Department of Pediatrics, Heart Research Center, Oregon Health Sciences University, NRC5, 3181 S.W. Sam Jackson Park Road, Portland, OR 97201, USA
*Author for correspondence (e-mail: kapiloff{at}ohsu.edu)
Accepted May 25, 2001
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SUMMARY |
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Key words: mAKAP, Protein kinase A, cAMP, Ryanodine receptor, Nuclear envelope, Heart
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INTRODUCTION |
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The PKA holoenzyme is composed of two regulatory (R) and two catalytic (C) subunits, which dissociate upon cAMP binding (Scott, 1991). PKA holoenzyme is sequestered in pools by constitutive binding of the R-subunit homodimer to AKAPs (Colledge and Scott, 1999). For example, AKAP18 is targeted to the cardiac myocyte plasma membrane, where it binds PKA that can phosphorylate and activate the L-type calcium channel (Fraser et al., 1998; Gray et al., 1998). AKAPs often serve as scaffolds for multi-protein complexes that include kinase substrates and other signaling enzymes. AKAP79/150 is one such scaffolding protein that associates in brain with PKA, protein kinase C, the protein phosphatase calcineurin, and the ß2-adrenergic receptor (Fraser et al., 2000). Rather than the concentration or overall abundance of signaling enzymes and substrates, it is the localization of a particular set of enzymes via association with a targeted scaffolding protein that confers specificity in function (Pawson and Nash, 2000; Pawson and Scott, 1997).
mAKAP (muscle A-Kinase Anchoring Protein) is a 255 kDa scaffolding protein present on the nuclear envelope (NE) of myocytes in heart and skeletal muscle that can bind PKA and phosphodiesterase type 4D3 (PDE4D3) (Dodge et al., 2001; Kapiloff et al., 1999). mAKAP targeting has been studied using recombinant fragments fused to green fluorescent protein (GFP). mAKAP is localized in the differentiated cardiomyocyte NE by two independent targeting domains, amino acid (aa) residues 772-915 and 915-1065, which contain spectrin-like repeat motifs (Kapiloff et al., 1999). mAKAP targeting is saturable, and endogenous mAKAP can be displaced by overexpression of an mAKAP fragment containing the targeting domains. mAKAP binds PKA via a putative amphipathic helix comprising aa residues 2055-2072, and PKA binding can be disrupted by substitution of isoleucine residue 2062 with a proline residue. This is consistent with structural data regarding the AKAP-RII interaction that the N-terminal domains of the PKA type II R-subunit homodimer form an X-type, four-helix bundle dimerization motif containing a hydrophobic groove that accommodates an AKAP amphipathic
-helix (Newlon et al., 2001). Through binding to a site within mAKAP residues 1286-1831, a rolipram-inhibited, cAMP-specific PDE4D3 is also associated with the mAKAP-PKA complex in heart (Dodge et al., 2001). PDE4D3-catalyzed degradation of cAMP is enhanced by cAMP-dependent PKA phosphorylation, thereby constituting a local, negative feedback loop to modulate NE-targeted, cAMP-dependent signaling.
mAKAP was initially identified by its ability to bind PKA (McCartney et al., 1995). Despite an understanding of its intracellular location and its association with PDE4D3, the function of mAKAP at the NE is yet uncertain. Using a candidate-directed approach, we have identified an association of mAKAP with the ryanodine receptor (RyR) and protein phosphatase 2A (PP2A). As shown below, mAKAP and RyR overlap in intracellular distribution at the NE of cardiac myocytes, a double membrane structure separating the cytoplasm, the perinuclear space and the nucleus. The perinuclear space holds a discrete store of intracellular Ca2+ that may be released into the surrounding area by NE-associated ion channels such as the RyR, with potential effects on gene expression (Abrenica and Gilchrist, 2000; Adebanjo et al., 1999; Adebanjo et al., 2000; Badminton et al., 1996; Chawla et al., 1998; Franco-Obregon et al., 2000; Gerasimenko et al., 1995; Malviya and Rogue, 1998; Rogue et al., 1998). Tetrameric with subunits of 560 kDa, the RyR is a high conductance Ca2+ channel, tightly regulated by multiple co-factors, notably Ca2+ itself, and by phosphorylation, including PKA-catalyzed phosphorylation (MacKrill, 1999). PP2A is a phospho-serine/threonine protein phosphatase that is involved in the regulation of many signaling pathways (Millward et al., 1999). PP2A has three subunits, a catalytic C-subunit and a scaffolding A-subunit that comprise a constitutive, core heterodimer and one of several possible regulatory B-subunits. We now describe the association of PP2A and RyR with the mAKAP complex, an assembly that is likely to be important to the integration of cAMP and Ca2+ signaling to the myocyte nucleus.
