Figure 1
Expression and functional equivalence of AKAR2-AKAP5 fusion protein to native AKAP5. HEK293 cells were transfected with an expression vector harboring one of the following: either AKAR2; AKAP5; AKAR2-AKAP5 or AKAR2(T/P)-AKAP5. Cell lysates were analyzed by SDS-PAGE and resolved proteins transferred to nitrocellulose membranes. Blots were probed with antibodies specific for AKAP5 or GFP (A). HEK 293 cells were transfected with antisense morpholino oligonucleotides (AKAP5 morph) targeting the 5′ untranslated region (UTR) of human AKAP5. At 24 h later, AKAP5-deficient cells were divided into two batches: one was transfected with empty plasmid; and the other transfected with plasmid encoding AKAR2-AKAP5 fusion protein. AKAP5-deficient cells and wild-type cells each were stimulated with 10 μM isoproterenol (ISO). Activation of the mitogen-activated kinases ERK1,2 was established by activation- and phospho-specific antibody staining of immunoblots. Blots were probed with antibodies specific for either activated- and phosphorylated- pERK or pan-ERK (C). The efficiency of the antisense morpholinos used to suppress endogenous AKAP5 in these experiments is shown (B). β-actin is used as internal control for loading equivalence and was stained with anti-actin antibodies. The changes in ISO-stimulated activation of ERK in celles in which AKAP5 was knocked-down (KD) and then rescued by exogenous expression of AKAR2-AKAP5 as displayed in Panel C are plotted (D). The results displayed are mean values and s.e.m. derived from at least three separate experiments. *, denotes significance of p < 0.05.
Figure 2
Spatial localization of AKAR2-AKAP5 and AKAR2-AKAP12 in resting cells. Fluorescence images of AKAR2, AKAR2-AKAP5, AKAR2-AKAP12 and were recorded at 473-495 nm for CFP and 527-591 nm for YFP, in HEK 293 cells. The nuclei were stained with Hoechst 33258. Individual panels display the signal from CFP only, YFP only, or Hoechst only. The merged image of all three inputs is provided. Experiments were repeated at least three times with equivalent results.
Figure 3
Continuous in vivo imaging of AKAP5 and AKAP12 phosphorylation by PKA following stimulation with β-adrenergic agonist, as reported by their AKAR2 biosensor derivatives. HEK 293 (A) and A431 cells (B) were transfected with the expression vectors for AKAP5 and AKAP12 biosensors, as well as the AKAR2 sensor itself. At 24 h post transfection, cells were washed twice with DMEM with 4.5 g/L glucose and sodium pyruvate lacking L-glutamine and phenol red. Cells were treated at time = 0 with 10 μM isoproterenol (ISO). The ratios of yellow-to-cyan signals were recorded every 5 s making use of METAFLUOR software. The data are normalized by dividing all ratios by the emission ratio captured in the basal sate before the stimulation. Experiments were repeated at least three times with equivalent results.
Figure 4
Activation of AKAP5 in vivo: analysis of RII-binding site disruption and chemical inhibition of PKA. HEK 293 cells were grown on glass bottom dishes. At 24 h later, cells were transfected with expression vectors encoding either AKAR2-AKAP5 (A) or AKAR2-AKAP12 (B). At 48 h, cells were washed twice with DMEM with 4.5 g/L glucose and sodium pyruvate lacking L-glutamine and phenol red. Cells were treated with either the sterated Ht-31 peptide (50 μM), or sterated Ht-31 control peptide (50 μM) for 30 min in a humidified atmosphere of 5% CO2 and 95% air at 37°C. For FRET data, black lines trace the biosensor signal in non-treated cells; grey lines trace the signal from Ht-31control peptide-treated cells; light grey lines trace the signal from Ht-31 peptide-treated cells. Likewise, cells expressing AKAR2-AKAP5 cells were treated with PKA inhibitors, either H89 (10 μM, dots trace the FRET signal) or KT-5720 (1 μM, dashes trace the FRET signal) (B) for 10 min before ISO stimulation. Non-treated cells represent the control. At time = -25 s the monitoring of the FRET signal commenced; at time = 0, cells were stimulated with 10 μM ISO. Experiments were repeated three times with equivalent results.
Figure 5
Imaging PKA-catalyzed phosphorylation via AKAP5 versus AKAP12: response to stimulation of the cells by β-adrenergic agonist. The YFP/CFP emission ratio images of HEK 293 cells was performed in cells expressing either AKAR2, AKAR2-AKAP5, or AKAR2-AKAP12. A time = 0, isoproterenol (ISO) was applied to the cultures. The first images displayed for each array are YFP-only images. Representative pseudocolor images display the FRET signal (PKC-catalyzed phosphorylation of the AKAR2 and two AKAP biosensors in response to ISO (10 μM). The sampling was performed at the time points indicated, recorded at every 5 s for a period of 2 min using METAFLUOR software. Experiments were repeated three times with equivalent results.
