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 [-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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