Dual wavelength photoactivation of cAMP- and cGMP-dependent protein kinase signaling pathways

Abstract

The spatial and temporal organization of biological systems offers a level of complexity that is challenging to probe with conventional reagents. Photoactivatable (caged) compounds represent one strategy by which spatiotemporal organizational complexities can be addressed. However, since the vast majority of caged species are triggered by UV light, it is not feasible to orthogonally control two or more spatiotemporal elements of the phenomenon under investigation. For example, the cGMP- and cAMP-dependent protein kinases are highly homologous enzymes, separated in time and space, which mediate the phosphorylation of both distinct and common protein substrates. However, current technology is unable to discriminate, in a temporally or spatially selective fashion, between these enzymes and/or the pathways they influence. We describe herein the intracellular triggering of a cGMP-mediated pathway with 360 nm light and the corresponding cAMP-mediated pathway with 440 nm light. Dual wavelength photoactivation was assessed in A10 cells by monitoring the phosphorylation of vasodilator-stimulated phosphoprotein (VASP), a known substrate for both the cAMP- and cGMP-dependent protein kinases. Illumination at 440 nm elicits a cAMP-dependent phosphorylation of VASP at Ser157, whereas 360 nm exposure triggers the phosphorylation of both Ser157 and Ser239. This is the first example of wavelength-distinct activation of two separate nodes of a common signaling pathway.

Fig. 1

Dual Wavelength Photoactivation of PKA and PKG Pathways. Top: coumarin-caged cAMP is photolyzed with 440 nm light resulting in the activation of PKA and subsequent phosphorylation of VASP at Ser157. Bottom: nitrobenzyl-caged PKG is photolyzed with 360 nm light resulting in phosphorylation of VASP at Ser157 and Ser239.

Fig. 2

UV scans of nitrobenzyl bromide (black) and the coumarin (grey) caging groups overlaid with the transmittance of the UV (blue) and 440 nm (green) bandpass filters. Absorbance of nitrobenzyl bromide and coumarin caging units was normalized setting the major peak at 100.

Fig. 3

Photoactivation of nitrobenzyl-caged PKG and coumarin-caged 8-Br-cAMP. (a) Fold increase in specific activity of caged PKG due to photolysis using either the 360 nm bandpass filter (solid ●) or the 440 nm bandpass filter (dashed ▼). (b) Fold increase in specific activity of PKA holoenzyme due to photoactivation of caged 8-Br-cAMP with either the 360 nm bandpass filter (solid ●) or the 440 nm bandpass filter (dashed ▼). Data are represented as averages with standard errors of three independent assays.

Fig. 3

Photoactivation of nitrobenzyl-caged PKG and coumarin-caged 8-Br-cAMP. (a) Fold increase in specific activity of caged PKG due to photolysis using either the 360 nm bandpass filter (solid ●) or the 440 nm bandpass filter (dashed ▼). (b) Fold increase in specific activity of PKA holoenzyme due to photoactivation of caged 8-Br-cAMP with either the 360 nm bandpass filter (solid ●) or the 440 nm bandpass filter (dashed ▼). Data are represented as averages with standard errors of three independent assays.

Fig. 4

Photoactivation of nitrobenzyl-caged PKG leads to phosphorylation of Ser157 and Ser239 on VASP in A10 cells. Quantification of the immunofluorescence of phospho157VASP (a) and phospho239VASP (b) in A10 cells microinjected with caged PKG. (c) Immunofluorescent images of A10 cells microinjected with caged PKG. A10 cells were either stimulated with 100 μM 8-Br-cGMP or microinjected with 50 μM caged PKG. Data are represented as averages of 5-25 cells with standard errors. Scale bar is 40 μm.

Fig. 4

Photoactivation of nitrobenzyl-caged PKG leads to phosphorylation of Ser157 and Ser239 on VASP in A10 cells. Quantification of the immunofluorescence of phospho157VASP (a) and phospho239VASP (b) in A10 cells microinjected with caged PKG. (c) Immunofluorescent images of A10 cells microinjected with caged PKG. A10 cells were either stimulated with 100 μM 8-Br-cGMP or microinjected with 50 μM caged PKG. Data are represented as averages of 5-25 cells with standard errors. Scale bar is 40 μm.