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MATERIALS AND METHODS |
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Immunohistochemistry of human heart tissue
Anonymous samples of paraffin-embedded normal human heart tissue were provided by the Cancer Pathology Shared Resource of the Oregon Cancer Center. 5 µm sections of these samples were prepared on Fisherbrand Plus slides (Fisher Scientific). Following de-paraffinization and rehydration, the slides were subjected to epitope retrieval by heating in a vegetable steamer (Black & Decker Inc.) containing 1 M Tris, pH 10, for 20 minutes and then cooling to room temperature. Treated slides were then washed with PBS (120 mM NaCl, 2.7 mM KCl, 10 mM sodium phosphate salts) containing 0.05% Tween (Sigma) and used for immunohistochemistry.
Antibodies were diluted into dilution buffer (PBS, 1% bovine serum albumin (BSA), 0.1% Tween 20, 0.1% sodium azide). Tris buffered saline (TBS; 10 mM Tris, pH 7.5, 150 mM NaCl) was used for all wash steps. Following a 10 minute incubation in dilution buffer, primary antibody (4 µg/ml VO56 affinity-purified antibody or rabbit whole IgG) was added for 45 minutes followed by washing. The slides were treated with quench solution (methanol, 6% H202) for 10 minutes, washed again, and then incubated with Envision anti-rabbit secondary reagent (Dako) for 30 minutes. After additional washes, premixed DAB solution (K3466, Dako) was added to the slides and allowed to react for 10 minutes. Slides were counterstained with hematoxylin prior to dehydration and coverslipping.
Immunoprecipitation from heart extracts
Rat hearts (Pel-freeze) were washed twice with cold PBS and then disrupted using a Polytron homogenizer at half-speed for 15 seconds in Buffer A (50 mM Hepes, pH 7.4, 10% glycerol, 1 µg/ml pepstatin, 1 µg/ml leupeptin, 1 mM 4-(2-Nethyl)benzenesulfonyl fluoride (AEBSF), 1 mM benzamidine, 1 mM dithiothreitol (DTT), 5 mM ethylenediaminetetraacetic acid (EDTA), 5 mM ethylene glycol-bis(ß-aminoethyl ether), 25 mM sodium fluoride, 40 mM ß-glycerophosphate, and 1 mM sodium pyrophosphate). A low-speed pellet was obtained by centrifugation of whole heart extract at 13,000 g for 5 minutes. This pellet was resuspended in Buffer A with 100 mM NaCl and 0.5% Triton X-100, mixed for 10 minutes at 4°C, and then a solubilized protein supernatant was generated by centrifugation at 20,000 g for 10 minutes. Solubilized protein supernatant was mixed at 4°C for 3 hours with 20 µl preimmune or VO54 anti-mAKAP immune antiserum previously bound to 20 µl protein-G-agarose (Upstate Biotechnology). Beads were washed for 5 minutes three times with Buffer A with 100 mM NaCl and 0.5% Triton X-100 before resuspension in sample buffer (12.5 mM Tris-HCl, pH 6.8, 1% mercaptoethanol, 2% glycerol, 0.4% sodium dodecyl sulfate, 6 µg/ml bromophenol blue).
