Orthogonal Dual Wavelength Photoactivation of cAMP and cGMP‐dependent Protein Kinase Signaling Pathways

The cAMP and cGMP‐dependent protein kinases (PKA and PKG) display the same consensus sequence phosphorylation preferences for protein substrates and these substrates are often members of the same signaling pathways. Although selective activators and inhibitors have been helpful in deconvoluting the downstream consequences of these highly homologous kinases, current technology is unable to discriminate, in a temporally or spatially selective fashion, between these enzymes and/or the pathways they influence. Photoactivatable (caged) compounds have been used to gain spatiotemporal control of cell signaling, however since the vast majority of these species respond to UV light it is not feasible to control two or more caged species and thus two or more nodes of a signaling network at variable time points. We describe herein the intracellular triggering of the PKA pathway with 440 nm light the corresponding PKG pathway with UV (360 nm) light. Orthogonal dual wavelength photoactivation of PKA and PKG was assessed in cell culture by monitoring the phosphorylation of vasodilator‐stimulated phosphoprotein (VASP). Activation at 440 nm elicits a PKA‐dependent phosphorylation of VASP at Ser157 while UV exposure triggers the phosphorylation of both Ser157 and Ser239. This is the first example of single‐photon, wavelength‐distinct, activation of two separate nodes of a common signaling pathway.