Fig. 5. Phosphorylated RII
binds with different affinity to the RII-binding motifs in AKAP450. Purified recombinant human RII was radiolabeled using the catalytic subunit of either PKA (A) or CDK1 (B) and used for overlays on blots with different AKAPs (100 ng). Lanes: 1, GST-AKAP79 (178-427); 2, GST-AKAP149 (285-387); 3, GST-AKAP450 (1390-1595); 4, GST-AKAP450 (2327-2602) (numbers indicate the amino acid extent of the recombinant fragments used). (C) Precipitation of RII isoforms with different GST-AKAPs (20 nM of both R and AKAP). In some experiments, RII was preincubated with CDK1 before the GST precipitation. Lanes: 1, RII (50 ng); 2, RII after CDK1 preincubation; 3, GST + RII (negative control); 4, GST-AKAP450 (1390-1595) + RII; 5, GST-AKAP450 (1390-1595) + RII-P; 6, GST-AKAP450 (2327-2602) + RII; 7, GST-AKAP450 (2327-2602) + RII-P; 8, GST-AKAP79 (178-427) + RII; 9, GST-AKAP149 (285-387) + RII. Immunodetection was with anti-hRII mAb.