Immunoblotting
Samples were size-fractionated by SDS-PAGE on 3% acrylamide stacking phase, 5% or higher percentage acrylamide resolving phase gels (Laemmli, 1970). 5% acrylamide resolving phase gels were transferred to nitrocellulose on a Biorad Semi-dry Transfer Unit in the absence of methanol, while 8-12% acrylamide resolving phase gels were transferred using a Biorad Liquid Transfer Unit in the presence of 10% methanol. Blots were blocked in 0.1% Blotto (0.1% BSA, 5% nonfat dry milk, TBS, 0.05% sodium azide) for 30 minutes and incubated with primary antibody diluted in 0.1% Blotto (polyclonal antibodies) or TTBS (TBS with 0.03% Tween; monoclonal antibodies) overnight at room temperature. Blots were washed four times for five minutes with TTBS, before incubation for 1 hour with 1:50,000 dilution of horseradish peroxidase-conjugated donkey anti-IgG antibody (Jackson Laboratories) in TTBS. After washing, bound antibody was detected with chemiluminescent substrate (Supersignal products, Pierce). Molecular weight markers include myosin (200 kDa), IgG heavy chain (48 kDa) and Kaleidoscope Prestained Standards (Biorad).
Ventriculocyte immunocytochemistry
Rat neonatal ventriculocytes were prepared as previously described (Kapiloff et al., 1999) and cultured on dual-well chamberslides coated with 1% gelatin and 1 mg/ml laminin solution at a density of about 100,000 myocytes/cm2. After one day in plating medium (Dulbeccos Modified Eagle Medium (DMEM) with 17% Media 199, 1% penicillin/streptomycin solution (Gibco/BRL), 10% horse serum, and 5% fetal bovine serum (FBS)), cells were incubated for two days in 80% DMEM, 20% Media 199 and 100 µM phenylephrine. For staining, cells were washed twice with PBS, fixed for 10 minutes in 3.7% formaldehyde in PBS, washed once with PBS, permeabilized with 0.3% Triton X-100 in PBS for 10 minutes, washed again with PBS and then blocked in PBS with 1% horse serum, 0.2% BSA. Slides were incubated with primary antibody (1 µg/ml) in blocking solution for 1 hour and washed several times with blocking solution. Slides were next incubated for 1 hour with Cy5 or FITC-conjugated donkey secondary antibodies (Jackson Laboratories) and Rhodamine Phalloidin (Molecular Probes), washed several times with PBS, and then mounted with coverslips and Slofade anti-fade solution (Molecular Probes). Hoechst 33258 stain (10 µg/ml) was included in the last PBS wash in order to locate nuclei. Specific immunofluorescence was detected in successive focal planes by laser-scanning confocal microscopy on an MRC1024 Biorad UV/Vis System.
Heart subcellular fractionation
The following procedure is a modification of established procedures for the isolation of sarcoplasmic reticulum (SR) and nuclei from heart tissue (Meissner, 1974; Tata, 1974). Two rat hearts (Pel-freeze) were washed twice with cold PBS and then disrupted using a Polytron PT10/35 Generator at half-speed for 15 seconds in 20 ml Buffer B (10 mM Hepes, pH 7.4, 1 µg/ml pepstatin, 1 µg/ml leupeptin, 1 mM AEBSF, 1 mM benzamidine, 5 mM EDTA) with 0.32 M sucrose. Whole heart homogenate was filtered through 2 layers and then 4 layers of cheesecloth, before low-speed centrifugation at 3800 g for 20 minutes. The supernatant fraction (S1) was clarified by centrifugation at 10,000 g for 20 minutes, before re-centrifugation at 100,000 g for 1 hour. The resulting pellet (P2 fraction), containing SR, Golgi apparatus and plasma membrane, was resuspended in 2 ml buffer B and 0.32 M sucrose. Purified SR was obtained from P2 fraction by sucrose step gradient centrifugation (8 parts 24%, 6 parts 40%, 2 parts 50% sucrose in 5 mM Hepes buffer) at 100,000 g for 90 minutes. Purified SR forms a layer at the interface between 24% and 40% sucrose.
