FIGURE 5.

mAKAP-bound PP2A contains B56δ subunit and is cAMP-activated. A, protein complexes were immunoprecipitated from clarified adult rat heart extracts (500 μg of total protein) using control (IgG) or mAKAP-specific antibody as in Fig. 2B and assayed for associated phosphatase activity. As indicated, the immunoprecipitates were preincubated with no addition or with 50 μm CPT-cAMP, 10 nm OA, or 50 nm PKI for 5 min before the addition of [³²P]histone substrate. n = 3; *, p < 0.05. B, endogenous protein complexes were immunoprecipitated from adult heart extract (2 mg of total protein) with B56δ and control (IgG) antibodies in 80 μg of extract, and 25% of the immunoprecipitates were loaded onto the gel. mAKAP was detected by immunoblotting (IB). n = 3. C, FLAG-tagged B56δ and/or GFP-tagged mAKAP were expressed in HEK293 cells. Protein complexes were immunoprecipitated (IP) using a mAKAP antibody. B56δ in the immunoprecipitates (25% loaded) and total extracts (5% loaded) was detected by immunoblotting with a FLAG antibody. n = 3. D, phosphatase activity associated with mAKAP-antibody immunoprecipitates prepared as in C was assayed using ³²P-labeled histone substrate. n = 3. E, HEK293 cells expressing mAKAP and B56δ were treated with 5 μm Fsk and 10 μm IBMX (Fsk/IBMX) for 10 min before immunoprecipitation of protein complexes with mAKAP antibody. Phosphatase activity associated with the immunoprecipitates was assayed using [³²P]histone substrate. n = 3. Note that PP2A B56δ and C subunit binding to mAKAP was not affected by Fsk/IBMX (see Fig. 6).