Figure 6
Activation of AKAP5 is reversed by tethered PDE. Wild-type HEK cells or cells stably transfected with Δ1-145,T/P395, an N-terminally truncated AKAP5 (lacking the PCDs that function in membrane localization) with a T/P395 mutation, were co-transfected with AKAR2-AKAP5 biosensor (A). At 24 h post biosensor transfection, cells were treated at time = 0 with 10 μM ISO (A,B,C). HEK293 cells were transiently transfected with AKAR2-AKAP5 and pretreated with either vehicle alone (DMSO) or 10 μM Rolipram (a chemical inhibitor of PDE4) for 10 min. These cells were treated with 10 μM ISO at time = 0 (B). Wild-type HEK293 and cells in which PDE4D5 was knocked down both were transiently transfected with AKAR2-AKAP5 biosensor. Cells were treated with 10 μM ISO at time = 0 (C). The ratios of yellow-to-cyan were recorded at every 5 s using METAFLUOR software and normalized by dividing all ratios by the emission ratio before stimulation. Experiments were repeated three times with equivalent results.
Figure 7
Probing the role of tethered PDE in AKAP5-based PKA-catalyzed phosphorylation. Wild-type HEK293 cells were untreated (Controls) or pretreated with siRNAs targeting PDE4D5, or stably transfected to express the Δ1-145,T/P395 AKAP fragment (a PDE-binding “sink”). These different cell treatments were followed by transfection with the AKAR2-AKAP5 biosensor. An addition batch of cells transfected with an expression vector harboring AKAR2-AKAP5 were pretreated with the PDE4-specific inhibitor Rolipram (10 μM for 10 min). The images display the YFP/CFP emission ratio images of HEK 293 cells derived from the AKAR2-AKAP5 biosensor in cells stimulated with ISO. The first images of each array are YFP-only images. Cells were treated with at time = 0 with 10 μM ISO and the imaging commenced. Representative pseudocolor images show the change in FRET in response to ISO (10 μM) stimulation at various time points recorded at every 5 s by METAFLUOR software.
Figure 8
Expression of the AKAP12 engineered with Δ1-145,T/P395 AKAP5 fragment which binds PDE effectively reverses PKA-catalyzed phosphorylation. HEK 293 cells were placed on glass bottom dishes. At 24 h later, cells were transfected with plasmids encoding either AKAR2-AKAP5 (A), AKAR2-AKAP12 (B) or AKAR2-AKAP12-[Δ1-145,T/P395] biosensors. At 48 h, cells were washed twice with DMEM with 4.5 g/L glucose and sodium pyruvate lacking L-glutamine and phenol red. At time = 0 cells were stimulated with 10 μM ISO and the FRET signal collected and monitored in real time. The ratios of yellow-to-cyan were recorded at every 5 s using METAFLUOR software and normalized by dividing all ratios by the emission ratio before stimulation. Experiments were repeated three times with equivalent results.
Figure 9
Probing the role of PDE in AKAP5–mediated signaling to ERK1,2. Wild-type HEK293 cells, cells treated with siRNAs targeting either AKAP5, AKAP12 or PDE4D5 or HEK293 cells stably transfected with an expression vector harboring Δ1-145,T/P395 AKAP5 fragment, or wild-type cells pretreated with the PDE4-specific inhibitor Rolipram (10 μM for 10 min) were stimulated with 10 μM ISO for different time points. Following the 10 min time course the cells were lysed and the lysates analyzed by SDS-PAGE. The resolved proteins were blotted and probed with activation- and phospho-specific antibodies to pERK1,2. Blots also were probed with antibodies to ERK1,2 (A). Expression of proteins targeted with siRNAs was established by immunoblotting of the same blots (B). Quantification of ERK1,2 activation in the three control and experimental conditions is displayed (C). The results shown are the activity ratio compared with the control and are the mean values and s.e.m. of three or more independent experiments. *, denotes significance of p < 0.05 of a ratio compare to AKAP5 deficient (AKAP5 KD) samples.
Figure 10
Time-courses of activation of AKAP biosensor, ERK1,2, and of beta-adrenergic receptor desensitization: differential responses of AKAP5 versus AKAP12. HEK 293 cells were stimulated with 10 μM ISO and sampled at times up to 30 min. HEK (A) or A431 (B) cells were sampled for three read-outs: ERK1,2 activation, AKAR2-AKAP biosensor FRET signaling, and beta-adrenergic receptor-mediated desensitization. In HEK cells, AKAR2-AKAP5 activation, ERK1,2 activation and receptor desensitization are displayed in Panel A. In A431 cells, AKAR2-AKAP12 activation, ERK1,2 activation and receptor desensitization are displayed in Panel B. “0% desensitization” denotes the magnitude of the cyclic AMP response to a second challenge with ISO compared to the first challenge. Desensitization data are plotted from earlier time-courses [47,48].