Fig. 6. Interaction of the AKAP450 N-terminal binding domain with wild-type hRII
(A,B) and hRII(T54D) (C). A sensor chip with 60 RUs of 8-AHA-cAMP immobilized on each surface was used to capture (A) 120 RU and (B,C) 500 RU of the RII subunit to a separate flow cell. (A) AKAP450 (1390-1595) (100 nM) was run over immobilized CDK1-phosphorylated (dotted line) and unphosphorylated (solid line) RII for 180 seconds. The indicated concentrations of AKAP450 (1390-1595) were run over the immobilized wild-type hRII (B) and hRII(T54D) (C) subunits for 300 seconds and the association phases of AKAP450 (1390-1595) were monitored in 20 mM MOPS, pH 7.0, 150 mM NaCl, 1 mM DTT and 0.005% surfactant P20. The dissociation phase was monitored for another 300 seconds after omitting the AKAP from the running buffer. The immobilization of the R subunits and the regeneration are not shown. (D) Aligned sequences of the RII binding domains in AKAP450 along with the consensus binding sequence (Vijayaraghavan et al, 1999). * denotes residues that differ between the two AKAP450 domains and from the consensus.
