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mAKAP and the ryanodine receptor are part of a multi-component signaling complex on the cardiomyocyte nuclear envelope

Michael S. Kapiloff*, Nicole Jackson and Nathan Airhart

Department of Pediatrics, Heart Research Center, Oregon Health Sciences University, NRC5, 3181 S.W. Sam Jackson Park Road, Portland, OR 97201, USA



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  		Fig. 1. The subcellular distribution of mAKAP in human heart tissue. Paraffin embedded, longitudinal sections of human heart tissue were stained with affinity-purified anti-mAKAP VO56 (A) or rabbit IgG (B) and Envision polymer horseradish-conjugated secondary antibody (brown stain). Nuclei were counter-stained with hematoxylin (blue). Scale is the same for each panel. Staining shown is representative of more than three separate experiments using sections from different individuals.

 


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  		Fig. 2. Immunoprecipitation of the ryanodine receptor (RyR), protein phosphatase 2A (PP2A) and PKA with mAKAP antiserum. A 13,000 g pellet fraction of whole heart extract was solubilized with 0.5% Triton X-100 and 150 mM NaCl and cleared by repeat centrifugation. The solubilized pellet fraction was used for immunoprecipitation experiments with control preimmune serum (lane 2) or specific mAKAP VO54 antiserum (lane 3). Whole heart extract (5 µg, lane 1) and immunoprecipitates were subjected to SDS-PAGE and immunoblotting with purified mAKAP antibody and specific RyR, PLB, PP2A C-subunit and PKA C-subunit monoclonal antibodies. The migration of the respective protein and molecular weight markers are indicated for each panel. Each panel is representative of three separate experiments.

 


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  		Fig. 3. Immunocytochemistry reveals the presence of mAKAP, RyR and PKA at the cardiomyocyte nuclear envelope. Primary cultures of rat neonatal ventriculocytes, maintained at high density in media containing 100 µM phenylephrine and exhibiting spontaneous contractile activity, were stained with anti-mAKAP VO54 polyclonal antibody (mAKAP, green, A,E), anti-RyR monoclonal antibody (RyR, blue, B), anti-PKA-RII{alpha} subunit monoclonal antibody (RII{alpha}, blue, F), rhodamine phalloidin, which detects F-actin in myofibrils (actin, red, D,H,L), mouse IgG (control, blue, J), or no primary antibody (control, green, I). FITC and Cy5-conjugated donkey anti-mouse and rabbit IgG secondary antibodies were used for specific detection. Panels C,G,K are composites of the preceding two images, and panels D,H,L are the same as panels C,G,K, except with the addition of the rhodamine channel. Nuclei, identified using Hoechst stain, occupied the area outlined by mAKAP staining (not shown). All images were acquired by confocal fluorescent microscopy and are presented at the same magnification as indicated by the bar in panel L. Staining shown is representative of greater than three separate experiments.

 


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  		Fig. 4. mAKAP-RyR complex is specific to the nucleus. (A) Adult rat hearts were fractionated for comparison of the constituents of different membrane compartments. Whole heart homogenate (WH, lane 1) was centrifuged at 3800 g. The resulting pellet (P1, lane 3) contained myofibrils, mitochondria and nuclei. Nuclei (lane 5) were purified from P1 by 2.4 M sucrose step gradient centrifugation at 50,000 g and repeat low-speed centrifugation. The 3800 g supernatant fraction (S1, lane 2) was clarified at 10,000 g, before centrifugation at 100,000 g. The 100,000 g pellet (P2, lane 4) contained sarcoplasmic reticulum, Golgi apparatus and plasma membrane. 5 µg protein for each fraction was subjected to SDS-PAGE and immunoblotting with purified mAKAP antibody and monoclonal RyR antibody. Antibodies for LAP-2, a nuclear matrix protein, and calsequestrin, a SR calcium-binding protein, served as indicators for the quality of the fractionations. (B) Comparison with pure SR. 5 µg whole heart homogenate (WH, lane 1), 25 µg purified nuclei (lane 2), and 25 µg purified sarcoplasmic reticulum (SR, lane 3) were subject to immunoblotting with anti-mAKAP VO54 antibody. SR fraction was prepared from P2 fraction by sucrose gradient centrifugation. (C) mAKAP and RyR are still associated after purification of nuclei. 75 µg of purified heart nuclei were treated as starting material in immunoprecipitation experiments with control preimmune serum (lane 1) or specific mAKAP VO54 antiserum (lane 2), and RyR was detected in immunoprecipitates by immunoblotting as performed in Fig. 2. As assessed by light microscopy, the nuclei used in these experiments remained intact throughout their purification (not shown). The migration of the respective protein and molecular weight markers are indicated for each panel. n=3 for the experiments represented in each panel.