The initial 3800 g pellet (P1), containing myofibrils, mitochondria and nuclei, was resuspended in 20 ml Buffer B with 2.4 M sucrose, and nuclei were sedimented by centrifugation at 50,000 g for 90 minutes. The nuclei-containing pellet was washed by resuspension in 1 ml buffer B and 0.32 M sucrose and repeat centrifugation at 3800 g for 20 minutes. The nuclei-containing pellet (Nuclei) was then resuspended in 1 ml buffer B and 0.32 M sucrose. mAKAP complex was immunoprecipitated from nuclei as above. Protein content was determined by a modification of the Lowry method (Lowry et al., 1951; Biorad DC Protein Assay kit).
Expression of full-length mAKAP and protein-protein interaction studies
A mAKAP cDNA encoding the full-length protein was subcloned from pEGFPN1 (Kapiloff et al., 1999) into the NheI and KpnI sites in pCDNA3.1(-)mychis B (Stratagene) in order to introduce a myc tag fusion. The fusion cDNA was transferred into pTRE-shuttle vector (Clontech) using SpeI and AflII restriction enzymes and recombinant adenovirus was produced using the Adeno-X Tet-Off Expression System (Clontech), according to the manufacturers protocols. mAKAP-expressing adenovirus was used to infect HEK293 and COS-7 cells for the production of mAKAP protein. RyR GST-fusion proteins were produced in E. Coli BL21 DE3 RIL (Stratagene) by way of pGEX-4T expression vectors (Pharmacia Biotech) containing rabbit RyR type II cDNA fragments that were inserted by ligation of restriction enzyme digested PCR products generated with Pfu Turbo high-fidelity enzyme (Stratagene), a RyR II full-length cDNA, and oligonucleotide primers designed to keep the cDNA in frame. GST-fusion proteins were purified from bacterial extracts using glutathione resin as suggested by the manufacturer (Clontech).
HEK293 or COS-7 cells were placed into culture at 50% confluence in DMEM with 10% FBS and 1% penicillin/streptomycin solution. The next day, cells were infected with mAKAP-expressing adenovirus and Adeno-X Tet-Off virus as suggested by the manufacturer (Clontech). 24-48 hours later, cells were lysed in Buffer A with 100 mM NaCl and 0.5% Triton X-100 (without phosphatase inhibitors). Soluble proteins were separated by centrifugation at 20,000 g for 10 minutes, divided into aliquots and incubated at 4°C with glutathione resin previously bound with GST-fusion proteins. Beads were washed three times for 5 minutes at 4°C with lysis buffer before resuspension in sample buffer, separation by SDS-PAGE and immunoblotting as described above.
Protein kinase A assay
Rat hearts were homogenized as above using Buffer C (50 mM Hepes, pH 7.4, 100 mM NaCl, 10 mM MgCl2, 0.5% Triton X-100, 1 mM DTT, 10% glycerol, 1 µg/ml pepstatin, 1 µg/ml leupeptin, 1 mM AEBSF, 1 mM benzamidine). Whole heart homogenate was cleared by centrifugation at 20,000 g for 10 minutes. mAKAP complex was immunoprecipitated as above using VO54 antiserum, and RyR complex using anti-RyR antibody. Beads containing immunoprecipitated complex were resuspended in 80 µl kinase buffer (50 mM MOPS, pH 6.8, 50 mM NaCl, 4 mM MgCl2, 1 mM DTT, 1 mM cAMP, 20 µM U0126, 50 nM microcystin and 10 µM rolipram) and pre-incubated for 30 minutes at room temperature with and without 10 µM protein kinase inhibitor (PKI). Phosphorylation was initiated by addition of 5 µl [-32P]ATP (7000 Ci/mmol, 13 µCi/µl). After incubation at 30°C for 30 minutes, reactions were stopped with 10 µl 5x sample buffer and 1 mM H4PO2. Kinase reactions were size-fractionated by SDS-PAGE and transferred to nitrocellulose as described above. Nitrocellulose filters were exposed to x-ray film and later incubated with antibody as described above. Control immunoprecipitations included addition of excess (100 µg) VO54 antigen (mAKAP residues 1401-2314) (Kapiloff et al., 1999) or use of non-immune immunoglobulin (2.5 µg IgG). The relative extent of phosphorylation was quantified by autoradiograph densitometry using a Biorad GS-700 Imaging Densitometer.
