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.