Fig. 4

Photoactivation of nitrobenzyl-caged PKG leads to phosphorylation of Ser157 and Ser239 on VASP in A10 cells. Quantification of the immunofluorescence of phospho157VASP (a) and phospho239VASP (b) in A10 cells microinjected with caged PKG. (c) Immunofluorescent images of A10 cells microinjected with caged PKG. A10 cells were either stimulated with 100 μM 8-Br-cGMP or microinjected with 50 μM caged PKG. Data are represented as averages of 5-25 cells with standard errors. Scale bar is 40 μm.

Fig. 5

Photoactivation of coumarin-caged 8-Br-cAMP leads to phosphorylation of Ser157 and Ser239 on VASP in A10 cells. Quantification of the immunofluorescence of phospho157VASP (a) and phospho239VASP (b) in A10 cells loaded with caged 8-Br-cAMP. (c) Immunofluorescent images of A10 cells loaded with caged 8-Br-cAMP. A10 cells were either stimulated with 100 μM 8-Br-cAMP, 100 μM 8-Br-cGMP or loaded with 100 μM caged 8-Br-cAMP. Data are represented as averages of 5-25 cells with standard errors. Scale bar is 40 μm.

Fig. 5

Photoactivation of coumarin-caged 8-Br-cAMP leads to phosphorylation of Ser157 and Ser239 on VASP in A10 cells. Quantification of the immunofluorescence of phospho157VASP (a) and phospho239VASP (b) in A10 cells loaded with caged 8-Br-cAMP. (c) Immunofluorescent images of A10 cells loaded with caged 8-Br-cAMP. A10 cells were either stimulated with 100 μM 8-Br-cAMP, 100 μM 8-Br-cGMP or loaded with 100 μM caged 8-Br-cAMP. Data are represented as averages of 5-25 cells with standard errors. Scale bar is 40 μm.

Fig. 5

Photoactivation of coumarin-caged 8-Br-cAMP leads to phosphorylation of Ser157 and Ser239 on VASP in A10 cells. Quantification of the immunofluorescence of phospho157VASP (a) and phospho239VASP (b) in A10 cells loaded with caged 8-Br-cAMP. (c) Immunofluorescent images of A10 cells loaded with caged 8-Br-cAMP. A10 cells were either stimulated with 100 μM 8-Br-cAMP, 100 μM 8-Br-cGMP or loaded with 100 μM caged 8-Br-cAMP. Data are represented as averages of 5-25 cells with standard errors. Scale bar is 40 μm.

Fig. 6

Wavelength-selective triggering of PKA and PKG in A10 cells. Quantification of (a) phospho157VASP and (b) phospho239VASP via immunofluorescence in A10 cells loaded with caged 8-Br-cAMP and/or microinjected with caged PKG. (c) Images of A10 cells loaded with caged 8-Br-cAMP and/or microinjected with caged PKG. A10 cells were either stimulated with 100 μM 8-Br-cAMP, 100 μM 8-Br-cGMP, loaded with 100 μM caged 8-Br-cAMP and/or microinjected with 50 μM caged PKG. Data are represented as averages of 5-25 cells with standard errors.

Fig. 6

Wavelength-selective triggering of PKA and PKG in A10 cells. Quantification of (a) phospho157VASP and (b) phospho239VASP via immunofluorescence in A10 cells loaded with caged 8-Br-cAMP and/or microinjected with caged PKG. (c) Images of A10 cells loaded with caged 8-Br-cAMP and/or microinjected with caged PKG. A10 cells were either stimulated with 100 μM 8-Br-cAMP, 100 μM 8-Br-cGMP, loaded with 100 μM caged 8-Br-cAMP and/or microinjected with 50 μM caged PKG. Data are represented as averages of 5-25 cells with standard errors.

Fig. 6

Wavelength-selective triggering of PKA and PKG in A10 cells. Quantification of (a) phospho157VASP and (b) phospho239VASP via immunofluorescence in A10 cells loaded with caged 8-Br-cAMP and/or microinjected with caged PKG. (c) Images of A10 cells loaded with caged 8-Br-cAMP and/or microinjected with caged PKG. A10 cells were either stimulated with 100 μM 8-Br-cAMP, 100 μM 8-Br-cGMP, loaded with 100 μM caged 8-Br-cAMP and/or microinjected with 50 μM caged PKG. Data are represented as averages of 5-25 cells with standard errors.