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RESULTS |
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Recombinant PP2A, RyR and mAKAP interaction
Given the association of RyR and mAKAP in cells, we were interested in studying their interaction at the molecular level using a pull-down assay (Fig. 5). Mutations have been found in the cytosolic, N-terminal domain of human type II RyR in patients with arrhythmogenic right ventricular dysplasia (ARVD), a cardiac disorder often presenting as sudden death (Tiso et al., 2001). Because the human mAKAP gene is linked to another genetic locus for ARVD (Kapiloff et al., 1999), we speculated that one of the RyR mutations might lie within the RyR domain responsible for mAKAP association, thereby facilitating the mapping analysis of this very large protein. Mutations have been found at RyR aa residues Arg176, Leu433, Asn2386 and Thr2504 in ARVD (Tiso et al., 2001), and in another genetic disorder, catecholaminergic polymorphic ventricular tachycardia (CPVT), at RyR aa residues Ser2246, Arg2474 and Arg4497 (Priori et al., 2001). GST-fusion proteins including large regions of RyR encompassing these mutations were produced in bacteria (Fig. 5A). Full-length mAKAP was expressed by infection with adenovirus vectors in HEK293 and COS-7 cells. Whole cell extracts containing mAKAP were then incubated with glutathione beads previously absorbed with GST-fusion proteins or GST alone (Fig. 5B). A GST-fusion protein containing RyR aa residues 1-568 specifically mediated the precipitation of mAKAP protein (lane 1). By contrast, GST-fusion proteins containing RyR aa residues 2080-2609 or 4332-4663 (lanes 2,3) and GST-alone (lane 4) were unable to bind mAKAP and serve as controls for these experiments. Similar amounts of GST fusion proteins were used in all experiments (data not shown). In parallel assays, it was apparent that a GST-PP2A A-subunit fusion protein was also effective at pulling down mAKAP (Fig. 5C). These data serve to map the mAKAP-interaction site on RyR type II to the N-terminal tenth of the RyR protein and to support the association of mAKAP with RyR and the core PP2A heterodimer in cells, in agreement with their co-immunoprecipitation with mAKAP antiserum from heart extracts.
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DISCUSSION |
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mAKAP staining has been shown to be limited to the NE of cardiomyocytes in frozen sections of rat cardiac tissue and in cultured myocytes using two independent, affinity-purified anti-mAKAP antibodies (Dodge et al., 2001; Kapiloff et al., 1999). We now extend those studies by showing that mAKAP is localized at the cardiomyocyte NE of paraffin-embedded human ventricular tissue (Fig. 1). Our results are strengthened by data that GFP fused to mAKAP is directed solely to the NE of transfected rat neonatal ventriculocytes and that two adjacent domains of mAKAP (aa residues 772-915 and 915-1065) containing spectrin-like repeats can also direct GFP to the NE (Kapiloff et al., 1999). These results differ from two reports that have found mAKAP in multiple myocyte compartments (Marx et al., 2000; Yang et al., 1998). We cannot exclude the possibility that mAKAP is targeted to other intracellular sites at levels beneath the threshold of detection in these experiments.
