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.Biological behavior is inherently dynamic, be it at the cellular, tissue, organ, or organismal level.(1) The response of living organisms and their component parts to environmental stimuli often reflects the spatiotemporal elements associated with that stimulus. For example, metastasis is driven by the directed migration of tumor cells toward spatially focused chemoattractant gradients.(2) However, spatiotemporal control of biological behavior is not limited to just environmental stimuli. As a result of evolution's efficiency, individual proteins, and the biochemical pathways they inhabit, are often used for more than one purpose. The activation of a specific protein can have a multitude of biological consequences yet, within the appropriate spatiotemporal context, elicits only a single response. Cytochrome c, as a key member of the mitochondrial electron transport chain, is essential for life in eukaryotes. However, upon release from the mitochondrion, it serves as a purveyor of death.(3) Only a micron separates life from death, a fact which highlights the control exerted by the spatiotemporal context under which the biological event occurs. Unfortunately, conventional probes of cellular biochemistry, such as inhibitors, activators, or sensors, typically lack the temporal or spatial resolution to adequately address biochemically-driven behaviors, especially those that transpire during a short time frame or occur within a restricted spatial environment.A wide variety of photoactivatable (caged) compounds and biomolecules have been described since their first in...

Tunable Visible and Near‐IR Photoactivation of Light‐Responsive Compounds by Using Fluorophores as Light‐Capturing Antennas

“…[2] Furthermore, the narrow wavelength-response range limits the ability to design a family of photo-activatable species that can be orthogonally triggered at distinct wavelengths. [3] We recently reported expansion of the photo-responsive toolkit to green light (approximately 560 nm), [4] however these compounds do not absorb light that falls within the optical window of tissue. With these challenges in mind, we report herein wavelength tunable photo-responsive species based on the vitamin B 12 cobalamin ring framework.…”

“…[1,3,4] This narrow region of the electromagnetic spectrum (a) lacks the desired tissue-penetrating depth displayed by 650 – 1000 nm wavelengths, (b) inflicts bio-molecular damage which can have undesirable short and long term in vivo consequences, and (c) can support only a limited number of orthogonal wavelength-responsive agents. Although the corrin ring of B 12 is unable to efficiently absorb light beyond 550 nm, we found that commercially available fluorophores can be used as antennas to capture long wavelength light, which promotes scission of the Co-C bond at wavelengths up to 800 nm.…”

How to control proteins with light in living systems

“…Clearly, improvements are needed if one wants to precisely monitor expression and stoichiometry in any cellular context. Except for the control of different photosensitive ion channels 72,73 , we are not aware of many examples of multiplexing in the photocontrol of cellular processes [74][75][76] , but this will certainly be one of the future challenges of that nascent field.…”

Chronic nicotine treatment enhances vascular smooth muscle relaxation in rats

“…Enzyme activity assay of PKG was performed according to the manufacturer's instructions (Genmed Scientifics Inc, Arlington, MA, Shanghai Branch) by coupling the formation of ADP to the reaction catalyzed by PK and LDH in the presence of phosphoenolpyruvate (PEP) with the oxidation of NADH [19] . Briefly, assays were conducted for 5 min at 30 °C in a reaction mixture of final volume 100 µL containing aortic medial lysate (8 μL), MOPS (pH 7.5), EDTA, cGMP, magnesium acetate, ATP, PEP, NADH, PK, LDH, selective heptapeptide glasstide RKRSRAE and QKRPRRKDTP [20] .…”