Figure 1
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.
Figure 2
Imaging of AKAR2 expressed in human epidermoid carcinoma A431 cells. Panel A, images of AKAR2 biosensor when expressed in A431 cells. The fluorescence images were recorded at 473-495 nm for CFP, and, 527-591 nm for YFP (citrine). Panel B, AKAR2-based fluorescence images were recorded at 473-495 nm for CFP and 527-591 nm for YFP (citrine), in A431 cells in which the nuclei were stained with DAPI. The DAPI recordings were performed at 435-485 nm. The images shown are representative of a large array of images collected for this purpose.
Figure 3
Expression of AKAR2-AKAP12 fusion protein in A431 cells: Mr, function, and imaging. A431 cells were transfected with an expression vector plasmid harboring one of the following cDNAs: AKAR2; AKAP12; and AKAR2-AKAP12. Panel A (top), the expression of AKAR2, AKAP12, and AKAR2-AKAP12 was examined. The stained immunoblots and the approximate Mr of each resolved band is indicated. Panel A (bottom), A431 cells were challenged with β-agonist (isoproterenol, 10 μm) for 5 min and the intracellular cAMP (pmol/105 cells) represents the normal response of naïve A431 cells (at time = "0"). Upon a 30 min challenge with isoproterenol, cells became desensitized to a second, subsequent challenge with the β-agonist (30). The agonist was then washed out and the cells were allowed to recover. The "resensitization" was measured in cells at 60 min following the washout (W60). This activation, desensitization, wash out and recovery were performed on A431 cells alone (Control), on AKAP12-deficient cells (i.e., KD AKAP12), and on AKAP12-deficient cells transiently expressing AKAR2-AKPA12. The results, displayed as mean values ± S.E., are of at least three separate experiments. Panel B, imaging of cellular distribution of AKAR2-AKAP12 in A431 cells. The fluorescence images were recorded at 473-495 nm for CFP and 527-591 nm for YFP. Nuclei stained with DAPI were recorded at 435-485 nm. The images shown are representative of a large array of images collected for this purpose.
Figure 4
AKAR2-AKAP12 fusion protein biosensor: FRET signaling in response to beta-adrenergic stimulation. Stably transfected A431 clones expressing AKAR-AKAP12 were serum starved over night and then stimulated with beta-adrenergic agonist (isoproterenol, 10 mM) for 30 min. Image and fluorescence density were recorded for CFP and YFP in the absence and following the 30 min challenge with isoproterenol (+ISO, 30 min). The recordings for CFP and YFP were sampled at 10 sec intervals. The images shown are representative of a large array of images collected for this purpose.
Figure 5
FRET-based "biosensing" by AKAR2-AKAP12 in response to beta-adrenergic agonist: effects of addition of PKA inhibitor KT5720. Stably transfected A431 clones expressing AKAR-AKAP12 were serum starved over night and then stimulated with beta-adrenergic agonist (isoproterenol, 10 μM) for 30 min. Upper panel, images were recorded for CFP and YFP in the absence (0), following the 30 min challenge with isoproterenol (+ISO, 30 min), and in the presence of isoproterenol for 30 min, then treated with PKA inhibitor KT5720 (+ISO 30 min, +KT5720) The measurements were made in the perinuclear, cytoplasmic area (open red circle) in which robust FRET signals from activated AKAR2-AKAP12 are observed. Lower panel, the fluorescence density measurements were collected from a large array of images collected for this purpose. The fluorescence density of CFP in the same area of the cells at basal state (-ISO) is highlighted by a red dashed line.
Figure 6
FRET-based "biosensing" by AKAR2(T/P) mutant-AKAP12 in response to beta-adrenergic agonist. Stably transfected A431 clones expressing AKAR2(T/P) mutant of AKAP12 (lacking the phosphorylation site for PKA in the substrate domain of the AKAR2 moiety) were serum starved over night and then untreated (-ISO) or treated with beta-adrenergic agonist (isoproterenol, 10 μM for 10 min (+ ISO 10 min). Upper panel, images and fluorescent densities recorded for CFP and YFP are displayed. The measurements were made in the perinuclear, cytoplasmic area (open red circle), an area in which robust FRET signals from activated AKAR2-AKAP12 are observed. Lower panel, the fluorescence density measurements were analyzed from a large array of images collected for this purpose. The fluorescence density of CFP in the same area of the cells at basal state (-ISO) is highlighted by a red dashed line.
Figure 7
FRET-based "biosensing" by AKAR2-AKAP12 in response to beta-adrenergic agonist: effects of HT-31 peptide interference of AKAP/RII subunit-binding. Stably transfected A431 clones expressing AKAR-AKAP12 were serum starved over night, pre-treated with liposomes loaded with the HT-31 peptide (50 μM loading), and then stimulated with beta-adrenergic agonist (isoproterenol, 10 μM) for 10 min. Upper panel, images were recorded for CFP and YFP from the HT-31 peptide-treated cells that were then incubated either without isoproterenol (-ISO), or with isoproterenol (+ISO, 10 μM) for 10 min. The measurements were made in the perinuclear, cytoplasmic area (open red circle), an area in which robust FRET signals from activated AKAR2-AKAP12 are observed. Lower panel, the fluorescence density measurements were collected from a large array of images collected for this purpose. The fluorescence density of CFP in the control cells (-HT31) at basal state (-ISO) is highlighted by a red dashed line.
Figure 8
FRET signaling from AKAR2-AKAP12 fusion protein "biosenses" dynamics of phosphorylated, activated AKAP. Panel A, A431 cells were stably transfected with AKAR2-AKAP12. Cells were put in serum starvation for 12 hrs before the recording. Images were recorded for CFP and YFP in absence and presence of isoproterenol (10 μM) and sampled from time = "0" min until time = 30 min. The sampling of the FRET was confined to two cellular locales: the cell membrane (open red rectangles) and the perinuclear, cytoplasmic regions (open yellow circles). The images shown are representative of a large array of images collected for this purpose. Panel B, the fluorescence densities in cell membrane and perinuclear areas were recorded for CFP and for YFP in absence and presence of beta-adrenergic agonist. The data sets are from many samplings, performed on individual cells, and displayed as a representative fluorescence density scan over time.