To investigate the function of the mAKAP targeting of a select pool of PKA (Fig. 3E-H) and PDE4D3 (Dodge et al., 2001) to the nuclear envelope, we have begun to define the other components of the mAKAP complex. Co-immunoprecipitation using anti-mAKAP antibodies revealed the association of mAKAP with PP2A and RyR, but not with a variety of other signaling proteins including calcineurin, PP1, SERCA2A and PLB (Fig. 2). PP2A is a heterotrimeric protein phosphatase implicated in the de-phosphorylation of multiple signaling enzymes, including protein kinase C, casein kinase, Ca2+/calmodulin-dependent protein kinases, mitogen activated protein kinases, cyclin-dependent protein kinases, and both PKA and RyR (Chu et al., 1990; Millward et al., 1999). In the heart, PP2A may be involved in contractile protein and ion-channel regulation as well. Interestingly, transgenic mice expressing a dominant negative form of PP2A A-subunit exhibit cardiac hypertrophy and die from dilated cardiomyopathy (Brewis et al., 2000). The regulation of PP2A is poorly understood and specificity is thought to be, in part, secondary to targeting by different B-subunits (Millward et al., 1999). PP2A C-subunit was detected in anti-mAKAP immunoprecipitates using heart extract (Fig. 2), and mAKAP expressed in HEK293 cells was precipitated with GST-A-subunit (Fig. 5C). It remains to be determined whether the mAKAP-PP2A interaction is indirect or whether mAKAP serves as a B-subunit to target the PP2A core heterodimer to the NE. PP2A has been found to associate with RyR aa residues 1451-1768, but whether this interaction is direct is not known (Marx et al., 2000). Given the extremely low levels of RyR in HEK293 cells (Querfurth et al., 1998), it is not likely that mAKAP bound PP2A through RyR in the pull-down experiments. Future studies will address the mechanism of binding and the role of PP2A in the mAKAP complex.
Having detected RyR in mAKAP immunoprecipitates, we investigated whether there was partial co-distribution of RyR and mAKAP at the cardiomyocyte NE. Immunocytochemistry of rat neonatal ventriculocytes revealed that RyR is principally an SR ion channel (Fig. 3B), consistent with its well-understood role in excitation-coupling (Franzini-Armstrong and Protasi, 1997). Importantly, there was a significant pool of RyR also at the NE, overlapping in distribution with mAKAP (Fig. 3A-D). These results were followed by experiments involving fractionation of adult rat heart tissue (Fig. 4). RyR was found in both microsomal and nuclear fractions (Fig. 4A), while mAKAP was present in nuclear, but not SR fractions (Fig. 4B). Confirming the presence of RyR-mAKAP complex at the NE, RyR and mAKAP were co-immunoprecipitated from purified nuclei (Fig. 4C). mAKAP-RyR co-immunoprecipitation has been independently demonstrated in a manuscript concerning the SR RyR macromolecular complex (Marx et al., 2000). One strength of the experiments presented here is that, in order to distinguish between pools of RyR, specific precaution that has not traditionally been warranted in RyR investigations was employed during homogenization to prevent nuclear disruption and mixing of NE and SR membranes (Meissner, 1974; Tata, 1974). The results of experiments involving immunohistochemistry and tissue fractionation lead us to conclude that mAKAP associates predominantly with a NE pool of RyR, and not significantly with RyR present at the SR.
Only recently has it become appreciated that a pool of RyR is resident on the NE (Bootman et al., 2000). Ca2+ currents sensitive to cADPr and anti-RyR staining have been detected on the NE of isolated nuclei (Adebanjo et al., 1999; Gerasimenko et al., 1995). Thus far, no molecular differences have been discerned between SR and NE RyRs. Type I RyR has been found at the NE of osteoclasts, cells that apparently do not contain type II or type III RyR (Adebanjo et al., 2000). mRNA for all three types of RyR have been detected in heart (Franzini-Armstrong and Protasi, 1997). Because the RyR antibody used in this study is selective, but not exclusive for type II RyR, and because the cytoplasmic domains of the RyR forms are highly similar, we cannot exclude the possibility that cardiac NE RyR is type I or III, rather than type II, the predominant form in the heart.
Our data suggest that an important difference between SR and NE RyR is the selective association of NE RyR with the mAKAP complex (Fig. 3). By RII-overlay assay, there is a highly abundant AKAP of about 140 kDa Mr in heart that is the major RII
-binding protein in SR preparations (data not shown). This putative SR-AKAP may be associated with SR RyR and may be important to excitation-coupling. More investigation is required to discern what other signaling components are associated with NE mAKAP-RyR complex, including those represented by the multiple phosphorylated bands found for mAKAP complex (Fig. 6, lanes 4,5). Although calcineurin and PP1 have been reported to be associated with RyR (Cameron et al., 1995; Marx et al., 2000), we detected neither in mAKAP immunoprecipitates (data not shown), reflecting the difficulty of higher order complex co-immunoprecipitation or, potentially, the different constituency of SR and NE RyR complexes. In addition, given the different concentrations of mAKAP and RyR in the microsomal (P2) and nuclear fractions (Fig. 4) and the very different staining patterns for mAKAP and RyR in cells (Fig. 3), a molecularly distinct subset of RyR or a protein other than RyR that is yet unidentified must serve as the NE anchor for mAKAP.