 


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  		Fig. 5. mAKAP is precipitated by an N-terminal fragment of RyR type II and the A-subunit of PP2A. mAKAP was expressed in HEK293 cells, and whole cell extracts were subjected to GST-pull down assay. Polypeptides fused to GST were full-length PP2A A-subunit and RyR aa residues 1-568, 2080-2609 and 4332-4663. (A) A schematic diagram showing the size of each RyR fragment and its location within the primary structure of the protein. For reference, the binding site for FKBP12.6 and the ion channel domain on type II RyR are indicated (see ‘Discussion’). Type II RyR is thought to be multiply phosphorylated by plural protein kinases, but only one site (Ser2809, denoted by asterisk) has been mapped, and by which kinase this residue is phosphorylated remains unclear (MacKrill, 1999; Marx et al., 2000). (B) Proteins pulled down by GST-RyR fusion proteins were subjected to SDS-PAGE, and mAKAP was detected by immunoblotting with purified VO56 antibody. Panel C. Identical experiment performed with GST-PP2A A-subunit and GST alone. The migration of mAKAP is indicated. The panels are representative of three individual experiments.

 


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  		Fig. 6. Ryanodine receptor (RyR) is phosphorylated by PKA in both anti-mAKAP and anti-RyR immunoprecipitates from whole heart. (A) Assay of endogenous protein kinase activity. Solubilized whole heart extracts were prepared as in Fig. 2, and protein complex was immunoprecipitated with mAKAP antiserum (lanes 1-5) or anti-RyR specific antibody (lanes 7-8). Immune complexes were incubated with [{gamma}-32P]ATP and cAMP in the absence (lanes 1,4,5,6,8) or presence (lanes 2,3,7) of the specific PKA inhibitor PKI. Controls (Con) included immunoprecipitation in the presence of excess mAKAP C-terminal antigen (lane 1) or with mouse IgG (lane 6). Lanes 2-5 represent duplicate reactions. Phosphorylation was detected by autoradiography after protein fractionation by SDS-PAGE and transfer to nitrocellulose. Bands containing PDE4D and RyR proteins were identified by subsequent immunodetection with the appropriate antibodies and are indicated respectively, although phosphorylated-PDE4D is only faintly detectable in the exposure shown. Molecular weights are as indicated. (B) RyR immunoblot. Equal loading was verified by immunodetection with anti-RyR antibodies. Panels are representative of three individual experiments.

 


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  		Fig. 7. Model of mAKAP complex. mAKAP associates with PKA, PDE4D3, PP2A and RyR at the cardiomyocyte NE. mAKAP is targeted to the NE by two spectrin-like repeat-containing domains (aa residues 772-915 and 915-1065) and binds PKA via an amphipathic helix at aa residues 2055-2072. mAKAP is associated with the N-terminal 568 aa of the RyR. PKA can phosphorylate RyR and PDE4D3, leading to increased ion channel and phosphodiesterase activity (a negative feedback loop; Dodge et al., 2001). PP2A may serve to reverse these phosphorylation events.

 

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