Figure 1From: AKAR2-AKAP12 fusion protein "biosenses" dynamic phosphorylation and localization of a GPCR-based scaffold Strategy for construction of plasmid encoding a targeting biosensor, AKAR2-AKAP12. Panel A, AKAR2 (a generous gift of the laboratory of Dr. Roger Tsien (UCSD) was engineered to the N-terminal of AKAP12, in two steps. The AKAP12 N-terminal half of the sequence (nucleotides 1-2180) was inserted as a Kpn1-BamH1 fragment in to the AKAR2 pcDNA3 hygro plasmid (pAKAR2-AKAP12'), as detailed in the Methods section. The C-terminal remainder of the AKAP12 molecule (nucleotides 2181-5346, AKAP12") was inserted as a BamH1-Not1 fragment into the pAKAR2-AKAP12' plasmid to generate the final construct, AKAR2-AKAP12. The AKAR2 moiety is composed of an N-terminal CFP, the Forkhead phosphoamino acid-binding domain (blue oval), docking site and substrate domain (red oval) with specificity for PKA-catalyzed phosphorylation, and a C-terminal YFP (citrine variant) which is fused to the N-terminus of the AKAP scaffold protein (i.e., the "targeting" moiety for the biosensor), AKAP12. Panel B, the docking of the kinase and phosphorylating the substrate site lead to a strong interaction with the Forkhead phosphoaminoacid-binding domain. This phosphate group-FHA domain binding brings the CFP and YFP moieties into close proximity, enabling FRET signaling. Phosphoprotein phosphatase hydrolyzes the phosphophate group and relaxes the interaction between CFP and YFP, attenuating the capacity for FRET.Back to article page