The RyR is a large ion channel with multiple functional domains (Fig. 5A). As much as the C-terminal fifth of the protein contributes to the ion channel core, while the extensive N-terminal foot domain and the C-terminus of RyR are cytosolic and can be involved in protein-protein interactions (Franzini-Armstrong and Protasi, 1997). Although the characterization of the interaction with most RyR modifiers remains incomplete or controversial (MacKrill, 1999), the site for FKBP12.6 has been mapped to within RyR aa residues 2361-2496 (Marx et al., 2000), near or overlapping where RyR type II mutations have been found in human disease (see Results). We have begun to define the mAKAP binding domain on RyR using GST-fusion proteins (Fig. 5A,B). A GST-fusion protein containing the N-terminal 568 aa residues of type II RyR can specifically mediate mAKAP precipitation, in contrast to two other large regions of the RyR or GST alone. This N-terminal region is also the site of mutations found in patients with ARVD (Tiso et al., 2001). It remains to be determined whether the RyR-mAKAP interaction is affected in ARVD or other forms of cardiomyopathy.
The RyR has been well studied as a mediator of calcium-induced calcium release. It is tightly regulated by the endogenous ligand cyclic ADP ribose (cADPr), by Ca2+ itself, by multiple protein-protein interactions, and by protein kinases including PKA and Ca2+/calmodulin-dependent protein kinase (MacKrill, 1999). By increasing the RyR sensitivity to Ca2+ and the rate of channel closure, PKA phosphorylation of the RyR can contribute in the heart to higher amplitude, faster cycling pulses of intracellular Ca2+ during states of increased inotropy and chronotropy (Valdivia et al., 1995). PKA-dependent phosphorylation has also been associated with decreased inhibition of the RyR by the constitutively high intracellular levels of magnesium ion (Hain et al., 1995) and with the dissociation of FKBP12.6 from the cardiac RyR (Marx et al., 2000). NE RyR is probably regulated in a similar manner by mAKAP-sequestered PKA in cells, for RyR is phosphorylated by endogenous PKA in isolated native mAKAP complex (Fig. 6).
Current ion channel theory holds that elementary, transient Ca2+ currents through individual channels called puffs and sparks can be locally important and may, if frequent, give rise to generalized changes in cellular Ca2+ levels (Bootman et al., 2000). In situ Ca2+ imaging has been used to demonstrate that nucleoplasmic Ca2+ levels in cultured cardiomyocytes and isolated nuclei can be affected autonomously by NE RyR channels (Abrenica and Gilchrist, 2000; Adebanjo et al., 1999; Adebanjo et al., 2000). Ca2+-currents derived from the NE are apparently involved in the regulation of nuclear import (Jans and Hubner, 1996) and Ca2+/calmodulin-dependent protein kinase-regulated cardiac gene transcription (Chawla et al., 1998; Heist and Schulman, 1998). The mAKAP complex that includes PKA, PDE4D3, PP2A and RyR is strategically located to modulate Ca2+-regulated nuclear events (Fig. 7). cAMP may increase perinuclear Ca2+ fluxes by PKA phosphorylation of the RyR, in a manner tightly controlled by the PKA-activated PDE4D3 and PP2A-mediated de-phosphorylation. Alternatively, because the RyR is Ca2+-sensitive, it is possible the RyR serves as a sensor for ambient Ca2+ levels, controlling signal transduction to the nucleus though the other mAKAP-bound signaling enzymes and participating in a complex web of phosphorylation-induced positive and negative feedback loops.
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ACKNOWLEDGMENTS |
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