S13A,S54A (lanes 5 and 6) were co-expressed with myc-tagged mAKAP in HEK293 (lanes 2–6). The transfected plasmids are indicated above each lane. The cells were incubated with 1 mM CPT-cAMP for 15 min before immunoprecipitation w
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